TRIPLES: a database of gene function in Saccharomyces cerevisiae

Using a novel multipurpose mini-transposon, we have generated a collection of defined mutant alleles for the analysis of disruption phenotypes, protein localization, and gene expression in Saccharomyces cerevisiae. To catalog this unique data set, we have developed TRIPLES, a Web-accessible database of TRansposon-Insertion Phenotypes, Localization and Expression in Saccharomyces. Encompassing over 250 000 data points, TRIPLES provides convenient access to information from nearly 7800 transposon-mutagenized yeast strains; within TRIPLES, complete data reports of each strain may be viewed in table format, or if desired, downloaded as tab-delimited text files. Each report contains external links to corresponding entries within the Saccharomyces Genome Database and International Nucleic Acid Sequence Data Library (GenBank). Unlike other yeast databases, TRIPLES also provides on-line order forms linked to each clone report; users may immediately request any desired strain free-of-charge by submitting a completed form. In addition to presenting a wealth of information for over 2300 open reading frames, TRIPLES constitutes an important medium for the distribution of useful reagents throughout the yeast scientific community. Maintained by the Yale Genome Analysis Center, TRIPLES may be accessed at http://ycmi.med.yale.edu/ygac/triples.htm     

A spontaneously active focus drives a model atrial sheet more easily than a model ventricular sheet

Tachycardias can be produced when focal activity at ectopic locations in either the atria or the ventricles propagates into the surrounding quiescent myocardium. Isolated rabbit atrioventricular nodal cells were coupled by an electronic circuit to a real-time simulation of an array of cell models. We investigated the critical size of an automatic focus for the activation of two-dimensional arrays made up of either ventricular or atrial model cells. Over a range of coupling conductances for the arrays, the critical size of the focus cell group for successful propagation was smaller for activation of an atrial versus a ventricular array. Failure of activation of the arrays at smaller focus sizes was due to the inhibition of pacing of the nodal cells. At low levels of coupling conductance, the ventricular arrays required larger sizes of the focus due to failure of propagation even when the focus was spontaneously active. The major differences between activation of the atrial and ventricular arrays is due to the higher membrane resistance (lower inward rectifier current) of the atrial cells.     

Vascular endothelial growth factor up-regulation via p21-activated kinase-1 signaling regulates heregulin-beta1-mediated angiogenesis

Heregulin-beta1 promotes the activation of p21-activated kinase 1 (Pak1) and the motility and invasiveness of breast cancer cells. In this study, we identified vascular endothelial growth factor (VEGF) as a gene product induced by heregulin-beta1. The stimulation by heregulin-beta1 of breast cancer epithelial cells induced the expression of the VEGF mRNA and protein and its promoter activity. Heregulin-beta1 also stimulated angiogenesis in a VEGF-dependent manner. Herceptin, an anti-HER2 antibody inhibited heregulin-beta1-mediated stimulation of both VEGF expression in epithelial cells and angiogenesis in endothelial cells. Because the activation of Pak1 and VEGF expression are positively regulated by heregulin-beta1, we hypothesized that Pak1 regulates VEGF expression, and hence explored the role of Pak1 in angiogenesis. We provide new evidence to implicate Pak1 signaling in VEGF expression. Overexpression of a kinase-dead K299R Pak1 leads to suppression of VEGF promoter activity, as well as VEGF mRNA expression and secretion of VEGF protein. Conversely, kinase-active T423E Pak1 promotes the expression and secretion of VEGF. Furthermore, expression of the heregulin-beta1 transgene, HRG, in harderian tumors in mice enhances the activation of Pak1 as well as expression of VEGF and angiogenic marker CD34 antigen. These results suggest that heregulin-beta1 regulates angiogenesis via up-regulation of VEGF expression and that Pak1 plays an important role in controlling VEGF expression and, consequently, VEGF secretion and function.     

CKbeta-8 [CCL23], a novel CC chemokine, is chemotactic for human osteoclast precursors and is expressed in bone tissues

We have previously demonstrated that a tartrate-resistant acid phosphatase (TRAP)-positive subpopulation of mononuclear cells isolated from collagenase digests of human osteoclastoma tissue exhibits an osteoclast phenotype and can be induced to resorb bone. Using these osteoclast precursors as a model system, we have assessed the chemotactic potential of 16 chemokines. Three CC chemokines, the recently described CKbeta-8, RANTES, and MIP-1alpha elicited significant chemotactic responses. In contrast, 10 other CC chemokines (MIP-1beta, MCP-1, MCP-2, MCP-3, MCP-4, HCC-1, eotaxin-2, PARC, SLC, ELC) and 3 CXC chemokines (IL-8, GROalpha, SDF-1) were inactive. None of these chemokines showed any chemotactic activity for either primary osteoblasts derived from human bone explants or the osteoblastic MG-63 cell line. The identity of the osteoclast receptor that mediates the chemotactic response remains to be established. However, all three active chemokines have been reported to bind to CCR1 and cross-desensitization studies demonstrate that RANTES and MIP-1alpha can partially inhibit the chemotactic response elicited by CKbeta-8. CKbeta-8, the most potent of the active CC chemokines (EC(max) 0.1-0.3 nM), was further characterized with regard to expression in human bone and cartilage. Although expression is not restricted to these tissues, CKbeta-8 mRNA was shown to be highly expressed in osteoblasts and chondrocytes in human fetal bone by in situ hybridization. In addition, CKbeta-8 protein was shown to be present in human osteophytic tissue by immunolocalization. These observations suggest that CKbeta-8, and perhaps other chemokines, may play a role in the recruitment of osteoclast precursors to sites of bone resorption.     

Elucidation of the solution structure of cardiotoxin analogue V from the Taiwan cobra (Naja naja atra)--identification of structural features important for the lethal action of snake venom cardiotoxins

The aim of the present study is to understand the structural features responsible for the lethal activity of snake venom cardiotoxins. Comparison of the lethal potency of the five cardiotoxin isoforms isolated from the venom of Taiwan cobra (Naja naja atra) reveals that the lethal potency of CTX I and CTX V are about twice of that exhibited by CTX II, CTX III, and CTX IV. In the present study, the solution structure of CTX V has been determined at high resolution using multidimensional proton NMR spectroscopy and dynamical simulated annealing techniques. Comparison of the high resolution solution structures of CTX V with that of CTX IV reveals that the secondary structural elements in both the toxin isoforms consist of a triple and double-stranded antiparallel beta-sheet domains. Critical examination of the three-dimensional structure of CTX V shows that the residues at the tip of Loop III form a distinct "finger-shaped" projection comprising of nonpolar residues. The occurrence of the nonpolar "finger-shaped" projection leads to the formation of a prominent cleft between the residues located at the tip of Loops II and III. Interestingly, the occurrence of a backbone hydrogen bonding (Val27CO to Leu48NH) in CTX IV is found to distort the "finger-shaped" projection and consequently diminish the cleft formation at the tip of Loops II and III. Comparison of the solution structures and lethal potencies of other cardiotoxin isoforms isolated from the Taiwan cobra (Naja naja atra) venom shows that a strong correlation exists between the lethal potency and occurrence of the nonpolar "finger-shaped" projection at the tip of Loop III. Critical analysis of the structures of the various CTX isoforms from the Taiwan cobra suggest that the degree of exposure of the cationic charge (to the solvent) contributed by the invariant lysine residue at position 44 on the convex side of the CTX molecules could be another crucial factor governing their lethal potency.     

Oxygen Consumption and Respiration Following Two Yoga Relaxation Techniques

The present study was conducted to evaluate a statement in ancient yoga texts that suggests that a combination of both calming and stimulating measures may be especially helpful in reaching a state of mental equilibrium. Two yoga practices, one combining calming and stimulating measures (cyclic meditation) and the other, a calming technique (shavasan), were compared. The oxygen consumption, breath rate, and breath volume of 40 male volunteers (group mean ± SD, 27.0 ± 5.7 years) were assessed before and after sessions of cyclic meditation (CM) and before and after sessions of shavasan (SH). The 2 sessions (CM, SH) were 1 day apart. Cyclic meditation includes the practice of yoga postures interspersed with periods of supine relaxation. During SH the subject lies in a supine position throughout the practice. There was a significant decrease in the amount of oxygen consumed and in breath rate and an increase in breath volume after both types of sessions (2-factor ANOVA, paired t test). However, the magnitude of change on all 3 measures was greater after CM: (1) Oxygen consumption decreased 32.1% after CM compared with 10.1% after SH; (2) breath rate decreased 18.0% after CM and 15.2% after SH; and (3) breath volume increased 28.8% after CM and 15.9% after SH. These results support the idea that a combination of yoga postures interspersed with relaxation reduces arousal more than relaxation alone does.     

The hPLIC proteins may provide a link between the ubiquitination machinery and the proteasome

Although there is a binding site on the proteasome for the polyubiquitin chains attached to degradation substrates by the ubiquitination machinery, it is currently unclear whether in vivo the activities of the ubiquitination machinery and the proteasome are coupled. Here we show that two human homologs of the yeast ubiquitin-like Dsk2 protein, hPLIC-1 and hPLIC-2, physically associate with both proteasomes and ubiquitin ligases in large complexes. Overexpression of hPLIC proteins interferes with the in vivo degradation of two unrelated ubiquitin-dependent proteasome substrates, p53 and IkappaBalpha, but not a ubiquitin-independent substrate. Our findings raise the possibility that the hPLIC proteins, and possibly related ubiquitin-like family members, may functionally link the ubiquitination machinery to the proteasome to affect in vivo protein degradation.     

Measurements of calcium transients in ventricular cells during discontinuous action potential conduction

The L-type calcium current (I(Ca)) is important in sustaining propagation during discontinuous conduction. In addition, I(Ca) is altered during discontinuous conduction, which may result in changes in the intracellular calcium transient. To study this, we have combined the ability to monitor intracellular calcium concentration ([Ca(2+)](i)) in an isolated cardiac cell using confocal scanning laser fluorescence microscopy with our "coupling clamp" technique, which allows action potential propagation from the real cell to a real-time simulation of a model cell. Coupling a real cell to a model cell with a value of coupling conductance (G(C) = 8 nS) just above the critical value for action potential propagation results in both an increased amplitude and an increased rate of rise of the calcium transient. Similar but smaller changes in the calcium transient are caused by increasing G(C) to 20 nS. The increase of [Ca(2+)](i) by discontinuous conduction is less than the increase of I(Ca), which may indicate that much of [Ca(2+)](i) is the result of calcium released from the sarcoplasmic reticulum rather than the integration of I(Ca).     

Identification and purification of the 69-kDa intracellular protease involved in the proteolytic processing of the crystal delta-endotoxin of Bacillus thuringiensis subsp. tenebrionis

The dynamics of appearance of intracellular proteases in relation to the synthesis of crystal delta-endotoxin was studied to identify the native intracellular protease(s) involved in the proteolytic processing of the 73-kDa protoxin of Bacillus thuringiensis subsp. tenebrionis. In vitro proteolytic activation of the 73-kDa protoxin indicated the possible role of 69-kDa protease in the proteolytic processing of 73-kDa protoxin. The purified 69-kDa protease was able to cause the proteolytic activation of the 73-kDa protoxin to 68-kDa toxin and this conversion was inhibited by ethylenediamine tetraacetic acid and 1,10-phenanthroline.