Hypothesis of Mechanistically Related Nitric Oxide Involvement of Heparan Sulphate in Degenerative Diseases & Animal Evolution Chronic stress

may alter RNA and DNA via an ascorbate modulated heparan sulphate/nitric oxide dependent mechanism. David Grant* To bottom *Address for correspondence Ashbank, 2a High Street, New Deer, Turriff, AB53 6SX, United Kingdom, This literature-based research (part funded by the Carnegie Foundation for the Universities of Scotland) was conducted after tenure of a Research Fellowship with Dr FB Williamson and Dr WF Long at the University of Aberdeen Department of Molecular and Cell Biology. Prof. KEL McColl (Univeristy of Glasgow, Department of Medicine & Therapeutics) is thanked for discussions. Draft by DG 7/02, 8/02 For Medical Hypothesis Post 30/7/02 modifications 1. Summary A necessarily tentative hypothesis is presented in which the already large range of of controls known to be exerted by the system of sulphated polysaccharides heparin/heparan sulphate proteoglycans(HSPGs) is extrapolated to include animal evolution and degenerative diseases, including cancer. These are suggested to be similarly promoted by stress events which stimulate a nitric oxide (NO)-dependent, postsynthetic non-enzymic processing of heparan sulphate (HS) allowing this, together with interactive effects generated with a parallel system dependent on enzymic postsynthetic HS processing, to enable cross reaction between the two HS processsing systems to generate amplification of long-term feedback loops. This enables altered Golgi HS biosynthesis for alteration genetic control element modulation (e.g. by HS fragment binding to clusters of basic amino acids in nucleic acid control elements). It is suggested that the existence of this dual control mechanism of HS processing, while permitting directed animal evolution, also permits, by disfunction of this system, the occurrence of various degenerative diseases. Directed alteration for stress minimization may be especially directed at in HS processing, sulphatase enzymes implicated in tissue morphogenesis. Additional discussion of HS-related controls includes a role for ascorbate in tissue protection both in by its effect modulating NO activity and by directly influencing heparin/HS biosynthsis and interaction of HS systems with cysteine and tyrosine based signalling systems.

2. Introduction. HS, biosynthesised exclusively as heparan sulphate proteoglycans (HSPGs) seems to have evolved coincidentally with the initital appearance of multicellular animals. This hint that the evolution of multicellular animals could somehow be dependent on HSPG biochemistry can now been seen to be consistent with the emerging knowledge of wide-ranging HSPG functional roles in cell-tocell interactions in developmnetal biology. Proposals for debate include the notion that HSPGs contibute to the response of genes to stress. An initial result of evolutionary pressures by NO and its metabolites is suggested to be an alteration in HS signalling producing a prompted targeted modulation of HS processing enzymes (which are known control the formation of animal development HS morphogens) via feedback loops linking genes with their eventual products via HS oligosaccharides which are formed both by the non-enzymic but highly stress-dependent NO processing and by parallel enzymic processing. Additional possible key mechanisms relevant to transmission of oxidative stress to genes, however, may include those proposed to be tranmitted by switching systems containing tyrosine and cysteine which are capable of interacting both with HS (including fragment) and with NO (and its metabolite) facilitating environmental and genetic interfacing. Cysteine and tyrosine (CY) groupings e.g. in SH2 protein domains are implicated in intracellular signalling. C..Y systems also occur in RNA processing enzymes including RNAase. Such (CY) systems are proposed to be subject to HS as well as a NO (and metabolites) dependence on (oxidative stress related) including trace redox metal catalysis of the formation of tyrosine nitrate.

3. General Background 3-1 Importance of HS in Multicellular Animal Biochemistry. HSPG is involved in multicelluar animal development, haematopoiesis and for mRNA control. The HS system of sulphated polysaccharides (cf Dietrich et al, 1983) occurring at all adherent animal cell surfaces as HSPGs (which an emerging consensus of recent work suggests to be precise-sugarsequence-dependent information carriers specifying each of cell type by unique HS patterns) has been so highly conserved throughout animal evolution, that this suggests that there must exist some major currently unknown function for HS, in addition to the growing number of known activities. It is now proposed, that this is the faculty of directly influencing animal evolution by the provision of feedback loops involving an influence of HSPG and its fragments upon selective alteration in gene activity. Such an HS-driven model of animal evolution is consistent with the HSPG modulation of kinase activities in growth factors and their receptors, involved in the processing of extracellular signalling, that are now known to pattern fields of cells in the developmental biology of animals (Perrimon & Bernfield 2000), including a dependence of embryogenesis on extracellular HSPG sulphatase activity (Dhoot et al 2001). (A corollary to this idea is that degenerative diseases including cancer are aberrant consequences of a similar process. Uncontrolled tumour growth may be caused by an alteration in HS gene control function (cf Dundas et al 2000) which is potentially subject to influence by the effects of oxidant/nitrant stress). 3-2 Effects of Reactive Oxygen Species Multicellular animal evolution seems to have been stimulated by the emergence of photosynthetic organisms capable of producing sufficient amounts of atmospheric oxygen and the possible formation of nitric oxides to promote the necessary stress factor-related catalysis of the generation of reactive oxygen and nitrogen intermediates capable of prompting environmental-directed alteration in RNA for eventual reverse transcription into genetic DNA. 3-3 Effects of Nitric Oxide & its Metabolites Oxidant stress has long been a central theme in medicine and biology, this having likely effects on animal lifespan and the occurrence of degenerative diseases such as cancer. In recent years, however, additional related effects of nitric oxide and its metabolites have been discovered which seem to uniquely involve HSPG biochemistry and which are also implicated in anti-pathogen, anti-crystal, and antioxidant defenses which all additionally potentially involve the effects of induced immunologically produced NO biochemical activity. A NO-dependent HSPG scission reaction is specifically directed towards HS chains in GAG mixtures. This reaction was employed in laboratory protocols for evaluating HS by oligosaccharide formation in GAG mixtures long before the discovery that such reactions had any physiological relevance. 3-4 Ascorbate in Control of HS and NO Critical control of reactive oxygen and reactive nitrogen oxide chemical species is clearly necessary for the maintenance of cellular integrity, evidently accomplished in animals by a number of complimentary systems which include important activities of superoxide dismutases (extracellularly bound to HSPG), as well as the major antioxidant ascorbate, which both demonstrates major influences on HSPG biochemistry, directly via enhancing the rate of biosynthesis and degree of sulphation, as well, indirectly, by controlling the rate of postsythetic deaminative cleavage by nitric oxide and its metabolites. NO itself is also a powerful antioxidant (e.g. Kanner et al 1991), it preventing the oxidative degradation of cell surface lipids more effectively than does alpha tocopherol (Rubbo et al 2000). Ascorbate efficiently reduces nitrite back to NO. 3.4.1 Effects of Lipids Lipids also signal for altered HSPG biosynthesis evidently by affecting Golgi protein transport. Deleterious effects of e.g. saturated lipids may be at least partly accounted for, it is suggested, by such effects, which may also explain beneficial effects of e.g. omega three lipids on alteration of HSPG in an anti-cancer role may partly be due to such HSPG alteration. In a related scenario oxidised lipids may signal for a deleterious pro-alteraiosclerosis alteration in HS biosynthesis at blood vessel walls. Excess serum glucose may similarly produce deleterious HS-dependent lipid procssing via biosynthetic modulatoin by excess glucose for reduction of sulphation of HS.

3-5 Effects of Polyamines. Another function of NO which together with ascorbate is apparently to boost Golgi-controlled HS biosynthesis and increase sulphation, which further contibute to a complex HS- antioxidant-related biofeedback mechanisms, apparently includes the scission of heparan sulphate at specific sites by NO metabolites specified by polyamines (Ding et al 2001)). 3-6 Effects of Cysteine-Tyrosine Coupling. A potential mode of coupling between oxidant stress signals, HSPG and genes is via (CY) signalling systems (cf Nakashima, 1996). Such coupled (CY) redox metal systems for the facilitation of signalling likely even preceded genetic use of DNA since ribonucleotide reductase which requires for its biosynthesis the pre-existence of this type of redox-based activity. RNAases also contain conserved (CY) groupings. The (CY) systems are also chemically related to the industrial antioxidant phenolic and bivalent sulphur systems, which can interact synergistically, and also to alpha tocopherol, glutathione and, it is believed, various clusters of cysteine and tyrosine (CY) in proteins including growth factors (discussed by Grant et al (1989)). An important antioxidant function proposed for (CY) containing apoliprotein a which apparently is uniquely elevated in animals such as man which lack the ability to synthesise ascorbic acid (Rath & Pauling 1990) but which also suggests that ascorbate-like HSPG control feedback signalling may also be an inherent property of such (C..Y) systems. HSPG/NO-ascorbate/(CY)- based systems are therefore suggested to be capable ot inducing initial alterations in Golgi apparatus biosynthesis of those HSPG sequences which are potentially designated, following fragmentation to competetivly bind to basic amino acid clusters in targeted RNA and DNA control elements and especially specifying those proteins which are most affected by the applied stress. Those genes specifiying e.g. heparanse but perhaps more especially sulfatase enzymes involved directly in organism development may be sensitive to such directed rearrangements. 3-7 HSPG Genetic Transfection? Genetic transfection processes may also potentially involve related HSPG activities since cell surface HSPG-gene polycation complexes facilitate the entry of genetic material into distant target cells (Mislick et al 1996; Mounkes et al 1998) provide a possible route for evolutionary directed change e.g. following apoptosis, to germ cells. Related effects of pathogens include HSPG binding bacterial (e.g. Helicobacter pylori) and HSPG binding viruses, which are further potential contributors to tranfer of genetic material via HSPG dependent pathways including reverse transcriptase activities of retroviruses. 3.7.1 Prion biochemistry seems to have a fundemental HSPG linked perspective, the details of which are yet, however, incompletely understod, but may be relevant to this discussion. The use by host organisms of viral reverse transcriptase activities also seems a suitable potential mechanism of gene modulation. 3-8 Such newly proposed heparin/HSPG functions, as well as the long-established roles in haemostasis, lipid processing, immune responses and angiogenesis will potentially be dependent on signalling by specific heparin/HS microstructures, especially sulphation patterns, which are apparently cell type specific but are subject to an evidently finely contolled alteration during biosynthesis being apparently potentially affected by lactate/pyruvate ratio, and the extracellular levels of glucose glucosamine, ascorbate, toxins, trace metal ions, types of lipids, lipid peroxidation products and perhaps oncogenic products (since transformed cells exhibt major alteration in HSPGs). Postsynthetic HSPG processing is also known to occur directly by action of reactive oxygen species as well as by the specific enzymic actions of heparanases and sulfatases. This is in addition to the well known, highly heparin/HSPG-specific non-enymic processing facilitated by NO (and its metabolite especially nitrous acid) afforded GlcNH2 deaminative cleavage reaction and which recent work has indicated, may be programmed via polyamine signalling (Ding et al 2001) but is probably hypersensitive to a direct catalytic influence by stress factors (especially by trace metals) and augmented by additional NO formation induced by immune responses. Acidosis, a form of stress is known to promote the deaminative cleavage of HS as is the (additional) presence of unprotected trace amounts of redox metal ions (such as those of iron and copper). These situations directly increases unsulphonated glucosamine moieties in HSPG primed for deaminative cleavage. These situations also likely increase

the incidence of tyrosine nitration (which although a hallmark of degenerative processes does not form by reaction of tyrosine with peroxynitrite at pH 7.4 in vitro in absence of catalysis). The individually specificfied cellular dependence of arranged anionic charge arrays present in HS polysaccharide chains seems to provide a system of extracellular communication allowing an unique specification of individual cell typing including patterning for organ and organism speciation. Rather than DNA biochemistry being the sole determinant of the aetilogy of degenerative diseases such as cancer and in determining how directed evolution might be possible, HSPG biochemistry is now suggested to participate in stress-directed controlled intracelluar nucleic acid rearrangements via the effect of NO modulated HSPG-dependent morphogenesis processes which are capable by amplified feedback signalling via alteration in the Golgi biosynthesis of HS to eventually influencing genetic controls. Since postsynthetic alteration of HSPG seems most higly perturable and an almost haphazard process, such rections are suggested be a useful focus of future research directed at more fully understanding the aetiology of degenerative diseases suvh as cancer. 3-9 Osmotic Stress & Hofmeister Effects. In addition to oxidant stress, osmotic stress may be countered by HSPG-related effects. There are hints (cf Graham, 1994) of an interrelationship between HSPG biochemistry and that of heat shock proteins (HSP) which share with HS the common property of having been conserved over long evolutionary times but have uncertain, but surely major, biological functions. Acute heat shock intiates the phenomenon of stress prompted glycosylation of proteins (cf Jethmalean et al 1994). Similar considerations perhaps also apply to the glucose regulation of gene expression. Modulation of aqueous solutions activities is relevant to their possible chaperone duties of prevention protein denaturation. Hofmeister ranked salts for such abilities. The aggregate structure of aqueous solutions follows the Hofmeister series (Luck 1965) of relevance to the nature of hydrated heparin HSPG/polysaccharides/counterion /water aggregate associations which may form with osmotic-related stress factors.

Part 2 More Specific Discussion of Research Reports An example of possible feedback opportunity between HSPG and gene structure is apparent in the extracellular sulfatase HSPG processing genes which are involved in postsynthetic HSPG processing and therefore openly exposed to stress factor effects. Extracellular processing of HSPG is known (Dhoot et al (2001) to occur by desulphation of GlcNAc,C6(-O-SO3-) for specifying Wnt signalling in avian embryo patterning. Qsulf sulphatase which contains a conserved C-Y motif at the active site cysteine (which when post-translationally modified to N-formylglycine, hydrolyses HSPG GlNAc, C6O-SO3- groups). C-Y motifs could also further permit modulation of Qsulf activity by ligand binding e.g. by heparin and related molecules which are known to bind to the C-Y motifs in epidermal growth factor (EGF) and related molecules (vide infra). Abnormal heparanase activity (experimentally induced) completely prevented CNS and mesodermal tissue development in Xenopus embryos (Furuya et al 1995). It is apparent that less severe effects of modulated heparanse activities could induce non-fatal perhaps beneficial alterations in tissue of relevance to evolutionary situations. An example of stress factor cellular activity subject to postsynthetic alteration in HSPG is provided by the cellular adhesion of alveolar cells which is dependent on cultured cell density, implicating interactions between HSPG GlcNSO3- groupings (Lin et al, 2000) which are subject to extracellular modification e.g. by NO or nitrous acid deaminative cleavage and/or desulphonation by acidosis. Both processes are also believed (Grant et al, unpublished) to be catalysed by those trace redox ions elevated during stress or intoxicantion which are suspected to promote cancer by induced alteration in DNA.

Regulatory oligosaccharides for angiogenic signalling evidently derive from severe post synthetic degradation of endothelial HSPG. These include unsulphated glucuronic acid rich fragments resembling a putative ancestral primitive bacterial capsular polysaccharide persisting in mature HSPGs. Hyaluronic acid with isomeric beta 1,3 and beta 1,4 linkages was inactive (Hahnenberger et al 1993).

Metal Ion HSPG Interactions. Divalent Cation Modulation of HSPG Activity Fibroblast growth factors (FGFs), which have accorded a particular focus of intensive research activity, are known to employ heparan sulphates in complex control mechanisms, including the action of the heparin-like segments for a putative divalent cation dependent control of autophosphorylation of tyrosine (Kan et al 1996, 1999) in receptors. Such divalent cation dependence suggests that possible stress via pathological alteration in the Ca/Mg ratio or Mn intoxication may influence such growth factor activity and be relevant to considerations of mechanisms of transmission of environmental stress signals. The adhesion of cells likely also includes HSPG, Ca2+-dependent coupling between receptors in different cells (cf Richard 1995) as well as HSPG-Ca2+-HSPG effects (cf transfection studies of syndecan-1 by Stanley et al 1995), a Ca2+ dependent interacton of L-selectin with heparin-like ligands (Norgard-Sumnicht et al 1993) and a Ca2+ dependent binding to immunoblogin superfamily platelet/endothelial cell adhesion molecule (PECAM) to HSPGs (and chondroitin sulphate) (DeLisser et al 1993) - Extracellular Ca2+ directs HSPG biosynthesis, at least for parathyroid cells (cf Takeuchi et al 1992). Such involvement of Ca2+ in HSPG biochemistry is consistent with the prediction (Long & Williamson 1979) of a focus for HSPG biochemistry involving iduronate conformation dependent bivalent cation signalling consistent with the original nmr results (Boyd et al 1979) which have been generally confirmed by later research. Anion effects include possible relevant effects of F- intoxication (reported by Susheela & Jha (1981) which were reported to increase the overall degree of sulphation but diminished the ratio of gluosamine/galactosamine incorporation into the glycosaminoglycans of bones). Chlorate is used in laboratory techniques to biosynthetically reduce HS sulphation . Selenate also alters HS biosynthesis (Dietrich et al DATA.) However, attempts to change the sulphation of BHK HS by alteration of cell culture medium sulphate concentration failed to show an effect (Grant et al unpublished). A mechanism whereby conformational effects of Na+/Ca2+ metal ion interaction at endothelial wall heparan sulphates may be used by the organism for flow sensing has been described (Siegel et al 1998); fluid shear forces (as applicable to blood flow) have also been found to alter heparan sulphate proteoglycan biosynthsis (Elhadj et al 2002) and nitric oxide production by bovine endothelial cells (Korenaga et al 1994) Such a mechanism may permit stress factors causeing abnormal flow patterns to alter HSPG biosythesis.

HSPG in Cell Nuclei The occurrence of glycosaminoglycans at the cell nuclei (e.g. Bhavanandan & Davidson, 1975), the alteration of nucleosome conformation and activity by heparin (Brotherton et al 1989) and the binding of nucleosomes at the cell surface by HSPGs (Watson et al 1999) provide evidence for a likely involvement of HSPGs in gene processing consistent with the known involvement of heparin/HSPG in fertilisation (including a chromatin decondensation effect of heparin) and embryology (Perrimon & Bernfield 2000) which may implicate conseved cysteines Zn fingers (Zn binding and transport might be facilitated by heparin like fragments since heparin has been known for some time to preferentially bind Zn (Parrish & Fair,1981)) (Zn also facilitates heparin kininogen binding (Renne/ et al, 2001). Reduced ability to control nuclear transcription activators (e.g. AP-1. SP-2, ETS-1 and nuclear factor kappa B) by defective HS in tumour cells may contibute to their uncontrolled proliferation (Dundas et al 2000). Interactions between HSPG and RNA involving destabilisation of the latter have been reported to modulate cytokine expression in primate bone marrow stromal fibroblasts (Yang et al 1998). Hence an involvement in multifunctional roles utilising a repertoire of uniquely flexible potential responses capable of generating biofeedback signals suggests that for the heparin/HSPG system a

functional capability exists similar to an assumed required complex fuzzy logic processor capable of organism stress minimisation over long-term coupled interactions between the environment, HSPG and DNA Such a smart sulphated polysaccharide physiochemical property is proposed to allow responsive gene restructuring directed by the unique HSPGs systems which contribute to long range communication and generate effective logical response to stress throughout the whole organism or even amongst groups of organisms (e.g. via the effect of genetic alteration produced by host organisms).

Addendum. Heparin binds with moderate strength under physiological saline conditions to the C-Y kringle domain in urokinase (Stephen et al 1992) suggesting that a related C-Y, but moderate strength binding to HS/HSPG may facilitate the storage and release of FGF from reservoirs in the extracellular matrix (Bashkin et al 1989) and in other growth factors related to epidermal growth factor (EGF which demonstrate well defined C-Y motifs (Grant et al in 1989) and demonstrate GAG enhancement of its angiogenic activity (Sato et al 1991) as well as heparin-dependent human cancinoma cell growth inhibition (Halpern & Carter 1989). Such C-Y structures are also present in the immunoglobin superfamily and may participate in relevant HSPG interactions. Heparin binding also occurs with the heparin binding epidermal like growth factor (HB-EGF) of macrophage origin, a smooth muscle cell mitogen, the transcription of which is markedly increased by TNFalpha and IL1, perhaps directly promoting atherosclerosis (as suggested by Yoshizumi et al 1992), however, since the expression of inducible NO synthase is also promoted by TNFalpha and IL-1 (as well as by IFN gamma, and IL-2 but suppressed by TGFbeta, IL4, IL8, IL10 and IL13) which might suggest that NO and its metabolites are also involved in the aetiology of this disease since NO is implicated in the promotion of related diseases (Stichentoch & Frolich 1998), the aetiology of which may include an inappropriate degradation of HSPG by excessive NO production. NO is established to be widely employed in various immune and signalling responses (which include inputs from heparin/HS biochemistry and gene activation. A characteristic marker of such NO activity in rheumatic diseases is the nitration of tyrosine (e.g. altering CY) dependent switching. Perhaps the least evolved effects of stress on HSPG is via NO and its metabolites and other oxidative stresses which are also generated by chronic inflammatory situations. The latter are associated with acidic pH conditions which cause inappropriate degradation of heparin and HS. Deaminative cleavage under acid conditions often associated with inflammation is primed by a removal of N-sulphonate groups, which normally stabilise HSPG against the occurrence of this process. Deleterious effects of excessive nitric oxide metabolite activity might be countered by inhibiting acidosis, or by glucosamine or glucosamine sulphate dietary supplementation, which tend to increase the rate of HSPG biosynthesis. (Reported benefit of dietary glucosamine supplementation in osteoarthritis patients therefore may arise by HSPG replacement therapy as discussed by McCarty 1995, 1997). Serum ferritin (related to iron overload) has been strongly correlated (Salonen et al 1990) with human mortality attributable to cardiovascular disfunction but this iron storage protein also (Jarrett et al 1989) increases with age in the absence of disease but its presence is associated with an increased risk of prooxidant damage. The deaminative cleavage of HSPG, is thought to be subject to catalysis by trace redox metals such as iron (Grant et al unpublished) made available under excess ferritin conditions, or by copper ions (Sorenson, 1984) augmented under stress response conditions (Grant 1998). This is relevant to HSPG-linked endothelial cell disfunction under iron overload conditions and may also account for the reported, unexpected and unexplained, in vitro deaminative cleavage observed (Vilar et al 1997) by direct reaction of NO with heparin (usually the formation of nitrous acid is required) at pH 7.4 (usually acidic pH is required) in phosphate but not in imidazole buffer). Later work by Fransson and coworkers has confirmed the endogenous utilisation of nitric oxide for deaminative cleavage of HSPG within endothelial cells, this process being putatively linked to polyamine signalling which alters HSPG biosynthesis by inserting unsulphonated glucosamine residues, pre-primed for rapid deaminative cleavage; these process are subject to control in laboratory studies via an alteration of the availabity of copper ions (which release nitric oxide bound at thiol storgage sites) via provision of exogenous copper chelating ligands (Mani et al 2000).

The induction of mouth cancer by betel nuts (Trivedy et al 1997) has been attributed to specific effects of copper. This may perhaps arise, it is now suggested, promoted via catalysis of the deaminative cleavage of HSPG. In the presence of EDTA or ascorbate copper and iron ions may stimulate redox cycling generating reactive nitrogen and oxygen species capable of degrading HSPG and DNA. A degradative acitivity of ascorbate on GAGs e.g. in vitreous humour was soon attributed to reactive oxygen species generated by the presence of trace copper ions (Robertson et al 1941). ESR detection of the extracellular release of ascorbyl radicals is a very reliable marker of the severity of myocardial ischemia and reperfusion injury (Pietri et al 1990) in which reactive oxygen free radicals are implicated. Ascorbate efficiently reduces nitrous acid back to NO. This may be an important biological function of ascorbate. The NO metabolite nitrous acid is a likely candidate as the major reactive agent for the deaminative cleavage of HS and hence ascorbate may spare HSPG from inappropriate degradation by this mechanism. The use of ascorbate dietary supplmentation for the inhibition of viral infections and combatting degenerative diseases such as cancer (Pauling discussed by Grant in 2000) may, be especially dependent on a specific signalling by ascorbate for a direct modulation of HSPG biosynthesis. An increase in sulphation of HS is produced under the influence of ascorbate for human fibroblasts (Edward & Oliver 1983, confirmed by Kao et al, 1990). (This is likely related to a wider effect of redox factors on HSPG biosynthsis as chlorate produces decreased sulphation). More highly sulphated heparin like fragments of HS demonstrate more potent antiviral (e.g. De Clercq E (1993), Choay et al 1993) as well as antiprion (Diringer 1985, Perez et al 1998) and anti-cancer activities (Engelberg in 2000). At least two members of the EXT family of tumour suppressors were found to encode Dglucosyl and N-acetylglucuronyl transferases required for the chain elongation of HS (Lin & Lindahl, 1998). HSPGs and their fragments are known to participate in antioxidant defence and defective HSPG antioxidative protection may therefore potentially promote tumorigenesis if depleted of this activity. Graham (1994) discussed a recently evolved heat shock protein (HSP) with putative heparanase activity. The puzzling detection of a 23 amino acid residue terminal fragment from a mammalian endo-heparanse recently incorporated into an anti-tumour heat shock protein (which only affected mouse but not analogous bacterial or yeast HSPs) and the putative role of heparin/HS in HSP70 (Furcht & McCarthy 1995) however concurs with the notion of a focus in animal evolution of mutually interactive HS and HSPG systems involved in gene alteration. HSPs and HSPGs, both strongly conserved throughout animal evolution and are implicated in the aetiology of degenerative diseases, which implies that coupled HSP HSPG effects, as well as post synthetic processing of GAGs and proteins, may be linked to stress response effects on genes induced by pathogenic microorganisms. This is consistent with known pro-cancer perturbation of heparan sulphate dependent growth factor activity generated as a consequence of binding to HSPG by Helicobacter pylori (associated with stomach cancer in a heparan sulphate binding-virulence correlated manner (Ascencio et al 1995, Chmiela et al 1995, Berq et al 1998).

Alteration in heparan sulphate microstructure via the effects of (saturated/polyunsaturated) lipids is apparent from reported effects of lipids on heparan sulphate biosynthesis (e.g. Paka et al 1999). (Oxidised lipids evidently signal for altered heparan sulphate production this being a possible major mechanism of cardiovascular damage (cf Paka et al 1999) perhaps partly due to the effects of such products (OBrien et al 1988) on the perturbation of heparin-like oligosaccharide modulation (Herbet & Maffrand 1989) of protein kinase C activity. Heparan sulphates including fragments evidently function in vivo for the inhibition of pathological crystallization (preventing calcification of blood and urinary systems, normally supersaturated with respect to Ca2+ salt precipitation) (cf Yamaguchi et al 1993, Grant et al 1992). Pathological plaque formation may include a related functionality failure of defective heparan sulphate proteoglycan systems (e.g. at artertial walls and in neurogenerative diseases and amyloidosis) perhaps due to aberrant self assembly of shed heparan sulphate proteoglycan fragments and their altered interaction with collagen, fibronectin etc under pathologcial conditons (including in cell culture models of cancer).

{Footnotes The original biochemical interest in heparin/ heparan sulphate biochemisty stemmed from the medical use of heparin as a blood anticoagulant. Haemostasis activity typically resides in proteolytic modulation via a highly sulphated heparin microstructure containing Glc C3,6(OSO3-)2NSO3- moieties which alter the conformation and activity of key proteins, especially antithrombin(III). Numerous additional activities of heparin/heparan sulphate have ultimately been discovered involving heparan sulphate proteoglycan dependent systems including an involvement in lipid metabolism and growth factor control. The latter was originally discovered by during research into angiogenesis by Folkman and coworkers for which a bivalent cation (e.g. Cu2+) dependence was also indicated. A wide variety of growth factors and cell types are now known to employ heparan sulphate-dependent control mechanisms (cf Bernfield et al 1999)}. A general statement of the mode of action of heparan sulphates in growth factor control is for recruitment of the growth factors and the assembly of cell surface receptors which includes the modulation of autophosphorylation kinase activity by the promotion growth factor receptor interaction. Such HSPG dependent systems are now known to critically influence haematopoeiesis (Gupta et al 1998, Drzeniek et al 1999) embryo development (Perrimon & Bernfield 1990) and wound healing. Environmentally-dependent effects transmitted by reactive oxygen and reactive nitrogen species during intracellular and extracellular stress coupling to C-Y signalling, enhanced by helper heparan sulphate proteoglycan (HSPGs) activities, agrees with the long-held notion that extracellular polysaccharides are involved in long range intracellular communication. This is in addition to their involvement in haemostasis, fibrinolysis, capillary permeability, matrix assembly, potentiation of mitotic, chemotactic, neurotrophic and angiogenic activities, cellular recognition, immunological signalling, adhesion,, differentation, proliferation, wound healing, apoptosis, gene expression, capacitation, morphogenesis, embryogenesis, nerve and brain development and function (including neurite outgrowth, synaptic function and myelination) phosphtidyl inositide anchoring and cytoskeletal function, lipid processing, glomerular filtration and antioxidant protection involving both the intact polymers and in some instances, fragments. Thus, although the biochemical details are far from clear, involvement in such wide-ranging processes could allow arrayed events affecting whole organisms to influence intracellular activity. Such processes could also allow complex extracellular communication events to occur over long time periods to stimulate altered gene structuring in a manner not producing lethal consequences. Signalling (by fragments of or by some other method dependent on cell type specific charge density array signalling) by heparin/heparan sulphate might fulfil this function which may involve nulcear elements such as nucleosomes bound to heparan sulphate proteoglycans at the cell surface and/or additional helper function of symbiotic organisms to facilitate non-lethal introduction of genetic material into host immunological cells communicating to stem cells. These types of cells involve HSPG directed recruitment and activation. Sensitivity of HS systems to various extracellular situations also includes direct effects of redox metal ions in the depolymerisation of heparin by complexed ferrous ions (Lahiri et al 1992) and induced biofeedback including a direct influence on heparan sulphate proteoglycan biosynthesis (both in causing altered biosynthesis producing altered microstructure and degree of glycosylation) as well as affecting post synthetic cleavage) such as the direct perturbation by hyperbaric oxygen of heparan sulphate biosynthsis (Karlinsky 1992). There is a possible heparan sulphate proteoglycan /iron biochemistry analogous to calcium biochemistry, including a helper function for tranferrin receptors, provision of an alternative ligand for iron entry into the cell as well as for modulation of morpholgical control of ironrich particles. Redox activity of Cu ions involved in immunological signalling (cf Sorenson 1984) may include a function of heparin/heparan processing including catalysis of nitric oxide reactions with heparin/HS. Nitric oxide and its metabolites, including tyrosine nitrate from peroxynitrite, activities are dependent on redox status (e.g. via ascorbate and thiol activities) and further subject to modulation by metal ion catalysis and osmotic stress. HS biosynthesis can be directly influenced by feedback from fragmented HS, redox status, metal ions and osmotic effects and additional polyamine signalling directs unsubstituted glucosamine positional biosynthesis (pre-primed for deaminative cleavage to specific oligosaccharides) allowing a fine-tuning of such HS structural modifications. Highly evolved heparanases and some heat shock proteins which seem likely also to generate HS oligosaccharides may provide a higher order of controlled HS oligosaccharide production and signalling relevant to gene modification

Nitric oxide is normally stored intracellularly at porphyrins and cysteine thiol groups where additional coordination to metal ions may occur. Release from thiol groups is subject to copper ion (Cu + being more effective than Cu2+ Singh et al (1992)) catalysis which can be diminished by the use of copper chelators in the laboratory to modulate in vivo intracellular HSPG processing by active nitric oxide metabolites (Mani et al 2000)) A further relevant related aspect of such nitric oxide dependent HSPG biochemistry is an important interlinking with polyamine signalling, recently established by Ding et al (2001). Clustering of acetyl choline receptors and related effects by heparin/ heparan sulphate proteoglycan (Baker et al 1992) suggests a related fundamental involvement of such biochemistry in neurological activity. The existence of biofeedback processes are the suggested from 1) the effect of exogenous heparin and nitric oxide in stimulation of the biosynthesis of heparin like HSPG by endothelial cells (Pinal et al 1994); 2) from the effects of extracellular metal ions on heparan sulphate biosynthesis (e.g. Ca2+ (for parathyroid cell heparan sulphate metabolic processing (Takeuchi et al 1990, 1992), the effects of toxic metal ions on kidney heparan sulphate proteoglycan biosynthesis, e.g. Pb (Fujiwara et al et al (1999) which diminishes HSPG sulphation much more than of other GAGs; cf also Kaji et al 1991), Cd (Cardenas et al 1992 ) Cu and Ni (Turnbull 1992); Different divalent metal ions are potentially required for two NacetylGlcN transferases TII and TII which initiate of polymerisation of HS. TI uses both Ca2+ and Mn2+ but TII uses only Mn2+ which effect may then influence HSPG structure. 3) Augmented extracellular glucose was found to markedly decrease heparan sulphate biosynthesis in glomerular epithelial cells (Morano et al 1999). 4) Ascorbate, on the other hand, markedly stimulated the biosynthesis of highly sulphated heparan sulphates (particularly those remaining at the cell surface following trypsinisation) in human embryonic epithelial cells (Edward & Oliver 1984) and in fibroblasts (Kao et al, 1990).

There is also believed to be involvement in cytoskeletal biochemistry and HSPG has been found at the mitotic spindle. (DATA)

References Abate C Patel L Rauscher FJ III Curran T (1990) Redox regulation of Fos and Jun DNA-binding activity in vitro Science 1157 DATA [The proto-oncogenes c-fos and c-jun function cooperatively in signal transduction via a heterdimeric complex of their protein products Fos and Jun which interact with the DNA regulators activator protein-1 (AP-1) leucine zipper domains direct positioning of clustered basic amino acid domains that bind to the DNA to which binding is modulated by redox alteration of a single conserved cysteine residue] cf Imai et al (1993) loc cit Heparin inhibits c-fos expression and endothelium 1 (ET-1) production in bovine endothelial cells Ascencio F Hansson HA Larm O Wadstroem T (1995) Helicobacter pylori interacts with heparin and heparin-depedent growth factors FEMS Immunol Med Microbiol 12 (3-4) 265-272 CA 124 109845f Cf Chmiela M et al (1995) and Berq DJ et al (1998) Baker LP Chen Q Peng HB (1992) Induction of acetylcholine receptor clustering by native polystyrene beads. Implication of an endogenous muscle-derived signalling system J Cell Sci 102 543-555 [A role of FGF2 -HSPG was implicated in the clustering of acetyl choline receptor clusters] cf Bullock S Rose SPR (1992)

Glycoproteins modulate changes in synaptic connectivity in memory function Biochem Soc Trans 20 412-414] Bashkin P Doctrow S Klagsburn M Svahn CM Folkman J Vlodavsky I (1989) Basic fibroblast growth factor binds to subendothelial extracellular matrix and is released by heparitinase and heparin-like molecules Biochemistry 28 1737-1743 Beckman JS Beckman TW Chen J Marshall PA Freeman BA (1990) Apparent hydroxyl radical production by peroxynitritie: implication for endothelial injury from nitric oxide and superoxide Proc Natl Acad Sci USA 87 1620-1624 [Peroxynitrite is a major reactive nitrogen reagent producing effects previously ascribed to hydroxyl radicals] cf however Pfeiffer S Mayer B (1998) Lack of tyrosine nitration by peroxynitrite generated at physiological pH J Biol Chem 273 (42) 27280-27285 high level of nitration of tyrosine a hallmark several disease degenerative diseases had been proudced by an additional factor ro by a different reagent (perhaps enhanced by CO2 {also redox metals such as trace active iron or copper ions as with the anomalous deaminative cleavage of HSPG by NO at pH 7.4}] Belting M Petersson S (CHECK) Fransson L-A (1999) Proteoglycan involvement in polyamine uptake Biochem j 338 317-323 [A specific role including sequence specific binding of HSPGs for the uptake of spermine by fibroblasts] Berq DJ et al (1998) Rapid development of severe hyperplastic gastritis with gastric epitheilial dedifferentiation in Helicobacter felis-infected IL-10(-/-) mice Am J Pathol 152 (5) 1377 CA 129 107408q Bernfield M Gotte M Park PW Reizes O Marilyn L Fitzgerald JL Zako M (1999) Functions of cell surface heparan sulfate proteoglycans Annu Rev Biochem 68 729-777 [Conserved cysteines in (GPI-anchored) glypicans and tyrosine(s) in (transmembrane cytoplasmic linked) syndecans may interact in growth factor regulation, cellular adhesion, activation, differentiation and motility in development, wound healing and immunolgical signalling in feedback loops involving kinase activities linked to post synthetic processing of HSPGs before and after shedding and re-uptake of HSPG units by cells. These events are known to be affected by mechanical, osmotic and redox stress.] Bhavanandan VP Davidson EA (1975) Mucopolysaccharides associated with nuclei of cultured mammalian cells Proc Natl Acad Sci USA 72 (6) 2032-2036 Boyd Long Williamson Getting DATA (1979) J Mol Biol DATA Bralet J Schreiber L Bouvier C Effect of acidosis and anoxia on iron delocalization from brain homogenates Brotherton TW Jagannadham MV Ginder GD (1989) Heparin binds to intact mononuleosomes and induces a novel unfolded structure Biochemistry 238 3518-3525 Brown GC (2000) Nitric oxide as a competetive inhibitor of oxygen consumption in the mitochoindrial respiratory chain Acta Physiol Scand 168 (4) 667-674 CA 133 149755y {NO diminsihes NF kappa beta which with the protein complex with MCP1 is believed to play an important role in atherosclerosis cf Takeshita A & Eto K CA 133 56741z}

Brown DR Qin K Herms JW Madlung A Manson J Strome R Fraser PE Kruck T von Boheln A Schulz-Schaeffer W Giese A Westaway D Kretzschmar H (1997) The cellular prion protein binds copper in vivo Nature 390 684-687 Cardenas A Bernard A Lauwerys R (1992) Incorporation of [35]sulfate into glomerular membranes of rats chronically exposed to cadmium and its relation with urinary glycosaminoglycans and proteinuria Toxicology 76 219-231 Cechowska Pasko M Palka J (2000) Age-dependent changes in glycosaminoglycan content in the skin of fasted rats. A possible mechanism Exp Toxicol Pathol 52 (2) 127-131 CA 133 264382k [NAD/NADH and lactate/pyruvate ratios, changed during ageing and fasting and lactate inhibits at least some GAG biosynthesis; the lactate content of blood and skin and lactate/pyruvate ratio was correlated with a 40% reduction in GAGs in the skin of old rats,and a redction in young but not old rats occurred with fasting. Lactate content was doubled in the skin of young fasted rats. An increase in the skin lactate/pyruvate and a decrease in this ratio in the blood was accompanied by a decrease in the skin GAG content suggesting that the effects were due to regulation of glucose (and glucosamine? CHE CK) for GAG biosynthesis (cori cycle? CHECK) Challis BC Edwards A Hunma RR Kyrtopoulos SA Outram JR (1978) IARC Sci Publ 19 (Environ. Aspects N-Nitroso Compd.) 127-142 CA 89 196583x [Fe and Cu etc catlayse nitrosamine formation] Chan RW (1992) Transparent ferromagnetic nonostructures Nature 359 591 [Fe2+ held at colloidal organic sulphonate(SO3-)n groups are hydrolysed and oxisised to give gamma Fe2O3 (cf Ziolo et a (1992)l Science 257 217-223) suggesting that Fe2+ bound by sulphated polysaccharides may behave similarly] Chmiela M Paziak-Domanska B Rudnicka W Wadstroem T (1995) The role of heparan sulphate-binding acitivity of Helicobacter pyroli in their adhesion to murine macrophages APMIS 103 (6) 469-474 CA 124 53455n [Pathologenic activity increases with strength of binding] Colburn P Dietrich CP Buonassisi T CHECK (1996) Arch Biochim Biophys 325 (1) 129 DATA CA 124 79179a [Endotoxin altered HSPG structure in endothelial cells by loss in a region rich in sulphate located at the non-reducing end of the GAG chain] Colin S Jeanny J-C Mascarelli F Vienet R Al-Mahmood S Courtois Y Labaree J (1999) In vivo involvement of heparan sulfate proteoglycan in the bioavailability, internalization, and catabolism of exogenous basic fibroblast growth factor Mol Pharm 1 74-82 Comper WD (1995) Interplay Genet Phys Dev Biol Forum. Workshop 199521 Ed Beysens D et al World Scientific Singapore CA 124 79513e [De novo occurrence of sulphated polysaccharides of ECM and cell surface has been correlated with the evolution of metazoa and differentiation in embryogensis] Cox JF Saravia F Briones M Maria HS (1995) Dose-dependent effect of heparin on fertilizing ability of goat spermatoza Theriogenology 44 (3) 451-460 CA 123 246357v {Cf Reyes R Rosado A Hernandez O Delgado NM (1989) Heparin and glutathione: physiological decondensing agents of human sperm nuclei

Gamete Research 23 39-47 and Valencia-Sanchez (1995) loc cit} Castillo GM Templeton DM (1992) Structure and metabolism of multiple heparan sulphate proteoglycans synthesized by the isolated rat glomerulus Biochem Biophys Acta 1136 119-128 [In contrast to other matrix components HSPGs are in a state of dynamic turnover] De Clercq E (1990) Targets and strategies for the antiviral chemotherapy of AIDS TiPS (11) 198-205 [Includes discussion of anti-HIV activity of sulphated polyanions including GAGs] De Lisser HM Yan HC Newman PJ Muller WA Buck CA Albelda SM (1993) Platelet/endothelial cell adhesion molecule-1 (CD31)-mediated cellular aggregation involves cell surface glycosaminoglycans J Biol Chem 268 (21) 16037-16046 Dhoot GK Gustafsson MK Ai Z Sun W Standiford DM Emerson CP Jr (2001) Regulation of Wnt singalling and embryo patterning by an extracellular sulfatase Science 293 1663-1666 [HSPG regulator function in developmental signalling via HSPG sulfatase Qsulf1 (which has homology with human lysosomal N-acetylglucosamine 6-sulfatase (HuG6Sulf)) expression of which is induced by Sonic hedgehog in myogenic ] Qsulf 1shares conserved C-Y, putativly stress-related, motifs with a human lysosomal 6 sulfatase). Stress factors are likely to influence such extracellular desulphation/desulphonation processes. The specific G6OSO3- patterns for Wnt signalling in quail embryo development are achieved by an extracellular sulphatase Qsulf1 Although Dhoot et al suggest that Qsulf1 (which has homology with Gln60sulphatas) regualates localized responses to widely distributed developmental signals for embryo patterning by achieving complete hydrolysis of GlcNAc6S in surface HSPG bound Wnts, perturbation via partial desulphation might also disrupt of embryo development.

{Comparison of the amino acid sequences of Qsulf 1 and HsG6Sulf show conserved C-Y motifs} cf Li et al (2000) HSPG expression of Nsulphotransferase (NST) during the transition of type I to type II rat alveolal cells was dependent on cell density and matrix and was intense in conditions where cells spread fully. 3-O sulphotransferase expression was however unchanged under these conditons) Dietrich CP Nader HB Strauss AH (1983) Structural difference of heparan sulfates according to the tissue and species of origin Biochem Biophys Res Commun 111 865-871 {cf Dietrich et al DATA (1996) Arch Biochem Biophys 325 (1) 129 Nader HB Ferrerira TMPC Toma L Chavante SF Dietrich CP Casu B Torri G (1988) Maintenance of heparan sulfate throughout evolution: chemical and enzymic degradation, and 13-Cn.m.r. spectral evidence Carbohydr Res 184 292-300 Cf Ferrerro et al (Dietrich CP) (1993) J Biochem 25 (6) 1219 DATA CA 120 50475a Ding K Sandgren S Mani K Belting M Fransson LA (2001) Modulations of glycpican-1 sulfate structure by inhibition of endogenous polyamine synthesis. Mapping of spermine-binding sites and heparanase, heparin lyase, and nitric oxide/nitrite cleavage sites J Biol Chem 276 50 46779-46791 Diringer H (1985) Funk Biol Med 4 129-141 cf Ehlers B Dringer HJ Gen Virol 65 1325-1330; Diringer H Ehler B (1991) J Gen Virol 72 457-460; Cf also WuDunn D Spear PG (1989)

J Virol 63 (1) 52-58 {Cf Polyamines (cf Ding et al 2001 loc cit), platelet factor 4 and poly L-lysine are known to block viral entry into cells presumably by a HS-dependent mechanism} Dow KE Riopelle RJ (1992) Influences of N-linked oligosaccharides in the presntation of neurite promoting activity of proteoglycans released by neurones in vitro [Castenospermine CHECK and swainsoline inhibited [3H] glucosamine incorporation into heparan sulphate and chondroitin sulphate proteoglycans] Cell Tisssue Res 268 (3) 553 DATA CA 117 45057z Drzeniek Z Stocker G Siebertz B Just U Schroeder T Ostertag W Haubeck H-D (1999) Heparan sulfate proteoglycan expression is induced during early erythroid differentiation of multipotent hematopoietic stem cells Blood 93 (9) 2884-2897 [HSPGs are involved including the binding and presentation of growth factors in cellular regulation via the direct contact interactions of primitive hematopoetic progenitor cells with stromal cells. A novel HSPG (not syndecan or glypican) which probably binds EPO growth factor was found on bone marrow (BM) stem cells of the erythroid lineage but not of the myeloid lineage. The HSPG is upregultedduring the early stages of erythroid differentation and downregulated when the cells mature into erythrocytes. HSPG may also be involved in the adhesion and homing of early erythroid precursors to the sites of erythropoiesis in the BM. Previous studies had shown HSPGs of bone marrow stromal cells or their ECM bind IL3 and granulocyte-macrophage colony stimulating factor GM-CSF) allowing biologically active presentation to hematopoietic cells] Cf Brunner G et al (1994) An endogenous glycosylphosphatidyl inositol specific phospholipase D releases b-FGF {FGF-2}-HSPG complexes from human bone marrow cells ibid 83(6) 2115 CA 120 261727x Dundas J et al DATA (2000) Effect of heparin and liver heparan sulphate on interaction of HepG2-derived transcription factors and their cis activating elements: altered potential of hepato cellular CHECK carcinoma heparan sulphate Biochem J 350 (1) 245 CA 133 320328 [PG assembly in malignant tumours is greatly altered and may promote growth via depeletion in gene control activity. In vitro and in vivo experiments indicated that heparin and HS are capable of inhibiting the induction of transcription factors (AP1, SO-1 ETS-1 and nuclear factor kappa B) wheras FFIID was hardly inhibited. HS from DATA liver was five times more effective than from liver carcinoma was less effective comparable to heparin in activity, indicating that altered HS microstructure may prmotoe uncontrolled cell proliferation] Edenber MJ Conrad HE Bizzo SV (1991) Heparin oligosaccharides enhance tissue type plasminogen activator: a relation between oligosaccharide length and stimulation of plasminogen activation Biochemistry 30 (4) 10999 DATA CA 115 229145s [Enhancement of plasminogen activation by the smaller oligosachharides is mediated exclusively via binding of tissue type plasminogen (Pg) activator (t-PA) whereas the larger oligosaccharides may interact with both t-PA and Pg] Edward M Oliver RF (1984) Ascorbate increases the sulphation of glycosaminoglycans synthesized by human skin fibroblasts Biochem Soc Trans 12 304 Cf These authors (1983) Changes in the synthesis and distribution of sulphated glycosaminoglycans in human skin fibroblasts upon ascorbate feeding ibid 11 383 [Upon increase in ascorbate from zero to 50microg/ml, cell associated HS increased by a factor of 19.2; Total GAGs in the pericellular/extracellular domain increased by 3.5 fold]. Elhadj S et al (2002) Chronic pulsatile shear stress impacts synthesis of proteoglycans by endothelial cells: Effect on platelet aggregation and coagulation J Cell Biochem 86 (2) 239-250

Engelberg H (1999) Actions of heparin that may affect the malignant process Cancer 85 (2) 257-272 Esko JD Rostand KS Weinke JL (1988) Tumor formation dependent on proteoglycan biosynthesis Science 241 1092-1096 Feyzi E Saldeen T Larsson E Lindahl U Salmivirta M (1998) Age-dependent modulation of heparan sulfate structure and function J Biol Chem 273 (22) 13395-13398 [HS from different species and cells differ in their pattern of sulphation in N-suphonate rich regions including a specific microstructure thought specific for platelet derived growth factor (PDGF) AA L and BBL binding which for a 76-year-old individual was 4-5 times greater than for a 21-year old individual (whereas binding of FGF-2 was only marginally dfiferent between the two ages); this was suggested to contribute to arteriosclerosis; The authors noted that their results were the first time discovery of a characteristic systematic change associated with the ageing process] Fisher RE Blumenthal Y (1982) J Biol Chem 257 (4) 1702-1704 CA 96 138847v [Heparin inhibits RNA polymerase and modifies the trypsin cleavage of RNA polymerase both alone and at a poly[dA.T.dA.P] template Folkman J Klagsburn M (1987) Angiogenic factors Science 235 442-447 [Heparin binding endothelial growth factors heparin affinity allowed their separation and purification FGF1 (acidic FGF) and FGF2 (basic FGF) originally studied 140 amino acid residues and 146 residue had 53% absolute sequence homology gene sequence data showed both had 154 amino acid residues; the receptors FGF1 mw 135-150,000, for FGF2 mw 125-145,000. Tyrosine phosphorylation was known only for the FGF1 receptor} {Heparin binding growth factor (vascular endothelial growth factor related to FGF1) activities are also implicated in glucose induced vascular disfunction (Stephan CC et al 1998, Diabetes 47 (11) 1771 CA130 64586f}. Fransson L-A (1982) Structural features of the contact zones for heparan sulphate self-association Carbohydr Res 110 127-133 Cf Fransson LA Havsmark B (1982) Correct secondary structure is required for self-association of heparan sulphate Carbohydr Res 110 (1) 135 CA 98 16981e [Modification of heparan sulphate by N-desulphonation and N-acetyaltion abolished chain-chain interaction] Frei B (1991) Ascorbic acid protects lipids in human plasma and low density lipoprotein against oxidative damage Am J Clin Nutr 54 1113S-1118S [Only ascorbic acid is reactive enough to intercept oxidants in the aqueous phase before they cause detectable oxidative damage to lipids being able to prevent the initiation process not achieved by other investigated antioxidant molecules (bilirubin, uric acdi, protein thiols, alpha tocopherol, ubiquinol-10, lycopene and beta carotene)] Fritz TA et al (1994) J Biol Chem 269 (46) 28809 CA 121 249190e [Differences in activation requires divalent cations: alpha-GlcNAcTI can use both Mn2+ and Ca2+ while alphaGlcNAcTII can only use Mn2+ provides a mechanism by which Mn2+ status might influence HSPG biochemistry]

Fujiwara Y Kaji T (1999) Lead inhibits the core protein synthesis of a large heparan sulfate proteolgycan perlecan by prolferating vascular endothelial cells in culture Toxicology 133 (2,3) 159-169 CA 131 154569c [Lead significantly decreased the accumulation of GAGs but more strongly affected HS than chondroitin or dermatan sulphates. Lead seems to decrease perlecan core protein sytnthesis without affecting the attached polysaccharide chain lengths. HSPGs are reduced but by a different mechansims between growing and growth arrested cells] Cf Kaji et al (1990) ibid 68 249-257 Furcht LT McCarthy JB (1995) Identification of homologous heparin binding peptide sequence present in fibronectin and 70kDFa family of heat-shock proteins Biochim Biophys Acta 1251 (1) 135 DATA CA 123 249546j Furuya S et al DATA (195) Elimination of heparan sulphate by heparitinases induces abnormal mesodermal and neural formation of Xenopus embryos Dev Growth Differ 37 (3) 337 CA 123 165404f [HS plays an indispensible role in establishing fundamental body building during early Xenopus development; heparanse deletion causes organism developments without CNS and mesodermal tissue but chondroitinase produced no similar effects] Gabizon R et al J Cell Physiol (1993CHECK)157 (2) 312-25 CA 120 28472a Cf also Perez et al loc cit; Gallagher JT Turnbull JE Lyon M (1992) Patterns of sulphation of heparan sulphate Int J Biochem 24 (4) 553 CA 117 42780u [cf Galagher JT Lyon M Steward WP (1986) Structure and function of heparan sulphate proteoglycans Biochem J 236 313-325] Gilat D et al (1995) Molecular behaviour adapts to context : heparanase functions as an ECM degrading enzyme or as a T cell adhesive molecule dependent on the local pH J Exp Med 181 (5) 1929-1934 CA 288881u [Migration of lymphocytes requires their adhesion. Penetration of the sub endothelial ECM is associated with expression of ECM degrading enzymes such as endo beta D glucouronidase (heparanase) which degrades HSPGs but only under relatively acidic conditions. At normal tissue pH the enzyme attaches to HS without degrading it and creates an anchor for CD4 +T cells] Graham LD (1994) Tumour region antigens to the hsp90 family (gp96) closely resemble tumour associated heparanase enzymes Biochem J 301 (3) 917 {cf Srivastava PK Endo beta D glucuronidase (heparanase) activity of heat shock protein.tumour region active agent gp96 ibid 301 (3) 915 Review with 21 refs} [Cf also comments following this paper by Nicholson JL & Nakajima M and Srivastava PK] The two most abundant HSPs (HSP70 and HSP90) are highly conserved among organism as diverse as bacterial flies and man (cf Pelham HRB (1986) Speculation on the functions of the major heat shock and glucose-regulated proteins, Cell 46 959-961). Grant D (2000) http://web.ukonline.co.uk/dgrant/dg4/ cf ibid dg/2 {Discussion sessions held with Prof KEL McColl and coworkers (Glasgow University)} [Ascorbate and nitric oxide modulation of heparan sulphate biochemistry. Tyrosine nitrate formation is indicative of the presence of NO metabolites DATA

Efffects of NO metabolites implicated in aetiology of rheumatic diseases (Stichentoch & Frolich 1998, loc cit); pathological deaminative cleavage of heparan sulphate chains also implicated Grant D Long WF Williamson FB (1987a) Pericellular heparans may contribute to the protection of cells from free radicals Med Hypoth 23 67-72 afforded by glycosaminoglycans, ibid 38 49-55] Cf These authors (1987b) Infrared spectroscopy of heparin-cation complexes Biochem J 244 143-149 (1989a) A comparison of the antioxidant requirements of proteins with those of synthetic polymers suggests an antioxidant function for clusters of aromatic and bivalent sulphur-containing amino aacid residues Med Hypoth 28 245-253 [A hypothesis similar to part of the suggestion of Nakashima(1996) that evolutionary conserved C-Y (and related serine and theonine as well as histidine) groupings in proteins are especially associated with antioxidant protection and also with ligand binding to RNA and DNA protooncogene function] (1990) The dependence on counter-cation of the degree of hydration of heparin Biochem Soc Trans 18 (6) 1283-1284 (1991) Heparin polypeptide interaction Biochem J 277 569-571 (1989b) Inhibiton by glycosaminoglycans of CaCO3 calcite) crystallization Biochem J 259 41-55 CA 110 171240x (1992a) Complexation of Fe2+ based ions by heparin is not a simple reversible thermodynamic process Biochem Soc Trans 20 (4) 361s Cf Biochem J 287 (3) 849-853 CA 117 226049u [Zn2+ heparin binding; evidence for phase change mechanism] (1992b) Degenerative and inflammatory diseases may result from defects in antimineralization mechanisms afforded by glycosaminoglycans Med Hypotheses 38 49-55 Grimm J Keller R de Groot PG (1988) Lamellar flow induces cell polarity and leads to rearrangement of proteoglycan metabolism in endothelial cells Thromb Haemostasis 60(3) 437-441 Gupta P et al DATA (1998) Structurally specific heparan sulphates support primitive haematopoiesis by forming multi molecular stem cell niches Blood 92 (12) 4641 CA 130 137148b Halpern J Carter BJ (1989) Modification of human carcinoma SW13 cells by heparin and growth factors J Cell Physiol 141 (16 DATA CA 111188341z [EGF and insulin like growth factor 1 (IGF 1) were not able to overcome heparin induced inhibition of cell growth (due to strong complexing by heparin) but such inhibition was partly overcome by TGFe and FGF-1 (weaker heparin complexes formed). Heparin-like substances present in the extracellular matrix play an important role in the control of epithelial growth]. Hahnenberger R Jakobson AM Ansan A Wehler T Svahn CM Lindahl U (1993) Low sulphated oligosaccharides derived from heparan sulphate inhibited normal angiogenesis CHECK Glycobiology 3 (6) 567-573 CA 120 235393r [Attempts to define a saccharide structure responsible for the anti-angiogenic effects of HSPG implicated the initial polymerisation product containing unsulphated and un-isomerized [GlcAbeta 1,4-GlcNAcalpha 1,4]n sequences which also occur in Escerichia coli K5 capsular polysaccharide of

anti-angiogenic activity. However isomeric hyaluoronic acid with [GlcA beta 1,3-GlcNAc beta 1,4]n structures was inactive] Hansen LK OLeary JJ Skubitz APN Furcht LT McCarthy JB (1995) Identification of a homologous heparin binding peptide sequence present in fibronectin and the 70kDa family of heat shock proteins Biochem Biophys Acta 1251 (1) 135-145 CA 123 249546s [LIGRK and LIGRR represent a common heparin binding motif in fibronectin , HSP70 and HSP70 and are consistent with the proposed role of heparins and similar polyanionic structures in the functions of 70kDa heat shock proteins] Heller R CHECK Munsche-Pauley F Grabner R Till U (1999) L-Ascorbic acid potentiates NO synthesis in endothelial cells J Biol Chem 274 (12) 8254-8260 [Ascorbic acid enhances NO synthesis in endothelial cells this being postulated to be the origin of the {Various linked signalling via HSPG systems suggested by this finding e.g. ascorbate (incr HSPG biosynthesis?) whicle NO also may modulate HSPG biosynthesis and cause HS deaminative cleavage (e.g. dependent on trace redox metal e.g. Cu availability) with resultant HS oligosaccharide or exogenous heparin(oid) feedback signalling and modulation of HSPG biosynthesis} Herbert J-M Maffrand J-P (1991) Effect of pentosan polysulfate , standard heparin and related compounds on protein kinase C activity Biochim Biophys Acta 1091 432-441 {Inhibiton of PKC by oligosaccharides is highly dependent on molecular weight} Hu W-L Regoeczi E (1992) Hepatic heparan sulphate proteoglycan and the recycling of transferrin Biochem Cell Biol 70 535-538 [Liver HSPG can some transferrin receptor functions; HSPG can facilitate the release of iron from transferin] Imai T et al (1993) Heparin inhibits ET-1 and protooncogene cfos expression in cultured bovine endothelial cells J Cardiovasc Pharmacol 22 (suppl 8) 549 DATA CA 120 69189g [Heparin probably inhibits proto ET-1 mRNA expression via a PTC dependent pathway] Ishii K et al (1982) Biochem Biophys Res Commun 104 (2) 541-547 [Heparin inhibited DNA topoisomerase ex mouse mammary carcinoma cells I 50 = 0.20 microg/ml. Other GAGs were much less effective] Jarrett DRJ Grosset AB Rowland Payne CME (1989) Ageing as a cause of raised serum ferritin in the absence of disease J Clin Exp Gerontol 11 (3-4) 145-54 Jethmalan SM et al (1994)DATA Heat shock induced prompt glycosylation. Identification of P-SG67 as calreticulin CHECK J Biol Chem 265 (38) 23602-23609 CA 121 224578z [Acute heat shock initiates the phenomenon of prompt glycosylation which is characterized by selective glycosylation of specific cellular proteins] Julian J Das SK Dey SK Baraniak D Ta V-T Carson DD (2001) Expression of heparin/heparan sulfate interaction protein/ribosomal protein L29 during the estrous cycle and early pregnancy in the mouse Biol Reprod 64 1165-1175 Klevit RE (1991) Recognition of DNA by Cys2His2 zinc fingers Science 253 1367-1393 [C2 containing zinc fingers are major DNA binding proteins]

[C2XXC2 also bind Cu ions as in metallothionenes] Kaji T Yamamoto C Sakamoto M (1991) Effect of lead on the glycosaminoglycan metabolism of bovine aortic endothelial cells in culture Toxicology 68 249-257 cf Fujiwara Y Kaji J (1999) Toxicology 133 (2,3) 159 CA 131 154569 Kan M Wang F Kan M DATA Gabriel JL McKeehan WL (1996) Divalent cations and heparin/heparan sulfate cooperate to control assembly and activity of the fibroblast growth factor receptor complex J Biol Chem 271 (42) 26143-26148 (cf also Kan M Wu X Wang F McKeegan WL (1999) Specificity of factors determined by heparan sulfate in a binary complex with receptor kinase Ibid 274 15946-15952) [cf also Colin S Jeanny JC Mascarelli F Vienet R Al-Makmood S Copurtois Y Labarre J (1999) In vivo involvement of heparan sulfate proteoglycan in the bioavailability, internalization, and catabolism of exogenous basic fibroblast growth factor Molecular Pharmacology 1 74-82 http://www.molpharm.org; Danmon DH Halegoua S DAmore P Wagner JA (1992) Fibroblast growth factors, like nerve growth factor induce morphological differentiation of PC12 cells regulated by glycosaminoglycans Exp Cell Res 201 154 CA 117 40895m David G Bernfield M (1998) The emerging roles of cell surface heparan sulfate proteoglycans Matrix biol 17 (7) 461-463 CA 130149916p Krufka A Guimond S RapraegerAC (1996) Two hierarchies of FGF-2 signalling by heparin: mitogenic stimulation and high-affinity binding/receptor transphosphorylation Biochemistry 351 11131-11141 (CHECKp) LaRochelle WJ Sakaguchi K Atabey N Cheon H-G Takagi Y Kinaia T Day RM Mike T Burgess WH Bottaro DP (1999) Heparan sulfate proteoglycan modulates keratinocyte growth factor signalling through interaction with both ligand and receptor DATA Johnson GR Wange L (1994) Heparan sulfate is essential to amphiregulin induced mitogenic signalling by the epidermal growth factor receptor J Biol Chem CHECK 269 (43) 27147 DATA CA 121 222877x [Heparan sulphate is essential to amphiregulin induced mitogen signalling by the EGF receptor tyrosine kinase] Lind T Tufaro F McCormick C Lindahl U Lidholr K (1998) The putative tumor suppressors EXT1 and EXT2 are glycosyltransferases required for the biosynthesis of heparan sulfate J Biol Chem 273 (41) 26265-26268 CA 130 64007t [McCormick C et al (1998) Nat Genet 19 152 had previously shown that EXT-1 rescues defective heparan sulphate biosynthesis and was now shown to elevate GlcA and GlcNAc transferase of mutant cells; Lin X et al (2000) also demonstrated that EXT-deficient mice show disruption of gastrulation and heparan sulphate biosynthesis (mutation in the human EXT-1 gene are responsible for multiple exotosis type. ] Lyon M Gallagher JT (1998) Biospecific sequences and domains of heparan sulphate and the regulation of cell growth and adhesion Matrix Biol 17 (7) 485 DSATA Fahem S Linhardt RJ Rees DC (1998) Diversity does make a difference : fibroblast growth factor-heparin interaction Curr Opin Struct Biol 8(5) 578-586 Richard C Liuzzo JP Moscatelli D (1995) Fibroblast growth factor 2 can mediate cell attachment by linking receptors and heparan sulphate proteoglycans in neighbouring cells J Biol Chem 270 (41) 24188-96

Kanner J Harel S Granit (1991) Nitric oxide as an antioxidant Arch Biochem Biophys 289 (1) 130-136 Cf Hinshelwood CN (1957) The Structure of Physical Chemistry, Oxford, the Clarenden Press cf e.g. p395 Cf also Brown GC loc cit Kao J Huey G Kao R Stern R (1990) Ascorbic acid stimulates production of glycosaminoglycans in cultured fibroblasts Experimental Mol Pathol 53 1-10 [Heparan sulphate biosynthesis is differentially increased by ascorbate. The most pronounced effect of altered GAGs upon ascorbate feeding at 25microg/ml (added 72 h prior to labelling and replendished every 24 h) on cell cultured human foreskin fibroblasts was in boosting HSPG formation] cf also Edward M Oliver RF (1994) loc cit who had previously published similar findings for human embryonic endothelial fibroblast cells, and Malemud CJ et al (1978) Connect Tissue Res 6(3) 171-9 who found that ascorbate boosts GAG biosynthesis in chondrocytes CA 9255968b Karlinsky JB Rounds S Faraber HW (1992) Effects of hypoxia on heparan sulphate in bovine aortic and pulmonary artery endothelial cells Circ Res 71 (4) 782-9 CA 118 78363v [Hypoxia induced decreased sulphation of heparan sulphate but increased the AT(III) binding site content] Karlsson K Marklund SL (1988) Extracellular superoxide dismutase association with cell surface bound sulphated glycosaminoglycans Basic Life Sci 49 647-650 Kishibe J Yamada S Okado Y Sato J Ito A Miyazaki K Sugahara K (2000) Structural requirements of heparan sulfate for the binding to the tumor-derived adhesion factor/angiomodulin that induces cord-like structures to ECV-304 human carcinoma cells J Biol Chem 275 (20) 15321-15329 Knaus H-G Scheffauer F Romanin C Schindler H-G Glossmann H (1990) Heparin binds with high affinity to voltage-dependent L-type Ca2+ channels. Evidence for an agonistic action J Biol Chem 265 11156-11166 Korengaga R Ando J Tsuboi H Yang W Sakumo I Toyoka T Kamiya A (1994) Biochem Biophys Res Commun 198 (1) 213-19 CA 120 103328p Lahiri B Lau PS Pousada M Stanton D Danishefsky I (1992) Arch Biochem Bioiphys 293 54-65 Linker A Hovingh P (1973) The hepartin sulfates (heparan sulfates) Carbohydr Res 29 41-62 [Amyloid heparan sulphates with high GlcNH2 contents] cf later work DATA( Lindahl et al) Liu Z Perlin AS (1994) Evidence of a selective free radical degradation of heparin mediated by cupric ion Carbohydr Res 225 183-191 Lahiri B Lai PS Pousada M Stanton D Danishefsky I DATA Depolymerization of heparin by complexed ferrous ions Arch Biochem Biophys 293 54-60 [Ascorbate promotes limited degradation of heparin

Previously heparin had been shown to be degraded by Fentons reagent and by activated phagocytes cf Metcalf DD et al (1990) loc cit Horner AA (1971) loc cit found that multichain rat skin heparin was degraded by ascorbic acid to single chain structures possibly due to the action of trace metal impurities] Li Z et al DATA (2000) Expression of N-deacetylase/sulfotransferase and 3-O sulfotransferase in rat alveolal CHECK type II cells Am J Physiol 279 (2 part 2) L292-301 CA 133 250129w [HSPG sulphation enzyme RNAs were studied as influencing accumulation in basal lamina beneath alveolal type I cells (e.g. during the transition from type II to type I cells); increased expression of Nsulphotransferatse showed that GN-sulphonation was dependent on cell density and matrix and was intense under condition where cells spread fully; the pattern of G 3 o sulphation was, however, was not changed under these conditions as indicated by expression of the 3OST. NaCl03 singificantl;y inhibited cultured type II cell spreading and the inhibition was reversed by Na2SO4] Libeu CO et al DATA (Linhardt RJ) (2001) J Biol Chem 276 42 39138-DATA [Defective binding of ApoE to HSPG associated with atherosclerosis via defective liver clearance alaso relevant to Alzheimers disease neural plaque formation] Lind T Tufaro F McCormick C Lindahl U Lidholt K (1998) The putative tumor suppressors EXT1 and EXT2 are glycosyltransferases required for the biosynthesis of heparan sulfate J Biol Chem 273 (41) 26265-26268 CA 130 64007t Liu Z Perlin AS (1994) Evidence of a selective free radical degradation of heparin mediated by cupric ion Carbohydr Res 255 183-191 Lindahl B Lindahl U (1997) Amyloid-specific heparan sulfate from human liver and spleen J Biol Chem 272 (42) 26091-26094 Long WF Williamson FB (1979) Glycosaminoglycans, calcium ions and the control of cell proliferation IRCS J Med Sci 7 429-434 Luck W (1965) Ber Bunsen Phys Chem 69 (1) 69 (also ibid 626) DATA Mandil AK et al DATA (1995) Kidney International 48 (5) 1508 [Heparin induced endothelial cell cytoskeletal reorganisation-possible mechanism for vascular relaxation] Mani K Jonsson M Edgren G Belting M Fransson L-A (2000) A novel role for nitric oxide in the endogenous degradation of heparan sulfate during recycling of glypican-1 in vascular endothelial cells Glycobiology 10 (6) 577-586 [This process can be affected by Cu ions via catalysis of release from thiol storage sites] cf Ishihari M (1998? DATA) A new degradative pathway for heparan sulphate proteoglycans Trends Glycosci Glycotechnol 10 (54) 331 DATA CA 130 63940e {NO and reactive oxygen species in regulation of the ECM metabolism Cf Heller R Munsche-Pauley F Graham R Till U () L ascorbic acid potentiates nitric oxide synthesis in endothelial cells J Biol Chem 274 (12) 8254-8260 CA 131 13677

McCarty MF (1997) Glucosamine may retard atherosclerosis by promoting endothelial production of heparan sulphate PG Med Hypoth 48 (3) 245 DATA CA 127 128534 [Glucosamine dietary supplementation aids arteriosclerosis etc cf also Theodosakis J Adderly B Fox B (1997) The Arthritis Cure Century Books ISBN 078123 7813 1]] Metcalf DD Thompson HL Klebanoff SJ Henderson WR (1990) Biochem J 272 51-57 Mislick KA Baldeschwieler JD (1996) Proc Natl Acad Sci 93 12349-12354 Mishra G Castellot JJ (1999CHECK) Heparin rapidly and selectively regulates protein tyrosine phosphorylation in vascular smooth muscle cells J Cell Physiol 187 (2) 205 Morano S Guidobaldi L Cipriani R Gabriele A Pantellini F Medici F DErme M DiMario U (1999) High glucose modifies heparan sulfate biosynthesis by mouse glomerular epithelial cells Diabetes.Metab Res Rev 15 (1) 13-20 CA 131 57278z [Effect of 30mmol glucose produced a 50% decrease in total cellular proteoglycan biosynthesis compared with physiological 5mmol; the effect was most centred on cell layer heparan sulphate proteoglycans, three identified species being reduced in amount by 81-91%- this is the opposite effect to what was found by Edward and Oliver (1984) loc cit for 50 mmol ascorbate on epithelial cell heparan sulphates where a 350% boost in total heparan sulphates but a 1950% increase in tyrpsin resistant cell surface heparan sulphate proteoglycan was observed in this case as speculated by these authors post synthetic degradation of heparan sulpahte might have occurred but this may have been decreased by the effect of ascorbate. This suggests that ascorbate may reduce back to NO metabolites such as nitrous acid known to efficiently deaminatively cleave heparan sulphate. Heparin administration however enhances NO production by injured endothelia cf CA 120 182713m CHECK as required for complex biofeedback heparan sulpahte processing/singalling] Cf Raats CJI et al DATA (2000) Glomerular heparan sulfate alteration. Mechanism and relevance for proteinuria Kidney Int 57 (2) 385 Ca 133 294275v [Excess glucose in diabetes caused down regulation of kidney HSPG biosynthesis and reduction in degree of sulphation of HSPG] Mounkes LC Zhong W Cipres-Palacin G Heath TD Debs RJ (1998) J Biol Chem 273 26164-26170 Murata K Yokoyama Y (1989) Acidic glycosaminoglycans in human atherosclerotic cerebral arterial tissues Atherosclerosis (Shannon Irel) 78(1) 69-79 CA 111 151343a CHECK [Data presented indicates a regular diminution of arterial wall heparan sulphate as a function of age Feyzi et al 1998 (loc cit) found that the microstructure of arterial wall heparan sulphate showed a systematic increase in the amount of trisulphated heparin-like sequence content; perhaps this is evidence for a biofeedback related attempt by the organ to limit deleteriaous calcification since more highly sulphated heparan sulphated would be predicted to provide such a function cf Grant et al 1989b 1992b)] Another age-dependent physiological marker is the reported human age (in absence of disease) increase in the serum ferritin DATA This has also been strongly implicated the aetiology of vascular damage linking Finnish mortality in eastern Finnish men (Salonen et al (1990) loc cit). Nader HB Ferreira TMPC Toma L Chavante SF Dietrich CP Casu B Torri G (1988) Maintenance of heparan sulphate through evolution Carbohydr Res 184 292

Nakashima I (1996) Can cysteine direct tyrosine in signal transduction for environment-oriented gene control? Nagoya J Med Sci 59 1-10 [A hypothesis that cysteine-oriented signalling potentially directs the tyrosine-oriented signalling in a mechanism of environmentally oriented control of internal signalling for gene control] {cf Koch CA Anderson D Moran MF Elis C Pawson T (1991) SH2 and SH3 domains: elements that control interactions of cytoplasimic signalling proteins Science 252 668-674 These domains have a number of conserved tyrosines and another highly conserved cluster of basic amino acids (a potential heparin/HSPG binding site, previously proposed binding site for tyrosine phosphates). C-Y motifs (Grant et al 1989) occur in SH2 domain S (at conserved positions) in c-Src, c-Yes, Fgr, Fyn, Lck, Lyn, Hck , Blk, at conserved Y but altered C in Vav, GAP-N (C deleted in GAP-C) PLC gamma 1N and gamma 2N (but with possible methionine juxtapositions in Nck (Fer), PLCgamma 1C, gamma 2C and GAP-C) and alternatively placed C-Y positions in p85 from same Y as above and from a different Y in cFps; in tensin there is a replacement of Y by H at the conserved position in the IV sub-domian) (but apparently absent in c-Abl, Arg, Dab1 where the conserved Y is replaced, as is also the case with p85 and in Nck with histidine; v-Crk lacks C-Y and also a putative heparin binding site)}. Nagasawa K Ogamo A Uchiyama H Matsuzaki K (1983) Hydrophobic interaction chromatography of glycosaminoglycuronans: the contribution of N-acetyl groups in heparin and heparan sulfate to the affinity for hydrophobic gels, and variety of molecular species in beef-kidney heparan sulfate Carbohydr Res 111 273-281 Nagasawa K Uchiyama H Sato N Hatano A (1992) Chemcial change involved in the oxidative-reduction depolymerization of heparin Carbohydr Res 236 165-180 CA 118 97491b Nicholson JL Nakajima M (1994) Similarity between heparanse enzyme and mammalian hsp90 like proteins [Hsp90 shares with heparanase occurrence at both cell surface and nuclear compartment. Attempts to demonstrate heparanse activites with purified hsp90 were however not successful ] cf Graham LD (1994) Srivastava PK (1994) Endo beta D glucouronidase (heparinase) activity of heat shock protein/tumour region antigen gp96 Ibid 301 (3) 915 {gp96 was suggested to be an evolutionary recent protein sharing multiple functions} Cf Nakajima M Irimura T Nicolson GL (1988) Heparanases and tumor metastasis J Cell Biochem 36 157-167 Nishinage M et al DATA (1993) J Clin Invest 192 (3) 1381 CA 119 24338u [Redox potential altered by H2O2 and cysteine which inhibit expression of anticoagulant heparan sulphate by endothelial cells] OBrien CA Ward NE Weinstein IB Bull AW Marnett LJ (1988) Activation of rat brain protein kinase C by lipid oxidation products Biochem Biophys Res Commun 155 (3) 1374 1380 [Activation of PKC by hydroperoxy fatty acids may be an early cellular response to oxidative stress] Olin KL Potter-Perigo S barrett HR Wight TN Chair A (1999) Lipoprotein lipase enhances the binding of native and oxidized low density liproteins to versican and biglycan synthesized by cultured arterial smooth muscle cells J Biol Chem 274 (49) 34629-34636 [Other GAGs, chondroitins and dermatan sulphates, may be cofactors in HSPG dependent lipid processin, arterial walls and be involved in adverse effects of oxidised lipids]

Paka L Kako Y Obunike JC Pillarisetti S (1999) Apolipprotein E containing high density lipoprotein stimulates endothelial production of heparan sulfate rich in biologically active heparin-like domains. A potent mechanism for the antiatherosclerotic action of vascular apolipoprotein E J Biol Chem 274 (8) 4816-4823 [Oxidised lipid adverse effect on heparan sulphate proteoglycans biosynthesis model of induction of arterosclerosis] cf Chang MY Potter-Perigo S Tsoi C Chair A Wight TN (DATA) Oxidised low density lipoproteins regulate synthesis of monkey aortic smooth muscle cell proteoglycans that have enhanced native low density lipoprotein binding properties J Biol Chem 275 (7) 4766-4773 Parrish RF Fair WR (1981) Selective binding of zinc ions to heparin rather than to other glycosaminoglycans Biochem J 193 407-41 [Copper ions have also been suggested to bind preferentially to heparin] Paudel HK et al (1999) Heparin induced conformational change in microtubule associated protein tau as detected by chemical and phosphopeptide mapping J Biol Chem 274 (12) 8029-8038 CA 131 13676y Pauling L (1991) In Block G Henson DE Levine M (Eds) (1991) Ascorbic acid: biologic functions and relation to cancer Proc Conf Natl Inst Health Bethesda MD Dept 112 1990 Am J Clin Nutr (1991) 54 1252S-1298S [Pauling, while producing evidence supporting beneficial anti-viral and anti-cancer activities of a therapeutic ascorbate dietary supplementation, lacked a convincing explanation for the observed effects which can now be attributed to this major influence of ascorbate in HSPG biochemisty, but this can be seen to be part of a wider dependence of HSPG biochemistry on redox status (e.g. as characterised by the effects of hypoxia and glucose status on HSPG biosynthesis)] Perez M Wandosell F Colaco C Avila J (1998) Sulphated glycosaminoglycans prevent the neurotoxicity of a human prion protein fragment [Prions bind copper physiologically cf Brown DR et al (1997)] Perrimon N Bernfield M 92000) Specificities of heparan sulphate proteoglycans in developmental processes Nature 404 725-728 Pietri S Culcasi M Stella L Cozzone PJ (1`990) Eur J Biochem 193 845-854 Pinhal MAS Walenga JM Jeske W Hoppenstaede D Dietrich CP Fareed J Nader HB (1994) Antithrombotic agents stimulate the synthesis and modify the sulphation pattern of a heparan sulphate proteoglycan from endothelial cells Thromb Res 74 (2) 143 DATA CA DATA 289743j Rahmoune H et al (Gallagher JT) DATA 1998 Biochemistry 37 (17) 6003 DATA [Mammary epithelial cells use distinct HSPGs for different sets of growth factors] Cf Brinkman YG et alm (Gallagher JT) Gycobiology 8 (5) 463 CHECK Shworah NM etr al (Rosenberg RD) (1998) Trends Glycosci Glycotechnol 10 (52) 175 Mishrc-Gories et al (1998) Ibid 10 (52) 193 Rath M Pauling L (1990) CHECK

Proc Natl Acad Sci USA 87 6204-6207 [cDNA for apo (a) shows a stong homolgy to plasminogen with multiple kringle stucture (C.Y) repeats with isoforms differing in kringle structure. It occurs especially in the plasma of those species which are unable to synthesise ascorbate and vice versa most mammalshaving an endogenous ascorbate lack detectable apo (a) in their plasma] Rees DA Steele IW Willliamson FB (1969) Conformational analysis of polysaccharides. III. The relation between stereochemistry and properties of some natural polysaccharide sulfates (I) J Polymer Sci (C) 261-276 Renne/ et al (David G) DATA J Biol Chem 275 (43) 33688-33696 Rej RN Holme KR Perlin AS (1990) Marked stereoselectivity in the binding of copper ions by heparin. Contrasts with the binding of gadolinium and calcium ions Carbohydr Res 207 143-152 Rider CC (1992) Sulphated glycoconjugates in the immune system Biochem Soc Trans 20 291-295 {cf Ianelle CJ De Lellis R Thorley-Lawson DA (1998) CD48 binds to a heparan sulfate on the surface of epithelial cells [CD48 is strikingly up-regulated on the surface of Epstein-Barr virus-infected B cells]} Lacy HM Sanderson RD (2001) Sperm protein 17 is expressed on normal and malignant lymphocytes and promotes heparan sulfatemediated cell-cell adhesion Blood 98 2160-2165 Robertson WvB Ropes MW Bauer W (1941) The degradation of mucins and polysaccharides by ascorbic acid and hydrogen peroxide Biochem J 35 903 DATA Robinson Mjet al DATA (2001) S100 family heterodimer MRP-8/14 binds with high affinity to heparin and HSPG on endothelial cells J Biol Chem 277 (5) 3658 DATA [S 100 expressed on nucleophils, monocytes and some secretory epithelia; involvement of hSPG in leucocyte adhesion to endothelium] Ross MA et al CHECK (1992) Heparin inhibits potentiation of thiobarbituric acid reactive substances in the presence of linoleic acid and Fe2+ ions Biochem Soc Trans 20 (4) 364s Effect of chemically modified heparins and of heparin fragments ion Fe2+ catlaysed perosicatio of linoleic acid [67-Ga mimics Fe heparan sulphate binding in tumor imaging Kojima et al 1983, Hama et al 1984, Sasaki et al 19856 DASTA Fe chelators block HIV kappa B activation (Schreck R Meier B Manner DN Drogue W Baeuele M (1992) J Exp Med 175 (5) 1181 DATA (cf also e.g. Albertinin RA et al (1995) FEBS Lett 377 (2) 240 Heparin as anti HIV agents and Albertini R (Varese) http://electra.chemistry.upatras.gr/fects/final/wshops.htm glycosaminoglycans inhibit metal-dependent oxidation of low and high density lipoproteins Ross W Salones J Holmes WM Goure RL (1999) Activation of E coli leu V transcription by FIS J Bacteriol 181 (12) 3864-3868 [RNA polymerase forms an unusually heparin-sensitive complex with leu V promoter] Rubo H Radi R Anselm D Kirk M Barnes S Butler J Eiserich JP Freeman BA (2000)

Nitric oxide reaction with lipid peroxyl radicals spares alpha-tocopherol during lipid peroxidation J Biol Chem 275 (15) 10812-10818 Ruponen M et al (2001) Extracellular glycosaminoglycans modify cellular trafficking of lipoplexes. J Biol Chem 276 (36) 33875 [Gene transfer by cationic lipids affected by binding to extracellular GAGs including HSPG] Roussel B et al (1990) A human monoclonal IgM with autoantibody activity against heparan sulphate and the mitotic spindle Clin Exp Immunol 1990 82(2) 294-298 Safaiyan F Kolset SO Prydz K Gottfridsson E Lindahl U Salmivirta M (1999) Selective effect of sodium chlorate treatment of the sulfation of heparan sulfate J Biol Chem 274 36267-36273 Salonen JT Nyyssones K Korpela H Yuomilehto J Seppanen R Salonen R (1992) High stored iron levels are associated with excess risk of myocardial infarction in Eastern Finnish men [Elevated seum ferritin and high dietary iron are risk factors and explains how iron chelators can prevent or limit myocardial ischemia and recovery in animal models ; iron may elevate the risk of myocardial infarction by promoting the oxidation of LDL protected against by dietary selenium required for glutathione peroxidase] [cf also inhibition of superoxide dismutase by aluminium (Shainkin-Kestenboum R et al 1989 Clin Sci (Lond) 77 (5) 463-6] Sampio LO Dietrich CP Colburn P Buonassisi V Nader HB (1992) Effect of monensin on the sulphation of heparan sulfate proteoglycan from endothelial cells J Cell Biochem 50 (1) 103 DATA CA 11 188863v [monesin affects the intracellular translocation of circulatory proteins at the level of trans Golgi cisternae. Exposure of endothelial cells to monesin causes biosynthesis of HS and chondroitin sulphate with a lower degree of sulphation, especially of 6-O sulphation] Sergeant Net al DATA (2000) Stimulaio of DNA synthesis and cell proliferation of human mamary myo epithelial cells by hepatocyte growth factor/scatter factor depends on heparan sulphate proteoglycans and cysteine phsphorylation of mitokine activated protein kinases p42/44 J Biol Chem 275 (22) 17094-17099 CA 133 100038b [The effect occurs when cells are grown on collagen I or fibronectin substrata but only mildly on plastic alone] Siegel G Malmsten M Lindman B (1998) Flow sensing at the endothelium-blood interface Colloids and Surfaces A : Physiochemical and Engineering Aspects 138 345-351 Sato E Tanaka T Takeya T Miyamoto H Koide SS (1991) Ovarian glycosaminoglycans potentiate angiogenic activity of epidermal growth factor in mice Endocrinolgy 128 (5) 2402-2406 Schroeder T Ostertag W Haubeck H-D (1999) Heparan sulfate proteoglycan expression is induced during early erythroid differentiation of multipotent hematopoietic stem cells Blood 93 (9) 2884-2897 Shainkin-Kestenbaum R Adler AJ Berlyne GM Caruso C (1989) Effect of aluminium on superoxide dismutase Clin Sci 77 463-466 [The combination of excess oxygen free radical production and aluminium intoxication may contribute to the aetiology of degenerative diseases] Siezlionski M Clarge DG Gordi MY (1992)

Binding of primitive haematopoeitic progenitor cells to marrow stromal cells involves heparan sulphate Blood 80 (4) 912 Singh RJ Hogg N Joseph J Kalyanaraman B (1996) Mechanism of nitric oxide release from S-nitrosothiols J Biol Chem 271 (31) 18596-18603 Cu+ decomposes S-nitrosothiols more rapidly than Cu2+ (cf also Nakon R Krishnamoorthy CR (1983) Science 221 749-750 which suggests that contamination of phosphate buffers by trace redox metals may allow deaminative cleavage of heparin by NO at physiological pH (reported by Vilar et al 1997, loc cit)) Sorenson JRJ (1984) Copper complexes in biochemistry and pharmacology Chemistry in Britain Dec 1110-1113 Stephens RW Bokman AM Myohanen HT Reisberg T Tapiovaara H Pedersen N GrondahlHansen J Llina/s M Vaheri A (1992) Heparin binding to the urokinase kringle domain Biochemistry 31 7572-7579 [Moderatly strong heparin binding to cysteine-tyrosine domain occurs under physiological salt conditions] cf Pucci M et al (2001) J Biol Chem 276 (7) 4756-4765 [Regulation of urokinase/urokinase receptor interaction by heparin-like GAGs, critical role in cell invasion] Stanley MJ et al (1995) Heparan sulphate mediated cell adhesion. Syndecans-1 and -4 mediate cellular adhesion folowing their transfection into human B lymphoid cells J Biol Chem 270 (10) 5077-5083 [The first confirmation of an involvement of syndecan heparan sulphate in cell-cell adhesion; this is further dependent on divalent cations and is inhibited by heparin or heparin-like GAGs, removal of HS from the cell surface or exogenous purified syndecan-1. A heterophilic adhesion mechanism involving binding of HSPG to a counter-receptor on the cell surface. Syndecan-4 transfected cells also aggregated in a HS-depedent manner in contrast betaglycan transfected cells which aggregated poorly] Stein PL van Zonneveld A-J Pannekoek H Strickland S (1989) Structural domains of human tissue-type plasminogen activator that confer stimulation by heparin J Biol Chem 264 (26) 15441-15444 Stichentoch DO Frolich JC (1998) Nitric oxide and inflammatory joint disease Brit J Rheumatol 37 246-257 Sugawara I Ishizaka S (1982) Polysaccharides with sulfate groups are human T-Cell mitogens and murine polyclonal B-cell activators (PBAS) Cellular Immunol 74 162-171 [Ref to prior findings of heparin activites included anticoagulant, antihyperlipidemic action, suppression of the secretory rate of aldosterone, increase in free concentration of thyroxine in plasma, inhibition of firinolytic activators, retardation of wound healing, depression of cell-mediated immunity, suppression of graft versus host reactions and acceleration of the healing of thermal burns] Susheela AK Jha M (1981) Effects of fluoride on glycosaminoglycans of cancellous and cortical bone of rabbits Experientia 37 1098-9 Ta T-V Baraniak D Julian J Korostoff J Carson DD Farach-Carson MC (2002)

Heparin sulfate interacting protein (HIP/L29) negatively regulates growth responses to basic fibroblast growth factor in gingival fibroblasts J Dental Res 81 247-252 Takeuchi Y Sakaguchi K Yanagashita M Aurbach GD Hascall VC (1990) Extracellular Ca2+ regulates distribution and transport of heparan sulfate proteoglycans in a rat parathyroid cell line J Biol Chem 265 (23) 13661-13668 Takeuchi Y Yanagishita M Hascall VC (1992) [The distribution of heparan sulphate proteoglycans in clonal rat parathyroid cells is regulated by the extracellular Ca2+ concentration which seems, however, only to regulate the distribution of heparan sulphate between the cell surface and intracellular compartments. Shed heparan sulphate proteolglycans are not internalised by these cells although these could conceivably be involved in signalling with other cell types which sense extracellular heparan sulphate (fragments)] Metabolic pathways of heparan sulfate proteoglycans in a rat parathyroid cell line Ibid 267 (21) 14677-14684 Cf Vanderwalle B et al DATA (1994) J Cancer Res Clin Oncol 120 (7) 389 CA 121 105487 [Ca2+ regulation of heparan sulphate proteoglycans in breast cancer cells; Ca 2+ also enhances the biosynthesis of such sulphated proteoglycans] Templeton DM (1991) Metal-proteoglycan interactions in the regulation of renal mesangial cells: implications for metal induced nephropathy Proc Trace Element Health Disease Aito A Ed Proc J Nord Trace Elem Soc/Union Pure Appl Chem Int Symp 1990 pp209-219 CA 111 1294071z CHECK [Paracrine role for HS in maintaining mesangial cell quiescence Inhibition by heparin of prolifertive response to phorbol esters but not EGF suggested PKC dependent mechanism countered by the presence of Ni; Inhibiton of insertion of HSPG into GBM by Ni and the prevention of cellular uptake of Ni by heparin; Appearance of HS in the nucleus correlated with inhibition of growth of log phase cells] {Another unusual metal ion dependent effect which may implicate Mn2+ toxicity and heparin/HSPG in the perturbation of soluble guanylate cyaclase activity was reported by Liebel MA & White AA (1982) (Biochem Biophys Res Commun 104 (3) 957-964)} Tiedemann DATA et al DATA (2001) J Biol Chem 276 (38) 36035-42 [Ca2+ dependence of extracellular microfibrils (fibrilin-1) binding to HSPG ] Theocharis DA (1992) Effect of glycosaminoglycans on peptide bond formation in bacterail ribosomes Int J Biochem 24 (5) 719-723 [In addition to the inhibiton of bacterial initiation complex (complex C) and peptide bond formation, heparin is a potent inhibitor of initiation of mammalian protein synthesis in cell free systems (Waldman & Goldstein, 1973 loc cit, and is also a potent inhibitor of elongation factor (EF-1) activity (Slobin 1976)inhibits the translation of poly (U) in extracts of E. coili and binds 30S ribosomal subunits and 70S ribosomes] Trivedy C et al (1997) Copper content in Areca catechu (betel nut) products and oral submucous fibrosis The Lancet 349 1447 Unnikrishnan VS Sudhakaran PR (1989) D(+) catechin enhances heparan sulphate content in rat liver Indian J Biochem Biophys 26 (6) 377-80 Valencia-Sanchez A et al (1995) Effect of heparin on the acrosome enzyme in rabbit spermatozoa

Mol Androl 7 (1,2) 57 CA 282178 [Heparin present in female reproductive tract fluids is likely involved in the acrosome release by binding to the spermatozoal surface and produces the Ca2+ influx necessary for fertilisation] Vilar RE Ghael D Li M Bhagat DD Arrigo LM Cowman MK Dweck HS Rosenfeld L (1997) Nitric oxide degradation of heparin and heparan sulphate Biochem J 324 473-479 [cf also Ghael D Li M Bhagat DD Arrigo LM Cowman MK Dweck HS Rosenfeld L (1997) Nitric oxide degradation of heparin and heparan sulfate DATA] Wang Y Cheong D Chan S Hooi SC (1999) Heparin/heparan sulfate interacting protein gene expression is up-regulated in human colorectal carcinoma and correlated with differentiation status and metastasis Cancer Res 59 2989-2994 Watson K Gooderham NJ Davies DS Edwards RJ (1999) Nucleosomes bind to cell surface proteoglycans J Biol Chem 274 31 21707-21713 Yoshizumi M Kourembanas S Temizer DH Cambria RP Quertermous T Lee M-E (1992) Tumor necrosis factor increases transcription of the heparin binding epidermal growth factor-like growth factor gene in vascular endothelial cells J Biol Chem 267 (14) 9467-9469 Heparin binding epidermal growth factor (HB-EGF) is a more potent mitogen than EGF than PDGF and is implicated as a vascular smooth muscle cell mitogen potentially implicated in the aetiology of atherosclerosis] Yang L Yang Y-C (1995) Heparin inhibits the expression of IL-11 and graunulocyte-macrophage colony stimulating factor GMC- SF in primate bone marrow stromal fibroblasts through mRNA destabilization Blood 86 (7) 2526-2533 [A novel mechanism of regulation of cytokine expression by heparin was detected since heparin by facilitation of the degradation of the mRNAs and did not alter the transcription of the genes] Zou S Magura ICE Hurley WL (1992) Heparin-binding properties of lactoferrin and lysozyme Comp Biochem Physiol 103B (4) 889-895 ADDENDUM Aspects of polysaccharide colloidal chemistry (including heparin/heparan sulphate) demonstrated nonclassical thermodynamic entropic effects, which may facilitate self-assembly of complex structures, in apparent contradiction to the second law of thermodynamics. Further studies of such activities may permit greater insight to be gained into this most central feature of biological systems. Self-association of complimentary HS chains may enable cell-cell signalling via extracellular HSPG interaction. Since the complex anionic (mainly sulphate half ester) charge density pattern seems to be highly functionally significant this will also permit coupling to nucleic acids. HSPG migration might then permit complex cross talk. HSPG-HSPG recognition may also include HS and core protein juxtaposition of syndecan and glypican units reinforced by de novo core protein disulphide bond formation. Nucleation of phase change may however similarly be required for this process as this apparently is a common situation for algal anionic polysaccharides (e.g. carrageenans) which have putatively related functions. Nucleation of phase change was also suggested by heparin cation binding studies (Grant et al 1992a). Signalling between heparan sulphate chains might be reinforced by electromagnetic communication. Divalent counterions apparently are delocalised along the chain undergoing hopping motions which appear to differ for different counterions (e.g. Cu2+ shows a characteristic intra carboxylate hopping especially under acidic conditions). Such movement will be associated with conformational movement of the polysaccharide chains and associated water molecules generating fluid waves or pulsing movements or hydrogen bonding arrangements between water molecules.

Iron ions may also bind strongly to heparin by a phase change mechanism (e.g. Grant et al 1992a) suggesting a possible HSPG modulation of iron-rich phase morphology which may conceivably influence the production of ferromagnetic particles believed to contribute to cellular activities (in species ranging from bacteria to mammals) including conferring orientation ability in the earths magnetic field.

That HSPG is a more evolved biopolymer than are other GAGs and could be considered to be specific sequence information-coded analogous to nucleic acids is entirely credible from current research results of HSPG from different cell and tissue types (e.g. Rahmoune et al 1998). However, involvement of HSPG in evolution according to the above hypothesis requires hypothetical functions of HSPG beyond what are present known (viz accurate microstructural differentation between HSPGs in cells with different nuclear DNA and accurate information feedback between DNA and HSPG, this being reversible) but accords with the original postulate of Dietrich et al that HSPG makeup determines each cell and organ type. This would require that any genetic change would cause an alteration of HSPG biosynthesis to reflect altered DNA sequence. Whilst current methodologies do not quite seem sufficiently advanced in sequencing HSPG polysaccharide chains to allow testing of this hypothesis, the consensus of current results confirms the possibility of such a notion in that each cell type is characterised by a customised polysaccharide charge density array particularly determined by the sulphate half ester anionic charge distribution, especially the GlcNC6OSO3- along the polymer chain. One might postulate that the packing of cells with externally arranged chemically different anionic density patterns could permit organisation of tissue in a manner somehow analogous to crystallization as indicated by Lima de Faria (DATA). Indeed in inorganic amorphous water-rich dispersion found in silicate chemistry, phenomena akin to such arrangements are known (as are also to phenomena akin to mitosis). In the case of HSPG, the coverage of cell surfaces is probably insufficient to justify such a simplistic notion of tissue morphogenesis based on arrangements of polysaccharide gel anionic clusters. Only if a very large amount of associated water is included in the polysaccharide gel would such a possibility be credible. Part of the problem of dealing with polysaccharide biochemistry is that current thinking tends to use methodologies developed from nucleic acid sequencing which might not be ideally suited to HSPG. Use of vibrational spectroscopy particularly using detectors (such as optoacoustic methods) not influenced by scattering and reflection which have been a deterrent to previous attempts at characterisation of sulphated polysaccharides, may be required. Oxidative and related stress is known to be capable of inducing genetic alteration. Although excessive uncontrolled oxidative stress may induce cancer by genetic damage, the imposition of properly functioning HSPG buffers between environmental stress and genes, as well as normally contributing to their protection (defects in which may promote degenerative diseases) may also promote a stress promoted alteration in genetic structure especially related to those genes which alter HSPG structures and may contribute to the occurrence of animal evolution to occur in response to a continuation of stress signal input to eventual alteration in RNA and DNA structure could occur via contributions from the proposed cytoplasmic switching systems acting in concert with appropriate HSPG microstructurs induced by external stress signalling and capable of communicating to genes for their eventual alterations What we are essentially suggesting that a signalling between heparan sulphate proteoglycans throughout the whole organism constitutes a computer-like control system to modify genes. HSPG acts as a fuzzy logic processor. Although such an idea may seem far-fetched it is suggested by a side rangeing assessment of the currently available information. The notion that genes respond to stress via a cysteine and tyrosine interlinked biochemistry (Nakashima 1996) and a hypothesis that oxidative stresses interact with juxtaposed cysteine and tyrosine residue (C-Y) clusters in proteins (Grant et al 1989) are now combined with the proposal that a relevant coupling between cellular activity and environmental stress may be activated via HSPG links. A CY/HSPG control of gene interaction could conceivably include activities of NO, its metabolites including their substitution of tyrosine, redox metal ions, ascorbate, thiols and polyamine signalling occurring at various levels of sophistication consistent with the existence of ranges of HSPG heparanases, sulfotransferase and sulfatase enzymes. Although oxidative stress may be eventually transmitted to selected genes by (CY) containing Src SH2 and similar cytoplasmic (CY) based switching systems suggested by Nakashima (1996) such an interface seems potentially suited chemically to inducing mild oxidative stress/+ electronic conduction along selective structures in RNA chains, perhaps allowing non-random rearrangements similar to

structural reorganization reactions which various phosphate-based polyester systems are prone (Van Wazer), i.e. involving an electronic bond rearrangemnt transfer with energy conduction along nucleic acid moieties without producing damaging free-radical mutations characterized by unchaperoned free-radical attack. Later reverse transcripiton of altered RNA incorporation into DNA may also be accomplished by coupled HSPG and NO interactions via binding of HSPG fragments produced by action of NO and its metobolites to histones and DNA control elelments. Such HSPG -controlled genetic rearrangement is suggested only to occur, upon an excessive prolongation of signalling by reactive oxygen species, crystals, pathogens, heatshock and trace toxic metals. The existence of possible biofeedback loops influencing cellular activity and capable of modifying genes, involving effects on HSPG and DNA structure of reactive oxygen and nitrogen molecule Perhaps aberrant evolutionary feedback loops of the kind now proposed, targeted inappropriately to non-stem cells, create the oncogenetic/protoongogenic perturbations which could conceivably be involved in the often suspected (Esko 1988, cf Engleberg 1990) relevance of heparin/HSPG to the aetiology of cancer, the origin of which while attributed to genetic alteration via unrepaired DNA damage, and while still not fully understood, seems to involve cellular activities additional to the creation of clones which disregard the protective signalling systems (including those provided by HSPGs) of the organism and for its progression require multiple failures of DNA repair, and various protective systems. signalling, redox status, redox metal ions and redox active molecules especially ascorbate are indicated The HSPG oligosaccharides formed by postsynthetic degradation are known to be capable of Reactive oxygen adducts (e.g. peroxynitrite, cf Beckman et al 1990)) and polyamines (Ding et al 2001) may influence HS activity in addition to the specifically modulation possible by action of NO sulphatases and heparanases. NO-based feedback loops may especially employ ascorbate and glucose signalling. The HSPG processing enzymes may be more evolutionary modulating HSPG biosynthesis by biofeedback NO and its metabolites also, especially under stress related situations, cause deaminative scission of HS chains to produce biologically active HS oligosaccharides. These effectors are proposed to allow amplification and direction of signalling for an alteration, initially in mRNA processing via the activity of competetively and further to redirect altered HSPG biosynthesis by HS-dependent morphogenic pathways. Gene alteration is then accomplished, it is suggested, as an eventual consequence of the long-term amplification of HSPG gated (C---Y)- HSPG modulated RNA loops stimulated by long-term stress impulses for the minimisation of such stress in organisms. To complete this hypothetical model of directed evolution requires an incorporation of novel sequences into germ DNA by hypothesised NO/ HSPG mediated apoptosis reverse trascription and transfection scenarios in which symbiotic or Amplification of non-enzymic postsynthetic processing of HS, which feeding into control loops. Alteration of genes specifying heparanase and morphogenic sulphatases may eventually redirect cellular activities and arrangements to enable a diminution of the effects of stress. enabling an amplification of HS signalling to be communicated to genes. athogenic organism biochemistry may participate.