The mitochondrial proteome of the model legume Medicago truncatula

Legumes carry out special biochemical functions, e.g. the fixation of molecular nitrogen based on a symbiosis with proteobacteria. At the cellular level, this symbiosis has to be implemented into the energy metabolism of the host cell. To provide a basis for future analyses, we have characterized the protein complement of mitochondria of the model legume Medicago truncatula using two-dimensional isoelectric focussing (IEF) and blue-native (BN)-SDS-PAGE. While the IEF reference map resulted mainly in resolution of those proteins associated with the mitochondrial matrix, the BN proteomic map allowed separation of protein subunits from the respiratory chain protein complexes, which are located in the organelle's inner membrane. The M. truncatula mitochondrial BN reference map revealed some striking similarities to the one from Arabidopsis thaliana but at the same time exhibited also some special features: complex II is of increased abundance and additionally represented by a low molecular mass form not reported for Arabidopsis. Furthermore three highly abundant forms of prohibitin complexes are present in the mitochondrial proteome of M. truncatula. Special features with respect to mitochondrial protein complexes might reflect adaptations of legumes to elevated cellular energy requirements enabling them to develop symbiotic interactions with rhizobial bacteria.     

Increased loss of CCR5+ CD45RA- CD4+ T cells in CD8+ lymphocyte-depleted Simian immunodeficiency virus-infected rhesus monkeys

Previously we have shown that CD8(+) T cells are critical for containment of simian immunodeficiency virus (SIV) viremia and that rapid and profound depletion of CD4(+) T cells occurs in the intestinal tract of acutely infected macaques. To determine the impact of SIV-specific CD8(+) T-cell responses on the magnitude of the CD4(+) T-cell depletion, we investigated the effect of CD8(+) lymphocyte depletion during primary SIV infection on CD4(+) T-cell subsets and function in peripheral blood, lymph nodes, and intestinal tissues. In peripheral blood, CD8(+) lymphocyte-depletion changed the dynamics of CD4(+) T-cell loss, resulting in a more pronounced loss 2 weeks after infection, followed by a temporal rebound approximately 2 months after infection, when absolute numbers of CD4(+) T cells were restored to baseline levels. These CD4(+) T cells showed a markedly skewed phenotype, however, as there were decreased levels of memory cells in CD8(+) lymphocyte-depleted macaques compared to controls. In intestinal tissues and lymph nodes, we observed a significantly higher loss of CCR5(+) CD45RA(-) CD4(+) T cells in CD8(+) lymphocyte-depleted macaques than in controls, suggesting that these SIV-targeted CD4(+) T cells were eliminated more efficiently in CD8(+) lymphocyte-depleted animals. Also, CD8(+) lymphocyte depletion significantly affected the ability to generate SIV Gag-specific CD4(+) T-cell responses and neutralizing antibodies. These results reemphasize that SIV-specific CD8(+) T-cell responses are absolutely critical to initiate at least partial control of SIV infection.     

Dorso-ventral polarity of the zebrafish embryo is distinguishable prior to the onset of gastrulation

We describe a set of observations on developing zebrafish embryos and discuss the main conclusions they allow:(1) the embryonic dorso-ventral polarity axis is morphologically distinguishable prior to the onset of gastrulation; and (2) the involution of deep layer cells starts on the prospective dorsal side of the embryo. An asymmetry can be distinguished in the organization of the blastomeres in the zebrafish blastula at the 30% epiboly stage, in that one sector of the blastoderm is thicker than the other. Dye-labelling experiments with DiI and DiO and histological analysis allow us to conclude that the embryonic shield will form on the thinner side of the blastoderm. Therefore, this side corresponds to the prospective dorsal side of the embryo. Simultaneous injections of dyes on the thinner side of the blastoderm and on the opposite side show that involution of deep layer cells during gastrulation starts at the site at which the embryonic shield will form and extends from here to the prospective ventral regions of the germ ring.     

RBM3 regulates temperature sensitive miR-142–5p and miR-143 (thermomiRs), which target immune genes and control fever

Fever is commonly used to diagnose disease and is consistently associated with increased mortality in critically ill patients. However, the molecular controls of elevated body temperature are poorly understood. We discovered that the expression of RNA-binding motif protein 3 (RBM3), known to respond to cold stress and to modulate microRNA (miRNA) expression, was reduced in 30 patients with fever, and in THP-1-derived macrophages maintained at a fever-like temperature (40°C). Notably, RBM3 expression is reduced during fever whether or not infection is demonstrable. Reduced RBM3 expression resulted in increased expression of RBM3-targeted temperature-sensitive miRNAs, we termed thermomiRs. ThermomiRs such as miR-142–5p and miR-143 in turn target endogenous pyrogens including IL-6, IL6ST, TLR2, PGE2 and TNF to complete a negative feedback mechanism, which may be crucial to prevent pathological hyperthermia. Using normal PBMCs that were exogenously exposed to fever-like temperature (40°C), we further demonstrate the trend by which decreased levels of RBM3 were associated with increased levels of miR-142–5p and miR-143 and vice versa over a 24 h time course. Collectively, our results indicate the existence of a negative feedback loop that regulates fever via reduced RBM3 levels and increased expression of miR-142–5p and miR-143.     

Similar mitochondrial activation kinetics in wild-type and creatine kinase-deficient fast-twitch muscle indicate significant Pi control of respiration

Past simulations of oxidative ATP metabolism in skeletal muscle have predicted that elimination of the creatine kinase (CK) reaction should result in dramatically faster oxygen consumption dynamics during transitions in ATP turnover rate. This hypothesis was investigated. Oxygen consumption of fast-twitch (FT) muscle isolated from wild-type (WT) and transgenic mice deficient in the myoplasmic (M) and mitochondrial (Mi) CK isoforms (MiM CK(-/-)) were measured at 20°C at rest and during electrical stimulation. MiM CK(-/-) muscle oxygen consumption activation kinetics during a step change in contraction rate were 30% faster than WT (time constant 53 ± 3 vs. 69 ± 4 s, respectively; mean ± SE, n = 8 and 6, respectively). MiM CK(-/-) muscle oxygen consumption deactivation kinetics were 380% faster than WT (time constant 74 ± 4 s vs. 264 ± 4 s, respectively). Next, the experiments were simulated using a computational model of the oxidative ATP metabolic network in FT muscle featuring ADP and Pi feedback control of mitochondrial respiration (J. A. L. Jeneson, J. P. Schmitz, N. A. van den Broek, N. A. van Riel, P. A. Hilbers, K. Nicolay, J. J. Prompers. Am J Physiol Endocrinol Metab 297: E774-E784, 2009) that was reparameterized for 20°C. Elimination of Pi control via clamping of the mitochondrial Pi concentration at 10 mM reproduced past simulation results of dramatically faster kinetics in CK(-/-) muscle, while inclusion of Pi control qualitatively explained the experimental observations. On this basis, it was concluded that previous studies of the CK-deficient FT muscle phenotype underestimated the contribution of Pi to mitochondrial respiratory control.     

Recombinant canarypox vaccine-elicited CTL specific for dominant and subdominant simian immunodeficiency virus epitopes in rhesus monkeys

Since virus-specific CTL play a central role in containing HIV replication, a candidate AIDS vaccine should generate virus-specific CTL responses. In this study, the ability of a recombinant canarypox virus expressing SIV Gag-Pol-Env (ALVAC/SIV gag-pol-env) was assessed for its ability to elicit both dominant and subdominant epitope-specific CTL responses in rhesus monkeys. Following a series of five immunizations, memory CTL responses specific for a dominant Gag epitope could be demonstrated in the peripheral blood of vaccinated monkeys. Memory CTL responses to a subdominant Pol epitope were undetectable in these animals. Following challenge with SIVmac251, the experimentally vaccinated animals developed high frequency CTL responses specific for the dominant Gag epitope that emerged in temporal association with the early containment of viral replication. Interestingly, the experimentally vaccinated, but not the control vaccinated animals, developed CTL responses to the subdominant Pol epitope that were detectable only after containment of early viremia. Thus, recombinant canarypox vaccination elicited low frequency, but durable memory CTL populations. The temporal association of the emergence of the dominant epitope-specific response with early viral containment following challenge suggests that this immune response played a role in the accelerated clearing of early viremia in these animals. The later emerging CTL response specific for the subdominant epitope may contribute to the control of viral replication in the setting of chronic infection.     

A locus on chromosome 10 influences C-reactive protein levels in two independent populations

High sensitivity C-reactive protein (hsCRP) is an independent risk factor for cardiovascular disease, such as stroke or coronary artery disease. Genetic factors influence significantly the inter-individual variability of hsCRP. The aim of this study was to identify genomic regions influencing hsCRP levels. A genome scan was performed in two independent studies of Caucasian populations, namely 513 Western-European families ascertained for myocardial infarction (n = 1,406) and 120 French-Canadian families diagnosed with hypertension (n = 758). In the myocardial infarction families, 31% of the inter-individual variation of hsCRP levels was explained by genetic factors (P = 0.0000015) and loci influencing hsCRP were identified on chromosomes 10 (at 141 cM) and 5 (at 150 cM) with multipoint LOD scores of 3.15 and 2.23, respectively. An additional suggestive signal was detected on chromosome 2 in subset analyses. A similar degree of heritability has been observed in a second independent population of French-Canadian hypertensive families for hsCRP (30%) and linkage results for chromosome 10 were confirmed with maximum LOD score of 2.7. We identified a chromosomal region in two independent populations which influences hsCRP in addition to several unique regions. This provides targets for the identification of genes involved in the regulation of hsCRP and the development and progression of vascular disease, including stroke. Ulrich Broeckel and Christian Hengstenberg contributed equally.     

An allele of gyrA prevents Salmonella enterica serovar Typhimurium from using succinate as a carbon source

A mutant gyrA allele resulting in an A271E substitution in the DNA gyrase protein generated a strain unable to grow on the C(4)-dicarboxylates succinate, malate, and fumarate. Bacteria harboring gyrA751 displayed decreased negative supercoiling in cells. Expression of the dctA gene, which encodes the C(4)-dicarboxylate transporter, was reduced in a gyrA751 mutant, providing the first evidence that dctA expression is supercoiling sensitive and uncovering a simple metabolic screen for lesions in gyrase that reduce negative supercoiling.     

Association study of GIT1 gene with attention‐deficit hyperactivity disorder in Brazilian children and adolescents

Attention‐deficit hyperactivity disorder (ADHD) is one of the most common psychiatric disorders in children with a worldwide prevalence of 5.3%. Recently, a Korean group assessed the G‐protein‐coupled receptor kinase‐interacting protein 1 (GIT1) gene that had previously been associated with ADHD. In their work, 27 single nucleotide polymorphisms SNPs in the GIT1 gene were tested; however, only the rs550818 SNP was associated with ADHD susceptibility. Moreover, the presence of the risk‐associated allele determined reduced GIT1 expression, and Git1‐deficient mice exhibit ADHD‐like phenotypes. The aim of this study was to determine if this association also occurs in a sample of Brazilian children with ADHD. No effect of GIT1 genotypes on ADHD susceptibility was observed in the case–control analysis. The odds ratios (ORs) were 0.75 (P = 0.184) for the CT genotype and 1.09 (P = 0.862) for the TT genotype. In addition, the adjusted OR of the CT+TT genotypes vs. the CC genotype was also estimated (P = 0.245). There were no dimensional associations between the GIT1 genotypes and both hyperactivity and /impulsivity, and only hyperactivity Swanson, Nolan and Pelham Scale‐Version IV (SNAP‐IV) scores (P = 0.609 and P = 0.247, respectively). The transmission/disequilibrium test indicated that there was no over‐transmission of rs550818 alleles from parents to ADHD children (z = 0.305; P = 0.761). We conclude that rs550818 is not associated with ADHD in this Brazilian sample. More studies are required before concluding that this polymorphism plays a role in ADHD susceptibility.