Preparation of a gap junction fraction from uteri of pregnant rats: the 28-kD polypeptides of uterus, liver, and heart gap junctions are homologous

A procedure for the preparation of a gap junction fraction from the uteri of pregnant rats is described. The uterine gap junctions, when examined by electron microscopy of thin sections and in negatively stained preparations, were similar to gap junctions isolated from heart and liver. Major proteins of similar apparent molecular weight (Mr 28,000) were found in gap junction fractions isolated from the uterus, heart, and liver, and were shown to have highly homologous structures by two-dimensional mapping of their tryptic peptides. An Mr 10,000 polypeptide, previously deduced to be a proteolytic product of the Mr 28,000 polypeptide of rat liver (Nicholson, B. J., L. J. Takemoto, M. W. Hunkapiller, L. E. Hood, and J.-P. Revel, 1983, Cell, 32:967-978), was also studied and shown by chymotryptic mapping to be homologous in the uterine, heart, and liver gap junction fractions. An antibody raised in rabbits to a synthetic peptide corresponding to an amino-terminal sequence of the liver gap junction protein recognized Mr 28,000 proteins in the three tissues studied, showing that the proteins shared common antigenic determinants. These results indicate that gap junctions are biochemically conserved plasma membrane specializations. The view that gap junctions are tissue-specific plasma membrane organelles based on previous comparisons of Mr 26,000-30,000 polypeptides is not sustained by the present results.     

Defective in vitro T cell colony formation in the acquired immunodeficiency syndrome

Depressed T cell immunity is a universal characteristic of the acquired immunodeficiency syndrome (AIDS). In the present study, 25 patients with AIDS and opportunistic infections, 22 individuals with AIDS-related complex (ARC, or chronic lymphadenopathy syndrome), and 20 healthy homosexuals were evaluated by means of the T cell colony assay. Forty-seven healthy heterosexual controls showed an average of 3964 +/- 319 colonies/7.5 X 10(5) cells, with a range of 880 to 9340. The mean in the 20 healthy homosexuals (3173 +/- 483) did not differ significantly from the controls; in this group, only three patients had values less than 1000 colonies/plate. By contrast, all AIDS patients and 14 ARC patients had colony counts less than 1000. The mean value for the AIDS patients was only 24 +/- 15 (p less than 0.0005 compared with either controls or healthy homosexuals); values in the ARC group were intermediate (1180 +/- 360). The addition of interleukin 2 to the plates promoted correction of the proliferative abnormality in ARC patients. This interleukin increased colony scores in the AIDS group, but the mean value was still significantly less than controls. Comparison indicated that the colony assay is a more sensitive indicator for detecting proliferative abnormalities than responses to PHA, Con A, or pokeweed mitogen in suspension cultures.     

Construction of novel class I histocompatibility antigens by interspecies exon shuffling

Human and mouse class I histocompatibility antigens share considerable structural homology at both the protein and DNA sequence level. This homology has allowed the production of hybrid class I molecules by the reciprocal exchange of DNA sequences corresponding to equivalent domains of HLA-B7 and either H-2Ld or H-2Dd. It is shown that these genes give rise to protein products that are stably expressed on the surface of murine L cells after DNA-mediated gene transfer. These proteins express only those monoclonal antibody-defined H-2 determinants that are expected based on their genetic construction. The molecules have allowed the localization of a number of polymorphic and monomorphic HLA-specific epitopes. In all but one case, expression of an epitope on a domain does not appear to be influenced by the replacement of adjacent human domains with their murine equivalents, suggesting a considerable degree of structural independence of the domains. Cells expressing the hybrid molecules have also been tested as targets for a panel of HLA-B7-specific cytotoxic T cell clones. The results show that the polymorphic determinants recognized by these clones map to the alpha 1 and alpha 2 domains of the HLA-B7 molecule. No evidence for an influence of species-related amino acid sequence differences in the third extracellular domain on T cell recognition was seen. The results are discussed in light of the proposed domain structure of the class I proteins and the potential use of such molecules for further functional studies.     

Identification of the host-range DNA which allows Rhizobium leguminosarum strain TOM to nodulate cv. Afghanistan peas

Summary The special ability of Rhizobium leguminosarum strain TOM to nodulate cv. Afghanistan peas had previously been shown to be determined by the symbiotic plasmid, pRL5JI, of this strain. A region of pRL5JI, 2.0 kb in size, was found to confer the ability to nodulate cv. Afghanistan peas when transferred to strains of R. leguminosarum which normally fail to nodulate this host. This region of pRL5JI, responsible for the extension of host-range, was closely linked to, but did not include, the genes required for root hair curling. Although extensive homology has been found between the R. leguminosarum nod genes on pRL5JI and those on the normal symbiotic plasmid pRL1JI, a fragment from the 2.0 kb region involved in nodulation of cv. Afghanistan has been identified, which was not homologous to DNA in strains which do not nodulate cv. Afghanistan.     

Chromosomal sex and distribution of functional germ cells in a series of chimeric mice

An unselected series of chimeric mice were test mated to determine the parental lineage of their functional gametes. The cytological sex of each animal was established and confirmed in all cases by karyological analysis of peripheral blood lymphocytes. The parental cell lineage for each cytological sex was unequivocally established by the presence or absence of the radiation-induced translocation 15(14) (T6). Eleven animals analysed, 10 of these were chimeric. Among the 10 chimeras, 3 were phenotypically female and 7 were phenotypically male. The cytological sex ratio (XY/XY:XX/XY:XX/XX) was 1:6:2. There were 646 offspring from test matings of these chimeras. The coat color analysis of these offspring demonstrated a concordance of cytological sex of the lineage resulting in functional gametes with the phenotypic sex of the animal.     

Optical difference spectrum of the electron acceptor Ao in photosystem I

Optical measurements were made during low temperature photoreduction of photosystem I acceptors, A₁ and A₀. In the presence of a significant amount ofA₁ (detected by EPR), no absorbance changes occurred between 750-350 nm, indicating that this species is not a chlorophyll or pheophytin molecule. Spectral changes in this region that may be correlated with the appearance of A⁻₀, suggest that this component is a chlorophyll a anion monomer. The species is present in reaction centres in a ratio of 0.94 A_o/P700.

Photosystem I Primary acceptor Optical difference spectrum Chlorophyll a monomer     

Experimental observations on the transmission of Schistosoma margrebowiei miracidia

Evans N. A. 1985. Experimental observations on the transmission of Schistosoma margrebowiei miracidia. International Journal for Parasitology15: 361–364. The survival and infectivity characteristics of Schistosoma margrebowiei miracidia at a temperature of 26°C are described. The maximum survival time and the time to 50% survival were approx. 13 and 9.5 h respectively. Miracidial infectivity toward Bulinus natalensis was relatively constant for the first 4 h of life but it then declined steadily to zero after 11–12 h. Snails exhibited age-dependent differences in their susceptibility to infection, individuals being most susceptible when 4 days old. Increases in snail age beyond 1 week were generally accompanied by an increased level of resistance to infection. Exposure of neonate (< 2 days old) snails produced high levels of mortality and a very low proportion of the survivors were infected at the time of parasitological examination.     

Photoaffinity labeling of a viral induced protein from tobacco. Characterization of nucleotide-binding properties

We have used the photoaffinity analogs 8-azidoadenosine 5'-triphosphate (8-N3ATP) and 8-azidoguanosine 5'-triphosphate (8-N3GTP) to investigate the relationship between a viral induced protein (Mr = 120,000) in tobacco mosaic virus (TMV)-infected tobacco and the TMV-induced RNA-dependent RNA polymerase activity. When the radioactive analogs [gamma-32P]8-N3ATP and [gamma-32P]8-N3GTP were incubated with the tobacco tissue homogenate from TMV-infected plants, incorporation of label occurred into the viral induced protein in the presence of UV light. The incorporation was found to be totally dependent on UV-illumination and greatly enhanced by Mg2+. Saturation of photoincorporated label indicates an apparent Kd of 16 microM (+/- 3 microM) and 12 microM (+/- 3 microM) for 8-N3ATP and 8-N3GTP, respectively. Protection against photolabeling by [gamma-32P]8-N3ATP and [gamma-32P]8-N3GTP with various nonradioactive nucleotides and nucleosides suggests that the photolabeled site is protected best by nucleoside triphosphates. At 200 microM both deoxyribonucleoside triphosphates and ribonucleoside triphosphates were very effective at protecting the site from photolabeling. These data suggest that the photolabeled protein may be part of an RNA-dependent RNA polymerase. The utility of nucleotide photoaffinity analogs as a method to study viral induced nucleotide-binding proteins is discussed.     

Infectious entry of murine retroviruses into mouse cells: evidence of a postadsorption step inhibited by acidic pH.

The entry into cells by many enveloped RNA viruses is accomplished by endocytosis and subsequent penetration of the endosomal membrane by an acidic pH-dependent fusion event. In the current study, we examined early events in the infectious entry of mouse retroviruses, using as a framework the observation that infection of a mouse tail skin cell line by the ecotropic virus Friend murine leukemia virus was inhibited at mildly acidic pH (pH 6). This inhibition operated on a postadsorption step, since binding of virus was unaffected at this pH. The rate of penetration of preadsorbed virus, which displayed first-order kinetics, was markedly affected by changes in the pH of the medium. The half-time for disappearance of infectious cell surface virus at 37 degrees C was approximately 10 min at pH 7.6. At pH 6.0, however, greater than 98% of the adsorbed infectivity remained at the cell surface after 45 min. This cell surface virus, though not infecting the cell at pH 6.0, retained its capacity to enter and infect the cell when the pH of the medium was raised. Acidic pH had little effect on the rate of fluid uptake by the cells, as measured by internalization of [3H]sucrose, indicating that global inhibition of endocytosis had not occurred. In contrast, cell fusion induced by Friend murine leukemia virus was optimal at pH 7.6 but markedly inhibited at a pH of less than 6.4. This inhibitory effect of acidic pH on membrane fusion is unique among the enveloped viruses which have been studied and would preclude entry of Friend murine leukemia virus from within acidified endocytic vesicles. Entry of other members of the ecotropic, mink cell focus-forming, and xenotropic host range groups displayed similar pH sensitivity. However, one xenotropic virus was relatively resistant to the effect of acidic pH, suggesting that differences might exist in the requirements for entry of different retroviruses.