Down-regulation of tricarboxylic acid (TCA) cycle genes blocks progression through the first mitotic division in Caenorhabditis elegans embryos

The cell cycle is a highly regulated process that enables the accurate transmission of chromosomes to daughter cells. Here we uncover a previously unknown link between the tricarboxylic acid (TCA) cycle and cell cycle progression in the Caenorhabditis elegans early embryo. We found that down-regulation of TCA cycle components, including citrate synthase, malate dehydrogenase, and aconitase, resulted in a one-cell stage arrest before entry into mitosis: pronuclear meeting occurred normally, but nuclear envelope breakdown, centrosome separation, and chromosome condensation did not take place. Mitotic entry is controlled by the cyclin B–cyclin-dependent kinase 1 (Cdk1) complex, and the inhibitory phosphorylation of Cdk1 must be removed in order for the complex to be active. We found that following down-regulation of the TCA cycle, cyclin B levels were normal but CDK-1 remained inhibitory-phosphorylated in one-cell stage-arrested embryos, indicative of a G2-like arrest. Moreover, this was not due to an indirect effect caused by checkpoint activation by DNA damage or replication defects. These observations suggest that CDK-1 activation in the C. elegans one-cell embryo is sensitive to the metabolic state of the cell, and that down-regulation of the TCA cycle prevents the removal of CDK-1 inhibitory phosphorylation. The TCA cycle was previously shown to be necessary for the development of the early embryo in mammals, but the molecular processes affected were not known. Our study demonstrates a link between the TCA cycle and a specific cell cycle transition in the one-cell stage embryo.The developmental program of any organism must be precisely executed. In Caenorhabditis elegans embryos, immediately after fertilization, two pronuclei form at opposite poles of the embryo: one containing the maternal chromosomes and the other containing the paternal ones (1, 2). These pronuclei then move toward each other, and at the same time centrosomes separate and begin to assemble a spindle. After pronuclear meeting, the cell enters its first mitosis, resulting in nuclear envelope breakdown, chromatin condensation, and the subsequent alignment of chromosomes on the metaphase plate, followed by chromosome segregation (2). Entry into mitosis depends on the mitotic cyclin B–cyclin-dependent kinase 1 (Cdk1) complex. The activity of this complex is regulated by both cyclin B levels and regulatory phosphorylation of Cdk1. In particular, Cdk1 activity is inhibited by Wee1 phosphorylation, which is removed at the onset of mitosis by the Cdc25 phosphatase (3, 4). Cdk1 activation is also subjected to various checkpoints that inhibit mitotic progression in the presence of intracellular damage (5). However, in organisms that undergo rapid embryonic divisions, including C. elegans, checkpoints are inoperative during the first few cell cycles (6).Although it is clear that cell cycle progression requires energy, the link, if any, between metabolic pathways and progression through mitosis is poorly understood. Genes and proteins involved in various aspects of metabolism (e.g., nucleotide biosynthesis and lipid metabolism) are regulated by the cell cycle machinery, and cells will not commit to a new cell cycle if nutrients are scarce (7). However, to what extent the metabolic state of the cell is sensed by the cell cycle machinery once cells have passed into S phase is not clear (8).We have previously conducted a visual screen in C. elegans embryos for genes that when down-regulated by RNAi lead to an abnormal nuclear morphology (9). Most genes whose inactivation affected early embryonic development did so without arresting cell cycle progression. It was therefore striking when we came across a set of genes, coding for enzymes of the tricarboxylic acid (TCA) cycle, that when down-regulated, led to a one-cell stage arrest with paired nuclei. The TCA cycle, also known as the Krebs cycle, uses the oxidation of acetate (in the form of acetyl CoA) derived from carbohydrates, proteins, or lipids, to generate intermediates (i.e., NADH and FADH₂) that are used by the electron transport chain for ATP production. Intermediates of the TCA cycle are also important for various anabolic pathways, such as fatty acid synthesis, and the synthesis of nucleotides. In this study, we examine the relationship between TCA cycle down-regulation and cell cycle progression in the one-cell C. elegans embryo. Our data suggest that down-regulation of the TCA cycle leads to a G2-like arrest at the one-cell stage embryo by preventing the activation of cyclin B–Cdk1.     

The trans-Golgi network-associated human ubiquitin-protein ligase POSH is essential for HIV type 1 production

HIV type 1 (HIV-1) was shown to assemble either at the plasma membrane or in the membrane of late endosomes. Now, we report an essential role for human ubiquitin ligase POSH (Plenty of SH3s; hPOSH), a trans-Golgi network-associated protein, in the targeting of HIV-1 to the plasma membrane. Small inhibitory RNA-mediated silencing of hPOSH ablates virus secretion and Gag plasma membrane localization. Reintroduction of native, but not a RING finger mutant, hPOSH restores virus release and Gag plasma membrane localization in hPOSH-depleted cells. Furthermore, expression of the RING finger mutant hPOSH inhibits virus release and induces accumulation of intracellular Gag in normal cells. Together, our results identify a previously undescribed step in HIV biogenesis and suggest a direct function for hPOSH-mediated ubiquitination in protein sorting at the trans-Golgi network. Consequently, hPOSH may be a useful host target for therapeutic intervention.     

Somatostatin analogue treatment attenuates histological findings of inflammation and increases mRNA expression of interleukin-1 beta in the articular tissues of rats with ongoing adjuvant-induced arthritis

Objective Somatostatin is a neuropeptide with modulatory effects on the immune system and the function of synovial cells; it has antiangiogenic and antiproliferative properties. This study aimed to evaluate the clinical, histological, and articular tissue cytokine mRNA response to somostatin treatment in rat adjuvant-induced arthritis (AIA).Methods Adjuvant-induced arthritis was induced in a total of 68 Lewis rats by immunization with complete Freunds adjuvant. Twenty-four rats were treated with a long-acting somostatin analogue 14 days after disease induction. Twenty-four untreated rats served as controls. The severity of arthritis was scored weekly for 42 days. In a second experiment, 20 rats (ten treated, ten controls) were killed 21 days after treatment for assessment of joint histopathology and articular tissue cytokine mRNA expression.Results Somatostatin analogue treatment significantly reduced histological scores of early inflammatory changes and increased articular tissue mRNA expression of interleukin-1 beta (IL-1). A trend toward improvement in physical scores of joint inflammation was seen in the treated group. Late destructive changes were not significantly different.Conclusion Treatment with a somostatin analogue attenuated early inflammatory changes in AIA joints and increased mRNA expression of IL-1 in the articular tissues of rats with ongoing arthritis. Improvement in the physical findings of joint inflammation was mild.     

Arsenite modulates cardiac substrate preference by translocation of GLUT4, but not CD36, independent of mitogen-activated protein kinase signaling.

The protein thiol-modifying agent arsenite, a potent activator of stress signaling, was used to examine the involvement of MAPKs in the regulation of cardiac substrate uptake. Arsenite strongly induced p38 MAPK phosphorylation in isolated rat cardiac myocytes but also moderately enhanced phosphorylation of p42/44 ERK and p70 S6K. At the level of cardiomyocytic substrate use, arsenite enhanced glucose uptake dose dependently up to 5.1-fold but failed to stimulate long-chain fatty acid uptake. At the substrate transporter level, arsenite stimulated the translocation of GLUT4 to the sarcolemma but failed to recruit CD36 or FABPpm. Because arsenite did not influence the intrinsic activity of glucose transporters, GLUT4 translocation is entirely responsible for the selective increase in glucose uptake by arsenite. Moreover, the nonadditivity of arsenite-induced glucose uptake and insulin-induced glucose uptake indicates that arsenite recruits GLUT4 from insulin-responsive intracellular stores. Inhibitor studies with SB203580/SB202190, PD98059, and rapamycin indicate that activation of p38 MAPK, p42/44 ERK, and p70 S6K, respectively, are not involved in arsenite-induced glucose uptake. In addition, all these kinases do not play a role in regulation of cardiac glucose and long-chain fatty acid uptake by insulin. Hence, arsenite's selective stimulation of glucose uptake appears unrelated to its signaling actions, suggesting that arsenite acts via thiol modification of a putative intracellular protein target of arsenite within insulin-responsive GLUT4-containing stores. Because of arsenite's selective stimulation of cardiac glucose uptake, identification of this putative target of arsenite within the GLUT4-storage compartment may indicate whether it is a target for future strategies in prevention of diabetic cardiomyopathy.     

Thionein can serve as a reducing agent for the methionine sulfoxide reductases

It has been generally accepted, primarily from studies on methionine sulfoxide reductase (Msr) A, that the biological reducing agent for the members of the Msr family is reduced thioredoxin (Trx), although high levels of DTT can be used as the reductant in vitro. Preliminary experiments using both human recombinant MsrB2 (hMsrB2) and MsrB3 (hMsrB3) showed that although DTT can function in vitro as the reducing agent, Trx works very poorly, prompting a more careful comparison of the ability of DTT and Trx to function as reducing agents with the various members of the Msr family. Escherichia coli MsrA and MsrB and bovine MsrA efficiently use either Trx or DTT as reducing agents. In contrast, hMsrB2 and hMsrB3 show <10% of the activity with Trx as compared with DTT, raising the possibility that, in animal cells, Trx may not be the direct hydrogen donor or that there may be a Trx-independent reducing system required for MsrB2 and MsrB3 activity. A heat-stable protein has been detected in bovine liver that, in the presence of EDTA, can support the Msr reaction in the absence of either Trx or DTT. This protein has been identified as a zinc-containing metallothionein (Zn-MT). The results indicate that thionein (T), which is formed when the zinc is removed from Zn-MT, can function as a reducing system for the Msr proteins because of its high content of cysteine residues and that Trx can reduce oxidized T.     

Cough ability measurements and recurrent respiratory symptoms in individuals with Ataxia Telangiectasia

Objectives: Ataxia-Telangiectasia (A-T) individuals often present with respiratory muscle weakness, causing recurrent respiratory system infections, asthma-like symptoms, and chronic cough life-threatening events. The cough flow volume maneuver may reveal powerless airflow needed for efficient cough. The study aims to explore cough ability in relation to the flow/volume maneuver. Methods: Data collected retrospectively from clinical charts of 35 A-T patients (age 12.7?±?4.9 years) included forced expiratory and cough flow/volume maneuvers performed on the same day. Analysis compared among the maneuvers matching indices, numbers of cough-spikes, flow rate decay, and the reference data of similar ages. Adjusted to age, BMI, and number of hospitalizations prior to the tests, values were correlated with the cough indices. Results: Cough peak-flow (C-PF) was propagated within 90?±?20?ms compared with peak expiratory flow (PEF?>?200?ms). C-PF measured values were higher than expiratory peak-flow measured values (3.27?±?1.53?L/s versus 3.02?±?1.52?L/s, respectively, but C-PF (%predicted) values were significantly lower than expiratory peak-flow (%predicted) (46?±?15 versus 68?±?20 %predicted, respectively, p?<?0.002). The number of spikes/maneuver was low when compared with reference (2.0?±?0.8 versus 6–12 spikes) and cough vital-capacity was lower than expiratory vital capacity (0.95?±?0.43 versus 1.03?±?0.47; p?<?0.01). Inefficient C-PF was more prevalent in patients suffering from recurrent respiratory illness. The length of wheelchair confinement duration mostly influenced the C-VC level. Conclusions: The cough flow–volume curve can be applied as a method to follow cough ability in patients with A-T who showed a significantly reduced cough capacity. Further studies are needed to establish if the findings may aid decisions regarding cough assistance.     

Synovial fluid levels of anti-cyclic citrullinated peptide antibodies and IgA rheumatoid factor in rheumatoid arthritis, psoriatic arthritis, and osteoarthritis

OBJECTIVE: To assess the levels of anti-cyclic citrullinated peptide (anti-CCP) and IgA rheumatoid factor (IgA-RF) in synovial fluids of patients with rheumatoid arthritis (RA), psoriatic arthritis (PsA), and osteoarthritis (OA). METHODS: Knee effusions of 29 patients with RA (23 women, 6 men; mean +/- SD age 60 +/- 15 years), 20 with PsA (6 women, 14 men; mean age 51 +/- 12 years), and 19 with OA (9 women, 10 men; mean age 73 +/- 11.8 years) were aspirated, tested for white blood cell (WBC) counts, centrifuged, and stored at -20 degrees . Sera of 22, 11, and 12 of these patients with RA, PsA, and OA, respectively, were similarly stored. IgG anti-CCP and IgA-RF were detected by enzyme-linked immunosorbent assay. Erythrocyte sedimentation rate and C-reactive protein levels were used as measures of disease activity. RESULTS: Mean levels of synovial fluid anti-CCP and IgA-RF were significantly increased in RA joint effusions compared with PsA and OA (anti-CCP: 150 +/- 134, 34 +/- 29, and 24 +/- 26 units, respectively [P < 0.003]; IgA-RF: 76 +/- 77, 15.7 +/- 10, and 18 +/- 20 units, respectively). No significant difference was noted between OA and PsA. A significant correlation was found between synovial fluid anti-CCP and serum anti-CCP and IgA-RF. In patients with RA, a significant correlation was found between synovial fluid WBC counts and IgA-RF (P = 0.03) and serum IgA-RF (P = 0.008), but not between synovial fluid and serum anti-CCP levels. In RA patients, C-reactive protein correlated with serum IgA-RF. CONCLUSION: Anti-CCP and IgA-RF were significantly increased in synovial fluid of RA in comparison with PsA and OA patients.