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.     

A study of anti-group A streptococcal monoclonal antibodies cross-reactive with myosin

Anti-group A streptococcal monoclonal antibodies were obtained from BALB c/BYJ mice immunized with purified membranes from M type 5 Streptococcus pyogenes. Two of the anti-streptococcal monoclonal antibodies were previously shown to cross-react with muscle myosin. In this study the monoclonal antibodies were reacted with tissue sections of normal human heart and skeletal muscle. Antibody binding was estimated by indirect immunofluorescence and immunoperoxidase techniques. Both of the monoclonal antibodies (36.2.2 and 54.2.8) investigated in this report reacted with heart and/or skeletal muscle sections. When evaluated by immunofluorescence, monoclonal antibody 54.2.8 demarcated the periphery of cardiac striated muscle cells and reacted to a lesser degree with subsarcolemmal components. Monoclonal antibody 36.2.2 failed to react with heart sections, but both of the monoclonal antibodies reacted strongly with skeletal muscle sections. Results similar to those observed with indirect immunofluorescence were obtained with the immunoperoxidase technique. By Western immunoblotting and competitive inhibition assays, monoclonal antibodies 36.2.2 and 54.2.8 both were found to react with the heavy chain of skeletal muscle myosin. However, only 54.2.8 reacted with the heavy chain of cardiac myosin. The specificity of the monoclonal antibodies for subfragments of skeletal muscle myosin indicated that monoclonal antibody 36.2.2 was specific for light meromyosin fragments, whereas 54.2.8 reacted with both heavy and light meromyosin. The data demonstrated that two monoclonal antibodies against streptococci were specific for skeletal muscle and/or cardiac myosin and for subfragments of the myosin molecule. The reactions of the monoclonal antibodies with human tissue sections were consistent with the immunochemical reactions of the monoclonal antibodies with both denatured and native myosin.     

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.     

Patterns of sequence variation in the mitochondrial D-loop region of shrews

Direct sequencing of the mitochondrial displacement loop (D-loop) of shrews (genus Sorex) for the region between the tRNA(Pro) and the conserved sequence block-F revealed variable numbers of 79-bp tandem repeats. These repeats were found in all 19 individuals sequenced, representing three subspecies and one closely related species of the masked shrew group (Sorex cinereus cinereus, S. c. miscix, S. c. acadicus, and S. haydeni) and an outgroup, the pygmy shrew (S. hoyi). Each specimen also possessed an adjacent 76-bp imperfect copy of the tandem repeats. One individual was heteroplasmic for length variants consisting of five and seven copies of the 79-bp tandem repeat. The sequence of the repeats is conducive to the formation of secondary structure. A termination-associated sequence is present in each of the repeats and in a unique sequence region 5' to the tandem array as well. Mean genetic distance between the masked shrew taxa and the pygmy shrew was calculated separately for the unique sequence region, one of the tandem repeats, the imperfect repeat, and these three regions combined. The unique sequence region evolved more rapidly than the tandem repeats or the imperfect repeat. The small genetic distance between pairs of tandem repeats within an individual is consistent with a model of concerted evolution. Repeats are apparently duplicated and lost at a high rate, which tends to homogenize the tandem array. The rate of D- loop sequence divergence between the masked and pygmy shrews is estimated to be 15%-20%/Myr, the highest rate observed in D-loops of mammals. Rapid sequence evolution in shrews may be due either to their high metabolic rate and short generation time or to the presence of variable numbers of tandem repeats.      

大鼠胼胝体内神经肽Y免疫反应阳性纤维的发育

本实验用免疫组织化学ＡＢＣ法研究了大鼠胼胝体内神经肽Ｙ免疫反应阳性（ＮＰＹ－ＩＲ）纤维的生后发育。结果发现,许多ＮＰＹ－ＩＲ纤维在大鼠出生时便存在于胼胝体内。ＮＰＹ－ＩＲ胼胝体纤维的密度在生后１周内继续逐渐增高,在第２周内达到最高峰。之后,ＮＰＹ－ＩＲ胼胝体纤维的密度逐渐下降,至第３周末时接近成年时的水平,即仅有少量ＮＰＹ－ＩＲ纤维存在于胼胝体内。这些结果提示在大鼠早期生后发育过程中许多ＮＰＹ－ＩＲ胼胝体纤维是暂时性的,其作用可能与大脑皮质的机能发育有关。     

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.