New structural proteins of Halobacterium salinarum gas vesicle revealed by comparative proteomics analysis

The Halobacterium salinarum gas vesicle (GV) is an extremely stable intracellular organelle with air trapped inside a proteinaceous membrane. Reported here is a comparative proteomics analysis of GV and GV depleted lysate (GVD) to reveal the membrane structural proteins. Ten proteins encoded by gvp-1 (gvpMLKJIHGFED-1 and gvpACNO-1) and five proteins encoded by gvp-2 (gvpMLKJIHGFED-2 and gvpACNO-2) gene clusters for the biogenesis of spindle- and cylindrical-, respectively, shaped GV were identified by LC-MS/MS. The peptides of GvpA1, I1, J1, A2, and J2 were exclusively identified in purified GV, GvpD1, H1, L1, and F2 only in GVD, and GvpC1, N1, O1, F1, H2, and O2 in both samples. The identification of GvpA1, C1, F1, J1, and A2 in GV is in agreement with their previously known structural function. In addition, the detection of GvpI1, N1, O1, H2, J2, and O2 in GV suggested they are new structural proteins. Among these, the structural role of GvpI1 and N1 in GV was further validated by immuno-detection of protein A-tagged GvpI1 and N1 fusion proteins in purified GV. Thus, LC-MS/MS could reveal at least a half dozen gas vesicle structural proteins in the predominant spindle-shaped GV that may be helpful for studying its biogenesis.     

Mechanisms of liver steatosis in rats with systemic carnitine deficiency due to treatment with trimethylhydraziniumpropionate

Rats with systemic carnitine deficiency induced by treatment with trimethylhydraziniumpropionate (THP) develop liver steatosis. This study aims to investigate the mechanisms leading to steatosis in THP-induced carnitine deficiency. Rats were treated with THP (20 mg/100 g) for 3 or 6 weeks and were studied after starvation for 24 h. Rats treated with THP had reduced in vivo palmitate metabolism and developed mixed liver steatosis at both time points. The hepatic carnitine pool was reduced in THP-treated rats by 65% to 75% at both time points. Liver mitochondria from THP-treated rats had increased oxidative metabolism of various substrates and of beta-oxidation at 3 weeks, but reduced activities at 6 weeks of THP treatment. Ketogenesis was not affected. The hepatic content of CoA was increased by 23% at 3 weeks and by 40% at 6 weeks in THP treated rats. The cytosolic content of long-chain acyl-CoAs was increased and the mitochondrial content decreased in hepatocytes of THP treated rats, compatible with decreased activity of carnitine palmitoyltransferase I in vivo. THP-treated rats showed hepatic peroxisomal proliferation and increased plasma VLDL triglyceride and phospholipid concentrations at both time points. A reduction in the hepatic carnitine pool is the principle mechanism leading to impaired hepatic fatty acid metabolism and liver steatosis in THP-treated rats. Cytosolic accumulation of long-chain acyl-CoAs is associated with increased plasma VLDL triglyceride, phospholipid concentrations, and peroxisomal proliferation.     

Cytokinin treatment of embryos inhibits the synthesis of chloroplast proteins in Norway spruce

A pulse treatment of Norway spruce (Picea abies (L.) Karst) embryos with the cytokinin N⁶-benzyladenine induces the formation of adventitious buds from subepidermal cells in the hypocotyl and cotyledons. In addition the treatment also inhibits elongation growth, a key process during germination. In this report we demonstrate that these effects on development of the plant are associated with a suppression of the accumulation of several major chloroplast proteins during germination. These proteins include the large subunit of ribulose bisphosphate/carboxylase oxygenase, two subunits of the chloroplast ATPase, protochlorophyllide reductase and a 23000-M_r component of photosystem II. For two nuclear-encoded proteins, the small subunit of ribulose bisphosphate carboxylase/oxygenase and the light-harvesting chlorophyll a/b-binding protein, a corresponding suppression of the increase in the steady-state amounts of mRNA is recorded. The suppression of chloroplast protein synthesis is consistant with the previously documented delay in greening that results from cytokinin treatment, but the effect is opposite to that found in other plants, where cytokinins promote the synthesis of chloroplast proteins, and stimulate chloroplast biogenesis. We believe that this difference is explained by the cytokinin primarily suppressing organ development, and a strict dependance of chloroplast biogenesis on the developmental state of the organs.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - CF1 coupling-factor 1 of chloroplast ATPase - LHCP light-harvesting chlorophyll a/b-binding protein - LSU large subunit of Rubisco - NADPH-protochlorophyllide oxidoreductase Pchlide reductase - SDS sodium dodecyl sulfate - SSU small subunit of Rubisco We thank K. Hutchison (Dept. of Biochemistry, University of Maine, Orono, Maine, USA) and P. Gustafsson (Dept. of Plant Physiology, University of Umeå, Sweden) for providing the Larix and Pinus clones, and M. Ryberg (Dept. of Plant Physiology, University of Göteborg, Sweden), R. Ölmüller (Botanisches Institut, Universität München, FRG) and W. Lockau (Institut für Botanik, Universität Regensburg, FRG), for the gift of antisera towards Pchlide reductase, RuBPCase and LHCP, and ATPase, respectively. Supported by the Swedish Council for Forestry and Agricultural Research and the Swedish Natural Sciences Research Council.     

The BD FACSPresto Point of Care CD4 Test Accurately Enumerates CD4+ T Cell Counts

Objective

Currently 50% of ART eligible patients are not yet receiving life-saving antiretroviral therapy (ART). Financial constraints do not allow most developing countries to adopt a universal test and offer ART strategy. Decentralizing CD4+ T cell testing may, therefore, provide greater access to testing, ART, and better patient management. We evaluated the technical performance of a new point-of-care CD4+ T cell technology, the BD FACSPresto, in a field methods comparison study.

Methods

264 HIV-positive patients were consecutively enrolled and included in the study. The BD FACSPresto POC CD4+ T cell technology was placed in two rural health care facilities and operated by health care facility staff. We compared paired finger-prick and venous samples using the BD FACSPresto and several existing reference technologies, respectively.

Results

The BD FACSPresto had a mean bias of 67.29 cells/ul and an r² of 0.9203 compared to the BD FACSCalibur. At ART eligibility thresholds of 350 and 500 cells/ul, the sensitivity to define treatment eligibility were 81.5% and 77.2% and the specificities were 98.9% and 100%, respectively. Similar results were observed when the BD FACSPresto was compared to the BD FACSCount and Alere Pima. The coefficient of variation (CV) was less than 7% for both the BD FACSCalibur and BD FACSPresto. CD4+ T cell testing by nurses using the BD FACSPresto at rural health care facilities showed high technical similarity to test results generated by laboratory technicians using the BD FACSPresto in a high functioning laboratory.

Conclusions

The BD FACSPresto performed favorably in the laboratory setting compared to the conventional reference standard technologies; however, the lower sensitivities indicated that up to 20% of patients tested in the field in need of treatment would be missed. The BD FACSPresto is a technology that can allow for greater decentralization and wider access to CD4+ T cell testing and ART.     

The application of new software tools to quantitative protein profiling via isotope-coded affinity tag (ICAT) and tandem mass spectrometry: I. Statistically annotated datasets for peptide sequences and proteins identified via the application of ICAT and tandem mass spectrometry to proteins copurifying with T cell lipid rafts

Lipid rafts were prepared according to standard protocols from Jurkat T cells stimulated via T cell receptor/CD28 cross-linking and from control (unstimulated) cells. Co-isolating proteins from the control and stimulated cell preparations were labeled with isotopically normal (d0) and heavy (d8) versions of the same isotope-coded affinity tag (ICAT) reagent, respectively. Samples were combined, proteolyzed, and resultant peptides fractionated via cation exchange chromatography. Cysteine-containing (ICAT-labeled) peptides were recovered via the biotin tag component of the ICAT reagents by avidin-affinity chromatography. On-line micro-capillary liquid chromatography tandem mass spectrometry was performed on both avidin-affinity (ICAT-labeled) and flow-through (unlabeled) fractions. Initial peptide sequence identification was by searching recorded tandem mass spectrometry spectra against a human sequence data base using SEQUEST software. New statistical data modeling algorithms were then applied to the SEQUEST search results. These allowed for discrimination between likely "correct" and "incorrect" peptide assignments, and from these the inferred proteins that they collectively represented, by calculating estimated probabilities that each peptide assignment and subsequent protein identification was a member of the "correct" population. For convenience, the resultant lists of peptide sequences assigned and the proteins to which they corresponded were filtered at an arbitrarily set cut-off of 0.5 (i.e. 50% likely to be "correct") and above and compiled into two separate datasets. In total, these data sets contained 7667 individual peptide identifications, which represented 2669 unique peptide sequences, corresponding to 685 proteins and related protein groups.     

Application of ion mobility spectrometry to the rapid screening of methamphetamine incorporated in hair

Using ion mobility spectrometry (IMS), a simple, sensitive and rapid screening for methamphetamine (MA) incorporated in user's hair has been developed. To completely unbind MA from hair matrix and to achieve its effective vaporization for the IMS detection, the hair sample was digested in 5 M NaOH (methanol-water, 4:1, v/v) solution prior to IMS measurement. MA in hair was semi-quantitatively detected by monitoring the digested hair sample employing dibenzylamine (DBA) as internal standard. The minimum amount of hair sample required was 2 mg and its digested sample was ample for four IMS measurements. Teh detection limit of MA in hair was 0.5 ng mg⁻¹. This proposed method was applicable to the semi-quantitative detection of MA in users' hair samples, and to the sectional analysis for MA in a limited amount of user's hair. The IMS results obtained were in good agreement with their GC-MS determination.     

Heterogeneous pattern of retinal nerve fiber layer in multiple sclerosis. High resolution optical coherence tomography: potential and limitations

Background

Recently the reduction of the retinal nerve fibre layer (RNFL) was suggested to be associated with diffuse axonal damage in the whole CNS of multiple sclerosis (MS) patients. However, several points are still under discussion. (1) Is high resolution optical coherence tomography (OCT) required to detect the partly very subtle RNFL changes seen in MS patients? (2) Can a reduction of RNFL be detected in all MS patients, even in early disease courses and in all MS subtypes? (3) Does an optic neuritis (ON) or focal lesions along the visual pathways, which are both very common in MS, limit the predication of diffuse axonal degeneration in the whole CNS? The purpose of our study was to determine the baseline characteristics of clinical definite relapsing-remitting (RRMS) and secondary progressive (SPMS) MS patients with high resolution OCT technique.

Methodology

Forty-two RRMS and 17 SPMS patients with and without history of uni- or bilateral ON, and 59 age- and sex-matched healthy controls were analysed prospectively with the high resolution spectral-domain OCT device (SD-OCT) using the Spectralis 3.5mm circle scan protocol with locked reference images and eye tracking mode. Furthermore we performed tests for visual and contrast acuity and sensitivity (ETDRS, Sloan and Pelli-Robson-charts), for color vision (Lanthony D-15), the Humphrey visual field and visual evoked potential testing (VEP).

Principal Findings

All 4 groups (RRMS and SPMS with or without ON) showed significantly reduced RNFL globally, or at least in one of the peripapillary sectors compared to age-/sex-matched healthy controls. In patients with previous ON additional RNFL reduction was found. However, in many RRMS patients the RNFL was found within normal range. We found no correlation between RNFL reduction and disease duration (range 9–540 months).

Conclusions

RNFL baseline characteristics of RRMS and SPMS are heterogeneous (range from normal to markedly reduced levels).     

Ultrastructure and Development of the Ooecial Walls in Some Calloporid Bryozoans (Gymnolaemata: Cheilostomata)

In the brood chambers (ovicells) of six calloporid cheilostomes studied each skeletal wall consists of four calcified layers: (1) a very thin superficial layer of planar spherulitic crystallites, (2) an upper (outer) layer with wall-perpendicular prismatic ultrastructure, (3) an intermediate lamellar layer, and (4) a lower (inner) wall-perpendicular prismatic layer. Comparative studies of both the ovicell wall ultrastructure and early ovicell formation showed a hypothetical opportunity for evolving complex (multilayered) skeletal walls by fusion of the initially separated gymnocystal and cryptocystal calcifications in Cheilostomata. In two species studied, a bilobate pattern in the final stage of the formation of the ooecial roof was encountered in specimens with the cuticle preserved. A possible explanation to this finding is discussed – the bilobate pattern is suggestive of the hypothetical origin of the brood chamber from (1) two flattened spines, or (2) reduction in spine number of an originally multispinous ovicell.     

Facies belts and communities of the arctic Vesterisbanken Seamount (Central Greenland Sea)

Summary The Arctic Vesterisbanken Seamount, situated far offshore in the central Greenland Sea, provides a unique facility for studing modern cold water siliceous carbonate deposits. A nearly year round sea ice cover, which retreats on average only during two months, and a rather constant temperature and salinity structure of the water column characterize the Arctic conditions of the area. Despite predominantly oligotrophic conditions with a pronounced food supply from the pelagic realm only during the ice-free season, the seamount is covered extensively by extended sponge-bryozoan constructions. Three distinct facies belts reveal a pronounced depth zonation which depends on variations in downslope food transfer and which is specifically effective due to the development of aTaylor current regime over the seamount: i) the crest facies from the summit at −133m to −260 m, ii) the shallow slope facies from −260 m to −400 m, iii) the deep slope facies from −400 m down to the abyssal plain at about— 3.000 m. Different biogenic structures and communities are found within these facies belts, including widely extended biogenic mats, sponge bryozoan-serpulid buildups with mounds, hedges, spurs and flatcake-like structures, bryozoan thickets and sponge-crinoid mounds. Depth zonation, internal structure and controlling parameters in the formation of these biogenic structures are discussed in the context of their significance as a modern end member of the Foramol facies and their implication for the fossil record. In addition, the younger volcanic and hydrothermal history of the seamount is presented with special reference to its bearing on Holocene biogenic colonization patterns.     

Differential roles of ArfGAP1, ArfGAP2, and ArfGAP3 in COPI trafficking

The formation of coat protein complex I (COPI)–coated vesicles is regulated by the small guanosine triphosphatase (GTPase) adenosine diphosphate ribosylation factor 1 (Arf1), which in its GTP-bound form recruits coatomer to the Golgi membrane. Arf GTPase-activating protein (GAP) catalyzed GTP hydrolysis in Arf1 triggers uncoating and is required for uptake of cargo molecules into vesicles. Three mammalian ArfGAPs are involved in COPI vesicle trafficking; however, their individual functions remain obscure. ArfGAP1 binds to membranes depending on their curvature. In this study, we show that ArfGAP2 and ArfGAP3 do not bind directly to membranes but are recruited via interactions with coatomer. In the presence of coatomer, ArfGAP2 and ArfGAP3 activities are comparable with or even higher than ArfGAP1 activity. Although previously speculated, our results now demonstrate a function for coatomer in ArfGAP-catalyzed GTP hydrolysis by Arf1. We suggest that ArfGAP2 and ArfGAP3 are coat protein–dependent ArfGAPs, whereas ArfGAP1 has a more general function.