Extending the Cellulosome Paradigm: the Modular Clostridium thermocellum Cellulosomal Serpin PinA Is a Broad-Spectrum Inhibitor of Subtilisin-Like Proteases

Clostridium thermocellum encodes a cellulosomal, modular, and thermostable serine protease inhibitor (serpin), PinA. PinA stability but not inhibitory activity is affected by the Fn(III) and Doc(I) domains, and PinA is a broad inhibitor of subtilisin-like proteases and may play a key role in protecting the cellulosome from protease attack.     

An efficient chemical approach to bispecific antibodies and antibodies of high valency

Irreversible chemical programming of monoclonal aldolase antibody (mAb) 38C2 has been accomplished with β-lactam equipped mono- and bifunctional targeting modules, including a cyclic-RGD peptide linked to either the peptide (d-Lys⁶)-LHRH or another cyclic RGD unit and a small-molecule integrin inhibitor SCS-873 conjugated to (d-Lys⁶)LHRH. We also prepared monofunctional targeting modules containing either cyclic RGD or (d-Lys⁶)-LHRH peptides. Binding of the chemically programmed antibodies to integrin receptors α(v)β(3) and α(v)β(5) and to the luteinizing hormone releasing hormone receptor were evaluated. The bifunctional and bivalent c-RGD/LHRH and SCS-783/LHRH, the monofunctional and tetravalent c-RGD/c-RGD, and the monofunctional bivalent c-RGD chemically programmed antibodies bound specifically to the isolated integrin receptor proteins as well as to integrins expressed on human melanoma M-21 cells. c-RGD/LHRH, SCS-783/LHRH, and LHRH chemically programmed antibodies bound specifically to the LHRH receptors expressed on human ovarian cancer cells. This approach provides an efficient, versatile, and economically viable route to high-valency therapeutic antibodies that target defined combinations of specific receptors. Additionally, this approach should be applicable to chemically programmed vaccines.     

Search for chromosomal variations among gas-exposed persons in Bhopal

Summary A chromosomal survey using standard lymphocyte cultures employing different media and G-banding techniques was initiated in 1984. This study became particularly important following the tragic gaseous exposure of the population in Bhopal at midnight on 2 December 1984. We have been able to formulate a chromosomal profile for each person whom we have studied; during 1986–1988, 154 persons were examined twice. Among seemingly normal individuals, as many as 20% might possess some chromosomal abnormality; of these, 50% may develop, at a later date, some kind of pathological complication (such as tumours, recurrent abortion or transmission of defects to their offspring). The people exposed to methyl isocyanate have repeatedly shown Robertsonian translocations, mostly in acrocentric chromosomes 13 and 21. Other types of translocations have been studied among all exposed (53) and normal (101) persons; the involvement of chromosomes 5, 9, 11, 14 and 16 is statistically significant (P= <0.001). One of the major clinical symptoms is dyspnoea; we have estimated that almost all seriously dyspnoeic patients have developed at least two categories of chromosomal aberrations, one of which is Robertsonian translocation, in at least 10% metaphases. Our chromosomal survey will be of significance because we are able to identify people with chromosomal aberrations that might be correlated with future pathological consequences of the accident. The chromosomal load that can be sustained with an apparently normal phenotype can also be measured.     

Effect of hypophysectomy and replacement therapy on fatty acid metabolism in the rat testis

1. The influence of pituitary gonadotrophins and of testosterone on the conversion of linoleic acid into other polyunsaturated fatty acids by rat testicular tissue was studied. 2. In immature hypophysectomized rats, follicle-stimulating hormone caused a threefold increase in the incorporation of radioactivity from [1-(14)C]linoleic acid into testicular lipids; the distribution of (14)C in the polyunsaturated fatty acids, however, was not significantly affected. 3. In mature hypophysectomized rats, the hormonal treatments had less pronounced effects on (14)C incorporation into testicular lipids, but caused a significant increase in the percentage of (14)C incorporated into polyunsaturated fatty acids of the omega-6 series, luteinizing hormone and testosterone having the more pronounced influences. 4. A time-course study of the appearance of radioactivity in the ejaculated spermatozoa of rabbits, after they had been given a tracer dose of [1-(14)C]linoleic acid, indicated that incorporation of radioactivity into spermatozoa occurred during all stages of spermatogenesis.     

Adherence of multiple serovars of Chlamydia trachomatis to a common receptor on HeLa and McCoy cells is mediated by thermolabile protein(s)

Several aspects of the adherence of purified elementary bodies (EB) of Chlamydia trachomatis to HeLa and to McCoy cells were examined using different techniques, including an ELISA. Serovar-specific, biotinylated monoclonal antibodies were used to detect cell-bound chlamydiae. In addition, purified chlamydiae were biotinylated and their adherence properties were studied. The assays were done at 4 degrees C to exclude the energy-dependent internalization of the cell-bound EB and host-cell membrane recycling that occur at 37 degrees C. Saturation kinetics were routinely observed at 4 degrees C, and the rate of adherence remained linear for approximately 60 min. Lineweaver-Burk analysis of the kinetics data showed that adherence of any one serovar was competitively inhibited by other serovars of C. trachomatis. This competition for the same receptor on the two alternative hosts, HeLa and McCoy, was also seen when the adherence assays were done at 37 degrees C in the presence of sodium azide, an energy poison that inhibits endocytosis of cell-bound chlamydiae. Chlamydiae exposed to 56 degrees C for 5 min, or treated with low doses of trypsin, failed to exhibit competitive inhibition, having suffered considerable loss of the ability to adhere to host-cells. These data suggest that heat- and trypsin-labile chlamydial moieties participate in the adherence reaction, and that oculo-genital serovars of C. trachomatis, including that of lymphogranuloma venereum, attach to the same receptor on the host-cell membrane.     

Construction of a cDNA for the human c-fes protooncogene protein-tyrosine kinase and its expression in a baculovirus system.

Previous studies have established that the 93-kDa protein-tyrosine kinase (PTK) encoded by the human c-fes protooncogene plays an active role in the induction of terminal myeloid differentiation. However, this enzyme is expressed at very low levels in myeloid cells, making isolation of sufficient quantities for detailed biochemical analysis difficult. To overcome this problem, we used the polymerase chain reaction to construct a full-length c-fes cDNA from overlapping 5' and 3' partial cDNA sequences. The c-fes cDNA was expressed at high levels in a baculovirus system, and the catalytically active recombinant c-fes gene product p93c-fes was partially purified by DEAE-Sepharose and tyrosine-agarose chromatography. Recombinant p93c-fes was indistinguishable from the native protein in terms of its apparent molecular weight following SDS-PAGE, catalytic activity, Km for poly(Glu,Tyr)4:1, antigenicity, and phosphopeptide pattern generated with Staphylococcus aureus protease.     

Antioxidant and Antidiabetic Properties of Extracts from Three Underutilized Food Plants of North East India

Three underutilized leafy vegetables Sarcochlamys pulcherrima (Roxb.) Gaudich (SP), Ipomoea aquatica Forssk. (IA) and Zanthoxylum rhetsa (Roxb.) DC (ZR) were extracted with different solvents viz. 95 % ethyl alcohol, methanol and hot water. The extracts were evaluated for their antioxidant potential via DPPH, ABTS and FRAP assay along with electroanalytical studies using cyclic voltammetry. The antidiabetic potential was determined by recording their α-amylase and α-glucosidase inhibitory assay. The total phenolic content (TPC), total flavonoid content (TFC) and the liquid chromatography-mass spectrometry (LC/MS) based phytochemical profiles of the extracts were also determined. All three extracts of SP exhibited significant antioxidant capacity. The antidiabetic potential of the IA and ZR extracts was found to be higher than or at par with that of standard acarbose. LC/MS studies reveal the presence of hitherto reported antioxidant and antidiabetic compounds like gamma-aminobutyric acid, cinnamic acid, caffeic acid, α-viniferin, piperlonguminine, niacin, kaempferol, etc., in the extracts.     

A preliminary assessment of possible mutagenicity of betel nut and ingredients of the betel quid when administered alone or in combinations to larvae of Drosophila melanogaster.

The "carcinogenic" betel nut and constituents of betel quid were tested for possible mutagenicity in Drosophila. The test compounds were administered either alone or in combinations by larval feeding. The data on sex-chromosome loss, sex-linked recessive lethals and autosomal translocations suggest lack of mutagenicity.     

Formation of vitamin A in a freshwater fish. Isolation of retinoic acid.

The intestines of freshly caught Saccobranchus fossilis (a freshwater fish that contains dehydroretinol) became free from carotenoids and from vitamin A when the fish were starved for about 20 days. When beta-carotene was administered to such fish, retinoic acid could be isolated from the intestines after approx. 4h. When lutein was administered to such fish, dehydroretinol and 3-hydroxyretinol could be isolated from the intestines after approx. 5h.