RANTES (CCL5) induces a CCR5-dependent accelerated shedding of syndecan-1 (CD138) and syndecan-4 from HeLa cells and forms complexes with the shed ectodomains of these proteoglycans as well as with those of CD44

We recently demonstrated that RANTES forms complexes with CCR5, syndecan-1 (SD-1), SD-4, and CD44 expressed by human primary macrophages and that SD-1 and SD-4 but neither CD44 nor SD-2 coimmunoprecipitate with CCR5. Here we show that RANTES directly binds in a glycosaminoglycan-dependent manner to SD-1, SD-4, and CD44. Moreover, RANTES accelerates the shedding of SD-1 and SD-4 ectodomains from HeLa cells expressing CCR5 and, by contrast, has no effect on the constitutive shedding of CD44 from these cells. These accelerated sheddings are prevented by the MEK1/2 inhibitor, U0126, and by the protein kinase C inhibitor bisindolylmaleimide I. This indicates that both MAP kinase--and protein kinase C-dependent signaling pathways are involved in these RANTES-induced accelerated sheddings. RANTES also induces a decreased expression of SD-1 and SD-4 by HeLa cells expressing CCR5 and on the contrary an increased expression of CD44 by these cells. By contrast, RANTES neither accelerates the shedding of SD-1 and SD-4 ectodomains from HeLa cells lacking CCR5, nor changes the SD-1-, SD-4-, and CD44-plasma membrane expressions of these cells. CCR5 is therefore involved in the RANTES-induced accelerated shedding of SD-1 and SD-4 ectodomains. Nevertheless, the fact that RANTES stimulates in Hela cells (expressing or lacking CCR5) the mRNA synthesis of SD-1 and SD-4 indicates that the molecular events that follow the synthesis of these proteoglycans differ, according to the presence or not of CCR5. Finally, RANTES forms GAG-dependent complexes with the shed ectodomains of SD-1 and SD-4 as well as with those of CD44. The role of these events in the pathophysiology of RANTES deserves further study.     

Synthesis and SAR of highly potent dual 5-HT1A and 5-HT1B antagonists as potential antidepressant drugs

Novel 5-HT₁ autoreceptor ligands based on the N-4-aryl-piperazinyl-N′-ethyl-5,6,7,8-tetrahydropyrido[4′, 3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one core are described. Aiming at antidepressants with a novel mode of action our objective was to identify potent antagonists showing balanced affinities and high selectivity for the 5-HT_1A and 5-HT_1B receptors. Strategies for the development of dual 5-HT_1A and 5-HT_1B antagonists based on 1 and 2 as leads and the corresponding results are discussed. Isoquinoline analogue 33 displayed high affinity and an antagonistic mode of action for the 5-HT_1A and the 5-HT_1B receptors and was characterized further with respect to selectivity, electrically stimulated [³H]5-HT release and in vivo efficacy.     

Human serum facilitates hepatitis C virus infection, and neutralizing responses inversely correlate with viral replication kinetics at the acute phase of hepatitis C virus infection

The factors leading to spontaneous clearance of hepatitis C virus (HCV) or to viral persistence are elusive. Understanding virus-host interactions that enable acute HCV clearance is key to the development of more effective therapeutic and prophylactic strategies. Here, using a sensitive neutralization assay based on infectious HCV pseudoparticles (HCVpp), we have studied the kinetics of humoral responses in a cohort of acute-phase patients infected during a single nosocomial outbreak in a hemodialysis center. The 17 patients were monitored for the spontaneous outcome of HCV infection for 6 months before a treatment decision was made. Blood samples were taken frequently (15 +/- 4 per patient). Phylogenetic analysis of the predominant virus(es) revealed infection by only one of two genotype 1b strains. While all patients seroconverted, their sera induced two opposing effects in HCVpp infection assays: inhibition and facilitation. Furthermore, the ability of sera to facilitate or inhibit infection correlated with the presence of either infecting HCV strain and divided the patients into two groups. In group 1, the progressive emergence of a relatively strong neutralizing response correlated with a fluctuating decrease in high initial viremia, leading to control of viral replication. Patients in group 2 failed to reduce viremia within the acute phase, and no neutralizing responses were detected despite seroconversion. Strikingly, sera of group 2, as well as naive sera, facilitated infection by HCVpp displaying HCV glycoproteins from different genotypes and strains, including those retrieved from patients. These results provide new insights into the mechanisms of viral persistence and immune control of viremia.     

The adaptor complex 2 directly interacts with the alpha 1b-adrenergic receptor and plays a role in receptor endocytosis

Using the yeast two-hybrid system, we identified the mu 2 subunit of the clathrin adaptor complex 2 as a protein interacting with the C-tail of the alpha 1b-adrenergic receptor (AR). Direct association between the alpha 1b-AR and mu 2 was demonstrated using a solid phase overlay assay. The alpha 1b-AR/mu 2 interaction occurred inside the cells, as shown by the finding that the transfected alpha 1b-AR and the endogenous mu 2 could be coimmunoprecipitated from HEK-293 cell extracts. Mutational analysis of the alpha 1b-AR revealed that the binding site for mu 2 does not involve canonical YXX Phi or dileucine motifs but a stretch of eight arginines on the receptor C-tail. The binding domain of mu 2 for the receptor C-tail involves both its N terminus and the subdomain B of its C-terminal portion. The alpha 1b-AR specifically interacted with mu 2, but not with the mu 1, mu 3, or mu 4 subunits belonging to other AP complexes. The deletion of the mu 2 binding site in the C-tail markedly decreased agonist-induced receptor internalization as demonstrated by confocal microscopy as well as by the results of a surface receptor biotinylation assay. The direct association of the adaptor complex 2 with a G protein-coupled receptor has not been reported so far and might represent a common mechanism underlying clathrin-mediated receptor endocytosis.     

Role of N-glycans and SDF-1alpha on the coassociation of CD4 with CXCR4 at the plasma membrane of monocytic cells and blood lymphocytes

CXCR4 is a coreceptor, along with CD4, for human immunodeficiency virus type 1 (HIV-1). Trimolecular complexes between HIV-1 glycoprotein (gp)120, CD4 and CXCR4 constitute a prerequisite for HIV entry. We studied whether CD4 is associated with CXCR4 on CD4+ CXCR4+ cells. Using the conformation-dependent anti-CXCR4 mAb 12G5, CD4 was coimmunoprecipitated with CXCR4 from the membrane of U937 cells which support HIV-1(LAI) efficient infection, and from that of peripheral blood lymphocytes (PBL). CD4 association with CXCR4 increased upon PBL coculture for 5 days with autologous monocytes, decreased upon treatment of the cells or the CD4-CXCR4 complex with either N-glycanase or stromal cell derived factor-1alpha (SDF-1alpha) and was abolished by incubation of the cells with both, N-glycanase and SDF-1alpha. This indicates that glycans are partly involved in CD4 association with CXCR4 and may partly explain the inhibitory effect of SDF-1alpha on HIV infection.     

Hemostasis imbalance in experimental hypertension

BACKGROUND: The rat model of chronic intoxication by N(G) -nitro-L-arginine methyl ester (L-NAME) induces severe systemic arterial hypertension and progressive ischemic lesions in the central nervous system and kidneys. We investigated the possible molecular basis of these thrombotic events. METHODS AND RESULTS: Administration of L-NAME increased plasma markers of thrombin generation, thrombin-antithrombin complexes, and soluble glycoprotein V, measured by specific ELISA. Thrombin generation in vivo was associated with ex vivo platelet desensitization to adenosine 5'-diphosphate and collagen-induced aggregation. In the aortic layers and renal arterioles, tissue factor mRNA (semi-quantitative RT-PCR) and activity (coagulation assay) were increased. In contrast, tissue factor activity was not modified in glomeruli. In parallel, an impairment of the fibrinolytic system was demonstrated by an increase in plasma levels and arterial secretion of plasminogen activator inhibitor-1. In the arterial wall, plasminogen activator inhibtor-1 mRNA was significantly increased. Moreover, antifibrinolytic activity, studied by fibrin reverse zymography, was increased whereas all tissue-plasminogen activator activity secreted by the hypertensive arterial wall was detected as complexes with its specific inhibitor. In animals treated with the angiotensin-converting enzyme (ACE) inhibitor Zofenil, all of these parameters remained at control levels. CONCLUSIONS: These results indicate that chronic blockade of nitric oxide production in rats results in enhancement of blood markers of thrombin generation associated with tissue factor induction and impairment of fibrinolysis in the vascular wall, which may contribute to the thrombotic complications associated with hypertension.     

Presence of angiotensin converting enzyme (ACE) interactive factors in ovaries of the grey fleshfly Neobellieria bullata

Angiotensin converting enzyme (ACE) activity, defined as a captopril-inhibitable dipeptidyl carboxypeptidase activity towards 3H-hippurylglycylglycine, was demonstrated in haemolymph, testes and ovaries of the grey fleshfly Neobellieria bullata, hereby suggesting a physiological role for ACE in these particular tissues. While the ACE activity in haemolymph and testes reached relatively high levels, only minute ACE activity could be detected in ovaries throughout the entire vitellogenic cycle. Ovarian extracts of Neobellieria bullata do contain, however, in addition to Neb-TMOF, the Neobellieria bullata trypsin modulating oostatic factor which is an in vitro and a putative in vivo substrate of ACE in circulation, several other heat-stable molecules which individually function either as an ACE substrate or ACE inhibitor. Presumably these ACE interactive factors mask ACE activity in the fly ovaries, as measured by a classic substrate-binding assay. Purification and characterisation of these ACE substrates/inhibitors is in progress and is likely to facilitate the elucidation of the enigmatic physiological relevance of ACE in insects.     

Disentangling ligand migration and heme pocket relaxation in cytochrome P450cam

In this work we show that ligand migration and active site conformational relaxation can occur independently of each other in hemoproteins. The complicated kinetics of carbon monoxide rebinding with cytochrome P450_cam display up to five distinct processes between 77 K and 300 K. They were disentangled by using a combination of three approaches: 1), the competition of the ligand with xenon for the occupation of internal protein cavities; 2), the modulation of the amount of distal steric hindrance within the heme pocket by varying the nature of the substrate; and 3), molecular mechanics calculations to support the proposed heme-substrate relaxation mechanism and to seek internal cavities. In cytochrome P450_cam, active site conformational relaxation results from the displacement of the substrate toward the heme center upon photodissociation of the ligand. It is responsible for the long, puzzling bimodal nature of the rebinding kinetics observed down to 77 K. The relaxation rate is strongly substrate-dependent. Ligand migration is slower and is observed only above 135 K. Migration and return rates are independent of the substrate.     

Nitrogen isotope ratios and fatty acid composition as indicators of animal diets in belowground systems

This study analyses trophic interactions between soil fungi, micro- and mesofauna in microcosm experiments. The trophic shift of ¹⁵N and fatty acids (FAs) was investigated in different food chains, which comprised either two (fungi and grazers) or three (fungi, nematodes and Collembola) levels. Contrary to the widely accepted assumption of ¹⁵N enrichment in trophic cascades the experiments revealed enrichment, depletion or no change in ¹⁵N of consumers compared to their diet. Factors responsible for this pattern were suggested to be: (1) the main metabolic pathway used for N excretion in ammonotelic nematodes to be similar or depleted in the heavier isotope, and uricotelic Collembola mostly enriched in the heavier isotope; (2) a higher shift in ¹⁵N with a high-protein diet (e.g. for predators); (3) compensation due to low-quality food altering the fractionation of ¹⁵N. Analysis of the lipid composition showed phospholipids to be generally unaffected and neutral lipids closely related to the FA pattern of the food source. Dietary routing of FAs into neutral lipids occurred, as evidenced by corresponding frequencies of FAs in host and consumer profiles. Additionally, several FAs were only detected in the grazer when present in the food source. Oleic acid showed a shift over three trophic levels, from fungi to nematodes to Collembola. The assimilation of dietary FAs resulted in a more diverse neutral lipid profile, i.e. animals higher in the food chain contained more individual FAs compared to animals lower in the food chain. The results indicate that monoenoic C18 and monoenoic C20 FAs have the potential to act as tools for the bioindication of feeding strategies in belowground systems. We suggest that primary consumers will have no or only trace amounts of monoenoic C20 acids in their neutral lipid profile, whereas consumers feeding on a eukaryote diet will show a considerably higher frequency.     

Teen at work: the burden of a double shift on daily activities

The purpose of this study was to the evaluate time spent by working and nonworking adolescents on daily activities (work, home duties, school, transportation, other activities, leisure, sleep, and naps). Twenty-seven students, 8 male workers, 8 female workers, 5 male nonworkers, and 6 female nonworkers, ages 14-18 yrs participated in the study. They attended evening classes Monday-Friday (19:00-22:30h) in a public school in the city of S?o Paulo, Brazil. The students answered a comprehensive questionnaire on the characterization of their life, work, and health conditions. Simultaneously, they wore actigraphs (Ambulatory Monitoring, Inc.) and completed a diary of their daily activities (time spent at work, on home duties, commuting, leisure, other activities) for a minimum of 10 to a maximum of 17 consecutive days. The means of the variables were tested for differences by a two-factor (work and sex) ANOVA and Student-t test applied to pair-wise samples (weekdays and weekends). The average duration during weekdays of working time was 7 h 09 min and home duties 0 h 48 min. As for commuting time, there was a work effect [F(1,23) = 4.9; p = 0.04]; mean commuting time was 2 h 22 min for workers (males and females) and 1 h 25 min for nonworkers. There was a significant difference between workers and nonworkers [F(1,23) = 4.6; p = 0.04] regarding extra-cirricular class activities; workers spent a mean of 3 min/day on them as opposed to 1 h 14 min by nonworkers. The average daily time spent on leisure activities by workers was 6h 31 min; whereas, for nonworkers it was 7h 38min. Time spent in school amounted to 2h 47min for workers in comparison to 3h 22min by nonworkers. There was a significant work effect upon sleep [F(1,23)= 10.0; p <0.01]. The work effect upon nighttime sleep duration was significant [F(1,23)= 16.7; p <0.01]. Male workers showed a mean night sleep of 6 h 57 min and female workers 07h 15min. The average nighttime sleep duration for nonworkers was 9 h 06 min. There was a significant interactive effect between work and sex [F(1,23)= 5.6; p=0.03] for naps. Female workers showed took shortest nap on average (36 min; SD = 26 min), and female nonworkers the longest naps (1 h 45min; SD= 35min). Study and employment exert significant impact on the life and activities of high school students. Work affects sleep and nap duration plus the amount of time spent in school and other extra-curricular activities.