The heat shock proteins (Hsps) play a positive role in lifespan determination, and histone acetylation has been shown to be involved in transcription of hsp genes in Drosophila. To further determine if hsp22 and hsp70 expression is correlated with lifespan, and if histone acetylation participates in this process, RNA levels for hsp22 and hsp70 were analyzed throughout the lifespan in the long-lived and short-lived iso-female lines. The results showed that hsp22 and hsp70 RNA levels were higher in long-lived line than in short-lived line and that the longlived flies responded more rapidly to heat but were more tolerant to high temperature. Moreover, we investigated the influences of histone acetylation modification on longevity and on hsp gene expression by using histone deacetylase (HDAC) inhibitors TSA and BuA. The results demonstrated that both inhibitors were able to extend the lifespan and promote hsp22 and hsp70 expression. However, the optimal concentrations of these inhibitors, and probably the mechanisms of their actions, vary with the genetic background. In addition, we showed that HDAC inhibitors caused the hyperacetylation of core histone H3, implicating the involvement of chromatin modulation in hsp gene transcription. These data suggested a close correlation among histone acetylation, hsp gene expression and longevity in D. melanogaster.

BackgroundAlthough sperm is transcriptionally and translationally quiescent, complex populations of RNAs, including mRNAs and non-coding RNAs, exist in sperm. Previous microarray analysis of germ cell mutants identified hundreds of sperm genes in Caenorhabditis elegans. To take a more comprehensive view on C. elegans sperm genes, here, we isolate highly pure sperm cells and employ high-throughput technologies to obtain sperm transcriptome and proteome.ResultsFirst, sperm transcriptome consists of considerable amounts of non-coding RNAs, many of which have not been annotated and may play functional roles during spermatogenesis. Second, apart from kinases/phosphatases as previously reported, ion binding proteins are also enriched in sperm, underlying the crucial roles of intracellular ions in post-translational regulation in sperm. Third, while the majority of sperm genes/proteins have low abundance, a small number of sperm genes/proteins are hugely enriched in sperm, implying that sperm only rely on a small set of proteins for post-translational regulation. Lastly, by extensive RNAi screening of sperm enriched genes, we identified a few genes that control fertility. Our further analysis reveals a tight correlation between sperm transcriptome and sperm small RNAome, suggesting that the endogenous siRNAs strongly repress sperm genes. This leads to an idea that the inefficient RNAi screening of sperm genes, a phenomenon currently with unknown causes, might result from the competition between the endogenous RNAi pathway and the exogenous RNAi pathway.ConclusionsTogether, the obtained sperm transcriptome and proteome serve as valuable resources to systematically study spermatogenesis in C. elegans.

Rossmann fold ͉ signal transduction N AD and its phosphorylated form, NADP, is a universal cofactor in a large number of redox reactions carried out by a variety of enzymes, especially dehydrogenases. It is widely recognized that NAD(P) is an essential molecule in energy metabolism in all organisms. More recently, mounting evidence suggests that, in addition to its central role in energy metabolism, NAD(P) is also involved in signaling pathways that regulate fundamental processes of cellular functions, including gene transcription and apoptosis (1,2). This realization generated a link between the energy metabolism and the regulated networks of biological processes.One of the NAD signaling mechanisms is through gene silencer proteins known as sirtuins, such as Sir2 in yeast, a NAD-dependent histone deacetylase. Increased activities of Sir2 induced by an increase in NAD production extended lifespan (3). It was shown that a product of NAD-dependent deacetylation by Sir2, nicotinamide, strongly inhibits the activity of Sir2 (4, 5). Similar mechanisms are present in other eukaryotes, including humans (6). NAD is also directly related to transcription regulation. PolyADP-ribose polymerases (PARPs) can modify the acceptor proteins by synthesizing a polyADP-ribose molecule using NAD ϩ as the substrate. The transcription factors that can be modified by PARPs include p53, YY1, NF-B, and TATA-binding protein (1). In other cases, transcription regulation is not carried out by any enzymatic reaction. For instance, the C-terminal-binding protein CtBP is a corepressor that has an increased affinity to its partners, such as adenovirus E1A or cellular repressor ZEB, when NADH binds to CtBP (7). Crystal structures showed that NADH binding to CtBP induced a conformational switch that stabilizes the dimerization of CtBP, which in turn promotes its binding to the repressors (8). In the case of the negative transcriptional regulator NmrA, NAD ϩ binding to NmrA controls the rate of nuclear entry of the GATA transcription-activating protein AreA (9, 10). NmrA has a similar structure to short-chain dehydrogenase/reductase (SDR) family proteins but no enzyme activities, because of the lack of conserved active-site residues (9). NAD(P) exerts its functions by association with proteins. The protein fold that binds NAD(P) was discovered by Rossmann when the crystal structure of lactate dehydrogenase was determined (11). The Rossmann fold is the most common fold, based on its predicted occurrence from the genes known today (12). This motif consists of six ␤-strands connected by ␣-helices with the NAD(P) molecule bound at the top [supporting information (SI) Fig. 5]. The Rossmann fold has always been present as a rigid-body domain in all other known crystal structures until the structure of HSCARG was determined. We found that the common ␣E that connects ␤5 to ␤6 in the Rossmann fold is deformed as an extended loop when NADP is not bound with HSCARG. This allows the formation of an asymmetric dimer between a subunit with NADP bound and an emp...

NADPH is an important cofactor in many biosynthesis pathways that control fundamental cellular processes. We recently determined the crystal structure of HSCARG, with functions previously unknown, and demonstrated it is an NADPH sensor, which undergoes restructuring and redistribution in response to changes of intracellular NADPH/NADP levels. In this study, we identified argininosuccinate synthetase (AS), a rate-limiting enzyme in nitric oxide synthesis, as capable of associating with HSCARG and demonstrated further that HSCARG decreased nitric oxide synthesis by down-regulating AS activity, whereas AS overexpression up-regulated hscarg mRNA transcription, suggesting a negative feedback mechanism. A decrease in the NADPH/NADP ؉ ratio, induced by dehydroepiandrosterone treatment, enhanced the interaction between HSCARG and AS, which resulted in stronger inhibition of AS activity and nitric oxide production. The dimerization region of HSCARG, amino acids 153-189, was identified to undergo critical interactions with AS. Furthermore, the viability of HSCARG RNA interference-treated epithelial cells decreased significantly, accompanied by an increase of the activity of caspase-3, which suggested that the loss of viability was because of apoptosis. These results indicate that HSCARG regulation of AS activity is crucial for maintaining the intracellular balance between redox state and nitric oxide levels.Nitric oxide (NO), 2 a cellular signaling molecule, has been shown to be involved in vascular regulation, autoimmunity, and neurotransmission and impacts diverse biological processes, including cell survival (1-7). The impaired production of NO can result in the vascular dysfunction, whereas overproduction of NO will induce some diseases such as the cerebral infarction, diabetes mellitus, and neurodegenerative disorders (7-11). Arginine is the sole amino acid substrate that is required for the production of NO (12, 13), and its regeneration from citrulline, the co-product of NO synthesis, is rate-limited by argininosuccinate synthetase (AS) (13-16). In addition, the reducing reagent donor, NADPH, and oxygen are necessary for NO production (13, 17). For this reason, NO production is not only limited by the regeneration of arginine but is also affected by the intracellular NADPH concentration, which requires cross-talk between the signaling pathways of NADPH and NO.In addition to its well known function in energy metabolism, NADH, along with its phosphorylated relative NADPH, has been recognized as an important regulatory molecule. Together, their roles are crucial in signaling pathways that control fundamental cellular processes, such as transcription, regulation of calcium homeostasis, and apoptosis (18 -20). NAD mainly exists in its oxidized state (NAD ϩ ), whereas NADP is largely found in its reduced form, NADPH (21, 22). The predominant function of NADP is to maintain a pool in its reduced form to ensure a rapid regeneration of the defense systems to protect cells from oxidative damage. NADPH holds a key position in...

No abstract

Preoptic-anterior hypothalamic (PO/AH) neurones sense and regulate body temperature. Although controversial, it has been postulated that warm-induced depolarization determines neuronal thermosensitivity. Supporting this hypothesis, recent studies suggest that temperature-sensitive cationic channels (e.g. vanilloid receptor TRP channels) constitute the underlying mechanism of neuronal thermosensitivity. Moreover, earlier studies indicated that PO/AH neuronal warm sensitivity is due to depolarizing sodium currents that are sensitive to tetrodotoxin (TTX). To test these possibilities, intracellular recordings were made in rat hypothalamic tissue slices. Thermal effects on membrane potentials and currents were compared in PO/AH warm-sensitive, temperature-insensitive and silent neurones. All three types of neurones displayed slight depolarization during warming and hyperpolarization during cooling. There were no significant differences in membrane potential thermosensitivity for the different neuronal types. Voltage clamp recordings (at −92 mV) measured the thermal effects on persistent inward cationic currents. In all neurones, resting holding currents decreased during cooling and increased during warming, and there was no correlation between firing rate thermosensitivity and current thermosensitivity. To determine the thermosensitive contribution of persistent, TTX-sensitive currents, voltage clamp recordings were conducted in the presence of 0.5 µM TTX. TTX decreased the current thermosensitivity in most neurones, but there were no resulting differences between the different neuronal types. The present study found no evidence of a resting ionic current that is unique to warm-sensitive neurones. This supports studies suggesting that neuronal thermosensitivity is controlled, not by resting currents, but rather by currents that determine rapid changes in membrane potential between successive action potentials.

Sperm activation is a fascinating example of cell differentiation, in which immotile spermatids undergo a rapid and dramatic transition to become mature, motile sperm. Because the sperm nucleus is transcriptionally silent, this transition does not involve transcriptional changes. Although Caenorhabditis elegans is a leading model for studies of sperm activation, the mechanisms by which signaling pathways induce this transformation remain poorly characterized. Here we show that a conserved transmembrane zinc transporter, ZIPT-7.1, regulates the induction of sperm activation in Caenorhabditis nematodes. The zipt-7.1 mutant hermaphrodites cannot self-fertilize, and males reproduce poorly, because mutant spermatids are defective in responding to activating signals. The zipt-7.1 gene is expressed in the germ line and functions in germ cells to promote sperm activation. When expressed in mammalian cells, ZIPT-7.1 mediates zinc transport with high specificity and is predominantly located on internal membranes. Finally, genetic epistasis places zipt-7.1 at the end of the spe-8 sperm activation pathway, and ZIPT-7.1 binds SPE-4, a presenilin that regulates sperm activation. Based on these results, we propose a new model for sperm activation. In spermatids, inactive ZIPT-7.1 is localized to the membranous organelles, which contain higher levels of zinc than the cytoplasm. When sperm activation is triggered, ZIPT-7.1 activity increases, releasing zinc from internal stores. The resulting increase in cytoplasmic zinc promotes the phenotypic changes characteristic of activation. Thus, zinc signaling is a key step in the signal transduction process that mediates sperm activation, and we have identified a zinc transporter that is central to this activation process.

Hybrid incompatibility (HI) prevents gene flow between species, thus lying at the heart of speciation genetics. One of the most common HIs is male sterility. Two superficially contradictory observations exist for hybrid male sterility. First, an introgression on the X Chromosome is more likely to produce male sterility than on autosome (so-called large-X theory); second, spermatogenesis genes are enriched on the autosomes but depleted on the X Chromosome (demasculinization of X Chromosome). Analysis of gene expression in Drosophila hybrids suggests a genetic interaction between the X Chromosome and autosomes that is essential for male fertility. However, the prevalence of such an interaction and its underlying mechanism remain largely unknown. Here we examine the interaction in nematode species by contrasting the expression of both coding genes and transposable elements (TEs) between hybrid sterile males and its parental nematode males. We use two lines of hybrid sterile males, each carrying an independent introgression fragment from Caenorhabditis briggsae X Chromosome in an otherwise Caenorhabditis nigoni background, which demonstrate similar defects in spermatogenesis. We observe a similar pattern of down-regulated genes that are specific for spermatogenesis between the two hybrids. Importantly, the down-regulated genes caused by the X Chromosome introgressions show a significant enrichment on the autosomes, supporting an epistatic interaction between the X Chromosome and autosomes. We investigate the underlying mechanism of the interaction by measuring small RNAs and find that a subset of 22G RNAs specifically targeting the down-regulated spermatogenesis genes is significantly up-regulated in hybrids, suggesting that perturbation of small RNA-mediated regulation may contribute to the X-autosome interaction.