Evidence for an essential histidine residue in the Neurospora crassa plasma membrane H+-ATPase

The Neurospora crassa plasma membrane H+-ATPase is rapidly inactivated in the presence of diethyl pyrocarbonate (DEP). The reaction is pseudo-first-order showing time- and concentration-dependent inactivation with a second-order rate constant of 385-420 M-1.min-1 at pH 6.9 and 25 degrees C. The difference spectrum of the native and modified enzyme has a maximum near 240 nm, characteristic of N-carbethoxyhistidine. No change in the absorbance of the inhibited ATPase at 278 nm or in the number of modifiable sulfhydryl groups is observed, indicating that the inhibition is not due to tyrosine or cysteine modification, and the inhibition is irreversible, ruling out serine residues. Furthermore, pretreatment of the ATPase with pyridoxal phosphate/NaBH4 under the conditions of the DEP treatment does not inhibit the ATPase and does not alter the DEP inhibition kinetics, indicating that the inactivation by DEP is not due to amino group modification. The pH dependence of the inactivation reaction indicates that the essential residue has a pKa near 7.5, and the activity lost as a result of H+-ATPase modification by DEP is partially recovered after hydroxylamine treatment at 4 degrees C. Taken together, these results strongly indicate that the inactivation of the H+-ATPase by DEP involves histidine modification. Analyses of the inhibition kinetics and the stoichiometry of modification indicate that among eight histidines modified per enzyme molecule, only one is essential for H+-ATPase activity. Finally, ADP protects against inactivation by DEP, indicating that the essential residue modified may be located at or near the nucleotide binding site.     

Properties of Neurospora crassa plasma membrane ATPase.

A variety of the biochemical properties of the electrogenic plasma membrane ATPase of Neurospora crassa are described. The enzyme catalyzes the hydrolysis of ATP, resulting in the formation of ADP and inorganic phosphate. Optimal activity is observed between pH 6 and 6.5. ATP hydrolysis approaches a maximum rate at an Mg-ATP concentration of 10–20 mm with a half-maximal velocity around 2 mm Mg-ATP. The enzyme requires a divalent cation for activity in the following order of preference at 10 mm: Mg²⁺, Co²⁺ > Mn²⁺ > Zn²⁺ > Fe²⁺, Ca²⁺, Cu²⁺. The enzyme is quite specific for ATP compared to the other nucleotides tested. Treatment of the plasma membranes with sodium deoxycholate inactivates the ATPase and the inactivation can be prevented by the addition of certain acidic phospholipids with the deoxycholate. Other classes of lipids cannot prevent the deoxycholate inhibition. The organic mercurials parachloromercuribenzoate and parachloromercuriphenylsulfonate are potent inhibitors of the ATPase, but N-ethylmaleimide at a similar concentration is not inhibitory. The organic mercurial inhibition is not reversed by mercaptoethanol. Under appropriate conditions, the inhibitory effect of p-chloromercuribenzoate is suppressed in the presence of ATP. Treatment of the plasma membranes with trypsin leads to a marked inhibition of the ATPase activity and this inhibition can be prevented by Mg-ATP. Neither the organic mercurial reactive site(s) nor the trypsin-sensitive site(s) are accessible from the outer surface of the plasma membranes. Some of the implications of the above findings are discussed.     

Direct evidence for the cytoplasmic location of the NH2- and COOH-terminal ends of the Neurospora crassa plasma membrane H+-ATPase

Reconstituted proteoliposomes containing Neurospora plasma membrane H+-ATPase molecules oriented predominantly with their cytoplasmic portion facing outward have been used to determine the location of the NH2 and COOH termini of the H+-ATPase relative to the lipid bilayer. Treatment of the proteoliposomes with trypsin in the presence of the H+-ATPase ligands Mg2+, ATP, and vanadate produces approximately 97-, 95-, and 88-kDa truncated forms of the H+-ATPase similar to those already known to result from cleavage at Lys24, Lys36, and Arg73 at the NH2-terminal end of the molecule. These results establish that the NH2-terminal end of the H+-ATPase polypeptide chain is located on the cytoplasmic side of the membrane. Treatment of the same proteoliposome preparation with trypsin in the absence of ligands releases approximately 50 water-soluble peptides from the proteoliposomes. Separation of the released peptides by high performance liquid chromatography and spectral analysis of the purified peptides identified only a few peptides with the properties expected of a COOH-terminal, tryptic undecapeptide with the sequence SLEDFVVSLQR, and NH2-terminal amino acid sequence analysis identified this peptide among the possible candidates. Quantitative considerations indicate that this peptide must have come from H+-ATPase molecules oriented with their cytoplasmic portion facing outward, and could not have originated from a minor population of H+-ATPase molecules of reverse orientation. These results directly establish that the COOH-terminal end of the H+-ATPase is also located on the cytoplasmic side of the membrane. These findings are important for elucidating the topography of the membrane-bound H+-ATPase and are possibly relevant to the topography of other aspartyl-phosphoryl-enzyme intermediate ATPases as well.     

Carbohydrate-containing endothelial cells lining the bulbus arteriosus of teleosts and the conus arteriosus of elasmobranchs (Pisces)

The study is a survey of the shape and carbohydrate histochemistry of the endothelial cells lining the conus arteriosus of 10 species of elasmobranchs and the bulbus arteriosus of 80 species of teleosts. Intensely PAS-positive cells that were often tall, were found in many teleosts and were typical of phylogenetically advanced species. They were not seen in any elasmobranch. Most of the teleosts with strongly PAS-positive cells were marine fish or euryhaline animals that were caught in freshwater. Beyond this, it is difficult to generalize on their life-style or habitat, for they included small fish, large fish, fast swimmers, bottom-living forms, deep-water fish, littoral species and Antarctic "bloodless" forms. It is likely that the PAS-positivity of the endothelial cells can be attributed to the moderately-dense granules revealed by E.M., but as yet the significance of these is unclear.     

High Mortality amongst Adolescents and Adults with Bacterial Meningitis in Sub-Saharan Africa: An Analysis of 715 Cases from Malawi

Mortality from bacterial meningitis in African adults is significantly higher than those in better resourced settings and adjunctive therapeutic interventions such as dexamethasone and glycerol have been shown to be ineffective. We conducted a study analysing data from clinical trials of bacterial meningitis in Blantyre, Malawi to investigate the clinical parameters associated with this high mortality.

Methods

We searched for all clinical trials undertaken in Blantyre investigating bacterial meningitis from 1990 to the current time and combined the data from all included trial datasets into one database. We used logistic regression to relate individual clinical parameters to mortality. Adults with community acquired bacterial meningitis were included if the CSF culture isolate was consistent with meningitis or if the CSF white cell count was >100 cells/mm³ (>50% neutrophils) in HIV negative participants and >5 cells/mm³ in HIV positive participants. Outcome was measured by mortality at discharge from hospital (after 10 days of antibiotic therapy) and community follow up (day 40).

Results

Seven hundred and fifteen episodes of bacterial meningitis were evaluated. The mortality rate was 45% at day 10 and 54% at day 40. The most common pathogens were S.pneumoniae (84% of positive CSF isolates) and N.meningitidis (4%). 607/694 (87%) participants tested were HIV antibody positive. Treatment delays within the hospital system were marked. The median presenting GCS was 12/15, 17% had GCS<8 and 44.9% had a seizure during the illness. Coma, seizures, tachycardia and anaemia were all significantly associated with mortality on multivariate analysis. HIV status and pneumococcal culture positivity in the CSF were not associated with mortality. Adults with community acquired bacterial meningitis in Malawi present with a severe clinical phenotype. Predictors of high mortality are different to those seen in Western settings. Optimising in-hospital care and minimising treatment delays presents an opportunity to improve outcomes considerably.     

Estimating the effect of moving meat-free products to the meat aisle on sales of meat and meat-free products: A non-randomised controlled intervention study in a large UK supermarket chain

BackgroundReducing meat consumption could bring health and environmental benefits, but there is little research to date on effective interventions to achieve this. A non-randomised controlled intervention study was used to evaluate whether prominent positioning of meat-free products in the meat aisle was associated with a change in weekly mean sales of meat and meat-free products.Methods and findingsWeekly sales data were obtained from 108 stores: 20 intervention stores that moved a selection of 26 meat-free products into a newly created meat-free bay within the meat aisle and 88 matched control stores. The primary outcome analysis used a hierarchical negative binomial model to compare changes in weekly sales (units) of meat products sold in intervention versus control stores during the main intervention period (Phase I: February 2019 to April 2019). Interrupted time series analysis was also used to evaluate the effects of the Phase I intervention. Moreover, 8 of the 20 stores enhanced the intervention from August 2019 onwards (Phase II intervention) by adding a second bay of meat-free products into the meat aisle, which was evaluated following the same analytical methods.During the Phase I intervention, sales of meat products (units/store/week) decreased in intervention (approximately −6%) and control stores (−5%) without significant differences (incidence rate ratio [IRR] 1.01 [95% CI 0.95–1.07]. Sales of meat-free products increased significantly more in the intervention (+31%) compared to the control stores (+6%; IRR 1.43 [95% CI 1.30–1.57]), mostly due to increased sales of meat-free burgers, mince, and sausages. Consistent results were observed in interrupted time series analyses where the effect of the Phase II intervention was significant in intervention versus control stores.ConclusionsProminent positioning of meat-free products into the meat aisle in a supermarket was not effective in reducing sales of meat products, but successfully increased sales of meat-free alternatives in the longer term.A preregistered protocol (https://osf.io/qmz3a/) was completed and fully available before data analysis.

Carmen Piernas and co-workers study positioning and marketing of meat-free products in supermarkets.     

Sulfate reduction and S-oxidation in a moorland pool sediment

In an oligotrophic moorland pool in The Netherlands, S cycling near the sediment/water boundary was investigated by measuring (1) SO₄ ^2– reduction rates in the sediment, (2) depletion of SO₄ ^2– in the overlying water column and (3) release of³⁵S from the sediment into the water column. Two locations differing in sediment type (highly organic and sandy) were compared, with respect to reduction rates and depletion of SO₄ ^2– in the overlying water.Sulfate reduction rates in sediments of an oligotrophic moorland pool were estimated by diagenetic modelling and whole core³⁵SO₄ ^2– injection. Rates of SO₄ ^2– consumption in the overlying water were estimated by changes in SO₄ ^2– concentration over time in in situ enclosures. Reduction rates ranged from 0.27–11.2 mmol m^–2 d^–1. Rates of SO₄ ^2– uptake from the enclosed water column varied from –0.5, –0.3 mmol m^–2 d^–1 (November) to 0.43–1.81 mmol m^–2 d^–1 (July, August and April). Maximum rates of oxidation to SO₄ ^2– in July 1990 estimated by combination of SO₄ ^2– reduction rates and rates of in situ SO₄ ^2– uptake in the enclosed water column were 10.3 and 10.5 mmol m^–2 d^–1 at an organic rich and at a sandy site respectively.Experiments with³⁵S^2– and³⁵SO₄ ^2– tracer suggested (1) a rapid formation of organically bound S from dissimilatory reduced SO₄ ^2– and (2) the presence of mainly non SO₄ ^2–-S derived from reduced S transported from the sediment into the overlying water. A³⁵S^2– tracer experiment showed that about 7% of³⁵S^2– injected at 1 cm depth in a sediment core was recovered in the overlying water column.Sulfate reduction rates in sediments with higher volumetric mass fraction of organic matter did not significantly differ from those in sediments with a lower mass fraction of organic matter.Corresponding author     

What can phylogeographic breaks in the livebearer Poeciliopsis turrubarensis tell about fish community boundaries in western Costa Rica?

This study asks if the geographic boundary delineating two fish communities in western Costa Rica is congruent with a phylogeographic break in a single widespread fish species Poeciliopsis turrubarensis (Poeciliidae) that spans this area. Such congruence would suggest that a common historical event (e.g. geological or climatic) could be responsible for both patterns. It was found that there was a shared break across a region in central Costa Rica suggesting a common cause may be responsible for both the abrupt shift in fish community composition and the genetic break in P. turrubarensis.     

Increase in the proportion of metabolically active bacteria along gradients of enrichment in freshwater and marine plankton: implications for estimates of bacterial growth and production rates

It is generally recognized that a fraction of all bacterioplanktoncells enumerated using conventional epifluorescence techniquesis neither growing, dividing nor metabolically active, but thevariation in the proportion of active cells among aquatic systemsis not well understood. Here, we hypothesize that the proportionof metabolically active cells increases systematically alonggradients of enrichment, and to test this hypothesis the numberand proportion of metabolically active planktonic bacteria wereinvestigated during the summer in a set of 24 temperate lakes,which span a considerable range in productivity. The tetrazoliumsalt 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was usedas an indicator of cells with an active electron transport system.The total number of bacteria ranged from 1.88x10⁶ to 7.70x10⁶ml^–1, whereas the number of active cells was more variableand ranged from 0.37x10⁶ to 2.18x10⁶ ml^–1 in the studylakes. The proportion of metabolically active cells ranged from15 to 33%, and tended to increase with nutrient and chlorophyllconcentrations, but not with dissolved organic carbon (DOC).Data on the number of total and active bacteria culled fromthe literature for marine, estuarine and freshwater systemsshow that the trends we measured in lakes are valid for pelagicsystems in general. Over a broad range of aquatic systems, thetotal number of bacteria varied by three orders of magnitude,whereas the number of active bacteria varied by four ordersof magnitude as system productivity increased. The proportionof active cells increased from ultraoligotrophic openocean areas(<5%) to highly productive estuaries (>50%). Our resultssuggest that in most aquatic systems there is a pool of rapidlygrowmg cells, embedded in a usually larger matrix of inactivebacteria, and that the relative size of the active and inactivepools varies systematically among bacterial populations alonggradients of enrichment.