Molecular Gene Profiling of Clostridium botulinum Group III and Its Detection in Naturally Contaminated Samples Originating from Various European Countries

We report the development of real-time PCR assays for genotyping Clostridium botulinum group III targeting the newly defined C. novyi sensu lato group; the nontoxic nonhemagglutinin (NTNH)-encoding gene ntnh; the botulinum neurotoxin (BoNT)-encoding genes bont/C, bont/C/D, bont/D, and bont/D/C; and the flagellin (fliC) gene. The genetic diversity of fliC among C. botulinum group III strains resulted in the definition of five major subgroups named fliC-I to fliC-V. Investigation of fliC subtypes in 560 samples, with various European origins, showed that fliC-I was predominant and found exclusively in samples contaminated by C. botulinum type C/D, fliC-II was rarely detected, no sample was recorded as fliC-III or fliC-V, and only C. botulinum type D/C samples tested positive for fliC-IV. The lack of genetic diversity of the flagellin gene of C. botulinum type C/D would support a clonal spread of type C/D strains in different geographical areas. fliC-I to fliC-III are genetically related (87% to 92% sequence identity), whereas fliC-IV from C. botulinum type D/C is more genetically distant from the other fliC types (with only 50% sequence identity). These findings suggest fliC-I to fliC-III have evolved in a common environment and support a different genetic evolution for fliC-IV. A combination of the C. novyi sensu lato, ntnh, bont, and fliC PCR assays developed in this study allowed better characterization of C. botulinum group III and showed the group to be less genetically diverse than C. botulinum groups I and II, supporting a slow genetic evolution of the strains belonging to C. botulinum group III.     

Composition and activity of beta-Proteobacteria ammonia-oxidizing communities associated with intertidal rocky biofilms and sediments of the Douro River estuary, Portugal

AIMS: To characterize the phylogenetic composition of ammonia-oxidizing bacteria (AOB) of the beta-subclass of the class Proteobacteria in intertidal sediment and rocky biofilms of the Douro estuary, and evaluate relationships with environmental variables and N-biogeochemistry. METHODS AND RESULTS: Cluster analysis of denaturing gradient gel electrophoresis profiles showed differences in beta-Proteobacteria AOB assemblage composition between rocky biofilms and sediments. All sequences obtained from intertidal rocky biofilm sites exhibited phylogenetic affinity to Nitrosomonas sp. lineages, whereas a majority of the sequences from the sediment sites were most similar to marine Nitrosospira cluster 1. Hierarchical cluster analysis based on environmental variables identified two main groups of samples. The first contained samples from rocky biofilm sites characterized by high concentrations of NO2- and NH4+, and high organic matter and chlorophyll a content. The second group contained all of the sediment samples; these sites were characterized by lower values for the variables above. In addition, rocky biofilm sites exhibited higher nitrification rates. CONCLUSIONS: Intersite differences in environmental and/or physical conditions led to the selection of different populations of beta-Proteobacteria AOB, supporting different magnitudes of N-cycling regimes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents an important step in establishing the influence of environmental factors on the distribution of beta-Proteobacteria AOB with possible consequences for N-biogeochemistry.     

HIV-1 VprB and C RNA cleavage by potent 10-23 DNAzymes that also cause reversal of G2 cell cycle arrest mediated by Vpr genes

Accessory Vpr protein of HIV-1 is known to influence several key cellular functions that also impacts on the HIV-1 replication cycle. Besides other activities, it alone causes cell cycle arrest at the G2 phase and thus potentially contribute to the overall pathology. We designed several 10-23 catalytic motifs containing DNAzymes (Dzs) against the full-length Vpr gene from subtype B and checked its activity against VprC gene from one of the Indian HIV-1 isolates. Among several Dzs that showed sequence-specific cleavage activities, Dz-94 was very potent and equally efficient in its ability to cleave full-length VprB and C RNA to completion under standard conditions of cleavage. Although Dz-90 target sequence was fully conserved between VprB and C genes, it was more effective on latter genes, suggesting that spatial structures of RNA at other regions of Vpr can also influence the cleavage activity for this Dz. HIV-1 VprB and C encoding genes under the powerful CMV promoter, when cotransfected into mammalian cells with Dz-94, a potent intracellular inhibition, was observed, which also resulted in reversing the G2 cell cycle arrest mediated by VprB and C proteins. Thus, Dz-94 could potentially be developed to prevent Vpr-mediated cytopathic effects caused by HIV-1 subtype B and C isolates.     

Expression of endothelin 1 and its receptors in the hypoxic pregnant rat

Endothelin 1 (EDN1) plays a primary role in the pathophysiology of hypoxia-induced fetal growth restriction in the rat. In this study we evaluated the effects of chronic maternal hypoxia on the expression of endothelin and its receptors and on receptor binding activity in the uterus and placenta of the rat, in order to elucidate their roles in hypoxia-induced fetal growth restriction. Timed-pregnant Sprague-Dawley rats were maintained in either a normoxic or a normobaric hypoxic (12% O(2)) atmosphere from Gestational Days 18-21. Uterine and placental tissues collected on Gestational Day 21 were assayed for Edn1, Ednra, and Ednrb (endothelin receptors) mRNA expression by real-time quantitative RT-PCR, for localization of EDN1 and its receptors by immunohistochemistry, for EDNRA and EDNRB protein expression by Western blot, and for receptor binding activity by homologous competitive binding assays. EDN1 mRNA expression was significantly increased in the hypoxic placenta, but not in the uterus, compared with normoxic controls. Immunohistochemistry revealed increased EDN1 specifically in the labyrinth of the placenta. Receptor mRNA levels were not significantly affected by hypoxia, but EDNRA protein expression was significantly decreased specifically in the uterine placental beds. Receptor binding decreased significantly in response to hypoxia in all tissues investigated, compared with controls. These results suggest that chronic maternal hypoxia results in increased expression of EDN1 in the placenta but not in the uterus, and that reduced binding activity, rather than regulation of receptor expression, is a mechanism by which these tissues regulate the local hemodynamic response to increased endogenous placental EDN1 in the setting of hypoxia.     

Impaired mitochondrial respiration promotes dendritic branching via the AMPK signaling pathway

Functional neuronal circuits require a constant remodeling of their network composed of highly interconnected neurons. The plasticity of synapses and the shaping of elaborated dendritic branches are energy demanding and therefore depend on an efficient mitochondrial oxidative phosphorylation (OXPHOS). The spatial and functional regulations of dendritic patterning occur also after cell fate specification; however, the molecular mechanisms underlying this complex process remain elusive. Here, we exploit the changes in dendritic architecture in highly branched neurons as a result of aberrant mitochondrial activity. In sensory neurons of Caenorhabditis elegans, genetic manipulations of mitochondrial complex I subunits cause an unexpected outgrowth of dendritic arbors and ectopic structures. The increased number of dendritic branches is coordinated through a specific signaling cascade rather than as a simple consequence of oxidative stress. On the basis of genetic and pharmacological evidence, we show that OXPHOS deficiency promotes branching through the activation of the AMP-activated protein kinase AMPK and the downstream target phosphoinositide 3-kinase PI3K. Taken together, our findings describe a well-defined signaling pathway that regulates dendritic outgrowth in conditions of compromised OXPHOS and the resulting AMPK activation.     

Mitigating Strategies of Gibberellins in Various Environmental Cues and Their Crosstalk with Other Hormonal Pathways in Plants: a Review

Plant Molecular Biology Reporter - Phytohormones are chemical substances that in minute concentration instruct a plethora of developmental and physiological responses in plants. These signal...     

Physicochemical properties of thiol proteinase inhibitor isolated from goat pancreas

Thiol proteinase inhibitors are crucial to proper functioning of all living tissues consequent to their cathepsin regulatory and myriad important biologic properties. Equilibrium denaturation of dimeric goat pancreas thiol proteinase inhibitor (PTPI), a cystatin superfamily variant has been studied by monitoring changes in the protein's spectroscopic and functional characteristics. Denaturation of PTPI in guanidine hydrochloride and urea resulted in altered intrinsic fluorescence emission spectrum, diminished negative circular dichroism, and loss of its papain inhibitory potential. Native like spectroscopic properties and inhibitory activity are only partially restored when denaturant is diluted from guanidine hydrochloride unfolded samples demonstrating that process is partially reversible. Coincidence of transition curves and dependence of transition midpoint (3.2M) on protein concentration in guanidine hydrochloride‐induced denaturation are consistent with a two‐state model involving a native like dimer and denatured monomer. On the contrary, urea‐induced unfolding of PTPI is a multiphasic process with indiscernible intermediates. The studies demonstrate that functional conformation and stability are governed by both ionic and hydrophobic interactions. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 708–717, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com