Development of mushrooms for thrombosis prevention by protoplast fusion

With thrombosis a major cause of death in Japan and the Western world, thrombin-inhibitory agents that constrain the formation of fibrin are sought. We screened for basidiomycetes showing anti-thrombin activity and isolated Laetiporus sulphureus. However, it was difficult to cultivate and its form was not satisfactory. We therefore used protoplast fusion between L. sulphureus and the commonly cultivated basidiomycete Hypsizygus marmoreaus to obtain cultivable basidiomycetes that produced an anti-thrombin substance. For the protoplast fusion of L. sulphureus and H. marmoreaus, the protoplast concentration, alternating electric field intensity, dielectrophoresis duration, and field pulse intensity used were of 1 x 10(7) protoplasts/ml, 100 V/cm.1 MHz, 60 s, and 8 kV/cm, respectively. The number of regenerated colonies obtained was 4961, from which 43 strains were selected for electrophoretic analysis. Four of the fusants were found to have a band from each parent in isozyme patterns obtained using their crude extract. The fruiting bodies of the fusants were very similar to those of H. marmoreaus. Crude extract from each of the fusants and from L. sulphureus showed anti-coagulative activity in terms of the thrombin clotting time. We thus obtained improved basidiomycetes that produce an anti-thrombin substance, are easily cultivated, and whose form resembles H. marmoreaus, a commonly used culinary mushroom.     

Efficient transformation of Mesorhizobium huakuii subsp. rengei and Rhizobium species

The transformation of Mesorhizobium huakuii subsp. rengei B3 with either pBBR122 or pKT230 was carried out. We determined the optimal conditions required for transformation by electroporation and obtained up to 10(5) CFU/microg pBBR122. Plasmids prepared from strain B3 yielded higher transformation efficiency than those from various dam or dcm mutant strains of Escherichia coli. This result suggests that a high transformation efficiency is not related to the methylation of plasmid DNA in E. coli. Using the optimal conditions for electroporation, we performed transformation of several species of Rhizobium, Mesorhizobium and Sinorhizobium. All tested strains of these species were transformed with pBBR122. Strains of M. huakuii and R. phaseoli AHU1133 were transformed at high efficiency, whereas transformation efficiencies of Rhizobium sp. NGR234 and S. meliloti strains were less than 2 x 10(3) CFU/microg plasmid DNA.     

Isolation of a thermophile degrading poly(butylene succinate-co-butylene adipate)

A thermophile, strain 73, which degrades poly(butylene succinate-co-butylene adipate) (PBSA) film was isolated from 95 soil samples obtained from different locations by cultivation using an enrichment culture technique at 60 degrees C. At this temperature, the strain grew on PBSA and the dissolved total organic carbon (TOC) concentration in the medium changed according to the growth stage, i.e., after the TOC concentration rapidly reached the minimum, it increased rapidly until it reached a peak and then decreased thereafter. During cultivation, PBSA was almost eliminated and the solution viscosity of the residual PBSA decreased markedly. Gel permeation chromatograms of the residual PBSA showed a significant decrease in the main peak and the appearance of a new peak at the low molecular weight region. The strain was identified as Bacillus stearothermophilus, which has an optimum growth temperature of approximately 63 degrees C.     

The absence of corpus luteum formation alters the endocrine profile and affects follicular development during the first follicular wave in cattle

We previously established a bovine experimental model showing that the corpus luteum (CL) does not appear following aspiration of the preovulatory follicle before the onset of LH surge. Using this model, the present study aimed to determine the profile of follicular development and the endocrinological environment in the absence of CL with variable nadir circulating progesterone (P(4)) concentrations during the oestrous cycle in cattle. Luteolysis was induced in heifers and cows and they were assigned either to have the dominant follicle aspirated (CL-absent) or ovulation induced (CL-present). Ultrasound scanning to observe the diameter of each follicle and blood collection was performed from the day of follicular aspiration or ovulation and continued for 6 days. The CL-absent cattle maintained nadir circulating P(4) throughout the experimental period and showed a similar diameter between the largest and second largest follicle, resulting in co-dominant follicles. Oestradiol (E(2)) concentrations were greater in the CL-absent cows than in the CL-present cows at day -1, day 1 and day 2 from follicular deviation. The CL-absent cows had a higher basal concentration, area under the curve (AUC), pulse amplitude and pulse frequency of LH than the CL-present cows. After follicular deviation, the CL-absent cows showed a greater basal concentration, AUC and pulse amplitude of growth hormone (GH) than the CL-present cows. These results suggest that the absence of CL accompanying nadir circulating P(4) induces an enhancement of LH pulses, which involves the growth of the co-dominant follicles. Our results also suggest that circulating levels of P(4) and E(2) affect pulsatile GH secretion in cattle.     

Direct ethanol production from barley beta-glucan by sake yeast displaying Aspergillus oryzae beta-glucosidase and endoglucanase

Three beta-glucosidase- and two endoglucanase-encoding genes were cloned from Aspergillus oryzae, and their gene products were displayed on the cell surface of the sake yeast, Saccharomyces cerevisiae GRI-117-UK. GRI-117-UK/pUDB7 displaying beta-glucosidase AO090009000356 showed the highest activity against various substrates and efficiently produced ethanol from cellobiose. On the other hand, GRI-117-UK/pUDCB displaying endoglucanase AO090010000314 efficiently degraded barley beta-glucan to glucose and smaller cellooligosaccharides. GRI-117-UK/pUDB7CB codisplaying both beta-glucosidase AO090009000356 and endoglucanase AO090010000314 was constructed. When direct ethanol fermentation from 20 g/l barley beta-glucan as a model substrate was performed with the codisplaying strain, the ethanol concentration reached 7.94 g/l after 24 h of fermentation. The conversion ratio of ethanol from beta-glucan was 69.6% of the theoretical ethanol concentration produced from 20 g/l barley beta-glucan. These results showed that sake yeast displaying A. oryzae cellulolytic enzymes can be used to produce ethanol from cellulosic materials. Our constructs have higher ethanol production potential than the laboratory constructs previously reported.     

Molecular Depolymerization of Moist Sweet Potato Starch Melt by Shearing Force under an Elevated Temperature

Sweet potato starch (SPS) with 20 or 25% moisture was heated and then fused at 150°C for 15 min under pressurization at 30–500 Kg/cm². At the same time, the heated SPS melt was extruded through a capillary tube under specified pressure in order to give shearing force. Thus, intact SPS, heated SPS and heated sheared SPS specimen were prepared and examined. The heated and heated sheared SPS did not contain any pyrolysis products because their spectrum profiles were the same as that of intact SPS. By heating and shearing, the intact SPS molecule was depolymerized into small molecules including a few amount of glucose. This finding was revealed by means of molecular weight estimation by viscometry and by gel filtration. The shearing toward SPS melt markedly stimulated the depolymerization while the effect of only heating on depolymerization of SPS was modulate in comparison with heated shearing. Water solubilities of intact SPS, heated SPS and heated sheared SPS with 20% moisture were 0.07, 3.5 and 51.5% respectively, and also the degrees of gelatinization of 1.8% for intact starch increased markedly to 29% for heated SPS and 80–95% (depending on the pressure at extrusion) for heated sheared SPS.     

Therapeutic Targets and Emerging Treatments in Advanced Chondrosarcoma

Simple SummaryChondrosarcomas develop chemoresistance to standard anticancer drugs, making it difficult to control unresectable or metastatic chondrosarcomas. To improve the clinical outcomes of chondrosarcoma, new treatment approaches, such as molecule-targeting agents and immunotherapy, are needed. Recent research has revealed promising biomarkers and therapeutic targets for chondrosarcoma. In addition, several molecule-targeting agents have shown favorable antitumor activities in several clinical studies in patients with advanced sarcomas, including chondrosarcoma. This review summarizes recent basic studies on biomarkers and therapeutic targets and recent clinical studies on treating chondrosarcomas.AbstractDue to resistance to standard anticancer agents, it is difficult to control the disease progression in patients with metastatic or unresectable chondrosarcoma. Novel therapeutic approaches, such as molecule-targeting drugs and immunotherapy, are required to improve clinical outcomes in patients with advanced chondrosarcoma. Recent studies have suggested several promising biomarkers and therapeutic targets for chondrosarcoma, including IDH1/2 and COL2A1. Several molecule-targeting agents and immunotherapies have shown favorable antitumor activity in clinical studies in patients with advanced chondrosarcomas. This review summarizes recent basic studies on biomarkers and molecular targets and recent clinical studies on the treatment of chondrosarcomas.     

Mechano-electrochemistry of a passive surface using an in situ micro-indentation test

Depassivation–repassivation of iron surfaces in boric–borate solutions were investigated by using the micro-indentation test. A pair of current peaks due to repair of the passive film following rupture of the film were observed during a series of indenter drives, i.e., loading and unloading of the indenter. The shape of the current peak depended on environmental conditions (conductivity and pH of the solution) and substrate conditions (mechanical processing history, alloyed element) as well as indentation conditions (repetition, maximum depth, and maximum load). Plastic deformation of the surface was accompanied by surface depassivation, while no depassivation occurred during the elastic deformation, indicating that the passive film on iron has a ductile property. The solution conditions did not affect the scale of depassivation but affected the rate of repassivation. Dislocations in the substrate made surface depassivation difficult but enhanced reactivity during the repassivation. The test also revealed that type-312L stainless steel has high corrosion resistance in a concentrated NaCl solution.     

Hydrogen concentration on mirror surfaces of neutron bottles

Concentrations and depth distributions of the surface hydrogen and the surface deuterium in vacuum evaporated nickel layers and beryllium plates have been investigated by the ERDA method. It is shown that if the evaporation vacuum pressure is kept better than 10⁻⁶ Torr, the hydrogen adsorption from the residual gas during the evaporation is negligible and the hydrogen from residual or atmospheric gases after the evaporation is the main source of the surface hydrogen. Commercial beryllium plates are found to have surface hydrogen of about 10¹⁶ atoms cm². Deuterium treated samples, either in D₂ gas or heavy water, do not show more than possibly a partial replacement of hydrogen. The influence of substrates is small if the thickness of the evaporated nickel layer is more than 2000 Å. The measurements are in good agreement with previously published results.     

Quantitative Visualization of the Interaction between Complement Component C1 and Immunoglobulin G: The Effect of CH1 Domain Deletion

Immunoglobulin G (IgG) adopts a modular multidomain structure that mediates antigen recognition and effector functions, such as complement-dependent cytotoxicity. IgG molecules are self-assembled into a hexameric ring on antigen-containing membranes, recruiting the complement component C1q. In order to provide deeper insights into the initial step of the complement pathway, we report a high-speed atomic force microscopy study for the quantitative visualization of the interaction between mouse IgG and the C1 complex composed of C1q, C1r, and C1s. The results showed that the C1q in the C1 complex is restricted regarding internal motion, and that it has a stronger binding affinity for on-membrane IgG2b assemblages than C1q alone, presumably because of the lower conformational entropy loss upon binding. Furthermore, we visualized a 1:1 stoichiometric interaction between C1/C1q and an IgG2a variant that lacks the entire C_H1 domain in the absence of an antigen. In addition to the canonical C1q-binding site on Fc, their interactions are mediated through a secondary site on the C_L domain that is cryptic in the presence of the C_H1 domain. Our findings offer clues for novel-modality therapeutic antibodies.