Merlin: hanging tumor suppression on the Rac.

Cancer can result from any number of abnormalities in the control of cell-cycle progression, intracellular signaling and transduction of extracellular cues. Many insights into the crucial events that govern the regulation of cell growth have derived from studies of the gene products mutated in inherited cancer syndromes. Recent work on the neurofibromatosis 2 (NF2) tumor suppressor gene suggests that this negative growth regulator might function by modulating growth factor and extracellular matrix (ECM) signals that trigger Rac1-dependent cytoskeleton-associated processes. In this article, we propose a molecular model for NF2 protein (merlin) function in the light of these and related new findings.     

Intracoronary radiation with gamma wire inhibits recurrent in-stent restenosis

To study the safety and efficacy of intracoronary gamma radiation delivered via a new high-activity (192)Ir source wire for the treatment of in-stent restenosis. In-stent restenosis results from neointimal tissue proliferation especially in its diffused form and presents a therapeutic challenge. Gamma radiation has been shown to decrease neointima formation within stents in animal models and in initial clinical trials. A total of 26 patients with in-stent restenosis underwent successful intervention and was treated with open-label (192)Ir using a high-activity line source. The specific activity of the source wire was 372+/-51 mCi, and the dwell time was 10.8+/-1.9 min. Primary endpoints were freedom from death, myocardial infraction (MI), and repeat target lesion revascularization (TLR) at 6 months. Secondary endpoints included angiographic restenosis and intravascular ultrasound (IVUS) neointimal hyperplasia. Procedural success was high (96.2%), and in-hospital and 30-day complications were low with no deaths, MI, or requirement for repeat revascularization. At 6 months, event-free survival was 85%: one patient required repeat PTCA, one underwent bypass surgery, and two had an MI. Baseline lesion length measured 15.77 mm. Follow-up angiography was available in 21/25 (84%) patients. The binary restenosis rates were 19.0% (4/21) in-stent and 23.8% (5/21) in-lesion. Follow-up IVUS was available in 20/25 patients. There was no increase in intimal hyperplasia from postintervention to follow-up (3.11.8 vs. 3.41.8 mm(2); P=.32). Eight patients had a reduction of neointimal intimal tissue at follow-up. These results indicate that intracoronary gamma radiation with the Angiorad source wire is safe and effective in preventing in-stent restenosis.     

Analysis of Transient and Catalytic Desosamine-binding Pockets in Cytochrome P-450 PikC from Streptomyces venezuelae

The cytochrome P-450 PikC from Streptomyces venezuelae exhibits significant substrate tolerance and performs multiple hydroxylation reactions on structurally variant macrolides bearing the deoxyamino sugar desosamine. In previously determined co-crystal structures (Sherman, D. H., Li, S., Yermalitskaya, L. V., Kim, Y., Smith, J. A., Waterman, M. R., and Podust, L. M. (2006) J. Biol. Chem. 281, 26289–26297), the desosamine moiety of the native substrates YC-17 and narbomycin is bound in two distinct buried and surface-exposed binding pockets, mediated by specific interactions between the protonated dimethylamino group and the acidic amino acid residues Asp⁵⁰, Glu⁸⁵, and Glu⁹⁴. Although the Glu⁸⁵ and Glu⁹⁴ negative charges are essential for maximal catalytic activity of native enzyme, elimination of the surface-exposed negative charge at Asp⁵⁰ results in significantly enhanced catalytic activity. Nevertheless, the D50N substitution could not rescue catalytic activity of PikC_E94Q based on lack of activity in the corresponding double mutant PikC_D50N/E94Q. To address the specific role for each desosamine-binding pocket, we analyzed the x-ray structures of the PikC_D50N mutant co-crystallized with narbomycin (1.85Å resolution) and YC-17 (3.2Å resolution). In PikC_D50N, the desosamine moiety of both YC-17 and narbomycin was bound in a catalytically productive “buried site.” This finding suggested a two-step substrate binding mechanism, whereby desosamine is recognized in the two subsites to allow the macrolide substrate to sequentially progress toward a catalytically favorable orientation. Collectively, the binding, mutagenesis, kinetic, and x-ray structural data suggest that enhancement of the catalytic activity of PikC_D50N is due to the facilitated relocation of substrate to the buried site, which has higher binding affinity, as opposed to dissociation in solution from the transient “surface-exposed site.”Macrolides are a large family of secondary metabolites belonging to the polyketide class of natural products generated by diverse genera of actinomycetes bacteria. The large macrolactone ring systems are derived from polymerization of simple carboxylic acid precursors catalyzed by modular polyketide synthases and often require further modification by specific tailoring enzymes (1) to acquire or enhance biological activity. The modular architecture of polyketide synthase gene clusters has led to the development of combinatorial biosynthetic approaches that aim to generate novel secondary metabolites through rational engineering of new combinations of polyketide synthase modules (2–4). Tailoring enzymes, including cytochrome P-450 monooxygenases (P-450),² are usually encoded within macrolide biosynthetic pathways (5). P-450 enzymes mainly serve to introduce hydroxyl or epoxide functional groups to nascent macrolactone structures or their glycosylated products (1, 3). To date, only three macrolide P-450 monooxygenases including EryF, EpoK, and PikC have been studied at both enzymatic and structural levels. Therefore, the principles of substrate recognition and regio- and stereochemical selectivity are just beginning to emerge for this intriguing group of biosynthetic enzymes.Streptomyces venezuelae P-450 PikC displays a relatively broad substrate and regiospecificity compared with EryF (6) and EpoK (7). This characteristic combined with robust catalytic efficiency as a single component engineered biocatalyst (8) has motivated us to further its development as a prototype P-450 monooxygenase directed toward metabolic engineering and synthetic chemical applications.³ Thus, PikC performs multiple hydroxylations of structurally variant macrolides including the 12-membered ring YC-17 and 14-membered ring narbomycin, leading to methymycin/neomethymycin and the natural ketolide antibiotic pikromycin, respectively (10) (Scheme 1). Ketolides are macrolide derivatives characterized by a C-3 keto group that have received significant attention recently because of their enhanced activity against drug-resistant microbial pathogens (11).Open in a separate windowSCHEME 1.Structures of the PikC native substrates and their hydroxylated products.Both endogenous PikC substrates are glycosylated with the 3-(dimethylamino)-3,4,6-trideoxy sugar desosamine that confers antibiotic activity to a number of macrolide antibiotics such as erythromycin, troleandomycin, mycinamicin, megalomicin (desosamine), tylosin, carbomycin, spiramycin (mycaminose, having an additional hydroxyl group at the C-4 position of the sugar ring), and a highly potent semisynthetic ketolide telithromycin (11–13). PikC catalyzes hydroxylation of variant macrolide substrates modified with altered sugar moieties through metabolic engineering (14–18) or with unnatural macrolactone ring systems (19, 20). PikC has also been shown to function effectively when immobilized on a microfluidic biochip (21), and when fused to a heterologous electron donor (8), the reductase domain of a self-sufficient P-450_RhF from Rhodococcus sp. NCIMB 9784 (22).Recent analysis of the x-ray crystal structures (23) revealed that YC-17 and narbomycin bind in the PikC active site via overlapping modes sharing the macrolactone-binding site and utilizing distinct desosamine binding regions, including buried and surface-exposed pockets, respectively. In both modes, the protonated dimethylamino group of desosamine binds between two negatively charged carboxyl groups of amino acid residues forming a salt bridge with the proximal (relative to the dimethyamino moiety) carboxyl and an ionic contact with the distal one. The triad of carboxylate residues Asp⁵⁰, Glu⁸⁵, and Glu⁹⁴ located in the BC loop provides this set of interactions. Elimination of the negative charge at Glu⁸⁵ or Glu⁹⁴ by site-directed mutagenesis virtually inactivates (Glu⁹⁴) or substantially reduces (Glu⁸⁵) conversion of both substrates (23). In contrast, elimination of the surface-exposed negative charge at Asp⁵⁰ via substitution of this residue with asparagine significantly enhances catalytic activity of PikC. To address the specific role for each desosamine-binding pocket, we analyzed the x-ray structures of the catalytically superior PikC_D50N mutant co-crystallized with narbomycin or YC-17. In PikC_D50N, YC-17 adopts the same binding mode as observed previously in the wild type, with desosamine bound in the buried pocket. In contrast to the previously observed binding mode in wild type PikC, narbomycin was also found predominantly in the buried pocket in the corresponding D50N mutant form, suggesting the possibility of initial substrate recognition in the “surface-exposed site,” with subsequent relocation to the catalytic “buried site.” We herein report PikC substrate binding, enzyme mutagenesis, and kinetic data to support this hypothesis and provide evidence for kinetic control over substrate dissociation versus relocation to the PikC catalytic pocket.     

Monthly day/night changes and seasonal daily rhythms of sexual steroids in Senegal sole (Solea senegalensis) under natural fluctuating or controlled environmental conditions

In this paper we attempted to investigate the existence of daily fluctuations on plasma sexual steroids (17beta-estradiol, E(2) and testosterone, T) in Senegal sole (Solea senegalensis) females. We described the monthly day/night concentrations and seasonal daily rhythms in animals reared under natural photo- and thermo-period. In addition, the influence of the natural annual fluctuation of the water temperature on the plasma concentration of these steroids was investigated, using one group of Senegal sole under a natural photoperiod, but with an attenuated thermal cycle (around 17-20 degrees C) for one year. Although no significant day/night differences were detected in monthly samplings, the existence of an annual rhythm of E(2) and T (p<0.01) with an acrophase in February was revealed by COSINOR analysis. Maximum values were reached in March for both steroids (6.1+/-1.7 ng mL(-1) at mid-dark, MD and 4.0+/-0.6 ng mL(-1) at mid-light, ML for E2 and 1.4+/-0.4 ng mL(-1) at MD and 0.8+/-0.1 ng mL(-1) at ML for T) in anticipation of the spawning season (May-June). As regards seasonal daily rhythms, the presence of daily oscillations was revealed. At the spring solstice (21st March) a daily rhythm was observed for both steroids (COSINOR, p<0.01), with an acrophase at 20:00 h (E(2)) and at 21:08 h (T). In summer, autumn and winter no daily rhythms were observed due to the low steroid levels at those seasons. When Senegal sole females were submitted to an attenuated annual thermal cycle, the steroid rhythm disappeared (there was no surge in spring, as in the control group) and these fish did not spawn, despite being subjected to natural photoperiod conditions. This result underlined the importance of the natural annual fluctuation of water temperature and photoperiod on the synchronization of the spawning season and on the onset of steroidogenesis.     

Cytokine expression during chronic versus occult hepatitis B virus infection in HIV co-infected individuals

Chronic hepatitis B virus infection is characterized by persistent detectable levels of hepatitis B surface antigen (HBsAg) and HBV DNA in the serum. In contrast, HBsAg is not detectable during occult HBV infection, despite the presence of HBV DNA. An altered host immune response could play a role in the development of occult HBV infection; however, potential differences in immune responses among chronic and occult HBV-infected patients have not been evaluated in vivo. In the current study, we evaluated serum levels of regulatory, apoptotic, and fibrotic/anti-fibrotic cytokines/markers as indicators of immune responses in 25 chronic and 12 occult HBV-infected patients. More than half of the patients in both chronic and occult HBV infection groups had IL-2, IL-4, IL-13, and IFN-γ levels below detectable limits. In contrast, most patients had detectable levels of IL-8, IL-10, IP-10, sFas, sFasL, and TGF-β1. Of these, only sFas was significantly different between the two groups, with lower levels observed during occult compared to chronic HBV infection (p = 0.01). As a surrogate marker of apoptotic inhibition, decreased sFas during occult HBV infection suggests that apoptosis occurs at different rates in occult compared to chronic HBV infection and therefore, may contribute to persistence of occult HBV infection.     

The LAT Story: A Tale of Cooperativity, Coordination, and Choreography

The adapter molecule LAT is a nucleating site for multiprotein signaling complexes that are vital for the function and differentiation of T cells. Extensive investigation of LAT in multiple experimental systems has led to an integrated understanding of the formation, composition, regulation, dynamic movement, and function of LAT-nucleated signaling complexes. This review discusses interactions of signaling molecules that bind directly or indirectly to LAT and the role of cooperativity in stabilizing LAT-nucleated signaling complexes. In addition, it focuses on how imaging studies visualize signaling assemblies as signaling clusters and demonstrate their dynamic nature and cellular fate. Finally, this review explores the function of LAT based on the interpretation of mouse models using various LAT mutants.