Host-vector systems for the thermotolerant methanol-utilizing bacteriaMethylophilus spp. KISRI-Strains

Fourteen recombinant plasmids were constructed by inserting fragments of pSAS, a naturally occurring plasmid ofMethylophilus spp. KISRI-5, into the multiple cloning sites of pUC19. Six recombinants and three knownEscherichia coli plasmids were used to transform three thermotolerant methylotrophic KISRI strains by use of an optimized protocol of electroporation. Analysis of transformants for plasmid DNA showed that all plasmids were stable in the methylotrophic hosts. These studies offer opportunities to developMethylophilus spp. as host-vector systems.     

Integration of bacteriophage SP24 into the chromosome of group A streptococci.

The group A streptococcal bacteriophage SP24 contains a unique phage att site and integrates into a common chromosomal locus in two unrelated group A streptococcal strains, CS24 and CS112. Southern blot analysis suggested that the terminally redundant phage DNA recombines to form the unit-length genome observed in the prophage state. Phage DNA integration appears to be required for stable lysogen formation and conversion to the M+ state; however, the precise role of the bacteriophage and the relationship of phage integration to increased M protein synthesis are unclear.     

Thromboxane and Prostacyclin Levels in Fetal Rabbit Brain and Placenta After Intrauterine Partial Ischemic Episodes

The appearance of arachidonic acid (AA) oxidation products in fetal rabbit brain and placenta under normal or partial short-term ischemic episodes induced by placental blood vessel restriction was examined. Intracerebral administration of [3H]AA into close-to-term rabbit fetuses gave rise to radioactively labeled prostaglandin (PG) E2, thromboxane B2, and 6-keto-PGF1 alpha metabolites as detected by HPLC analysis. A significant increase of 20-30% of [3H]AA precursor into eicosanoids was detected in brain of fetuses after 2-h restriction. The thromboxane B2 and 6-keto-PGF1 alpha levels were determined by radioimmunoassay technique over a period of 48 h following ischemic episodes. Thromboxane B2 content in affected animals was higher by five- and twofold at 3 h over control fetal brain and placental tissue values, respectively, and remained significantly higher for 24 h. 6-Keto-PGF1 alpha levels reached a peak value that was greater by 2.5- and 1.5-fold at 6 h for the ischemic brain and placental tissue, respectively, compared with control fetuses. PGE2 levels were less affected, attaining a maximum of 1.9- and 1.1-fold in brain and placenta correspondingly. The thromboxane/prostacyclin ratio reached a maximum in the brain after approximately 3 h, while that in the placenta continued to rise even after 20 h. Persisting high levels of thromboxane are indicative of cerebral vasoconstriction and may suggest possible damaging effects.     

The asymmetric effect of lanthanides on Na+-gradient-dependent Ca2+ transport in synaptic plasma membrane vesicles

Lanthanides (La3+, Pr3+ and Tb3+) inhibit Na+-gradient-dependent Ca2+ influx into synaptic plasma membrane vesicles. 50% inhibition is obtained by 7 microM lanthanide concentration. The inhibition of the Na+-gradient-dependent Ca2+ uptake exhibits competitive kinetic behaviour. The apparent Km of the Ca2+ influx is increased from 50 microM in the absence of lanthanides to 118 microM in the presence of La3+, 170 microM in the presence of Pr3+ and 130 microM in the presence of Tb3+. The maximal reaction velocity is not altered (8.35 nmol Ca2+ transported per mg protein per min in the absence of lanthanides and 8.16 nmol/mg per min in the presence of lanthanides). Lanthanides also inhibited Na+-gradient-dependent Ca2+ efflux from synaptic plasma membrane vesicles that were preloaded with Ca2+ in a Na+-gradient-dependent manner. Introduction of La3+ into the interior of the synaptic plasma membrane vesicles by rapid freezing of the vesicles in liquid N2 and slow thawing had no effect on either Na+-gradient-dependent Ca2+ influx or efflux. Synaptic plasma membrane vesicles can be preloaded with Ca2+ also in an ATP-dependent manner. This form of Ca2+ uptake is also inhibited by La3+ though at higher concentrations than the Na+-gradient-dependent Ca2+ uptake. Na+-gradient-dependent efflux from synaptic plasma membrane vesicles preloaded in an ATP-dependent fashion ('inside-out' vesicles) unlike efflux from synaptic plasma membrane vesicles preloaded in a Na+-gradient-dependent manner was not inhibited by La3+. These findings suggest that the inhibition by La3+ is manifested asymmetrically on both sides of the synaptic plasma membrane. Lanthanides are probably not transported via the Na+-Ca2+ exchanger since Tb3+ entry measured by fluorescence of Tb3+-dipicolinic acid complex formation occurred at high Tb3+ concentrations only (1.5 mM or above) and was not Na+-gradient dependent.     

Specific localization and quantification of biotin transport components in yeast by use of a biotin-conjugated,impermeant, electron-dense label

Summary Two approaches are described for the localization and quantification of biotin transport components in yeast cells. One approach is based on tracing the fate of a radioactive affinity label for the biotin transport system, [¹⁴C]-biotinyl-p-nitrophenyl ester (pBNP), through various stages of subcellular fractionations. A complementary method involves the use of a biotinderivatized, impermeant, electron-dense, affinity-cytochemical label (ferritin-biotin conjugates) for subsequent visualization by electron microscopy. Values of approximately 8,000 and 4,000 sites/cell, respectively, were achieved by the two methods. Complicating factors, future perspectives and the relevance of the two methods to the isolation of transport components are discussed.     

Cell lineage analysis of the mammalian female germline

Fundamental aspects of embryonic and post-natal development, including maintenance of the mammalian female germline, are largely unknown. Here we employ a retrospective, phylogenetic-based method for reconstructing cell lineage trees utilizing somatic mutations accumulated in microsatellites, to study female germline dynamics in mice. Reconstructed cell lineage trees can be used to estimate lineage relationships between different cell types, as well as cell depth (number of cell divisions since the zygote). We show that, in the reconstructed mouse cell lineage trees, oocytes form clusters that are separate from hematopoietic and mesenchymal stem cells, both in young and old mice, indicating that these populations belong to distinct lineages. Furthermore, while cumulus cells sampled from different ovarian follicles are distinctly clustered on the reconstructed trees, oocytes from the left and right ovaries are not, suggesting a mixing of their progenitor pools. We also observed an increase in oocyte depth with mouse age, which can be explained either by depth-guided selection of oocytes for ovulation or by post-natal renewal. Overall, our study sheds light on substantial novel aspects of female germline preservation and development.