Amino acid sequence of the amphiphilic phosphocarrier protein factor IIILac of the lactose-specific phosphotransferase system of Staphylococcus

The lactose-specific factor III of the phosphotransferase system of Staphylococcus aureus is an amphiphilic trimeric protein composed of identical subunits. It is hydrophilic in its unphosphorylated state and can be isolated from the cytoplasmic protein fraction. It becomes a constituent of the membrane-bound phosphotransferase complex upon phosphorylation of a single histidyl residue. The sequence of S. aureus factor IIILac was determined and revealed that the subunits consist of 103 residues corresponding to a Mr of 11 367 and of 34 101 for the native trimer: (sequence; see text) According to this sequence and previous work histidine residue 82 located in the C-terminal part of the polypeptide chain is phosphorylated at the N-3 position by phosphoenolpyruvate, enzyme I, and histidine-containing phosphocarrier protein. The N-terminal part of the protein comprising approximately one-third of the chain exhibits in vitro affinity toward membrane-bound enzyme IILac.     

Treatment of dialysis membranes for simultaneous dialysis and concentration

Dialysis membranes used for simultaneous dialysis-concentration required pretreatment to remove uv-absorbing compounds leached from the membranes and to reduce the absorption of protein to the membranes. This was accomplished with sodium carbonate and ethanol or with "sulfur-removal solutions." Protein determinations were made with a micro-Bradford protein reaction and with uv absorbance at 280 nm. Soluble membrane components contributed to aberrant uv spectra and altered the ratio of 280/260-nm absorbance. Simultaneous dialysis and concentration in the micro protein dialyzer-concentrator apparatus, combining aspects of thin-layer dialysis and ultrafiltration, resulted in rapid removal of salts from the protein solutions. Prior treatment of membranes reduced uncertainties in retentate recoveries, eliminated uv-absorbing components of membranes, and improved recoveries of protein.     

Growth of epizootic hemorrhagic disease, akabane, and ephemeral fever viruses in Aedes albopictus cells maintained at various temperatures

The growth curves of one epizootic hemorrhagic disease (EHD) virus serotype (Reoviridae), two Akabane virus strains (Bunyaviridae) and three bovine ephemeral fever (BEF) group viruses (Rhabdoviridae) were determined in Aedes albopictus cells maintained at 15, 20, 28 and 33 degrees C. Ae albopictus cells supported the growth of all the viruses although not necessarily at all temperatures. Because none of the viruses exhibited cytopathic effect in Ae albopictus cells, growth was assayed in baby hamster kidney 21 (BHK21) cells maintained at 37 degrees C. The temperature at which the Ae albopictus cells were maintained had a marked effect on the growth and yield for each virus studied. EHD virus was heat-stable and grew after 4 days at 28 and 33 degrees C, and after 8 days at 20 degrees C. No growth was recorded up to 12 days at 15 degrees C. The two Akabane viruses were heat-sensitive and exhibited different growth patterns. One strain (B8935) showed no growth at 15 degrees C and only minimal growth at 20, 28 and 33 degrees C. The other strain (CSIRO 16) showed growth after 1-2 days at all temperatures with higher titres reached at 15 and 20 degrees C than at 28 and 33 degrees C. The BEF group viruses grew to approximately the same titres at all temperatures. At the higher temperatures (28 and 33 degrees C) most of BEF group viruses had disappeared within 9 days. In contrast at the lower temperatures (15 and 20 degrees C), there was still virus present 18 days after inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Augmentation of mouse natural killer cell activity by LS 2616, a new immunomodulator

The quinoline-3-carboxamide LS 2616 administered to mice in drinking water increased spontaneous cytotoxicity against YAC-1 cells in a dose-dependent manner. The enhancement of spontaneous cytotoxicity was found to be mediated by NK cells, as judged by their lack of adherence to nylon wool columns, relative resistance to treatment with antibodies to Thy-1.2 and complement, and almost total abrogation after depletion of asialo-GM1+ cells. Enhancement of NK activity was evident after 2 days of treatment, was maximal after 4 days, and remained elevated during the 14-day exposure period studied. NK activity returned to control levels 4 days after cessation of treatment. NK activity was significantly increased in spleen, peripheral blood, lymph nodes, and bone marrow of LS 2616-treated mice, while activity in peritoneal exudate cells and thymus remained low. LS 2616 was able to elevate NK activity in several mouse strains studied, including mice homozygous for the beige gene. Serum interferon levels were not increased during treatment with LS 2616. Combined injection of the interferon inducer Poly I:C and LS 2616 did not increase NK activity above that of animals injected with Poly I:C alone. However, Poly I:C, in contrast to LS 2616, increased NK activity in peritoneal exudate cells. Studies at the single cell level revealed that LS 2616 increased NK activity by increasing the number of lytically active cells via recruitment of new target-binding cells and not by increasing the lytic activity of pre-existing binders.     

Relation between macronuclear DNA and total protein content and generation time in thechilodonella steini (Ciliata) sister cells

Summary Using the monotone dependence function (mdf) together with correlation coefficient it was found that the Ma-DNA content as well as total protein content are regularly, linearly, positively and strongly dependent in sister cells (proter-opisthe) ofChilodonella steini. Additionally it was shown that proter-opisthe ordering is irrelevant to Ma-DNA and protein contents.Analysis of sister cell generation times (TG) confirmed the existence of regular, linear, positive and strong codependence.The relations between Ma-DNA and total protein contents, between protein content and TG, and between Ma-DNA content and TG were also described. There is a weak, linear dependence between Ma-DNA and total protein contents. Relations of TG and Ma-DNA content or TG and total protein content are non-linear and not even monotone. Low and high levels of DNA or proteins are connected with long generation times.     

Late enzymes of vindoline biosynthesis

From differentiated plants of Catharanthus roseus (L.) G. Don we have isolated a specific enzyme of the vindoline biosynthetic pathway catalysing the S-adenosylmethionine-dependent methylation of 11-O-demethyl-17-O-deacetyl-vindoline. The enzyme we named S-adenosyl-L-methionine : 11-O-demethyl-17-O-deacetylvindoline 11-O-methyltransferase. This transferase exhibits a high substrate specificity. Obviously the O-methylation at C-11 precedes the O-acetylation at the C-17 position during the biosynthesis of vindoline.A second enzyme was detected which hydrolyses the acetyl function of vindoline. The distribution of this acetylesterase in C. roseus plants demonstrates that the enzyme is not specifically associated with the vindoline distribution in the plant material. Most probably this enzyme plays no essential role in the biosynthesis of vindoline.     

Late enzymes of vindoline biosynthesis. Acetyl-CoA: 17-O-deacetylvindoline 17-O-acetyl-transferase

From differentiated plants of Catharanthus roseus (L.) G. Don, a specific enzyme was isolated and named acetyl-CoA : 17-O-deacetylvindoline 17-O-acetyltransferase, acting on the biosynthetic formation of the Aspidosperma type alkaloid vindoline.The enzyme shows a high selectivity towards different substrates. The acetyl-CoA-dependent transferase also catalyses the reverse reaction by hydrolysis of the 17-O-acetyl group of vindoline in the presence of free CoA. This enzyme is localized only in vindoline-containing plant parts, but was so far not detectable in cell suspension cultures of C. roseus. The enzyme allows the synthesis of labelled vindoline with high specific activity, applicable for instance as tracer for radioimmunoassays of vindoline.     

Impaired natural killer cell function in hemophiliacs with or without continuous substitution

In the present study, 28 hemophiliacs substituted continuously and 5 hemophiliacs who had received almost no blood products were investigated. Cells of OKT 3+, OKT 4+, and OKT 8+ subsets were counted. Percoll separated fractions of peripheral blood mononuclear cells were examined by morphological criteria and were tested for NK cell activity. We found that the NK cell activity of both groups of hemophiliacs was decreased on testing Ficoll separated cells or low density Percoll separated cells. Normal NK cell activity was found in medium density cells of hemophiliacs. Two possible explanations are discussed: first, the NK cell activity may be suppressed in hemophiliacs and secondly, there may be a block in maturation of NK cell activity. It is unlikely that chronic substitution by blood products counts for these alterations. The possible role of chronic infections is discussed.     

Analysis and detection of chlamydial DNA

Elementary bodies of lymphogranuloma venereum (LGV) strains of Chlamydia trachomatis contain, in addition to the genomic DNA, a 6.7 kb plasmid. The plasmid from serovar L2 (434-B) was cloned at the BamHI site of pBR327 into Escherichia coli and a restriction cleavage map of this pLGV125 recombinant plasmid determined. All 15 C. trachomatis serovars contained DNA sequences that hybridized with pLGV125. When total DNA from L2 elementary bodies was used as a probe in Southern blotting and spot hybridization, serovars L1, L2 and L3 exhibited significant homology. The detection level of homologous DNA was 100 pg and LGV DNA was detectable in infected cells when total L2 probe was used in the nucleic acid hybridization test. These DNA probes may be useful as investigative and diagnostic reagents for C. trachomatis.