Assimilatory Sulfur Metabolism in Marine Microorganisms: Considerations for the Application of Sulfate Incorporation into Protein as a Measurement of Natural Population Protein Synthesis

The sulfur content of residue protein was determined for pure cultures of Nitrosococcus oceanus, Desulfovibrio salexigens, 4 mixed populations of fermentative bacteria, 22 samples from mixed natural population enrichments, and 11 nutritionally and morphologically distinct isolates from enrichments of Sargasso Sea water. The average 1.09 ± 0.14% (by weight) S in protein for 13 pure cultures agrees with the 1.1% calculated from average protein composition. An operational value encompassing all mixed population and pure culture measurements has a coefficient of variation of only 15.1% (n = 41). Short-term [³⁵S]sulfate incorporation kinetics by Pseudomonas halodurans and Alteromonas luteoviolaceus demonstrated a rapid appearance of ³⁵S in the residue protein fraction which was well modelled by a simple exponential uptake equation. This indicates that little error in protein synthesis determination results from isotope dilution by endogenous pools of sulfur-containing compounds. Methionine effectively competed with sulfate for protein synthesis in P. halodurans at high concentrations (10 μM), but had much less influence at 1 μM. Cystine competed less effectively with sulfate, and glutathione did not detectably reduce sulfate-S incorporation into protein. [³⁵S]sulfate incorporation was compared with [¹⁴C]glucose assimilation in a eutrophic brackish-water environment. Both tracers yielded similar results for the first 8 h of incubation, but a secondary growth phase was observed only with ³⁵S. Redistribution of ¹⁴C from low-molecular-weight materials into residue protein indicated additional protein synthesis. [³⁵S]sulfate incorporation into residue protein by marine bacteria can be used to quantitatively measure bacterial protein synthesis in unenriched mixed populations of marine bacteria.     

Effects of excess selenomethionine on selenium status indicators in pregnant long-tailed macaques (Macaca fascicularis)

Forty pregnant long-tailed macaques were treated daily for 30 d with 0, 25, 150 or 300 μg selenium as L-selenomethionine/kg body weight. Erythrocyte and plasma selenium and glutathione peroxidase specific activities, hair and fecal selenium, and urinary selenium excretion were increased by and were linearly related to L-selenomethionine dose. Hair selenium was most sensitive to L-selenomethionine dose, with an 84-fold increase in the 300 μg selenium/(kg-d) group relative to controls (r=0.917). Daily urinary selenium excretion (80-fold,r=0.958), plasma selenium (22-fold,r=0.885), erythrocyte selenium (24-fold,r=0.920), and fecal selenium (18-fold,r=0.911) also responded strongly to L-selenomethionine. Erythrocyte and plasma glutathione peroxidase specific activities increased 154% and 69% over controls, respectively. Toxicity was associated with erythrocyte selenium >2.3 μg/mL, plasma selenium >2.8 μg/mL, and hair selenium >27 μg/g. Plasma, erythrocyte, and hair selenium concentrations may be useful for monitoring and preventing the toxicity of L-selenomethionine administered to humans in cancer chemoprevention trials.     

Aerial web-weaving spiders: Linking molecular and organismal processes in evolution

Aerial web-weaving spiders display a wide variety of foraging behaviors that can be tied to the evolution of one family of proteins, the silks. In some cases, the physical structure and mechanical properties of silks alone determine the ecology of spiders: the habitats in which they forage, the prey they capture and their subsequent reproductive success. Future studies that integrate research on the physical structure of silks, the molecular genetics of silk synthesis and the foraging ecology of spiders in primitive and derived phylogenetic groups could reveal how molecular and organismal processes interact in evolution.     

Characterization of an insulin receptor mutant lacking the subunit processing site

An insulin receptor mutant was constructed utilizing site-directed mutagenesis to delete the Arg-Lys-Arg-Arg basic amino acid cleavage site (positions 720-723) from the cDNA encoding the human insulin proreceptor. This mutant was transfected into Chinese hamster ovary cells. Immunoprecipitation of metabolically labeled cells revealed a 205-kDa proreceptor which bound to wheat germ agglutinin. Processed 130-kDa alpha and 95-kDa beta subunits were also observed and contained approximately 20% as much protein as the proreceptor on a molar basis. Trypsin digestion of intact metabolically labeled cells decreased the proreceptor band by 80%. Pulse-chase studies revealed a half-life of 28 h for the proreceptor. When cells were photolabeled with 125I-B2(2-nitro-4-azidophenylacetyl)-des-PheB1 (NAPA)-insulin, the proreceptor incorporated 10% as much label as the 130-kDa alpha subunit in spite of a 5-fold molar excess. Incubation of NAPA-labeled cells at 37 degrees C for 20 min resulted in 60% of the labeled subunits, but little labeled proreceptor, becoming resistant to trypsin degradation. Immunoprecipitation of NAPA-insulin-stimulated cells with anti-phosphotyrosine antibodies revealed that 62% of the processed labeled receptors, but very little proreceptor, contained phosphotyrosine. Thus, this mutant receptor is synthesized, glycosylated, and expressed on the cell surface as uncleaved proreceptor, although some processing to alpha and beta subunits still occurs. It exhibits a markedly decreased affinity for insulin, and when insulin is bound to, demonstrates defective internalization, down-regulation, and autophosphorylation. These data suggest that cleavage of the mutant proreceptor into subunits is required not only for the development of high affinity binding sites, but also for normal transduction of the signal which activates the beta subunit tyrosine kinase.     

Biological Control of Damping-Off of Alfalfa Seedlings with Bacillus cereus UW85

We explored the potential of biological control of alfalfa (Medicago sativa L.) seedling damping-off caused by Phytophthora megasperma f. sp. medicaginis by screening root-associated bacteria for disease suppression activity in a laboratory bioassay. A total of 700 bacterial strains were isolated from the roots of field-grown alfalfa plants by using Trypticase soy agar. A simple, rapid assay was developed to screen the bacteria for the ability to reduce the mortality of Iroquois alfalfa seedlings that were inoculated with P. megasperma f. sp. medicaginis zoospores. Two-day-old seedlings were planted in culture tubes containing moist vermiculite, and each tube was inoculated with a different bacterial culture. Sufficient P. megasperma f. sp. medicaginis zoospores were added to each tube to result in 100% mortality of control seedlings. Of the 700 bacterial isolates tested, only 1, which was identified as Bacillus cereus and designated UW85, reduced seedling mortality to 0% in the initial screen and in two secondary screens. Both fully sporulated cultures containing predominantly released spores and sterile filtrates of these cultures of UW85 were effective in protecting seedlings from damping-off; filtrates of cultures containing predominantly vegetative cells or endospores inside the parent cell had low biocontrol activity. Cultures grown in two semidefined media had significantly greater biocontrol activities than cultures grown in the complex tryptic soy medium. In a small-scale trial in a field infested with P. megasperma f. sp. medicaginis, coating seeds with UW85 significantly increased the emergence of alfalfa. The results suggest that UW85 may have potential as a biocontrol agent for alfalfa damping-off, thus providing an alternative to current disease control strategies.     

Technique for Simultaneous Determination of [35S]Sulfide and [14C]Carbon Dioxide in Anaerobic Aqueous Samples

A technique for the simultaneous determination of [³⁵S]sulfide and [¹⁴C]carbon dioxide produced in anaerobic aqueous samples dual-labeled with [³⁵S]sulfate and a ¹⁴C-organic substrate is described. The method involves the passive distillation of sulfide and carbon dioxide from an acidified water sample and their subsequent separation by selective chemical absorption. The recovery of sulfide was 93% for amounts ranging from 0.35 to 50 μmol; recovery of carbon dioxide was 99% in amounts up to 20 μmol. Within these delineated ranges of total sulfide and carbon dioxide, 1 nmol of [³⁵S]sulfide and 7.5 nmol of [¹⁴C]carbon dioxide were separated and quantified. Correction factors were formulated for low levels of radioisotopic cross-contamination by sulfide, carbon dioxide, and volatile organic acids. The overall standard error of the method was ±4% for sulfide and ±6% for carbon dioxide.     

Dissolved Hydrocarbons and Related Microflora in a Fjordal Seaport: Sources, Sinks, Concentrations, and Kinetics

The continuous addition of toluene as a solute of treated ballast water from oil tankers into a well-defined estuary facilitated the study of the dynamics of dissolved hydrocarbon metabolism in seawater. Most rates of toluene oxidation were in the range of 1 to 30 pg/liter per h at 0.5 μg of toluene per liter. Near the ballast water injection point, a layer of warm ballast water, rich in bacteria, that was trapped below the less-dense fresh surface water was located. Toluene residence times were approximately 2 weeks in this layer, 2 years elsewhere in Port Valdez, and 2 decades in the surface water of a more oceanic receiving estuary adjacent. Mixing was adequate for a steady-state treatment which showed that 98% of the toluene was flushed from Port Valdez before metabolism and gave a steady-state concentration of 0.18 μg/liter. Total bacterial biomass from direct counts and organism size data was usually near 0.1 mg/liter, but ranged up to 0.8 mg/liter in the bacteria-rich layer. The origin of bacteria in this layer was traced to growth in oil tanker ballast during shipments. The biomass of toluene oxidizers in water samples was estimated from the average affinity of pure-culture isolates for toluene (28 liters per g of cells per h) and observed toluene oxidation kinetics. Values ranged from nearly all of the total bacterial biomass within the bacteria-rich layer down to 0.2% at points far removed. Because the population of toluene oxidizers was large with respect to the amount of toluene consumed and because water from a nearby nonpolluted estuary was equally active in facilitating toluene metabolism, we searched for an additional hydrocarbon source. It was found that terpenes could be washed from spruce trees by simulated rainfall, which suggested that riparian conifers provide an additional and significant hydrocarbon source to seawater.     

Partial characterization of a Trypanosoma cruzi-released decomplementing factor

Trypanosoma cruzi releases a factor (SCAF) when grown in vitro which decomplements normal mouse, human, and guinea pig sera. The production and potency of SCAF was dependent on the density of cultured parasites, parasite viability and proliferative capacity, and duration of culture. The in vitro interaction between SCAF and serum complement (C') occurred rapidly and was complete within 30 min of mixing. The administration of SCAF to normal mice resulted in up to 50% reduction in hemolytic C' activity, whereas SCAF had no effect on the C' levels in mice infected wit T. cruzi for more than 10 days. The active moiety of SCAF was shown to be a nonproteinaceous substance(s) with a molecular weight of approximately 23,000 daltons.     

The biotransformation of α-(2, 4, 6-trimethylphenyl) ethylamine by rabbit liver preparations

Microsomal incubation of the parent amine, α-(2, 4, 6-trimethylphenyl) ethylamine, VI, produced imine, II, alcohol, IV, oxime, V, and several unknowns. The isolation of imine, II, produces evidence that imines may be involved in the microsomal deamination of primary amines. Separate incubations of the imine, II, produced oxime, V, and several unknowns, one of which is tentatively identified as the nitro derivative. Incubation of the oxime, V, gave the alcohol, IV, the “nitro” metabolite as above and 2 unknowns. Incubation of the hydroxylamine, VII, gave oxime, V, alcohol, IV, 2 unknowns and the “nitro” metabolite.     

Inhibitory effects of different quantitative compositions of Hymenaea leaf resins on a generalist herbivore Spodoptera exigua

The hypotheses that quantitative variation in leaf resins in the leguminous genus Hymenaea may partially be a response to insect predation was tested in feeding experiments with the generalist herbivore, the beet armyworm, Spodoptera exigua Hübn (Noctuidae). Leaf resins of all Hymenaea species are comprised of essentially the same suite of sesquiterpene hydrocarbons, but discrete quantitative patterns have been classified as Compositional Types based on the amounts of major components. In this work pure extracts of leaf resins of Type I (high - and β-selinene), Type II (intermediate amounts of -and β-selinene and caryophyllene) and Type III (high caryophyllene) were incorporated into an artificial diet for the insects at 1 and 3.2% (dry wt). Resin Type treatments produced differential dose-dependent effects on growth rate (lower larval weights and increased time to pupation) and in mortality. More significant inhibitory effects occurred in Compositional Types with a predominance of a single compared (i.e. Types I and III). Significantly higher mortality occurring in Type III treatments suggests that caryophllene may have higher potential toxicity than - and β-selinene for S. exigua. These experiments indicate that feeding by generalist herbivores could be a factor determining quantitative compositional variation: (a) among populations; (b) during the development of leaves; (c) in the spatial distribution within the leaves; and (d) between parent tree and seedling progeny.