Drosophila BubR1 is essential for meiotic sister-chromatid cohesion and maintenance of synaptonemal complex

The partially conserved Mad3/BubR1 protein is required during mitosis for the spindle assembly checkpoint (SAC). In meiosis, depletion causes an accelerated transit through prophase I and missegregation of achiasmate chromosomes in yeast [1], whereas in mice, reduced dosage leads to severe chromosome missegregation [2]. These observations indicate a meiotic requirement for BubR1, but its mechanism of action remains unknown. We identified a viable bubR1 allele in Drosophila resulting from a point mutation in the kinase domain that retains mitotic SAC activity. In males, we demonstrate a dose-sensitive requirement for BubR1 in maintaining sister-chromatid cohesion at anaphase I, whereas the mutant BubR1 protein localizes correctly. In bubR1 mutant females, we find that both achiasmate and chiasmate chromosomes nondisjoin mostly equationally consistent with a defect in sister-chromatid cohesion at late anaphase I or meiosis II. Moreover, mutations in bubR1 cause a consistent increase in pericentric heterochromatin exchange frequency, and although the synaptonemal complex is set up properly during transit through the germarium, it is disassembled prematurely in prophase by stage 1. Our results demonstrate that BubR1 is essential to maintain sister-chromatid cohesion during meiotic progression in both sexes and for normal maintenance of SC in females.     

Thrombopoietin inhibits nerve growth factor-induced neuronal differentiation and ERK signalling

Thrombopoietin (TPO), a hematopoietic growth factor regulating platelet production, and its receptor (TPOR) were recently shown to be expressed in the brain where they exert proapoptotic activity. Here we used PC12 cells, an established model of neuronal differentiation, to investigate the effects of TPO on neuronal survival and differentiation. These cells expressed TPOR mRNA. TPO increased cell death in neuronally differentiated PC12 cells but had no effect in undifferentiated cells. Surprisingly, TPO inhibited nerve growth factor (NGF)-induced differentiation of PC12 cells in a dose- and time-dependent manner. This inhibition was dependent on the activity of Janus kinase-2 (JAK2). Using phospho-kinase arrays and Western blot we found downregulation of the NGF-stimulated phosphorylation of the extracellular signal-regulated kinase p42ERK by TPO with no effect on phosphorylation of Akt or stress kinases. NGF-induced phosphorylation of ERK-activating kinases, MEK1/2 and C-RAF was also reduced by TPO while NGF-induced RAS activation was not attenuated by TPO treatment. In contrast to its inhibitory effects on NGF signalling, TPO had no effect on epidermal growth factor (EGF)-stimulated ERK phosphorylation or proliferation of PC12 cells. Our data indicate that TPO via activation of its receptor-bound JAK2 delays the NGF-dependent acquisition of neuronal phenotype and decreases neuronal survival by suppressing NGF-induced ERK activity.     

The fixation of locally beneficial alleles in a metapopulation

Extinction, recolonization, and local adaptation are common in natural spatially structured populations. Understanding their effect upon genetic variation is important for systems such as genetically modified organism management or avoidance of drug resistance. Theoretical studies on the effect of extinction and recolonization upon genetic variance started appearing in the 1970s, but the role of local adaptation still has no good theoretical basis. Here we develop a model of a haploid species in a metapopulation in which a locally adapted beneficial allele is introduced. We study the effect of different spatial patterns of local adaptation, and different metapopulation dynamics, upon the fixation probability of the beneficial allele. Controlling for the average selection pressure, we find that a small area of positive selection can significantly increase the global probability of fixation. However, local adaptation becomes less important as extinction rate increases. Deme extinction and recolonization have a spatial smoothing effect that effectively reduces spatial variation in fitness.     

A novel Cas family member, HEPL, regulates FAK and cell spreading

For over a decade, p130Cas/BCAR1, HEF1/NEDD9/Cas-L, and Efs/Sin have defined the Cas (Crk-associated substrate) scaffolding protein family. Cas proteins mediate integrin-dependent signals at focal adhesions, regulating cell invasion and survival; at least one family member, HEF1, regulates mitosis. We here report a previously undescribed novel branch of the Cas protein family, designated HEPL (for HEF1-Efs-p130Cas-like). The HEPL branch is evolutionarily conserved through jawed vertebrates, and HEPL is found in some species lacking other members of the Cas family. The human HEPL mRNA and protein are selectively expressed in specific primary tissues and cancer cell lines, and HEPL maintains Cas family function in localization to focal adhesions, as well as regulation of FAK activity, focal adhesion integrity, and cell spreading. It has recently been demonstrated that upregulation of HEF1 expression marks and induces metastasis, whereas high endogenous levels of p130Cas are associated with poor prognosis in breast cancer, emphasizing the clinical relevance of Cas proteins. Better understanding of the complete protein family should help inform prediction of cancer incidence and prognosis.     

1H, 13C and 15N resonance assignments of the Calmodulin-Munc13-1 peptide complex

Ca²⁺-Calmodulin binding to the variable N-terminal region of the diacylglycerol/phorbol ester-binding UNC13/Munc13 family of proteins modulates the short-term synaptic plasticity characteristics in neurons. Here, we report the sequential backbone and side chain resonance assignment of the Ca²⁺-Calmodulin/Munc13-1^458–492 peptide complex at pH 6.8 and 35°C (BMRB No. 15470).     

Differential and common leukemogenic potentials of multiple NUP98-Hox fusion proteins alone or with Meis1

NUP98-Hox fusion genes are newly identified oncogenes isolated in myeloid leukemias. Intriguingly, only Abd-B Hox genes have been reported as fusion partners, indicating that they may have unique overlapping leukemogenic properties. To address this hypothesis, we engineered novel NUP98 fusions with Hox genes not previously identified as fusion partners: the Abd-B-like gene HOXA10 and two Antennepedia-like genes, HOXB3 and HOXB4. Notably, NUP98-HOXA10 and NUP98-HOXB3 but not NUP98-HOXB4 induced leukemia in a murine transplant model, which is consistent with the reported leukemogenic potential ability of HOXA10 and HOXB3 but not HOXB4. Thus, the ability of Hox genes to induce leukemia as NUP98 fusion partners, although apparently redundant for Abd-B-like activity, is not restricted to this group, but rather is determined by the intrinsic leukemogenic potential of the Hox partner. We also show that the potent leukemogenic activity of Abd-B-like Hox genes is correlated with their strong ability to block hematopoietic differentiation. Conversely, coexpression of the Hox cofactor Meis1 alleviated the requirement of a strong intrinsic Hox-transforming potential to induce leukemia. Our results support a model in which many if not all Hox genes can be leukemogenic and point to striking functional overlap not previously appreciated, presumably reflecting common regulated pathways.     

The ingi and RIME non-LTR retrotransposons are not randomly distributed in the genome of Trypanosoma brucei

The ingi (long and autonomous) and RIME (short and nonautonomous) non--long-terminal repeat retrotransposons are the most abundant mobile elements characterized to date in the genome of the African trypanosome Trypanosoma brucei. These retrotransposons were thought to be randomly distributed, but a detailed and comprehensive analysis of their genomic distribution had not been performed until now. To address this question, we analyzed the ingi/RIME sequences and flanking sequences from the ongoing T. brucei genome sequencing project (TREU927/4 strain). Among the 81 ingi/RIME elements analyzed, 60% are complete, and 7% of the ingi elements (approximately 15 copies per haploid genome) appear to encode for their own transposition. The size of the direct repeat flanking the ingi/RIME retrotransposons is conserved (i.e., 12-bp), and a strong 11-bp consensus pattern precedes the 5'-direct repeat. The presence of a consensus pattern upstream of the retroelements was confirmed by the analysis of the base occurrence in 294 GSS containing 5'-adjacent ingi/RIME sequences. The conserved sequence is present upstream of ingis and RIMEs, suggesting that ingi-encoded enzymatic activities are used for retrotransposition of RIMEs, which are short nonautonomous retroelements. In conclusion, the ingi and RIME retroelements are not randomly distributed in the genome of T. brucei and are preceded by a conserved sequence, which may be the recognition site of the ingi-encoded endonuclease.     

Disruption of ptLPD1 or ptLPD2, Genes That Encode Isoforms of the Plastidial Lipoamide Dehydrogenase,Confers Arsenate Hypersensitivity in Arabidopsis

Arsenic is a ubiquitous environmental poison that inhibits root elongation and seed germination to a variable extent depending on the plant species. To understand the molecular mechanisms of arsenic resistance, a genetic screen was developed to isolate arsenate overly sensitive (aos) mutants from an activation-tagged Arabidopsis (Arabidopsis thaliana) population. Three aos mutants were isolated, and the phenotype of each was demonstrated to be due to an identical disruption of plastidial LIPOAMIDE DEHYDROGENASE1 (ptLPD1), a gene that encodes one of the two E3 isoforms found in the plastidial pyruvate dehydrogenase complex. In the presence of arsenate, ptlpd1-1 plants exhibited reduced root and shoot growth and enhanced anthocyanin accumulation compared with wild-type plants. The ptlpd1-1 plants accumulated the same amount of arsenic as wild-type plants, indicating that the aos phenotype was not due to increased arsenate in the tissues but to an increase in the innate sensitivity to the poison. Interestingly, a ptlpd1-4 knockdown allele produced a partial aos phenotype. Two loss-of-function alleles of ptLPD2 in Arabidopsis also caused elevated arsenate sensitivity, but the sensitivity was less pronounced than for the ptlpd1 mutants. Moreover, both the ptlpd1 and ptlpd2 mutants were more sensitive to arsenite than wild-type plants, and the LPD activity in isolated chloroplasts from wild-type plants was sensitive to arsenite but not arsenate. These findings show that the ptLPD isoforms are critical in vivo determinants of arsenite-mediated arsenic sensitivity in Arabidopsis and possible strategic targets for increasing arsenic tolerance.Arsenic (As) is a naturally occurring metalloid found in soil, water, and air, but anthropogenic activities, including smelting and fossil fuel combustion, have led to increased environmental exposure (Mandal and Suzuki, 2002). In the environment, As exists in both organic and inorganic forms. Arsenate [As(V)] is the principal inorganic form of As in aerobic soils, while arsenite [As(III)] is the main form found under anaerobic conditions (Marin et al., 1993; Onken and Hossner, 1995, 1996; Mandal and Suzuki, 2002; Masscheleyn et al., 2002).Both As(V) and As(III) are toxic to plants, inducing symptoms ranging from poor seed germination and inhibited root growth to death (Meharg and Hartley-Whitaker, 2002; Lee et al., 2003; Ahsan et al., 2008; Smith et al., 2010). The modes of action of As(V) and As(III) differ, owing to their distinct chemical properties. As(V), with its structural similarity to phosphate, can compete with phosphate in oxidative phosphorylation, leading to the production of ADP-As(V) (Gresser, 1981). However, half-maximal stimulation of ADP-As(V) formation requires physiologically unlikely concentrations of approximately 0.8 mm As(V) (Moore et al., 1983). As(V) has been recently shown to enhance membrane fluidity, and thus membrane permeability, by binding and replacing phosphate or choline head groups (Tuan et al., 2008). The resulting damage to the membrane would disrupt the transport of mineral nutrients and water (Smith et al., 2010). As(V) can be promptly reduced in plants, including Arabidopsis (Arabidopsis thaliana), to As(III) by endogenous As(V) reductases, so that often more than 90% of As in plant cells is in the form of As(III) (Zhao et al., 2009). As(III) readily forms covalent bonds with sulfhydryl groups, especially vicinal dithiols. Binding to the free thiols of proteins is believed to be the basis of As(III) toxicity, either by inhibiting activity directly or by disrupting protein structure. Many enzymes have been proposed to be targets leading to As(III) toxicity, and the As(III) sensitivity of some of these enzymes has been investigated in nonplant systems (Adamson and Stevenson, 1981; Cavigelli et al., 1996; Lynn et al., 1997; Hu et al., 1998; Kitchin and Wallace, 2008). Of the many potential protein targets, only the pyruvate dehydrogenase complex (PDC) has been shown to be inactivated by physiologically relevant micromolar concentrations of As(III) (Hu et al., 1998), suggesting that PDC may be the primary target for As(III)-mediated cytotoxicity. However, little is known about the mechanism of As toxicity in vivo, especially in plants.Although As is phytotoxic, some plants species are resistant to high levels of As through avoidance mechanisms, while species of the Pteridaceae family of ferns hyperaccumulate As without toxic effects (Verbruggen et al., 2009; Zhao et al., 2009). As an analog of phosphate, As(V) is readily taken up by plants through high-affinity phosphate transporters encoded by the PHOSPHATE TRANSPORTER1 (PHT1) gene family (Shin et al., 2004; González et al., 2005; Catarecha et al., 2007). Except for the hyperaccumulating ferns, avoidance of As toxicity by resistant species is often accomplished by a decrease in phosphate uptake activity (Meharg and Hartley-Whitaker, 2002). Unlike As(V), the transport of As(III) is facilitated by aquaporin nodulin 26-like intrinsic proteins (Bienert et al., 2008; Isayenkov and Maathuis, 2008; Ma et al., 2008; Kamiya et al., 2009). In roots and fronds of hyperaccumulating ferns, As(III) is sequestered in the vacuole (Lombi et al., 2002; Pickering et al., 2006). Much of the As(III) taken up by nonaccumulating resistant species may be released back to the rhizosphere through an undefined efflux pathway (Zhao et al., 2009). As(III) that remains in tissues reacts with thiol-containing molecules, such as glutathione or phytochelatins, both of which are usually produced in greater abundance in response to As (Grill et al., 1987; Sneller et al., 1999; Schmöger et al., 2000; Schulz et al., 2008). As(III)-glutathione adducts can be sequestered in the vacuole (Dhankher et al., 2002; Bleeker et al., 2006). However, increased synthesis of glutathione or phytochelatins alone is unlikely to confer a very high level of tolerance (Zhao et al., 2009).To identify genes essential for As resistance in plants, we used a genetic screen to identify mutants of Arabidopsis that were hypersensitive to As(V). The screen was analogous to that used to isolate the salt overly sensitive (sos) mutants of Arabidopsis (Wu et al., 1996) that led to the identification of the SOS pathway for salt tolerance (Zhu, 2000, 2003). Our hypothesis was that arsenate overly sensitive (aos) mutants would reveal a different set of genes from those identified in mutants showing increased resistance to As(V).     

Long-chain acyl CoA synthetase in microsomes from rat brain gray matter and white matter

Long-chain acyl coenzyme A (CoA) synthetase in homogenates and microsomes from rat brain gray and white matter was studied. The formation of the thioesters of CoA was studied upon addition of [1-¹⁴C]-labeled fatty acids. The maximal activities were seen with linoleic acid, followed by arachidonic, palmitic, and docosahexaenoic acids in both gray and white matter homogenates and microsomes. The specific activities in microsomes were 3–5 times higher than in homogenates. The presence of Triton X-100 in the assay system enhanced the activity of long-chain acyl CoA synthetase in homogenates. The effect was more pronounced in palmitic and docosahexaenoic acid activation. The apparentK _m values andV _max values for palmitic and docosahexaenoic acids were much lower than for linoleic and arachidonic acids. The presence of Triton X-100 in the medium caused a definite decrease in the apparentK _m and Vmax values for all the fatty acid except palmitic acid in which case the reverse was true. There were no significant differences observed in the kinetic measurements between gray and white matter microsomes. These findings are similar to those resulting from the known interference of Triton X-100 in the measurement of kinetic variables of long-chain acyl CoA synthetase of liver microsomes. In this work, no correlation was observed between the fatty acid composition of gray and white matter and the capacity of these tissues for the activation of different fatty acids.