NMR structures of loop B RNAs from the stem-loop IV domain of the enterovirus internal ribosome entry site: a single C to U substitution drastically changes the shape and flexibility of RNA

The 5'-untranslated region of positive-strand RNA viruses harbors many cis-acting RNA structural elements that are important for various viral processes such as replication, translation, and packaging of new virions. Among these is loop B RNA of the stem-loop IV domain within the internal ribosomal entry site (IRES) of enteroviruses, including Poliovirus type 1 (PV1). Studies on PV1 have shown that specific recognition of loop B by the first KH (hnRNP K homology) domain of cellular poly(rC)-binding protein 2 (PCBP2) is essential for efficient translation of the viral mRNA. Here we report the NMR solution structures of two representative sequence variants of enteroviral loop B RNA. The two RNA variants differ at only one position (C vs U) within a six-nucleotide asymmetric internal loop sequence that is the binding site for the PCBP2 KH1 domain. Surprisingly, the two RNAs are drastically different in the overall shape and local dynamics of the bulge region. The RNA with the 5'-AUCCCU bulge sequence adopts an overall L shape. Its bulge nucleotides, especially the last four, are highly flexible and not very well defined by NMR. The RNA with the 5'-AUUCCU bulge sequence adopts an overall U shape, and its bulge sequence exhibits only limited flexibility. A detailed analysis of the two RNA structures and their dynamic properties, as well as available sequence data and known KH domain-RNA complex structures, not only provides insights into how loop B RNA might be recognized by the PCBP2 KH1 domain but also suggests a possible correlation between structural flexibility and pre-existing structural features for protein recognition.     

Genetics of metabolic variations between Yersinia pestis biovars and the proposal of a new biovar, microtus

Yersinia pestis has been historically divided into three biovars: antiqua, mediaevalis, and orientalis. On the basis of this study, strains from Microtus-related plague foci are proposed to constitute a new biovar, microtus. Based on the ability to ferment glycerol and arabinose and to reduce nitrate, Y. pestis strains can be assigned to one of four biovars: antiqua (glycerol positive, arabinose positive, and nitrate positive), mediaevalis (glycerol positive, arabinose positive, and nitrate negative), orientalis (glycerol negative, arabinose positive, and nitrate positive), and microtus (glycerol positive, arabinose negative, and nitrate negative). A 93-bp in-frame deletion in glpD gene results in the glycerol-negative characteristic of biovar orientalis strains. Two kinds of point mutations in the napA gene may cause the nitrate reduction-negative characteristic in biovars mediaevalis and microtus, respectively. A 122-bp frameshift deletion in the araC gene may lead to the arabinose-negative phenotype of biovar microtus strains. Biovar microtus strains have a unique genomic profile of gene loss and pseudogene distribution, which most likely accounts for the human attenuation of this new biovar. Focused, hypothesis-based investigations on these specific genes will help delineate the determinants that enable this deadly pathogen to be virulent to humans and give insight into the evolution of Y. pestis and plague pathogenesis. Moreover, there may be the implications for development of biovar microtus strains as a potential vaccine.     

A glucose biosensor based on chitosan-glucose oxidase-gold nanoparticles biocomposite formed by one-step electrodeposition

An amperometric biosensor for the quantitative measurement of glucose is reported. The biosensor is based on a biocomposite that is homogeneous and easily prepared. This biocomposite is made of chitosan hydrogel, glucose oxidase, and gold nanoparticles by a direct and facile electrochemical deposition method under enzyme-friendly conditions. The resulting biocomposite provided a shelter for the enzyme to retain its bioactivity at considerably extreme conditions, and the decorated gold nanoparticles in the biocomposite offer excellent affinity to enzyme. The biosensor exhibited a rapid response (within 7s) and a linear calibration range from 5.0 microM to 2.4 mM with a detection limit of 2.7 microM for the detection of glucose. The combination of gold nanoparticles affinity and the promising feature of the biocomposite with the onestep nonmanual technique favor the sensitive determination of glucose with improved analytical capabilities.     

Increased seizure susceptibility and cortical malformation in beta-catenin mutant mice

Beta-catenin has been implicated in epilepsy because of its altered post seizure expression and the role of Wnt2 signaling in autism. To determine beta-catenin's role in seizure susceptibility, we injected penetylenetetrazol intraperitoneally in beta-catenin cerebral cortex- and hippocampus-specific knockout mice. We then analyzed the latency, number, and duration of four phases of seizure behaviors: (I) non-seizure activity, (II) myoclonic jerks, (III) generalized clonic seizures, and (IV) tonic seizures. The latencies to both death and Phase IV were significantly reduced in mutant mice. Mutant mice also spent significantly more time in Phases III and IV and showed significantly less time in the non-convulsive state (Phase I). Nissl and gold chloride staining indicated that the knockout mice had underdeveloped cortices, lacked a corpus callosum, and were missing hippocampal structures. This suggests that dysfunction of beta-catenin-mediated signaling pathways in mice leads to cortical malformation and increased seizure susceptibility.     

Endogenous hydrogen sulfide regulation of myocardial injury induced by isoproterenol

Previous work has shown that the endogenous cystathionine γ-synthase (CSE)/hydrogen sulfide (H₂S) pathway participates in the regulation of cardiac contraction. We hypothesized that the pathway might participate in the pathophysiological regulation of ischemic heart disease. Isoproterenol injection of rat hearts induced a myocardial ischemic injury model, with reduced myocardial and plasma H₂S levels, decreased CSE activity, and upregulated CSE gene expression. Exogenous administration of the H₂S donor NaHS reduced the mortality rate; increased left-ventricular pressure development and left-ventricular-end systolic pressure; and decreased left-ventricular-end diastolic pressure (LVEDP) and subendocardial necrosis, capillary dilatation, leukocytic infiltration, fibroblast swelling, and fibroblastic hyperplasia. As well, production of lipid peroxidation, including myocardial malondialdehyde (MDA), and plasma MDA and conjugated diene, was reduced. Oxidative stress injury is an important mechanism of isoproterenol-induced myocardial injury. In vitro experiments revealed that NaHS might antagonize myocyte MDA production by oxygen-free radicals and that NaHS directly scavenged hydrogen peroxide and superoxide anions. Our results suggest that the endogenous CSE/H₂S pathway contributes to the pathogenesis of isoproterenol-induced myocardial injury. Administration of exogenous H₂S effectively protects myocytes and contractile activity, at least by its direct scavenging of oxygen-free radicals and reducing the accumulation of lipid peroxidations.     

New Aspects on Lanosterol 14α-Demethylase and Cytochrome P450 Evolution: Lanosterol/Cycloartenol Diversification and Lateral Transfer

Sterol 14-demethylase (CYP51) is a member of the cytochrome P450 superfamily, widely found in animals, fungi, and plants but present in few prokaryotic groups. CYP51 is currently believed to be the ancestral cytochrome P450 that has been transferred from prokaryotes to eukaryotic kingdoms. We propose an alternate view of CYP51 evolution that has an impact on understanding the evolution of the entire CYP superfamily. Two hundred forty-nine bacterial and four archaeal CYP sequences have been aligned and a bacterial CYP tree designed, showing a separation of two branches. Prokaryotic CYP51s cluster to the minor branch, together with other eukaryote-like CYPs. Mycobacterial and methylococcal CYP51s cluster together (100% bootstrap probability), while Streptomyces CYP51 remains on a distant branch. A CYP51 phylogenetic tree has been constructed from 44 sequences resulting in a ((plant, bacteria),(animal, fungi)) topology (100% bootstrap probability). This is in accordance with the lanosterol/cycloartenol diversification of sterol biosynthesis. The lanosterol branch (nonphotosynthetic lineage) follows the previously proposed topology of animal and fungal orthologues (100% bootstrap probability), while plant and D. discoideum CYP51s belong to the cycloartenol branch (photosynthetic lineage), all in accordance with biochemical data. Bacterial CYP51s cluster within the cycloartenol branch (69% bootstrap probability), which is indicative of a lateral gene transfer of a plant CYP51 to the methylococcal/mycobacterial progenitor, suggesting further that bacterial CYP51s are not the oldest CYP genes. Lateral gene transfer is likely far more important than hitherto thought in the development of the diversified CYP superfamily. Consequently, bacterial CYPs may represent a mixture of genes with prokaryotic and eukaryotic origin.     

Ischemia impairs the association between connexin 43 and M3 subtype of acetylcholine muscarinic receptor (M3-mAChR) in ventricular myocytes.

We used Western blot analysis to examine the expression of connexin 43 and M2/M3 acetylcholine muscarinic receptors (mAChR) and their interaction in ventricular myocytes from control and the ischemic heart. We confirmed that the connexin 43 and M2/ M3-mAChR were expressed in ventricular myocytes. Moreover, we showed that M3-mAChR was expressed in non-glycosylated (72 kDa) and glycosylated forms (115 kDa). Immunostaining showed that connexin 43 is closely associated with M3-mAChR in parts of cell membranes of myocytes. Immunoprecipitation of lysate of cardiac myocytes with M2/M3-mAChR antibody pulled down a 44 kDa protein recognized by connexin 43 antibody. Ischemia increased the expression of M3-mAChR in myocytes. The ischemiainduced increase in the M3-mAChR expression was specific because ischemia did not affect the expression of M1, M2, M4 and M5- mAChR in the heart. On the other hand, ischemia decreased the expression of connexin 43 in myocardium. We also examined the effect of ischemia on the interaction between M2/M3-mAChR and connexin 43. Ischemia suppressed the association of M3-mAChR with connexin 43 but did not affect the association of connexin 43 with M2-mAChR. Administration of choline before ischemia not only partially restored the expression of connexin 43 but also attenuated the ischemia-induced suppression of the association between connexin 43 and M3-mAChR. We conclude that connexin 43 interacts with M2/M3-mAChR and that ischemia specifically impairs the association between M3-mAChR and connexin 43.     

Cloned endangered species takin (Budorcas taxicolor) by inter-species nuclear transfer and comparison of the blastocyst development with yak (Bos grunniens) and bovine

Interspecies cloning might be used as an effective method to conserve endangered species and to support the study of nuclear-cytoplasm interaction. In this study, we describe the development of takin-bovine embryos in vitro produced by fusing takin ear fibroblasts with enucleated bovine oocytes and examine the fate of mitochondrial DNA in these embryos. We also compare the blastocyst development of takin-bovine embryos with yak-bovine and bovine-bovine embryos and compare the cell numbers of the blastocyst. Our results indicate that: (1) takin-bovine cloned embryos can develop to the blastocyst stage in vitro (5%), (2) blastocyst mitochondria DNA are derived primarily from bovine oocytes in spite of a little takin donor cell mitochondrial DNA, (3) using the same cloned protocol, development efficiency is significantly different between bovine-bovine cloning, yak-bovine, and takin-bovine cloning (48 vs. 28% vs. 5%, P < 0.01), and (4) cell numbers in the blastocysts of the three species of embryos were not different. These results suggest that the bovine oocytes can reprogram the takin, yak, and bovine fibroblast nuclei. However, the development efficiency of intra-species cloning tends to be higher than inter-species cloning; the more close the species of the donor cell is to the recipient oocyte (yak versus takin), the greater the blastocyst development in vitro.     

Characterization of the structures involved in localization of the SUN proteins to the nuclear envelope and the centrosome

The nuclear envelope forms a selective barrier that separates the cytoplasm from the nucleus. During mitosis the nuclear envelope breaks down so that the microtubule network can form contacts with the kinetochore and guide chromosome segregation. Previous studies have suggested a model in which the centrosome and the microtubule network may play a role in nuclear envelope breakdown through as yet unidentified interactions with proteins localized to the nuclear envelope. In the current study we characterized a nuclear envelope protein SUN2 and identified a substructure involved in its localization to the nuclear envelope. We found that a structurally related protein, SUN1, may be localized to the nuclear envelope through a different mechanism. Furthermore, the SUN2 protein can form different assemblies, including homodimers and heterodimers with SUN1. Finally, we provide evidence indicating that SUN1 and SUN2 may form a physical interaction between the nuclear envelope and the centrosome.