Synthesis and SAR of novel oxazolidinones: discovery of ranbezolid

Novel oxazolidinones were synthesized containing a number of substituted five-membered heterocycles attached to the 'piperazinyl-phenyl-oxazolidinone' core of eperezolid. Further, the piperazine ring of the core was replaced by other diamino-heterocycles. These modifications led to several compounds with potent activity against a spectrum of resistant and susceptible gram-positive organisms, along with the identification of ranbezolid (RBx 7644) as a clinical candidate.     

Epstein-Barr virus latent membrane protein 1 (LMP-1) half-life in epithelial cells is down-regulated by lytic LMP-1

This study examined the effect of naturally occurring Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) gene sequence variation on the LMP-1 half-life in epithelial cells. The LMP-1 half-life was not influenced by sequence variation in amino acids 250 to 307 or amino acids 343 to 352. The LMP-1 half-life was short when the amino acid encoded at position 129 was methionine, the initiation codon product of lytic LMP-1 (lyLMP-1). The mutation of amino acid 129 to isoleucine greatly increased the LMP-1 half-life. Expression of lyLMP-1 in trans down-regulated the LMP-1 half-life in a dose-dependent manner and restored a short-half-life phenotype to the mutated LMP-1 construct lacking the cis ability to express lyLMP-1. This observed dominant negative effect of lyLMP-1 expression on the LMP-1 half-life in epithelial cells in vitro may have implications for EBV epithelial oncogenesis in vivo.     

The muscular Dystrophy Surveillance Tracking and Research Network (MD STARnet): surveillance methodology

BACKGROUND: This report focuses on the common protocol developed by the Muscular Dystrophy Surveillance Tracking and Research Network (MD STARnet) for population-based surveillance of Duchenne and Becker muscular dystrophy (DBMD) among 4 states (Arizona, Colorado, Iowa, and New York). METHODS: The network sites have developed a case definition and surveillance protocol along with software applications for medical record abstraction, clinical review, and pooled data. Neuromuscular specialists at each site review the pooled data to determine if a case meets the case criteria. Sources of potential cases of DBMD include neuromuscular specialty clinics, service sites for children with special healthcare needs, and hospital discharge databases. Each site also adheres to a common information assurance protocol. RESULTS: A population-based surveillance system for DBMD was created and implemented in participating states. CONCLUSIONS: The development and implementation of the population-based system will allow for the collection of information that is intended to provide a greater understanding of DBMD prevalence and health outcomes.     

Zebrafish transgenic Enhancer TRAP line database (ZETRAP)

Background  

The zebrafish, Danio rerio, is used as a model organism to study vertebrate genetics and development. An effective enhancer trap (ET) in zebrafish using the Tol2 transposon has been demonstrated. This approach could be used to study embryogenesis of a vertebrate species in real time and with high resolution.     

NMR metabolomics of MTLn3E breast cancer cells identifies a role for CXCR4 in lipid and choline regulation

The alpha chemokine receptor CXCR4 is up-regulated in certain types of breast cancer. Truncation of the C-terminus of this receptor alters cell morphology and increases invasiveness and metastatic potential. Here, to better understand the effects of CXCR4 expression and truncation in breast cancer cells, we have used high resolution magic angle spinning (HR-MAS) NMR studies of rat breast carcinoma MtLn3E cells to characterize the metabolite complement of cells heterologously expressing human CXCR4 or its C-terminal truncation mutant, Δ34-CXCR4. Notable reductions in choline levels were detected when either cells expressing wild-type CXCR4 or Δ34-CXCR4 were compared with cells containing an empty expression vector. Cells expressing CXCR4-Δ34 had reduced lipid content when compared with either the wild-type CXCR4 expressing cells or those containing the empty expression vector. Taken together, our results show that distinct effects on the metabolite complement can be linked to either CXCR4 expression or CXCR4 regulation. The metabolite markers for these two effects identified in the present study can, in turn, be used to further investigate the role of CXCR4 in metastasis.     

Arv1 lipid transporter function is conserved between pathogenic and nonpathogenic fungi

The lipid transporter Arv1 regulates sterol trafficking, and glycosylphosphatidylinositol and sphingolipid biosyntheses in Saccharomyces cerevisiae. ScArv1 contains an Arv1 homology domain (AHD) that is conserved at the amino acid level in the pathogenic fungal species, Candida albicans and Candida glabrata. Here we show S. cerevisiae cells lacking Arv1 are highly susceptible to antifungal drugs. In the presence of drug, Scarv1 cells are unable to induce ERG gene expression, have an altered pleiotrophic drug response, and are defective in multi-drug resistance efflux pump expression. All phenotypes are remediated by ectopic expression of CaARV1 or CgARV1. The AHDs of these pathogenic fungi are required for specific drug tolerance, demonstrating conservation of function. In order to understand how Arv1 regulates antifungal susceptibility, we examined sterol trafficking. CaARV1/CgARV1 expression suppressed the sterol trafficking defect of Scarv1 cells. Finally, we show that C. albicansarv1/arv1 cells are avirulent using a BALB/c disseminated mouse model. We suggest that overall cell survival in response to antifungal treatment requires the lipid transporter function of Arv1.     

Co-Factor Binding Confers Substrate Specificity to Xylose Reductase from Debaryomyces hansenii

Binding of substrates into the active site, often through complementarity of shapes and charges, is central to the specificity of an enzyme. In many cases, substrate binding induces conformational changes in the active site, promoting specific interactions between them. In contrast, non-substrates either fail to bind or do not induce the requisite conformational changes upon binding and thus no catalysis occurs. In principle, both lock and key and induced-fit binding can provide specific interactions between the substrate and the enzyme. In this study, we present an interesting case where cofactor binding pre-tunes the active site geometry to recognize only the cognate substrates. We illustrate this principle by studying the substrate binding and kinetic properties of Xylose Reductase from Debaryomyces hansenii (DhXR), an AKR family enzyme which catalyzes the reduction of carbonyl substrates using NADPH as co-factor. DhXR reduces D-xylose with increased specificity and shows no activity towards “non-substrate” sugars like L-rhamnose. Interestingly, apo-DhXR binds to D-xylose and L-rhamnose with similar affinity (K_d∼5.0–10.0 mM). Crystal structure of apo-DhXR-rhamnose complex shows that L-rhamnose is bound to the active site cavity. L-rhamnose does not bind to holo-DhXR complex and thus, it cannot competitively inhibit D-xylose binding and catalysis even at 4–5 fold molar excess. Comparison of K_d values with K_m values reveals that increased specificity for D-xylose is achieved at the cost of moderately reduced affinity. The present work reveals a latent regulatory role for cofactor binding which was previously unknown and suggests that cofactor induced conformational changes may increase the complimentarity between D-xylose and active site similar to specificity achieved through induced-fit mechanism.     

Connexin 26 (GJB2) mutations in the Turkish population: implications for the origin and high frequency of the 35delG mutation in Caucasians

Mutations in the Connexin 26 (GJB2/Cx26) gene are responsible for more than half of all cases of prelingual non-syndromic recessive deafness in many Caucasian populations. To determine the importance of Cx26 mutations as a cause of deafness in Turks we screened 11 families with prelingual non-syndromic deafness, seven (64%) of which were found to carry the 35delG mutation. We subsequently screened 674 Turkish subjects with no known hearing loss and found twelve 35delG heterozygotes (1.78%; 95% confidence interval: 0.9%-3%) but no examples of the 167delT mutation. To search for possible founder effects, we typed chromosomes carrying the 35delG mutation for closely linked polymorphic markers in samples from Turkey and United States and compared the allele frequencies with those of hearing subjects. The data showed a modest degree of disequilibrium in both populations. Analyses of two pedigrees from Turkey demonstrated both conserved and different haplotypes, suggesting possible founder effects and multiple origins of the 35delG mutation.