ContentsPolyanion water activity regulation &

Preliminary notes dealing with the enthalpy-entropy compensation effect0. Sub-transition state free energy flow chanelling may generate enthalpy entropy compensation01. The isokinetic temperature phenomenon may give rise to stochastic structural reorganization phenomena 02. 03. Chemical affinity re-defined for stochastic chemical reactivityThe Fluctuation Theorem suggests that F (rather than F) is the chemical affinity in compensated systems

1. Brownian Motion2. Biological Nucleation 3. Compensation effects elucidate water structure

4. Liu & Guo (2001) Review5. Aberdeen U. (1984) Review

6. Putative Time Reversal & Compressed Time 7. Coveney (1988) Review 8. Vacuum Space and Extrathermodynamic Phenomena 9. Reverse Entropy & Vacuum Space 10. Vacuum Time 11., 12 Dehydrochlorination compensation data etc. 13 Redefining the nature of chemical reactivity 14. Soubility & Nucleation 14a. Nucleaton inhibitors ---------------------------------------------------------------------------------15. Van Wazer structural reorgnization ------------------------------------------------------------------------------16. (Heparan Sulfate & Degenerative Diseases) Mss. ex University of Aberdeen, Etc.

Manuscript POLYANION WATER ACTIVITY REGULATION A Hypothesis: Heparin/Heparan Sulphates Modulate Protein Activities by Water Activity Regulation Including Nanohole Induced Non-Equilibrium Water and Hofmeister-Like Effects Generated by Interfacial Aqueous Phases with High Ionic Strength, Multi-Element Inorganic Salt Compositions David Grant PhD MRSC* Aberdeenshire UK AB53 6SX (& University of Aberdeen) Heparan sulphate microstructure, evidently strictly regulated both temporarily and positionally during development (being responsible amongst numerous other established functions for regulating several growth factor activities [(1) cf. Bernfield et al. 1999; Hacker et al 2005] which are apparently dependent on the existence in syndecans, glypicans, agrin and other protoglycans of heparan sulphate sidechains having a great variety different linearly information-encoded sequences (dependent on the occurrence in this uronic acid (1->4) D glucosamine repeat disaccharides of sulphated iduronate, glucouronate and N acetyl, N-sulphonate or unsubstituted glucosamine 2-deoxyglucosamine, residues) which are believed to enable several types of signalling including those involving binding to basic sites

in proteins via specific information encoded sequences of sugars analogous to the pentasaccharide antithrombin-binding site heparin (Bernfield et al 1999; Lyon & Gallagher 1997) (such an information code is more complex than that of DNA to which it is obviously is however similar in principle although the heparan sulphate system apparently lacks any ability to act as a template for its self replication). It is now further suggested that binding of polysaccharides to proteins may require correct interstitial aqueous solutions (e.g. containing separate soft-ice like phases) which are now believed to contribute to the inorganic co-solute multi-ionic environments** . With heparan sulphate it seems that a system other than molecular recognition by an antithrombin (AT)-like-fine tuned electrostatic quaternary N+ protein link to individual codon sulphate mineral-like anionic patterns must allow fine structure discrimination to be established. A required high degree of selectivity by different microstructures present in different heparan sulphates seems to be the basis for their ability to select different proteins for their normal (e.g. growth factor orchestration) functions. The existence of some previously unsuspected unconventional molecular recognition system is however apparently required to facilitate this. This possibility is suggested by the work of Kreuger et al 2005) who found that most probable signalling oligosaccharides failed to discriminate between their individual FGF growth factor isoforms target binding sites when they were present as individual molecular signals (obtained from the high molecular weight by scission, separation and purification) . This finding is starkly contrary to the selectivity apparently achieved by the parent high molecular weigh proteoglycans in their in vivo environments. This situation might have arisen (as suggested by Kreuger et al) from the ability of such larger molecules (but not the smaller segments) to form additional hydrogen-bonding, Van der Waals forces and salt links (including those with inorganic ion bridges) and perhaps involving the whole heparan sulphate proteoglycan chain which may be involved. It is now proposed that the ultimate basis of heparan sulphate signalling is actually the induced water structure (e.g. this is acknowledged to be promoted in some non-specific manner by the extracellular matrix) and that this is the ultimate cause of tissue integrity. Tissue development may also require input form correct water structures associated with the longer polysaccharide segments molecules. This correct water structure for the proposed heparan sulphate signalling etc. functions could involve both hydrophilic and hydrophobic repulsive and attractive forces similar to the often long range effects which have been identified to occur in aqueous solutions between hydrophobic surfaces and hydrophobic and hydophilic mica surfaces (cf. Christenson & Claesson 2001) for which such long range effects have been identified might conceivably mimic the high anionic density + hydrophobic N-Ac region systems of heparan sulphates). These types of water structures are also believed to critically depend on the presence of sub-microscopic sized nano holes which have been in water structures (micro-bubbles) (e.g. adjacent to mica and silicate surface studies by atomic force microscopy) pointing to the possible key role of such a mechanism in the of formation of (presumable non-equilibrium, thermostatically unstable) water microstructures in the biochemical mechanism of tissue generation and its upkeep. Myelin basic protein integrity may depend on the hydrophobic effect on water structure and depend on microbubbles for its existence as it is associated with lipid induced hydrophobic waters structuring effects (Muelle et al 1999). It should be noted that the integrity of this protein has also been associated with heparan sulphate proteoglycans which enable the repair of damaged myelin sheaths, defects in which can be argued to give rise to neurological conditions such as multiple sclerosis. A scenario by which is this process becomes disrupted by a UV-vitamin D-thyroid factor dependent sulphate transporter which facilitates heparan sulphate sulphation could explain the geographical incidence of multiple sclerosis in Australia and perhaps also the possible promotion of this disease by barium intoxication (Purdey, 2004). It can also be logically suggested (e.g. Dr 2008) that protein folding must depend largely on the existence and the activities of interstitial water; The Hofmeister series can be explained by the effects of high concentrations of protein stabilisers or denaturant solutes on the surface tension of the interfacial water. This concept should also be extended it is now proposed to include the effect of nanobubbles on water structure. Since heparin and heparin-like sites in heparan sulphate are ultra-anionic, high ionic strength co-solute generating environments to those which promote the Hofmeister effect, it is also expected that the interstitial water which occurs adjacent to heparin/heparan sulphate polysaccharides could likewise

generate local Hofmeister effects which could determine how heparan sulphate attaches to and alters the conformation of target proteins. Direct evidence that such interstitial water might participates in such actions was obtained from studies of the binding of heparin to poly-L-lysine and poly-L-arginine (Grant et al. 1991) which indicated that such binding was accompanied by large changes in the overtone stretching frequencies of the water molecules attached to heparin. Related studies by these authors of the binding of inorganic ions and inorganic solids to heparin** and heparan sulphate tended to confirm that such binding depended on hydration changes (rather than electrostatic attractive forces, e.g. as had previously been believed to control this activity (cf. the Manning hypothesis). Related studies by the same authors that interstitial water and entropy changes thereof also apparently determined how a range of inorganic counterions bound to heparin. Further direct evidence for the existence of a high ionic strength environment adjacent to heparin is that crystalline akaganeite fibers (identified by X-ray diffractions) are produced at heparin surfaces (following binding of Fe2+ to heparin, its oxidation to Fe3+ Co2+ - dependent (such ions were also apparently commonly co-purified with heparin). [Formation of this fibrillar crystalline material {FeO.OH} is known to require the high ionic strength conditions]; (Williamson FB Aberdeen, personal communication of unpublished work). Biological Fluids are Seawater-Like Multielement Matrices from which Heparin and Anionic Polysaccharides Selectively Sequester the Least Abundant Solute Ions. In an internet paper (which is now no longer accessible) which discussed how water structure influences protein folding G Wilse Robinson quoted Szengt Gorgi who had earlier pointed out that humans are essentially bags of skin filled with seawater. Haraguchi, (2004) included heparin in his tabulation of topics for which a suggested that the new science of metallomics [a concept which had earlier been introduced by RJP Williams] which deals with the multi-inorganic nature of geological and biological phenomena including the seawater range of inorganic elements, which should be potentially be considered of fundamental importance to biochemistry since biological fluids are multi-inorganic ion solutions which are usually approximately similar in their multi-inorganic element compositions to seawater and other natural waters. It might further be suggested that since heparan sulphate seems to have co-evolved with multicellular animals in the sea some 109 years ago a primitive role of cell surface heparan sulphates was to act as a nutrient gatherer and buffer for bio-friendly seawater-like multi inorganic element containing salt solutions. This notion seems to be confirmed by a report from the Dietrich group of the existence of an exact mathematical relationship (Nader et al. 1983) between the amounts of tissue heparan sulphates (and other sulphated polysaccharides) and the salinities of the habitats of fifteen species of aquatic invertebrates, where habitat water might be required to directly bathe the heparan -sulphateproteoglycan-lined tissues. Anionic polysaccharides (and proteins) in animals when bathed in the multi-inorganic ion salt solution biological fluids will generate multi-inorganic ion/hydration water complexes similar to the anionic polysaccharides abundantly present in the cell walls of marine algae had been established (Wassemannn, 1949) most likely to exist in this form in vivo rather than being present, as was originally supposed, as free alginic acid Although less chemically definable than the pure polyanionc polysaccharides (alginates, carrageenans, and the polysaccharide side-chains of glycosaminoglycans etc.) but nevertheless of considerable importance to the homoeostasis of inorganic ions including carbonate, bicarbonate and Ca2+ ions in natural waters is the system of humic/fulvic polymers (a system of polymethylene, polycarbonyl, caboxylated) material which comprises the largest system of organic polymers on earth. These natural polyanions bind numerous metal ions present in seawater etc. via abundant -COO- groups, which apparently gave rise to the geological deposits of fulvate organic matter. Determination by spark source mass spectrometry (SSMS) of the multi-inorganic element contents of geological fulvates and marine alginate showed obvious qualitative similarities to the SSMS results for the multi-element contents of the animal polysaccharide heparin (Grant et al. 1987). Less information is currently available from the literature of similar studies of heparan sulphates (which are more difficult to obtain in large amounts) but studies conducted in the context of scitigraphic imaging (e.g. of tumours) has indicated that this procedure may depend upon the binding of the radionuclides to heparan sulphate proteoglycans e.g. at cell surfaces; side experiments

established that 45Ca in heparan sulphate could be replaced by a range of multivalent metal counterions in a manner consistent with heparan sulphate being normally present in vivo in the form of a multiinorganic matrix. The apparent differences in the observed biochemical/physical properties of different brands of heparin seems to at least in part have its origin in the different degrees of purification achieved by different manufacturers. It might even be suggested that such attempts at purification actually achieve inappropriate forms of heparin, at least from the requirement of biochemical if not from the standpoint of pharmacological research. What is obviously required for fundamental biochemical researches is the actual form of polyanions which are present in vivo. That the achievement of the equilibrium between the polyanion and the multi-element bathing fluids is not re-established rapidly may be deduced form the reports that the different single salt forms of heparin have different in vivo activities. The traditional view was that the multi-element character of unrefined heparin was of little scientific interest and that samples used for biochemical researches should be as free from such multi-elements as possible. [A similar hypothesis was applied to chitosan research, where the existence of multielements had been attributed to uptake from a final washing in tap water]. This idea is suggested to be incorrect and the status of the inorganic/water co-sphere around anionic polysaccharides which could potentially be part of an organometallic signalling system needs to be re-evaluated.

*This hypothesis was generated from private discussions (including with FB Williamson PhD and Professor WF Long** of the former University of Aberdeen, Marischal College Polysaccharide Laboratory, correspondence with Professor RJP Williams , Oxford University) and extensive internet literature studies made privately from Ashbank, Turriff AB53 6SX, UK as well as by use of the facilities kindly provided by Queen Mother Library Kings College and Forresterhill Medical Library, University of Aberdeen. **The majority of the published papers of this polysaccharide research group we posted by Professor WF Long at http://www/abdn.ac.uk/~bch118/publicatuions2003march.doc References arranged alphabetically Bernfield M et al 1999, Ann Rev Biochem. 68, DATA72907777; Cf. Hacker U Nybakken K Perrison N Nature Reviews Molecular Cell Biology 6 530-54- doi: 10.1038/nrm1681 Christenson HK Claesson PM 2001 Adv Colloid Interface Sci 91 (3) 391-436 Dr A 2008 Salts, interfacial water and protein conformation Biotechnol & Biotechnol Eq. 22 (1) 629-633 [Cf. Dr A Kelemen L Fbin L Taneva SG Fodor E Pli T Cupane A Cacace MG Ramsden JJ 2007 J Phys Chem 111, 5344-5350] Protein folding was traditionally viewed as an intrinsic property of the amino acid sequence in which the solvent had a secondary role; inherent hydrophilic/hydrophobic effects were believed to be directed by the amino acid sequences alone. This view has recently been challenged. Dr et al re-evaluated how proteins fold and suggested that the principal influence (or driving force) for protein folding originates from a dominant effect of water structure/activity on protein supramolecular structure/conformation. Such an influence of water structure also explains the Hofmeister or lyotropic series a phenomenon of protein denaturation characteristic of high solute (including inorganic salt) molecule concentrations. [Hofmeister salts change the hydophobic/hydrophilic properties of protein-water interfaces, kosmotropes making them more hydrobphobic while chaotropes make them more hydrophilic (these

terms were used in the context of their surface free energies). Surface tension*** of salt solutions in air had long been regarded as being related to the Hofmeister effect and indicated that this most likely arose from changes in water structure produced by the presence of high concentrations of salts (this was also suggested by overtone bands attributed to water molecule aggregates {cf., Kleeberg 1987; the ability of salt to influence the H-bonding in water is only seen in the immediate environment of the ions (Omta et al. 2003 Science 301, 347-34) which explains why high salt concentrations are needed affect the overall water structure and promote Hofmeister activities}. Grant D et al 1987 Biochem J. 244, 143-149 (Abbreviated list of multi-elements in heparin; the single ion form of heparin should also have been reported to have a list of these elements, albeit present in a much lesser amounts) Cf CPS 2000 Chemistry Preprint Archive , 2000 Oct, 94-104 available at http://preprint.chemweb.com/biochem.001002Multi element content of heparin (permanent internet file) The sample of sodium heparin chosen for evaluation by SSMS had been donated by a major pharmaceutical company presumably because of it was considered to be highly suitable for conducting academic biochemical researches. A highly purified sample using a standard industrial single salt heparin preparatory procedure was also studied by SSMS evaluation. Both heparins were found to have SSMS profiles similar to other natural polyanions (i.e. alginate or fulvate-like) multi-element matrices. These multi-element contents correlated (in log-log plots similar to those shown by Haraguchi, 2004) with those of human blood serum, human hair, marine alginates, seawater and the natural fulvates from geological deposits. A considerably body of literature was later found which supported the notion that commercial heparins are always contaminated to varying extents by such elements as Si ,Al, Cu, Zn, Mn, As, V, Ca, Sr and Ba. Cf. Harrison GE Sutton A Nature 1963 (4869) 809; Bowen HJM in Trace Element in Biochemistry, Academic Press, London, 1966; Heineman G Vogt W Biol Trace Elem Res. 2000, 75, 227-234; Alcock NM Serum versus plasma for trace metal analysis Elem Metab Man Ani Proc Int Symp 4th 1981 (Pub 1982) Eds JM Gawthorme, JMMMC Howell CL White p 678-680, Springer Verlag Berlin; Schwarz KA PNAS USA 1973. 70, 1608-1612; Bohrer D et al J Parenteral Enteral Nutrition 2005, 29 Bohrer D et al RBA 2004 36 (2) 99-103. The original report of these SSMS data was made by Moffat CF Ph D Thesis Synthesis, Characterizsation and Applications of Chemically Modified Heparins University of Aberdeen 1987 p 187-18 Grant D Tait MI Long WF Williamson FB Microstructure-dependent crystallization modulation by alginates (unpublished) Grant D Somers JA Tait MI Long WF Williamson FB Anti-calcite crystallisation activities of carrageenans Posters presented by MI Tait at the XIIIth International Seaweed Symposium Vancouver 13-18 August, 1989 Grant D et al (heparin-polypeptide interaction involvement of polymer-associated water) Biochem J 1991 277, 569-571 Grant D 2000 web.ukonline.co.uk/dgrant/dg Grant D et al (dependence on counter-ion of the degree of hydration of heparin) Biochem Soc Trans 1990, 18, 1293-1294 Haraguch H 2004 J Anal At Spectrom. 19, 5-14 Israelachvile J 1987 PNAS USA 84 4722-4724 Kleeberg H 1987 Proc Symposium in Honor of WAP Luck Marburg FRG

Kreuger J et al 2005 J Biol Chem 389, 145-150 [the oligosaccharides studied had the same charge densities, this seemed to suggest that charge density was the important factor for target protein binding not the way the charges were disposed along the oligosaccharides] Lyon M Gallagher JT Matrix Biol 1998, 17, 485-493 Cf Esko JD Lindahl U. Suggested reading list entitled Molecular diversity of heparan sulfate available at http://www.jci.org/content/full/108/2/169/DC1 Muelli H et al. 1999 Biophys J 76 (2) 1072-1079 Nader HB Medeiros MGL Paiva JF Paiva VMP Jeronimo SMB Ferreira TMPC Dietrich CP (1983) Comp Biochem Physiol. 76, 433-436 (Relationship between habitat salinity and the average total sulphated glycsoaminoglycan contents in fifteen species of Crustacea, Pelecypoda and Gastropoda) Park PW et al 2000 J Biol Chem 275 29923-29926 Purdey M (2004) Barium and multiple sclerosis (heparan sulphate mechanism) Sasisekharan R Shriver Z Venkataraman G Narayanasanu U Nat Rev Cancer 2002 2(7) 521-528 Wassermann A (1949) Cation adsorption by brown algae. The mode of occurrence of alginic acid Annals of Botany N.S. XIII (49) 79-88 Cf Black WAP Mitchell RL (1952) Trace elements in the common algae and in sea water J Mar Biol Asoc UK 30 (3) 575-584 (Alginates also bind to inorganic surfaces in a highly microstructure-discriminated manner (1); this seems also to be the case for animal polysaccharides and such binding could conceivable by utilized for sequence evaluation purposes). --------------------------------------------------------------------------------------------------------------

MANUSCRIPTS Preliminary Notes DEALING WITH THE ENTHALPY-ENTROPY COMPENSATION EFFECT Ms. 0Consideration Of How Constancy Of A Sub-Transition State Free Energy Flow Process Arising From Channeling of Energy May Generate Exact Enthalpy Entropy Compensation so that in a set of rate constants for chemically related processes

kr (where lnkr=AexpEa/RT) Ea becomes a linear function of logA] If chemical reactions proceed via the formation of transition state complexes in which the driving force for the reaction is provided by the (Gibbs-Helmholtz) Free Energy (F)

F = H-TS[H (enthalpy) T (absolute Temperature) x S (entropy)]

(1)

and if this driving force is exerted only at the actual part of the transition state complex which becomes changed* then for any set of chemical reactions (in which e.g. for a wide range of starting molecules which all contain the same (or a similar) sub-structure on which chemical transformation is performed) the consequence of this restriction to control the actual reacting centre by a related process over the whole set of reaction rate determining processes for which the values of

Fare (approximately) constant,then comparison (of F, H or S) between members of a set of such selected chemicalreactions must show that such values of

F are (approximately) zero,viz.substituting equation (2) in equation (1) gives

F 0

(2) ; H TS (3)

i.e. an (approximate) enthalpy ( H)-entropy ( S) compensation then exists for appropriate sets of rate constantsThis simple argument shows how the poorly understood enthalpy-entropy compensation process may arise simply from a conventional free energy driving mechanism which acts only upon a restricted reaction channel in the transition state complex.

In equation (2) T (=, the Leffler isothermal temperature at which all reactions in the selected set proceed at equal rate).(*This differs from the model of the transition state complex which was proposed by some later variations of the transition state theory which suggested that coupling of vibrations from distant parts of this molecule could contribute to energy flow) The above argument uses a single value of free energy, F , i.e. only one part of the conventional free energy driving force, ie. not, (as convention dictates) the change in free energy F which is associated with the completed reaction ; this use of partial time invariant free energy per se and not its variation time over the process of chemical change indicates that the role of time as a processor of free energy is being changed from that conceived in the clasical thermodynamics-derived expression of chemical affinity.

Ms 0-1

The Isokinetic Temperature Phenomenon May Give Rise To A General-Throughout-Chemistry Stochastic Structural Reorganization Phenomena.It should be noted that enthalpy-entropy compensation/ isokinetic temperature occurs widely throughout chemistry (and also throughout physics) and similarly random structural reorganization of chemical substances arise under controlled pyrolysis conditions. These two sets of phenomena are now putatively directly linked together.

Stochastic Structural Reorganization The process by which during heating above a critical temperature, all types of chemical substances throughout the periodic table can under their individally appropriately (pyrolysis) temperature conditions undergo complex structural reorganizations which lead to the formation of apparently highly complex mixtures of final products. While pyrolysis of non-carbon (inorganic) systems tend to occur at lower temperature that do those for carbon centered (organic) structures a similar underlying randomization of side chains about a core structure mechanism may determine the outcomes of both kinds of pyrolysis processes (conducted e.g. under sealed tube conditions). Examples include the various poly inorganic oxy acid esters (e.g. the polysilicic acid esters and the polyphosphoric acid esters for which the process of P-O-P bond formation and redistribution in the set of [P(O)(OR)3 (P)-O-P(O)(OR)2 {(P)-O-}2P(O)(OR) {(P)-O-}3P(O) give rise to ortho, (monomer) and condensed phosphates of linear chain, crosslinked (branched) chain and ring structures via the ring/chain ratio plus the operation of the sets of reactions: monomer + middles = 2 ends; end + branch = 2 middles]. The above behavior putatively arise simply because of the occurrence of a general enthalpy-entropy compensation behavior facilitated by the exitence of a critical temperature. (In sets of chemcial reactions which exhibit enthalpy-entropy compensation there exists an isokinetic temperature at which all rates within these sets of such reactions becomes equal and the products of these sets of compensated reaction processes (especially if they are conducted in a system which leads to polymeric structures) tend to show very clearly identifiable stochastic outcomes (or for a range of progressively near isokinetic conditions a range of progressively near-stochastic outcomes arise).

MSS02

Chemical Affinity, The Driving Force of Chemical Reactivity Re-Defined for Stochastic Chemical Reactivity It should be noted that historically the original idea proposed by Bertholet and Thomsen in the early nineteenth century was that the heat produced during a reaction was a useful indicator of chemical affinity or the driving force behind chemical reactions. This could not, however, explain why endothermic reactions can occur spontaneously. Hence it was suggested (by Gibbs VantHoff and Le Chatelier) that the proper definition of the chemical reactivity (athe driving force or chemical affinity) is the maximum work which a chemical reaction can perform. Hence the Gibbs-Helmholz concept of free energy F=H-TS Work & Free Energy The classical concept of chemical reactivity, the Gibbs-Helmholtz free energy F (=H-TS) change in F as a driving force was ultimately derived from the hypothesis that reactions could occur spontaneously only if such reactions could be made to perform useful work. However, the stochastic reorganization (scrambling) reactions which came to light only after such modern analytical techniques as NMR were introduced in the twentieth century, seemed to indicate that chemical structure alteration could also arise spontaneously under conditions where the overall free energy in the reaction vessel does not change, and therefore the idea that the magnitude of potential free energy change is what always primarily causes chemical transformations to occur (and always thereby potentially performs work) seems to be flawed. What then can give a more universally valid index of chemical reactivity? (It should be n oted that the general occurrence of certain extrathermodynamic free energy relationships which were not predictable from classical thermodynamics also questions the original ideas relating to how the overall free energy change equates to chemical reactivity). Stochastic (reversible reaction quasi-equilibria) chemical reactivity during which in the overall stoichiometric process, free energy is conserved perhaps should be distinguished from the type of nonstochastic chemical reactivity where free energy change remains a valid concept as a driving force for chemical reactivity. It now seems worthwhile instead, for the elucidation of the nature stochastic processes to re-focus more on the isokinetic temperature to consider how this relates to stochastic chemical reactivity (which is putatively a central consequence of the existence of enthalpy-entropy compensation) which putatively dictates the outcome of the (free-energy change-free) stochastic processes.

This draws attention to temperature per se and the isokinetic temperature (T) in particular as a possible usely focus for deriving a more general driving force for chemical transformation. MSS03The Fluctuation Theorem

suggests why F (and not F) may be a useful measure for the chemical reactivity driving force in compensated chemical systems.It should be noted that S and not S appears in the Evans equation which may be written S = P/t

(where P = expSt; antilog P = P= St where t is time and P is the ratio of the probabilities that S will take a positive or a negative value (i.e. that the direction of time will be positive or negative)) Substituting Evans S in F =H-TS, F = H-TP/t From the Leffler equation (H=ST, T = H/S (where T is the isokinetic temperature))

when TTF H-[H/S]P/t If H1 chloride dehydrochlorination and (similarly also to C=C isomerisations of Clcontaining olefins). Actual rate determining (perhaps heterogeneous) transition state complexes all lead to the (future) formation of >C=C< from HC-Cl (this includes chlorinated alipahtic molecule dehydrochlorination as well as chlorinated aliphatic substance isomerisation) in which the set of log A values are not restricted [as required by simple application of classical thermodynamics-based theories to a value of ca. 13.5] but vary over a wide range of values e.g. 2-20 (for kr sec-1 (apparent unimolecular rate processes) and even if the actual value is influenced by the surface polyion catalyst assisted processing) the (overall) value of Ea for any particular starting substance in the set is determined solely by the value of log A (which requires to obey the Leffler isokinetic relationship logA = Ea, [where is the isothermal temperature at which all reactions in the set occur at the same rate]). Reverse Time Theory The Fluctuation Theorem (Evans 1992, cf. Yang et al. 2002) proposed that for small entities over small time scales reverse time flow will occur to a varying extent according to the probability ratio of forward vs.

reverse time = exp. [entropy] . [time]. This time reversal was proposed to be exponentially size-related so that only forward time need be considered for larger particles such as the machines for which classical thermodynamics was designed, but for small-sized, short lifetime entities such as typical chemical molecules and transition state complexes these are now predicted to experience a real reverse time processing. This may be a critical requirement for all chemical reactivity. It is also then the ultimate origin of the enthalpy-entropy compensation phenomenon observed in sets of (chemical logic future state probability) rate constants. For individual series of chemical reactions (irrespective of the reaction mechanism) the products of such reactions can be considered to feedback virtual information (as it were from the future) into the series of their transition states so that the precise rate by which these products can be generated is achieved by a balance between the overall observed logA and Ea values. These entropy and enthalpy changes are then not independent variables but are determined by how matter interacts with virtual time in the virtual space of the transition state complex where the effect of temperature on a virtual conjoined chemical potential [the enthalpy-entropy] is determined by such a compensation process. This redefines what chemical reactivity actually is.

______________________________________________ ______________________________________________ _____________________________________________

Other ManuscriptsDG Note 27/4/12

15. Van Wazer Structural Reorganization

A review (which was originally drafted in 1975) of ideas generated from my participation in J.R. Van Wazers Structural- Reorganization-Throughout-The-Periodic-Table research program (which had been offered but not published or otherwise put to use) is now re-formatted below.

---------------------------------------------------------------------------------------SCRAMBLING REACTIONS David Grant, B.Sc., Ph.D. During the latter part of the eighteenth century, Berthollet and Proust debated the constancy of the combining proportions of elements in chemical species, (1). Berthollet recognized the importance of equilibria, regarding constant proportions as being the exception. There is a grain of truth in this, e.g., polymeric and amorphous species can exhibit variable compositions, and, while numerous compounds including those based on carbon backbones have a high probability of remaining unchanged for a long time, some substances having central atoms other than carbon at structural centers are intrinsically of lower stability than are purely organic molecular species. This applies to the liquid phase which can facilitate rapid molecular rearrangements to occur (and most especially if this process is fast with respect to the time required to separate the individual molecular species then the scrambled product is what is most commonly encountered). This scrambling, may, however, not be detected by vibrational spectroscopy or by X-ray diffraction. Numerous reaction products, previously though to be single compounds have been found to be the scrambled mixtures which, if at equilibrium will exhibit some reproducible physical properties in a manner similar to that of pure compounds. The most general form of scrambling was established by Van Wazer, (2) (ca. 1955) who extended the earlier concepts of Flory, and demonstrated that equilibria of which the following is typical, are established:(RO)2>P(O)(OR) (neso) + -O-P(O)(OR)-O- (middles) 2 O-P(O)Ge< (4 co-ord.) (11), >Sn< (4 co-ord.) (12), >Si< (4 co-ord.) (13), -B< (3 co-ord.) (14), [and also, e.g., RnMX4-n (R = alkyl etc., X = halogen, M = Ge, Sn, Si, etc.)]. Recent interest has been shown in CO exchange in cluster compounds, (15); different sites may exhibit markedly different exchange rates. Cf., Fe3 (CO)12 scrambling (15a). Examples of scrambling in polymer systems are: in polysiloxanes, (16); -silicates, (17); -sulphates, (18); -sulphones, (19); -sulphides, (20); -selenides, (20); -borates, (21); -phosphates, (22); -phosphonates, (23); -ethane, oxy,diphosphonates, (24); -arsenites, (25); -germthioxanes, (26); -(fluoro)arsenious methylimides, (27) and -tin sulphides, (28). Scrambling (at ambient temperature), of alkoxyl vs. Cl groups on -V(O)< is some 107 time faster than on -P(O)< but both systems are similarly random, (19). An example of scrambling on V(O)< (V(O)Cl3 / (CH3)3Si-O-Si(CH3)3) is shown in Fig. 1

Fig.1

[R=2Si/(V+2Si)]

The greater rates of scrambling on transition-metal centers may give rise to fluxional activity (rapid, often intramolecular NMR-detected scrambling) (29). The use of transition-metal compounds as catalysts for organic reactions, e.g., in hydrogenation, isomerization and polymerization, seems to be afforded by the scrambling potential at the metal, e.g., scrambling-related polymer generation arises from the -olefin monomer additions to the metalcentered scrambling centers which are the active sites of the Ziegler-Natta polymerization catalysts. In

the polymer chain propagation process the transition-metal forms metal-carbon bonds into which bonded olefinic bonds insert. This is likely to be a reversible (equilibrium) reaction, but is far displaced towards the polymer form at a polymerization temperature of ca. 70oC (30). Whether isotactic or stereospecific polymers arise during this insertion process seems to be dependent on the rate of structural reorganization of metal bound co-ligands. A further example of organic reaction catalysis at metal centers is the metathesis of olefins at W and Mo centers, which leads to an apparent scrambling of the alkyl groups about the double bond, (31). Scrambling may be catalyzed both positively or negatively, e.g., phosphorus halides scrambling is accelerated by H2O (32); for boron halides, scrambling is inhibited by bases, (33). There is probably no single type of mechanism which can explain scrambling reactions. Under scrambling conditions a mechanistic approach to the rationalization of chemical reaction products obtained at sub-scrambling temperature, so useful for organic reactions at near-ambient temperatures, is much less helpful for achieving an understanding of scrambling process. The important criteria are now knowledge of the equilibria (perhaps more accurately described as [enthalpy-entropy compensated] extrathermodynamic pseudoequilibria) which govern this behavior, (34). A general (first order approximation) principle of scrambling is that the type of equilibrium (e.g. random or non-random) is dependent on the ligands and is independent of the sites; the rate of scrambling, however, depends on the site, (35). Carbon is the slowest site. Scrambling of hydrocarbons (e.g. at 1000oC) gives CH4, C2H6, C3H8 and char (closed system). Phosphorus hydrides behave similarly (char now being red phosphorus), as do silicon hydrides, (36). The outcome of high temperature scrambling behavior of hydrocarbons is derivable from the accurate thermodynamic data which is available for these compounds, and the above scrambled corresponds to a thermodynamic equilibrium. However, at lower temperatures, reversible exchange between aliphatic and aromatic carbon-based systems, occurs. These are likely to be pseudo-equilibria, where the forward and reverse rates are not quite equal, and proceed by different mechanisms; this situation is illustrated by the scrambling of chlorocarbons in sealed-tubes, where all chlorocarbons which have been studied give CCl4, C2Cl6 and C6Cl6, with inappreciable char formation, (37). The phenomenon of aromaticity may be considered to be a double bond scrambling around the ring. Negative catalysis (Fe(CO)3 complexing sites) slow the process sufficiently for individual cyclohexatrienes to be distinguished, (38). In boranes and related compounds, a variety of BHB scrambling reactions have been reported , e.g. with B3H8 , (39),

Silicates scramble near-randomly, (17), therefore giving rise to appreciable amounts of a large number of structures. Nucleation of crystallization of a particular structure can remove it form the equilibrium, eventually converting all of the molecules present. [Bond exchange also can lead to flow in compositions beyond the gel point. In inorganic polymers such as silicates bond exchange can lead to flow whereas flow in organic polymers flow most often involves the sliding of the intact molecules over each other].

The formation of quite complicated structures in high yield in pre-biotic conditions could have been an outcome of scrambling reactions and subsequent nucleation of specific sub-types of molecules allowing the formation of specific proteinoid and poly-sugar-triphosphate molecules to be achieved in high yield.

References (1) -Cf. A Short History of Chemistry , J.R. Partington, Macmilllan, London, 1957, p. 153 (2) Phosphorus and Its Compounds Vol I, J.R. Van Wazer, Interscience, New York, 1959; C.F. Callis , J. R Van Wazer, J.N. Schoolery and W.A. Anderson, J Amer. Chem. Soc., (1957),79, 2719; J.R. Van Wazer, C.F. Callis and J.N. Schoolery, (1955), 77, 4945 (3) E. Schwarzman and J.R. Van Wazer, J. Amer. Chem. Soc., (1960), 82, 6009 (3a) cf., e.g., J.R. Van Wazer, Amer. Scientist, ref. (36) (4) G. Caligingaert and H.A. Beatty, J. Amer. Chem. Soc. (1939), 61, 2748 G. Calingaert, H.A. Beatty and H.R. Neal, ibid., (1939), 61, 2755 G. Calingaert and H Soros, ibid., (1939), 61, 2758 G. Calingaert, H.A. Beatty and H. Soroos, ibid. (1940), 62, 1099 (5) P.J. Flory, J. Amer Chem. Soc., (1942), 64, 2205 (6) G.S. Forbes and H.H. Anderson, J. Amer. Chem. Soc., (1944), 66, 931 (7) R.J.H. Clark and P.D. Mitchell, J.Chem. Soc. Dalton, 1972, 2429 (8) L.C.D. Groenwege and J.H. Payne Jr., J. Amer Chem. Soc., (1959), 81, 6357 (9) P.A.W. Dean and D.F. Evans, J. Chem. Soc., A, 1970, 2569

(10) M. D. Rausch and J.R. Van Wazer. Inorg. Chem. (1964), 3,761 Cf. J.C. Lockart, Chem. Rev., (1965), 65, 131 (11) G.M. Burch and J.R.Van Wazer, J. Chem. Soc. A. 1966, 586 cf. Inorg. Chem., 1964, 3, 268 (12) J.J. Burke and P.C. Lauterbur, J. Amer. Chem. Soc. (1961), 83, 326; G.S. Forbes and H.H. Anderson, ibid., (1945), 67, 1911; (1944), 66, 931, G. Calingaert, H. Soroos and V. Hnizda, ibid., (1940), 62, 1107 D. Grant and J.R. Van Wazer, J. Organometal. Chem., (1965), 4, 229 (13) K. Moedritzer and J.R. Van Wazer Inorg. Chem. (1964), 3, 268 and refs. cited; cf J.R. Van Wazer and K. Moedritzer, J. Inorg. Nucl. Chem. (1964), 24, 73 (14) H.K Hofmeister and J.R. Van Wazer, J Inorg. Nucl. Chem., (1964), 26, 1209 M.F. Lappert, M.R. Litzow et al., J. Chem Soc.(A) 1971, 383; (15) L. Milone, S. Aime, E.W. Randall and E Rosenberg J.C.S. Chem. Commun., 1975, 452 T.J. Marks and G.W. Grynkewich, J. Organometallic Chem., (1975), 91, C9-12,

F.A. Cotton, D.L. Hunter and P. Lahuerti, Inorg. Chem., (1975), 14, 511. (15a) B.F.G. Johnson, J.C.S. Chem. Commun., 1976, 703 (16) K. Moedritzer and J. R. Van Wazer, J. Amer. Chem. Soc. (1964), 86, 802 (17) D. Grant, J. Inorg. Nucl. Chem., (1967), 29, 69 R.O. Gould, B.M. Lowe and N.A. MacGilp, J.C.S. Chem. Commun., 1974, 720 (18) J.R. Van Wazer, D. Grant and C.H. Dungan, J. Amer. Chem. Soc., (1965), 87, 3333 (19) Unpublished work of D. Grant and J.R. Van Wazer (20) J.R. Van Wazer, D. Grant, J. Amer. Chem. Soc., (1964), 86, 3012 (21) H.K. Hofmeister and J.R. Van Wazer, J Inorg. Nucl. Chem., (1964), 26, 1201 (22) J.R. Van Wazer, C.F. Callis, J.N. Shoolery and R.C. Jones, J. A Amer. Chem. Soc., (1956), 78, 5709 and 5715 C.F. Callis, J.R. Van Wazer, J.N. Shoolery and W.A. Anderson, J. Amer. Chem. Soc. (1957), 79, 2719 D.P. Ames, S. Ohashi, C.F. Callis and J.R. Van Wazer ibid., (1959), 81, 6350 M. M. Crutchfield, C.F. Callis, R.R. Irani and G.C. Roth, Inorg. Chem., (1962), 1, 813 L.C.D. Groenweghe, J.H. Payne and J.R. Van Wazer, J. Amer. Chem. Soc., (1960), 82, 5305 E. Schwarzmann and J.R. Van Wazer, ibid., (1961), 83, 365 D.R. Cooper and J.A. Semlyen, Polymer, (1972), 13, 414 J.R. Van Wazer and S. Norval, J. Amer. Chem. Soc., (1966), 88, 4415 L.C.D. Groenweghe and J.H. Payne Jr., J. Amer. Chem. Soc., (1959), 81, 6357 (23) D. Grant, J.R. Van Wazer and C.H. Dungan , J. Polymer Sci., (1967),A-1,5, 57 (24) Unpublished work of D. Grant (Glasgow University manuscript in preparation [(added later: eventually published in Eur. Polym. J. (1979), 15, 1161)] (25) J.R. Van Wazer, K. Moedritzer and D.W. Matula, J. Amer. Chem. Soc., (1964), 86, 807 (26) K. Moedritzer and J.R. Van Wazer, J. Amer. Chem. Soc., (1968), 90, 1520 (27) M.D. Rausch, J.R. Van Wazer and K. Moedritzer, J. Amer. Chem. Soc., (1964), 86, 814 (28) K. Moedritzer and J.R. Van Wazer, Inorg. Chem., (1964), 3, 943 (29) F.A. Cotton, Chem. Brit., (1968), 4, 345 Cf. S. Cradock, E.A.V. Ebsworth, H. Moretto and D.W.H. Rankin, J.C.S. Dalton, 1975, 390; A.J. Campbell, C.A. Fyfe and E. Maslowsky Jr., Chem Commun., 1971, 1032; P.C. Angus and

S.R. Stobart, J.C.S. Dalton, 1973, 2374 (30) Cf. D. Grant , J Polymer Sci., Polymer Letters , (1975), 13,1 (31) Cf. N. Calderon and R.N. Hinrichs, Chemtech., (1974), 4, 627 E.L. Muetterties and M.A. Busch, J.C.S. Chem. Commun., 1974, 754 and refs. cited; A.J. Amass, Br. Polymer J. (1972), 4, 327

(32) A.D. Jordan and R.G. Cavell, Inorg. Chem., (1972), 11, 564 (33) B. Benton-Jones, M.E.A. Davidson, J.S. Hartman, J.J. Klassen and J.M. Miller, J.C.S. Dalton, 1972, 2603 Cf. M.J. Bula, J.S. Hartman and C.V. Raman, ibid., 1974, 725 (34) D.W. Matula, L.C.D. Groenweghe and J.R. Van Wazer, J Chem. Phys. (1964), 41, 3105 R.M. Levy and J.R. Van Wazer, ibid., (1966), 45, 1824 L.C.D. Groenweghe, J.R. Van Wazer and A.W. Dickenson, Anal. Chem., (1964), 36, 303 J.R. Van Wazer and K. Moedritzer, Angew. Chem. Internat. Edit., (1966), 5, 341 K. Moedritzer, Inorg. Chim. Acta, 1970, 4, 613 J.R. Van Wazer and L.C.D. Groenweghe, Nuclear Magnetic Resonance in Chemistry, B. Pesce, Ed., (1965), 283 J.R. Van Wazer Inorganic Polymer Chemistry J. Macromol. Sci., 1967, A1, 29 J.C. Lockart, Chem. Rev., (1965), 65, 131Note added to ms. later: the Van Wazer scrambling phenomena are likely to be afforded by extrathermodynamic pseudoequilibria associated with enthalpy-entropy compensation phenomena, the general occurrence of which throughout biology, chemistry and physics putatively requires a re-think of classical thermodynamics, an e.g. involves application of reverse time and vacuum energy concepts ]

(35) J.R. Van Wazer, Proc. Conf. Coord. Chem., 8th, Vienna, 1964, Springer-Verlag, Vienna, Ed. V. Gutman, p.162 (36) J.R. Van Wazer, Amer. Scientist, (1962) , 50, 450 (37) N.E. Aubrey and J.R. Van Wazer, J. Amer Chem Soc., (1964), 86, 4380 D. Grant, J. Appl. Chem. Biotechnol., (1974), 24, 49 (38) R. Victor, R. Ben-Shoshan and S. Sarel, Chem. Commun., 1970, 1680 (39) H. Beall and C.H. Bushweller, Chem. Rev., (1973), 73, 465 cf, E.L. Muetterties, E.L. Hoel, C.G. Salentine and M.F. Hawthorne, Inorg. Chem., (1975), 14, 950 Further References, Scrambling Centre Indicated. J.R. Van Wazer et al, J. Inorg. Nucl. Chem. (1964), 26, 1209 (boron); Inorg. Chem., (1964), 3, 139 (arsenic); J. Amer. Chem. Soc., (1964), 86, 811; Inorg. Chem., (1964), 3, 280 (phosphorus); Ibid., (1965), 4, 1294 (silicon) (silicon, germanium) J. Inorg. Nucl. Chem., (1964), 26, 737 (silicon) J. Organometal. Chem., (1968), 12, 69 (silicon)

Ibid., (1975), 85, 41 ([silicon] phosphorus) Inorg. Chim. Acta. (1967), 1, 407; Ibid., (1967), 1, (1967), 152 (silicon, germanium) (cf. K. Moedritzer ibid. (1971), 5, 547; (1974), 10, 163 (silicon, germanium K.M. Abraham and J.R. Van Wazer, J. Inorg. Nucl. Chem., (1975), 37, 541 (silicon, germanium) E. Fluck, J.R. Van Wazer and L.C.D. Groenweghe, J. Amer. Chem. Soc., (1959), 81, 6363 (phosphorus) J. Inorg. Nucl. Chem., (1967), 29,1571 (germanium); Ibid. (1964), 26, 737; Ibid., (1967), 29, 1851 (silicon) Inorg. Chem., (1965), 4, 1294 (review) J. Chem. Phys. (1964), 41, 3122 (several elements) J.G. Reiss and S.C. Pace, Inorg. Chim. Acta, (1974), 9, 61 (silicon) M.W. Grant and R.H. Prince, J. Chem. Soc., (A), 1969, 1138 (silicon)(germanium) F. Glocking, S.R. Stobart and J.J. Sweeney, J.C.S. Dalton, 1973, 2029 (mercury); A.G. Lee and G.M. Sheldrick, ibid., (A), 1969, 1055 (thallium); J.A.S. Howell and K.C. Moss, ibid., (A), 1971, 2483 (tantalum); R. Davis, M.N.S. Hill, C.E. Holloway, B.F.G. Johnson and K.H. Al-Obaidi, ibid., (A), 1971, 994 (molybdenum and tungsten); H. Hagnauer, G.C. Stocco and R.S. Tobias, J. Organometal. Chem., (1972), 46, 179 (gold); C.E. Holloway, J. Coord. Chem., 1971,1, 253 (tantalum); J. Evans, B.F.G. Johnson, J. Lewis and J.R. Norton, J.C.S. Chem, Commun., 1973, 807 (rhodium); J.F. Nixon, B. Wilkins and D.A. Clement, J.C.S. Dalton, 1974, 1993 (rhodium); J. Evans, B.F.G. Johnson, J. Lewis and R.Watt, ibid., 1974, 2368 (rhodium); M. Green and G.J. Parker, ibid., 1974, 333 (rhodium and iridium); A.J.P. Domingos, B.F.G. Johnson and J. Lewis, ibid., 1974, 145 (ruthenium); T.H. Whitesides and R.A. Budnik, J.C.S. Chem. Commun., 1974, 302 (ruthenium); F. Calderazzo, M. Pasquali and T. Salvatori, J.C.S. Dalton, 1974, 1102 (uranium); R.J. Cross and N.H. Tennent, ibid., 1974, 1444 (platinium); T. Mole, Organometal. Reactions, (1970),1,1, (review, aluminum); N.S. Ham and T. Mole, Progr. Nuclear Magnetic Resonance Spectrosocpy, Ed. J.W. Feeney and L.H. Sutcliff, Pergamon Press, London, (1969), 4, 91 (review); F.A. Cotton, Accounts Chem. Res., (1968), 1, 251 (review). (Examples of scrambling on carbon); K. Moedritzer and J.R. Van Wazer, J. Org. Chem., (1965), 30, 3920 (polyoxymethylenes); Ibid., (1965), 30, 3925 (acetals and orthoformates) J.T. Bursey , M.M. Bursey and D.G.I. Kingston, Chem. Rev., (1973), 73, 191 (intramolecular hydrogen transfer on carbon during mass spectrometery).Added Later Following my postgraduate research (which had been funded by an Albright & Wilson Mfg. U.K. Studentship and supervised by D.S. Payne at the University of Glasgow, Scotland, U.K, (earning Ph.D. in 1962 for a thesis A Study of Phosphites [cf. D. Grant et al., J. Inorg. Nucl. Chem., 1964, 26, 1985 and ibid., 26, 2103) and a study of the use of Ce(IV) for oxidative analytical chemistry [Anal. Chim. Acta, 1961, 25, 422 ]), I had the honor to work as a Postdoctoral Fellow with John R Van Wazer in his (Monsanto Co, St Louis) research group (which conducted fundamental researches into the phenomenon of stochastic structural reorganization; this processes, which had been found to determine the chemical constitution of condensed phosphates (and further seemed to offer insight into the in biological use of condensed phosphates for energy transfer and as a basic determinant of the structure and activity of nucleic

acids), also seemed to allow for a fuller understanding of the thermal stability of numerous kinds of substances (including inorganic, organometallic and organic polymers). I later contributed to I.S.R. U.K. research activities (which ceased in 1975) [cf., e.g. E.W. Duck et al. Eur. Polymer J., 1974, 10, 77; ibid., 481; ibid., 1979, 15, 625) and, The Pertinence Of The Scrambling Behaviour of Ligands on Transition-Metal Centres to Ziegler-Natta Catalyst Activities (J. Polymer Sci., Polymer Lett., 1975, 13, 1 ) which suggested that the polymerization of olefins (as well as the related process of olefin metathesis) occurred by a process akin to Van Wazer structural reorganization at the catalytic center monomer adducts].

Mss. ex University of Aberdeen

Archival Literature Surveys from personal files retained from Marischal College Polysaccharide Research GroupD. Grant, Ph.D., Turriff AB53, Scotland, UK

Contents

A Survey Conducted by Nancy EWoodhead

B Survey Conducted by David GrantA A Perspective Suggested by Peer-Reviewed Literature a of How Glycosaminoglycans May Mediate Cellular Activities Including Proliferation and Transformation

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A topic which is believed to be of public interest was the subject of a presentation made in 1982 (to the W.F. Long, F.B. Williamson Polysaccharide Research Group at Marischal College Aberdeen) by Nancy E. Woodhead. This document contains an edited transcript of my shorthand notes made at the time to which I now append an update of the peer-reviewed literature in this field and also an edited version of a hypothesis

(Ascorbate and Nitric Oxide in Redox Control of Heparan Sulphate) which has originally posted on the internet in 2000 on a site which is no longer active. ===================================================================== Glycosaminoglycans and Cellular Transformation Prior to 1982 literature reports in this field were classified by Nancy Woodhead as follows: 1. 2. 3. 4. 5. Experiments directly relating glycosaminoglycans (GAGs) to cell growth control and Transformation. Alterations in GAG contents of cultured cells after transformation. Alterations of GAG contents of mammalian tumors compared to normal tissue; [this provides more material than is available from cells in culture]. Changes in complex composition of certain cell surface GAGs; [especially of HS)] Searches for a specific changes in function of cell surface GAGs; [possible functional change due to regional alterations in GAG structure].

1. Includes reports of experiments relating GAGs to cell growth control and cellular transformation; starting from 1932: Year Author 1932 Zakrezewski 1957 Sister M. Lippman Reported that heparin suppresses growth of normal embryonic tissue and Jensen sarcoma tissue. Reported experimental evidence that heparin can act as a mitotic inhibitor. [This was an in vivo study of the effect of how administration of subcutaneous heparin affected the measured size of Ehrlich Ascites tumors in rats. It was shown that heparin, under the conditions studied, produced a 40-50% regression of tumors].

1960 Ozzello et al.

Umbilical cord extracted hyaluronate (HA) or chondroitin sulfate (CS) promoted the growth of human mammary carcinoma cells in culture. Any of these substance taken singly promoted tumor growth. Heparin prevented mitosis. Evidence for intracellular action of heparin. Heparin also caused formation of microvili on cell surfaces. (Is this an abnormal effect of heparin or is at an apparent normal intracellular function of heparin-like molecules?) It was apparent that the phenomenon was probably not a unique property of EDTA (a high affinity Ca2+ chelator) which had been known to produce microvilli formation. This outcome may simply be caused by the removal of Ca2+ from the cell surface. (Heparin-like molecules may have this function in vivo).

1964 Costachel et al.

1966 Takeuchi

CS enhanced of tumor growth. Hydrocortisone inhibited the growth of tumors, but this inhibition was prevented by the presence of CS. Heparin CS and HA : maintained cells in culture after they degenerated in normal culture medium. Some effect of CS and HA on cell surfaces was suggested to

1974 Takeuchi et al.

increase their potential for affecting cell growth. 1975 Olin et al. ? Salt effects ? (and related sources of salt e.g. heparin etc.) can apparently [link to this document lost] can exert a concentration-dependent control effect on tumor cell growth. Large doses inhibited epithelial cell growth but low cell surface doses accelerated growth. E.g. 50-100g/ml level (at) cell surface inhibited cell growth while 0.5g/ml promoted growth. Summary of the pre-1975 findings 1. Polyanions can enhance tumor growth by protecting cell surface antigenic sites. 2. The production of new connective tissue giving GAGs is favorable to cancer cell growth More GAGs (if present) they can be chosen for cancers to (allow cells to stick?) 3. Effect of heparin on nucleoproteins. Evidence shows that heparin interacts with proteins in the nucleus (and thereby affects DNA) and so (affects) protein synthesis. 4. Heparin removes Ca from cell surfaces. Cellular proliferation might be affected by this mechanism. Studies which report an Alteration in GAG content of cultured cells after transformation 1966 Ishimoto et al. Avian sarcoma virus in chicken embryo fibroblasts led to (i) 5x increase in HA synthesized, (ii) extracellular HA increases. 1973 Satoh et al. Herpes type II or SV40 virus transforming virus in hamster embryo fibroblasts led to (i) increased HA (ii) HS proportion of total GAGs

1977

Hopwood and Dorfman SV40 in human skin fibroblasts led to (i) increased HA; increased HS, decreased dermatan sulfate (DS). (ii) HA acid synthesis is inversely proportional to cell density in normal but not in transformed cells. (i) Primary and permanent cell lines (Associated) increased CS and increased HS in permanent cell lines (ii) (Associated) increased DS and increased HS in primary cell lines. These results were similar for transformed (cell lines) (these are equivalent to permanent?) Rous sarcoma virus (RSV) transformed chondrocytes, showed: (i) less cell surface GAGs; (ii) HS (was) shed into the cell culture medium. (1) Chemical transformation of liver parenchymal cell clones led to: Increased CS; production of (usually less) HS.

1978 Dietrich et al.

1979 Mikuni-Takagali and Toole i 1980 Ninomaya et al.

Suggested reason/(consequences) for these changes: (1) CS acts as stimulant for cell division, with HS and DS having different roles such as recognition and adhesiveness. (2) HS may act as a negative control element of growth. (3) Loss of activity of GAG degrading enzymes or increase in GAG synthesizing enzymes: not a direct effect of transformation. 1978 Chiarugi et al. Assessed Mammalian Tumors 100 cases of human cancer studied: normal and neoplastic GAG content compared: (i) All neoplastic tissue showed changed GAG contents. (ii) Malignant tissue showed larger changes than non invasive tumors (iii) Most common effects - increase in CS or increase in HA or decrease in HA or CS. Often an increase (occurs in) total GAG

(1) Increase in HA and/or CS is a characteristic abnormality of GAGs in cancerous tissue. (2) Increase in total GAG in tumor tissue gives overall change in negative charge associated with the tissue. ------Studies Reporting Changes in Chemical Structure of Cell Surface GAG Following Cellular Transformation 3TS cell and SV40 transformed cells (i) (ii) 1978 Nakamura et al. HA and CS not changed. HS from transformed cells elutes at lower ionic strength from anion exchange column.

1975 Underhill and Keller

AH130 Ascites hepatoma Cell-associated GAG 93% HS (extracellular) fluid 58% HS, 26% HA, 16% CS Cell associated (i) HS less sulfated. (ii) HS (is) highly heterogeneous (as indicated by electrophoresis).

1978 Winterbourne and Mora 3TS/SV3TS cells (i) HS elutes from anion exchanger at lower anionic strength. 35 3 (ii) S/ H ratio lower. (But) no change in overall turnover rate of HS. 1979 Chandreschan and Davidson Normal human breast cell line (compared with) human breast carcinoma cell lines.

Cancer cell lines (i) Mainly CS and HS. (i) Heterogeneous HS. (ii) Size Charge (and) NAc/N-SO3 altered.

1980 Keller et al.

3TS/SV3T3

(Following transformation) (i) HS charge density is lower. (ii) 8% decrease (occurs) in O-sulfate 1981 Winterbourne and Mora 3TS/SV3TS (i) (ii) (iii) (iv) HS charge-density is lower. O-sulfate containing disaccharides of HNO2 degraded HS; the same. O-sulfate-containing oligosaccharides of HNO2 degraded HS show lower 6-O sulfation. Overall sulfation a decrease occurs in 6 (-O-sulfate).

CONCLUSION(S TO BE) DRAWN FROM THE ABOVE EXPERIMENTS 1. 2. 3. 4. Change in sulfate incorporation into GAGs may reflect change in substrate PAPS pool sizes. T antigen* (SV40 early gene product) may be responsible for change in HS structure. Ca2+ binding of the cell surface may be altered by lower HS charge. Changes in HS structure may lead to altered binding to cell surface protein e.g. fibronectin (this is of interest since fibronectin is not found on many transformed cells).

* Binds heparin therefore could possibly change the structure of HS on the cell surface HS was found only on cells expressing T antigen. -------

Specific Change in Properties of Cell Surface GAGs 1982 Fransson et al. (i) (ii) (iii) (comparing) 3T3/8V3TS and PyY 3TS cells HS (is) heterogeneous HS has a lower change density (in transformed cells) HS iduronate/glucuronate bearing N-sulfate segments (are less common in transformed cells).

Increased heterogeneity is (also) associated with reduction in self-aggregation properties. 1982 1983 Culp and Dorfman Highly N-sulfated HS sequences (bind) most efficiently to fibronectin affinity columns.

Stamatoglou and Keller 3TS/8V3TS HS elution from fibronectin (or) collagen. No difference between normal and transformed cells (when using physiol.) NaCl Only heparin will displace HS from collagen (but) heparin and DS can displace HS from fibronectin.

1982 Castellot et al.

Smooth muscle cell growth is inhibited by heparin like substances. Endothelial cells exposed to heparinoids are released from growth inhibition.

Heparitinase released from cells or platelets may cause stimulation of mitosis by degrading heparin like compounds. If you get damage to an endothelial surface to a blood vessel then the platelets will adhere to this surface (but only if they) release heparinase and so stimulate cell growth. The release of thrombin also will occur under these conditions. It should be noted that: Cancer (n.b. is the) uncontrolled growth of cells. Transformation - (is the) event leading to cancerous growth of cells. Metastasis (is the critical-for-cancer-mortality) loss of cells from tumor surface to form subsidiary tumors elsewhere. N.b. Inflammatory conditions - (are those conditions where) CS and HS also tend to increase N.b. also: GAGs structures become altered before or during cellular transformation. But Validity of Considertion of Fibronectin as a Driving Force ? Could such evidence point in the wrong direction? The important events in cellular transformation may be associated (most specifically) with the GAGs. {Note added later. The current paradigm is that (usually multiple) mutation(s) of DNA produce cellular transformation and neoplasia. Whilst this mechanism is undoubtedly the primary cause of cancer the follow-on effect on the glycosylation system is putatively also an additional critical part of the etiologies of these diseases; especially the transformation-associated alteration of extracellular proteoglycan glycosylation regulatory functions afforded by HS may actually cause the greatest damage to the organism by allowing uncontrolled cellular growth angiogeneiss and metastasis to occur}. N.b. The HS information encoded processing system is now thought to be a major epigenetic driver. Chiarugi et al. had noted in 1974 (Biochim Biophys Acta. 345 283-293) that The effect of N-sulfated polyanions on tissue growth in vivo and in vitro has been recognized since 1932 (this was intimated by Zakrezewki loc. cit } But (at what stage) in the transformation process are the HS or other GAGs related to the change in events involved ? The initial important change must be there - the insertion of (some) new genetic interaction. Perhaps a clue can be gained from the circumstances where Ca is chelated out (cf. thrombin, antithrombin and the heparin effect) Mammalian GAGs - can be anti-inflammatory -- can be anti-cancer.

(Cf.) A consideration which might be of relevance is how cross-linked carrageenan beads can (behave) like proteoglycan complexes; (they are set in cells but they are not actually in the cells).

a Adapted from notes made during a lecture/discussion literature survey of how GAGs (especially heparan sulfate (HS)) participated in the etiology of cellular transformation and neoplasia presented to the Marishcal College (Aberdeen) Polysaccharide Research Group* on 18 November 1982 by Nancy E. Woodhead (then a graduate student member of the W.F. Long and F.B. Williamson Polysaccharide Research Group) written down in a University of Aberdeen Lab. Notebook 16/3/82-26/11/82 [Page heading Nancys Talk]

retained by D Grant AB53 UK.

Cf also NE Woodhead et al., IRCS Medical Science : Biochemistry ; Cancer; Cell and Molecular Biology; Connective Tissue, Skin and Bone ; Pathology [IRCS Med. Sci., 14 427428 (1986) (Heparan sulfates from fibroblasts exhibiting a temperature-dependent transformed growth trait). (This was a comparative study of normal vs. chemicallytransformed cells which exhibited a transformed growth trait at 37oC. The transformed cells reverted to a normal growth pattern at a lower temperature. The observed changes in heparan sulfates may have mediated this phenotype change.These results remain of topical interest.

*[The hypothesis that cellular proliferation is, at least in part, determined by the binding of Ca2+ ions to cell surface glycosaminogycans was, for about thirty years, a principal researchfocus for a polysaccharide research group headed by W.F. Long and F.B. Williamson at the University of Aberdeen. Barr L. et al. reported in 2006 (FASEB J. 20 E963-E975) that a Ca2+ dependent pathway (involving the completion of the glycosaminogycan protein linkage tetrasaccharide formation) controls chondroitin and heparan sulfate proteoglycan biosynthesis].

The Addendum (vide infra) lists a fairly random selection of more recent similar topic peer-reviewed literature

References Citations given in the 1982 lecture by N.E. Woodhead are listed below in the order in

which they appear in my notes (which were taken at the time); these are also listed in the The Heparan Sulphates of Control, Virally-Transformed and ChemicallyTransformed Fibroblasts Nancy Elizabeth Woodhead Ph.D. Thesis University of Aberdeen, , 1985. It should be noted that this thesis also reported for the first time on how a chemical carcinogen (N-methyl-N-nitro-N-nitrosoguandine)transformation of cells gave rise to similar diminution of HS sulphation to that observed with viral transformation or as is commonly found in HS extracted from cancer tissue. A previous Aberdeen University Ph.D. Thesis by H.H.K Watson (1980) had also dealt with related researches.Additional references to those discussed in the 1982 lecture and which further support the hypothesis that the etiology of neoplasia could depend on alteration of GAG composition following cellular transformation which were Chapter 1.8 [Glycosaminoglycans in Cancer] cited of the above Thesis are: Kuroda et al. (1974) [Rat tumors] Cancer Res. 34 308-312;

Kupchella et al. (1981) [Rat tumors ] ibid., 41 419-424 Kojima at al. (1975) Horai et al. (1981) [Human hepatic tumor] ibid., 502-547 [Human lung tumor] Cancer. 48 2016-2021

Knudsen et al. (1984) [Murine tumors synthesized 20x the amounts of GAGs in vivo/in vitro] J Cell Biochem. 25 183-196 David and Van den Berg (1983) [Transformed mouse epithelial cells] J Biol Chem. 258 7338-7344; Eur J Biochem. 1989

178 609-617 Robinson et al. (1984) ibid., 253 668-793 Ohkuboka (1983) Cancer Res. 43 2712-7 Additional Related-Topic Older References Mitotic Gelation (Water Structure?) Cf. Chiarugi V.P. et al. (1974) Biochim Biophys Acta. 345 283-293: Heparin has been found to prevent mitotic gelation ; (a periodic release of free heparin occurs in synchrony with the cell cycle); Augusti-Tocco G. and Chiarugi V.P. (1976) Cell Differentiation 5 (3)161-170 (Surface glycosaminoglycans as a differentiation cofactor in neuroblastoma cell culture) the switch from the round to the neuron-like cells can be obtained by a simple change of the culture conditions, which causes an increase of cell adhesion this is accompanied by an increasing ability of cells to retain heparan sulfate. Control of Cellular Activities by Cell Surface GAG Selective Binding of Ca2+ Cf. Long W.F. and Williamson W.F. loc. cit. and Cf. Vannucchi S. et al. Biochem J. 1978 170 185-187. Table 4 of this article indicates that the relative Ca2+ binding capacity of commercial GAGs was found to be HA 1.00, heparin 2.76, HS 2.00, CS(A) 1.60, CS(B) 1.67 and CS(C) 1.80. Role of Ascorbate as a Heparan Sulfation Control Agent Watson and Edward (1980) Biochem Soc Trans. 8 134-136 (Cf. Edward and Oliver (1983) [Ascorbate boosts HS sulfation] ibid., 11 383; ibid. 12 304 {this was confirmed by Kao et al. (1990) Exp Mol Pathol. 1990 53 1-10}and may be part of the mechanism by which ascorbate demonstrates anti-cancer activity as indicated, e.g. by Cameron and Pauling (1985) PNAS 75 4538-4542 cf. ibid., 79 3685-3689}) -------------------------------------------------------------------------------------------------------------------------------------------------Esko J.D. Rostand K.S. Weinke J.L.(1988) (Tumor formation dependent on proteoglycan biosynthesis) Science. 241 1092-1096; cf. Barr L. et al., (2006) 20 E963-E975 (Evidence of calcium-dependent pathways in the regulation of human 1,3glucuronosyltransferase-1 (GlcAT-I) gene expression : a key enzyme in proteoglycan synthesis) FASEB J. [These GAG chains are important regulators in a wide range of biological events, such as matrix deposition, intracellular signaling, morphogenesis, cell migration normal and tumor cell growth] Fedarko N.S. Ishihara M and Conrad H.E. (1989) (Control of cell division in hepatoma cells by exogenous heparan sulfate proteoglycan) J Cell Physiol. 139 287-294 PMID 2715188 (Additional related references accessed by Marion Ross a later member of the Aberdeen University polysaccharide group)) Zimina N.P. et al. (1987) Biokhimia. 52 (5) 856-861 [All types of sulfated GAGs in actively proliferating tissues (except

regenerating tissues) have a reduced degree of sulfation)Kosir M.A. and Culp L.A. (1988) Surg Forum Med. 39 424-426 Matuoka K et al. (1984) Cell Structure and Function 9 357-367 [1. HS plays a particular function in contact regulation of cell proliferation. 3. Transformation-related changes in the structure of HS molecules do not much affect the function of HS. 3. The cellular transformation, however, is accompanied by alteration in the growth regulating system sensitive to extracellular HS. Heparin inhibited growth in both normal and transformed cells]

Cf. also Long WF and Williamson FB (1979)

(Glycosaminoglycans, calcium ions and control of cell proliferation)

IRCS Journal Med Sci. 7 429-434; Cf. also Med Hypoth. 11 285-308 and ibid., 13 385-394 Most of the relevant Aberdeen University Polysaccharide Research Group publications including those of

N.E. Woodhead (up to 2003) which had been undertaken in part to advance the above hypothesis were listed by Professor W.F. Long atweb. abdn.ac.uk/~bch118/publications2003march.doc NE Woodhead et al. also conducted in vivo and in vitro studies relating to the Long-Williamson GAG divalent metal ion animal cellular control hypothesis (cf., Biochem J. 1986 237 281-284)

HS, the ubiquitous component cell surfaces and extracellular matrices of animals, may affect transformation of cells and also be a controller of their proliferation, at least in part, via HS-mediated control of the activities of (Ca2+ and Zn2+) metal ion signaling activities.Zakrezewki Z. (1933) Z Krebsforsch. 36 513-521 Lippman M. (1957) Cancer Res. 17, 11-14 Ozzello L. et al. (1960) ibid., 20, 600-605 Costachel O et al. (1964) Exptl Cell Res. 34 542-547 Takeuchi J. (1966) ibid., 26, 797-802 Takeuchi J. et al. (1976) ibid., 36, 2133-2139 Olin (source not found) Obrink B. et al. (1975) Conn Tiss Res. 3 187-193 Ishimoto N. et al. (1966) J Biol Chem. 241 2052-2057 Satoh C. et al. (1973) Proc Natl Acad Sci USA. 70 54-63 Hopwood J.J. and Dorfman A. (1977) J Biol Chem. 252 4771-4785 Dietrich C.P. and Armelin H.A. (1978) Biochem Biophys Res Commun. 84 794-801; Cf. Dietrich C.P. and DeOca H .M. ibid., 80 805-812; Mikuni-Takagaki Y. and Toole B.P. (1979) J Biol Chem. 254 8409-8415 Chiarugi V.P. et al. (1978) Cancer Res. 38 4717-4721 Underhill C.B. and Keller J.M. (1975) Biochem Biophys Res Commun. 63 448-454; cf., J Cell Physiol. 89 53-64 Nakamura N. et al. (1978) Biochim Biophys Acta. 538 445-457 Winterbourne D.J. and Mora P.T. (1978) J Biol Chem. 253 5109-5120 (1981) ibid. 256 4310-4320 Keller L. et al. (1980) Biochemistry. 19 2529-2536 Fransson L.-. and Havsmark B. (1982) Carbohydr Res. 110 135-144 Stamatoglou S.C. and Keller J.M. (1982) J Cell Biol. 96 1820-1823 Castellot J.J. et al. (1982) J Biol Chem. 257 11256-11260 ADDENDUM

Selected Later Referenceswhich further extend knowledge of the (heparin/HS involvement in animal cell proliferation and transformation) research topic

Zhou H. et al. (M402, a novel heparan sulfate mimetic, targets multiple pathways implicated in tumor progression) PloS ONE. 2011 6 (6) e21106 Chao B.H. et al. (Clinical use of the low-molecular-weight heparins in cancer patients: focus on the improved patient outcomes) Thrombosis. 2011 2011:530183; PMID 22084664 Borsing L. et al. (Sulfated hexasaccharides attenuate metastasis by inhibition of P-selectin and heparanase) Neoplasia. 2011 13 (5) 445-452 Casu B. et al. (Heparin-derived HS mimics that modulate inflammation and cancer) Matrix Biol. 2010 29 (16) 442-452 Raman K and Kuberan B (Chemical tumor biology of HS proteoglycan) Curr Chem Biol. 2010 4 (10) 20-31 [Heparan sulfate is a profound target for developing novel cancer therapeutics because modifying HS chains would affect Hpa, Hsulf-1, Hsulf-2, H-sulf-2, and 3-OST {these are key enzymes involved in HS biosynthesis and catabolism} activity in tumor cells, which in turn would affect angiogenesis, growth factor signal over amplification, and tumor growth, invasion and metastasis] Barash U. et al. (Proteoglycans in health and disease: new concepts for heparanase function in tumor progression and metastasis) FEBS J. 2010 277 (19) 3890-3903 Lee Y.D. et al. (Antiangiogenic activity of orally absorbable heparin derivatives in different types of cancer cells) Pharm Res. 2009 26 (12) 2667-76; PMID 19830530; Zacharski L.R. and Lee A.Y. (Heparin as an anticancer theraapeutic) Expert Opin Investgated Drugs. 2008 17 (7) 1029-1037 Pumphrey CY et al. (Neoglycans, carbodiimide-modified glycosaminoglycans: a new class of anticancer agents that inhibit cancer cell proliferation and induce apoptosis) Cancer Res. 2002 62 (13) 3722-3728 Engelberg H. Cancer. 1999 85 (23) 257-272 [Heparin is a potential anti-cancer drug] Cf. e.g. Goldberg I.D. Ann N Y Acad Sci 1986 463 289-291 Coombe D.R. and Kett W.C. (Heparan sulfate-protein interactions: therapeutic potential through structure-function insights) Cell Mol Life Sci. 2005 62 410-424 [Cf. also the later review by Casu et al. loc. cit.) is of especially relevance as it outlines why GAGs and particularly heparin/heparin -HS-like structures are now attracting considerable interest as a source of new therapeutics for the treatment of infectious diseases, inflammation and allergic diseases and cancers; attempts to provide a critical review of the historical development emphasises the complexity of the biochemistry of HS (which is confirmed to be a major master system which inter alia controls embryo development wound healing, hemostasis and the immune system) and points out that in vitro

studies may not reproduce the in vivo conditions especially as regards the present of metal cation cofactors and pH both of which can have profound effects on the microstructure of HS chains on which outcome of the interactions with the target proteins depend]. ANGIOGENESIS Ritchie J.P. et al., (SST0001, a chemically modified heparin, inhibits myeloma growth and angiogeneis via disruption of the heparanase/syndecan-1 axis) Clin Cancer Res. 2011 17 (6) 1382-1393 Logie J.J. et al. (Glucocorticoid-mediated inhibition of angiogenic changes in human endothelial cells is not caused by reductions in cell proliferation or migration) PloS ONE. 2010 5 (12) e14476; [Anti-angiogenic actions of glucocorticoids may be in part mediated by induction of thrombospondin-1 (TSP-1); this in turn implicates a key role of HS in this process as cf. Feitsma K. et al. who had indicated in J Biol Chem. 2000 275 (13) 9396-9402 (Interaction of thrombospondin-1 and heparan sulfate from endothelial cells. The microstructure of the HS cell surface binding sites for thrombospondin-1(TSP-1) (which are responsible for TST-1 endocytosis) contains a trisulfated 2-Osulfated iduronic acid-N-sulfated 6-O-sulfated glucosamine disaccharide unit which is distinct from the HS structure which is required for e.g. basic fibroblast growth factor binding]. Jakobsson L. et al., (Heparan sulfate in trans potentiates VEGFR-mediated angiogenesis) Developmental Cell. 2006 10 625-634[Cf. Medical News Today (www.medicalnewstoday.com/articles/43115.php (New discovery about role of sugar in cell communication,,, A research team from Uppsala Univeristy has uncovered an entirely new mechanism for how communication between cells

is regulated. By functioning like glue, a certain type of sugar in the body can make cell communication more effective and stimulate the generation of new blood vessels. The discovery paves the way for the development of drugs for cancer and rheumatism, for example)]Linhardt R.J. (Heparin-induced cancer cell death) Chem Biol. 2004 11 (4) 420-422 Blackhall F.H. et al. (Binding of endostatin to endothelial HS shows a differential requirement for specific sulfates) Biochem J. 2003 375 (1) 131-139 [Endostatin is believed to inhibit angiogenesis (and hence tumorigeneis) by a mechanism which appears to involve the binding of endostatin to HS and for which 6-O sulfates played a dominant role in site selectivity]; Folkman J. (Angiogeneiss inhibition and tumor regression caused by heparin or a heparin fragment in the presence of cortisol) Science. 1983 221 719-725; Cf. Crum R. et al., (A new class of steroids inhibits angiogenesis in the presence of heparin or HS fragments) Ibid., 1985, 230, 1375-1378 Cf. Schachtschabel D.O. and Sluke G. (Effect of cortisol on glycosaminoglycan synthesis and growth of diploid, human fibroblasts (WI-38) in relation to in vitro aging) Z Gerontol. 1984 17 (3) 141-149 PMID 6475191 [Contrary to the medium the pattern of the cell surface GAGs was changed by 1.4x10-7M cortisol with an increase in HA synthesis and a decrease in that of sulfated GAGs; this effect of cortisol is equivalent to a counter aging influence]

HEPARIN AFFIN REGULATORY PEPTIDE (HARP) [Pleiotrophin] Vacherot F. et al., (Involvement of heparin affin regulatory peptide in human prostate cancer) Prostate. 1999 38 126-136 Cf. Heroult M et al. (HARP binds to VEGF and inhibits VEGF-induced angiogenesis) Oncogene. 2004 23 1745-1753 Lee T-Y. Folkman J. and Javaherian K. (HSPG (heparan sulfate proteoglycan)-binding peptide corresponding to the exon 6a-encoded domain of VEGF (vascular endothelial growth factor [VEGF]) inhibits tumor growth by blocking angiogenesis in murine model) PloS ONE. 2010 5 (4) e9945; Cf. Chen J.-L et al. (Effect of non-anticoagulant N-desulfated heparin on expression of vascular endothelial growth factor (VEGF), angiogenesis and metastasis of arthotopic implantation of human gastric cancer} World J Gastroenterol. 2007 13 (3) 457-461 [N-desulfated heparin inhibits tumor metastasis and angiogeneis via an inhibition of the expression of VEGF (Possible Therapeutic Potential of (Non-Animal-Sourced) HEPARINOIDS (HEPARIN-LIKE SULFATED POLYSACCHARIDE) Zaslau S et al., Amer J Surg. 2006 192 (5) 640-643 (Pentosan polysulfate (Elmiron): In vitro effects on prostate cancer cells regarding cell growth and vascular endothelial growth factor production); [This heparinoid is a sulfated beech wood xylan also known as SP54] Cf. Noda H et al. (Antitumor activity of polysaccharides and lipids from marine algae) Nippon Suisan Gakkaishi 1989 55 (7) 1265-1271 EXT (Heparan sulfate related) TUMOR SUPPRESSOR Kitagawa H. et al. (The tumor suppressor EXT-like gene EXTL2 encodes a key enzyme for the chain initiation of heparan sulfate) J Biol Chem. 1999 274 (20) 13933-13937; Lind T. et al. (The putative tumor suppressors EXT1 and EXT2 are glycosyltrqansferases required for the biosynthesis of heparan sulfate) J Biol Chem 1998 273 (41) 26265-26268 Rahmoune H. et al. Biochem Soc Trans. 1996 24 (3) 355S [While the usual effect of cellular transformation is a lower production of heparan sulfate of decreased degree of sulfation, cellular transformation can also be accompanied by an augmentation of heparan sulfate biosynthesis (with increasing cell surface as well as culture medium released heparan sulfate) relative to normal cells)] Cf. Biochem J. 1998 273 33 21111-21114 [A MCF-7 tumor cell HS microstructure which binds laminin-1 (implicated in tumor-host adhesion) was identified (IdoA(2-O-SO3-)-GlcNSO3-(6-O-SO3-)]5[IdoA(2-O-SO3-)-AManR(6S-O-SO3-was generated using ahydrazinolysis/deaminative procedure which cleaves deacetylated N-acetylglucosaminic bonds).

Liuzzo J.P. and Moscatelli D. (Human leukemia cell lines bind basic fibroblast growth factor (FGF) on FGF receptors and HS: Down modulation of FGF receptors by phorbol ester) Blood. 1996 87 (1) 245-255

HEPARAN SULFATE AT CELL NUCLEUS Buczek-Thomas J.A. et al. (Inhibition of histone acetyltransferase by glycosaminoglycans) J Cell Biochem. 2008 105 (1) 108-120 Hsia E. et al. (Nuclear localization of basic fibroblast growth factor is mediated by HS proteoglycans through protein kinase C signaling) J Cell Biochem. 2003 88 (6) 1214-1225 Richardson T.P. et al. (Regulation of HS protoglycan nulcear localization by fibronectin) J Cell Sci. 2001 114 (9) 1613-1623 HEPARANASE (Effect at Nucleus) Purushothaman A et al., (Heparanase-mediated loss of nuclear syndecan-1 enhances histone acetyltransferase (HAT) activity to promote expression of genes that drive an aggressive phenotype J Biol Chem. 2011 286 (35) 30377-30383 Yang Y. et al. (Heparanase enhances local and systemic osteolysis in multiple myeloma by upregulating the expression and secretion of RANKL) Cancer Res. 2010 70 (21) 8329-8338 Chen L. and Sanderson R.D. (Heparanase regulates levels of syndecan-1{HS proteoglycan} in the nucleus) PloS ONE. 2009 4 (3) e4947 [Although HS function within the nucleus is not well understood there is emerging evidence that it may act to repress transcriptional activity] Mani K et al. (Tumor attenuation by 2(hydroxynapthyl)--D-xylopyranoside requires priming of heparan sulfate and nuclear targeting of the products) Glycobiology. 2004 14 (5) 387-397[N.b. the HS oligomers which were found to signal to the nucleus in these studies showed anhydroMan end groups. These are formed during (nitric oxide ascorbate Cu/Zn facilitated) nitrosative cleavage i.e. the (partly) non-enzymic cleavage of HS (to give putative hormone-like HS fragments) from un-substituted GlcNH2 groups in HS pre-primed for nitrosative cleavage. (While such structures are known to occur in HS although the mechanism of their insertion e.g. during primarly biosynthesis details are still unknown but putatively may aberrantly be augmented as a result of non-enzymic de-N sulfonation during acidosis or redox metal ion dyshomeosasis of precursor GlcN-SO3- groups. Or perhaps by other mechanisms which putatively contribute to the aetiologies of neoplasia and other degenerative diseases}]

HEPARANASE - GROWTH FATOR PHOSPHORYLATIION Cohen-Kaplan V. et al., (Heparanase augments epidermal growth factor receptor phosphorylation: correlation with head and neck tumor progression) Cancer Res. 2008 68 (24) 10077-85 HEPARANASE -METASTASIS Cohen E. et al. (Heparanase is overexpressed in lung cancer and correlates inversely with patient survival) Cancer. 2008 113 (5) 1004-1011; Faye C. et al.

(Molecular interplay between endostatin, integrins and heparan sulfate) J Biol Chem. 2009 284 (33) 22029-22040 Liu D et al. (Tumor cell surface heparan sulfate as cryptic promoters or inhibitors of tumor growth and metastasis) PNAS USA 2002 99 (2) 568-573 [Specific different HS microstructure mixtures generated in vivo by exogenous heparinase apparently signal for opposite growth effects on tumor cells was suggested by the observation of the effect of difference between heparinases I and III when injected into mice with B16BL6 melanoma; while the HS oligomer mixture arising from heparinase III digestion caused tumour cell inhibition that produced by heparinase I digestion caused tumor cell growth to be enhanced] Vlodavsky I and Friedman Y. (Molecular properties and involvement of heparanase in cancer metastasis and angiogenesis) J Clin Invest. 2001 108 (3) 341-347 CELLULAR IMMUNE ANTI-TUMOR RESPONSE Dziarski R. (Enhancement of mixed leukocyte reaction and cytotoxic antitumor responses by heparin) J Immunol. 1989 143 (1) 356-365 INORGANIC CALCIUM ION : COFACTOR IN HEPARAN SULFATE ACTION Ca2+ etc. chelation, heparan sulfate/chondroitin sulfate ratio METASTASIS Cf. Tmr J et al. (Modulation of heparan-sulfate/chondroitin-sulfate ratio by glycosaminoglycan biosynthesis inhibitors affects liver metastatic potential of tumor cells) Int J Cancer. 1995 62 755-761 [Ethane-1-hydroxyl-1-1-diphosphonate [EHDP] a well tolerated pharmaceutical (and a Ca2+ binding ligand) and other (different mechanism) GAG biosynthesis inhibitors, were apparently able to diminish tumor metastasis (by putatively Ca2+ dependent) inhibition of heparan sulfate biosynthesis. It should be noted that EHDP and related bisphosphonates osteoporosis therapeutic use also has been indicated to produce an extended life expectance by an unknown mechanism; it should also be noted that bisphosphonates per se have been reported to demonstrate anti-metastatic effects in e.g. in prostate cancer cf. e.g. Montaque R et al., Eur Urol. 2004 46 (3) 389-401] Kan M. et al. J Biol Chem. 1996 271 26143-26148 [Divalent metal ions are essential cofactors for basic fibroblast growth factor assembly] for correct interaction between heparan sulfate and L-selectin as well as 2 and 3 integrins and for annexinV assembly on cell surfaces]; Valencia-Snchez A. et al. Mol Androl. 1995 7 (1,2) 57 [Heparan sulfate Ca2+ flux control during capacitation and modulation of acrosome reaction by heparin] Takeuchi Y. et al. J Biol Chem. 1990 265 (23) 13661-13668 [Extracellular Ca2+ concentration regulates the distribution and transport of heparan sulfate proteglycans and heparan fragments in a rat parathyroid cell line] Vandewalle B. et al. J Cancer Res Clin Oncol. 1994 120 (7) 389 Ca2+ enhanced HS proteoglycan activity which modulate tumor cell growth Hayashi M. and Yamada K.M. J Biol Chem. 1982 257 5263-5267 [Divalent cations are required for heparin binding to fibronectin] Boehm T. et al. (Zinc binding of endostatin is essential for its anti-angiogenic activity)

Biochem Biophys Res Commun. 1990 252 (1) 190-194;The roles of Ca2+ and other metal ions in the inorganic biochemistry of heparan sulfateallows for a system of Ca2+ Zn2+, Cun+ etc. activity regulation (and perturbation of this toxic Mn+ metal ions) could be relevant inter alia to a fuller understanding of how GAGs contribute to animal tissue homeostasis and non-specific immune protection and wound healing.

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