Photoproduction of indole 3-acetic acid by Rhodobacter sphaeroides from indole and glycine

Rhodobacter sphaeroides photoproduced indole 3-acetic acid (IAA) when the precursor L-tryptophan was added to the basal medium. Of the other organic carbon sources that influenced IAA formation from L-tryptophan, -ketoglutaric acid gave the maximum formation of 530 mg 1^–1 in less than 24 h of illuminated anaerobic incubation. IAA was also produced from indole, glycine and glucose together by Rb. sphaeroides under photoanaerobic conditions yielding 61 mg l^–1 within 30 fh.     

Effect of ammonium and nitrate on growth and appearance of nitrate reductase and nitrite reductase in dark- and light-grown mustard seedlings

Nitrate-induced and phytochrome-modulated appearance of nitrate reductase (NR; EC 1.6.6.1) and nitrite reductase (NIR; EC 1.7.7.1) in the cotyledons of the mustard (Sinapis alba L.) seedling is strongly affected by externally supplied ammonium (NH ₄ ⁺ ). In short-term experiments between 60 and 78 h after sowing it was found that in darkness NH ₄ ⁺ —simultaneously given with NO ₃ ^- —strongly inhibits appearance of nitrate-inducible NR and NIR whereas in continuous far-red light—which operates exclusively via phytochrome without significant chlorophyll formation —NH ₄ ⁺ (simultaneously given with NO ₃ ^- ) strongly stimulates appearance of NR. The NIR levels are not affected. This indicates that NR and NIR levels are regulated differently. In the absence of external NO ₃ ^- appearance of NR is induced by NH₄ in darkness as well as in continuous far-red light whereas NIR levels are not affected. On the other hand, in the absence of external NO ₃ ^- , exogenous NH ₄ ⁺ strongly inhibits growth of the mustard seedling in darkness as well as in continuous far-red light. This effect can be abolished by simultaneously supplying NO ₃ ^- . The adverse effect of NH ₄ ⁺ on growth (NH ₄ ⁺ -toxicity) cannot be attributed to pH-changes in the medium since it was shown that neither the growth responses nor the changes of the enzyme levels are related to pH changes in the medium. Non-specific osmotic effects are not involved either.Abbreviations c continuous - D darkness - FR far-red light - NIR nitrite reductase (EC 1.7.7.1) - NR nitrate reductase (EC 1.6.6.1)     

<Emphasis Type="Italic">Anoxybacillus sediminis</Emphasis> sp. nov., a novel moderately thermophilic bacterium isolated from a hot spring

A Gram-stain positive, moderately thermophilic, aerobic, spore-forming and rod-shaped bacterium, designated YIM 73012^T, was isolated from a sediment sample collected from a hot spring located in Tibet, China, and was characterized by using a polyphasic taxonomy approach. The strain is oxidase positive and catalase negative. Growth occurred at 37–65 °C (optimum, 45–50 °C), at pH 6.0–8.5 (optimum, pH 7.0–7.5) and with 0.5–3.5% NaCl (optimum, 0.5–1.0%, w/v). The major fatty acids were iso-C_15:0, iso-C_16:0 and C_16:0. The major polar lipids comprised of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine and phosphatidylglycerol. The cell wall peptidoglycan contained meso-diaminopimelic acid. The respiratory quinone was MK-7. The G+C content of genomic DNA was 43.6 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strain YIM 73012^T forms a distinct lineage with respect to the genus Anoxybacillus in the family Bacillaceae. Based on 16S rRNA gene sequence identities the closely related phylogenetic neighbours are Anoxybacillus caldiproteolyticus DSM 15730^T (96.7%) and Saccharococcus thermophilus DSM 4749^T (96.6%). Strain YIM 73012^T was distinguishable from the closely related reference strains by the differences in phenotypic, chemotaxonomic and genotypic characteristics, and represents a novel species of the genus Anoxybacillus, for which the name Anoxybacillus sp. nov. is proposed. The type species is Anoxybacillus sediminis sp. nov., with the type strain YIM 73012^T (=?KCTC 33884^T?=?DSM 103835^T).     

<Emphasis Type="Italic">Phenylobacterium terrae</Emphasis> sp. nov., isolated from a soil sample of Khyber-Pakhtun-Khwa,Pakistan

A Gram-stain negative, aerobic, motile, non-spore-forming and rod-shaped bacterial strain, designated YIM 730227^T, was isolated from a soil sample, collected from Karak district, Khyber-Pakhtun-Khwa, Pakistan. The bacterium was characterized using a polyphasic taxonomic approach. Pairwise comparison of the 16S rRNA gene sequences showed that strain YIM 730227^T is closely related to Phenylobacterium lituiforme FaiI3^T (97.5% sequence similarity), Phenylobacterium muchangponense A8^T (97.4%), Phenylobacterium panacis DCY109^T (97.1%), Phenylobacterium immobile E^T (97.1%) and Phenylobacterium composti 4T-6^T (97.0%), while also sharing 98.0% sequence similarity with Phenylobacterium hankyongense HKS-05^T after NCBI blast, showing it represents a member of the family Caulobacteraceae. The major respiratory quinone was Q-10 and the major fatty acids were C_16:0, summed feature 8 (comprising C_18:1ω7c and/or C_18:1ω6c), C_18:1ω7c 11-methyl and C_17:0. The polar lipids were phosphatidylglycerol, unidentified glycolipids, phospholipid and unidentified lipid. The G?+?C content of the genomic DNA was 68.2 mol%. The DNA–DNA relatedness values of strain YIM 730227^T with P. hankyongense HKS-05^T, P. lituiforme FaiI3^T, P. muchangponense A8^T, P. panacis DCY109^T, P. immobile E^T and P. composti 4T-6^T were 31.3?±?0.6, 26.1?±?0.2, 24.3?±?0.1, 21.8?±?0.9, 19.8?±?0.6 and 18.2?±?1.1%, respectively, values lower than 70%. Besides the morphological and chemotaxonomic characteristics, phylogenetic analyses of 16S rRNA gene sequences and the biochemical characteristics indicated that the strain YIM 730227^T represents a novel member of the genus Phenylobacterium, for which the name Phenylobacterium terrae sp. nov. (type strain YIM 730227^T =?KCTC62324^T?=?CGMCC 1.16326^T) is proposed.     

Developmental effects of fumonisin B1 in mice

Developmental and toxic effects of aqueous extracts of F. moniliforme culture material containing known levels of fumonisin B₁ were recently reported in mice and included maternal hepatotoxicity and lethality, maternal body weight gain reduction, increased embryonic resorptions, reduced offspring body weights, and fetal malformations including cleft palate, hydrocephalus, malformed ribs and incomplete digital and sternal ossification. These studies also suggested that the effects of the fungal extract on the mouse offspring may be mediated via maternal effects. The contribution of fumonisin B₁ (FB₁), a major toxic metabolite of F moniliforme, in the induction of these effects was evaluated in this study by administering 0 to 100 mg pure FB₁/kg of body weight on gestational days (GD) 7 through 15 to pregnant Charles River CD1 mice and assessing maternal health and fetal development till the end of gestation. Doses of 25 mg/kg or higher of pure FB₁ induced maternal liver lesions (mostly necrotic changes), associated with ascites and increased hepatocytic nuclear diameter. Fumonisin doses of 50 mg/kg or higher also resulted in significantly increased maternal ALT on GD12, and reduced offspring bodyweights on GD 18. Increased resorptions and decreased numbers of live offspring were only evident at 100 mg FB₁/kg body weight. Offspring exhibited dose-dependent increase in the incidence and severity of hydrocephalus of both the lateral and third ventricles at doses of 25 mg/kg or higher. Doses of 25 mg/kg or higher also increased the sphinganine/sphingosine (Sa/So) ratios in maternal but not fetal livers. These results suggest that FB₁ may be a developmental toxicant accounting for most but not all earlier reported effects of F. moniliforme culture extract. Association of FB₁ effects on the offspring with maternal hepatoxicity and with alteration of Sa/So ratio in maternal but not fetal liver supported the earlier claim that FB₁ effects on the mouse offspring are mediated by maternal hepatotoxicity.     

Developmental effects of fumonisin B1-containingFusarium moniliforme culture extract in CD1 mice

Pregnant Charles River CD1 mice were treated with a semipurified extract ofFusarium moniliforme culture containing 0, 12.5, 25, 50 or 100 mg FB₁/kg each day orally (diluted in distilled water) between gestational days (GD) 7 and 15 to evaluate the developmental toxicity of FB₁. Following sacrifice of dams on GD 18, litters were examined for gross abnormalities and divided equally for skeletal or visceral examination by routine techniques. Significant maternal mortality was observed at doses of 50 and 100 mg FB₁/kg. Dose-dependant decreases in maternal body weight gains, number of live offsprings per litter, and mean body weight of the offspring were produced at FB₁ doses of 25 mg/kg or higher. The percentage of implants resorbed increased at all doses in a dose-dependant manner. A dose-dependant increase, except at the lowest dose tested, in the incidence of ossification deficits involving digits and sternum, short and wavy ribs, and hydrocephalus of lateral and third ventricles was also evident. Cleft palate was seen only at the highest FB₁ dose. Maternal intoxication manifested as a dose-dependant increase in the severity of ascites associated mainly with increased histopathologic scores reflecting hepatocellular damage at day 18. Concommittant increases in serum alanine amino transferase (ALT) on GD 12, reflecting parenchymal liver cell damage, was also observed at all doses above 12.5 mg of FB₁/kg. These results suggest that FB₁-containingF. moniliforme culture extract is developmentally toxic in mice, and that this toxicity may be mediated by maternal hepatotoxicity.     

Liver fatty acid-binding protein initiates budding of pre-chylomicron transport vesicles from intestinal endoplasmic reticulum

The rate-limiting step in the transit of absorbed dietary fat across the enterocyte is the generation of the pre-chylomicron transport vesicle (PCTV) from the endoplasmic reticulum (ER). This vesicle does not require coatomer-II (COPII) proteins for budding from the ER membrane and contains vesicle-associated membrane protein 7, found in intestinal ER, which is a unique intracellular location for this SNARE protein. We wished to identify the protein(s) responsible for budding this vesicle from ER membranes in the absence of the requirement for COPII proteins. We chromatographed rat intestinal cytosol on Sephacryl S-100 and found that PCTV budding activity appeared in the low molecular weight fractions. Additional chromatographic steps produced a single major and several minor bands on SDS-PAGE. By tandem mass spectroscopy, the bands contained both liver and intestinal fatty acid-binding proteins (L- and I-FABP) as well as four other proteins. Recombinant proteins for each of the six proteins identified were tested for PCTV budding activity; only L-FABP and I-FABP (23% the activity of L-FABP) were active. The vesicles generated by L-FABP were sealed, contained apolipoproteins B48 and AIV, were of the same size as PCTV on Sepharose CL-6B, and by electron microscopy, excluded calnexin and calreticulin but did not fuse with cis-Golgi nor did L-FABP generate COPII-dependent vesicles. Gene-disrupted L-FABP mouse cytosol had 60% the activity of wild type mouse cytosol. We conclude that L-FABP can select cargo for and bud PCTV from intestinal ER membranes.     

P2Y1 Receptor Activation of the TRPV4 Ion Channel Enhances Purinergic Signaling in Satellite Glial Cells

Transient receptor potential (TRP) ion channels of peripheral sensory pathways are important mediators of pain, itch, and neurogenic inflammation. They are expressed by primary sensory neurons and by glial cells in the central nervous system, but their expression and function in satellite glial cells (SGCs) of sensory ganglia have not been explored. SGCs tightly ensheath neurons of sensory ganglia and can regulate neuronal excitability in pain and inflammatory states. Using a modified dissociation protocol, we isolated neurons with attached SGCs from dorsal root ganglia of mice. SGCs, which were identified by expression of immunoreactive Kir4.1 and glutamine synthetase, were closely associated with neurons, identified using the pan-neuronal marker NeuN. A subpopulation of SGCs expressed immunoreactive TRP vanilloid 4 (TRPV4) and responded to the TRPV4-selective agonist GSK1016790A by an influx of Ca²⁺ ions. SGCs did not express functional TRPV1, TRPV3, or TRP ankyrin 1 channels. Responses to GSK1016790A were abolished by the TRPV4 antagonist HC067047 and were absent in SGCs from Trpv4^−/− mice. The P2Y₁-selective agonist 2-methylthio-ADP increased [Ca²⁺]_i in SGCs, and responses were prevented by the P2Y1-selective antagonist MRS2500. P2Y₁ receptor-mediated responses were enhanced in TRPV4-expressing SGCs and HEK293 cells, suggesting that P2Y₁ couples to and activates TRPV4. PKC inhibitors prevented P2Y₁ receptor activation of TRPV4. Our results provide the first evidence for expression of TRPV4 in SGCs and demonstrate that TRPV4 is a purinergic receptor-operated channel in SGCs of sensory ganglia.     

Luminescent trimethoprim-polyaminocarboxylate lanthanide complex conjugates for selective protein labeling and time-resolved bioassays

Labeling proteins with long-lifetime emitting lanthanide (III) chelate reporters enables sensitive, time-resolved luminescence bioaffinity assays. Heterodimers of trimethoprim (TMP) covalently linked to various cs124-sensitized, polyaminocarboxylate chelates stably retain lanthanide ions and exhibit quantum yields of europium emission up to 20% in water. A time-resolved, luminescence resonance energy transfer (LRET) assay showed that TMP-polyaminocarboxylates bind to Escherichia coli dihydrofolate reductase (eDHFR) fusion proteins with nanomolar affinity in purified solutions and in bacterial lysates. The ability to selectively impart terbium or europium luminescence to fusion proteins in complex physiological mixtures bypasses the need for specific antibodies and simplifies sample preparation.