Higher plants produce seed through pollination, using specific interactions between pollen and pistil. Self-incompatibility is an important mechanism used in many species to prevent inbreeding; it is controlled by a multi-allelic S locus. 'Self' (incompatible) pollen is discriminated from 'non-self' (compatible) pollen by interaction of pollen and pistil S locus components, and is subsequently inhibited. In Papaver rhoeas, the pistil S locus product is a small protein that interacts with incompatible pollen, triggering a Ca(2+)-dependent signalling network, resulting in pollen inhibition and programmed cell death. Here we have cloned three alleles of a highly polymorphic pollen-expressed gene, PrpS (Papaver rhoeas pollen S), from Papaver and provide evidence that this encodes the pollen S locus determinant. PrpS is a single-copy gene linked to the pistil S gene (currently called S, but referred to hereafter as PrsS for Papaver rhoeas stigma S determinant). Sequence analysis indicates that PrsS and PrpS are equally ancient and probably co-evolved. PrpS encodes a novel approximately 20-kDa protein. Consistent with predictions that it is a transmembrane protein, PrpS is associated with the plasma membrane. We show that a predicted extracellular loop segment of PrpS interacts with PrsS and, using PrpS antisense oligonucleotides, we demonstrate that PrpS is involved in S-specific inhibition of incompatible pollen. Identification of PrpS represents a major advance in our understanding of the Papaver self-incompatibility system. As a novel cell-cell recognition determinant it contributes to the available information concerning the origins and evolution of cell-cell recognition systems involved in discrimination between self and non-self, which also include histocompatibility systems in primitive chordates and vertebrates.

Multiple members of the let-7 family of miRNAs are often repressed in human cancers, thereby promoting oncogenesis by derepressing targets such as HMGA2, K-Ras and c-Myc. However, the mechanism by which let-7 miRNAs are coordinately repressed is unclear. The RNA-binding proteins LIN28 and LIN28B block let-7 precursors from being processed to mature miRNAs, suggesting that their overexpression might promote malignancy through repression of let-7. Here we show that LIN28 and LIN28B are overexpressed in primary human tumors and human cancer cell lines (overall frequency approximately 15%), and that overexpression is linked to repression of let-7 family miRNAs and derepression of let-7 targets. LIN28 and LIN28b facilitate cellular transformation in vitro, and overexpression is associated with advanced disease across multiple tumor types. Our work provides a mechanism for the coordinate repression of let-7 miRNAs observed in a subset of human cancers, and associates activation of LIN28 and LIN28B with poor clinical prognosis.

In lead discovery, libraries of 10(6) molecules are screened for biological activity. Given the over 10(60) drug-like molecules thought possible, such screens might never succeed. The fact that they do, even occasionally, implies a biased selection of library molecules. We have developed a method to quantify the bias in screening libraries toward biogenic molecules. With this approach, we consider what is missing from screening libraries and how they can be optimized.

Regulatory T cells (T(reg) cells) are critically involved in maintaining immunological tolerance, but this potent suppression must be 'quenched' to allow the generation of adaptive immune responses. Here we report that sphingosine 1-phosphate (S1P) receptor type 1 (S1P1) delivers an intrinsic negative signal to restrain the thymic generation, peripheral maintenance and suppressive activity of T(reg) cells. Combining loss- and gain-of-function genetic approaches, we found that S1P1 blocked the differentiation of thymic T(reg) precursors and function of mature T(reg) cells and affected T(reg) cell-mediated immune tolerance. S1P1 induced selective activation of the Akt-mTOR kinase pathway to impede the development and function of T(reg) cells. Dynamic regulation of S1P1 contributed to lymphocyte priming and immune homeostasis. Thus, by antagonizing T(reg) cell-mediated immune suppression, the lipid-activated S1P1-Akt-mTOR pathway orchestrates adaptive immune responses.

Huntington's disease (HD) is an inherited, progressive neurological disorder caused by a CAG/polyglutamine repeat expansion, for which there is no effective disease modifying therapy. In recent years, transcriptional dysregulation has emerged as a pathogenic process that appears early in disease progression. Administration of histone deacetylase (HDAC) inhibitors such as suberoylanilide hydroxamic acid (SAHA) have consistently shown therapeutic potential in models of HD, at least partly through increasing the association of acetylated histones with down-regulated genes and by correcting mRNA abnormalities. The HDAC enzyme through which SAHA mediates its beneficial effects in the R6/2 mouse model of HD is not known. Therefore, we have embarked on a series of genetic studies to uncover the HDAC target that is relevant to therapeutic development for HD. HDAC7 is of interest in this context because SAHA has been shown to decrease HDAC7 expression in cell culture systems in addition to inhibiting enzyme activity. After confirming that expression levels of Hdac7 are decreased in the brains of wild type and R6/2 mice after SAHA administration, we performed a genetic cross to determine whether genetic reduction of Hdac7 would alleviate phenotypes in the R6/2 mice. We found no improvement in a number of physiological or behavioral phenotypes. Similarly, the dysregulated expression levels of a number of genes of interest were not improved suggesting that reduction in Hdac7 does not alleviate the R6/2 HD-related transcriptional dysregulation. Therefore, we conclude that the beneficial effects of HDAC inhibitors are not predominantly mediated through the inhibition of HDAC7.

Alexander disease is a rare disorder of the central nervous system with characteristic symmetric white matter abnormalities with frontal predominance on magnetic resonance (MR) images. Histopathology shows a lack of myelin in the affected white matter, variably interpreted as hypomyelination or demyelination. To increase our insight into the nature of the pathology leading to the MR imaging findings in Alexander disease, we applied serial MR imaging, spectroscopy, magnetization transfer (MT) imaging (MTI), and diffusion tensor imaging (DTI) in six patients with juvenile Alexander disease.

Previous studies revealed that upon salinity challenge, milkfish (Chanos chanos), the euryhaline teleost, exhibited adaptive changes in branchial Na(+)/K(+)-ATPase (NKA) activity with different Na(+) and K(+) affinities. Since alteration of activity and ion-affinity may be influenced by changes in different isoforms of NKA alpha-subunit (i.e., the catalytic subunit), it is, thus, intriguing to compare the patterns of protein abundance of three major NKA alpha-isoform-like proteins (i.e., alpha1, alpha2, and alpha3) in the gills of euryhaline milkfish following salinity challenge. The protein abundance of three NKA alpha-isoform-like proteins in gills of milkfish reared in seawater (SW), fresh water (FW), as well as hypersaline water (HSW, 60 per thousand) were analyzed by immunoblotting. In the acclimation experiments, the SW group revealed significantly higher levels of NKA alpha1- and alpha3-like proteins than the FW or HSW group. Time-course experiments on milkfish that were transferred from SW to HSW revealed the abundance of branchial NKA alpha1-like and alpha3-like proteins decreased significantly after 96 and 12 hr, respectively, and no significant difference was found in NKA alpha2-like protein. Furthermore, when fish were transferred from SW to FW, the amounts of NKA alpha1- and alpha3-like proteins was significantly decreased after 96 hr. Taken together, acute and chronic changes in the abundance of branchial NKA alpha1- and alpha3-like proteins may fulfill the requirements of altering NKA activity with different Na(+) or K(+) affinity for euryhaline milkfish acclimated to environments of various salinities.

Acute kidney injury frequently accompanies sepsis. Endotoxin is known to reduce tissue levels of cAMP and low levels of cAMP have been associated with renal injury. We, therefore, hypothesized that endotoxin induced renal injury by activating phosphodiesterase 3 (PDE3) which metabolizes cAMP and that amrinone an inhibitor of PDE3 would prevent the renal injury.

Histone lysine methylation plays a fundamental role in chromatin organization and marks distinct chromatin regions. In particular, trimethylation at lysine 9 of histone H3 (H3K9) and at lysine 20 of histone H4 (H4K20) governed by the histone methyltransferases SUV39H1/2 and SUV420H1/2 respectively, have emerged as a hallmark of pericentric heterochromatin. Controlled chromatin organization is crucial for gene expression regulation and genome stability. Therefore, it is essential to analyze mechanisms responsible for high order chromatin packing and in particular the interplay between enzymes involved in histone modifications, such as histone methyltransferases and proteins that recognize these epigenetic marks.

The persistence of cooperative relationships is an evolutionary paradox; selection should favor those individuals that exploit their partners (cheating), resulting in the breakdown of cooperation over evolutionary time. Our current understanding of the evolutionary stability of mutualisms (cooperation between species) is strongly shaped by the view that they are often maintained by partners having mechanisms to avoid or retaliate against exploitation by cheaters. In contrast, we empirically and theoretically examine how additional symbionts, specifically specialized parasites, potentially influence the stability of bipartite mutualistic associations. In our empirical work we focus on the obligate mutualism between fungus-growing ants and the fungi they cultivate for food. This mutualism is exploited by specialized microfungal parasites (genus Escovopsis) that infect the ant's fungal gardens. Using sub-colonies of fungus-growing ants, we investigate the interactions between the fungus garden parasite and cooperative and experimentally-enforced uncooperative ("cheating") pairs of ants and fungi. To further examine if parasites have the potential to help stabilize some mutualisms we conduct Iterative Prisoner's Dilemma (IPD) simulations, a common framework for predicting the outcomes of cooperative/non-cooperative interactions, which incorporate parasitism as an additional factor.

The genomes of pathogenic Escherichia coli may contain several different fimbrial operons. How bacteria regulate and coordinate the choice of fimbrial expression under different circumstances remains largely unanswered. In this report we have investigated the role of the sfaX(II) gene associated to the Sfa(II) fimbrial determinant in the E. coli isolate IHE3034. sfaX(II) belongs to a subfamily of genes, the 17k Da genes, located near different fimbrial operons in uropathogenic and newborn meningitis E. coli (NMEC) strains. Using the NMEC isolate IHE3034 and non-pathogenic E. coli strains we found that the sfaX(II) gene had an inhibitory effect on type 1 fimbriae expression. Down-regulation of type 1 fimbriae was exerted at transcriptional level both by inhibiting expression from the fimA promoter and by reducing the frequency of OFF-to-ON switching. The effect of sfaX(II) on expression of the recombinase FimB that catalyzes OFF-to-ON switching might explain the described reduction in percentage of ON cells. Moreover, expression of the sfaX(II) gene strongly influenced motility and flagella production of the NMEC isolate IHE3034. We propose that the sfaX(II) gene, and presumably other members in the 17 kDa gene family, may play a role in the control of virulence related gene expression in pathogenic E. coli.

To assess the role of the glycolytic enzyme phosphofructokinase (Pfk) in the in vivo fitness of the pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) we have generated single and double gene deletion mutants of the two known isoforms of this enzyme, pfkA and pfkB. In a mouse model of typhoid fever, bacterial counts in the spleen and liver were similar between wild type and single pfkA and pfkB mutant-infected mice. However, a double pfkAB mutant was significantly attenuated for growth in vivo. This defect was complemented by provision of either pfkA or pfkB on pBR322. Together these data show that Pfk activity is required for the full in vivo fitness of S. Typhimurium with functional redundancy between pfkA and pfkB. The level of attenuation of the pfkAB double mutant was not sufficient for its consideration as a live attenuated vaccine strain.

No abstract available

Forkhead box protein A1 (Foxa1) is an evolutionarily conserved winged helix transcription factor. In this study, the effect of Foxa1 on the expression of HSP72 was examined by RT-PCR and Western blot in Foxa1 overexpression or deficient cells. The results showed overexpression of Foxa1 promoted the expression of HSP72, while Foxa1 depletion, induced by antisense oligonucleotides, decreased the expression of HSP72 in MCF-7 cells under normal and heat stress condition. Electrophoretic mobility shift assay and chromatin immunoprecipitation revealed that Foxa1 bound to HSP72 promoter, and heat stress promoted its DNA binding activity. Luciferase reporter showed that Foxa1 also increased the transcription activity of HSP72 promoter. These results indicate an important role for Foxa1 as a novel regulator of expression of HSP72.

The Type IV Secretion System (T4SS) is an efficient pathway with which bacteria can mediate the transfer of DNA and/or proteins to eukaryotic cells. In Wolbachia pipientis, a maternally inherited obligate endosymbiont of arthropods and nematodes, two operons of vir genes, virB3-B6 and virB8-D4, encoding a T4SS were previously identified and characterized at two separate genomic loci. Using the largest data set of Wolbachia strains studied so far, we show that vir gene sequence and organization are strictly conserved among 37 Wolbachia strains inducing various phenotypes such as cytoplasmic incompatibility, feminization, or oogenesis in their arthropod hosts. In sharp contrast, extensive variation of genomic sequences flanking the virB8-D4 operon suggested its distinct location among Wolbachia genomes. Long term conservation of the T4SS may imply maintenance of a functional effector translocation system in Wolbachia, thereby suggesting the importance for the T4SS in Wolbachia biology and survival inside host cells.

Dietary flavonoids have been shown to reduce risk of cardiovascular disease, but the underlying molecular mechanisms are not known. The objective of this study was to investigate the effect of nobiletin, a dietary phytochemical belonging to polymethoxy flavonoid from the peel of Citrus fruit, on vascular smooth muscle cells (VSMCs) proliferation and its mechanisms. VSMCs proliferation was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-dephenyl tetrazolium bromide (MTT) and [(3)H]thymidine incorporation assay. The activity of extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and p38 mitogen-activated protein kinases (MAPK) were determined by western blotting. [Ca(2+)](i) was measured by laser scanning confocal microscopy. Our results showed that angiotensin II-induced VSMCs proliferation was inhibited by nobiletin. While no effect on ERK1/2 and p38 MAPK, nobiletin markedly inhibited angiotensin II-induced activation of JNK. Anthra[1-9-cd]pyrazol-6(2H)-one (SP600125), an inhibitor of JNK, decreased the [(3)H]thymidine incorporation induced by angiotensin II. Nobiletin also attenuated both the intracellular Ca(2+) mobilization and the extracellular Ca(2+) influx induced by angiotensin II. Furthermore, intracellular Ca(2+) chelation by BAPTA-AM, extracellular Ca(2+) chelation by EGTA or blockade of L-type Ca(2+) channel with verapamil inhibited angiotensin II-induced JNK activation. These findings suggest that the preventing effect of nobiletin on angiotensin II-induced VSMCs proliferation is attributed, in part, to its inhibitory effect on Ca(2+)-dependent JNK activation in VSMCs. Thus, inhibition of JNK by nobiletin may imply its usefulness for the treatment of cardiovascular diseases relevant to VSMCs growth.

Several signaling pathways are differentially activated during apoptotic cell death. We have previously found that during apoptotic death of cerebellar granule neurons (CGN) induced by potassium deprivation (K5) and staurosporine there is an increase in the generation of reactive oxygen species (ROS). The inhibition of ROS generation reduces the extent of cell death. However, remain to be elucidated the mechanisms by which ROS participate in this apoptotic process. On the other hand, it is well known that c-Jun amino-terminal kinase (JNK) pathway plays a pivotal role in cell death of several cell types. In the present study we found that K5 activated the JNK pathway and that its inhibition with SP600125 markedly prevented caspase 3 activation, nuclear condensation and cell death induced by K5. In contrast, JNK pathway was not activated by staurosporine and the JNK inhibitor did not affect cell death induced by this stimulus. We also found that JNK inhibition did not affect ROS levels induced by K5 or staurosporine, suggesting that ROS are upstream of JNK pathway activation. Antioxidants increased ASK1 phosphorylation and decreased JNK1/2 and c-Jun phosphorylation induced by K5. According to these results, we suggest that apoptosis induced by K5 is JNK-dependent and mediated by ROS, but apoptosis induced by staurosporine is not dependent on JNK and that the observed ROS generation by staurosporine seems not to be involved in the activation of this signaling pathway.

The distribution of neurons that are immunoreactive to latexin, which is an endogenous inhibitor of the A/B subfamily of metallocarboxypeptidases, was investigated in the adult cat telencephalon. Latexin-immunoreactive neurons were distributed in the lower layers of the neocortex and adjacent ventral mesocortex, as well as in the claustrum/endopiriform formation. There were marked regional and laminar differences in density and distribution of latexin-immunoreactive neurons in the cerebral cortex. The density followed a roughly lateral-to-medial decreasing gradient: it was high in lateral cortical regions, which included the insular, second somatosensory, and anterior sylvian areas, and in the temporal auditory field; moderate in laterodorsal cortical regions, which included the primary and second auditory fields; and low in dorsal cortical regions, which included visual areas 18 and 19. Latexin-immunoreactive neurons were absent in medial cortical regions, which included the motor, premotor, prefrontal, prelimbic, cingulate, and retrosplenial areas. The lateral-to-medial gradient was apparent even within a single cytoarchitectonic area in certain cortical regions. The allocortex was devoid of latexin-immunoreactive neurons, with the exception of the anteroventral part of the dentate gyrus. The majority of cortical latexin-immunoreactive neurons were localized in layers V and VI and appeared to correspond to the "modified pyramidal cells in the infragranular layers." The remaining latexin-immunoreactive neurons were localized in layer IV, as well as in lower layer III and in the white matter. There were no latexin-immunoreactive neurons from layer I through upper layer III. Latexin-immunoreactive neurons were present in telencephalic structures outside the cerebral cortex, with particularly high density in the claustrum/endopiriform formation. All these features, with the exception of that detected in the archicortex, are compatible with the features observed previously in the rat telencephalon. The similar pattern of distribution of latexin-immunoreactive neurons in several mammalian species from different orders suggests that latexin plays an important role in a specific cortical network.

It is acknowledged that neurotransmission in the mouse vas deferens is predominantly mediated by ATP and noradrenaline (NA) released from sympathetic nerves while cholinergic transmission in the rodent vas deferens is often overlooked despite early literature. Recently we have characterized a cholinergic component of neurogenic contraction of mouse isolated vas deferens. In the present paper, by confocal imaging of Ca(2+) dynamics we detected acetylcholine (ACh) action at muscarinic cholinergic neuroeffector junctions at high-resolution. Experiments were carried out in the presence of prazosin (100 nM) and alpha,beta methylene ATP (alpha,beta-MeATP) (1 microM) to inhibit responses to NA and ATP respectively. Exogenous ACh (10 microM) elicited Ca(2+) transients, an effect blocked by the muscarinic receptor antagonist, cyclopentolate (1 microM). Ca(2+) transients were evoked by electrical stimulation of intrinsic nerves in the presence of the cholinesterase inhibitor neostigmine (10 microM). Stimulation produced a marked increase in the frequency and number of Ca(2+) transients. Cyclopentolate reduced the frequency of occurrence of spontaneous and evoked events to control levels. The alpha(2)-adrenoceptor antagonist yohimbine (300 nM) did not affect the spontaneous Ca(2+) transients, but increased the frequency of occurrence of evoked transients, an effect inhibited by cyclopentolate. The postjunctional effects of neuronally-released ACh are limited by the action of cholinesterase. Release of ACh appears to be tonically inhibited by NA released from sympathetic nerve terminals through action at prejunctional alpha(2)-adrenoceptors. Tetrodotoxin (TTX, 300 nM) abolished the nerve-evoked Ca(2+) events, with no effect on Ca(2+) transients elicited by exogenous ACh. In conclusion, the presence of spontaneous and evoked cholinergic Ca(2+) transients in smooth muscle cells of the mouse isolated vas deferens has been revealed. These events are mediated by ACh acting at M(3) muscarinic receptors. This action stands in marked contrast to the lack of effect of neuronally-released NA on smooth muscle Ca(2+) dynamics in this tissue.

Yellow head virus (YHV) is a highly virulent pathogen of Penaeus monodon shrimp. It is one of six known genotypes in the yellow head complex of nidoviruses which also includes mildly pathogenic gill-associated virus (GAV, genotype 2) and four other genotypes (genotypes 3-6) that have been detected only in healthy shrimp. In this study, comparative phylogenetic analyses conducted on replicase- (ORF1b) and glycoprotein- (ORF3) gene amplicons identified 10 putative natural recombinants amongst 28 viruses representing all six genotypes from across the Indo-Pacific region. The approximately 4.6 kb genomic region spanning the two amplicons was sequenced for three putative recombinant viruses from Vietnam (genotype 3/5), the Philippines (genotype 5/2) and Indonesia (genotype 3/2). SimPlot analysis using these and representative parental virus sequences confirmed that each was a recombinant genotype and identified a recombination hotspot in a region just upstream of the ORF1b C-terminus. Maximum-likelihood breakpoint analysis predicted identical crossover positions in the Vietnamese and Indonesian recombinants, and a crossover position 12 nt upstream in the Philippine recombinant. Homologous genetic recombination in the same genome region was also demonstrated in recombinants generated experimentally in shrimp co-infected with YHV and GAV. The high frequency with which natural recombinants were identified indicates that genetic exchange amongst genotypes is occurring commonly in Asia and playing a significant role in expanding the genetic diversity in the yellow head complex. This is the first evidence of genetic recombination in viruses infecting crustaceans and has significant implications for the pathogenesis of infection and diagnosis of these newly emerging invertebrate pathogens.