Preparation of C-6 substituted chitin derivatives under homogeneous conditions

Tosylation of chitin under homogeneous conditions was achieved by the reaction of tosyl chloride with chitin in a DMAc/LiCl solvent system. The resultant tosyl-chitin was fully N-acetylated with acetic anhydride in methanol. The fully acetylated tosyl-chitin was subsequently reacted with the sodium salts of ethyl p-hydroxybenzoate, diethyl malonate, and diethyl phosphite in DMAc to give the corresponding chitin derivatives of 6-O-ethyl benzoate-chitin, 6-deoxy-diethyl malonate-chitin, and 6-(deoxydiethyl) phosphite-chitin, respectively. Subsequent hydrolysis of the chitin-ester derivatives with tert-butoxide in dimethyl sulfoxide (DMSO) generated 6-O-carboxyphenyl-chitin and 6-(deoxydicarboxy)methyl-chitin. The structures of the chitin derivatives were assessed by FT-IR, (13)C NMR, and (31)P NMR, while the degree of substitution of the S(N)2 reaction was estimated by elemental analysis. All the chitin derivatives were found to be soluble or swellable in water, DMAc, or DMSO.     

Rapid homogeneous lauroylation of wheat straw hemicelluloses under mild conditions

Hemicellulose-based hydrophobic biomaterials with degrees of substitution ranging from 0.46 to 1.54 were synthesized under mild conditions in homogeneous media (N,N-dimethylformamide-lithium chloride) by reacting the native wheat straw hemicellulosic polymers with lauroyl chloride using 4-dimethylaminopyridine as a catalyst. Other catalysts such as N-bromosuccinimide, N-methyl pyrrolidine, N-methyl pyrrolidinone, and pyridine were also investigated. Under optimum reaction conditions (2 equiv of lauroyl chloride and triethylamine per hydroxyl group, 5% 4-dimethylaminopyridine, 40 °C, 35 min), a high DS value of 1.54 was obtained. The biomaterials were characterized by FT-IR spectroscopy and ¹³C NMR spectroscopy as well as by thermal analysis. The results showed that the lauroylation occurred preferably at the C-3 hydroxyl group of β-d-Xylp units in the hemicelluloses, and the thermal stability of the hydrophobic polymers increased by esterification.     

Kinetic behaviour of acid phosphatase-albumin co-polymers in homogeneous phase and under gel-immobilized conditions.

1. Antiserum raised to purified human liver monoamine oxidase was used to characterize the monoamine oxidase from human liver, brain cortex, placenta and platelets. 2. Antibodies to monoamine oxidase were purified by adsorption with a mitochondrial preparation. 3. Monoamine oxidase was present in liver particle-free supernatant as measured by enzyme activity and immunodiffusion. 4. Multiple precipitin lines were obtained on immunodiffusion analysis against the purified liver enzyme. It is proposed that this is due to either aggregation or to differential lipid binding. 5. The results suggest that the functionally different enzymes found in liver, brain cortex, platelets and placenta are immunochemically related and may be identical.     

Chlorination of cellulose with N-chlorosuccinimide-triphenylphosphine under homogeneous conditions in lithium chloride-N,N-dimethylacetamide.

Microcrystalline cellulose was chlorinated with N-chlorosuccinimide-triphenylphosphine under homogeneous conditions in LiCl-N,N-dimethylacetamide. At the early stage of the reaction only replacement of the 6-hydroxyl groups with chlorine was observed, and 3-hydroxyl groups were replaced at a lower rate with Walden inversion. The effects of reaction conditions on the extent of chlorination were studied in detail. More than two equivalents of chlorination reagents per glucose residue were necessary to attain a high degree of substitution (ds) by chlorine, and the maximum ds attained was 1.86. Chlorinated disaccharides were found in the hydrolyzates of chlorodeoxycelluloses hydrolyzed under mild conditions, and their structures were studied by mass spectrometry.     

Preparation of proteoglycan from salmon nasal cartilage under nondenaturing conditions

Salmon nasal cartilage was micronized in ethanol using a rotor–stator homogenizer for the high yield of proteoglycan extraction. This procedure also brought about depressing the degradation of proteoglycan and the contamination of collagens. Proteoglycan was extracted by 4 M magnesium chloride and isolated by anion-exchange chromatography. The gel filtration HPLC and the antibody reactivity showed that the core protein was intact.     

Damaged-bone adaptation under steady homogeneous stress

In this work an extension of the adaptive-elasticity theory is proposed in order to include the contribution of bone microdamage as a stimulus. Some aspects of damaged-bone tissue adaptation, brought about by a change of the daily loading history, are investigated. In particular, under the assumption of a small strain approximation and isothermal conditions, the solution of the remodeling rate equation for steady homogeneous stress is discussed and the damage effect upon the remodeling time constant is shown. The result is both theoretical and numerical, based on a recent theory of internal damaged-bone remodeling (Ramtani, S., and Zidi, M., 1999, "Damaged-Bone Remodeling Theory: Thermodynamical Approach, " Mechanics Research Communications, Vol. 26, pp. 701-708. Ramtani, S., and Zidi, M., 2001, "A Theoretical Model of the Effect of Continum Damage on a Bone Adaption Model," Journal of Biomechanics, Vol. 34, pp. 471-479) and motivated by the works of Cowin, S. C., and Hegedus, D. M., 1976, "Bone Remodeling I: Theory and Adaptive Elasticity," Journal of Elasticity, Vol. 6, pp. 471-479 and Hegedus, D. H., and Cowin, S. C., 1976, "Bone Remodeling II: Small Strain Adaptive Elasticity," Journal of Elasticity, Vol. 6, pp. 337-352.     

Preparation of erythrocyte ghosts by a glycol-induced osmotic lysis under isoionic conditions

A procedure has been developed for obtaining haemoglobin-free, erythrocyte ghosts under ionic conditions approximating that of the cell cytoplasm. Haemolysis was effected by incorporating glycol into cells suspended in the isoionic medium and then diluting with a large volume of glycol-free medium.The ghosts were of uniform spherical shape throughout the preparative procedure and were impermeable to macromolecules.Analysis of polypeptides by sodium dodecyl sulphate-gel elecrophoresis at each stage of preparation and comparison with ghosts prepared under hypo-ionic conditions served to distinguish membrane components from those of cytoplasm.     

Chlorella pyrenoidosa 7-11-05 in batch and in homogeneous continuous culture under autotrophic conditions

The growth ofChlorella pyrenoidosa 7-11-05 in batch and in continuous culture using two types of cultivation vessels was compared. The high values of specific growth rate obtained in batch deep stirred tank culture containing small amount of inoculum are not easily attainable in continuous culture with regard to the high number of autospores formed throughout the ontogenetic cycle ofChlorella. The obtained surface and volume productivities prove that in both types of cultivation vessels a high growth intensity can be achieved. The deep tank perfectly mixed cultivation may become of significance in the future.     

Hydrotrispyrazolylborate-copper complexes as catalysts for the styrene cyclopropanation reaction with ethyl diazoacetate under homogeneous and heterogeneous conditions

A series of complexes of general formulae Tp^xCu have been employed as the catalyst for the styrene cyclopropanation reaction with ethyl diazoacetate, both under homogeneous and heterogeneous conditions. Several solid supports have been tested, with silica gel being the most convenient for this transformation. A comparison of the results obtained under both conditions has shown that the catalytic pocket is affected by the support.     

Chlorella pyrenoidosa 7-11-05 in batch and in homogeneous continuous culture under autotrophic conditions

The cell content of substances (proteins, nucleic acids and chlorophylls) which play a significant role in growth processes in algae was used to characterize the physiological state of a continuous culture ofChlorella pyrenoidosa. The ratio of the various components of the cell content did not alter significantly with changes in the dilution rate. An increase in the mean cell volume was accompanied by a proportional increase in the amount of the various components. Their respective dry weight concentrations rose with the dilution rate and after reaching a maximum either fell or remained constant. The specific rates of synthesis of the given substances tended to rise, i.e. maximum activity of the culture was not attained. Deoxyribonucleic acid synthesis appears to be the endogenous factor limiting growth of the culture. The proportion of the individual nucleic acid fractions was compared with protein synthesis.     

Genetic variations in root morphology and phosphorus efficiency of Pinus massoniana under heterogeneous and homogeneous low phosphorus conditions

Background and aims

The vertical distribution of available phosphorus (P) in the soil is usually heterogeneous with soil depth. However, little is known about the P efficiency of conifer species under vertically heterogeneous low-P conditions. The purpose of this study was to investigate the genetic variations in growth traits and P efficiency of Pinus massoniana, under heterogeneous and homogeneous low-P conditions.

Methods

Pot experiments consisting of low-P (a low P level in all soil layers), layered-P (a high P level in the topsoil and a low P level in the bottom soil), and high-P (high P levels in all soil layers) conditions were designed and conducted. Three-way ANOVA was used to investigate genetic variations in P efficiency and the major growth traits under these three types of P conditions.

Results

There were substantial genetic variations in the major growth traits, including tree height, stem diameter and seedling dry weight, under both heterogeneous and homogeneous low-P conditions. The heritability for major growth traits was high under both types of low-P condition. Moreover, there were significant genotype × P interaction effects for growth parameters.

Conclusions

Our results indicate that it may be possible to select Masson pine genotypes with high P efficiency and productivity. The significant genotype × environment interactions should be exploited in breeding, and genotypes showing specific adaptations to certain nutrient environments should be bred and used within that environment.     

Fluorescence resonance energy transfer from pyrene to perylene labels for nucleic acid hybridization assays under homogeneous solution conditions

We characterized the fluorescence resonance energy transfer (FRET) from pyrene (donor) to perylene (acceptor) for nucleic acid assays under homogeneous solution conditions. We used the hybridization between a target 32mer and its complementary two sequential 16mer deoxyribonucleotides whose neighboring terminals were each respectively labeled with a pyrene and a perylene residue. A transfer efficiency of ~100% was attained upon the hybridization when observing perylene fluorescence at 459 nm with 347-nm excitation of a pyrene absorption peak. The Förster distance between two dye residues was 22.3 Å (the orientation factor of 2/3). We could change the distance between the residues by inserting various numbers of nucleotides into the center of the target, thus creating a gap between the dye residues on a hybrid. Assuming that the number of inserted nucleotides is proportional to the distance between the dye residues, the energy transfer efficiency versus number of inserted nucleotides strictly obeyed the Förster theory. The mean inter-nucleotide distance of the single-stranded portion was estimated to be 2.1 Å. Comparison between the fluorescent properties of a pyrene–perylene pair with those of a widely used fluorescein–rhodamine pair showed that the pyrene–perylene FRET is suitable for hybridization assays.     

Lithoautotrophic growth of the freshwater strain Beggiatoa D-402 and energy conservation in a homogeneous culture under microoxic conditions

The freshwater filamentous bacterium Beggiatoa D-402 was shown to grow lithoautotrophically in a homogeneous culture under microoxic conditions only, the growth yield being the highest at 0.1 mg O(2) l(-1). High activities of the Calvin cycle key enzymes and of the dissimilatory path thiosulfate oxidation enzymes were found in the bacterial cells. The rate of CO(2) fixation above 112 nmol min(-1) (mg protein)(-1), an about 90% increase in the protein carbon at the expense of CO(2) carbon and an increase in the molar yield up to 12 mg dry weight (mmol oxidized thiosulfate)(-1) indicate the bacterial growth was autotrophic. Thiosulfate was oxidized by the strain almost completely into sulfate. The metabolically useful energy was conserved by oxidative phosphorylation that was coupled to oxidation of sulfur compounds. The bacterial membranes were found to contain CO-binding cytochromes b and two cytochromes c with M(r) 23 and 26 kDa, the terminal part of the respiratory chain containing presumably a cbb(3)-type oxidase. A cytochrome c with M(r) 12 kDa was detected in the soluble fraction.     

Preparation of electrophoretically homogeneous erythrocuprein and its thermodenaturation]

The procedure for isolation of electrophoretically homogeneous preparation of erythrocuprein from 17 1 blood batch was described. The preparation had absorbtion value, A260/A680, of 25-26. The protein completely retains superoxide dismutase activity after treatment 7 min. at 75 degrees C. The heating of protein solution at 100 degrees C bring about autoreduction of protein copper as demonstrated by decreasing optical and EPR-spectral intensities. The reduced protein was oxidized in aerobic condition and oxidized preparation had optical and EPR-spectra differ from those of the native protein the activy being decreased.     

Preparation of highly 3H-labeled S-100 protein under nondenaturing conditions

A method for obtaining a tritium-labeled S-100 protein of high specific radio-activity (〉~ 10 Ci/mmol) under mild conditions is described. The method is based on the reductive methylation of lysine residues; the labeling procedurs does not appreciably alter the physical and chemical properties of 8–100 protein, as measured by studies of intrinsic fluorescence enhancement, ⁴⁵Ca binding, electrophoretic mobility, titrations of sulfydryl groups, and intramolecular crosslinking of S-100 via disulfide bond formation. Alternative labeling procedures based on chemical or enzymatie iodination with ¹²⁵I, invelving the use of powerful oxidizing agents, cause an irreversible exidation of the sulfydryl groups and affect the above-mentioned properties of the S-100 protein.     

Preparation of a homogeneous soluble D-beta-hydroxybutyrate apodehydrogenase from mitochondria.

D-beta-Hydroxybutyrate dehydrogenase of bovine heart mitochondria has been purified to apparent homogeneity. The membrane-bound enzyme is first released by phospholipase A digestion of the mitochondria. Lithium bromide, 0.4 M, is used to aid release, and dithiothreitol is required to stabilize the enzyme. The membranous material is removed by centrifugation, and the apoenzyme is recovered in the supernatant and precipitated with ammonium sulfate to 50 percent of saturation. The main purification (100-fold) is achieved by selective adsorption and elution on controlled pore glass beads. The purified enzyme has been purified approximately 250-fold from the mitochondria. The purified enzyme is homogeneous as shown by poly-acrylamide gel electrophoresis in sodium dodecyl sulfate or acid-urea systems; a sharp band is obtained which is equivalent to a subunit molecular weight of 31,500. The apoenzyme is devoid of lipid and is completely inactive as isolated. It can be reactivated by adding aqueous microdispersions of lecithin or phospholipids containing lecithin. The apoenzyme is stable, i.e. it has a half-life of about 450 hours at 0-2 degrees in 0.4 M lithium bromide, containing 5 mM dithiothreitol at pH 7, and is soluble at these conditions, existing mainly as a monomer and dimer in dilute solution. It has a tendency to associate into larger aggregates when the salt concentration is lowered. The enzyme does not have a distinctive amino acid composition as compared with other proteins or soluble dehydrogenases. The purified apodehydrogenase is well suited for study of specific protein-lipid interaction, as well as the molecular basis for the role of phospholipid in this lipid-requiring enzyme.     

Preparation of a homogeneous coated vesicle fraction from bean leaves

Summary Using a procedure previously developed for suspension-cultured carrot cells, we have been able to isolate two different coated vesicle-containing fractions from green bean leaves (Vicia faba). The two fractions differ in their isopycnic densities in D₂O-Ficoll as well as in their diameters. One of the fractions (the less dense of the two) is almost 100% pure as judged by negative staining. Because of this the polypeptide pattern obtained from SDS-PAGE is most clear and has enabled a clear recognition of clathrin light chains, in addition to the 190 kDa heavy chain coat component. Significantly the 100k Da and 50k Da polypeptides typical of brain coated vesicles are absent from bean leaf coated vesicles. Due to a) the high degree of vacuolation b) the presence of large amounts of ribulose bisphosphate carboxylase in the postmicrosomal supernatant, the yield of coated vesicles from bean leaves, as compared to nongreen plant cells, or to bovine brain tissue is extremely low (1 mg coated vesicles from 2.4 kg leaf tissue).Abbreviations D₂O deuterium oxide - EGTA ethylene glycol-bis ( amino ethyl ether) N,N,N,N tetraacetic acid - MES, 2 (N-morpholino)-ethanesulfonic acid - PMSF phenyl methylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TRIS Tris-hydroxy methyl amino methane     

Preparation of chitin nanofibers from squid pen beta-chitin by simple mechanical treatment under acid conditions

A procedure for preparing individualized chitin nanofibers 3-4 nm in cross-sectional width and at least a few microns in length was developed. The key factors to prepare the chitin nanofibers with such high aspect ratios are as follows: (1) squid pen beta-chitin is used as the starting material and (2) ultrasonication of the beta-chitin in water at pH 3-4 and 0.1-0.3% consistency for a few minutes. Transparent and highly viscous dispersions of squid pen beta-chitin nanofibers in water can be obtained by this method. No N-deacetylation occurs on the chitin molecules during the nanofiber conversion procedure. Moreover, the original crystal structure of beta-chitin is maintained, although crystallinity index decreases from 0.51 to 0.37 as a result of the nanofiber conversion. Cationization of the C2 amino groups present on the crystallite surfaces of the squid pen beta-chitin under acid conditions is necessary for preparing the nanofibers.     

Preparation of giant liposomes in physiological conditions and their characterization under an optical microscope.

Unilamellar liposomes with diameters of 25-100 microns were prepared in various physiological salt solutions, e.g., 100 mM KCl plus 1 mM CaCl2. Successful preparation of the giant liposomes at high ionic strengths required the inclusion of 10-20% of a charged lipid, such as phosphatidylglycerol, phosphatidylserine, phosphatidic acid, or cardiolipin, in phosphatidylcholine or phosphatidylethanolamine. Three criteria were employed to identify unilamellar liposomes, yielding consistent results. Under a phase-contrast microscope those liposomes that showed the thinnest contour and had a vigorously undulating membrane were judged unilamellar. When liposomes were stained with the lipophilic fluorescent dye octadecyl rhodamine B, fluorescence intensities of the membrane of individual liposomes were integer multiples (up to four) of the lowest ones, the least fluorescent liposomes being those also judged unilamellar in the phase-contrast image. Micropipette aspiration test showed that the liposomes judged unilamellar in phase and fluorescence images had an area elastic modulus of approximately 160 dyn/cm, in agreement with literature values. The giant liposomes were stable and retained a concentration gradient of K+ across the membrane, as evidenced in fluorescence images of the K(+)-indicator PBFI encapsulated in the liposomes. Ionophore-induced K+ transport and associated volume change were observed in individual liposomes.