A functional interaction between CPI-17 and RACK1 proteins in bronchial smooth muscle cells

CPI-17 is a phosphorylation-dependent inhibitor of smooth muscle myosin light chain. Using yeast two-hybrid system, we have identified the receptor for activated C kinase 1 (RACK1) as a novel interaction partner of CPI-17. The direct interaction and co-localization of CPI-17 with RACK1 were confirmed by immunoprecipitation and confocal microscopy analysis, respectively. An in vitro assay system using recombinant/purified proteins revealed that the PKC-mediated phosphorylation of CPI-17 was augmented in the presence of RACK1. These results suggest that RACK1 may play a role in PKC/CPI-17 signaling pathway.     

Dietary restriction suppresses inflammation and delays the onset of stroke in stroke-prone spontaneously hypertensive rats

Subjects with high blood levels of inflammatory markers and patients with chronic inflammatory disorders are at high risk for stroke. Dietary restriction (DR) suppresses systemic inflammation to deter age-related chronic diseases. To examine whether DR delays the onset of stroke, 10-week-old stroke-prone spontaneously hypertensive rats (SHRSP) were assigned to either a control (ad libitum) or DR (50% diet of control) group, and day of stroke onset and lifespan were observed. DR markedly delayed the onset of stroke in SHRSP compared to control without affecting blood pressure. Day of stroke onset (median) in the control group was 34 days, whereas it was 70 days in the DR group. After 2 weeks of DR and before the onset of stroke, plasma levels of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and monocyte chemoattractant protein-1 (MCP-1) and their mRNA expression levels in adipose tissue were significantly lower in the DR rats than in the control rats. Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) mRNA expression levels in cerebrovascular endothelial cells (CVECs), and macrophage infiltration into brain were lower in the DR rats than in the control rats. IL-1β and TNF-α treatment in CVECs increased MCP-1, C-reactive protein, ICAM-1, and VCAM-1 mRNA and their protein levels in vitro. In conclusion, suppression of inflammation in response to DR may lead to a delay in the onset of stroke independent of any effect on blood pressure in SHRSP.     

Impaired Replication Capacity of Acute/Early Viruses in Persons Who Become HIV Controllers

Human immunodeficiency virus type 1 (HIV-1) controllers maintain viremia at <2,000 RNA copies/ml without antiretroviral therapy. Viruses from controllers with chronic infection were shown to exhibit impaired replication capacities, in part associated with escape mutations from cytotoxic-T-lymphocyte (CTL) responses. In contrast, little is known about viruses during acute/early infection in individuals who subsequently become HIV controllers. Here, we examine the viral replication capacities, HLA types, and virus sequences from 18 HIV-1 controllers identified during primary infection. gag-protease chimeric viruses constructed using the earliest postinfection samples displayed significantly lower replication capacities than isolates from persons who failed to control viremia (P = 0.0003). Protective HLA class I alleles were not enriched in these early HIV controllers, but viral sequencing revealed a significantly higher prevalence of drug resistance mutations associated with impaired viral fitness in controllers than in noncontrollers (6/15 [40.0%] versus 10/80 [12.5%], P = 0.018). Moreover, of two HLA-B57-positive (B57⁺) controllers identified, both harbored, at the earliest time point tested, signature escape mutations within Gag that likewise impair viral replication capacity. Only five controllers did not express “protective” alleles or harbor viruses with drug resistance mutations; intriguingly, two of them displayed typical B57 signature mutations (T242N), suggesting the acquisition of attenuated viruses from B57⁺ donors. These data indicate that acute/early stage viruses from persons who become controllers have evidence of reduced replication capacity during the initial stages of infection which is likely associated with transmitted or acquired CTL escape mutations or transmitted drug resistance mutations. These data suggest that viral dynamics during acute infection have a major impact on HIV disease outcome.Human immunodeficiency virus type I (HIV-1)-infected individuals who control viremia spontaneously without antiviral therapy have been termed HIV controllers (3, 18, 21, 48, 52). Unraveling the mechanisms associated with this phenotype should provide important insights regarding HIV pathogenesis and could contribute to vaccine development.Host and viral genetics, as well as host innate and adaptive immune responses, influence the rate of disease progression in HIV-1 infection (reviewed in reference 18). Several studies have reported the correlation between in vitro HIV replication capacity and level of plasma virus loads or disease progression in individuals with chronic infection (6, 13, 35, 45, 50, 55). Studies of HIV-1 elite controllers (EC), who control viremia to below the limit of detection in commercial assays, have revealed the presence of replication-competent viruses in these individuals (7), although these viruses appear to be less fit based on studies of envelope (35) and Gag-protease (45). This fitness defect in the chronic phase of infection is due at least in part to fitness-impairing mutations induced by cytotoxic-T-lymphocyte (CTL) responses restricted by “protective” HLA class I alleles (46).In contrast, little is known about viruses obtained from the acute/early phase of infection in persons who subsequently become HIV-1 controllers, largely due to the difficulty in enrolling such people during the acute/early phase of infection. The characterization of acute/early-phase viruses in individuals who subsequently achieve low set-point virus loads is of paramount importance to our understanding of the mechanisms of HIV-1 control.In the present study, we analyzed acute/early-phase plasma HIV RNA sequences from 18 untreated individuals who were diagnosed during the acute/early phase and subsequently became controllers (<2,000 RNA copies/ml). We compared these to sequences from a group of HIV-1 noncontrollers enrolled similarly during acute/early infection. We also generated chimeric viruses carrying patient-derived gag-protease sequences from acute/early-phase infection and compared the viral replication capacities of the chimeric viruses from controllers and from noncontrollers.We observed that the chimeric viruses derived from controllers have significantly reduced replicative capacities compared to those from noncontrollers. Moreover, we observed that at least 80% of these individuals who go on to become controllers featured transmission of attenuated drug-resistant viruses, transmission of HLA-B57-restricted CTL escape variants to HLA-mismatched recipients, selection of attenuated CTL escape variants in HLA-B57-positive (B57⁺) recipients, or combinations of these factors. Taken together, these results indicate that the initial viral dynamics have a major influence on the subsequent course of disease.     

Interspecific interference competition alters habitat use patterns in two species of land snails

Focus has been placed on the relative importance of environmental heterogeneity and biological interactions on community structure. For land snails, abiotic factors have commonly been assumed to be the most important factors for shaping communities because resources for land snails are commonly not limiting, and because interspecific resource competition would not be strong enough to promote ecological divergence. However, clear divergence in habitat use is often observed among sympatric land snail species. Such an ecological divergence would be promoted not by resource competition but by other interactions such as aggression. To test this hypothesis, laboratory experiments were designed to explore aggressive behaviour in the land snails Euhadra quaesita and E. peliomphala and field surveys were conducted to examine their habitat use. In the laboratory experiments, we examined how the presence of one species affects the growth of the other species by supplying sufficient amounts of food and calcium for both species. Experimental trials showed that adult E. quaesita decreased the growth of E. peliomphala under constant diet conditions. In contrast, E. peliomphala did not affect the growth of E. quaesita. Because E. peliomphala was often attacked by E. quaesita and its shell was often eroded by E. quaesita gnawing, aggression by E. quaesita appears to be the primary factor reducing the growth of E. peliomphala. When the two species coexist in nature, adults and juveniles of E. quaesita are terrestrial, while those of E. peliomphala are arboreal. When these species occurred alone, E. quaesita were still terrestrial, but E. peliomphala were more terrestrial than when they were in sympatry. Our results suggest that habitat use of these species in the natural communities is affected by interspecific interference. These findings further suggest that the divergence of habitat use between the species can occur by aggression even in environments with unlimited resources.     

Metabolic activation of N-thiazol-2-yl benzamide as glucokinase activators: Impacts of glutathione trapping on covalent binding

Glucokinase activators (GKAs) are currently under investigation as potential antidiabetic agents by many pharmaceutical companies. Most of GKAs reported previously possess N-aminothiazol-2-yl amide moiety in their structures because the aminothiazole moiety interacts with glucokinase (GK) and shows strong GK activation. During the development of N-aminothiazol-2-yl amide derivatives, we identified a bioactivation and metabolic liability of 2-aminothizole substructure of GKA 3 by assessing covalent binding, metabolites in liver microsomes and glutathione (GSH) trap assay.     

Discovery of 6-benzyloxyquinolines as c-Met selective kinase inhibitors

A novel quinoline derivative that selectively inhibits c-Met kinase was identified. The molecular design is based on a result of the analysis of a PF-2341066 (1)/c-Met cocrystal structure (PDB code: 2wgj). The kinase selectivity of the derivatives is discussed from the view point of the sequence homology of the kinases, the key interactions found in X-ray cocrystal structures, and the structure–activity relationship (SAR) obtained in this work.     

Structural characteristics of green tea catechins for formation of protein carbonyl in human serum albumin

Catechins are polyphenolic antioxidants found in green tea leaves. Recent studies have reported that various polyphenolic compounds, including catechins, cause protein carbonyl formation in proteins via their pro-oxidant actions. In this study, we evaluate the formation of protein carbonyl in human serum albumin (HSA) by tea catechins and investigate the relationship between catechin chemical structure and its pro-oxidant property. To assess the formation of protein carbonyl in HSA, HSA was incubated with four individual catechins under physiological conditions to generate biotin-LC-hydrazide labeled protein carbonyls. Comparison of catechins using Western blotting revealed that the formation of protein carbonyl in HSA was higher for pyrogallol-type catechins than the corresponding catechol-type catechins. In addition, the formation of protein carbonyl was also found to be higher for the catechins having a galloyl group than the corresponding catechins lacking a galloyl group. The importance of the pyrogallol structural motif in the B-ring and the galloyl group was confirmed using methylated catechins and phenolic acids. These results indicate that the most important structural element contributing to the formation of protein carbonyl in HSA by tea catechins is the pyrogallol structural motif in the B-ring, followed by the galloyl group. The oxidation stability and binding affinity of tea catechins with proteins are responsible for the formation of protein carbonyl, and consequently the difference in these properties of each catechin may contribute to the magnitude of their biological activities.