Diagnostic test systems based on the immunoenzyme method in leprosy

Diagnostic test systems for the detection of IgG and IgM to Mycobacterium leprae in the blood sera of leprosy patients and armadillos experimentally infected with M. leprae have been developed on the basis of the indirect immunoperoxidase assay. The possibility has been shown of prognosing the activity of the leprotic process in leprosy patients and the results of the experimental infection of armadillos by the dynamic increase of antibody reactions with the development of the infection.     

Ganglioside abnormalities associated with failed neural differentiation in a T-locus mutant mouse embryo

The T-locus on mouse chromosome 17 contains a number of mutations that disrupt cellular differentiation and embryonic development. Because of their purported role in neuronal differentiation and brain development, gangliosides were studied in mouse embryos homozygous for two T-locus mutations: T and twl. Mice homozygous for the dominant T mutation die from failed mesodermal differentiation in the notochord, whereas mice homozygous for the recessive twl mutation die from failed neural differentiation in the ventral portion of the neural tube. No major ganglioside abnormalities were found in T/T mutant embryos at Embryonic Day 10 (E-10). In contrast, E-11 twl/twl mutants expressed a marked deficiency of the tetrasialoganglioside GQ1. Since this ganglioside migrates with GQ1b in three different thin-layer solvent systems, it may have the same structure as GQ1b. To gain insight into regional distribution, gangliosides were examined in head regions and body regions of normal (+/+) E-11 embryos. The ganglioside composition of these regions was the same as that of the whole embryo, with GM3 and GD3 comprising about 75% of the total ganglioside distribution. Moreover, N-acetylneuraminic acid was the only sialic acid species detectable in the E-10 and the E-11 embryos. These findings indicate that N-acetylneuraminic acid-containing gangliosides are synthesized actively in E-10 and E-11 mouse embryos and also suggest that the GQ1 deficiency in the twl/twl mutants is closely associated with failed neural differentiation.     

In vitro modulation using a synthetic antioxidant of the stimulus-induced formation of cyclic AMP

The in vitro treatment of membranes isolated from different rat organs with a water-soluble synthetic antioxidant has resulted in the change of basal and stimulus-induced adenylate cyclase activity. It is believed that the antioxidant effect is realized rather at the level of signal transfer from activated receptor to adenylate cyclase than at the level of agonist-receptor interaction.     

Production of tetanolysin preparations and characteristics of their properties

As the result of the study of tetanolysin-producing Clostridium tetani strains, their populations have been found to be markedly heterogeneous with respect to the hemolytic activity of clone cultures. On the basis of normal and dialyzed cultures of selected variants with maximum activity the preparations of tetanolysin have been obtained, and their hemolytic activity and antigenic properties have been studied. Antihemolytic rabbit sera have also been obtained and characterized. Partially purified preparations of tetanolysin with high hemolytic activity have been obtained by the fractionation of C. tetani dialyzed cultures with ammonium sulfate.     

Cloning and expression of the Erwinia chrysanthemi asparaginase gene in Escherichia coli and Erwinia carotovora

A genomic library of Erwinia chrysanthemi DNA was constructed in bacteriophage lambda 1059 and recombinants expressing Er. chrysanthemi asparaginase detected using purified anti-asparaginase IgG. The gene was subcloned on a 4.7 kb EcoRI DNA restriction fragment into pUC9 to generate the recombinant plasmid pASN30. The position and orientation of the asparaginase structural gene was determined by subcloning. The enzyme was produced at high levels in Escherichia coli (5% of soluble protein) and was shown to be exported to the periplasmic space. Purified asparaginase from E. coli cells carrying pASN30 was indistinguishable from the Erwinia enzyme on the basis of specific activity [660-700 units (mg protein)-1], pI value (8.5), and subunit molecular weight (32 X 10(3]. Expression of the cloned gene was subject to glucose repression in E. coli but was not significantly repressed by glycerol. Recombinant plasmids, containing the asparaginase gene, when introduced into Erwinia carotovora, caused increased synthesis of the enzyme (2-4 fold higher than the current production strain).     

Isolation of mutants of the obligate methanotroph Methylomonas albus defective in growth on methane

A strain of Methylomonas albus BG8WM, a type 1 obligate methanotroph, which had been maintained for 2 ycars by serial passage on solid medium containing methanol as a carbon source was found to mutate at a frequency of 10^-5-10^-6 to resistance to dichloromethane (DCM^R), the parental strain BG8 did not give rise to DCM^R colonies. DCM^R strains were no longer capable of growth on methane as a carbon cource and exhibited greatly reduced or undetectable methane mono-oxygenase activity. The mutants fell into three groups on the basis of SDS-PAGE analysis of the polypeptide profiles of the particulate fraction of cell extracts. One or more of four polypeptides of Mr 70,000, 50,000, 25,000 and 23,000 were implicated as being components of the methane mono-oxygenase. Spontaneous reversion to growth on methane and sensitivity to dichloromethane occurred at a frequency of about 10^-8. The loss of methane mono-oxygenase activity was not associated with loss of the resident 55 kb plasmid.Abbreviations DCM^R dichloromethane-resistant - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - NMS nitrate minimal salts medium     

The ossification centers in the lower end of tibiotarsus in domestic fowl.

The morphogenesis of lower end of tibia in chick is studied commonly but the process of ossification of the same has received very little attention so far. The present study is directed to throw some light on the appearance of ossification centers in the lower end of tibiotarsus of chick. The histology of lower end of tibiotarsus was studied by procuring developing tibiotarsi from chick embryos (20) of 6th day incubation till hatching and 3 post hatched chicks. The transparancies of chick embryos at different incubation periods and post hatched chicks were prepared by Dawson's Alizarin staining method. Three cartilage center (tibial, fibulare and intermedium) appeared in 6...9 days of incubation period in the tarsal region. These gradually fused with the lower end of tibia. Three ossification centres developed in the lower end of tibiotarsus. One for intermedium appeared on 16th day and two fotibial and fibulare on 20th day. All these three centres could be located in the transparancies of the chick embryos in tarsal region. The present study proves that the three cartilages centres maintain their individuality during the ossification process even though those fuse completely with the lower end of tibia in chick. The centers for tibial and fibulare are similar to epiphyseal centres of mammals in histological details.     

Quantifying Biomass Changes of Single CD8+ T Cells during Antigen Specific Cytotoxicity

Existing approaches that quantify cytotoxic T cell responses rely on bulk or surrogate measurements which impede the direct identification of single activated T cells of interest. Single cell microscopy or flow cytometry methodologies typically rely on fluorescent labeling, which limits applicability to primary cells such as human derived T lymphocytes. Here, we introduce a quantitative method to track single T lymphocyte mediated cytotoxic events within a mixed population of cells using live cell interferometry (LCI), a label-free microscopy technique that maintains cell viability. LCI quantifies the mass distribution within individual cells by measuring the phase shift caused by the interaction of light with intracellular biomass. Using LCI, we imaged cytotoxic T cells killing cognate target cells. In addition to a characteristic target cell mass decrease of 20–60% over 1–4 h following attack by a T cell, there was a significant 4-fold increase in T cell mass accumulation rate at the start of the cytotoxic event and a 2–3 fold increase in T cell mass relative to the mass of unresponsive T cells. Direct, label-free measurement of CD8+ T and target cell mass changes provides a kinetic, quantitative assessment of T cell activation and a relatively rapid approach to identify specific, activated patient-derived T cells for applications in cancer immunotherapy.