Plasmid content and homology of 16 strains of filamentous,nonheterocystous cyanobacteria

We have developed several strain-specific, rapid, small-scale plasmid isolation procedures in order to characterize the plasmid profiles of 16 filamentous, nonheterocstous cyanobacteria. At least one distinct plasmid was found in eight strains, with seven of these containing two or more different plasmids. Eight strains were found to be without plasmid DNA. Both the large, 12.9 kb, and the small, 1.6 kb, plasmids fromPlectonema boryanum 581 were isolated, purified, and cloned. Southern blots of plasmid DNAs from the eight strains were probed with these cloned DNAs and also with ultra-pure plasmid DNA fromPhormidium liridum 426. Four strains ofP. boryanum (485, 581, 594, 1542) andP. luridum 426 have identical plasmid profiles, and plasmid homology is extensive.     

Production of the penicillin precursor δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine (ACV) by cell-free extracts from Streptomyces clavuligerus

Abstract Glycerol-stabilised cell extracts of Streptomyces clavuligerus contain an enzyme activity which synthesises ACV from the individual amino acids L -α-aminoadipic acid, L -cysteine and L -valine. Enzyme activity was optimum in reaction mixtures containing 1 mM ATP together with an ATP regenerating system. The ACV synthetase enzyme formed ACV analogs when provided with L - carboxymethylcysteine in place of L -α-aminoadipic acid or when provided with L - allo isoleucine or L -α-aminobutyrate in place of L -valine. Multistep conversion of individual amino acids to penicillin and cephalosporin antibiotics was restricted as a result of the inhibitory effects of L -α-aminoadipic acid and L -cysteine on isopenicillin N synthetase.     

Rapid hydrophobic grid membrane filter-enzyme-labeled antibody procedure for identification and enumeration of Escherichia coli O157 in foods

An O-antigen-specific monoclonal antibody, labeled by horseradish peroxidase-protein A, was used in a hydrophobic grid membrane filter-enzyme-labeled antibody method for rapid detection of Escherichia coli O157 in foods. The method yielded presumptive identification within 24 h and recovered, on average, 95% of E. coli O157:H7 artificially inoculated into comminuted beef, veal, pork, chicken giblets, and chicken carcass washings. In food samples from two outbreaks involving E. coli O157:H7, the organism was isolated at levels of up to 10(3)/g. The lower limit of sensitivity was 10 E. coli O157 per g of meat. Specific typing for E. coli O157:H7 can be achieved through staining with labeled H7 antiserum or tube agglutination.     

Nanoplanktonic coccolithophorids (Prymnesiophyceae, Haptophyceae) from the Weddell Sea, Antarctica

The examination of whole mounts prepared for transmission electron microscopy has resulted in the finding of thirteen taxa of nanoplanktonic coccolithophorids from the Weddell Sea, Antarctica. The material was collected as part of the AMERIEZ programme, March 1986. Cold-water adapted nanoplanktonic coccolithophorids have previously been shown to constitute a recurrent plankton element at subarctic and arctic localities. Three of the Weddell Sea species, Wigwamma annulifera, W. arctica , and Papposphaera sagittifera , are conspecific with northern hemisphere material, while two species, Calciarcus alaskensis and Turrisphaera arctica , are possibly identical with previously described arctidsubarctic material. Six taxa new to science have been described from the Weddell Sea, Wigwamma antarctica, W. triradiata, Trigonaspis melvillea, Pappomonas weddellensis, Papposphaera obpyramidalis , and P. simplicissima . The cooccurrence of identical forms at the two poles, and the fact that the species described are allocated to "arctic" genera, indicate a geologically relatively recent exchange of biological material between the poles.     

Flow cytometric detection of herpes simplex virus type 2 infected and transformed cells by immunoenzymatic and by indirect immunofluorescence staining

The glucose oxidase antiglucose oxidase (GAG) immunoenzymatic staining procedure has been used to detect herpes simplex virus (HSV) antigens microscopically. In this study, the GAG procedure was adapted to cells in suspension, and its potential usefulness in flow cytometry was examined. HSV-2 infected monkey kidney and HSV-2 transformed mouse cells were stained using antisera to HSV-2 or to an HSV-2 specific protein with a molecular weight of 38 Kd, respectively, with the GAG procedure. Flow cytometric analysis of the GAG stained cells was then performed by the measurement of scattered light intensity in the angular intervals 1 degree-2 degrees, 2.5 degrees-19 degrees, and 3 degrees-6 degrees. The greatest scattered light intensity decrement caused by staining occurred in the 3 degrees-6 degrees angular interval, as predicted by previous work. In infected cells, which stain intensely by immunofluorescence, the difference between positively and negatively stained cells was adequate for detecting infected cells using the GAG method; however, this was not the case for the lightly staining transformed cells. The indirect immunofluorescence method of analysis of the same populations was superior to the scattered light method of analysis of the GAG stained infected and transformed cells.     

Stereoselective Nicotine-Induced Release of Dopamine from Striatal Synaptosomes: Concentration Dependence and Repetitive Stimulation

Using a sensitive perfusion system we have studied the nicotine-induced release of [3H]dopamine ([( 3H]DA) from striatal synaptosomes. Nicotine-evoked release was concentration dependent with an EC50 of 3.8 microM. The response to 1 microM nicotine was comparable to that to 16 mM K+; 10 microM veratridine evoked a larger response. All three stimuli were Ca2+ dependent but only the response to veratridine was blocked by tetrodotoxin. Repetitive stimulations by 1 microM (-)-nicotine (100 microliters) at 30-min intervals resulted in similar levels of [3H]DA release; higher concentrations of (-)-nicotine resulted in an attenuation of the response particularly following the third stimulation. This may reflect desensitisation or tachyphylaxis of the presynaptic nicotinic receptor. The action of nicotine was markedly stereoselective: a 100-fold higher concentration of (+)-nicotine was necessary to evoke the same level of response as 1 microM (-)-nicotine. It is proposed that these presynaptic nicotinic receptors on striatal terminals are equivalent to high-affinity nicotine binding sites described in mammalian brain.     

Differences in the Biophysical Properties of the Benzodiazepine/γ-Aminobutyric Acid Receptor Chloride Channel Complex in the Long-Sleep and Short-Sleep Mouse Lines

Significant differences in the thermal stability of benzodiazepine receptors were found in cerebral cortical membranes prepared from the long-sleep (LS) and short-sleep (SS) selected mouse lines. Thus, benzodiazepine receptors from LS mice were heat inactivated (55 degrees C) at a significantly faster rate than those from SS mice. Although gamma-aminobutyric acid (GABA) reduced the rate of heat inactivation in both lines, the more rapid rate of inactivation in the LS line was maintained. Furthermore, the potency of GABA to enhance [3H]flunitrazepam binding decreased threefold in membranes from LS mice as the incubation temperature was increased from 0 degrees to 37 degrees C, but was unaltered in membranes from SS mice. These differences in the biophysical properties of the benzodiazepine/GABA receptor chloride channel complex ("supramolecular complex"), together with a higher KD for t-[35S]butylbicyclophosphorothionate in membranes from LS compared to SS mice, suggest that the supramolecular complex may modulate the differential sensitivity to some depressants and convulsants in these lines.     

One-step gene disruption by cotransformation to isolate double auxotrophs in Candida albicans

Summary The Candida albicans LEU2 gene was disrupted by substituting lambda DNA for a small deletion within the LEU2 gene. Cotransformation with a selectable URA3 ARS vector was used to introduce a linear fragment containing the disruption into the genome of a C. albicans ura3 deletion mutant. Cotransformants containing the lambda DNA were identified by colony hybridization and the URA3 plasmid was subsequently cured. Leu2 disrupted heterozygotes were detected by Southern hybridization and one disruptant was subsequently treated with UV irradiation. Only one leu2 ura3 mutant (SGY-484) was isolated out of 11,000 mutagenized cells. SGY-484 was transformed to Leu⁺ with either the C. albicans or Saccharomyces cerevisiae LEU2 gene. Southern hybridization analysis revealed that the mutant is not homozygous for the disruption; the leu2 mutation reverts and is most likely a point mutation. Unexpectedly, an ade2 ura3 mutant was isolated from the same mutagenesis.     

Deglycosylation of a lectin intermediate during assembly of ConA

Summary Metabolic labelling of immature jackbeans (Canavalia ensiformis) has been used in a pulse-chase study to determine changes in the glycosylation pattern of polypeptides during the assembly of Concanavalin A. In an analysis that allowed the identification of 7 intermediates, only the first precursor form of the lectin was labelled with D-[U-¹⁴C]-glucosamine. These results indicate that processing of the lectin involves a novel deglycosylation event in which an N-linked oligosaccharide is removed from a protein in the absence of proteolysis.Abbreviations endo H endo -N-acetylglucosaminidase H - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - ConA Concanavalin A