Blockade of PI3Kgamma suppresses joint inflammation and damage in mouse models of rheumatoid arthritis

Phosphoinositide 3-kinases (PI3K) have long been considered promising drug targets for the treatment of inflammatory and autoimmune disorders as well as cancer and cardiovascular diseases. But the lack of specificity, isoform selectivity and poor biopharmaceutical profile of PI3K inhibitors have so far hampered rigorous disease-relevant target validation. Here we describe the identification and development of specific, selective and orally active small-molecule inhibitors of PI3Kgamma (encoded by Pik3cg). We show that Pik3cg(-/-) mice are largely protected in mouse models of rheumatoid arthritis; this protection correlates with defective neutrophil migration, further validating PI3Kgamma as a therapeutic target. We also describe that oral treatment with a PI3Kgamma inhibitor suppresses the progression of joint inflammation and damage in two distinct mouse models of rheumatoid arthritis, reproducing the protective effects shown by Pik3cg(-/-) mice. Our results identify selective PI3Kgamma inhibitors as potential therapeutic molecules for the treatment of chronic inflammatory disorders such as rheumatoid arthritis.     

Human attitudes towards herpetofauna: The influence of folklore and negative values on the conservation of amphibians and reptiles in Portugal

Background

Human values and folklore of wildlife strongly influence the effectiveness of conservation efforts. These values and folklore may also vary with certain demographic characteristics such as gender, age, or education. Reptiles and amphibians are among the least appreciated of vertebrates and are victims of many negative values and wrong ideas resulting from the direct interpretation of folklore. We try to demonstrate how these values and folklore can affect the way people relate to them and also the possible conservation impacts on these animals.

Methods

A questionnaire survey distributed to 514 people in the district of Évora, Portugal, was used to obtain data regarding the hypothesis that the existence of wrong ideas and negative values contributes to the phenomenon of human-associated persecution of these animals. A structural equation model was specified in order to confirm the hypothesis about the possible relationships between the presence of perceptions and negative values about amphibians and reptiles and persecution and anti-conservation attitudes. Sociodemographic variables were also added.

Results

The results of the model suggest that the presence of folklore and negative values clearly predicts persecution and anti-conservation attitudes towards amphibians and reptiles. Also, the existence of folklore varies sociodemographically, but negative values concerning these animals are widespread in the population.

Conclusions

With the use of structural equation models, this work is a contribution to the study of how certain ideas and values can directly influence human attitudes towards herpetofauna and how they can be a serious conservation issue.     

Membrane transport in Caenorhabditis elegans: an essential role for VPS34 at the nuclear membrane

Here we present a detailed genetic analysis of let-512/vps34 that encodes the Caenorhabditis elegans homologue of the yeast phosphatidylinositol 3-kinase Vps34p. LET-512/VPS34 has essential functions and is ubiquitously expressed in all tissues and developmental stages. It accumulates at a perinuclear region, and mutations in let-512/vps34 result in an expansion of the outer nuclear membrane as well as in a mislocalization and subsequent complete lack of expression of LRP-1, a C.elegans LDL receptor normally associated with the apical surface of hypodermal cells. Using a GFP::2xFYVE fusion protein we found that the phosphatidylinositol 3-phosphate (PtdIns 3-P) product of LET-512/VPS34 is associated with a multitude of intracellular membranes and vesicles located at the periphery, including endocytic vesicles. We propose that LET-512/VPS34 is required for membrane transport from the outer nuclear membrane towards the cell periphery. Thus, LET-512/VPS34 may regulate the secretory pathway in a much broader range of compartments than was previously suggested for the yeast orthologue.     

Sampling properties of DNA sequence data in phylogenetic analysis

We inferred phylogenetic trees from individual genes and random samples of nucleotides from the mitochondrial genomes of 10 vertebrates and compared the results to those obtained by analyzing the whole genomes. Individual genes are poor samples in that they infrequently lead to the whole-genome tree. A large number of nucleotide sites is needed to exactly determine the whole-genome tree. A relatively small number of sites, however, often results in a tree close to the whole-genome tree. We found that blocks of contiguous sites were less likely to lead to the whole-genome tree than samples composed of sites drawn individually from throughout the genome. Samples of contiguous sites are not representative of the entire genome, a condition that violates a basic assumption of the bootstrap method as it is applied in phylogenetic studies.      

The promoter of filamentation (POF1) protein from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> is an ATPase involved in the protein quality control process

Background

The gene YCL047C, which has been renamed promoter of filamentation gene (POF1), has recently been described as a cell component involved in yeast filamentous growth. The objective of this work is to understand the molecular and biological function of this gene.

Results

Here, we report that the protein encoded by the POF1 gene, Pof1p, is an ATPase that may be part of the Saccharomyces cerevisiae protein quality control pathway. According to the results, Δpof1 cells showed increased sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, heat shock and protein unfolding agents, such as dithiothreitol and tunicamycin. Besides, the overexpression of POF1 suppressed the sensitivity of Δpct1, a strain that lacks a gene that encodes a phosphocholine cytidylyltransferase, to heat shock. In vitro analysis showed, however, that the purified Pof1p enzyme had no cytidylyltransferase activity but does have ATPase activity, with catalytic efficiency comparable to other ATPases involved in endoplasmic reticulum-associated degradation of proteins (ERAD). Supporting these findings, co-immunoprecipitation experiments showed a physical interaction between Pof1p and Ubc7p (an ubiquitin conjugating enzyme) in vivo.

Conclusions

Taken together, the results strongly suggest that the biological function of Pof1p is related to the regulation of protein degradation.

     

Marmoset phylogenetics, conservation perspectives, and evolution of the mtDNA control region

Marmosets (genus Callithrix) are a diverse group of platyrrhine primates with 13-15 purported taxa, many of them considered endangered. Morphological analyses constitute most of the basis for recognition of these forms as distinct taxa. The purpose of this study was to provide a molecular view, based on mitochondrial control region sequences, of the evolutionary history of the marmosets, concomitant with a molecular phylogenetic perspective on species diversity within the group. An additional purpose was to provide the first comparative examination of a complete New World monkey control region sequence with those of other mammals. The phylogenetic analyses provide convincing support for a split between the Atlantic forest and Amazonian marmosets, with the inclusion of the pygmy marmoset (Cebuella pygmaea) at the base of the Amazonian clade. The earliest branch of the Atlantic forest group was C. aurita. In the Amazonian group, the analyses do not support the recognition of C. humeralifer and the recently described C mauesi as distinct taxa. They do, however, support a clear distinction between C. argentata and a strongly supported mixed clade of C. humeralifer and C. mauesi. In the Atlantic forest group, the phylogenetic tree suggests mixing between C. penicillata, C. kuhli, and possibly C. jacchus. Most of the sequence features characteristic of other mammal control regions were also evident in marmosets, with the exception that conserved sequence blocks (CSBs) 2 and 3 were not clearly identifiable. Tandem repeat units often associated with heteroplasmy in a variety of other mammals were not evident in the marmoset sequences.      

The human collagen beta(1-O)galactosyltransferase,GLT25D1, is a soluble endoplasmic reticulum localized protein

Background  

Glycosyl transferases transfer glycosyl groups onto their substrate. Localization partially defines their function. Glycosyl transferase 25 domain 1 (GLT25D1) was recently shown to have galactosyltransferase activity towards collagens and another well known substrate, mannose binding lectin (MBL). To gain more insight in the role of galactosylation of lysines in the Gly-X-Lys repeats of collagenous proteins, we investigated the subcellular localization of GLT25D1.     

Respiratory burst oscillations in human neutrophils and their correlation with fluctuations in apparent cell shape

Neutrophils pretreated with phorbol 12-myristate 13-acetate (1-10 nM) and stimulated with low concentrations of chemotactic agonists (1-10nM) exhibited a marked increase in respiratory burst activity that was characterized by regular oscillations. These were accompanied by parallel oscillations in turbidity having the same phase and period. Four different agonists, f-Met-Leu-Phe, complement fragment C5a, platelet-activating factor, and leukotriene B4, induced virtually identical oscillations, with mean periods of 7.9 +/- 0.6 s (respiratory burst) and 7.9 +/- 0.8 s (turbidity) at 37 degrees C. No burst oscillations were observed at high agonist concentrations (50-100 nM) unless the fungal metabolite 17-hydroxywortmannin was added prior to stimulation. In the absence of phorbol 12-myristate 13-acetate, the respiratory burst activity was inhibited by 17-hydroxywortmannin, the protein kinase C inhibitor staurosporine, and calcium depletion, while agonist-dependent turbidity changes including the oscillations were unaffected. Turbidity changes reflect corresponding changes in cell size and/or shape, suggesting that cyclic alterations in morphology such as lamellipod extension and retraction physically affect the catalytic efficiency of the membrane-bound burst enzyme NADPH-oxidase. The oscillations appear to be controlled via receptor-dependent activation mechanisms which do not involve PKC activation or the rise in internal calcium presumably derived from phospholipase C activation.     

Phylogenetic Analysis of Heterothallic Neurospora Species

We examined the phylogenetic relationships among five heterothallic species of Neurospora using restriction fragment polymorphisms derived from cosmid probes and sequence data from the upstream regions of two genes, al-1 and frq. Distance, maximum likelihood, and parsimony trees derived from the data support the hypothesis that strains assigned to N. sitophila, N. discreta, and N. tetrasperma form respective monophyletic groups. Strains assigned to N. intermedia and N. crassa, however, did not form two respective monophyletic groups, consistent with a previous suggestion based on analysis of mitochondrial DNAs that N. crassa and N. intermedia may be incompletely resolved sister taxa. Trees derived from restriction fragments and the al-1 sequence position N. tetrasperma as the sister species of N. sitophila. None of the trees produced by our data supported a previous analysis of sequences in the region of the mating type idiomorph that grouped N. crassa and N. sitophila as sister taxa, as well as N. intermedia and N. tetrasperma as sister taxa. Moreover, sequences from al-1, frq, and the mating-type region produced different trees when analyzed separately. The lack of consensus obtained with different sequences could result from the sorting of ancestral polymorphism during speciation or gene flow across species boundaries, or both.     

Anonymous nuclear DNA markers in the American oyster and their implications for the heterozygote deficiency phenomenon in marine bivalves

A puzzling population-genetic phenomenon widely reported in allozyme surveys of marine bivalves is the occurrence of heterozygote deficits relative to Hardy-Weinberg expectations. Possible explanations for this pattern are categorized with respect to whether the effects should be confined to protein-level assays or are genomically pervasive and expected to be registered in both protein- and DNA-level assays. Anonymous nuclear DNA markers from the American oyster were employed to reexamine the phenomenon. In assays based on the polymerase chain reaction (PCR), two DNA-level processes were encountered that can lead to artifactual genotypic scorings: (a) differential amplification of alleles at a target locus and (b) amplification from multiple paralogous loci. We describe symptoms of these complications and prescribe methods that should generally help to ameliorate them. When artifactual scorings at two anonymous DNA loci in the American oyster were corrected, Hardy-Weinberg deviations registered in preliminary population assays decreased to nonsignificant values. Implications of these findings for the heterozygote-deficit phenomenon in marine bivalves, and for the general development and use of PCR-based assays, are discussed.