Integrin engagement suppresses RhoA activity via a c-Src-dependent mechanism

The Rho family GTPases Cdc42, Rac1 and RhoA control many of the changes in the actin cytoskeleton that are triggered when growth factor receptors and integrins bind their ligands. Rac1 and Cdc42 stimulate the formation of protrusive structures such as membrane ruffles, lamellipodia and filopodia. RhoA regulates contractility and assembly of actin stress fibers and focal adhesions. Although prolonged integrin engagement can stimulate RhoA, regulation of this GTPase by early integrin-mediated signals is poorly understood. Here we show that integrin engagement initially inactivates RhoA, in a c-Src-dependent manner, but has no effect on Cdc42 or Rac1 activity. Additionally, early integrin signaling induces activation and tyrosine phosphorylation of p190RhoGAP via a mechanism that requires c-Src. Dynamic modulation of RhoA activity appears to have a role in motility, as both inhibition and activation of RhoA hinder migration. Transient suppression of RhoA by integrins may alleviate contractile forces that would otherwise impede protrusion at the leading edge of migrating cells

The protein tyrosine phosphatase Shp-2 regulates RhoA activity

Remodeling of filamentous actin into distinct arrangements is precisely controlled by members of the Rho family of small GTPases [1]. A well characterized member of this family is RhoA, whose activation results in reorganization of the cytoskeleton into thick actin stress fibers terminating in integrin-rich focal adhesions [2]. Regulation of RhoA is required to maintain adhesion in stationary cells, but is also critical for cell spreading and migration [3]. Despite its biological importance, the signaling events leading to RhoA activation are not fully understood. Several independent studies have implicated tyrosine phosphorylation as a critical event upstream of RhoA [4]. Consistent with this, our recent studies have demonstrated the existence of a protein tyrosine phosphatase (PTPase), sensitive to the dipeptide aldehyde calpeptin, acting upstream of RhoA [5]. Here we identify the SH2 (Src homology region 2)-containing PTPase Shp-2 as a calpeptin-sensitive PTPase, and show that calpeptin interferes with the catalytic activity of Shp-2 in vitro and with Shp-2 signaling in vivo. Finally, we show that perturbation of Shp-2 activity by a variety of genetic manipulations results in raised levels of active RhoA. Together, these studies identify Shp-2 as a PTPase acting upstream of RhoA to regulate its activity and contribute to the coordinated control of cell movement

The protein tyrosine phosphatase Shp-2 regulates RhoA activity

Remodeling of filamentous actin into distinct arrangements is precisely controlled by members of the Rho family of small GTPases [1]. A well characterized member of this family is RhoA, whose activation results in reorganization of the cytoskeleton into thick actin stress fibers terminating in integrin-rich focal adhesions [2]. Regulation of RhoA is required to maintain adhesion in stationary cells, but is also critical for cell spreading and migration [3]. Despite its biological importance, the signaling events leading to RhoA activation are not fully understood. Several independent studies have implicated tyrosine phosphorylation as a critical event upstream of RhoA [4]. Consistent with this, our recent studies have demonstrated the existence of a protein tyrosine phosphatase (PTPase), sensitive to the dipeptide aldehyde calpeptin, acting upstream of RhoA [5]. Here we identify the SH2 (Src homology region 2)-containing PTPase Shp-2 as a calpeptin-sensitive PTPase, and show that calpeptin interferes with the catalytic activity of Shp-2 in vitro and with Shp-2 signaling in vivo. Finally, we show that perturbation of Shp-2 activity by a variety of genetic manipulations results in raised levels of active RhoA. Together, these studies identify Shp-2 as a PTPase acting upstream of RhoA to regulate its activity and contribute to the coordinated control of cell movement

Baseline resistance to nucleoside reverse transcriptase inhibitors fails to predict virologic response to combination therapy in children (PACTG 338)

BACKGROUND: The association between baseline drug resistance mutations and subsequent increase in viral failure has not been established for HIV-infected children. We evaluated drug resistance mutations at 39 codon sites (21 protease inhibitor (PI) resistant codons and 18 nucleoside reverse transcriptase inhibitor (NRTI) resistant codons) for 92 clinically stable NRTI-experienced, PI-naive HIV-infected children 2 to 17 years of age who were initiating new therapy with ritonavir plus zidovudine (ZDV) and lamivudine or plus stavudine. The association between baseline drug resistance mutations and subsequent viral failure after 12 and 24 weeks of highly active antiretroviral therapy (HAART) was studied. RESULTS: There were few primary PI associated mutations in this PI-naïve population, but 84% had NRTI mutations – codons 215 (66%), 41 (42%), 67 (37%), 210 (33%) and 70 (32%). None of the specific baseline drug resistance mutations were associated with a higher rate of virologic failure after 12 or 24 weeks of HAART. Median week 12 viral load decreased as the total number of NRTI mutations at baseline increased (P = 0.006). Specifically, a higher level of baseline ZDV resistance mutation was associated with a decrease in viral failure after 12 weeks on a ZDV-containing HAART regimen (P = 0.017). CONCLUSION: No increase was seen in the rate of viral failure after HAART associated with the presence of resistance mutations at baseline. This paradoxical result may be due to adherence, replicative capacity, or ZDV hypersusceptibility to the new regimen

Genotypic Susceptibility Scores and HIV Type 1 RNA Responses in Treatment-Experienced Subjects with HIV Type 1 Infection

This study compared the role of genotypic susceptibility scores (GSS) as a predictor of virologic response in a group (n = 234) of HIV-infected, protease inhibitor (PI)-experienced subjects. Two scoring methods [discrete genotypic susceptibility score (dGSS) and continuous genotypic susceptibility score (cGSS)] were developed. Each drug in the subject's regimen was given a binary susceptibility score using Stanford inferred drug resistance scores to calculate the dGSS. In contrast to the dGSS, the cGSS model was designed to reflect partial susceptibility to a drug. Both GSS were independent predictors of week 16 virologic response. We also compared the GSS to a phenotypic susceptibility score (PSS) model on a subset of subjects that had both GSS and PSS performed, and found that both models were predictive of virologic response. Genotypic analyses at enrollment showed that subjects who were virologic nonresponders at week 16 revealed enrichment of several mutated codons associated with nucleoside reverse transcriptase inhibitors (NRTI) (codons 67, 69, 70, 118, 215, and 219) or PI resistance (codons 10, 24, 71, 73, and 88) compared to subjects who were virologic responders. Regression analyses revealed that protease mutations at codons 24 and 90 were most predictive of poor virologic response, whereas mutations at 82 were associated with enhanced virologic response. Certain NNRTI-associated mutations, such as K103N, were rapidly selected in the absence of NRTIs. These data indicate that GSS may be a useful tool in selecting drug regimens in HIV-1-infected subjects to maximize virologic response and improve treatment outcomes

Baseline resistance to nucleoside reverse transcriptase inhibitors fails to predict virologic response to combination therapy in children (PACTG 338)

Abstract Background The association between baseline drug resistance mutations and subsequent increase in viral failure has not been established for HIV-infected children. We evaluated drug resistance mutations at 39 codon sites (21 protease inhibitor (PI) resistant codons and 18 nucleoside reverse transcriptase inhibitor (NRTI) resistant codons) for 92 clinically stable NRTI-experienced, PI-naive HIV-infected children 2 to 17 years of age who were initiating new therapy with ritonavir plus zidovudine (ZDV) and lamivudine or plus stavudine. The association between baseline drug resistance mutations and subsequent viral failure after 12 and 24 weeks of highly active antiretroviral therapy (HAART) was studied. Results There were few primary PI associated mutations in this PI-naïve population, but 84% had NRTI mutations – codons 215 (66%), 41 (42%), 67 (37%), 210 (33%) and 70 (32%). None of the specific baseline drug resistance mutations were associated with a higher rate of virologic failure after 12 or 24 weeks of HAART. Median week 12 viral load decreased as the total number of NRTI mutations at baseline increased (P = 0.006). Specifically, a higher level of baseline ZDV resistance mutation was associated with a decrease in viral failure after 12 weeks on a ZDV-containing HAART regimen (P = 0.017). Conclusion No increase was seen in the rate of viral failure after HAART associated with the presence of resistance mutations at baseline. This paradoxical result may be due to adherence, replicative capacity, or ZDV hypersusceptibility to the new regimen

An overview of the taxonomy, phylogeny, and typification of nectriaceous fungi in Cosmospora, Acremonium, Fusarium, Stilbella, and Volutella

A comprehensive phylogenetic reassessment of the ascomycete genus Cosmospora (Hypocreales, Nectriaceae) is undertaken using fresh isolates and historical strains, sequences of two protein encoding genes, the second largest subunit of RNA polymerase II (rpb2), and a new phylogenetic marker, the larger subunit of ATP citrate lyase (acl1). The result is an extensive revision of taxonomic concepts, typification, and nomenclatural details of many anamorph- and teleomorph-typified genera of the Nectriaceae, most notably Cosmospora and Fusarium. The combined phylogenetic analysis shows that the present concept of Fusarium is not monophyletic and that the genus divides into two large groups, one basal in the family, the other terminal, separated by a large group of species classified in genera such as Calonectria, Neonectria, and Volutella. All accepted genera received high statistical support in the phylogenetic analyses. Preliminary polythetic morphological descriptions are presented for each genus, providing details of perithecia, micro- and/or macro-conidial synanamorphs, cultural characters, and ecological traits. Eight species are included in our restricted concept of Cosmospora, two of which have previously documented teleomorphs and all of which have Acremonium-like microconidial anamorphs. A key is provided to the three anamorphic species recognised in Atractium, which is removed from synonymy with Fusarium and epitypified for two macroconidial synnematous species and one sporodochial species associated with waterlogged wood. Dialonectria is recognised as distinct from Cosmospora and two species with teleomorph, macroconidia and microconidia are accepted, including the new species D. ullevolea. Seven species, one with a known teleomorph, are classified in Fusicolla, formerly considered a synonym of Fusarium including members of the F. aquaeductuum and F. merismoides species complex, with several former varieties raised to species rank. Originally a section of Nectria, Macroconia is raised to generic rank for five species, all producing a teleomorph and macroconidial anamorph. A new species of the Verticillium-like anamorphic genus Mariannaea is described as M. samuelsii. Microcera is recognised as distinct from Fusarium and a key is included for four macroconidial species, that are usually parasites of scale insects, two of them with teleomorphs. The four accepted species of Stylonectria each produce a teleomorph and micro- and macroconidial synanamorphs. The Volutella species sampled fall into three clades. Pseudonectria is accepted for a perithecial and sporodochial species that occurs on Buxus. Volutella s. str. also includes perithecial and/or sporodochial species and is revised to include a synnematous species formerly included in Stilbella. The third Volutella-like clade remains unnamed. All fungi in this paper are named using a single name system that gives priority to the oldest generic names and species epithets, irrespective of whether they are originally based on anamorph or teleomorph structures. The rationale behind this is discussed