Soluble methionine enhances accumulation of a 15 kDa zein, a methionine-rich storage protein, in transgenic alfalfa but not in transgenic tobacco plants

With the general aim of elevating the content of the essential amino acid methionine in vegetative tissues of plants, alfalfa (Medicago sativa L.) and tobacco plants, as well as BY2 tobacco suspension cells, were transformed with a beta-zein::3HA gene under the 35S promoter of cauliflower mosaic virus encoding a rumen-stable methionine-rich storage protein of 15 kDa zein. To examine whether soluble methionine content limited the accumulation of the 15 kDa zein::3HA, methionine was first added to the growth medium of the different transgenic plants and the level of the alien protein was determined. Results demonstrated that the added methionine enhanced the accumulation of the 15 kDa zein::3HA in transgenic alfalfa and tobacco BY2 cells, but not in whole transgenic tobacco plants. Next, the endogenous levels of methionine were elevated in the transgenic tobacco and alfalfa plants by crossing them with plants expressing the Arabidopsis cystathionine gamma-synthase (AtCGS) having significantly higher levels of soluble methionine in their leaves. Compared with plants expressing only the 15 kDa zein::3HA, transgenic alfalfa co-expressing both alien genes showed significantly enhanced levels of this protein concurrently with a reduction in the soluble methionine content, thus implying that soluble methionine was incorporated into the 15 kDa zein::3HA. Similar phenomena also occurred in tobacco, but were considerably less pronounced. The results demonstrate that the accumulation of the 15 kDa zein::3HA is regulated in a species-specific manner and that soluble methionine plays a major role in the accumulation of the 15 kDa zein in some plant species but less so in others.     

Heat shock protein 60 activates cytokine-associated negative regulator suppressor of cytokine signaling 3 in T cells: effects on signaling, chemotaxis, and inflammation

Previously, we reported that treatment of T cells with the 60-kDa heat shock protein (HSP60) inhibits chemotaxis. We now report that treatment of purified human T cells with recombinant human HSP60 or its biologically active peptide p277 up-regulates suppressor of cytokine signaling (SOCS)3 expression via TLR2 and STAT3 activation. SOCS3, in turn, inhibits the downstream effects of stromal cell-derived-1alpha (CXCL12)-CXCR4 interaction in: 1) phosphorylation of ERK1/2, Pyk2, AKT, and myosin L chain, required for cell adhesion and migration; 2) formation of rear-front T cell polarity; and 3) migration into the bone marrow of NOD/SCID mice. HSP60 also activates SOCS3 in mouse lymphocytes and inhibits their chemotaxis toward stromal cell-derived factor-1alpha and their ability to adoptively transfer delayed-type hypersensitivity. These effects of HSP60 could not be attributed to LPS or LPS-associated lipoprotein contamination. Thus, HSP60 can regulate T cell-mediated inflammation via specific signal transduction and SOCS3 activation.     

Identification of a new glycerol-3-phosphate acyltransferase isoenzyme, mtGPAT2, in mitochondria

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and rate-limiting step of glycerolipid synthesis. Two distinct GPAT isoenzymes had been identified in mammalian tissues, an N-ethylmaleimide (NEM)-sensitive isoform in the endoplasmic reticulum membrane (microsomal GPAT) and an NEM-resistant form in the outer mitochondrial membrane (mtGPAT). Although only mtGPAT has been cloned, the microsomal and mitochondrial GPAT isoforms can be distinguished, because they differ in acyl-CoA substrate preference, sensitivity to inhibition by dihydroxyacetone phosphate and polymixin B, temperature sensitivity, and ability to be activated by acetone. The preponderance of evidence supports a role for mtGPAT in synthesizing the precursors for triacylglycerol synthesis. In mtGPAT(-/-) mice, PCR genotyping and Northern analysis showed successful knockout of mtGPAT; however, we detected a novel NEM-sensitive GPAT activity in mitochondrial fractions and an anti-mtGPAT immunoreactive protein in liver mitochondria, but not in microsomes. Rigorous analysis using two-dimensional gel electrophoresis revealed that the anti-mtGPAT immunoreactive proteins in wild type and mtGPAT(-/-) liver mitochondria have different isoelectric points. These results suggested the presence of a second GPAT in liver mitochondria from mtGPAT(-/-) mice. Characterization of this GPAT activity in liver from mtGPAT null mice showed that, unlike the mtGPAT activity in wild type samples, activity in mtGPAT knockout mitochondria did not prefer palmitoyl-CoA, was sensitive to inactivation by NEM, was inhibited by dihydroxyacetone phosphate and polymixin B, was temperature-sensitive, and was not activated by acetone. We conclude that a novel GPAT (mtGPAT2) with antigenic epitopes similar to those of mtGPAT is detectable in mitochondria from the livers of mtGPAT(-/-) mice.     

Gibberellic Acid (GA3) Inhibits ROS Increase in Chloroplasts During Dark-Induced Senescence of Pelargonium Cuttings

The temporal and spatial changes in reactive oxygen species (ROS) during dark treatment of Pelargonium cuttings and the effect of gibberellic acid (GA₃) on ROS levels were studied. ROS-related fluorescence was detected in mitochondria and cytoplasm of epidermal cells and in chloroplasts. By monitoring dichlorofluorescein (DCF) fluorescence, an initial decrease in ROS was observed under darkness in the epidermal cell cytoplasm and the chloroplasts, which was followed by an increase on the third day. Following 3 days under darkness, the size and the structure of the chloroplasts also changed, and they became more sensitive to illumination as judged by a higher accumulation of ROS. Pretreatment of leaves with GA₃ did not prevent the structural changes in the chloroplasts, but it inhibited the increase in ROS levels in all cell compartments, including the chloroplasts. It is suggested that the inhibition of ROS increase by GA₃ prevented complete disintegration of chloroplasts during dark-induced senescence and thereby enabled the maintenance of chlorophyll levels in the tissue.     

Occupancy of lymphocyte LFA-1 by surface-immobilized ICAM-1 is critical for TCR- but not for chemokine-triggered LFA-1 conversion to an open headpiece high-affinity state

Lymphocyte arrest and spreading on ICAM-1-expressing APCs require activation of lymphocyte LFA-1 by TCR signals, but the conformational switches of this integrin during these critical processes are still elusive. Using Ab probes that distinguish between different LFA-1 conformations, we found that, unlike strong chemokine signals, potent TCR stimuli were insufficient to trigger LFA-1 extension or headpiece opening in primary human lymphocytes. Nevertheless, LFA-1 in these TCR-stimulated T cells became highly adhesive to both anchored and mobile surface-bound ICAM-1, although it failed to bind soluble ICAM-1 with measurable affinity. Rapid rearrangement of LFA-1 by immobilized ICAM-1 switched the integrin to an open headpiece conformation within numerous scattered submicron focal dots that did not readily collapse into a peripheral LFA-1 ring. Headpiece-activated LFA-1 microclusters were enriched with talin but were devoid of TCR and CD45. Notably, LFA-1 activation by TCR signals as well as subsequent T cell spreading on ICAM-1 took place independently of cytosolic Ca(2+). In contrast to LFA-1-activating chemokine signals, TCR activation of LFA-1 readily took place in the absence of external shear forces. LFA-1 activation by TCR signals also did not require internal myosin II forces but depended on intact actin cytoskeleton. Our results suggest that potent TCR signals fail to trigger LFA-1 headpiece activation unless the integrin first gets stabilized by surface-bound ICAM-1 within evenly scattered actin-dependent LFA-1 focal dots, the quantal units of TCR-stimulated T cell arrest and spreading on ICAM-1.     

An evolutionary space-time model with varying among-site dependencies

It is now widely accepted that sites in a protein do not undergo independent evolutionary processes. The underlying assumption is that proteins are composed of conserved and variable linear domains, and thus rates at neighboring sites are correlated. In this paper, we comprehensively examine the performance of an autocorrelation model of evolutionary rates in protein sequences. We further develop a model in which the level of correlation between rates at adjacent sites is not equal at all sites of the protein. High correlation is expected, for example, in linear functional domains. On the other hand, when we consider nonlinear functional regions (e.g., active sites), low correlation is expected because the interaction between distant sites imposes independence of rates in the linear sequence. Our model is based on a hidden Markov model, which accounts for autocorrelation at certain regions of the protein and rate independence at others. We study the differences between the novel model and models which assume either independence or a fixed level of dependence throughout the protein. Using a diverse set of protein data sets we show that the novel model better fits most data sets. We further analyze the potassium-channel protein family and illustrate the relationship between the dependence of rates at adjacent sites and the tertiary structure of the protein.     

maSigPro: a method to identify significantly differential expression profiles in time-course microarray experiments

MOTIVATION: Multi-series time-course microarray experiments are useful approaches for exploring biological processes. In this type of experiments, the researcher is frequently interested in studying gene expression changes along time and in evaluating trend differences between the various experimental groups. The large amount of data, multiplicity of experimental conditions and the dynamic nature of the experiments poses great challenges to data analysis. RESULTS: In this work, we propose a statistical procedure to identify genes that show different gene expression profiles across analytical groups in time-course experiments. The method is a two-regression step approach where the experimental groups are identified by dummy variables. The procedure first adjusts a global regression model with all the defined variables to identify differentially expressed genes, and in second a variable selection strategy is applied to study differences between groups and to find statistically significant different profiles. The methodology is illustrated on both a real and a simulated microarray dataset.     

Anaerobic Ammonium-Oxidizing (Anammox) Bacteria and Associated Activity in Fixed-Film Biofilters of a Marine Recirculating Aquaculture System

Microbial communities in the biological filter and waste sludge compartments of a marine recirculating aquaculture system were examined to determine the presence and activity of anaerobic ammonium-oxidizing (anammox) bacteria. Community DNA was extracted from aerobic and anaerobic fixed-film biofilters and the anaerobic sludge waste collection tank and was analyzed by amplifying 16S rRNA genes by PCR using anammox-selective and universal GC-clamped primers. Separation of amplified PCR products by denaturing gradient gel electrophoresis and sequencing of the different phylotypes revealed a diverse biofilter microbial community. While Planctomycetales were found in all three communities, the anaerobic denitrifying biofilters contained one clone that exhibited high levels of sequence similarity to known anammox bacteria. Fluorescence in situ hybridization studies using an anammox-specific probe confirmed the presence of anammox Planctomycetales in the microbial biofilm from the denitrifying biofilters, and anammox activity was observed in these biofilters, as detected by the ability to simultaneously consume ammonia and nitrite. To our knowledge, this is the first identification of anammox-related sequences in a marine recirculating aquaculture filtration system, and our findings provide a foundation for incorporating this important pathway for complete nitrogen removal in such systems.     

The P1812A and P25T BRCA1 and the 5164del4 BRCA2 mutations: occurrence in high-risk non-Ashkenazi Jews

Founder mutations in the BRCA1 and BRCA2 genes have been discovered in the Ashkenazic Jewish population, but a founder mutation(s) has not been discovered among non-Ashkenazi Jews (NAJ). Two BRCA1 mutations (P1812A, P25T), and a BRCA2 mutation (5164del4) have been detected in NAJ high-risk families. We studied the prevalence of these three mutations in 270 high-risk NAJ families, including 85 from Iraq/Iran, 67 from North Africa, 27 from Yemen, 50 from the Balkan region, and 41 with mixed ancestry. The three mutations were detected only in individuals related to the original families. We conclude that the P1812A and P25T BRCA1 and 5164del4 BRCA2 mutations are not likely to be founder mutations in NAJ high-risk families. We also assessed the pathogenicity of the BRCA1 P1812A mutation in vitro using reporter gene assays in yeast and mammalian cells. We found that the BRCA1 P1812A variant activity assays yielded a slightly reduced reporter gene activity. Thus, there is some uncertainty as to the pathogenicity of BRCA1 P1812A.