On the subunit composition of the Neurospora plasma membrane H+-ATPase.

The resolution-reconstitution approach has been employed in order to gain information as to the subunit composition of the Neurospora plasma membrane H+-ATPase. Proteoliposomes prepared from sonicated asolectin and a highly purified, radiolabeled preparation of the 105,000-dalton hydrolytic moiety of the H+-ATPase by a freeze-thaw procedure catalyze ATP hydrolysis-dependent proton translocation as indicated by the extensive 9-amino-6-chloro-2-methoxyacridine fluorescence quenching that occurs upon the addition of MgATP to the proteoliposomes, and the reversal of this quenching induced by the H+-ATPase inhibitor, vanadate, and the proton conductors, carbonyl cyanide m-chlorophenylhydrazone and nigericin plus K+. ATP hydrolysis is tightly coupled to proton translocation into the liposomes as indicated by the marked stimulation of ATP hydrolysis by carbonyl cyanide m-chlorophenylhydrazone and nigericin plus K+. The maximum stimulation of ATPase activity by proton conductors is about 3-fold, which indicates that at least two-thirds of the hydrolytically active ATPase molecules present in the reconstituted preparation are capable of translocating protons into the liposomes. Furthermore, as estimated by the extent of protection of the reconstituted 105,000-dalton hydrolytic moiety against tryptic degradation by vanadate in the presence of Mg2+ and ATP, the fraction of the total population of ATPase molecules that are hydrolytically active is at least 91%. Taken together, these data indicate that at least 61% of the ATPase molecules present in the reconstituted preparation are able to catalyze proton translocation. This information allows an estimation of the amount of any polypeptide in the preparation that must be present in order for that polypeptide to qualify as a subunit that is required for proton translocation in addition to the 105,000-dalton hydrolytic moiety, and an analysis of the radiolabeled ATPase preparation by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea rules out the involvement of any such polypeptides larger than 2,500 daltons. This indicates that the Neurospora plasma membrane H+-ATPase has no subunits even vaguely resembling any that have been found to be associated with other transport ATPases and that if this enzyme has any subunits at all other than the 105,000-dalton hydrolytic moiety, they must be very small

Chemical state of the cysteine residues in the Neurospora crassa plasma membrane H (+)-ATPase.

The plasma membrane H(+)-ATPase of Neurospora crassa was treated with 5,5'-dithiobis(2-nitrobenzoate) to determine its cysteine content and with 2-nitro-5-thiosulfobenzoate to determine its cystine content. Six and seven mol of thiols/mol of H(+)-ATPase were detected in the 5,5'-dithiobis(2-nitrobenzoate) and 2-nitro-5-thiosulfobenzoate reactions, respectively, indicating that 6 of the 8 cysteine residues in the molecule are present as free cysteines and that 2 are present in disulfide linkage. The results of quantitative carboxymethylation experiments using [14C]iodoacetate under nonreducing and reducing conditions fully support this conclusion. Preparations of the ATPase 14C carboxymethylated under the above conditions were treated with trypsin, and the tryptic digests were resolved into hydrophilic and hydrophobic peptide fractions by our recently published procedure (Rao, U.S., Hennessey, J.P., Jr., and Scarborough, G.A. (1988) Anal. Biochem. 173, 251-264). Five of the six labeled free cysteine peptides partitioned into the hydrophilic peptide fraction and were purified and established to contain Cys376, Cys409, Cys472, Cys532, and Cys545. The labeled free cysteine residue in the hydrophobic peptide fraction was identified as either Cys840 or Cys869 by virtue of its presence in a large approximately 21-kDa hydrophobic peptide established previously to begin at Ser660. This in turn identified either Cys840 or Cys869 as one of the disulfide bridge cysteines. The other disulfide bridge cysteine was identified as Cys148 by purification and NH2-terminal sequencing of an additional peptide labeled in the reduced enzyme. The disulfide bridge is therefore between Cys148 and either Cys840 or Cys869. Because Cys148 is present in a putative membrane-embedded sector near the NH2 terminus of the ATPase molecule and Cys840 and Cys869 are present in a similar sector near the COOH terminus, it is possible that the disulfide bridge plays an important structural role in holding the two major membrane-embedded sectors of the molecule, distant in the linear sequence, together

Identification of the major cytoplasmic regions of the Neurospora crassa plasma membrane H (+)-ATPase using protein chemical techniques.

The transmembrane topography of the Neurospora crassa plasma membrane H(+)-ATPase has been investigated using purified, reconstituted components and direct protein chemical techniques. Reconstituted proteoliposomes containing H(+)-ATPase molecules oriented predominantly with their cytoplasmic surface facing outward were treated with trypsin to liberate peptides present on the cytoplasmic surface of the H(+)-ATPase as recently described (Hennessey, J.P., Jr., and Scarborough, G. (1990) J. Biol. Chem. 265, 532-537. The released peptides were then separated from the proteoliposomes by gel filtration chromatography and further purified by high performance liquid chromatography. Fourteen such peptides were identified by NH2-terminal amino acid sequence analysis, directly defining these parts of the molecule as present on the cytoplasmic surface of the membrane. Moreover, this information identified several additional flanking stretches as likely to be cytoplasmically located by virtue of the fact that they are too short to cross the membrane and return. These results and the results of other recent experiments establish 417 residues of the 919 present in the ATPase molecule, at positions 2-100, 186-256, 441-663, and 897-920, as cytoplasmically located. Taken together with the results of our preliminary investigations of the membrane embedded sectors of the ATPase, this information allows the formulation of a reasonably detailed model for the transmembrane topography of the ATPase polypeptide chain

Identification of the membrane-embedded regions of the Neurospora crassa plasma membrane H (+)-ATPase.

Reconstituted proteoliposomes containing functional Neurospora crassa plasma membrane H(+)-ATPase molecules oriented predominantly with their cytoplasmic surface exposed were treated with trypsin and then subjected to Sepharose CL-6B column chromatography to remove the liberated peptides. The peptides remaining associated with the liposomes were then separated from the phospholipid by Sephadex LH-60 column chromatography and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Six H(+)-ATPase peptides with approximate molecular masses of 7, 7.5, 8, 10, 14, and 21 kDa were found to be tightly associated with the liposomal membrane. Amino acid sequencing of the 7-, 7.5-, and 21-kDa peptides in the LH-60 eluate identified them as H(+)-ATPase fragments beginning at residues 99 or 100, 272, and 660, respectively. After further purification, the approximately 10- and 14-kDa peptides were also similarly identified as beginning at residues 272 and 660. The approximately 8-kDa fragment was purified further but could not be sequenced, presumably indicating NH2-terminal blockage. To identify which of the liposome-associated peptides are embedded in the membrane, H(+)-ATPase molecules in the proteoliposomes were labeled from the hydrophobic membrane interior with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine and cleaved with trypsin, after which the membrane-associated peptides were purified and assessed for the presence of label. The results indicate that the approximately 7-, 7.5-, and 21-kDa peptides are in contact with the lipid bilayer whereas the approximately 8-kDa peptide is not. Taken together with the results of our recent analyses of the peptides released from the proteoliposomes, this information establishes the transmembrane topography of nearly all of the 919 residues in the H(+)-ATPase molecule

Fast Adaptation in Vestibular Hair Cells Requires Myosin-1c Activity

SummaryIn sensory hair cells of the inner ear, mechanical amplification of small stimuli requires fast adaptation, the rapid closing of mechanically activated transduction channels. In frog and mouse vestibular hair cells, we found that the rate of fast adaptation depends on both channel opening and stimulus size and that it is modeled well as a release of a mechanical element in series with the transduction apparatus. To determine whether myosin-1c molecules of the adaptation motor are responsible for the release, we introduced the Y61G mutation into the Myo1c locus and generated mice homozygous for this sensitized allele. Measuring transduction and adaptation in the presence of NMB-ADP, an allele-specific inhibitor, we found that the inhibitor not only blocked slow adaptation, as demonstrated previously in transgenic mice, but also inhibited fast adaptation. These results suggest that mechanical activity of myosin-1c is required for fast adaptation in vestibular hair cells

Applications of nutrient profiling : potential role in diet-related chronic disease prevention and the feasibility of a core nutrient-profiling system

Background/objectives: A number of different nutrient-profiling models have been proposed and several applications of nutrient profiling have been identified. This paper outlines the potential role of nutrient-profiling applications in the prevention of diet-related chronic disease (DRCD), and considers the feasibility of a core nutrient-profiling system, which could be modified for purpose, to underpin the multiple potential applications in a particular country.Methods: The &lsquo;Four &lsquo;P&rsquo;s of Marketing&rsquo; (Product, Promotion, Place and Price) are used as a framework for identifying and for classifying potential applications of nutrient profiling. A logic pathway is then presented that can be used to gauge the potential impact of nutrient-profiling interventions on changes in behaviour, changes in diet and, ultimately, changes in DRCD outcomes. The feasibility of a core nutrient-profiling system is assessed by examining the implications of different model design decisions and their suitability to different purposes.Results and conclusions: There is substantial scope to use nutrient profiling as part of the policies for the prevention of DRCD. A core nutrient-profiling system underpinning the various applications is likely to reduce discrepancies and minimise the confusion for regulators, manufacturers and consumers. It seems feasible that common elements, such as a standard scoring method, a core set of nutrients and food components, and defined food categories, could be incorporated as part of a core system, with additional application-specific criteria applying. However, in developing and in implementing such a system, several country-specific contextual and technical factors would need to be balanced.<br /

Associated reading skills in children with a history of Specific Language Impairment (SLI)

A large cohort of 200 eleven-year-old children with Specific Language Impairment (SLI) were assessed on basic reading accuracy and on reading comprehension as well as language tasks. Reading skills were examined descriptively and in relation to early language and literacy factors. Using stepwise regression analyses in which age and nonverbal IQ were controlled for, it was found that a single word reading measure taken at 7 years was unsurprisingly a strong predictor of the two different types of reading ability. However, even with this measure included, a receptive syntax task (TROG) entered when reading accuracy score was the DV. Furthermore, a test of expressive syntax/narrative and a receptive syntax task completed at 7 years entered into the model for word reading accuracy. When early reading accuracy was excluded from the analyses, early phonological skills also entered as a predictor of both reading accuracy and comprehension at 11 years. The group of children with a history of SLI were then divided into those with no literacy difficulties at 11 and those with some persisting literacy impairment. Using stepwise logistic regression, and again controlling for IQ and age, 7 years receptive syntax score (but not tests of phonology, expressive vocabulary or expressive syntax/narrative) entered as a positive predictor of membership of the ‘no literacy problems’ group regardless of whether early reading accuracy was controlled for in step one. The findings are discussed in relation to the overlap of SLI and dyslexia and the long term sequelae of language impairment

Systematic review and meta-analysis of reduction in all-cause mortality from walking and cycling and shape of dose response relationship

BACKGROUND AND OBJECTIVE: Walking and cycling have shown beneficial effects on population risk of all-cause mortality (ACM). This paper aims to review the evidence and quantify these effects, adjusted for other physical activity (PA). DATA SOURCES: We conducted a systematic review to identify relevant studies. Searches were conducted in November 2013 using the following health databases of publications: Embase (OvidSP); Medline (OvidSP); Web of Knowledge; CINAHL; SCOPUS; SPORTDiscus. We also searched reference lists of relevant texts and reviews. STUDY ELIGIBILITY CRITERIA AND PARTICIPANTS: Eligible studies were prospective cohort design and reporting walking or cycling exposure and mortality as an outcome. Only cohorts of individuals healthy at baseline were considered eligible. STUDY APPRAISAL AND SYNTHESIS METHODS: Extracted data included study population and location, sample size, population characteristics (age and sex), follow-up in years, walking or cycling exposure, mortality outcome, and adjustment for other co-variables. We used random-effects meta-analyses to investigate the beneficial effects of regular walking and cycling. RESULTS: Walking (18 results from 14 studies) and cycling (8 results from 7 studies) were shown to reduce the risk of all-cause mortality, adjusted for other PA. For a standardised dose of 11.25 MET.hours per week (or 675 MET.minutes per week), the reduction in risk for ACM was 11% (95% CI = 4 to 17%) for walking and 10% (95% CI = 6 to 13%) for cycling. The estimates for walking are based on 280,000 participants and 2.6 million person-years and for cycling they are based on 187,000 individuals and 2.1 million person-years. The shape of the dose-response relationship was modelled through meta-analysis of pooled relative risks within three exposure intervals. The dose-response analysis showed that walking or cycling had the greatest effect on risk for ACM in the first (lowest) exposure interval. CONCLUSIONS AND IMPLICATIONS: The analysis shows that walking and cycling have population-level health benefits even after adjustment for other PA. Public health approaches would have the biggest impact if they are able to increase walking and cycling levels in the groups that have the lowest levels of these activities. REVIEW REGISTRATION: The review protocol was registered with PROSPERO (International database of prospectively registered systematic reviews in health and social care) PROSPERO 2013: CRD42013004266

Regge behaviour of distribution functions and t and x-evolutions of gluon distribution function at low-x

In this paper t and x-evolutions of gluon distribution function from Dokshitzer-Gribov-Lipatov-Altarelli-Parisi(DGLAP) evolution equation in leading order(LO) at low-x, assuming the Regge behaviour of quark and gluon at this limit, are presented. We compare our results of gluon distribution function with MRST 2001, MRST 2004 and GRV '98 parameterizations and show the compatibility of Regge behaviour of quark and gluon distribution functions with perturbative quantum chromodynamics(PQCD) at low-x. We also discuss the limitations of Taylor series expansion method used earlier to solve DGLAP evolution equations, in the Regge behaviour of distribution functions.Comment: 19 pages, 7 figure