Assimilatory sulfur metabolism in marine microorganisms: Sulfur metabolism,protein synthesis,and growth of Pseudomonas halodurans and Alteromonas luteo-violaceus during unperturbed batch growth

Analysis of the distribution of ³⁵S-sulfate and ¹⁴C-glutamate in major biochemical components of the two marine bacteria, Pseudomonas halodurans and Alteromonas luteo-violaceus, was compared with cell density and total cellular protein during exponential growth in batch culture. For both organisms, the sulfur distribution was restricted principally to the low molecular weight organic and protein fractions, which together accounted for over 90% of the total sulfur. Carbon was more widely distributed, with these two fractions containing only 70% of the total label.Growth rate constants calculated from increases in cell numbers, protein, and ³⁵S and ¹⁴C in the various fractions indicated nearly balanced growth in A. luteo-violaceus, with constants derived from all biosynthetic parameters agreeing within 5% during the exponential phase. In contrast, protein synthesis and ³⁵S incorporation into residue protein constants were 30% higher than constants derived from cell counts and incorporation of ¹⁴C in P. halodurans. Therefore the cellular protein content P. halodurans varied over a two-fold range, with maximum protein per cell in the late exponential phase. A distinct reduction in the rate constants for total protein and ³⁵S incorporation into residue protein foreshadowed entry into the stationary phase more than one generation before other parameters.Incorporation of ³⁵S-sulfate into residue protein paralleled protein synthesis in both bacteria. The weight percent S in protein agreed well with the composition of an average protein derived from the literature. Sulfur incorporation into protein may be a useful measurement of marine bacterial protein synthesis.Abbreviations L.M.W. low molecular weight - TCA trichloroacetic acid - CFU colony forming unit     

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.     

Microbial Activities in Undecompressed and Decompressed Deep-Seawater Samples

Microbial transformations of ¹⁴C-labeled substrates (sodium glutamate, Casamino Acids, glucose, and sodium acetate) were measured in undecompressed seawater samples collected from depths of 1,800 to 6,000 m, during 14- to 21-day incubation periods at in situ temperature (3°C). Each substrate was tested at two concentrations (ca. 0.5 and 5.0 μg/ml) and two in situ pressures. The data were compared to 1-atmosphere (ca. 1.013 × 10² kPa) controls. The rates of ¹⁴C incorporation and ¹⁴CO₂ production as well as the amounts of total substrate utilization were generally lower at pressure than in the decompressed controls but were significantly different for each of the four substrates used. The utilization of acetate was the least affected by pressure; rates were similar to those measured at 1 atmosphere in two out of four experiments. In contrast, transformation rates of the amino acids at pressure averaged to only 38% of those in the controls. A single but reproducible “barophilic” response was observed with glucose as a substrate in samples collected from a depth of 4,500 m at a specific area in the northwestern Atlantic Ocean. Except for this latter set of experiments, the transformation of all substrates showed an increased lag period at pressure as compared to the 1-atmosphere controls.     

Cell recovery by continuous flotation

Summary Cell recovery by means of continuous flotation of the Hansenula polymorpha cultivation medium without additives was investigated as a function of the cultivation conditions as well as of the flotation equipment construction and flotation operational parameters. The cell enrichment and separation is improved at high liquid residence times, high aeration rates, small bubble sizes, increasing height of the aerated column, and diameter of the foam column. Increasing cell age and cultivation with nitrogen limitation reduce the cell separation.Symbols C_P cell mass concentration in medium g·l^–1 - C_R cell mass concentration in residue g·l^–1 - C_S cell mass concentration in foam liquid g·l^–1 - V equilibrium foam volume cm³ - V gas flow rate through the aerated liquid column cm³·s^–1 - V_F feed rate to the flotation column ml/min - ¹ V _S/V foaminess s - mean liquid residence time in the column s     

Characterization and regulation of the expression of scyllatoxin (Leiurotoxin I) receptors in the human neuroblastoma cell line NB-OK 1.

125I-[Tyr2]scyllatoxin allowed to label a single class of high-affinity receptors in membranes from the human neuroblastoma cell line NB-OK 1. The Kd of these receptors was 60 pM for scyllatoxin (Leiurotoxin I) and 20 pM for apamin and the Bmax was low (3.8 fmol/mg membrane protein). K+ increased toxin binding at low concentrations but exerted opposite effects at high concentrations. Ca2+, guanidinium and Na+ exerted only inhibitory effects on binding. Scyllatoxin binding sites were overexpressed 2.5-fold after a 24-h cell pretreatment with 2 mM butyrate. This effect was suppressed by cycloheximide.     

Syntheses of branched-chain sugars with push-pull functionality

The reaction of methyl 4,6-O-benzylidene-3(2)-deoxy-- -erythro-hexopyranosid-2(3)-ulose with carbon disulfide, alkyl iodide, and sodium hydride gave methyl 4,6-O-benzylidene-3(2)-[bis(alkylthio)methylene]-3(2)-deoxy-- -erythro-hexopyranosid-2(3)-uloses. Methyl 4,6-O-benzylidene-2-[bis(methylthio)methylene]-2-deoxy-- -erythro-hexopyranosid-3-ulose (5) reacted with aromatic amines to give, in a rearrangement process, N-aryl-2-aryliminomethyl-4,6-O-benzylidene-2-deoxy-- -erythro-hex-1-enopyranosylamin-3-uloses. The reaction of 5 which hydrazine hydrate afforded 5-methylthio-(methyl-4,6-O-benzylidene-2,3-dideoxy-- -erythro-hexopyranosido)[3,2-c]pyrazole.     

The activation of adenylate cyclase by pituitary adenylate cyclase activating polypeptide (PACAP) via helodermin-preferring VIP receptors in human SUP-T1 lymphoblastic membranes.

Competition binding curves, using [125I-acetyl-His1]PACAP-27 as radioligand and dose-effect curves of adenylate cyclase activation in human SUP-T1 lymphoblastic membranes showed that PACAP-27 and PACAP-38 stimulate the enzyme through a single class of helodermin-preferring VIP receptors with the following order of potency: helodermin = [acetyl-His1]PACAP-27 greater than PACAP-38 greater than PACAP-27 greater than VIP. PACAP (6-27) (Ki 0.5-0.8 microM) and [Des-His1, Asn3]PACAP-27 (Ki 1-2 microM) acted as competitive antagonists. Using a series of 13 PACAP-27 analogues and fragments and three VIP analogues, we identified positions 1, 2, 3, 9 and 13 in PACAP-27 as being of importance for high-affinity binding. Thus, we added further evidence for considering that the present helodermin-preferring VIP receptors, when compared to a majority of VIP receptors and PACAP receptors, exhibit an original specificity pattern.     

Levels of hypoxanthine phosphoribosyltransferase RNA in human cells

The gene for the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT) is expressed at a low level in many cells. As is the case with several other “housekeeping genes,” thorough studies of hprt gene regulation have been hampered by the low levels of its mRNA. We have used RNA/RNA hybridization in solution to determine the concentration of hprt-RNA in human cells. The sensitivity and specificity of the method have been validated, and it is shown that hprt-RNA can be accurately determined at a level of a few mRNA molecules per cell. As expected for a housekeeping gene, low and relatively constant hprt-RNA levels (0.3–0.8 pg/μg DNA) were found in primary cultures of normal amnion cells and fibroblasts, EBV-transformed lymphoblastoid cell lines, neuroblastoma, glioblastoma, and melanoma cell cultures. While resting lymphocytes were found to contain very low amounts of hprt-RNA, lymphocytes stimulated with phytohemagglutinin (PHA) showed a 10-fold increase to about 0.8–1.2 pg/μg DNA, which corresponds to 6–10 hprt-RNA molecules per cell. The level started to increase about 20 h after PHA stimulation, 5–10 h before the onset of DNA synthesis, and a steady-state level was reached after 2–3 days in culture. In PHA-stimulated lymphocytes from two brothers with inherited HPRT deficiency (LeschNyhans syndrome), the hprt-RNA level in PHA-stimulated lymphocytes was only about 25% of that in normal subjects. In T-cells selected for HPRT deficiency by growth in 6-thioguanine medium, the levels of hprt-RNA were either normal or very low, which probably reflects the different nature of the mutations involved. These results demonstrate the sensitivity of this method for determinations of low levels of RNA and clearly show induction of hprt-RNA after mitogenic stimulation of human lymphocytes.     

Influence of different data cleaning solutions of point‐occurrence records on downstream macroecological diversity models

Digital point‐occurrence records from the Global Biodiversity Information Facility (GBIF) and other data providers enable a wide range of research in macroecology and biogeography. However, data errors may hamper immediate use. Manual data cleaning is time‐consuming and often unfeasible, given that the databases may contain thousands or millions of records. Automated data cleaning pipelines are therefore of high importance. Taking North American Ephedra as a model, we examined how different data cleaning pipelines (using, e.g., the GBIF web application, and four different R packages) affect downstream species distribution models (SDMs). We also assessed how data differed from expert data. From 13,889 North American Ephedra observations in GBIF, the pipelines removed 31.7% to 62.7% false positives, invalid coordinates, and duplicates, leading to datasets between 9484 (GBIF application) and 5196 records (manual‐guided filtering). The expert data consisted of 704 records, comparable to data from field studies. Although differences in the absolute numbers of records were relatively large, species richness models based on stacked SDMs (S‐SDM) from pipeline and expert data were strongly correlated (mean Pearson''s r across the pipelines: .9986, vs. the expert data: .9173). Our results suggest that all R package‐based pipelines reliably identified invalid coordinates. In contrast, the GBIF‐filtered data still contained both spatial and taxonomic errors. Major drawbacks emerge from the fact that no pipeline fully discovered misidentified specimens without the assistance of taxonomic expert knowledge. We conclude that application‐filtered GBIF data will still need additional review to achieve higher spatial data quality. Achieving high‐quality taxonomic data will require extra effort, probably by thoroughly analyzing the data for misidentified taxa, supported by experts.