Tissue-specific differences in activation of atypical protein kinase C and protein kinase B in muscle, liver, and adipocytes of insulin receptor substrate-1 knockout mice

Insulin receptor substrates (IRSs) 1 and 2 are postulated to control the activation of phosphatidylinositol 3-kinase (PI3K)-dependent signaling factors, namely, atypical protein kinase C (aPKC) and protein kinase B (PKB)/Akt, which mediate metabolic effects of insulin. However, it is uncertain whether aPKC and PKB are activated together or differentially in response to IRS-1 and IRS-2 activation in insulin-sensitive tissues. Presently, we examined insulin activation of aPKC and PKB in vastus lateralis muscle, adipocytes, and liver in wild-type and IRS-1 knockout mice, and observed striking tissue-specific differences. In muscle of IRS-1 knockout mice, the activation of both aPKC and PKB was markedly diminished. In marked contrast, only aPKC activation was diminished in adipocytes, and only PKB activation was diminished in liver. These results suggest that IRS-1 is required for: 1) activation of both aPKC and PKB in muscle; 2) aPKC, but not PKB, activation in adipocytes; and 3) PKB, but not aPKC, activation in liver. Presumably, IRS-2 or other PI3K activators account for the normal activation of aPKC in liver and PKB in adipocytes of IRS-1 knockout mice. These complexities in aPKC and PKB activation may be relevant to metabolic abnormalities seen in tissues in which IRS-1 or IRS-2 is specifically or predominantly down-regulated.     

Spontaneous neural activity is required for the establishment and maintenance of the olfactory sensory map

We have developed a genetic approach to examine the role of spontaneous activity and synaptic release in the establishment and maintenance of an olfactory sensory map. Conditional expression of tetanus toxin light chain, a molecule that inhibits synaptic release, does not perturb targeting during development, but neurons that express this molecule in a competitive environment fail to maintain appropriate synaptic connections and disappear. Overexpression of the inward rectifying potassium channel, Kir2.1, diminishes the excitability of sensory neurons and more severely disrupts the formation of an olfactory map. These studies suggest that spontaneous neural activity is required for the establishment and maintenance of the precise connectivity inherent in an olfactory sensory map.     

Increased cell death in osteopontin-deficient cardiac fibroblasts occurs by a caspase-3-independent pathway

Reperfusion-induced oxidative injury to the myocardium promotes activation and proliferation of cardiac fibroblasts and repair by scar formation. Osteopontin (OPN) is a proinflammatory cytokine that is upregulated after reperfusion. To determine whether OPN enhances fibroblast survival after exposure to oxidants, cardiac fibroblasts from wild-type (WT) or OPN-null (OPN(-/-)) mice were treated in vitro with H(2)O(2) to model reperfusion injury. Within 1 h, membrane permeability to propidium iodide (PI) was increased from 5 to 60% in OPN(-/-) cells but was increased to only 20% in WT cells. In contrast, after 1-8 h of treatment with H(2)O(2), the percent of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-stained cells was more than twofold higher in WT than OPN(-/-) cells. Electron microscopy of WT cells treated with H(2)O(2) showed chromatin condensation, nuclear fragmentation, and cytoplasmic and nuclear shrinkage, which are consistent with apoptosis. In contrast, H(2)O(2)-treated OPN(-/-) cardiac fibroblasts exhibited cell and nuclear swelling and membrane disruption that are indicative of cell necrosis. Treatment of OPN(-/-) and WT cells with a cell-permeable caspase-3 inhibitor reduced the percentage of TUNEL staining by more than fourfold in WT cells but decreased staining in OPN(-/-) cells by approximately 30%. Although the percentage of PI-permeable WT cells was reduced threefold, the percent of PI-permeable OPN(-/-) cells was not altered. Restoration of OPN expression in OPN(-/-) fibroblasts reduced the percentage of PI-permeable cells but not TUNEL staining after H(2)O(2) treatment. Thus H(2)O(2)-induced cell death in OPN-deficient cardiac fibroblasts is mediated by a caspase-3-independent, necrotic pathway. We suggest that the increased expression of OPN in the myocardium after reperfusion may promote fibrosis by protecting cardiac fibroblasts from cell death.     

A high molecular mass cranberry constituent reduces mutans streptococci level in saliva and inhibits in vitro adhesion to hydroxyapatite

Previous investigations showed that a high molecular mass, non-dialyzable material (NDM) from cranberries inhibits the adhesion of a number of bacterial species and prevents the co-aggregation of many oral bacterial pairs. In the present study we determined the effect of mouthwash supplemented with NDM on oral hygiene. Following 6 weeks of daily usage of cranberry-containing mouthwash by an experimental group (n = 29), we found that salivary mutans streptococci count as well as the total bacterial count were reduced significantly (ANOVA, P < 0.01) compared with those of the control (n = 30) using placebo mouthwash. No change in the plaque and gingival indices was observed. In vitro, the cranberry constituent inhibited the adhesion of Streptococcus sobrinus to saliva-coated hydroxyapatite. The data suggest that the ability to reduce mutans streptococci counts in vivo is due to the anti-adhesion activity of the cranberry constituent.     

Pig-tailed macaques (<Emphasis Type="Italic">Macaca nemestrina</Emphasis>) possess six MHC-E families that are conserved among macaque species: implication for their binding to natural killer receptor variants

MHC loci encode highly polymorphic molecules involved in the presentation of self and non-self peptides to cells of the adaptive and innate immune systems. Although variable, MHC-E genes are well conserved among primates and provide signals to natural killer cells. In this study, we sequenced and analyzed MHC-E alleles of pig-tailed macaque (Macaca nemestrina), a nonhuman primate used for HIV pathogenesis and vaccine studies. Among a group of seven macaques, the characterization of eight Mane-E alleles revealed an increased number of polymorphic sites compared with human HLA-E alleles. Phylogenetic analyses of MHC-E alleles from pig-tailed macaque, rhesus monkey (Macaca mulatta) and cynomolgus macaque (Macaca fascicularis) demonstrated that the three macaque species shared six families of macaque MHC-E alleles and indicated that these families existed in the common ancestor 5.5 million years ago. Polymorphic Mane-E sites were not concentrated within the peptide-binding pockets, but were distributed throughout the entire ORF. The peptide-binding domain of Mane-E is similar to its human analogue, and peptide substrates theoretically capable of binding to Mane-E molecules were found in the leader sequence of classical Mane-A and -B molecules. Additionally, the polymorphic amino acids located in the ₁ and ₂ domains of Mane-E molecules have side chains expected to be oriented toward solvent and away from the peptide-binding groove, suggesting that some of them (positions 19, 73, 79 and 145) might be available for interaction with polymorphic receptors of natural killer cells.     

The modified super-ellipsoid yield criterion for human trabecular bone

Despite the importance of multiaxial failure of trabecular bone in many biomechanical applications, to date no complete multiaxial failure criterion for human trabecular bone has been developed. By using experimentally validated nonlinear high-resolution, micromechanical finite-element models as a surrogate for multiaxial loading experiments, we determined the three-dimensional normal strain yield surface and all combinations of the two-dimensional normal-shear strain yield envelope. High-resolution finite-element models of three human femoral neck trabecular bone specimens obtained through microcomputed tomography were used. In total, 889 multiaxial-loading cases were analyzed, requiring over 41,000 CPU hours on parallel supercomputers. Our results indicated that the multiaxial yield behavior of trabecular bone in strain space was homogeneous across the specimens and nearly isotropic. Analysis of stress-strain curves along each axis in the 3-D normal strain space indicated uncoupled yield behavior whereas substantial coupling was seen for normal-shear loading. A modified super-ellipsoid surface with only four parameters fit the normal strain yield data very well with an arithmetic error +/-SD less than -0.04 +/- 5.1%. Furthermore, the principal strains associated with normal-shear loading showed excellent agreement with the yield surface obtained for normal strain loading (arithmetic error +/- SD < 2.5 +/- 6.5%). We conclude that the four-parameter "Modified Super-Ellipsoid" yield surface presented here describes the multiaxial failure behavior of human femoral neck trabecular bone very well.     

How prevalent is functional alternative splicing in the human genome?

Comparative analyses of ESTs and cDNAs with genomic DNA predict a high frequency of alternative splicing in human genes. However, there is an ongoing debate as to how many of these predicted splice variants are functional and how many are the result of aberrant splicing (or 'noise'). To address this question, we compared alternatively spliced cassette exons that are conserved between human and mouse with EST-predicted cassette exons that are not conserved in the mouse genome. Presumably, conserved exon-skipping events represent functional alternative splicing. We show that conserved (functional) cassette exons possess unique characteristics in size, repeat content and in their influence on the protein. By contrast, most non-conserved cassette exons do not share these characteristics. We conclude that a significant portion of cassette exons evident in EST databases is not functional, and might result from aberrant rather than regulated splicing.     

PIVOT: protein interacions visualizatiOn tool

Protein Interaction VisualizatiOn Tool (PIVOT) is a visualization tool for protein-protein interactions. It allows the user to create personal data sets of interactions by combining information from private and public data sources. The user can gradually access the interactions' data using a clear interactive map that is focused on the researcher's protein of interest, and is reshaped and expanded in response to his/her queries. It also offers several visual enhancements and intelligent queries that help the user efficiently study it. PIVOT allows the user to search the interactions data set for paths connecting proteins that are expected to co-operate. The user can also employ PIVOT to predict unknown interactions among proteins, based on interactions among their homologous proteins in other species.