HIV envelope-CXCR4 signaling activates cofilin to overcome cortical actin restriction in resting CD4 T cells

Binding of the HIV envelope to the chemokine coreceptors triggers membrane fusion and signal transduction. The fusion process has been well characterized, yet the role of coreceptor signaling remains elusive. Here, we describe a critical function of the chemokine coreceptor signaling in facilitating HIV infection of resting CD4 T cells. We find that static cortical actin in resting T cells represents a restriction and that HIV utilizes the Galphai-dependent signaling from the chemokine coreceptor CXCR4 to activate a cellular actin-depolymerizing factor, cofilin, to overcome this restriction. HIV envelope-mediated cofilin activation and actin dynamics are important for a postentry process that leads to viral nuclear localization. Inhibition of HIV-mediated actin rearrangement markedly diminishes viral latent infection of resting T cells. Conversely, induction of active cofilin greatly facilitates it. These findings shed light on viral exploitation of cellular machinery in resting T cells, where chemokine receptor signaling becomes obligatory.     

MutL homologs in restriction-modification systems and the origin of eukaryotic MORC ATPases

   

The provenance and biochemical roles of eukaryotic MORC proteins have remained poorly understood since the discovery of their prototype MORC1, which is required for meiotic nuclear division in animals. The MORC family contains a combination of a gyrase, histidine kinase, and MutL (GHKL) and S5 domains that together constitute a catalytically active ATPase module. We identify the prokaryotic MORCs and establish that the MORC family belongs to a larger radiation of several families of GHKL proteins (paraMORCs) in prokaryotes. Using contextual information from conserved gene neighborhoods we show that these proteins primarily function in restriction-modification systems, in conjunction with diverse superfamily II DNA helicases and endonucleases. The common ancestor of these GHKL proteins, MutL and topoisomerase ATPase modules appears to have catalyzed structural reorganization of protein complexes and concomitant DNA-superstructure manipulations along with fused or standalone nuclease domains. Furthermore, contextual associations of the prokaryotic MORCs and their relatives suggest that their eukaryotic counterparts are likely to carry out chromatin remodeling by DNA superstructure manipulation in response to epigenetic signals such as histone and DNA methylation.     

Active site characteristics of CYP4B1 probed with aromatic ligands.

The active site topography of rabbit CYP4B1 has been studied relative to CYP2B1 and CYP102 using a variety of aromatic probe substrates. Oxidation of the prochiral substrate cumene by CYP4B1, but not CYP2B1 or CYP102, resulted in the formation of the thermodynamically disfavored omega-hydroxy metabolite, 2-phenyl-1-propanol, with product stereoselectivity for the (S)-enantiomer. Reaction of CYP4B1, CYP2B1, and CYP102 with phenyldiazene produced spectroscopically observable sigma-complexes for each enzyme. Subsequent oxidation of the CYP2B1 and CYP102 complexes followed by LC/ESI--MS analysis yielded heme pyrrole migration patterns similar to those in previous literature reports. Upon identical treatment, no migration products were detected for CYP4B1. Intramolecular deuterium isotope effects for the benzylic hydroxylation of o-xylene-alpha-(2)H(3), p-xylene-alpha-(2)H(3), 2-(2)H(3),6-dimethylnaphthalene, and 4-(2)H(3),4'-dimethylbiphenyl were determined for CYP4B1 and CYP2B1 to further map their active site dimensions. These probes permit assessment of the ease of equilibration, within P450 active sites, of oxidizable methyl groups located between 3 and 10 A apart [Iyer et al. (1997) Biochemistry 36, 7136--7143]. Isotope effects for the CYP4B1-mediated benzylic hydroxylation of o- and p-xylenes were fully expressed (k(H)/k(D) = 9.7 and 6.8, respectively), whereas deuterium isotope effects for the naphthyl and biphenyl derivatives were both substantially masked (k(H)/k(D) approximately equal to 1). In contrast, significant suppression of the deuterium isotope effects for CYP2B1 occurred only with the biphenyl substrate. Therefore, rapid equilibration between two methyl groups more than 6 A apart is impeded within the active site of CYP4B1, whereas for CYP2B1, equilibration is facile for methyl groups distanced by more than 8 A. Collectively, all data are consistent with the conclusion that the active site of CYP4B1 is considerably restricted relative to CYP2B1.     

Characterization of Biofilm Formation by Borrelia burgdorferi In Vitro

Borrelia burgdorferi, the causative agent of Lyme disease, has long been known to be capable of forming aggregates and colonies. It was recently demonstrated that Borrelia burgdorferi aggregate formation dramatically changes the in vitro response to hostile environments by this pathogen. In this study, we investigated the hypothesis that these aggregates are indeed biofilms, structures whose resistance to unfavorable conditions are well documented. We studied Borrelia burgdorferi for several known hallmark features of biofilm, including structural rearrangements in the aggregates, variations in development on various substrate matrices and secretion of a protective extracellular polymeric substance (EPS) matrix using several modes of microscopic, cell and molecular biology techniques. The atomic force microscopic results provided evidence that multilevel rearrangements take place at different stages of aggregate development, producing a complex, continuously rearranging structure. Our results also demonstrated that Borrelia burgdorferi is capable of developing aggregates on different abiotic and biotic substrates, and is also capable of forming floating aggregates. Analyzing the extracellular substance of the aggregates for potential exopolysaccharides revealed the existence of both sulfated and non-sulfated/carboxylated substrates, predominately composed of an alginate with calcium and extracellular DNA present. In summary, we have found substantial evidence that Borrelia burgdorferi is capable of forming biofilm in vitro. Biofilm formation by Borrelia species might play an important role in their survival in diverse environmental conditions by providing refuge to individual cells.     

An improved intrafemoral injection with minimized leakage as an orthotopic mouse model of osteosarcoma

Osteosarcoma, the most common type of primary bone cancer, is the second highest cause of cancer-related death in pediatric patients. To understand the mechanisms behind osteosarcoma progression and to discover novel therapeutic strategies for this disease, a reliable and appropriate mouse model is essential. For this purpose, osteosarcoma cells need to be injected into the bone marrow. Previously, the intratibial and intrafemoral injection methods were reported; however, the major drawback of these methods is the potential leakage of tumor cells from the injection site during or after these procedures. To overcome this, we have established an improved method to minimize leakage in an orthotopic mouse model of osteosarcoma. By taking advantage of the anatomical benefits of the femur with less bowing and larger medullary cavity than those of the tibia, osteosarcoma cells are injected directly into the femoral cavity following reaming of its intramedullary space. To prevent potential leakage of tumor cells during and after the surgery, the injection site is sealed with bone wax. This method requires a minor surgery of approximately 15 min under anesthesia. Our established orthotopic osteosarcoma model could serve as a valuable and reliable tool for examining progression of various types of bone tumors.     

Reconstructing the ubiquitin network - cross-talk with other systems and identification of novel functions

Background  

The ubiquitin system (Ub-system) can be defined as the ensemble of components including Ub/ubiquitin-like proteins, their conjugation and deconjugation apparatus, binding partners and the proteasomal system. While several studies have concentrated on structure-function relationships and evolution of individual components of the Ub-system, a study of the system as a whole is largely lacking.     

Novel,achiral aminoheterocycles as selective monoamine reuptake inhibitors

A variety of novel aminoheterocycle scaffolds as selective monoamine reuptake inhibitors have been prepared and one of these scaffolds is achiral. The main elements responsible for hERG channel, CYP2D6 and CYP3A4 inhibition were identified.