A widely distributed hydrogenase oxidises atmospheric H2 during bacterial growth

Diverse aerobic bacteria persist by consuming atmospheric hydrogen (H₂) using group 1h [NiFe]-hydrogenases. However, other hydrogenase classes are also distributed in aerobes, including the group 2a [NiFe]-hydrogenase. Based on studies focused on Cyanobacteria, the reported physiological role of the group 2a [NiFe]-hydrogenase is to recycle H₂ produced by nitrogenase. However, given this hydrogenase is also present in various heterotrophs and lithoautotrophs lacking nitrogenases, it may play a wider role in bacterial metabolism. Here we investigated the role of this enzyme in three species from different phylogenetic lineages and ecological niches: Acidithiobacillus ferrooxidans (phylum Proteobacteria), Chloroflexus aggregans (phylum Chloroflexota), and Gemmatimonas aurantiaca (phylum Gemmatimonadota). qRT-PCR analysis revealed that the group 2a [NiFe]-hydrogenase of all three species is significantly upregulated during exponential growth compared to stationary phase, in contrast to the profile of the persistence-linked group 1h [NiFe]-hydrogenase. Whole-cell biochemical assays confirmed that all three strains aerobically respire H₂ to sub-atmospheric levels, and oxidation rates were much higher during growth. Moreover, the oxidation of H₂ supported mixotrophic growth of the carbon-fixing strains C. aggregans and A. ferrooxidans. Finally, we used phylogenomic analyses to show that this hydrogenase is widely distributed and is encoded by 13 bacterial phyla. These findings challenge the current persistence-centric model of the physiological role of atmospheric H₂ oxidation and extend this process to two more phyla, Proteobacteria and Gemmatimonadota. In turn, these findings have broader relevance for understanding how bacteria conserve energy in different environments and control the biogeochemical cycling of atmospheric trace gases.Subject terms: Environmental microbiology, Biogeochemistry     

sn-1,2-diacylglycerols and phorbol diesters: uptake, metabolism, and subsequent assimilation of the diacylglycerol metabolites into complex lipids of cultured cells

The cell-permeable diacylglycerol mediators have been shown to mimic partially the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on cultured cells. In order to evaluate the metabolic stability of the lipid mediators, several radiolabeled diacylglycerols were synthesized and their uptake and intracellular fate in cultured HL-60 (human promyelocytic leukemia) cells was compared with TPA. In addition to whole cell assessment, the stability of diacyl lipids and TPA was evaluated in a buffer/water system and in the presence of serum and subcellular fractions. The compounds studied include 1,2-dioleoyl-sn-glycerol (DiOG), 1-oleoyl-2-acetyl-sn-glycerol (OaG), 1-palmitoyl-2-acetyl-sn-glycerol (PaG), the ether-linked analog 1-palmityl-2-acetyl-sn-glycerol (ePaG), and TPA. TPA was comparatively stable to lipase hydrolysis in all systems examined. First, the data show that within 5 min at pH 7.9, nearly 50% of the PaG (originally greater than 92% 1,2-isomer) had isomerized, and rapid formation of the 1,3-isomer also occurred with OaG and ePaG. The metabolism of OaG and PaG by serum hydrolases, using a reaction medium containing 10% serum, was chiefly by acetate hydrolysis; however, fatty acid was also liberated. After a 60-min incubation 68% of the [14C]OaG was converted, by serum enzymes, to monooleoylglycerol plus oleic acid. Heat-inactivation of serum reduced the enzymatic formation of fatty acid by 60-70%. ePaG was also metabolized by serum enzymes, but the ether-linked alkylglycerol product was stable. The results of cell-free studies (postmitochondrial supernatant) showed that cellular enzymes were present that could, like serum, convert the diacylglycerols to monoacylglycerols and free fatty acids. Studies using cultured cells showed that radiolabeled OaG, PaG, and ePaG were rapidly taken up by the cells and metabolized. Labeled metabolic products from the diacylglycerols appeared, in a time-dependent manner, in cellular phospholipids and triacylglycerols. The results from experiments employing 1-acyl-2-acetyl-sn-[3H]glycerol and [3H]acyl-2-acetyl-sn-glycerol indicate that the intracellular mode of mediator metabolism is via complete hydrolysis with subsequent incorporation of 3H-acyl groups into complex lipids. Data are also presented which show that a substantial amount of cellular lipid acyl group modification occurs and large amounts of glycerol are produced when cells are cultured with OaG. Collectively, these results demonstrate that the diacylglycerol mediators, when compared with TPA, are not stable and are metabolized by both serum and cellular enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Respiration and insulin release in mouse pancreatic islets. Effects of L-leucine and 2-ketoisocaproate in combination with D-glucose and L-glutamine

In addition to the 7-, 5- and 2-carboxyglutamyl varieties of dicoumarol-induced prothrombins (Malhotra, O.P. (1979) Thromb. Res. 15, 427-463), we have isolated two more atypical prothrombins, one containing 1.1 +/- 0.1 gamma-carboxyglutamic acid, '1-carboxyglutamyl prothrombin,' and the other less than 0.2, '0-carboxyglutamyl prothrombin.' Both variants showed a single component by analytical polyacrylamide-gel electrophoresis in the absence and in the presence of sodium dodecyl sulfate and contained antigenic activity indistinguishable from that of normal prothrombin. The pI of both proteins as assessed by electrofocusing was 4.835 +/- 0.015, compared with 4.58 for 10- and 7-, 4.75 for 5- and 4.81 for 2-carboxyglutamyl materials. By the two-stage prothrombin assay procedure, the 1- and 0-carboxyglutamyl variants generated thrombin, respectively 19 and 13% of normal prothrombin, and their activation times ranged from 4 to 7 h, compared with 7 min for normal. Kinetic studies, utilizing the one-stage coagulation assay, showed that both Km and tmin (minimal clotting time) increase proportionally with the decrease of gamma-carboxyglutamyl residues (from 10 to 7, 5, 2, 1 and 0 gamma-carboxyglutamic acids). Each of the five (partially) acarboxy prothrombins owe their clotting activity to gamma-carboxyglutamyl residues and not to the presence of some normal prothrombin molecules.     

Patterns of free-radical production after tourniquet ischemia: implications for the hand surgeon.

Since use of the pneumatic tourniquet is standard procedure for the hand surgeon, ischemic and reperfusion injury is a risk. To determine optimal periods of ischemia, 100 rabbit hindlimbs were subjected to various ischemic insults and analyzed for malondialdehyde (an indicator of free-radical production). Group 1 (3 hours of continuous ischemia) had 12.5 percent more reperfusion damage than controls (p less than 0.05). Group 2 (three 1-hour ischemic insults) had 10 percent more damage than controls (p less than 0.05). Group 3 (two 90-minute ischemic episodes) had 21 percent more damage than controls (p = 0.0001). Group 4 (4 1/2 hours of continuous ischemia) had 14.5 percent more damage than controls (p less than 0.01). Group 5 (three 90-minute ischemic episodes) had 10.8 percent more damage than controls (p less than 0.01). Group 6 (6 hours of continuous ischemia) had 17.5 percent more damage than controls (p less than 0.002). Group 7 (four 90-minute ischemic episodes) had 14 percent more damage than controls (p less than 0.01). Group 8 (three 2-hour ischemic episodes) had 22.5 percent more damage than controls (p less than 0.003). And group 9 (two 3-hour ischemic episodes) had 42 percent more damage than controls (p less than 0.0001). These results suggest a direct correlation in reperfusion injury with duration of tourniquet ischemia. Additionally, allowing specific reperfusion periods in some groups ultimately increased the amount of reperfusion injury.     

Intraovarian mechanisms in the hormonal control of granulosa cell differentiation in rats

The present review summarizes our studies on the hormonal control of the differentiation of granulosa cells in vitro. Studies on the hormonal regulation of granulosa cell oestrogen and progestagen biosynthesis by various regulatory agents facilitate our understanding of the control processes involved in follicular maturation and luteinization in vivo. This multiple hormonal control mechanism also provides an interesting model for future studies on the diverse mode of hormone action.     

HSP27 expression regulates CCK-induced changes of the actin cytoskeleton in CHO-CCK-A cells

We investigated how heat shock protein 27 (HSP27)and its phosphorylation are involved in the action of cholecystokinin(CCK) on the actin cytoskeleton by genetic manipulation of Chinesehamster ovary (CHO) cells stably transfected with the CCK-A receptor. In these cells, as in rat acini, CCK activated p38 mitogen-activated protein (MAP) kinase and increased the phosphorylation of HSP27. Thiseffect could be blocked with the p38 MAP kinase inhibitor SB-203580.Examination by confocal microscopy of cells stained with rhodaminephalloidin showed that CCK dose-dependently induced changes of theactin cytoskeleton, including cell shape changes, which were coincidentwith actin cytoskeleton fragmentation and formation of actin filamentpatches in the cells. To further evaluate the role of HSP27, CHO-CCK-Acells were transfected with expression vectors for either wild-type(wt) or mutant (3A, 3G, and 3D) human HSP27. Overexpression of wt-HSP27and 3D-HSP27 inhibited the effects on the actin cytoskeleton seen afterhigh-dose CCK stimulation. In contrast, overexpression ofnonphosphorylatable mutants, 3A- and 3G-HSP27, or inhibition ofphosphorylation of HSP27 by preincubation of wt-HSP27 transfected cellswith SB-203580 did not protect the actin cytoskeleton. These resultssuggest that phosphorylation of HSP27 is required to stabilize theactin cytoskeleton and to protect the cells from the effects of highconcentrations of CCK.

     

Renal disease,epidermal necrosis,and epithelial cell antibodies.

OBJECTIVE--To describe the association between epithelial cell IgM, which has previously been associated with an increased incidence of loss of renal graft in children, with a novel cutaneous eruption and unexplained native renal disease. DESIGN--Observational study on children with epithelial cell antibody presenting with unexplained renal or skin disease. SETTING--General paediatric department and regional paediatric nephrology unit. PATIENTS--Six children (five girls, one boy), who presented to the unit in 1989-90. RESULTS--Three children, two of whom had a history of a hyperpigmented rash, presented with hypertension, proteinuria, and impaired renal function. Renal biopsy specimens from two of these children showed severe arteriolar endothelial cell swelling with arteriolar occlusion. These children fully recovered after treatment with antihypertensive drugs. The third child developed end stage renal failure and required dialysis. Three other children presented with an unusual cutaneous eruption but no evidence of renal disease. Histology of the skin lesions showed acute epidermal necrosis and features consistent with a viral infection. CONCLUSIONS--The aetiology and pathogenesis of the epithelial cell antibody are unknown. These cases indicate that it may have a role in native kidney disease and focal epidermal necrosis. Clinical and histological features suggest that the antibody may be associated with a viral infection.