A novel method for the evaluation of proximal tubule epithelial cellular necrosis in the intact rat kidney using ethidium homodimer

Background  

Ethidium homodimer is a cell-membrane impermeant nuclear fluorochrome that has been widely used to identify necrotic cells in culture. Here, we describe a novel technique for evaluating necrosis of epithelial cells in the proximal tubule that involves perfusing ethidium homodimer through the intact rat kidney. As a positive control for inducing necrosis, rats were treated with 3.5, 1.75, 0.87 and 0.43 mg/kg mercuric chloride (Hg²⁺, intraperitoneal), treatments which have previously been shown to rapidly cause dose-dependent necrosis of the proximal tubule. Twenty-four h after the administration of Hg²⁺, ethidium homodimer (5 μM) was perfused through the intact left kidney while the animal was anesthetized. The kidney was then removed, placed in embedding medium, frozen and cryosectioned at a thickness of 5 μm. Sections were permeabilized with -20°C methanol and then stained with 4',6-diamidino-2-phenylindole (DAPI) to label total nuclei. Total cell number was determined from the DAPI staining in random microscopic fields and the number of necrotic cells in the same field was determined by ethidium homodimer labeling.     

Development of sporeless/low sporing strains of Pleurotus through mutation

Summary The sporophores of Pleurotus are gymnocarpous and continuously release spores in the atmosphere causing respiratory allergies like hay fever and farmer’s lung disease among workers. The allergy is caused by the antigens present on the walls of the spores. Apart from this, during commercial production, these spores settle on the fruit bodies, germinate and form a velvety film which gives an unpleasant appearance to the mushrooms. The spores emitted may include new genotypes likely to attack wood or trees. Spore allergy is one of the most important limiting factors for the large scale cultivation of this species. Different approaches are being adopted at IIHR for the production of commercial sporeless/low-sporing strains of Pleurotus to alleviate the spore allergy problem. Attempts were made during the present investigation to produce sporeless or low-sporing mutants through u.v. mutation. Mutation of the mycelium did not yield the desired results. Mutation of the spores of Pleurotus sajor-caju yielded an extremely low-sporing mutant after 75 min exposure. The character has been found to be stable for more than 10 generations of subculturing.     

An influenza A virus-specific and HLA-DRw8-restricted T cell clone cross-reacting with a transcomplementation product of the HLA-DR2 and DR4 haplotypes

The clone TA10 is a T3+ T4+ T8- proliferative and cytolytic human T cell clone. This clone has been shown to be specific for the hemagglutinin of influenza A Texas virus and restricted by an HLA class II molecule associated with the DRw8-Dw8.1 phenotype. Here we show that TA10 and all of its subclones can also react with eight HLA-DRw8 negative, Epstein-Barr virus (EBV)-transformed cell lines or phytohemagglutinin blasts in the absence of influenza antigens. All of these cell lines are HLA-DR2/DR4 with a classic DR2 long haplotype. The only nonreactive HLA-DR2/DR4 cell line observed bears a DR2 short haplotype. Only heterozygous HLA-DR2/DR4 but not parental DR2 or DR4 EBV-transformed cell lines can be recognized by TA10, indicating that the cross-reacting determinant is a transcomplementation product between HLA-DR2 and HLA-DR4 haplotypes. DR-specific, but not DQ- or DP-specific monoclonal antibodies, inhibit in the proliferation assay and in the chromium release test both the DRw8-Dw8.1-restricted and the anti-DR2/DR4 reactions. These results show that HLA-DR-restricted, anti-viral human T cell clone can evidence cross-reactivity for allospecific class II molecules of the major histocompatibility complex, and human CTL can recognize transcomplementation products of class II HLA genes. In addition, the results suggest that a beta-chain coded for by an HLA-DR gene and associated with an alpha-chain coded for by a still unidentified but possibly HLA-DQ gene constitute this functional transcomplementation product.     

Dynamics of macrozoobenthos productivity characteristics on shelves of the Far Eastern seas in the 2000s

The trophic structure of benthic communities in the Sea of Okhotsk and Bering Sea was analyzed based on the results of macrobenthic survey catches conducted in the Far Eastern seas in the 2000s. The production of trophic levels and entire communities was determined. It was shown that production of benthic communities is still at a high level and has almost not changed since the 1980s. The arrangement of the communities of some shelf areas by productivity characteristics was made.     

Use of EDTA to prevent spontaneous cell-to-cell transformation provides a more accurate estimation of gene transfer frequencies

Summary Spontaneous cell-to-cell transformation between naturally competent bacteria on selective media resulted in an overestimation of the transferability of genetic information. EDTA effectively prevented transformation on selective media whereas DNaseI did not reliably inhibit cell-to-cell transformation. An improved method to estimate gene transfer frequencies is described.