Occurrence of Horizontal Gene Transfer of PIB-type ATPase Genes among Bacteria Isolated from the Uranium Rich Deposit of Domiasiat in North East India

Uranium (U) tolerant aerobic heterotrophs were isolated from the subsurface soils of one of the pre-mined U-rich deposits at Domiasiat located in the north-eastern part of India. On screening of genomic DNA from 62 isolates exhibiting superior U and heavy metal tolerance, 32 isolates were found to be positive for P_IB-type ATPase genes. Phylogenetic incongruence and anomalous DNA base compositions revealed the acquisition of P_IB-type ATPase genes by six isolates through horizontal gene transfer (HGT). Three of these instances of HGT appeared to have occurred at inter-phylum level and the other three instances indicated to have taken place at intra-phylum level. This study provides an insight into one of the possible survival strategies that bacteria might employ to adapt to environments rich in uranium and heavy metals.     

Spinal ascending projections to the nucleus tractus spinalis nervi trigemini of the dog

Seven dogs were subjected 30 min to ligation of the thoracic aorta and were then kept alive 6-7 days after the ligature had been removed. Their spinal cord and brain stem were treated by the Nauta-Gygax method and the extent and appearance of preterminal and terminal degeneration of certain ascending spinal systems were analysed. In the medulla oblongata region, marked degenerating fibres from the lower thoracic and lumbosacral cord segments were found in the nucleus tractus spinalis nervi trigemini. Preterminal and terminal degenerating fibres were visualized in the caudal part of the trigeminal nuclear complex. Comparison with the literature showed these to be previously unknown projections with a relationship to the nucleus tractus spinalis nervi trigemini.     

Visualization of multiple protein bands on the same nitrocellulose membrane by double immunoblotting

A method has been developed which allows the simultaneous immunodetection of more than one type of protein on the same nitrocellulose membrane. This procedure does not require special labeling of samples or elution of antibodies from the membrane as do the alternatives cited in the literature (1,2). Proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to the membrane before specific immunostaining with either peroxidase/4-chloro-1-naphthol or immunogold/silver staining. Antigen identity is visually determined by the formation of different-colored precipitates on the membrane. This innovation in protein blotting offers a savings in time and reagents as well as permitting identification of closely spaced bands with certainty.     

Amino acid sequence of rabbit ventricular myosin light chain-2: Identity with the slow skeletal muscle isoform

Many studies have established a correlation of differences in the activities of various muscle types with differences in the expression of myosin isoforms. In this paper we report the sequence determination of myosin light chain-2 from rabbit slow skeletal (LC2s) and ventricular (LC2v) nmscles. We sequenced tryptic peptides from LC2v which account for all except a few terminal amino acid residues. The major part (87 residues) of the rabbit LC2s sequence, obtained from tryptic and cyanogen bromide (CNBr) peptides, was found to be identical to rabbit LC2v. Our results provide the first sequence information on LC2s from any species, and lend strong support to the hypothesis that LC2s and LC2v are identical. Comparisons of rabbit LC2v and LC2s with rabbit LC2f (from fast skeletal muscle), and also with chicken LC2f and LC2v, show clearly that LC2s and LC2v from mammalian and avian species are more closely related to each other than they are to LC2f isoforms from the same species.     

Permeability Increase Induced by Escherichia coli Hemolysin A in Human Macrophages is Due to the Formation of Ionic Pores: A Patch Clamp Characterization

Escherichia coli hemolysin is known to cause hemolysis of red blood cells by forming hydrophilic pores in their cell membrane. Hemolysin-induced pores have been directly visualized in model systems such as planar lipid membranes and unilamellar vesicles. However this hemolysin, like all the members of a related family of toxins called Repeat Toxins, is a potent leukotoxin. To investigate whether the formation of channels is involved also in its leukotoxic activity, we used patch-clamped human macrophages as targets. Indeed, when exposed to the hemolysin, these cells developed additional pores into their membrane. Such exogenous pores had properties very different from the endogenous channels already present in the cell membrane (primarily K⁺ channels), but very similar to the pores formed by the toxin in purely lipidic model membranes. Observed properties were: large single channel conductance, cation over anion selectivity but weak discrimination among different cations, quasilinear current-voltage characteristic and the existence of a flickering pre-open state of small conductance. The selectivity properties of the toxin channels appearing in phospholipid vesicles were also investigated, using a specially adapted polarization/depolarization assay, and were found to be completely consistent with that of the current fluctuations observed in excised macrophage patches. Received: 14 August 1995/Revised: 2 October 1995     

Effect of the cumulus on in vitro fertilization of bovine matured oocytes

The effect of the cumulus on in vitro fertilization in bovines was examined. Follicular oocytes were cultured in medium 199 plus OCS and extra granulosa cells. Frozen-thawed bovine spermatozoa was separated by the swim-up technique, suspended in Talp medium and capacitated with heparin. Fresh sheep and goat semen was incubated for 4 h at room temperature, washed and spermatozoa were then suspended in Talp medium and capacitated by incubation at 38.5 °C and 5% CO₂ in air and heparin.

In experiment 1, cumulus-enclosed oocytes, denuded oocytes and denuded oocytes plus additional cumulus cells were incubated with a reduced concentration of bovine spermatozoa for 8 or 18 h. In Experiment 2, cumulus enclosed and denuded oocytes were incubated with bovine spermatozoa for 4, 6, 8 and 18 h using a sperm concentration adjusted to secure high fertilization rates. In Experiment 3, cumulus-enclosed and denuded bovine oocytes were incubated with either sheep or goat spermatozoa for 18 h. Fertilization rates were then calculated and compared statistically. The results showed that 1) the cumulus improved the fertilization rate only when cumulus cells were associated with the oocytes 2) the timing of sperm penetration was not modified by the cumulus and started at 4 h after sperm incubation and 3) the presence of the cumulus improved the heterologous fertilization rate only when sheep spermatozoa were used. The results suggest that the cumulus improves fertilization rate by providing a capacitation-inducing mechanism and by facilitating the interaction between capacitated spermatozoa and the zona pellucida surface.     

L cells expressing DQ molecules of the DR3 and DR4 haplotypes: reactivity patterns with mAbs

cDNAs coding for the HLA class II DR and DQ and chains of the diabetogenic haplotypes DR3 and DR4 were introduced into a mammalian expression vector and transfected into L-cell mouse fibroblasts to produce cells expressing individual human class II molecules. Stable L transfectants were generated expressing each of the DR or DQ isotypes of the cis-encoded and chains of the DR3 or DR4 haplotypes, as well as the trans-encoded and chains of the DQ molecules of the two haplotypes. However, isotype mismatched combinations (DR/DQ or DQ/DR) did not result in any stable transfectants. The stable DQ L-cell transfectants obtained, along with homozygous B-cell lines expressing the DQ2 and DQ8 specificities, were tested against a large panel of twentyone anti-HLA class II monoclonal antibodies (mAbs). Their unusual reactivity patterns are described including the failure of most pan-DQ mAbs to react with all DQ expressing L-cell transfectants. Interestingly, some mAbs react with certain heterodimers expressed on B-LCL but fail to recognize the same heterodimers expressed on the transfectants. This is suggestive of minor structural modifications that class II molecules undergo depending on the cells they are expressed on.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number U07848. The name DQB1 ^* 0202 was officially assigned by the WHO Nomenclature Committee in April 1994. This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report (Bodmer et al. 1992), names will be assigned to new sequences as they are identified. Lists of such new names will be published in the following WHO Nomenclature Report     

RAM, a gene of yeast required for a functional modification of RAS proteins and for production of mating pheromone a-factor

We have identified a gene (SUPH) of S. cerevisiae that is required for both RAS function and mating by cells of a mating type. supH is allelic to ste16, a gene required for the production of the mating pheromone a-factor. Both RAS and a-factor coding sequences terminate with the potential acyltransferase recognition sequence Cys-A-A-X, where A is an aliphatic amino acid. Mutations in SUPH-STE16 prevent the membrane localization and maturation of RAS protein, as well as the fatty acid acylation of it and other membrane proteins. We propose the designation RAM (RAS protein and a-factor maturation function) for SUPH and STE16. RAM may encode an enzyme responsible for the modification and membrane localization of proteins with this C-terminal sequence.