Conformational study of 13C-enriched fibroin in the solid state, using the cross polarization nuclear magnetic resonance method

Silk fibroin with the alanyl carboxyl carbon enriched with 13C was obtained by giving a diet containing 13C-enriched alanine to the larvae of Bombyx mori and Antheraea pernyi at the fifth instar. Sericin-free fibroin fibers were prepared from cocoons, and gut was made from the liquid silk in the gland. The final 13C content was about 13%. Cross polarization/magic angle sample spinning spectra at 25 MHz and 75 MHz were measured for each sample at different orientations. Spectra were simulated using the principal values and orientations of the shielding tensor in the alanine crystal. The results indicate that the beta-structure of the fibroin may be a little more flattened than the typical pleated sheet beta-structure.     

Formation of a Tree having a Low Lignin Content

Received 30 September 2001/ Accepted in revised form 26 October 2001     

Purification of a fibrolast-inhibitory factor from Actinobacillus actinomycetemcomitans Y4

Abstract A factor showing inhibitory activity against human gingival fibrolasts was extracted from the cytosol fraction of Actinobacillus actinomycetemcomitans Y4. The activity markedly inhibited the proliferation of human gingival fibrolasts, but had no effect on cell viability or gross morphology. No such activity was found in cytosol fractions from either Porphyromonas gingivalis 381 or Escherichia coli HB101. The extract from A. actinomycetemcomitans Y4 was then purified by anion-exchange chromatography, hydroxyapatite chromatography and gel-filtration chromatography to give a single band on SDS-PAGE with an apparent molecular mass of 65 kDa. The purification ratio was 183-fold with a recovery rate of 5% compared with the crude extract (starting material) when the activity was assessed by direct cell counts.     

Repeated streptozotocin injections cause early onset of glomerulosclerosis in mice.

Diabetic nephropathy (DN), a major cause of end-stage chronic renal failure, is histologically characterized by glomerulosclerosis. To investigate the molecular mechanisms of DN, it is important to establish a stable model of glomerulosclerosis in mice, because genomic manipulation techniques (such as gene destruction or transgene insertion) are well established in rodent species. In this study, we found that repeated administrations of streptozotocin led to early onset of glomerular sclerotic lesions in C57BL/6 mice, accompanied with renal dysfunction. During the natural course of DN, glomerular endothelial cells decreased at 10 weeks after the start of streptozotocin-injections, whereas myofibroblastic mesangial cells became evident. Our results provide an animal tool to elucidate the molecular mechanisms of DN, for example to investigate vascular pathology in diabetic glomerular diseases.     

Separation of dilute aqueous butanol and acetone solutions by pervaporation through liquid membranes

A simultaneous extraction-stripping process is proposed for separating volatile products from fermentation broths, it is based on pervaporation through a liquid membrane supported with a hydrophobic porous membrane. The liquid membrane prepared with oleyl alcohol was selected as the most suitable for separating volatile products resulting from acetone-butanol fermentation. The separation performance and stability of the oleyl alcohol liquid membrane were investigated by using dilute aqueous butanol and acetone solutions. The oleyl alcohol liquid membrane was found to be superior by far in both selectivity and permeability of butanol to the better known silicone rubber membrane in its high selectivity for alcohols. Using the oleyl alcohol liquid membrane, the dilute aqueous butanol solutions of around 4 g/L obtained in acetone-butanol fermentation could be concentrated up to 100 times. The stability of this liquid membrane was also quite good as long as the surface tension of the feed solution was less than the critical surface tension of the support membrane. No change in the separation performance was found after the continuous usage in a long period of 100 h.     

Augmentation of the generation of cell-mediated cytotoxicity in culture by mitomycin C

Summary The effects of mitomycin C (MMC) on the generation of cell-mediated cytotoxicity in primary stimulation culture of human peripheral blood mononuclear cells (PBM) with the B lymphoblastoid Raji cell line were assessed. The cell-mediated cytotoxicity induced in culture was significantly augmented when MMC was added to cultures on day –1 to day 3 for 24 h at concentrations of 2.5×10^–2 g/ml and 2.5×10^–3 g/ml. To identify the cell populations affected by MMC, PBM were separated by adherence to plastic after treatment with MMC for 24 h (day –1). The two populations were recombined with untreated separated cells and stimulated with antigen. The ability to develop an augmented cell-mediated cytotoxicity was associated with the adherent cell fraction of MMC-treated PBM. Therefore, the ability of MMC-treated adherent cells to produce interleukin 1 (IL 1) was examined. Significantly higher levels of IL 1 were produced by treated cells as compared to untreated adherent cells. The results appear to indicate that the selective effects of MMC on the adherent cell fraction, especially the modification of IL 1 production, may be involved in the mechanisms of MMC-induced augmented cell-mediated cytotoxicity.     

Functions of the COOH-terminal region of cyclodextrin glucanotransferase of alkalophilic Bacillus sp. #1011: relation to catalyzing activity and pH stability

Cyclodextrin glucanotransferase (beta-CGTase) of alkalophilic Bacillus sp. #1011 degrades starch to mainly beta-cyclodextrin (beta-CD). This enzyme is considered to contain an extra-polypeptide in its COOH-terminal region in addition to its NH2-terminal domain which exhibits the starch-degrading activity. To analyze the functions of this extra-polypeptide in the beta-CGTase, two mutated enzymes, in which DNA regions encoding 10 or 13 amino acids from the COOH-terminus were deleted, were obtained. The mutated enzymes degraded starch to glucose, maltooligosaccharides and alpha-CD, in addition to beta-CD. Furthermore, the pH stability of the mutated enzymes in the alkaline pH range (pH 9-11) was reduced.     

Denaturation of bacteriorhodopsin by organic solvents

The denaturation of bacteriorhodopsin by various organic solvents was studied using absorption, circular dichroism (CD) and fluorescence measurements. Organic solvents with a hydrogen-bonding group caused the release of retinal. The CD measurements showed that the helical structure was maintained even in the denatured state, whereas its tertiary structure was destroyed. The change in fluorescence intensity of tryptophan and fluorescent retinal also confirmed that the tertiary structure was destroyed. Comparison of the denaturation efficiency of various organic solvents showed that the concentration at denaturation was inversely proportional to the partition coefficient of the denaturant. This inverse proportionality clearly indicated that denaturation was determined by the concentration of denaturants which partitioned into the hydrophobic region of the membrane. It was discussed from the experimental results that the tertiary structure of bacteriorhodopsin was stabilized by the hydrogen-bonding networks between side chains of the helices. The results obtained from analysis of the amino acid sequence were also consistent with the hydrogen-bonding mechanism for the formation of the tertiary structure.     

Temperature dependence of the structure of aggregates of tobacco mosaic virus protein at pH 7.2. Static synchrotron small-angle X-ray scattering

The small-angle X-ray scattering (SAXS) method using a synchrotron radiation source was applied to the study of the self-aggregation process of tobacco mosaic virus protein (TMVP) at a concentration of 5.0 or 12.0 mg ml-1 in 50 mM or 100 mM-phosphate buffer (ionic strengths approx. 0.1 and 0.2, respectively) at pH 7.2 in the temperature region of 4.8 to 25.0 degrees C. This paper presents the results of static measurements of SAXS. Sedimentation velocity experiments were performed simultaneously under the same conditions. These results are qualitatively parallel to those of the SAXS measurements, although the size of stacked disks derived from the SAXS measurements is larger than that derived from the sedimentation experiments, suggesting a change in the equilibrium conditions in the centrifugal field. Qualitative analysis of the SAXS data with model simulation calculations implies that the aggregation of TMVP consists of two steps: (1) the aggregation of A-protein comprising a few subunits to form double-layered disks; and (2) the random polymerization of double-layered disks by disk-stacking. Increase in temperature, ionic strength or protein concentration induced TMVP to polymerize to form a double-layered disk or a quadruple-layered short rod with consumption of A-proteins, accompanied by a small number of multi-layered short rods. The SAXS results indicate that the A-protein and the multilayered short rods are polydisperse with respect to size and shape, i.e. the mixture of A-protein, double-layered disks and multi-layered short rods coexists in the equilibrium state without pressure-induced partial dissociation of TMPV as observed during normal ultracentrifugation, and even under solution conditions in which the formation of double-layered disks or higher-order aggregates is favored.     

Energy saving effect of pervaporation using oleyl alcohol liquid membrane in butanol purification

The liquid membrane prepared with oleyl alcohol was used in pervaporation of dilute aqueous butanol solutions. The selectivity of this liquid membrane was found to be superior than that of silicone rubber membrane, and the separation factor for butanol was 180. Energy saving effect of pervaporation in butanol purification was investigated by comparing the energies required to purify a butanol solution of 0.5 wt.% in the following three separation systems; a conventional distillation system, a separation system combining pervaporation with distillation, and a pervaporation system using a hydrophobic membrane and a hydrohylic membrane in series. When the pervaporation using oleyl alcohol liquid membrane was employed as a pretreatment process of butanol purification, the energy requirement was found to be around one-tenth of that of conventional distillation.List of Symbols E _D MJ/kg Specific energy requirement of butanol purification by distillation - J kg/(m² · h) Total permeation flux - J _B kg/(m² · h) Permeation flux of butanol - P ₁, P ₂ MPa Pressure at inlet and outlet of vacuum pump - Q kJ/h Energy transfer rate - Q _C Q _W kJ/h Energy consumption rate of condenser and vacuum pump - R J/K · mol Gas constant - t, T °C, K Temperature - W-g/h Mass flow rate of butanol/water binary mixture - (W) _F1 ,-kg/h Mass flow rate of aqueous butanol solution - (W) _F2 at inlet and outlet of permeation cell - W* kJ/mol Energy requirement of adiavatic expansion - X _B Butanol mass fraction of aqueous butanol solution - (X _B ) _F Butanol mass fraction of aqueous butanol solution supplied into distillation column - (X _B ) _F1 Butanol mass fraction of aqueous butanol - (X _B ) _F2 solution at inlet and outlet of permeation cell - Y _B Butanol mass fraction in permeate - Separation factor of butanol - Adiavatic constant