Secretory expression of a glutamic-acid-specific endopeptidase (SPase) from Staphylococcus aureus ATCC12600 in Bacillus subtilis

Summary In order to obtain a large quantity of glutamic-acid-specific endopeptidase of Staphylococcus aureus ATCC12600 (SPase) without cultivating its pathogenic host bacterium, expression plasmids enabling secretion of SPase from Bacillus subtilis were constructed by inserting the SPase gene into B. subtilis-Escherichia coli shuttle vectors. B. subtilis harbouring a simple recombinant plasmid containing the coding and the 5-flanking regions of SPase in the shuttle vector pHY300PLK secreted 22 mg/l of SPase into the medium. As this level was lower than that of the natural strain (45 mg/l), we tried to increase the expression level by constructing a series of hybrid plasmids with the following features: (1) the terminator sequence of the alkaline protease gene from B. subtilis, (2) the promoter and the leader sequences of the -amylase gene or of alkaline protease gene from B. amyloliquefaciens, (3) the vector pHY300PLK and the fused vector of pHY300PLK and pUB110. By using a variety of hybrid plasmids, the resulting transformants secreted SPase at levels of 33–120 mg/l. The recombinant SPase isolated from the medium was indistinguishable from the natural one with respect to its behaviour on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting as well as its enzyme activity.Correspondence to: S. Kakudo     

Acceleration of the Decay of Membrane Potential after Energization of Chloroplasts in Intact Zea mays Leaves

The relationship between dissipation of the flash-induced membranepotential across the thylakoid membrane and the high energystate was studied in Zea mays leaves. The dark decay of theflash-induced 515-nm absorbance change was accelerated by shortpreillumination of the leaf. No acceleration of the decay bypreillumination was observed when leaves were incubated in argonor CO² gas or treated with DCMU. These effects of preilluminationand incubation were reversible. The delayed fluorescence from chlorophyll a was reversibly decreasedby incubating leaves in argon or CO² gas, though the modes ofdepression were somewhat different from each other. In leavesincubated in argon or CO² gas, the phase of slow decrease ofthe intensity of prompt fluorescence during illumination reversiblydisappeared. The results suggested that the dissipation of membrane potentialgenerated by a flash was accelerated after the energizationof chloroplasts in leaves, probably by increased H^– permeabilityof the thylakoid membrane. O2 was important in maintaining (indarkness) and forming (under illumination) the high energy statein chloroplasts in intact leaves. (Received October 1, 1980; Accepted December 15, 1980)     

Cartilage-derived factor (CDF) : II. Somatomedin-like action on cultured chondrocytes

Previously, we showed that fetal bovine cartilage contains a polypeptide that stimulates the incorporation of [³⁵S]sulfate into proteoglycans synthesized by rat and rabbit costal chondrocytes in culture. In this paper, we report that the cartilage-derived factor (CDF) increases not only [³⁵S]sulfate incorporation but also [³H]thymidine incorporation into rabbit chondrocytes in monolayer culture. The dose-response curve of CDF stimulation of DNA synthesis was similar in profile to that of CDF stimulation of proteoglycan synthesis. In addition, CDF markedly enhanced [³H]uridine incorporation into rabbit chondrocytes and significantly enhanced [³H]serine incorporation into total protein. These findings indicate that fetal bovine cartilage contains a factor that shows somatomedin-like activity in monolayer cultures of rabbit chondrocytes.     

Selective degradation of altered tomato β-fructofuranosidase molecules by neutral protease

Evidence is presented for the selective breakdown of altered tomato β-fructofuranosidase molecules by a neutral protease from Bacillus subtilis.     

Grouping Antigens of Four Lactobacillus Species and Their Characteristics

Antigenic analyses of Lactobacillus bulgaricus, Lactobacillus lactis, Lactobacillus brevis and Lactobacillus buchneri were carried out by double immunodiffusion in agar. Antigens were extracted from whole cells and cell wall preparations with cold trichloroacetic acid. Most strains of the four species possessed antigen 9 in their cell walls. Another antigen, antigen 10, was found in the cell walls of all the strains of L. brevis and L. buchneri, and in some strains of L. lactis, but not in L. bulgaricus. Fractionation of the antigens was attempted using the cell wall extracts of L. lactis L-10 with only antigen 9 and of L. brevis X-1 with both antigens 9 and 10. The partially purified fractions of antigen 9 and of the complex of antigens 9 and 10 were obtained by zone electrophoresis. However, antigen 10 from the complex could not be separated by the same method or gel filtration on Sephadex G-100 since the two antigens 9 and 10 of the complex always behaved together. The fraction of antigen 9 consisted almost entirely of glycerol and glucose as sugar components, the molar ratio being 2:1. The complex of antigens 9 and 10 also consisted of the same sugars, and the molar ratio of glycerol: glucose was 4:1. Inhibition tests indicated that the immunodominant component of antigen 9 was α-methylglucoside (glucose), and most probably the determinant is a glucosylated glycerol teichoic acid. It was considered that the determinant of antigen 10 is a glycerol teichoic acid although glucosamine and galactosamine inhibited effectively the reaction between antigen 10 and its antibody.     

Clinical relevance of oncogenic driver mutations identified in endometrial carcinoma

PurposeEndometrial carcinoma (EC) is a clinically heterogeneous disease characterized by a number of different histological subtypes, and its heterogeneity may be involved in the accumulation of multiple genetic alterations. The aim of this work was to investigate the comprehensive mutational profile of EC tumors, and examine the associations between somatic mutations and clinicopathological features or survival in EC patients.MethodsA total of 100 surgical tumors were obtained from EC patients who had previously undergone surgery. Genomic DNA samples extracted from fresh-frozen tissues were analyzed using the Ion AmpliSeq Cancer Hotspot Panel v2 Kit, covering 50 tumor-related genes.ResultsValidated mutations were detected in 91 of the 100 tumors (91%) and identified in eight of the most frequently mutated genes, namely PTEN (57%), PIK3CA (51%), TP53 (30%), KRAS (23%), CTNNB1 (21%), FBFR2 (13%), FBXW7(10%) and RB1 (9%). PTEN mutations were found to associated with young age (< 60), early-stage, endometrioid histology, non-recurrence and better overall survival (OS). CTNNB1 mutations were associated with young age, endometrioid histology and better OS. On the other hands, TP53 mutations were associated with late-stage, non-endometrioid histology, high-grade, recurrence and worse OS. FBWX7 mutations were associated with late-stage, vascular invasion and lymph node metastasis. FGFR2 mutations correlated with deep (≥ 1/2) myometrial invasion.ConclusionOur comprehensive mutational profile will be useful for understanding and evaluating the molecular characteristics of EC tumors, and may lead to the establishment of novel treatment strategies that improve the survival of patients with EC in the future.     

Influences of the priming procedure and saline circulation conditions on polyvinylpyrrolidone in vitro elution from polysulfone membrane dialyzers

In hemodialysis (HD), the patient's blood is purified via circulation in an extracorporeal circuit containing a dialyzer. In the manufacturing process of polysulfone (PSu) membrane dialyzers, the membranes are hydrophilized via the addition of the hydrophilic agent polyvinylpyrrolidone (PVP) to increase their hydraulic permeability. The elution of PVP from the membrane reduces the membrane's hydraulic permeability, and the eluted PVP could cause adverse effects in the human body. Therefore, it is important to identify the factors that induce PVP elution from PSu dialyzer membranes to improve the efficiency and safety of HD. In the present study, experimental circuits connecting each of the three types of PSu membrane dialyzers that had been sterilized, using gamma irradiation, autoclaving, or in-line steam methods, were prepared. After the dialyzers were primed, saline was circulated in the circuits at a flow rate of 100 mL/min or 200 mL/min. At 0, 2, 4, 6, and 8 h after circulation was initiated, the amount of PVP eluted from the PSu membranes in vitro was determined. In this experimental setting, longer the circulation duration, greater the amount of PVP eluted from the PSu membranes of the tested dialyzers; however, the flow rate did not influence the in vitro elution of PVP. Furthermore, the immersion of the dialyzer membranes in saline for 24 h strongly facilitated the in vitro elution of PVP. In sum, these results suggest that the duration of PSu membrane incubation in saline is a determinant of the level of PVP elution from the PSu membrane dialyzers.     

Copper-dependent DNA damage induced by hydrazobenzene,an azobenzene metabolite

Hydrazobenzene is carcinogenic to rats and mice and azobenzene is carcinogenic to rats. Hydrazobenzene is a metabolic intermediate of azobenzene. To clarify the mechanism of carcinogenesis by azobenzene and hydrazobenzene, we investigated DNA damage induced by hydrazobenzene, using ³²P-5′-end-labeled DNA fragments obtained from the c-Ha-ras-1 proto-oncogene and the p53 tumor suppressor gene. Hydrazobenzene caused DNA damage in the presence of Cu(II). Piperidine treatment enhanced the DNA damage greatly, suggesting that hydrazobenzene caused base modification and liberation. However, azobenzene did not cause DNA damage even in the presence of Cu(II). Hydrazobenzene plus Cu(II) caused DNA damage frequently at thymine residues. Catalase and a Cu(I)-specific chelator inhibited Cu(II)-mediated DNA damage by hydrazobenzene. Typical ^·OH scavengers did not inhibit the DNA damage. The main active species is probably a metal oxygen complex, such as Cu(I)-OOH. Formation of 8-oxo-7, 8-dihydro-2′-deoxyguanosine was increased by hydrazobenzene in the presence of Cu(II). Oxygen consumption and UV-Visible spectroscopic measurements have shown that hydrazobenzene is autoxidized to azobenzene with H₂O₂ formation. It is considered that the metal-mediated DNA damage by hydrazobenzene through H₂O₂ generation may be relevant for the expression of carcinogenicity of azobenzene and hydrazobenzene.