Construction of a cosmid library of Spirulina platensis as an approach to DNA physical mapping

Abstract A Spirulina platensis gene library has been constructed using cosmid vector pMMB34. The cosmid bank was controlled for its random gene distribution by colony hybridization. Genes were identified using either homologous or heterologous probes of genes involved in photosynthesis (large and small subunit of d -ribulose 1,5-bisphosphate carboxylase, 32 kDa thylakoid protein, α, β subunits of C-phycocyanin) and protein synthesis (elongation factors EF-Tu, EF-G).     

Identification of a deletion in the low density lipoprotein (LDL) receptor gene in a patient with familial hypercholesterolaemia

Summary DNA samples from 60 unrelated UK patients with familial hypercholesterolaemia (FH) were screened by Southern blot hybridisation to detect gross alterations in the low density lipoprotein (LDL) receptor gene. One patient was found to have a 2kb deletion in the 3 part of the gene. The deletion cosegregates with the FH phenotype in his family. This finding is compatible with the deletion being the cause of FH in this case and makes a presymptomatic test based on DNA analysis available for this family. The defects in most of the other patients are likely to be due to point mutations.     

Properties of action potentials carried by divalent cations in identified leech neurons

Properties of divalent cation potentials carried by either Sr2+ or Ca2+ ions in Na+-free, TEA-Ringer solution were characterized in identified neurons of two species of leeches (Macrobdella and Haementeria). In Macrobdella, the overshoot of the potentials varied logarithmically with [Sr2+]0 (28.5 mV per 10-fold change). The overshoot, Vmax, and duration of the potentials increased with increasing divalent cation concentration and saturated at about 20 to 30 mM [Sr2+]0. The Vmax, amplitude, and duration of the potentials were reversibly blocked by Co2+ and Mn2+. The block by Mn2+ could be well-fitted by a reverse Langmuir-curve with an apparent KI of 100 micromolar. The local anesthetic procaine also reversibly inhibited the Vmax and duration of the potentials. The inhibition was greater at alkaline pH suggesting that procaine blocks the calcium channel from inside the membrane. The identified leech neurons examined in Macrobdella varied considerably in their ability to sustain somatic divalent cation potentials. Stimulation of T cells and most motoneurons produced no or only weak potentials, whereas stimulation of Retzius, N, Nut, and AP cells evoked overshooting potentials of several seconds' duration. Stimulation of the ALG cell of Haementeria in normal Ringer solution evoked a slowly-rising, purely Ca2+-dependent potential of approximately 100 ms duration. This response was TTX-resistant, unaffected by complete removal of Na+ from the Ringer solution, and abolished by 1 mM Mn2+. The overshoot varied logarithmically with a slope of 28 mV/decade change in [Ca2+]0.     

Permeability of the boundary layers of Bdellovibrio bacteriovorus 109J and its bdelloplasts to small hydrophilic molecules.

Measurements of the sucrose-permeable and -impermeable volumes during Bdellovibrio bacteriovorus attack on Escherichia coli or Pseudomonas putida showed that the volume of the bdelloplast increased over that of the substrate cell. Although the pattern of the increase differed with the two organisms, the volumes reached maximum at about 60 min into the bdellovibrio growth cycle. By this time, the cytoplasmic membranes of the attacked cells were completely permeable to sucrose. The kinetics of increase in sucrosepermeable volumes were similar to the kinetics of attachment and penetration (Varon and Shilo, J. Bacteriol. 95:744-753, 1968). These data show that the original cytoplasmic and periplasmic compartmentalization of the substrate cell ceases to exist with respect to small hydrophilic molecules during bdellovibrio attack. In contrast, the effective pore size of the outer membrane of the substrate cell to small oligosaccharides remains unaltered during bdelloplast formation as was shown by direct measurements of its exclusion limits. The major porin protein of E. coli, OmpF, was recoverable from the bdelloplast outer membrane fraction until the onset of lysis. The Braun lipoprotein was removed from the bdelloplast wall early, and OmpA was lost in the terminal part of the bdellovibrio growth cycle.     

High affinity, calcium-stimulated adenosine triphosphatase activity in the particulate fraction of rat pancreatic acini

Adenosine triphosphatase activity which is Mg2+-dependent and stimulated by submicromolar concentrations of Ca2+ (as Ca . ATP) was identified in the total particulate fraction of rat pancreatic acini. Half-maximal activity (V0.5) is obtained at 100.1 +/- 6 nM Ca . ATP with a Hill coefficient of 2.2 +/- 0.1 (mean +/- S.E.; n = 4). Maximal activity was 75 +/- 19 pmol of Pi released from ATP minute-1 microgram of membrane protein-1 (mean +/- S.E.; n = 7). High affinity Ca2+-ATPase activity was unaffected by ouabain, Na+, K+, La3+, and added calmodulin. Activity was slightly reduced by ruthenium red (0.1 mM) and by oligomycin (80 micrograms/ml) but was reduced almost 50% by the phenothiazine derivative fluphenazine in a dose-related and Ca2+-dependent manner. Hydrolysis of p-nitrophenyl phosphate was 9% of the rate of ATP hydrolysis and was independent of Ca2+ concentration. However, ADP, GTP, UTP, and ITP were hydrolyzed at 76-93% the rate that ATP was hydrolyzed with V0.5 values and Hill coefficients similar to those of Ca . ATP. We conclude that rat pancreatic acini contain an enzyme for active Ca2+ translocation: ATPase activity that is Mg2+-dependent and stimulated by submicromolar concentrations of Ca . ATP. Substrate hydrolysis appears to involve positive cooperative interactions of multiple ligand-binding sites and may be regulated in part by calmodulin.     

Monocyte suppression of antigen-specific lymphocyte responses in diffuse cutaneous leishmaniasis patients from the Dominican Republic

Patients from the Dominican Republic with diffuse cutaneous leishmaniasis showed in vivo and in vitro anergy to leishmanial antigen. Relatives of these DCL patients living in the same endemic area frequently showed skin test and lymphocyte reactivity to leishmanial antigens. This further supports the concept of specific anergy in patients with diffuse cutaneous leishmaniasis. Adherent suppressor cells modulate the antigen-specific lymphocyte proliferative response. Suppressor cells could also be isolated by Percoll gradient centrifugation. Co-culturing of lymphocytes and monocytes from HLA-identical leishmanin responders and nonresponders also identified the suppressor cell as a monocyte. In one patient, this suppression disappeared when clinical cure had been accomplished.     

The Electrical Conductivity of Bovine Milk in Mastitis Diagnosis

The electrical conductivity (EC) of milk is mainly a function of the electrolyte concentration in the milk and therefore raised in mastitis. The present investigation was aimed at elaborating, if possible, a diagnostic model for screening purposes based on EC determinations and consistent with the diagnostic procedures and interpretations commonly used in laboratory milk diagnosis in the Nordic countries (Klastrup 1975). According to this diagnosis (here called reference diagnosis) cell numbers above 300,000/ml (cell count or the corresponding CMT-score) in foremilk quarter samples during the main part of the lactation period and significantly above the lowest value on within-udder comparison during late lactation are considered indicative of mastitis and bacteriological examinations are made when called for.     

RAM, a gene of yeast required for a functional modification of RAS proteins and for production of mating pheromone a-factor

We have identified a gene (SUPH) of S. cerevisiae that is required for both RAS function and mating by cells of a mating type. supH is allelic to ste16, a gene required for the production of the mating pheromone a-factor. Both RAS and a-factor coding sequences terminate with the potential acyltransferase recognition sequence Cys-A-A-X, where A is an aliphatic amino acid. Mutations in SUPH-STE16 prevent the membrane localization and maturation of RAS protein, as well as the fatty acid acylation of it and other membrane proteins. We propose the designation RAM (RAS protein and a-factor maturation function) for SUPH and STE16. RAM may encode an enzyme responsible for the modification and membrane localization of proteins with this C-terminal sequence.     

Role of uronic acids present in phytopathogenic fungi as inducers of polygalacturonases during autolysis

The presence of uronic acids in the culture fluid and mycelium of the fungi: Alternaria alternata, Botrytis cinerea, Drechslera halodes, Fusarium culmorum, Fusarium oxysporum, Monilinia fructigena, Mucor mucedo, Rhizopus stolonifer and Trichoderma hamatum was detected and quantified. In these fungi the concentration of uronic acids increased during the growth phase and the maximal concentrations were found at the end of the growth phase or onset of autolysis both in the mycelium as well as in the culture fluid. The uronic acids were metabolized during the first days of autolysis decreasing to constant levels until the end of the autolytic period studied.The variations in the activity of polygalacturonase and polymethylgalacturonase present in the culture fluid were determined at the onset and during autolysis in these fungi. These enzymic activities were found in the culture fluid of these fungi, with exception of M. rouxii, and they showed an increasing activity in the first days of autolysis and later a slight increase or decrease was observed. The presence of uronic acids in these phytopathogenic or saprophytic fungi and the low levels detected during autolysis could be related to the induction of pectic enzymes and the pathogenicity of these fungi.