Identification of a calmodulin-regulated Ca2+-ATPase in the endoplasmic reticulum

A unique subfamily of calmodulin-dependent Ca2+-ATPases was recently identified in plants. In contrast to the most closely related pumps in animals, plasma membrane-type Ca2+-ATPases, members of this new subfamily are distinguished by a calmodulin-regulated autoinhibitor located at the N-terminal instead of a C-terminal end. In addition, at least some isoforms appear to reside in non-plasma membrane locations. To begin delineating their functions, we investigated the subcellular localization of isoform ACA2p (Arabidopsis Ca2+-ATPase, isoform 2 protein) in Arabidopsis. Here we provide evidence that ACA2p resides in the endoplasmic reticulum (ER). In buoyant density sucrose gradients performed with and without Mg2+, ACA2p cofractionated with an ER membrane marker and a typical "ER-type" Ca2+-ATPase, ACA3p/ECA1p. To visualize its subcellular localization, ACA2p was tagged with a green fluorescence protein at its C terminus (ACA2-GFPp) and expressed in transgenic Arabidopsis. We collected fluorescence images from live root cells using confocal and computational optical-sectioning microscopy. ACA2-GFPp appeared as a fluorescent reticulum, consistent with an ER location. In addition, we observed strong fluorescence around the nuclei of mature epidermal cells, which is consistent with the hypothesis that ACA2p may also function in the nuclear envelope. An ER location makes ACA2p distinct from all other calmodulin-regulated pumps identified in plants or animals.     

Physiological factors in stretcher carriage performance

In this study we examined the anthropometric and physiological factors that may account for the ability to carry a casualty on a stretcher. Eleven young soldiers were pretested to obtain their anthropometry, body composition, physical fitness, and muscle cross-sectional areas. They then performed a two-person manual carry of a stretcher containing an 82-kg manikin while walking on a treadmill at a speed of 4.8 km/h. Subjects walked until volitional fatigue, as indicated by slippage of the stretcher from their hands. Average (SD) carriage time was 2.7(1.4) min with a range of 1.4-6.4 min. A stepwise multiple linear regression revealed that forearm bone-plus-muscle cross-sectional area, thigh muscle cross-sectional area, and push-up performance accounted for most of the variance in hand carriage time (r2 = 0.99, P < 0.001). These data suggest that muscle cross-sectional area and upper-body muscular endurance are important physiological factors in the ability to carry a loaded stretcher by hand.     

Lateral gene transfer and the complex distribution of insertions in eukaryotic enolase

Insertions and deletions in protein-coding genes are relatively rare events compared with sequence substitutions because they are more likely to alter the tertiary structure of the protein. For this reason, insertions and deletions which are clearly homologous are considered to be stable characteristics of the proteins where they are found, and their presence and absence has been used extensively to infer large-scale evolutionary relationships and events. Recently, however, it has been shown that the pattern of highly conserved, clearly homologous insertions at positions with no other detectable homoplasy can be incongruent with the phylogeny of the genes or organisms in which they are found. One case where this has been reported is in the enolase genes of apicomplexan parasites and ciliates, which share homologous insertions in a highly conserved region of the gene with the apparently distantly related enolases of plants. Here we explore the distribution of this character in enolase genes from the third major alveolate group, the dinoflagellates, as well as two groups considered to be closely related to alveolates, haptophytes and heterokonts. With these data, all major groups of the chromalveolates are represented, and the distribution of these insertions is shown to be far more complicated than previously believed. The incongruence between this pattern, the known evolutionary relationships between the organisms, and enolase phylogeny itself cannot be explained by any single event or type of event. Instead, the distribution of enolase insertions is more likely the product of several forces that may have included lateral gene transfer, paralogy, and/or recombination. Of these, lateral gene transfer is the easiest to detect and some well-supported cases of eukaryote-to-eukaryote lateral transfer are evident from the phylogeny.     

A bifunctional 3,5-epimerase/4-keto reductase for nucleotide-rhamnose synthesis in Arabidopsis

l-Rhamnose is a component of plant cell wall pectic polysaccharides, diverse secondary metabolites, and some glycoproteins. The biosynthesis of the activated nucleotide-sugar form(s) of rhamnose utilized by the various rhamnosyltransferases is still elusive, and no plant enzymes involved in their synthesis have been purified. In contrast, two genes (rmlC and rmlD) have been identified in bacteria and shown to encode a 3,5-epimerase and a 4-keto reductase that together convert dTDP-4-keto-6-deoxy-Glc to dTDP-beta-l-rhamnose. We have identified an Arabidopsis cDNA that contains domains that share similarity to both reductase and epimerase. The Arabidopsis gene encodes a protein with a predicated molecular mass of approximately 33.5 kD that is transcribed in all tissue examined. The Arabidopsis protein expressed in, and purified from, Escherichia coli converts dTDP-4-keto-6-deoxy-Glc to dTDP-beta-l-rhamnose in the presence of NADPH. These results suggest that a single plant enzyme has both the 3,5-epimerase and 4-keto reductase activities. The enzyme has maximum activity between pH 5.5 and 7.5 at 30 degrees C. The apparent K(m) for NADPH is 90 microm and 16.9 microm for dTDP-4-keto-6-deoxy-Glc. The Arabidopsis enzyme can also form UDP-beta-l-rhamnose. To our knowledge, this is the first example of a bifunctional plant enzyme involved in sugar nucleotide synthesis where a single polypeptide exhibits the same activities as two separate prokaryotic enzymes.     

A role for hTRPC1 and lipid raft domains in store-mediated calcium entry in human platelets

We have previously suggested that store-mediated Ca2+ entry (SMCE) in human platelets may be activated by a secretion-like coupling model, involving de novo coupling of the type II inositol 1,4,5-trisphosphate receptor (IP(3)RII) to the putative Ca2+ entry channel, hTRPC1. In other cells, hTRPC1 has been reported to be associated with cholesterol-rich lipid raft domains (LRDs) in the plasma membrane. Here we have shown that hTRPC1 is largely associated with detergent-resistant platelet membranes, from which it is partially released when the cells are depleted of cholesterol by treatment with methyl-beta-cyclodextrin (MBCD). MBCD treatment inhibited thapsigargin (TG)-evoked SMCE in a concentration-dependent manner, reducing it to 38.1+/-4.1% at a concentration of 10mM. Similarly, the Ca2+ entry evoked by thrombin (1unit/ml) was reduced to 48.2+/-4.5% of control following MBCD (10mM) treatment. Thrombin- and TG-evoked coupling between IP(3)RII and hTRPC1 was also reduced following cholesterol depletion. These results suggest that hTRPC1 is associated with LRDs in human platelets and that these domains are important for its participation in SMCE.     

Chlorophyllide a Oxygenase mRNA and Protein Levels Correlate with the Chlorophyll a/b Ratio in Arabidopsis thaliana

Plants can change the size of their light harvesting complexes in response to growth at different light intensities. Although these changes are small compared to those observed in algae, their conservation in many plant species suggest they play an important role in photoacclimation. A polyclonal antibody to the C-terminus of the Arabidopsis thaliana chlorophyllide a oxygenase (CAO) protein was used to determine if CAO protein levels change under three conditions which perturb chlorophyll levels. These conditions were: (1) transfer to shaded light intensity; (2) limited chlorophyll synthesis, and (3) during photoinhibition. Transfer of wild-type plants from moderate to shaded light intensity resulted in a slight reduction in the Chl a/b ratio, and increases in both CAO and Lhcb1 mRNA levels as well as CAO protein levels. CAO protein levels were also measured in the cch1 mutant, a P642L missense mutation in the H subunit of Mg-chelatase. This mutant has reduced total Chl levels and an increased Chl a/b ratio when transferred to moderate light intensity. After transfer to moderate light intensity, CAO mRNA levels decreased in the cch1 mutant, and a concomitant decrease in CAO protein levels was also observed. Measurements of tetrapyrrole intermediates suggested that decreased Chl synthesis in the cch1 mutant was not a result of increased feedback inhibition at higher light intensity. When wild-type plants were exposed to photoinhibitory light intensity for 3 h, total Chl levels decreased and both CAO mRNA and CAO protein levels were also reduced. These results indicate that CAO protein levels correlate with CAO mRNA levels, and suggest that changes in Chl b levels in vascular plants, are regulated, in part, at the CAO mRNA level.     

Histone deacetylase inhibitors potentiate TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in lymphoid malignancies

New therapies are required for chronic lymphocytic leukemia (CLL), an incurable disease characterized by failure of mature lymphocytes to undergo apoptosis. Activation of cell surface death receptors, such as via TRAIL receptor ligation, may provide a novel therapeutic target for various malignancies. However, CLL and other lymphoid malignancies are resistant to TRAIL. We report that low concentrations of histone deacetylase (HDAC) inhibitors, such as depsipeptide, which alone failed to induce apoptosis, markedly sensitize CLL cells and other primary lymphoid malignancies to TRAIL-induced apoptosis. These combinations caused little or no toxicity to normal lymphocytes. HDAC inhibitors sensitized resistant cells to TRAIL-induced apoptosis by facilitating formation of an active death-inducing signalling complex (DISC), leading to the rapid activation of caspase-8. The facilitated DISC formation also occurred in the absence of TRAIL-R2 upregulation. Thus, the combination of HDAC inhibitors and TRAIL may be valuable in the treatment of various hemopoietic malignancies.     

P-type ATPase heavy metal transporters with roles in essential zinc homeostasis in Arabidopsis

Arabidopsis thaliana has eight genes encoding members of the type 1_B heavy metal–transporting subfamily of the P-type ATPases. Three of these transporters, HMA2, HMA3, and HMA4, are closely related to each other and are most similar in sequence to the divalent heavy metal cation transporters of prokaryotes. To determine the function of these transporters in metal homeostasis, we have identified and characterized mutants affected in each. Whereas the individual mutants exhibited no apparent phenotype, hma2 hma4 double mutants had a nutritional deficiency phenotype that could be compensated for by increasing the level of Zn, but not Cu or Co, in the growth medium. Levels of Zn, but not other essential elements, in the shoot tissues of a hma2 hma4 double mutant and, to a lesser extent, of a hma4 single mutant were decreased compared with the wild type. Together, these observations indicate a primary role for HMA2 and HMA4 in essential Zn homeostasis. HMA2promoter- and HMA4promoter-reporter gene constructs provide evidence that HMA2 and HMA4 expression is predominantly in the vascular tissues of roots, stems, and leaves. In addition, expression of the genes in developing anthers was confirmed by RT-PCR and was consistent with a male-sterile phenotype in the double mutant. HMA2 appears to be localized to the plasma membrane, as indicated by protein gel blot analysis of membrane fractions using isoform-specific antibodies and by the visualization of an HMA2-green fluorescent protein fusion by confocal microscopy. These observations are consistent with a role for HMA2 and HMA4 in Zn translocation. hma2 and hma4 mutations both conferred increased sensitivity to Cd in a phytochelatin-deficient mutant background, suggesting that they may also influence Cd detoxification.