Distribution ofVibrio cholerae in two Florida estuaries

The distribution ofVibrio cholerae was examined in 2 Florida estuaries, Apalachicola and Tampa Bay.Vibrio cholerae serotype non-01 was the most abundant serotype, being isolated from 45% of the oyster samples, 30% of the sediments, 50% of the waters, and 75% of the blue crabs.Vibrio cholerae serotype 01 was isolated from only one oyster sample. Strong linear correlations betweenV. cholerae and temperature, salinity, or the other physical/chemical parameters measured,Escherichia coli, or fecal coliforms were not observed, but a range of temperatures and salinities appeared relevant to the distribution of the organism. The organism was present in the highest concentrations when salinities were 10‰–25‰ and temperatures were 20?C–35?C.In vitro growth curves of 95V. cholerae environmental isolates further supported that 10‰–25‰ was an ideal salinity range for the organisms. The results suggest thatV. cholerae is a widely distributed organism in the nutrient-rich warm waters of the Gulf Coast estuaries.     

Relationship among fecal coliforms, Escherichia coli, and Salmonella spp. in shellfish

The relationship of fecal coliforms, Escherichia coli, and Salmonella spp. was examined in freshly harvested and stored shellfish. In 16 of 40 freshly collected oyster samples, fecal coliform levels were above the recommended wholesale level suggested by the National Shellfish Sanitation Program (less than or equal to 230/100 g), and Salmonella spp. were present in three of these samples. Salmonella spp. were not, however, present in any sample containing less than 230 fecal coliforms per 100 g. Analysis of the data suggests that low fecal coliform levels in both fresh and stored oysters are good indicators of the absence of Salmonella spp., but that high levels of fecal coliforms are somewhat limited in predicting the presence of Salmonella spp. E. coli levels correlated very strongly with fecal coliform levels in both fresh and stored oysters and clams, suggesting that there is no advantage in replacing fecal coliforms with E. coli as an indicator of shellfish quality.     

Adrenal modulation of the inhibitory effect of corticotropin releasing factor on feeding

Corticotropin releasing factor (CRF) reduces food intake in rats after central administration. In these studies we examined whether the adrenal gland and the vagus were involved in CRF suppression of intake. One hour intake was reduced by a 5 μg (ICV) injection of CRF in sham but not adrenalectomized rats maintained on 0.9% NaCl. In a separate experiment on rats maintained on tap water, the inhibitory effect of CRF (5 μg) lasted at least 4 hours in sham rats whereas adrenalectomized rats did not significantly differ from controls. These experiments suggest that the adrenal gland modulates the feeding response to CRF. As replacement with corticosterone (0.75 mg/kg) in total adrenalectomized rats did not restore responsiveness to 5 or 10 μg of CRF, we next studied whether the adrenal medulla was responsible for the decreased responsiveness to CRF. In rats lacking the adrenal medulla only, food intake was reduced by a 5 μg injection of CRF; in sham rats, intake was significantly reduced by doses as low as 0.1 μg of CRF. An additional experiment examined the effect of gastric vagotomy on the CRF feeding response. Vagotomized rats were as responsive to 5 and 10 μg injections of CRF as sham rats, which suggests that the effect is not dependent on the vagus nerve. These findings indicate that the adrenal gland, primarily the medulla, plays an intermediate role in the reduction of food intake caused by central injections of CRF. This conclusion is consistent with the known effect of CRF on adrenomedullary discharge.     

Inhibition of neutrophil superoxide production by human plasma alpha 1-antitrypsin.

We report here that human plasma alpha 1-antitrypsin (alpha 1-AT) inhibited human neutrophil O2.- release elicited by a variety of stimulants. In comparison, the inhibitory capacities of two serine protease inhibitors, L-1-tosylamide 2-phenylethyl chloromethyl ketone (TPCK) and soybean trypsin inhibitor (SBTI), and the human recombinant alpha 1-AT mutant, alpha 1-AT-Arg358 were in the order: alpha 1-AT = TPCK much greater than alpha 1-AT-Arg358 greater than SBTI when cells were stimulated with concanavalin A plus cytochalasin E. These data suggest that, in human inflammatory fluids containing relatively high concentrations of alpha 1-AT (such as rheumatoid arthritis synovial fluid), (i) alpha 1-AT may down-regulate the inflammatory process by inhibiting the neutrophil respiratory burst and (ii) serpin oxidation by neutrophil-released reactive oxygen species is unlikely to occur.     

The influence of nearest neighbors on the rate and pattern of spontaneous point mutations

Summary The numbers and local sequence environments of the two types of substitution mutation plus additions and deletions have been obtained directly in this study from differences between a large number of extant primate gene and pseudogene sequences. A total of 3786 mutations were scored in regions where similarities between pseudogene and corresponding gene sequences is 85%, comprising 30% of the pseudogene database of 80,584 bp. The pattern of mutations obtained in this fashion is almost identical to that obtained by Li et al. (1984) using a slightly different, more direct approach and with a smaller database. When mutations were scored, the neighbor pairs on the 5 and 3 sides were also noted, leading to a large 16 × 12 matrix of transitions and transversions. Biases of varying magnitude are found in the rates of substitution of the same base pair in different local sequence environments. The overall order for the effect of the 5 neighbor on the rates of substitution mutation of a pyrimidine is A > C T > G, and G > A > T > C for the 3 neighbor; where these results represent the average of substitution rates for the complement purine with complement neighbors of bases ordered above. The order for the 3 neighbor is essentially the same for the two transitions and most of the four transversions as well; however, the order for the 5 neighbor is more variable. The overall rate for the C · G T · A transition is not unusual, however the presence of a 3 neighboring G · C pair boosts the rate substantially, presumably due to specific cytosine methylation of the CG doublet in primate DNAs. The rate of the T · A C · G transition is also well above average when the 3 neighbor is an A · T, and to a lesser extent a G · C, pair. The latter bias is typical in that it reflects the association of alternating pyrimidine-purine sequences with increasing mutation rates. The substitution of the pyrimidine in a 5 purine-pyrimi-dine-purine3 sequence generally occurs much faster than in a pyrimidine tract and points to the local conformation as a major determining factor of the substitution rate. An apparent inverse relationship is found between starting and product doublet frequencies of base pairs undergoing mutations with specific 3 neighbors, indicating that differences in intrinsic substitution rates of base pairs with specific neighbors are a key factor in producing the familiar biases of nearest-neighbor frequencies.Offprint requests to: R. D. Blake     

Group selection, individual selection, and the evolution of genetic drift.

In a subdivided population, genetic drift affects variation between groups, and thus it can have an important effect on the outcome of evolution (Wright, 1978). The rate of genetic drift is determined, in part, by the behaviour of population members. This paper presents three mathematical models in which behavioural traits that affect the rate of genetic drift are allowed to coevolve with traits that are under selection at the group and individual levels. The results show that if group selection is strong relative to individual selection, then behavioural traits that enhance the rate of genetic drift will tend to increase in frequency. The strength of this effect depends, in part, on the way in which vacant sites are colonized.     

On-line fluorescence-monitoring of the methanogenic fermentation

On-line in situ fluorescence measurements of the methanogenic fermentation were conducted with reactors receiving either glucose or a mixture of volatile fatty acids as the substrate. The reactors were perturbed from steady-state conditions in order to assess the response of fluorescencemonitoring probes. Two fluorescence-monitoring probes were evaluated over a period of 8 months; they performed in a consistent manner, and their response was not significantly affected by the changes in pH and redox potential encountered during routine reactor operation. A commercially available probe, designed to measure NAD(P)H, demonstrated particular promise for detecting imbalance caused by the entry of air, inhibitor addition and was capable of distinguishing between different substrates. This fluorescence-monitoring probe detected imbalance more rapidly than other on-line measurements such as pH, Eh, or gas production, or off-line measurements such as volatile fatty acid concentration or gas composition. An experimental fluorescence-monitoring probe, designed to measure coenzyme F(420), also showed some promise in this regard. The response of the fluorescence-monitoring probes also revealed details of the metabolic routes in the reactors and the probes represent a useful research tool. For example, a failure to observe the characteristic response of the NAD(P)H-monitoring probe to formate addition during the metabolism of acetate, propionate, or glucose strongly suggests that any formate liberated during their catabolism is degraded via a different route to exogenously added formate.     

Drought tolerance in faster- and slower-growing black spruce (Picea mariana) progenies: II. Osmotic adjustment and changes of soluble carbohydrates and amino acids under osmotic stress

The possibility was considered that osmotic adjustment, the ability to accumulate solutes in response to water stress, may contribute to growth rate differences among closely-related genotypes of trees. Progeny variation in osmotic adjustment and turgor regulation was investigated by comparing changes in osmotic and pressure potentials, soluble carbohydrates, and amino acids in osmotically stressed seedlings in 4 full-sib progenies of black spruce [ Picea mariana (Mill.) B. S. P.] that differed in growth rate under drought. Osmotic stress was induced by a stepwise increase in the concentration of polyethylene glycol (PEG)-3350 from 10 (w/v) to 18 and 25%, which provided osmotic potentials in solution culture of -0.4, -1.0 and -2.0 MPa each for 3 days. All 4 progenies maintained a positive cell turgor even at 25% PEG, due to a significant decline in osmotic potential. Although total amino acids, principally proline, increased, ca 60% of the decrease in osmotic potential was attributable to soluble carbohydrates and glucose was the major osmoregulating solute. There was little progeny variation in any of measured parameters in unstressed seedlings. Compared to two slower-growing progenies, the two progenies capable of more vigorous growth under drought in the field accumulated more soluble carbohydrates (mainly glucose and fructose), developed lower osmotic potential and maintained higher turgor pressure when osmotically-stressed in solution culture. The ability to adjust osmotically and maintain turgor under drought stress could thus be a useful criterion for the early selection of faster-growing, drought-tolerant genotypes.     

The active centers of adenylylsulfate reductase from Desulfovibrio gigas. Characterization and spectroscopic studies

In order to utilize sulfate as the terminal electron acceptor, sulfate-reducing bacteria are equipped with a complex enzymatic system in which adenylylsulfate (AdoPSO4) reductase plays one of the major roles, reducing AdoPSO4 (the activated form of sulfate) to sulfite, with release of AMP. The enzyme has been purified to homogeneity from the anaerobic sulfate reducer Desulfovibrio gigas. The protein is composed of two non-identical subunits (70 kDa and 23 kDa) and is isolated in a multimeric form (approximately 400 kDa). It is an iron-sulfur, flavin-containing protein, with one FAD moiety, eight iron atoms and a minimum molecular mass of 93 kDa. Low-temperature EPR studies were performed to characterize its redox centers. In the native state, the enzyme showed an almost isotropic signal centered at g = 2.02 and only detectable below 20 K. This signal represented a minor species (0.10-0.25 spins/mol) and showed line broadening in the enzyme isolated from 57Fe-grown cells. Addition of sulfite had a minor effect on the EPR spectrum, but caused a major decrease in the visible region of the optical spectrum (around 392 nm). Further addition of AMP induced only a minor change in the visible spectrum whereas major changes were seen in the EPR spectrum; the appearance of a rhombic signal at g values 2.096, 1.940 and 1.890 (reduced Fe-S center I) observable below 30 K and a concomitant decrease in intensity of the g = 2.02 signal were detected. Effects of chemical reductants (ascorbate, H2/hydrogenase-reduced methyl viologen and dithionite) were also studied. A short time reduction with dithionite (15 s) or reduction with methyl viologen gave rise to the full reduction of center I (with slightly modified g values at 2.079, 1.939 and 1.897), and the complete disappearance of the g = 2.02 signal. Further reduction with dithionite produces a very complex EPR spectrum of a spin-spin-coupled nature (observable below 20 K), indicating the presence of at least two iron-sulfur centers, (centers I and II). M?ssbauer studies on 57Fe-enriched D. gigas AdoPSO4 reductase demonstrated unambiguously the presence of two 4Fe clusters. Center II has a redox potential less than or equal to 400 mV and exhibits spectroscopic properties that are characteristic of a ferredoxin-type [4Fe-4S] cluster. Center I exhibits spectra with atypical M?ssbauer parameters in its reduced state and has a midpoint potential around 0 mV, which is distinct from that of a ferredoxin-type [4Fe-4S] cluster, suggesting a different structure and/or a distinct cluster-ligand environment.     

Hexaheme nitrite reductase from Desulfovibrio desulfuricans. M?ssbauer and EPR characterization of the heme groups

M?ssbauer and EPR spectroscopy were used to characterize the heme prosthetic groups of the nitrite reductase isolated from Desulfovibrio desulfuricans (ATCC 27774), which is a membrane-bound multiheme cytochrome capable of catalyzing the 6-electron reduction of nitrite to ammonia. At pH 7.6, the as-isolated enzyme exhibited a complex EPR spectrum consisting of a low-spin ferric heme signal at g = 2.96, 2.28, and 1.50 plus several broad resonances indicative of spin-spin interactions among the heme groups. EPR redox titration studies revealed yet another low-spin ferric heme signal at g = 3.2 and 2.14 (the third g value was undetected) and the presence of a high-spin ferric heme. M?ssbauer measurements demonstrated further that this enzyme contained six distinct heme groups: one high-spin (S = 5/2) and five low-spin (S = 1/2) ferric hemes. Characteristic hyperfine parameters for all six hemes were obtained through a detailed analysis of the M?ssbauer spectra. D. desulfuricans nitrite reductase can be reduced by chemical reductants, such as dithionite or reduced methyl viologen, or by hydrogenase under hydrogen atmosphere. Addition of nitrite to the fully reduced enzyme reoxidized all five low-spin hemes to their ferric states. The high-spin heme, however, was found to complex NO, suggesting that the high-spin heme could be the substrate binding site and that NO could be an intermediate present in an enzyme-bound form.