Complex formation with heavy meromyosin of the isolated actin-like component of thrombosthenin,the contractile protein from blood platelets

Heavy meromyosin from rabbit skeletal muscle myosin was added on grids to the filamentous polymer of highly purified thrombosthenin A, tho actin-like protein in blood platelets. This resulted in an arrowhead complex formation between heavy meromyosin and the polymer, providing evidence that the polymer has a helical tertiary structure similar to muscle actin. The complex formation was inhibited by ATP.     

Characterization of a glucose polymer from PC12 cells and neuronal cells of rat embryo

A large-sized glucose polymer was isolated by pronase digestion from line PC12 pheochromocytoma cells metabolically labeled with [1-3H]galactose. The polymer was included on a column of concanavalin A-Sepharose and could be eluted with 10 mM methyl-alpha-mannoside. Its slight retention in a column of Bio-Gel A-5m suggested that its molecular weight was in the several millions. Glucose was the component monosaccharide and there were two minor lipophilic components present. The polymer was digested with alpha-amylase into a series of oligosaccharides and was cleaved by glucoamylase into glucose residues. The disaccharide obtained by digestion with alpha-amylase was identified as maltose in several HPLC systems and by NMR spectroscopy. NMR measurement revealed the trisaccharide to be maltotriose. Susceptibility of the polymer molecule to alpha-amylase, and the digestion products obtained, indicated a resemblance to glycogen. An analysis for saccharide compositions before and after reduction of the polymer suggested the presence of an aglycon part. Contrary to expectations based on the presence of this moiety, the polymer displayed good solubility in neutral organic solvents. Two-thirds of the glucose polymer was also soluble in 10% TCA. A similar glucose polymer was isolated from neuronal cells of rat embryos metabolically labeled with [1-3H]galactose. Mouse neuroblastoma cells did not synthesize the polymer.     

Oxidation of a glucose polymer during exercise: comparison with glucose and fructose

The purpose of this study was to compare the oxidation of 13C-labeled glucose, fructose, and glucose polymer ingested (1.33 g.kg-1 in 19 ml.kg-1 water) during cycle exercise (120 min, 53 +/- 2% maximal O2 uptake) in six healthy male subjects. Oxidation of exogenous glucose and glucose polymer (72 +/- 15 and 65 +/- 18%, respectively, of the 98.9 +/- 4.7 g ingested) was similar and significantly greater than exogenous fructose oxidation (54 +/- 13%). A transient rise in plasma glucose concentration was observed with glucose ingestion only. However, plasma insulin levels were similar with glucose and glucose polymer ingestions and significantly higher than with water or fructose ingestion. Plasma free fatty acid and glycerol responses to exercise were blunted with carbohydrate ingestion. However, fat utilization was not significantly different with water (82 +/- 14 g), glucose (60 +/- 3 g), fructose (59 +/- 11 g), or glucose polymer ingestion (60 +/- 8 g). Endogenous carbohydrate utilization was significantly lower with glucose (184 +/- 22 g), glucose polymer (187 +/- 31 g), and fructose (211 +/- 18 g) than with water (239 +/- 30 g) ingestion. Plasma volume slightly increased with water ingestion (7.4 +/- 4.5%), but the decrease was similar with glucose (-7.6 +/- 5.1%) and glucose polymer (-8.2 +/- 4.6%), suggesting that the rate of water delivery to plasma was similar with the two carbohydrates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polycondensation of thermal precursors of amino acids and characterization of constituent amino acids.

As a model reaction of polyamino acid formation from non-amino acid precursors, diammonium citraconate (I), ammonium citraconamate(II) and ammonium itaconamate(III) were converted to polyimide type polymers by thermal polycondensation by heating at 130–210°C. The imide type polymer was partially hydrolyzed to the corresponding peptide type polyamino acid. The polymer was composed of α-methylaspartic acid (IV), threo- and erythro-ß-methylaspartic acid (V) and α-(aminomethyl) succinic acid (VI). On the other hand, IV was thermally polycondensed to the corresponding polymer. It was found that the amino acid composition of the polymer was similar to that of the polymer prepared from I, II and III. The formation and isomerization of amino acids during the thermal polycondensation are described.     

Immobilized beta-cyclodextrin polymer coupled to agarose gel properly refolding recombinant Staphylococcus aureus elongation factor-G in combination with detergent micelle

A novel artificial chaperone system using a combination of interactions between the unfolded protein, a detergent and a chromatographic column packed with immobilized beta-cyclodextrin (beta-CD) polymer coupled to an agarose gel, was introduced to refold recombinant Staphylococcus aureus elongation factor-G (EF-G). Pre-mixing of 10% Triton X-100 and unfolded EF-G at 24 mg/ml followed by a 20-fold dilution into refolding buffer led to successful capturing of EF-G by Triton X-100 resulting in formation of a detergent-protein complex at 1.2mg/ml of final protein concentration. The complex was subsequently applied to the immobilized beta-CD polymer column resulting in correct refolding of EF-G at a concentration of 530 microg/ml with 99% mass recovery. Detergent concentrations above critical micelle concentration were required for efficient capturing of EF-G at high protein concentration. Other detergents with hydrophile-lipophile-Balance values similar to that of Triton X-100 (Triton N-101, Noindet P40 (NP40), and Berol 185) also produced similar result. Soluble polymerized beta-CD was more efficient than the monomer to remove the detergent from the protein complex in a batch system. Immobilized beta-CD polymer column further improved the capability of detergent removal and was able to prevent aggregation that occurred with the addition of soluble beta-CD polymer at high protein concentration in the batch system. The mechanism for this system-assisted refolding was tentatively interpreted: the released protein could correctly refold in an enclosed hydrophilic environment provided by the integration of matrix and beta-CD polymer, and thus avoided aggregation during detergent removal.     

Isolation of Halotolerant, Thermotolerant, Facultative Polymer-Producing Bacteria and Characterization of the Exopolymer

Over 200 bacterial strains were selected for anaerobic growth at 50°C and extracellular polysaccharide production in a sucrose-mineral salts medium with NaNO₃ and up to 10% NaCl. The predominant cell type was an encapsulated gram-positive, motile, facultative sporeforming rod similar to Bacillus species. Strain SP018 grew and produced the polysaccharide on a variety of substrates at salinities up to 12% NaCl. Good polymer production only occurred anaerobically and was optimal between 4 and 10% NaCl. The ethanol-precipitated SP018 polymer was a charged heteropolysaccharide that contained glucose, mannose, arabinose, ribose, and low levels of allose and glucosamine. The SP018 polymer showed pseudoplastic behavior, was resistant to shearing, and had a higher viscosity at dilute concentrations and at elevated temperatures than xanthan gum. High-ionic-strength solutions reversibly decreased the viscosity of SP018 polymer solutions. The bacterium and the associated polymer have many properties that make them potentially useful for in situ microbially enhanced oil recovery processes.     

Molecularly imprinted polymers from nicotinamide and its positional isomers

Imprinted polymers were prepared for nicotinamide and its positional isomers. The influence of porogenic solvent and functional monomer on recognition properties of the polymer was compared. The results indicated that two functional groups, the heterocyclic nitrogen and the amide group, in the nicotinamide or isonicotinamide molecule have a synergistic effect in binding to the polymer. The polymers prepared with nicotinamide and isonicotinamide can be used as HPLC stationary phase for the separation of positional isomers of nicotinamide or isonicotinamide, while the polymer prepared with picolinamide showed no specificity toward the template. The mechanisms for the differences in recognition are discussed. In addition to the retention of polymers to their templates the polymers also displayed excellent retention to nicotinic acid and isonicotinic acid, compounds structurally similar to the template. This dual recognition property of the polymer may be useful in circumstances where the preparation of a polymer for a specific template may be problematic because of poor stability or solubility.     

Microtubule dynamics at the G2/M transition: abrupt breakdown of cytoplasmic microtubules at nuclear envelope breakdown and implications for spindle morphogenesis

We recently developed a direct fluorescence ratio assay (Zhai, Y., and G.G. Borisy. 1994. J. Cell Sci. 107:881-890) to quantify microtubule (MT) polymer in order to determine if net MT depolymerization occurred upon anaphase onset as the spindle was disassembled. Our results showed no net decrease in polymer, indicating that the disassembly of kinetochore MTs was balanced by assembly of midbody and astral MTs. Thus, the mitosis-interphase transition occurs by a redistribution of tubulin among different classes of MTs at essentially constant polymer level. We now examine the reverse process, the interphase-mitosis transition. Specifically, we quantitated both the level of MT polymer and the dynamics of MTs during the G2/M transition using the fluorescence ratio assay and a fluorescence photoactivation approach, respectively. Prophase cells before nuclear envelope breakdown (NEB) had high levels of MT polymer (62%) similar to that previously reported for random interphase populations (68%). However, prophase cells just after NEB had significantly reduced levels (23%) which recovered as MT attachments to chromosomes were made (prometaphase, 47%; metaphase, 56%). The abrupt reorganization of MTs at NEB was corroborated by anti- tubulin immunofluorescence staining using a variety of fixation protocols. Sensitivity to nocodazole also increased at NEB. Photoactivation analyses of MT dynamics showed a similar abrupt change at NEB, basal rates of MT turnover (pre-NEB) increased post-NEB and then became slower later in mitosis. Our results indicate that the interphase-mitosis (G2/M) transition of the MT array does not occur by a simple redistribution of tubulin at constant polymer level as the mitosis-interphase (M/G1) transition. Rather, an abrupt decrease in MT polymer level and increase in MT dynamics occurs tightly correlated with NEB. A subsequent increase in MT polymer level and decrease in MT dynamics occurs correlated with chromosome attachment. These results carry implications for understanding spindle morphogenesis. They indicate that changes in MT dynamics may cause the steady-state MT polymer level in mitotic cells to be lower than in interphase. We propose that tension exerted on the kMTs may lead to their lengthening and thereby lead to an increase in the MT polymer level as chromosomes attach to the spindle.     

Modulation of tubulin-nucleotide interactions by metal ions: comparison of beryllium with magnesium and initial studies with other cations.

With microtubule-associated proteins (MAPs) BeSO4 and MgSO4 stimulated tubulin polymerization as compared to a reaction mixture without exogenously added metal ion, while beryllium fluoride had no effect (E. Hamel et al., 1991, Arch. Biochem. Biophys. 286, 57-69). Effects of both cations were most dramatic at GTP concentrations in the same molar range as the tubulin concentration. We have now compared effects of beryllium and magnesium on tubulin-nucleotide interactions in both unpolymerized tubulin and in polymer. Polymer formed with magnesium had properties similar to those of polymer formed without exogenous cation, except for a 20% lower stoichiometry of exogenous GTP incorporated into the latter. In both polymers the incorporated GTP was hydrolyzed to GDP. Stoichiometry of GTP incorporation into polymers formed with beryllium or magnesium was identical, but much of the GTP in the beryllium polymer was not hydrolyzed. The beryllium polymer was more stable than the magnesium polymer. Beryllium also differed from magnesium in only weakly enhancing the binding of GTP in the exchangeable site of unpolymerized tubulin, while neither cation affected GDP exchange at the site. If both cations were present in a reaction mixture, polymer stability was little changed from that of the beryllium polymer, but most of the GTP incorporated into polymer was hydrolyzed. Six additional metal salts (AlCl3, CdCl2, CoCl2, MnCl2, SnCl2, and ZnCl2) also stimulated MAP-dependent tubulin polymerization, but enhanced polymer stability did not correlate with polymer GTP content. We postulate that enhanced polymer stability is a consequence of cation binding directly to tubulin and/or polymer while deficient GTP hydrolysis in the presence of beryllium, as well as aluminum and tin, is a consequence of tight binding of cation to GTP in the exchangeable site.     

Response of microbial adhesives and biofilm matrix polymers to chemical treatments as determined by interference reflection microscopy and light section microscopy.

The polymers involved in the adhesion of Pseudomonas fluorescens H2S to solid surfaces were investigated to determine whether differences between cell surface adhesives and biofilm matrix polymers could be detected. Two optical techniques, i.e., interference reflection microscopy (IRM) and light section microscopy (LSM), were used to compare the responses of the two types of polymer to treatment with electrolytes, dimethyl sulfoxide (DMSO), and Tween 20. To evaluate initial adhesive polymers, P. fluorescens H2S cells were allowed to attach to glass cover slip surfaces and were immediately examined with IRM, and their response to chemical solutions was tested. With IRM, changes in cell-substratum separation distance between 0 and ca. 100 nm are detectable as changes in relative light intensity of the image; a contraction of the polymer would be detected as a darkening of the image, whereas expansion would appear as image brightening. To evaluate the intercellular polymer matrix in biofilms, 3-day-old biofilms were exposed to similar solutions, and the resultant change in biofilm thickness was measured with LSM, which measures film thicknesses between 10 and 1,000 microns. The initial adhesive and biofilm polymers were similar in that both appeared to contract when treated with electrolytes and to expand when treated with Tween 20. However, with DMSO treatment, the initial adhesive polymer appeared to contract, whereas there was no change in thickness of the biofilm polymer. These results indicate that both polymers bear acidic groups and thus act electrostatically with cations and are able to enter into hydrophobic interactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polyethyleneglycol molecular mass and polydispersivity effect on protein partitioning in aqueous two-phase systems

The partitioning of model proteins (bovine serum albumin, ovalbumin, trypsin and lysozyme) was assayed in aqueous two-phase systems formed by a salt (potassium phosphate, sodium sulfate and ammonium sulfate) and a mixture of two polyethyleneglycols of different molecular mass. The ratio between the PEG masses in the mixtures was changed in order to obtain different polymer average molecular mass. The effect of polymer molecular mass and polydispersivity on the protein partition coefficient was studied. The relationship between the logarithm of the protein partition coefficient and the average molecular mass of the phase-forming polymer was found to depend on the polyethyleneglycol molecular mass, the salt type in the bottom phase and the molecular weight of the partitioned protein. The polymer polydispersivity proved to be a very useful tool to increase the separation between two proteins having similar isoelectrical point.     

Exocellular glycopeptide from a Penicillium charlesii mutant incapable of growth on D-galactose.

The compositions of exocellular saccharide-containing polymers from six mutants of Penicillium charlesii incapable of growing on galactose were investigated. The polymers from the mutants contain a much smaller percentage of galactose than that reported for the peptidophosphogalactomannan (PPGM) from the wild-type organism (Gander et al. 1974). A polymer containing only one galactosyl residue per 49 mannosyl residues was investigated in detail. This polymer is a glycopeptide (peptidomannan) with an amino acid composition similar to that of peptidophospogalactomannan and a mass of about 23,000 daltons. Treatment of peptidomannan with 0.4 N NaOH releases mannan, mannopentaose, mannotetraose, mannotriose, mannobiose, and mannose residues, which are attached to the peptide by O-glycosidic linkage to seryl and threonyl groups. The quantity of glycerol and threitol, derived from mannosyl and internal galactofuranosyl residues, respectively, following Smith degradation, showed that peptidomannan contains 2 mol of internal galactofuranosyl residues per mol of polymer. The polymer contains only 3 mol of (1 yields 5)-linked galactofuranosyl residues per mol of polymer, as described by analysis of the methylation products. Methylation analysis also indicates that the polysaccharide contains primarily (1 yields 2)-linked (67.5%) and (1 yields 6)-linked (20.2%) mannopyranosyl residues. However, acetolysis of the polymer suggests that 37% of the residues are (1 yields 6)-linked. Mannopentaose, mannotetraose, mannotriose, mannobiose, and mannose in a molar ratio of 0.30:0.11:0.15:0.39:0.06, respectively, are released by acetolysis. This result is similar to that obtained with peptidophosphogalactomannan. We conclude that the occurrence of large numbers of galactofuranosyl residues in the major extracellular glycopeptide is not an obligatory requirement for glycopeptide formation.     

Movement and regeneration of epicuticular waxes through plant cuticles

Regeneration of plant epicuticular waxes was studied in 24 plant species by high-resolution scanning electron microscopy. According to their regeneration behaviour, four groups could be distinguished: (i) regeneration occurs at all stages of development; (ii) regeneration occurs only during leaf expansion; (iii) regeneration occurs only in fully developed leaves; (iv) plants were not able to regenerate wax. Wax was removed from the leaves with water-based glue and a liquid polymer, i.e. water-based polyurethane dispersion. In young leaves these coverings could not be removed without damaging the leaves. After a few days, waxes appeared on the surface of these polymer films, which still adhered to the leaves. It is concluded that waxes move through the cuticle in a process similar to steam distillation. This hypothesis could be further substantiated in refined in vitro experiments. Wax isolated from Eucalyptus globulus was applied to a filter paper, subsequently covered with a liquid polymer and fixed onto a diffusion chamber filled with water. The diffusion chamber was put into a desiccator. After 8-10 days at room temperature, crystals similar in dimensions and shape to in situ crystals appeared on the surface of the polyurethane film. This indicates that waxes in molecular dimensions move together with the water vapor that permeates through the polymer membrane. Based on these results, we propose a new and simple hypothesis for the mechanism of wax movement: the molecules that finally form the epicuticular wax crystals are moved in the cuticular water current.     

Studies on J-chain biosynthesis in tumours producing immunoglobulins in NZB mice.

Investigation of biosynthesis of J chain in plasmacytomas induced in NZB mice revealed that this protein was not only synthesized in the cells that produce polymer immunoglobulin A but also in those that produce immunoglobulin G monomer. It was also found that protein similar to J chain of BALB/c-mice was associated with polymer immunoglobulin A but not with immunoglobulin G of NZB mouse myeloma proteins.     

Aggregation of phospholipid vesicles by water-soluble polymers.

Water-soluble polymers such as dextran and polyethylene glycol are known to induce aggregation and size growth of phospholipid vesicles. The present study addresses the dependence of these processes on vesicle size and concentration, polymer molecular weight, temperature, and compartmentalization of the vesicles and polymers, using static and dynamic light scattering. Increasing the molecular weight of the polymers resulted in a reduction of the concentration of polymer needed for induction of aggregation of small unilamellar vesicles. The aggregation was fully reversible (by dilution), within a few seconds, up to a polymer concentration of at least 20 wt %. At relatively low phosphatidylcholine (PC) concentrations (up to approximately 1 mM), increasing the PC concentration resulted in faster kinetics of aggregation and reduced the threshold concentration of polymer required for rapid aggregation (CA). At higher PC concentrations, CA was only slightly dependent on the concentration of PC and was approximately equal to the overlapping concentration of the polymer (C*). The extent of aggregation was similar at 37 and 4 degrees C. Aggregation of large unilamellar vesicles required a lower polymer concentration, probably because aggregation occurs in a secondary minimum (without surface contact). In contrast to experiments in which the polymers were added directly to the vesicles, dialysis of the vesicles against polymer-containing solutions did not induce aggregation. Based on this result, it appears that exclusion of polymer from the hydration sphere of vesicles and the consequent depletion of polymer molecules from clusters of aggregated vesicles play the central role in the induction of reversible vesicle aggregation. The results of all the other experiments are consistent with this conclusion.     

Ex situ bioremediation of phenol contaminated soil using polymer beads

Polymer beads have been used to absorb high concentrations of phenol from soil decreasing the initial concentration of 2.3 g kg⁻¹ soil to 100 mg kg⁻¹ soil and achieving a phenol loading within the polymer beads of 27.5 mg phenol g⁻¹ beads. The phenol-loaded polymer beads were removed from the soil and placed in a bioreactor, which was then inoculated with a phenol-degrading microbial consortium. All of the phenol contained within the polymer beads was shown to desorb from the polymer matrix and was degraded by the microbial consortium. The beads were used again (twice) in a similar manner with no loss in performance.     

Elastic constants of polymer-grafted lipid membranes

The surface expansion that is induced by the lateral pressure in the brush region of lipid membranes containing grafted polymers is deduced from the scaling and mean-field theories for the polymer brush, together with the equation of state for a lipid monolayer at the equivalence pressure with fluid lipid bilayers. Depending on the length and mole fraction of the polymer lipid, the membrane expansion can be appreciable. Direct experimental evidence for this lateral expansion comes from recent spin-label measurements with lipid membranes containing poly(ethylene glycol)-grafted lipids. The expansion in lipid area modifies the elastic constants of the polymer-grafted membranes in a way that opposes the direct elastic response of the polymer itself. Calculations as a function of polymer lipid content indicate that the net change in isothermal area expansion modulus of the membrane is negative but small, in contrast to previous predictions. A similar situation applies to the curvature elastic moduli of membranes containing short polymer lipids. For longer polymer lipids, however, the direct contribution of the polymer brush to the bending elastic constants dominates, and the increase in bending moduli with increasing polymer lipid content rapidly exceeds the basal values of the bare lipid membrane. The spontaneous (or intrinsic) curvature of the component monolayer of polymer lipid-containing membranes is calculated for the first time. The polymer brush contribution to spontaneous curvature scales quadratically with the polymer length, and at least quadratically with the mole fraction of polymer lipid.     

Coupling of nuclear morphometry to cell geometry and growth in human fibroblasts

The nuclear morphology of WI-38 cells plated on increasing thicknesses of the polymer, poly-2-hydroxyethylmethacrylate, was studied. Changes in nuclear morphometry were found to parallel changes in cellular morphometry: namely a transition to a more rounded and compact conformation, with increasing thicknesses of the polymer. Such changes in nuclear morphometry were opposite to those already observed after stimulation of proliferation by serum addition and similar to those observed with increasing confluency. These results suggest that coupling between cell and nuclear geometry may represent a mechanism for the control of proliferation by cell shape in nontransformed cells.     

Isolation and Characterization of a Galactosamine Wall from Spores and Spherules of Physarum polycephalum

The myxomycete, Physarum polycephalum, can be induced under laboratory conditions to form two different hard-walled forms, spores and spherules. Characterization of both types of walls revealed only a single sugar, galactosamine. It was identified after acid hydrolysis of the isolated walls by chromatography in three solvent systems, by its positive reaction with ammoniacal silver nitrate, ninhydrin, Galactostat, and the Elson-Morgan test, and by ninhydrin degradation to lyxose. Galactosamine was present as a polymer with solubility characteristics the same as the β1-4–linked glucosamine polymer (chitosan). The walls were also found to contain about 2% protein. Spherule walls revealed a single glycoprotein on gel electrophoresis. Spore walls contained a similar protein component. The phosphate content of isolated spherule walls was 9.8%, and that of spore walls was 1.4%. Spore walls also contained about 15% melanin which was shown to be similar to fungal melanin. A novel method was used to measure the rate of mature spherule formation based on the loss of extractability of P. polycephalum natural pigment. The presence of a rare galactosamine polymer in P. polycephalum spore and spherule walls as the only carbohydrate suggests that the myxomycetes are not closely related to the fungi or the protozoa.     

Effect of nitrogen limitation on enrichment of activated sludge for PHA production

Polyhydroxyalkanoates (PHA) are good candidates to plastics because of their material properties similar to conventional plastics and complete biodegradability. The use of activated sludge can be a cheaper alternative to pure cultures for PHA production. In this study, effect of nitrogen limitation during acclimatization period of biomass on production of polyhydroxyalkanoate was investigated. Activated sludge was selected in two sequencing batch reactors operated with and without nitrogen limitation. Batch tests were performed to examine polymer productions of activated sludges acclimatized to different nitrogen regimes. Responses of biomass to different organic loading rates, organic acids, and carbon to nitrogen (C/N) ratios were studied by determining specific polymer storage rate, polymer storage yield, and sludge polymer content of biomasses. Results obtained from batch experiments showed that concentrations of polymer accumulated by two different sludges increased directly with initial substrate concentration. Observed highest polymer yields for the biomasses enriched with and without nitrogen deficiency were 0.69 g COD PHA g(-1) COD S and 0.51 g COD PHA g(-1) COD S, and corresponding polymer contents of biomasses were 43.3% (g COD PHA g(-1) COD X) and 38.3% (g COD PHA g(-1) COD X), respectively. Polymer yields for both biomasses decreased with substrate shift however, biomass enriched with nitrogen deficiency adapted well to acetate-propionate mixture. The results presented in this study showed that polymer storage ability of biomass was improved more under dynamic conditions with nitrogen deficiency when compared to that without nitrogen deficiency. Limiting ammonia availability during batch experiments also caused higher polymer production by suppressing growth, as well as during enrichment of biomass.