The effect of growth conditions on adenylate cyclase activity and virulence-related properties of Bordetella pertussis

Growth of Bordetella pertussis in Stainer & Scholte medium in which the NaCl had been replaced by one of several inorganic or organic salts resulted in a large decrease in adenylate cyclase activity, histamine-sensitizing activity and in the amounts of two cell-envelope polypeptides of Mr 28000 and 30000. Although some variation between strains was observed, there was never a case where one of these properties was lost independently of the others. Cultures in which these properties were lost had decreased amounts of extracellular cAMP when compared to NaCl-grown cultures. Adenylate cyclase activity was detected in three locations of B. pertussis cultures (extracellular, extracytoplasmic but cell-associated, and cytoplasmic). After growth in medium containing high concentrations of MgSO4, enzyme activity was decreased to a similar extent in all three locations.     

Structural organization of the beta-globin locus of B-haplotype sheep

Goats and some sheep synthesize a juvenile hemoglobin, Hb C (alpha 2 beta C2), at birth and produce this hemoglobin exclusively during severe anemia. Sheep that synthesize this juvenile hemoglobin are of the A haplotype. Other sheep, belonging to a separate group, the B haplotype, do not synthesize hemoglobin C and during anemia continue to produce their adult hemoglobin. To understand the basis for this difference we have determined the structural organization of the beta- globin locus of B-type sheep by constructing and isolating overlapping genomic clones. These clones have allowed us to establish the linkage map 5' epsilon I-epsilon II-psi beta I-beta B-epsilon III-epsilon IV- psi beta II-beta F3' in this haplotype. Thus, B sheep lack four genes, including the BC gene, and have only eight genes, compared with the 12 found in the goat globin locus. The goat beta-globin locus is as follows: 5' epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F3'. Southern blot analysis of A-type sheep reveals that these animals have a beta- globin locus similar to that of goat, i.e., 12 globin genes. Thus, the beta-globin locus of B-haplotype sheep resembles that of cows and may have retained the duplicated locus of the ancestor of cows and sheep. Alternatively, the B-sheep locus arrangement may be the result of a deletion of a four-gene set from the triplicated locus.      

Inhibition of sporulation in Bacillus subtilis by bromodeoxyuridine and the effect on DNA replication

A 5-bromo-2'-deoxyuridine (BUdR)-tolerant derivative of a thymidine (TdR)-requiring strain of Bacillus subtilis was used to examine the effect of BUdR, an analogue of TdR, on sporulation. At a TdR:BUdR ratio which had little effect on growth, sporulation was inhibited if cells were exposed to BUdR during the period of DNA synthesis at the onset of the process. Cells recovered from BUdR inhibition of sporulation if the analogue was removed and DNA replication allowed to continue with TdR alone. BUdR prolonged the period of DNA synthesis during sporulation and experiments with chloramphenicol suggested that this was due in part to unscheduled initiation of new rounds of replication.     

Tolerance to bromodeoxyuridine in a thymidine-requiring strain of Bacillus subtilis

Mutations which allow tolerance to 5-bromo-2'-deoxyuridine (BUdR) in a thymidine (TdR)-requiring strain of Bacillus subtilis have been examined. Differences in sensitivity to BUdR existed between isogenic strains harbouring the mutations. Those mutations originally isolated as BUdR-tolerant also bestowed tolerance to 5-bromouracil and vice versa. The strain exhibiting the greatest tolerance to BUdR maintained a normal rate of replication in the presence of BUdR whereas the parent strain did not, but the tolerant strain incorporated less analogue into DNA than the parent strain. The basis of the tolerance mutation appeared to lie at the point of uptake of the analogue into the cell as the tolerant mutant preferentially took up TdR over BUdR into whole cells. DNA polymerase activity measured in vitro did not distinguish between TdR and BUdR in either the parent or the mutant strain and although TdR kinase activity showed a preference for TdR over BUdR as a substrate, the extent of discrimination was similar in both strains.     

Adenylate cyclase activity during phenotypic variation of Bordetella pertussis

During MgSO4-induced modulation of Bordetella pertussis, adenylate cyclase activity, histamine-sensitizing activity (HSA) and the major cell-envelope polypeptides with Mr 28000 and 30000 (X polypeptides) were lost synchronously at a rate which could be accounted for by a simple growth-dilution effect. MgSO4 and other compounds which induced the above phenotypic change caused little inhibition of adenylate cyclase activity. Nicotinic acid was the sole exception and at 4.1 mM-caused 60% inhibition of activity. Lysates of modulated cells, mixed with lysates of unmodulated cells, had no effect on either adenylate cyclase activity or HSA. Protein synthesis was a prerequisite for MgSO4-induced modulation and also for the reversal of this process. Exogenous cAMP and dibutyryl cAMP (5 mM) had no counteracting effect on MgSO4- or nicotinic acid-induced modulation. The concentration of MgSO4 required to induce loss of the X polypeptides (10 to 11 mM) was not altered by promoting adenylate cyclase activity by including an activator in the growth medium. In one culture containing 10 mM-MgSO4 and activator, partial loss of the X polypeptides occurred and yet the extracellular cAMP concentration was twice that of cultures without activator and where full expression of the X polypeptides occurred. [3H]cAMP-binding activity was detected in cell extracts of several strains of B. pertussis, but antiserum against purified Escherichia coli catabolite repressor protein gave no reaction with B. pertussis cell extracts. Respiration rates with amino acids were similar for modulated and unmodulated variants and an avirulent strain of B. pertussis. These results are discussed in relation to a possible causal role for adenylate cyclase in modulation of B. pertussis.     

Density gradient analysis of DNA replicated during Bacillus subtilis sporulation.

Density gradient centrifugation was used to monitor DNA replication during sporulation of a 5-bromo-2'-deoxyuridine-tolerant, thymidine-requiring strain of Bacillus subtilis. DNA of heavy, intermediate, and light density was found in cells induced to sporulate in the presence of bromodeoxyuridine, but only intermediate DNA was detected in mature spores. Cells grown with bromodeoxyuridine until DNA was in the heavy form formed spores containing intermediate and light DNA when sporulated with thymidine alone.     

The influence of temperature on the fluorine and calcium composition of fish scales

Proton microprobe studies of the scales of the kahawai (Arripis trutta) and the snapper (Chrysophrys auratus) showed non-linear changes in the fluorine to calcium ratio that increase with increasing temperature, but both species showed a different temperature sensitivity. Fluorine and calcium levels vary within years from summer to winter by up to 1000%, making fluorine and calcium levels good markers of seasonal events.     

A substrate-mediated assay of bacterial proton efflux/influx to predict the degree of spoilage of beef mince stored at chill temperatures

A method was developed to predict spoilage of minced meat at chill temperatures, based on the difference in proton efflux from and influx into bacterial cells. This difference depends on the number of organisms present, the available glucose in the meat sample and the ability of the organisms to metabolize amino acids. The proton efflux/influx of a meat filtrate containing bacteria was measured at 25°C with a pH/ion meter in the presence of peptone with or without glucose. There was a noticeable rate of change of mV h_-1 of the meat filtrate prior to the organoleptic detection of spoilage which may be used semi-predictively to determine the remaining shelf-life of meat at different storage temperatures. The method could be investigated further, encompassing type and relative numbers of organisms, incubation temperature, meat type and composition (i.e. available glucose) to produce a spoilage prediction model. The method does not require sophisticated equipment, only a standard pH/ion meter, is cheap, needing only peptone and glucose, is relatively simple, and takes less than 2 h to perform.     

Selective inhibition of prostaglandin biosynthesis by gold salts and phenylbutazone

Gold salts and phenylbutazone selectively inhibit the synthesis of PGF_2α and PGE₂ respectively. Lowered production of one prostaglandin species is accompanied by an increased production of the other. Selective inhibition by these drugs was observed in the presence of adrenaline, reduced glutathione and copper sulphate under conditions when most anti-inflammatory compounds inhibited PGE₂ and PGF_2α syntheses equally. It is postulated that selective inhibitors may have a different mode of action and beneficial effects may be related to the endogenous ratio of PGE to PGF required for normal function.     

Two silicone nontoxic fouling release coatings: Hydrosilation cured PDMS and CaCO3 filled,ethoxysiloxane cured RTV11

Two silicone coatings have been evaluated for barnacle adhesion. One coating is an unfilled hydrosilation cured polydimethylsiloxane (PDMS) network, while the other is a room temperature vulcanized (RTV), filled, ethoxysiloxane cured PDMS elastomer, RTV11^?. The adhesion strength of one species of barnacle, Balanus eburneus, to the hydrosilation coatings is in the range of 0.37–0.60 kg cm^‐2 while the corresponding range for RTV11 is 0.64–0.90 kg cm^‐2. The easier release of B. eburneus from the hydrosilation cured network compared to RTV11 is discussed in relationship to differences in bulk and surface properties. Preliminary results suggest bulk modulus may be the most important parameter in determining barnacle adhesion strength. In light or mechanical property analysis, a re‐evaluation of surface properties and chemical stability is presented.