Association of syntaxin 3 and vesicle-associated membrane protein (VAMP) with H+/K(+)-ATPase-containing tubulovesicles in gastric parietal cells.

H+/K(+)-ATPase is the proton pump in the gastric parietal cell that is responsible for gastric acid secretion. Stimulation of acid secretion is associated with a reorganization of the parietal cells resulting in the incorporation of H+/K(+)-ATPase from a cytoplasmic membrane pool, the tubulovesicle compartment, into the apical canalicular membrane. To better characterize the role of membrane trafficking events in the morphological and physiological changes associated with acid secretion from parietal cells, we have characterized the expression and localization of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in these cells. Each of the six different SNARE proteins examined [syntaxins 1 through 4 of 25-kDa synaptosome-associated protein, and vesicle-associated membrane protein] were found to be expressed in parietal cells. Furthermore, two of these SNAREs, vesicle-associated membrane protein and syntaxin 3, were associated with H+/K(+)-ATPase-containing tubulovesicles while the remainder were excluded from this compartment. The expression of syntaxin 1 and synaptosome-associated protein of 25 kDa in parietal cells, two SNAREs previously thought to be restricted to neuroendocrine tissues, suggests that parietal cells may utilize membrane trafficking machinery that is similar to that utilized for regulated exocytosis in neurons. Furthermore, the localization of syntaxin 3, a putative target membrane SNARE, to the tubulovesicle compartment indicates that syntaxin 3 may have an alternative function. These observations support a role for intracellular membrane trafficking events in the regulated recruitment of H+/K(+)-ATPase to the plasma membrane after parietal cell stimulation.     

A simple chlorophyll fluorescence parameter that correlates with the rate coefficient of photoinactivation of Photosystem II

A method of partitioning the energy in a mixed population of active and photoinactivated Photosystem II (PS II) complexes based on chlorophyll fluorescence measurements is presented. There are four energy fluxes, each with its quantum efficiency: a flux associated with photochemical electron flow in active PS II reaction centres (J_{PS II}), thermal dissipation in photoinactivated, non-functional PS IIs (J_NF), light-regulated thermal dissipation in active PS IIs (J_NPQ) and a combined flux of fluorescence and constitutive, light-independent thermal dissipation (J_f,D). The four quantum efficiencies add up to 1.0, without the need to introduce an ‘excess’ term E, which in other studies has been claimed to be linearly correlated with the rate coefficient of photoinactivation of PS II (k_pi). We examined the correlation of k_pi with various fluxes, and found that the combined flux (J_NPQ + J_f,D= J_pi) is as well correlated with k_pi as is E. This combined flux arises from F_s/F_m^′, the ratio of steady-state to maximum fluorescence during illumination, which represents the quantum efficiency of combined non-photochemical dissipation pathways in active PS IIs. Since F_s/F_m^′ or its equivalent, J_pi, is a likely source of events leading to photoinactivation of PS II, we conclude that F_s/F_m^′ is a simple predictor of k_pi.     

Calcium-sensing receptor modulates extracellular Ca(2+) entry via TRPC-encoded receptor-operated channels in human aortic smooth muscle cells

Ca-sensing receptor (CaSR), a member of the G protein-coupled receptor family, regulates the synthesis of parathyroid hormone in response to changes in serum Ca(2+) concentrations. The functions of CaSR in human vascular smooth muscle cells are largely unknown. Here we sought to study CaSR activation and the underlying molecular mechanisms in human aortic smooth muscle cells (HASMC). Extracellular Ca(2+) ([Ca(2+)](o)) dose-dependently increased free cytosolic Ca(2+) ([Ca(2+)](cyt)) in HASMC, with a half-maximal response (EC(50)) of 0.52 mM and a Hill coefficient of 5.50. CaSR was expressed in HASMC, and the [Ca(2+)](o)-induced [Ca(2+)](cyt) rise was abolished by dominant negative mutants of CaSR. The CaSR-mediated increase in [Ca(2+)](cyt) was also significantly inhibited by pertussis toxin, the phospholipase C inhibitor U-73122, or the general protein kinase C (PKC) inhibitor chelerythrine, but not by the conventional PKC inhibitor, G?6976. Depletion of membrane cholesterol by pretreatment with methyl-β-cyclodextrin markedly decreased CaSR-induced increase in [Ca(2+)](cyt). Blockade of TRPC channels with 2-aminoethoxydiphenyl borate, SKF-96365, or La(3) significantly inhibited [Ca(2+)](o) entry, whereas activation of TRPC6 channels with flufenamic acid potentiated [Ca(2+)](o) entry. Neither cyclopiazonic acid nor caffeine or ionomycin had any effect on [Ca(2+)](cyt) in [Ca(2+)](o)-free solutions. TRPC6 and PKCε mRNA and proteins were detected in HASMC, and [Ca(2+)](o) induced PKCε phosphorylation, which could be prevented by chelerythrine. Our data suggest that CaSR activation mediates [Ca(2+)](o) entry, likely through TRPC6-encoded receptor-operated channels that are regulated by a PLC/PKCε cascade. Our study therefore provides evidence not only for functional expression of CaSR, but also for a novel pathway whereby it regulates [Ca(2+)](o) entry in HASMC.     

Faster Rubisco is the key to superior nitrogen-use efficiency in NADP-malic enzyme relative to NAD-malic enzyme C4 grasses

In 27 C4 grasses grown under adequate or deficient nitrogen (N) supplies, N-use efficiency at the photosynthetic (assimilation rate per unit leaf N) and whole-plant (dry mass per total leaf N) level was greater in NADP-malic enzyme (ME) than NAD-ME species. This was due to lower N content in NADP-ME than NAD-ME leaves because neither assimilation rates nor plant dry mass differed significantly between the two C4 subtypes. Relative to NAD-ME, NADP-ME leaves had greater in vivo (assimilation rate per Rubisco catalytic sites) and in vitro Rubisco turnover rates (k(cat); 3.8 versus 5.7 s(-1) at 25 degrees C). The two parameters were linearly related. In 2 NAD-ME (Panicum miliaceum and Panicum coloratum) and 2 NADP-ME (Sorghum bicolor and Cenchrus ciliaris) grasses, 30% of leaf N was allocated to thylakoids and 5% to 9% to amino acids and nitrate. Soluble protein represented a smaller fraction of leaf N in NADP-ME (41%) than in NAD-ME (53%) leaves, of which Rubisco accounted for one-seventh. Soluble protein averaged 7 and 10 g (mmol chlorophyll)(-1) in NADP-ME and NAD-ME leaves, respectively. The majority (65%) of leaf N and chlorophyll was found in the mesophyll of NADP-ME and bundle sheath of NAD-ME leaves. The mesophyll-bundle sheath distribution of functional thylakoid complexes (photosystems I and II and cytochrome f) varied among species, with a tendency to be mostly located in the mesophyll. In conclusion, superior N-use efficiency of NADP-ME relative to NAD-ME grasses was achieved with less leaf N, soluble protein, and Rubisco having a faster k(cat).     

Daphnia magna microRNAs respond to nutritional stress and ageing but are not transgenerational

Maternal effects, where the performance of offspring is determined by the condition of their mother, are widespread and may in some cases be adaptive. The crustacean Daphnia magna shows strong maternal effects: offspring size at birth and other proxies for fitness are altered when their mothers are older or when mothers have experienced dietary restriction. The mechanisms for this transgenerational transmission of maternal experience are unknown, but could include changes in epigenetic patterning. MicroRNAs (miRNAs) are regulators of gene expression that have been shown to play roles in intergenerational information transfer, and here, we test whether miRNAs are involved in D. magna maternal effects. We found that miRNAs were differentially expressed in mothers of different ages or nutritional state. We then examined miRNA expression in their eggs, their adult daughters and great granddaughters, which did not experience any treatments. The maternal (treatment) generation exhibited differential expression of miRNAs, as did their eggs, but this was reduced in adult daughters and lost by great granddaughters. Thus, miRNAs are a component of maternal provisioning, but do not appear to be the cause of transgenerational responses under these experimental conditions. MicroRNAs may act in tandem with egg provisioning (e.g., with carbohydrates or fats), and possibly other small RNAs or epigenetic modifications.     

A Novel Method to Evaluate the Community Built Environment Using Photographs – Environmental Profile of a Community Health (EPOCH) Photo Neighbourhood Evaluation Tool

Background

Previous research has shown that environments with features that encourage walking are associated with increased physical activity. Existing methods to assess the built environment using geographical information systems (GIS) data, direct audit or large surveys of the residents face constraints, such as data availability and comparability, when used to study communities in countries in diverse parts of the world. The aim of this study was to develop a method to evaluate features of the built environment of communities using a standard set of photos. In this report we describe the method of photo collection, photo analysis instrument development and inter-rater reliability of the instrument.

Methods/Principal Findings

A minimum of 5 photos were taken per community in 86 communities in 5 countries according to a standard set of instructions from a designated central point of each community by researchers at each site. A standard pro forma derived from reviewing existing instruments to assess the built environment was developed and used to score the characteristics of each community. Photo sets from each community were assessed independently by three observers in the central research office according to the pro forma and the inter-rater reliability was compared by intra-class correlation (ICC). Overall 87% (53 of 60) items had an ICC of ≥0.70, 7% (4 of 60) had an ICC between 0.60 and 0.70 and 5% (3 of 60) items had an ICC ≤0.50.

Conclusions/Significance

Analysis of photos using a standardized protocol as described in this study offers a means to obtain reliable and reproducible information on the built environment in communities in very diverse locations around the world. The collection of the photographic data required minimal training and the analysis demonstrated high reliability for the majority of items of interest.     

Purification and characterization of proteolytic fragments of lipocortin I that inhibit phospholipase A2

Human lipocortin I is a 38.5-kDa phospholipase A2 inhibitor that has been produced in Escherichia coli in large quantities by recombinant DNA technology (Wallner, B.P., Mattaliano, R.J., Hession, C., Cate, R. L., Tizard, R., Sinclair, L.K., Foeller, C., Chow, E.P., Browning, J.L., Ramachandran, K.L., and Pepinsky, R.B. (1986) Nature 320, 77-80). To localize the region within the protein responsible for its inhibitory activity, we generated a series of fragments of the recombinant product by limited proteolysis with elastase and characterized their structure by sequencing and peptide mapping. Five active fragments have been analyzed in detail. The smallest is an 18-kDa fragment derived from the amino-terminal half of lipocortin. Three of the larger fragments contain this region. The fifth fragment is missing 83 amino acids from the amino terminus. A region common to all the active fragments (amino acid residues 97-178) is 70% homologous with the corresponding region from a second member of the lipocortin family which recently was cloned (Huang, K-S., Wallner, B.P., Mattaliano, R.J., Tizard, R., Burne, C., Frey, A., Hession, C., McGray, P., Sinclair, L.K., Chow, E.P., Browning, J.L., Ramachandran, K.L., Tang, J., Smart, J.E., and Pepinsky, R.B. (1986) Cell 46, 191-199) and thus presumably is important for activity. In addition to inhibitory fragments, we have isolated a 3-kDa proteolytic fragment from the amino terminus of lipocortin I that contains the known phosphorylation site for protein-tyrosine kinases. Because of sequence homology of the 3-kDa fragment with biologically active synthetic peptides from pp60v-src and middle T antigen, its release by proteases may represent an important part of the activity of lipocortin.