Isolation and in vitro translation of polysomes from mature rye leaves

Cytoplasmic polysomes have been prepared from mature leaves of winter rye (Secale cereale L. cv Puma). This is the first time a method has been developed for isolation of highly polymerized polysomes from mature leaves. The degree of intactness of isolated plant polysomes has been determined by two independent but complementary methods: size class distribution by sucrose gradient centrifugation and in vitro translation. The polymerization of isolated polysomes was estimated by the ratio of the proportion of large polysomes to the proportion of small polysomes obtained from the profiles. Our results show that the composition of the optimal polysome isolation buffer for mature rye leaves is different from that reported for young tobacco and pea leaves. Polysomes were translated in vitro with the S-105 wheat germ fraction. The degree of polysome polymerization has a significant effect on their in vitro translation since both the incorporation of amino acid and the presence of high molecular weight polypeptides are proportional to the large polysomes/small polysomes ratio. This study emphasizes the need to evaluate isolation conditions carefully before proceeding with polysome studies in any particular tissue or in tissues under different physiological status.     

Effect of chlorhexidine on molecular weight distribution of fructans produced by fructosyltransferase in solution and immobilized on surface

The effect of chlorhexidine (CHX), a potent antibacterial agent, was tested on the molecular weight distribution (MWD) of fructans synthesized by cell-free fructosyltransferase (FTF) in solution in comparison to FTF immobilized onto hydroxyapatite (HA). Size-exclusion chromatography (SEC) analysis has shown that cell-free FTF, both in solution and immobilized on HA, produces both low MW (1.9-2.2 kDa) and high MW (913-1047 kDa) fructans. CHX at a concentration of 0.02% altered the MWD of the fructans by reducing the polydispersity ratio and changing the MWD of the fructans synthesized both by immobilized FTF and by FTF in solution. These changes of the fructans in the presence of CHX adds a new prospective to the anticaries effect of CHX in addition to its antibacterial properties.     

Analysis of cell-wall polymers during cotton fiber development

Although the fibers of cotton (Gossypium hirsutum L.) are single cells with a secondary wall composed primarily of cellulose, the cell-wall polymers of the fibers are technically difficult to characterize with respect to molecular weights. This limitation hinders understanding how the fiber wall composition changes during development, particularly with respect to genotypic variations, and how the molecular composition is related to physical properties. We analyzed cell-wall polymers from cotton fibers (cultivar, Texas Marker-1) at several developmental stages (8–60 days post-anthesis; DPA) by gel-permeation chromatography of components soluble in dimethyl acetamide and lithium chloride. This procedure solubilizes fiber cell-wall components directly without prior extraction or derivatization, processes that could lead to degradation of high-molecular-weight components. Cellwall polymers from fibers at primary cell-wall stages had lower molecular weights than the cellulose from fibers at the secondary wall stages; however, the high-molecularweight cellulose characteristic of mature cotton was detected as early as 8 DPA. High-molecular-weight material decreased during the period of 10–18 DPA with concomitant increase in lower-molecular-weight wall components, possibly indicating hydrolysis during the later stages of elongation.Abbreviations DMAC dimethyl acetamide - DP degree of polymerization - DPA days post anthesis - GPC gel-permeation chromatography - MW molecular weight - MWD molecular-weight distribution - TM-1 Texas Marker 1     

Combination of SEC/MALS experimental procedures and theoretical analysis for studying the solution properties of macromolecules

Size-exclusion chromatography (SEC) with dual detection, i.e., employing refractive index (RI) and multiangle light-scattering (MALS) detectors, has been applied to study the solution properties of two very different polymer-solvent systems at 25 degrees C: poly(N-vinylcarbazole) (PVCz) in an organic solvent THF that is a very good solvent and a system under theta conditions that is formed by polyvinylpyrrolidone (PVP) in water containing a 0.1 M concentration of NaNO(3). In both cases, the analysis of a single highly polydisperse sample obtained by free radical polymerization is enough for obtaining molecular weight and radius of gyration calibration curves, molecular weight distributions (MWD) (and thus, molecular weight averages), molecular dimensions, scaling laws coefficients and unperturbed dimensions. Extrapolation to theta conditions produces values of the characteristic ratio of the unperturbed dimensions C(n)=(o)/nl(2)=15.9 and 14, respectively, for PVCz and PVP. Unperturbed dimensions are also theoretically calculated with different models such as Kuhn equivalent chain, worm-like chain and rotational isomeric states model. Conformational parameters required for this last model were taken from the literature in the case of PVCz; however, they are calculated by molecular dynamics simulations in the case of PVP. Theoretical values obtained with the RIS model are in good agreement with the experimental results.     

Degradation of the parasporal crystal produced by Bacillus thuringiensis var. kurstaki

Degradation products of the parasporal crystals of Bacillus thuringiensis var. kurstaki obtained by treatment with alkali, gut juice from larvae of Bombyx mori, and various plant and mammalian enzymes were compared for elution pattern, approximate molecular weight (MW), and toxicity. The results indicated that with alkaline treatment the most toxic extract was obtained with 0.05–0.1 M NaOH. Toxicity was found associated mainly with a protein peak of 230,000 MW although other toxic peaks were found in the tailing. Heat-treated midgut juice from larval B. mori gave similar results. After digestion of parasporal crystals with clarified midgut juice, five peaks causing toxicity and having MW of approximately 235,000, 67,000, 30,200, 5000, and 1000, respectively, were identified. Treatment of B. thuringiensis δ-endotoxin with α-chymotrypsin gave peaks causing mortality of approximate MW 235,000, 34,000, 5000, and 1000. Trypsin, pronase, carboxypeptidase, and enterokinase digests of the B. thuringiensis δ-endotoxin gave toxic components ranging from 235,000 to 30,000 MW. The protein protoxin molecules are digested to give small toxic subunits that may be of practical value for structural determinations and for molecular mode of action studies.     

Diene polymerizations with the lanthanide coordination catalysts. I. Piperylene polymerizations effected by the type of lanthanide

The present paper concerns studies into piperylene polymerizations with the catalytic systems LnHal₃· 3TBP-Al(i-C₄H₉)₃ (Ln=Ce, Pr, Nd, Gd, Tb, Dy; Hal=Cl, Br, I; TBP=tributylphosphate) occurring in various solvents. The activity and stereospecificity of these catalysts have been found to depend on the composition of the initial lanthanide component LnHal₃·3TBP and mostly on the lanthanide itself. With other conditions being equal, the highest polymerization rates are provided by the Nd catalyst, and the Gd catalyst favours the formation of polypiperylene with the maximum content of cis-1,4-units. The data obtained have proved the dependence of anti-sin isomerization of π-allyl active centres on the types of the lanthanide, halogen, hydrocarbon solvent and diene monomer used.     

Protein composition of the cell envelope of the bacterium <Emphasis Type="Italic">Shewanella frigidimarina</Emphasis> Pi 2-35 (Gammaproteobacteria: Shewanellaceae)

The protein composition of the outer (OM) and cytoplasmic (CM) membranes of the cell envelope of the psychrotolerant marine bacterium Shewanella frigidimarina Pi 2-35, which was isolated from a sample of sea ice from the Amur Bay (Sea of Japan), was analyzed using SDS-PAGE. The cell envelope of this microorganism has been found to contain high molecular weight proteins, cytochromes, which were extracted with detergents of different types. It is shown that the composition of the CM of S. frigidimarina Pi 2-35 does not depend on the cultivation temperature. Proteins with a molecular weight (MW) within the range of 30–50 kDa, which is characteristic for nonspecific porins of terrestrial Gram-negative bacteria, were detected only in the OM of bacteria that were cultivated at a low temperature (6–8°C). Two porin-like proteins, P_ZW (MW 40 kDa) and P_LYS (MW 34.5 kDa), were isolated from the OM by extraction with the 0.5% non-ionic detergent Zwittergent 3-14 and by digestion of the peptidoglycan layer of the cell envelope with lysozyme, respectively. The use of the bilayer lipid membrane (BLM) technique showed that both proteins exhibit a pore-forming activity. The efficiency of channel formation and the pattern of fluctuations of current in the BLM indicate that the isolated porins are not identical in their functional properties.     

Clinical dextran purified by fractional ultrafiltration coupled with water washing

Dextran with extremely narrow molecular weight distribution (MWD) is demanded for clinical use. To elucidate the effect of fractional ultrafiltration on the purification of clinical dextran, a range of ultrafiltration membranes with 100, 30, 5 and 1 kDa molecular weight cut-off (MWCO) were applied. High Performance Gel Permeation Chromatography (HPGPC) analysis indicated that the MWD of ultrafiltrated dextran fractions are wide, with polydispersity index (D) between 2.2 and 5.4, suggesting that the MWD of dextran is hard to be strictly controlled by fractional ultrafitration. However, when coupled with water washing during ultrafiltration process, the homogeneity of dextran was greatly improved. An ultrafiltration fraction of 5-30 kDa (Mw ≈ 35 kDa) with narrow MWD (D = 1.2) was obtained after 5 times of water washing. The results show that fractional ultrafiltration coupled with water washing can be used as a simple and effective method to improve the quality of clinical dextran.     

Cetyltrimethylammonium bromide polyacrylamide gel electrophoresis: estimation of protein subunit molecular weights using cationic detergents.

Polyacrylamide gel electrophoresis in the presence of the cationic detergent, cetyltrimethylammonium bromide (CTAB), has been previously used to obtain more accurate estimates of the molecular weight of certain highly charged and membrane protein subunits that exhibit anomalous electrophoretic behavior in the presence of sodium dodecyl sulfate (SDS). The improved method reported herein is comparable to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) method in simplicity, time, and quality of gels, but the CTAB-PAGE method appears to have a wider range of application for diverse types of proteins. The technique may also be used for verification of molecular weight data and thus detection of possible anomalous results obtained using the anionic SDS-PAGE method. The described method eliminates the precipitates formed between ammonium persulfate and cationic detergents during gel polymerization and between cationic detergents and the protein dyes during staining that have complicated previous methods. The reliability of the technique is indicated by the high correlation coefficient (?0.97) between R_f and molecular weight. Data are presented to indicate that the method can be used to estimate the subunit molecular weight of unknown proteins with a 95% level of confidence.     

Crystallization of deoxyhemoglobin S by fiber alignment and fusion.

Fibers of deoxyhemoglobin S obtained directly from lysed sickled red blood cells have been compared with fibers from chromatographically pure deoxyhemoglobin S solutions of known chemical composition. Electron micrographs of negatively stained specimens reveal that the molecular packing within the fibers remains largely invariant with changes in pH, ionic strength, Mg²⁺ concentration, 2,3-diphosphoglycerate concentration, temperature or the method of deoxygenation.When solutions of chromatographically pure deoxyhemoglobin S are stirred, the fibers align into well defined fascicles. After several hours of stirring, long needles and twisted ribbons develop and in a relatively short time replace the fascicles in solution. With continued stirring all forms are replaced by small crystals. By use of electron microscopy and low-angle X-ray diffraction we have found these crystals to have cell parameters indistinguishable from those of crystals grown in polyethylene glycol and citrate/phosphate buffer at pH 5 to 6 (Wishner et al., 1975a).Our evidence indicates that crystal formation in stirred solutions of deoxyhemoglobin S is the result of a progressive alignment and fusion of the fibers, and that the molecular arrangement within the fibers is closely related to that within the crystal. The remarkable pH invariance of the molecular packing within the fiber and crystal structures is consistent with the dominance of hydrophobic bonding between molecules. The β6-valine contact observed by Wishner et al. (1975b) is apparently the pathological contact responsible for the polymerization of deoxyhemoglobin S in vivo. On the basis of our observations and knowledge of the crystal structure we propose that the deoxyhemoglobin S fiber consists of eight molecular double strands, four of which run in each direction along the length of the fiber.     

The human LT system. V. A comparison of the relative lytic effectiveness of various MW human LT classes on 51Cr-labeled allogeneic target cells in vitro: enhanced lysis by LT complexes associated with Ig-like receptor(s).

The present studies examine the in vitro cell-lytic capacity of various molecular weight (MW) human lymphotoxin (LT) classes obtained from lectin-activated normal or immune lymphocytes on allogeneic target cells. The findings reveal that the high-MW complex class of LT is up to 100 times more effective than the smaller MW LT forms (α, β, and γ) in causing lysis of various allogeneic cell types including lymphoid cells in vitro. Moreover, the data suggest that lectin-stimulated alloimmune cells (MLC sensitized) release complex LT forms in association with a specific antigen-binding receptor(s), and that these complexes are from 3 to 10 times more effective on the sensitizing target cell than complexes obtained from lectin-stimulated nonimmune cells. Positive evidence that complex-induced lysis involved LT was indicated by the finding that lysis was completely neutralized by incubation with heterologous antisera directed against a refined human α₂-LT subclass (anti-α₂) and partially neutralized with anti-human Fab₂′ serum. These findings support the concept that LT molecules may represent a system of related cell-lytic molecules. While the smaller MW forms are only weakly lytic by themselves, they can be assembled into highly lytic complexes which may be focused or directed by an antigen-binding receptor(s).     

The regulation of growth in the mesonephric kidney of adult Xenopus laevis by an endogenous inhibitor of proliferation.

The effect of dorsal lymph sac implanted tissue fragments, of a 100,000g kidney supernatant, and of various kidney-derived ultrafiltrate fractions on the percentage of DNA synthesizing cells in the mesonephric kidney of Xenopus laevis following partial unilateral nephrectomy was investigated autoradiographically. Using Amicon filters with cut-off values of MW 50,000 and 10,000, three ultrafiltrate fractions were obtained: a fraction containing molecules of MW 50,000 and less, a fraction containing molecules of MW 10,000 and less, and one containing molecules in the range of MW 10,000 to 50,000. The ultrafiltrates containing molecules of less than 10,000 MW were found to depress DNA synthetic activity on the sixth postoperative day by 30 to 40%, while the fraction containing molecules between MW 10,000 and 50,000 showed no significant effect. It has been concluded that an endogenous inhibitor of proliferation, with the attributes of a chalone, is present in the fraction of less than 10,000 MW. The loss of inhibitor action following Pronase treatment of the ultrafiltrate suggests that the inhibitor substance may be a protein or polypeptide, or that such constituent may be the carrier for the active agent. Since a depression in DNA synthetic activity of 60% was obtained in normal adult mesonephric kidneys following the injection of the ultrafiltrate, it is concluded that both compensatory growth and reparative growth in the kidney of Xenopus laevis are regulated by a G₁ kidney chalone of less than 10,000 MW.     

Measurement of the molecular weight distribution of debranched starch

Combining molecular weight distribution (MWD) data for linear chains of debranched starch from capillary electrophoresis and from size exclusion chromatography (SEC) with detection by differential refractive index and by multi-angle laser light scattering has enabled Mark-Houwink parameters to be determined for linear starch chains. For accurate results, it was found to be important to take SEC band broadening into account, and a methodology for implementing this is presented. This deconvolution technique can be used to reveal features such as maxima or shoulders in the MWD which have qualitative as well as quantitative significance. Remarkably, these data show that the empirical Mark-Houwink relation between molecular weight and hydrodynamic volume is, for linear debranched starch, valid for much lower molecular weights than synthetic polymers. This implies that these Mark-Houwink parameters can be used with "universal calibration" to enable SEC to be used with relative ease to provide MWDs for debranched starch for essentially any degree of polymerization.     

Physico-chemical characterization of lignins from different sources for use in phenol-formaldehyde resin synthesis

During the last decades lignin has been investigated as a promising natural alternative to petrochemicals in phenol-formaldehyde (PF) resin production, due to their structural similarity. Physico-chemical characterization of three types of lignin, namely kraft pine lignin (L1), soda-anthraquinone flax lignin (L2), and ethanol-water wild tamarind lignin (L3) has been evaluated to determine which one is the most suitable chemical structure for above purpose. Characterization has been performed using Fourier transform infrared spectroscopy (FT-IR) and proton nuclear magnetic resonance spectrometry ((1)H NMR) to analyse the chemical structure, gel permeation chromatography (GPC) for determining molecular weight (MW) and molecular weight distribution (MWD), differential scanning calorimetry (DSC) to measure the glass transition temperature and thermogravimetric analysis (TGA) to follow the thermal degradation. Both structural and thermal characteristics suggest that kraft pine lignin (L1) would be a better phenol (P) substitute in the synthesis of lignin-phenol-formaldehyde (LPF) resins, as it presents higher amounts of activated free ring positions, higher MW and higher thermal decomposition temperature.     

Studies on Guizotia abyssinica L. oil: Biodiesel synthesis and process optimization

Guizotia abyssinica seeds, a common bird feedstock, have been explored for the potential of biodiesel synthesis. The oil was extracted from the seeds by solvent extraction and composition of G. abyssinica oil was examined. The reaction parameters for biodiesel synthesis have been optimized. Temperature, oil: methanol ratio, catalyst type and catalyst concentration were found to have significant role on ester conversion. According to this study, the maximum yield of ester (98.7%) can be obtained with optimized sodium methoxide catalyst dosage (0.6%) at an operational temperature of 65 °C. Methyl ester of G. abyssinica oil was also studied for its oxidation stability and low temperature properties. Further, the synthesized product was blended in diesel at 5–20% ratios and evaluated for physico-chemical properties.     

Asymmetric binuclear titanocenes linked by alkyl benzyl ethers: Synthesis, characterization and use as catalysts for ethylene polymerization

A series of novel asymmetric binuclear titanocenes linked with alkyl benzyl ethers p-[(C₅H₅TiCl₂)C₅H₄CH₂]C₆H₄O(CH₂)_n[C₅H₄(TiCl₂C₅H₅)] (n = 2-5) (13-16) have been synthesized by treating p-(LiC₅H₄CH₂)C₆H₄O(CH₂)_n(C₅H₄Li) (n = 2-5) (9-12) with C₅H₅TiCl₃. The new complexes have been characterized by elemental analysis and NMR spectra. Their catalytic activity for ethylene polymerization was investigated in the presence of aluminoxane (MAO). The results show that 13-16 are efficient catalysts for producing polyethylene (PE) with a broad molecular weight distribution (MWD). Their catalytic activity is highly dependent on the length of the alkyl chain and the polymerization conditions. A longer alkyl chain increases the catalytic activity, whereas the molecular weight of the produced polyethylene decreases.     

Structural characterization of rubber from jackfruit and euphorbia as a model of natural rubber

A structural study of low molecular weight rubbers from Jackfruit (Artocarpus heterophyllus) and Painted spurge (Euphorbia heterophylla) was carried out as model compounds of natural rubber from Hevea brasiliensis. The rubber content of latex from Jackfruit was 0.4-0.7%, which is very low compared with that of 30-35% in the latex from Hevea tree. The rubber from Jackfruit latex was low molecular weight with narrow unimodal molecular weight distribution (MWD), whereas that obtained from E. heterophylla showed very broad MWD. The 1H and 13C NMR analyses showed that Jackfruit rubber consists of a dimethylallyl group and two trans-isoprene units connected to a long sequence of cis-isoprene units. The alpha-terminal group of Jackfruit rubber was presumed to be composed of a phosphate group based on the presence of 1H NMR signal at 4.08 ppm corresponding to the terminal =CH-CH2OP group.     

Synthesis of biodegradable poly-epsilon-caprolactone microspheres by dispersion ring-opening polymerization in supercritical carbon dioxide

A series of fluorinated diblock and triblock copolymers of poly(epsilon-caprolactone) and poly(heptadecafluorodecylacrylate) were prepared by combining ring-opening polymerization of epsilon-CL and atom transfer radical polymerization of the acrylate. These copolymers with well-controlled molecular weight and composition were characterized by (1)H NMR spectroscopy and used as stabilizers for the dispersion ring-opening polymerization of epsilon-CL in supercritical carbon dioxide. The effect of composition and architecture of the polymeric stabilizers on the stabilization of PCL microspheres was investigated. Finally, purification of PCL was successfully implemented by reactive supercritical fluid extraction of the tin catalyst.     

Absolute molecular weight and molecular weight distribution of guar by size exclusion chromatography and low-angle laser light scattering

A procedure to determine the absolute weight-average molecular weight (M_w) and molecular weight distribution (MWD) of guar by aqueous size exclusion chromatography coupled with low angle laser scattering is described. It is shown that for a rigorously purified sample of guar solution the values for M_w and MWD are 2·2×10⁶ and 1·9 respectively. The effect of sample preparation and purification on these molecular parameters are discussed. Limitations and challenges in the aqueous size exclusion chromatography of complex water soluble polymers such as guar are also explored.     

Rheological and microstructural investigation of oat β-glucan isolates varying in molecular weight

The rheological properties and microstructure of aqueous oat β-glucan solutions varying in molecular weight were investigated. The structural features and molecular weights (MW) were characterized by ¹³C NMR spectroscopy and high performance size-exclusion chromatography (HPSEC), respectively. The microstructure of the β-glucans dispersions was also examined by atomic force microscopy (AFM). The samples with β-glucan content between 78 and 86% on a dry weight basis had MW, intrinsic viscosity ([η]) and critical concentration (c*) in the range of 142-2800 × 10³ g/mol, 1.7-7.2 dl/g and 0.25-1.10 g/dl, respectively. The flow and viscoelastic behaviour was highly dependent on MW and on the concentration of the β-glucans dispersions. Pseudoplastic behaviour was exhibited at high concentrations and Newtonian behaviour was evident at low concentrations. At the same concentration, the viscosity was higher for higher MW samples. The Cox-Merz rule was applicable for the lower molecular weight samples at higher concentrations whereas the high molecular weight sample deviated at concentrations greater than 1.0%, w/v. The mechanical spectra with variation of both MW and concentration were typical of entangled biopolymer solutions. AFM images revealed the formation of clusters or aggregates linked via individual polymer chains scattered heterogeneously throughout the system. The aggregate size increased with the molecular weight of the samples investigated and has been linked to the rheological behaviour of the samples.