Hydroxylated analogues of the orally active broad spectrum antifungal, Sch 51048 (1), and the discovery of posaconazole [Sch 56592; 2 or (S,S)-5

As part of a detailed study, the syntheses, biological activities, and pharmacokinetic properties of hydroxylated analogues of the previously described broad spectrum antifungal agents, Sch 51048 (1), Sch 50001 (3), and Sch 50002 (4), are described. Based on an overall superior profile, one of the alcohols, Sch 56592 (2), was selected for clinical studies.     

A Soybean C2H2-Type Zinc Finger Gene GmZF1 Enhanced Cold Tolerance in Transgenic Arabidopsis

Zinc finger proteins were involved in response to different environmental stresses in plant species. A typical Cys2/His2-type (C2H2-type) zinc finger gene GmZF1 from soybean was isolated and was composed of 172 amino acids containing two conserved C2H2-type zinc finger domains. Phylogenetic analysis showed that GmZF1 was clustered on the same branch with six C2H2-type ZFPs from dicotyledonous plants excepting for GsZFP1, and distinguished those from monocotyledon species. The GmZF1 protein was localized at the nucleus, and has specific binding activity with EP1S core sequence, and nucleotide mutation in the core sequence of EPSPS promoter changed the binding ability between GmZF1 protein and core DNA element, implying that two amino acid residues, G and C boxed in core sequence TGACAGTGTCA possibly play positive regulation role in recognizing DNA-binding sites in GmZF1 proteins. High accumulation of GmZF1 mRNA induced by exogenous ABA suggested that GmZF1 was involved in an ABA-dependent signal transduction pathway. Over-expression of GmZF1 significantly improved the contents of proline and soluble sugar and decreased the MDA contents in the transgenic lines exposed to cold stress, indicating that transgenic Arabidopsis carrying GmZF1 gene have adaptive mechanisms to cold stress. Over-expression of GmZF1 also increased the expression of cold-regulated cor6.6 gene by probably recognizing protein-DNA binding sites, suggesting that GmZF1 from soybean could enhance the tolerance of Arabidopsis to cold stress by regulating expression of cold-regulation gene in the transgenic Arabidopsis.     

The SARS Coronavirus E Protein Interacts with PALS1 and Alters Tight Junction Formation and Epithelial Morphogenesis

Intercellular tight junctions define epithelial apicobasal polarity and form a physical fence which protects underlying tissues from pathogen invasions. PALS1, a tight junction-associated protein, is a member of the CRUMBS3-PALS1-PATJ polarity complex, which is crucial for the establishment and maintenance of epithelial polarity in mammals. Here we report that the carboxy-terminal domain of the SARS-CoV E small envelope protein (E) binds to human PALS1. Using coimmunoprecipitation and pull-down assays, we show that E interacts with PALS1 in mammalian cells and further demonstrate that the last four carboxy-terminal amino acids of E form a novel PDZ-binding motif that binds to PALS1 PDZ domain. PALS1 redistributes to the ERGIC/Golgi region, where E accumulates, in SARS-CoV–infected Vero E6 cells. Ectopic expression of E in MDCKII epithelial cells significantly alters cyst morphogenesis and, furthermore, delays formation of tight junctions, affects polarity, and modifies the subcellular distribution of PALS1, in a PDZ-binding motif-dependent manner. We speculate that hijacking of PALS1 by SARS-CoV E plays a determinant role in the disruption of the lung epithelium in SARS patients.     

Regulatory BC1 RNA and the fragile X mental retardation protein: convergent functionality in brain

Background

BC RNAs and the fragile X mental retardation protein (FMRP) are translational repressors that have been implicated in the control of local protein synthesis at the synapse. Work with BC1 and Fmr1 animal models has revealed that phenotypical consequences resulting from the absence of either BC1 RNA or FMRP are remarkably similar. To establish functional interactions between BC1 RNA and FMRP is important for our understanding of how local protein synthesis regulates neuronal excitability.

Methodology/Principal Findings

We generated BC1−/− Fmr1−/− double knockout (dKO) mice. We examined such animals, lacking both BC1 RNA and FMRP, in comparison with single knockout (sKO) animals lacking either one repressor. Analysis of neural phenotypical output revealed that at least three attributes of brain functionality are subject to control by both BC1 RNA and FMRP: neuronal network excitability, epileptogenesis, and place learning. The severity of CA3 pyramidal cell hyperexcitability was significantly higher in BC1−/− Fmr1−/− dKO preparations than in the respective sKO preparations, as was seizure susceptibility of BC1−/− Fmr1−/− dKO animals in response to auditory stimulation. In place learning, BC1−/− Fmr1−/− dKO animals were severely impaired, in contrast to BC1−/− or Fmr1−/− sKO animals which exhibited only mild deficits.

Conclusions/Significance

Our data indicate that BC1 RNA and FMRP operate in sequential-independent fashion. They suggest that the molecular interplay between two translational repressors directly impacts brain functionality.     

Genome Sequence of Hybrid Vibrio cholerae O1 MJ-1236, B-33, and CIRS101 and Comparative Genomics with V. cholerae

The genomes of Vibrio cholerae O1 Matlab variant MJ-1236, Mozambique O1 El Tor variant B33, and altered O1 El Tor CIRS101 were sequenced. All three strains were found to belong to the phylocore group 1 clade of V. cholerae, which includes the 7th-pandemic O1 El Tor and serogroup O139 isolates, despite displaying certain characteristics of the classical biotype. All three strains were found to harbor a hybrid variant of CTXΦ and an integrative conjugative element (ICE), leading to their establishment as successful clinical clones and the displacement of prototypical O1 El Tor. The absence of strain- and group-specific genomic islands, some of which appear to be prophages and phage-like elements, seems to be the most likely factor in the recent establishment of dominance of V. cholerae CIRS101 over the other two hybrid strains.Vibrio cholerae, a bacterium autochthonous to the aquatic environment, is the causative agent of cholera, a life-threatening disease that causes severe, watery diarrhea. Cholera bacteria are serogrouped based on their somatic O antigens, with more than 200 serogroups identified to date (6). Only toxigenic strains of serogroups O1 and O139 have been identified as agents of cholera epidemics and pandemics; serogroups other than O1 and O139 have the potential to cause mild gastroenteritis or, rarely, local outbreaks. Genes coding for cholera toxin (CTX), ctxAB, and other virulence factors have been shown to reside in bacteriophages and various mobile genetic elements. In addition, V. cholerae serogroup O1 is differentiated into two biotypes, classical and El Tor, by a combination of biochemical traits, by sensitivity to biotype-specific bacteriophages, and more recently by nucleotide sequencing of specific genes and by molecular typing (5, 17, 19).There have been seven pandemics of cholera recorded throughout human history. The seventh and current pandemic began in 1961 in the Indonesian island of Sulawesi and subsequently spread to Asia, Africa, and Latin America; the six previous pandemics are believed to have originated in the Indian subcontinent. Isolates of the sixth pandemic were almost exclusively of the O1 classical biotype, whereas the current (seventh) pandemic is dominated by the V. cholerae O1 El Tor biotype as the causative agent, a transition occurring between 1923 and 1961. Today, the disease continues to remain a scourge in developing countries, confounded by the fact that V. cholerae is native to estuaries and river systems throughout the world (8).Over the past 20 years, several new epidemic lineages of V. cholerae O1 El Tor have emerged (or reemerged). For example, in 1992, a new serogroup, namely, O139 of V. cholerae, was identified as the cause of epidemic cholera in India and Bangladesh (25). The initial concern was that a new pandemic was beginning; however, the geographic range of V. cholerae O139 is currently restricted to Asia. Additionally, V. cholerae O1 hybrids and altered El Tor variants have been isolated repeatedly in Bangladesh (Matlab) (23, 24) and Mozambique (1). Altered V. cholerae O1 El Tor isolates produce cholera toxin of the classical biotype but can be biotyped as El Tor by conventional phenotypic assays, whereas V. cholerae O1 hybrid variants cannot be biotyped based on phenotypic tests and can produce cholera toxin of either biotype. These new variants have subsequently replaced the prototype seventh-pandemic V. cholerae O1 El Tor strains in Asia and Africa, with respect to frequency of isolation from clinical cases of cholera (27).Here, we report the genome sequence of three V. cholerae O1 variants, MJ-1236, a Matlab type I hybrid variant from Bangladesh that cannot be biotyped by conventional methods, CIRS101, an altered O1 El Tor isolate from Bangladesh which harbors ctxB of classical origin, and B33, an altered O1 El Tor isolate from Mozambique which harbors classical CTXΦ, and we compare their genomes with prototype El Tor and classical genomes. From an epidemiological viewpoint, among the three variants characterized in this study, V. cholerae CIRS101 is currently the most “successful” in that strains belonging to this type have virtually replaced the prototype El Tor in Asia and many parts of Africa, notably East Africa. This study, therefore, gives us a unique opportunity to understand why V. cholerae CIRS101 is currently the most successful El Tor variant.     

Characterization of fibroblasts recruited from bone marrow-derived precursor in neonatal bronchopulmonary dysplasia mice

We sought to determine whether the extrapulmonary origin of fibroblasts derived from bone marrow (BM) progenitor cells is essential to lung fibrosis in bronchopulmonary dysplasia (BPD). Neonate mice were durably engrafted with BM isolated from transgenic reporter mice that expressed green fluorescent protein (GFP). Such chimera mice were subjected to 60% O(2) exposure for 14 days. A large number of fibroblast-specific protein-1 (FSP1) and GFP-positive fibroblasts were identified in active fibrotic lesions. More surprisingly, however, FSP1(+) fibroblasts also arose in considerable numbers from BM-derived alveolar type II cells (AT2) through epithelial-mesenchymal transition (EMT) during lung fibrogenesis. Cultured lung fibroblasts could express the CXC chemokine receptor (CXCR4) and responded chemotactically to their cognate ligand, chemokine (C-X-C motif) ligand 12 (CXCL12), which were elevated in the serum of BPD mice. These data suggest that lung fibroblasts in BPD fibrosis could variably arise from BM progenitor cells. This finding, which suggests the pathophysiological process of fibrosis, could contribute to a therapy for BPD that is characterized by extensive interstitial fibrosis.     

A highly selective and colorimetric assay of lysine by molecular-driven gold nanorods assembly

In this contribution, a simple, rapid, colorimeteric and selective assay for lysine was achieved by a controllable end-to-end assembly of gold nanorods (AuNRs) in the presence of Eu(3+) and lysine. This one-pot end-to-end assembly of 11-mercaptoundecanoic acid (MUA) modified AuNRs was occurred in Britton-Robinson buffer of pH 6.0, which involves the coordination binding between Eu(3+) and COO(-) groups as well as the electrostatic interaction of the COO(-) groups of MUA with the -NH(3)(+) group of lysine. As monitored by absorption spectra, scanning electron microscopic (SEM) images and dynamic light scattering (DLS) measurement, the end-to-end chain assembly results in large red-shift in the longitudinal plasmon resonance absorption (LPRA), giving red-to-blue color change of AuNRs. Importantly, it was found that the red-shift of LPRA is linearly proportional to the concentrations of lysine in the range of 5.0×10(-6)-1.0×10(-3)M with the limit of detection (LOD) being 1.6×10(-6)M (3σ/k). This red-shift of LPRA is highly selective, making it possible to develop a rapid, selective and visual assay for lysine in food samples.     

Effects of caffeine on phosphatidylinositide turnover and calcium mobilization in human neuroblastoma SK-N-SH cells

The effects of caffeine on receptor-controlled Ca2+ mobilization and turnover of inositol phosphates in human neuroblastoma SK-N-SH cells were studied. Caffeine inhibited both the rise in cytosolic Ca2+ concentration ([Ca2+]i) evoked by muscarinic receptor agonists and the total production of inositol phosphates in a dose-dependent manner, but to different extents. At 10 mM, caffeine inhibited agonist-evoked generation of inositol phosphates almost completely, whereas the agonist-evoked [Ca2+]i rise remained observable after caffeine treatment, in the absence or presence of extracellular Ca2+. Raising the cytosolic cAMP concentration increased the carbachol-induced [Ca2+]i rise, and this effect was abolished in the presence of caffeine. Our data suggested that caffeine may exert two effects on receptor-controlled Ca2+ mobilization: 1) inhibition of inositol phosphate production, 2) augmentation of the size of the releasable Ca2+ pool by elevating cytosolic cAMP concentration.