Characteristics of a novel high viscosity polysaccharide,methylan, produced byMethylobacterium organophilum

Summary A new anionic high molecular weight polysaccharide, Methylan, was produced byMethylobacterium organophilum from methanol as a sole source of carbon and energy under the specific culture conditions. By GPC and light scattering, the molecular weight was determined to be 2–4×10⁶ dalton and the distribution of molecular weight was very homogeneous. Methylan was composed of carbohydrate (80%), uronic acid (12%), protein (6%) and pyruvic acid (5%). The sugar composition of Methylan was identified as glucose, galactose and mannose with the approximate molar ratio of 232. Methylan solution showed a pseudoplastic non-Newtonian fluid property at the concentration above 0.05%. At the concentration of 1% Methylan solution, the consistency index was 18,000 centipoise which was almost 10 times higher than that of Xanthan and the flow behavior index was 0.15.     

Antitumor activity of methylan polysaccharide derivatives

Methylan polysaccharide derivatives were prepared by dialkylaminoalkylation and reductive amination followed by quaternization. Their antitumor activity was investigated and a relationship between structure and activity is suggested. For quaternized DEAE-methylan at only 75 μg ml^?1, tumor cell proliferation was suppressed by 58–84% in three cell lines tested in the order Colo < Hela < HepG2.     

Preparation and structural analysis of actinidain-processed atelocollagen of yellowfin tuna (Thunnus albacares)

Pepsin-hydrolyzed collagen (atelocollagen) is a trimer, consisting of alpha 1 and alpha 2 monomers, and shows molecular species corresponding to a monomer, dimer (beta chain), and trimer (gamma chain) by SDS-polyacrylamide gel electrophoresis. Atelocollagen was purified from yellowfin tuna (Thunnus albacares) by salt precipitation and cation-exchange chromatography. Enzymatic hydrolysis of the atelocollagen by actinidain, a cysteine protease purified from kiwifruit, was analyzed by SDS-polyacrylamide gel electrophoresis. The triple helical structure unique to collagen was retained in the atelocollagen as judged by circular dichroism spectra. The actinidain-processed atelocollagen showed only monomeric alpha 1 and alpha 2 chains, with no beta and gamma chains, by SDS-polyacrylamide gel electrophoresis; nevertheless, it retained the typical triple helical structure. It is suggested that actinidain cleaved the atelocollagen molecule at specific sites on the inside of the inter-strand cross-linking peptides.     

Effect of acyl chain composition on salt-induced lamellar to inverted hexagonal phase transitions in cardiolipin

Salt-induced fluid lamellar (L alpha) to inverted hexagonal (HII) phase transitions have been studied in diphosphatidylglycerols (cardiolipins) with different acyl chain compositions, using 31P nuclear magnetic resonance (NMR) spectroscopy. Cardiolipins with four myristoyl chains, tetramyristoyl cardiolipin (TMCL), and with four oleoyl chains, tetraoleoyl cardiolipin (TOCL), were synthesized chemically. TMCL was found to undergo a thermotropic lamellar gel to lamellar liquid-crystalline phase transition at 33-35 degrees C. This lipid exhibited an axially symmetric 31P-NMR spectrum corresponding to a lamellar phase at all NaCl concentrations between 0 and 6 M. In the case of TOCL, formation of an HII phase was induced by salt concentrations of 3.5 M NaCl or greater. These observations, taken together with earlier findings that bovine heart cardiolipin aqueous dispersions adopt an HII phase at salt concentrations of 1.5 M NaCl or greater, indicate that increasing unsaturation and length of the acyl chains favour formation of the HII phase in diphosphatidylglycerols.     

Isolation and characterization of a native placental basement-membrane collagen and its component alpha chains.

Native type IV collagen was isolated from human placenta using pepsin solubilisation followed by fractional salt precipitation and chromatogarphic purification. The native preparation was characterised using amino acid analyses, disc gel electrophoresis, segment-long-spacing crystallites and immunological methods. Two component alpha chains were isolated with molecular weights of approximately 95000 and 70000. Cyanogen bromide digests of these chains indicated that they are not related to any of the known alpha chains of interstitial collagens or to the recently described collagen containing alphaA and alphaB chains. They are also not related to one another and are therefore probably fragments of two genetically distinct type IV collagen alpha chains.     

Characterization of a type IV procollagen synthesized by human amniotic fluid cells in culture.

Fetal epithelioid cells, isolated from human amniotic fluid, synthesize and secrete a type IV-like procollagen characterized by a unique pattern of cyanogen bromide (CNBr)-produced peptides. The procollagen is disulfide-bonded and, after reduction, migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a doublet between collagen beta components and pro-alpha 1(I) chains. No conversion of the procollagen to collagen or to procollagen intermediates is observed in cell culture. The procollagen was purified by salt fractionation and ion exchange chromatography; its amino acid composition resembles that of collagenous proteins extracted from basement membranes, with a high 3- and 4-hydroxyproline and hydroxylysine content and low levels of alanine and arginine. The major products obtained after limited proteolytic digestion of the protein retain interchain disulfide bonds and, after reduction, migrate on sodium dodecyl sulfate-polyacrylamide gel electrophoresis near intact pro-alpha 1(I) chains. The procollagen is secreted efficiently by amniotic fluid cells despite almost complete inhibition of peptidyl hydroxylation but, unlike type I procollagen, the secreted underhydroxylated chains lack interchain disulfide bonds. Since these cells also secrete fibronectin and elaborate an extensive extracellular matrix, the system should prove useful in the study of cell-matrix interactions.     

Two heavy chains of 21S dynein from sea urchin sperm flagella

The biochemical properties of 21S dynein derived from sea urchin sperm flagella and of its components dissociated by low salt treatment were studied. SDS-urea gel electrophoresis and two-dimensional gel electrophoresis showed that the 21S dynein preparation contains two distinct heavy chains. These two heavy chains, termed A alpha and A beta, had apparently the same molecular weight of 500,000 but showed different mobilities on SDS-urea gels. The isoelectric points of A alpha and A beta heavy chains were 5.7 and 5.2, respectively, in the presence of urea. Proteolytic digestion patterns of these two heavy chains were clearly different, but the amino acid compositions were similar. Low salt treatment and sucrose density gradient centrifugation could partially separate the components of 21S dynein into two fractions: the one with larger sedimentation coefficient contained the A alpha heavy chain, and the other with smaller sedimentation coefficient contained the A beta heavy chain and three intermediate chains. These two fractions showed distinctly different kinetic properties, and thus may play different roles in dynein-microtubule interaction.     

NMR investigations of the 4-ethyl guaicol self-diffusion in iota (ι)-carrageenan gels

Self-diffusion coefficient of an aroma molecule (4-ethyl guaicol) was measured using the pulsed field gradient spin echo NMR (PGSE-NMR) method in order to investigate the influence of a macromolecular matrix on its diffusion and release processes. Iota (ι)-carrageenan was used for its ability to form thermoreversible gels in aqueous salt solutions. Variations of the ι-carrageenan and the salt concentrations permitted various gels with different thermal and rheological properties to be obtained. These latter were modified by an isotope effect obtained by preparing gels in D₂O. The NMR self-diffusion measurements realised for water and the aroma molecules indicated neither chemical interactions with ι-carrageenan, nor obstruction effects from the polysaccharide chains. In ι-carrageenan gels, the diffusional phenomenon was highly dependent on the heterogeneous gel structure and controlled by hydrodynamic interactions due to frictional drag between each molecule of the system and water microviscosity changes.     

Drying and Rehydration of Calcium Alginate Gels

In this paper, we study the rehydration properties of air-dried calcium alginate gel beads. Rehydration is shown to depend on alginate source (i.e. mannuronic to guluronic acid ratio) and the salt concentration in the rehydration medium. Rehydration curves are described adequately by the empirical Weibull equation. Wide-angle X-ray diffraction measurements are performed to obtain information on the microstructure of dried alginate gels. The X-ray diffraction patterns provide evidence for formation of ordered domains in which alginate polymers are laterally associated. Formation of ordered structures during drying is found to have a large impact on rehydration properties. Lateral association of alginate chains is reduced (and rehydration improved) by removing excess calcium ions from the gel beads in a washing step prior to air drying. In addition, rehydration properties of mixed alginate–carboxymethyl cellulose (CMC) gel beads are investigated. The presence of CMC in the gel matrix is found to reduce lateral association of alginate chains during drying and to improve rehydration properties.     

A gel network constituted by rigid schizophyllan chains and nonpermanent cross-links

This work reports a gel network formed by rigid schizophyllan (SPG) chains with Borax as a cross-linking agent. The formed cross-links are non-permanent and somewhat dynamic in nature because the cross-linking reaction is governed by a complexation equilibrium. Gelation processes are traced by dynamic viscoelastic measurements to examine the effects of Borax content, SPG concentration, temperature, salt concentration, salt type, and strain. The first-order kinetic model containing three parameters, t(0) (induction time), 1/tau(c) (gelation rate), and (saturated storage modulus), is successfully applied to describe the gelation of the SPG-Borax system. Gelation occurs faster at higher Borax content, higher SPG concentration, higher salt concentration, or lower temperature. Moreover the gelation is cation-type-specific. Storage modulus is a linear function of both Borax content and SPG concentration. The linear relationship between storage modulus and Borax content can be explained by a modified ideal rubber elasticity theory with a front factor alpha to take into account the presence of ineffective cross-links and the effect of SPG chain rigidity. On the other hand, the linear dependence of storage modulus on SPG concentration could be explained on the basis of chain-chain contacting behavior of extended SPG chains. Apparent activation energy and cross-linking enthalpy are calculated to be -74.5 and -32.4 kJ/mol for the present system. Strain sweep measurements manifest that the elasticity behavior of this gel starts to deviate from Gaussian-chain network at a small strain of 10%.     

Characterization of a novel collagen chain in human placenta and its relation to AB collagen.

A novel collagen chain, termed alpha C, has been isolated from human placenta by limited pepsin digestion. The collagen containing the alpha C chain copurifies with placental AB collagen during selective salt precipitation but is virtually absent from fetal birth membranes, which contain relatively larger amounts of AB. Both native AB and alpha C-containing collagens are resistant to human skin collagenase under conditions that support cleavage of type I by greater than 90%. The alpha C chain was separated from alpha B by phosphocellulose chromatography and subsequently from alpha P by chromatography on CM-cellulose. Its amino acid composition is distinct from alpha A and alha B although all three chains posses compositional features in common; the carbohydrate content of the alpha C chain was intermediate between those of alpha A and alpha B. Analysis by NaDodSO4-polyacrylamide gel electrophoresis of peptides produced by CNBr cleavage and by limited digestion with the enzyme mast cell protease indicated different and unique products for the alpha A, alpha B, and alpha C chains. The data support the existence of another collagen chain which is related to the alpha A and alpha B chains but which is structurally unique. The proteins containing these chains may in turn comprise a subfamily of collagen isotypes which represents a divergence from and/or specialization of the type IV basement membrane collagens.     

Production of a polysaccharide, methylan, in Methylobacterium organophilum by controlling ammonium ion

Summary Cell growth increased proportionally to the initial concentration of ammonium ion, however, methylan production was significantly inhibited at the high concentration of ammonium ion. The control of ammonium ion within the desired level(usually 0.45 g/l) was needed to reduce the inhibition. Methylan production was increased to 12.5 g/l by maintaining ammonium ion below 0.15 g/l.     

Isolation and characterization of myosin from amoebae of Physarum polycephalum

Myosin was isolated from amoebae of Physarum polycephalum and compared with myosin from plasmodia, another motile stage in the Physarum life cycle. Amoebal myosin contained heavy chains (Mr approximately 220,000), phosphorylatable light chains (Mr 18,000), and Ca2+-binding light chains (Mr 14,000) and possessed a two-headed long-tailed shape in electron micrographs after rotary shadow casting. In the presence of high salt concentrations, myosin ATPase activity increased in the following order: Mg-ATPase activity less than K-EDTA-ATPase activity less than Ca-ATPase activity. In the presence of low salt concentrations, Mg-ATPase activity was activated approximately 9-fold by skeletal muscle actin. This actin-activated ATPase activity was inhibited by micromolar levels of Ca2+. Amoebal myosin was indistinguishable from plasmodial myosin in ATPase activities and molecular shape. However, the heavy chain and phosphorylatable light chains of amoebal myosin could be distinguished from those of plasmodial myosin in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peptide mapping, and immunological studies, suggesting that these are different gene products. Ca2+-binding light chains of amoebal and plasmodial myosins were found to be identical using similar criteria, supporting our hypothesis that the Ca2+-binding light chain plays a key role in the inhibition of actin-activated ATPase activity in Physarum myosins by micromolar levels of Ca2+.     

Toxic effect of dimethoate on the crosslinking of gingival and uteral collagen in female albino rats fed with normal and high protein diets

The effect of dimethoate on gingival and uteral collagen crosslinking was studied in normal and high protein diets fed female albino rats. The gingival and uteral samples were collected from all the groups of rats and percent reversibility of neutral salt soluble collagen gel and the solubility of insoluble collagen in KCNS or urea were determined. The analysis of gingival and uteral samples showed alpha 1 and alpha 2 sub units of neutral salt soluble collagen appreciably increased beta chains and aldehyde content significantly decreased in dimethoate treated animals compared to controls. The results indicate that due to the effect of dimethoate, the crosslinking and maturation of collagen are impaired.     

A Fourier transform infrared spectroscopic study of the interaction of alkaline earth cations with the negatively charged phospholipid 1, 2-dimyristoyl-sn-glycero-3-phosphoglycerol

The interaction of aqueous phospholipid dispersions of negatively charged 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol, sodium salt (DMPG) with the divalent cations Mg(2+), Ca(2+) and Sr(2+) at equimolar ratios in 100 mM NaCl at pH 7 was investigated by Fourier transform infrared spectroscopy. The binding of the three cations induces a crystalline-like gel phase with highly ordered and rigid all-trans acyl chains. These features are observed after storage below room temperature for 24 h. When the gel phase is heated after prolonged incubation at low temperature phase transitions into the liquid crystalline phase are observed at 58 degrees C for the DMPG:Sr(2+), 65 degrees C for the DMPG:Mg(2+), and 80 degrees C for the DMPG:Ca(2+) complex. By subsequent cooling from temperatures above T(m) these complexes retain the features of a liquid crystalline phase with disordered acyl chains until a metastable gel phase is formed at temperatures between 38 and 32 degrees C. This phase is characterized by predominantly all-trans acyl chains, arranged in a loosely packed hexagonal or distorted hexagonal subcell lattice. Reheating the DMPG:Sr(2+) samples after a storage time of 2 h at 4 degrees C results in the transition of the metastable gel to the liquid crystalline phase at 35 degrees C. This phase transition into the liquid crystalline state at 35 degrees C is also observed for the Mg(2+) complex. However, for DMPG:Mg(2+) at higher temperatures, a partial recrystallization of the acyl chains occurs and the high temperature phase transition at 65 degrees C is also detected. In contrast, DMPG:Ca(2+) exhibits only the phase transition at 80 degrees C from the crystalline gel into the fluid state upon reheating. Below 20 degrees C, the rate of conversion from the metastable gel to a thermodynamically stable, crystalline-like gel phase decreases in the order Ca(2+)&z. Gt;Mg(2+)>Sr(2+). This conversion into the crystalline gel phase is accompanied by a complete dehydration of the phosphate groups in DMPG:Mg(2+) and by a reorientation of the polar lipid head groups in DMPG:Ca(2+) and in DMPG:Sr(2+). The primary binding sites of the cations are the PO(2)(-) groups of the phosphodiester moiety. Our infrared spectroscopic results suggest a deep penetration of the divalent cations into the polar head group region of DMPG bilayers, whereby the ester carbonyl groups, located in the interfacial region of the bilayers, are indirectly affected by strong hydrogen bonding of immobilized water molecules. In the liquid crystalline phase, the interaction of all three cations with DMPG is weak, but still observable in the infrared spectra of the DMPG:Ca(2+) complex by a slight ordering effect induced in the acyl chains, when compared to pure DMPG liposomes.     

Physical and chemical properties of the major envelope glycoprotein gp85 from avian myeloblastosis virus

The major envelope glycoprotein gp85 of avian myeloblastosis virus, observed by electron microscopy as nearly spherical knobs projecting from the virus surface, was purified to homogeneity by gel filtration in 6 M guanidinium chloride followed by ion-exchange chromatography. The purified glycoprotein has a molecular weight of 80 000 from sedimentation equilibrium analysis. Glycoprotein gp85 contains approx. 45% carbohydrate including 25% N-acetylglucosamine, while the remaining weight consists of a polypeptide chain of approx. 45 000 daltons. Based on the oligosaccharide chain molecular weight data of Lai and Duesberg (Lai, M.M.C. and Duesberg, P.H. (1972) Virology 50, 359-372), the carbohydrate is calculated to be distributed between seven to nine oligosaccharide side chains. No self-association of gp85 was observed up to 2.0 mg/ml in dilute salt solution. The hydrodynamic properties of gp85 in dilute salt solution indicate a highly elongated molecule with an axial ratio of 7. One structural model which reconciles the hydrodynamic properties of gp85 with the nearly spherical architecture observed by electron microscopy requires the organization of the polypeptide chain and approx. 50% of the carbohydrate into a globular form. The remaining covalently linked oligosaccharides would by necessity extend outwardly from the globular structure as randomly oriented chains.     

Interaction of double-stranded T4 DNA with cationic gel of poly(diallyldimethylammonium chloride)

Interaction between duplex T4 DNA and a slightly cross-linked cationic gel of poly(diallyldimethylammonium chloride) in aqueous media was studied by fluorescent microscopy. While short DNA chains such as plasmid DNAs penetrate into the gel and form a phase of polyelectrolyte complex with the cationic network, the genomic giant DNA chains of T4 phages form complexes only on local areas of the gel surface. The DNA/gel complex exhibited different characteristic morphologies depending on the conditions for preparing the complex, such as the DNA concentration, flux of the solution, and surface geometry of the gel: (1) In the interaction with the flat surface of film-type gel, compact round objects, which reflected a condensed state of single DNA chains, were observed. (2) In the interaction with partly dried gel, a characteristic pattern similar to propagating waves was formed on the gel surface. (3) When flux is generated for a concentrated DNA solution, long oriented fiberlike structures were formed, which consisted of ensembles of chains. The interaction with small pieces of mechanically decomposed gel leads to complete covering of their surface by the DNA.     

Molecular dynamic simulations study of the effect of salt valency on structure and thermodynamic solvation behaviour of anionic polyacrylate PAA in aqueous solutions

The effect of salt concentration and valency on intermolecular structure and solvation thermodynamic properties of aqueous solution containing polyacrylicacid (PAA) chains and multi-valent salts calcium chloride (CaCl₂) and aluminium chloride (AlCl₃) as a function of charge density was investigated using atomistic molecular dynamic simulations with explicit solvent. Salt-free solution favours the self-association of uncharged (acidic form) PAA chains facilitated by inter-chain hydrogen bonds. The ionised (charged) PAA chains are not associated in salt-free aqueous solutions and undergo self-association in the salt solutions due to bridging effect induced by condensed salt ions in agreement with scattering investigations available in literature. The collapse behaviour of PAA in presence of CaCl₂ and re-expansion behaviour of PAA chains in case of AlCl₃ salt solutions are observed. The rigidity of PAA chains decrease with increase in salt concentration, in agreement with experimental results available in literature. The trivalent salt favours relatively the greater extent of shrinking of PAA chains as well as inter-chain interactions as compared to divalent salts as evident from radius-of-gyration, H-bond and pair-wise solvation enthalpy data. The conformation and hydration behaviour of the acid form of PAA chains are not significantly altered by added salt ions. The hydration behaviour of ionised PAA chains is significantly reduced by added salts due to screening effect of the condensed salt ions. The pair correlation functions of solutions species such as Ca²⁺, Al³⁺, Na⁺ and Cl^? with respect to PAA oxygen show the greater affinity of PAA units with the higher valency Al³⁺ ions over Ca²⁺ and Na⁺ in solution. With increase in concentration of AlCl₃ and CaCl₂ salts, a decrease in effective charge density of ionised PAA chains is observed from the existence of unfavourable PAA–water, PAA–Ca²⁺ and PAA–Al³⁺ interactions.     

Divalent cations affect chain mobility and aggregate structure of lipopolysaccharide from Salmonella minnesota reflected in a decrease of its biological activity

The physicochemical properties and biological activities of rough mutant lipopolysaccharides Re (LPS Re) as preformed divalent cation (Mg²⁺, Ca²⁺, Ba²⁺) salt form or as natural or triethylamine (Ten⁺)-salt form under the influence of externally added divalent cations were investigated using complementary methods: Differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopic (FT-IR) measurements for the β ↔ α gel to liquid crystalline phase behaviour of the acyl chains of LPS, synchrotron radiation X-ray diffraction studies for their aggregate structures, electron density calculations of the LPS bilayer systems, and LPS-induced cytokine (interleukin-6) production in human mononuclear cells. The divalent cation salt forms of LPS exhibit considerable changes in physicochemical parameters such as acyl chain mobility and aggregate structures as compared to the natural or monovalent cation salt forms. Concomitantly, the biological activity was much lower in particular for the Ca²⁺- and Ba²⁺-salt forms. This decrease in activity results mainly from the conversion of the unilamellar/cubic aggregate structure of LPS into a multilamellar one. The reduced activity also clearly correlates with the higher order - lower mobility - of the lipid A acyl chains. Both effects can be understood by an impediment of the interactions of LPS with binding proteins such as lipopolysaccharide-binding protein (LBP) and CD14 due to the action of the divalent cations.