Molecular Detection of Ehrlichia canis in Dogs in Malaysia

An epidemiological study of Ehrlichia canis infection in dogs in Peninsular Malaysia was carried out using molecular detection techniques. A total of 500 canine blood samples were collected from veterinary clinics and dog shelters. Molecular screening by polymerase chain reaction (PCR) was performed using genus-specific primers followed by PCR using E. canis species-specific primers. Ten out of 500 dogs were positive for E. canis. A phylogenetic analysis of the E. canis Malaysia strain showed that it was grouped tightly with other E. canis strains from different geographic regions. The present study revealed for the first time, the presence of genetically confirmed E. canis with a prevalence rate of 2.0% in naturally infected dogs in Malaysia.     

Impact of H216 on the DNA Binding and Catalytic Activities of the HIV Restriction Factor APOBEC3G

APOBEC3G has an important role in human defense against retroviral pathogens, including HIV-1. Its single-stranded DNA cytosine deaminase activity, located in its C-terminal domain (A3Gctd), can mutate viral cDNA and restrict infectivity. We used time-resolved nuclear magnetic resonance (NMR) spectroscopy to determine kinetic parameters of A3Gctd''s deamination reactions within a 5′-CCC hot spot sequence. A3Gctd exhibited a 45-fold preference for 5′-CCC substrate over 5′-CCU substrate, which explains why A3G displays almost no processivity within a 5′-CCC motif. In addition, A3Gctd''s shortest substrate sequence was found to be a pentanucleotide containing 5′-CCC flanked on both sides by a single nucleotide. A3Gctd as well as full-length A3G showed peak deamination velocities at pH 5.5. We found that H216 is responsible for this pH dependence, suggesting that protonation of H216 could play a key role in substrate binding. Protonation of H216 appeared important for HIV-1 restriction activity as well, since substitutions of H216 resulted in lower restriction in vivo.     

Population Shift Underlies Ca2+-induced Regulatory Transitions in the Sodium-Calcium Exchanger (NCX)

In eukaryotic Na⁺/Ca²⁺ exchangers (NCX) the Ca²⁺ binding CBD1 and CBD2 domains form a two-domain regulatory tandem (CBD12). An allosteric Ca²⁺ sensor (Ca3–Ca4 sites) is located on CBD1, whereas CBD2 contains a splice-variant segment. Recently, a Ca²⁺-driven interdomain switch has been described, albeit how it couples Ca²⁺ binding with signal propagation remains unclear. To resolve the dynamic features of Ca²⁺-induced conformational transitions we analyze here distinct splice variants and mutants of isolated CBD12 at varying temperatures by using small angle x-ray scattering (SAXS) and equilibrium ⁴⁵Ca²⁺ binding assays. The ensemble optimization method SAXS analysis demonstrates that the apo and Mg²⁺-bound forms of CBD12 are highly flexible, whereas Ca²⁺ binding to the Ca3–Ca4 sites results in a population shift of conformational landscape to more rigidified states. Population shift occurs even under conditions in which no effect of Ca²⁺ is observed on the globally derived D_max (maximal interatomic distance), although under comparable conditions a normal [Ca²⁺]-dependent allosteric regulation occurs. Low affinity sites (Ca1–Ca2) of CBD1 do not contribute to Ca²⁺-induced population shift, but the occupancy of these sites by 1 mm Mg²⁺ shifts the Ca²⁺ affinity (K_d) at the neighboring Ca3–Ca4 sites from ∼ 50 nm to ∼ 200 nm and thus, keeps the primary Ca²⁺ sensor (Ca3–Ca4 sites) within a physiological range. Thus, Ca²⁺ binding to the Ca3–Ca4 sites results in a population shift, where more constraint conformational states become highly populated at dynamic equilibrium in the absence of global conformational transitions in CBD alignment.     

Salamander diversity alters stream macroinvertebrate community structure

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Distinct roles for ROCK1 and ROCK2 in the regulation of cell detachment

This study, using mouse embryonic fibroblast (MEF) cells derived from ROCK1^−/− and ROCK2^−/− mice, is designed to dissect roles for ROCK1 and ROCK2 in regulating actin cytoskeleton reorganization induced by doxorubicin, a chemotherapeutic drug. ROCK1^−/− MEFs exhibited improved actin cytoskeleton stability characterized by attenuated periphery actomyosin ring formation and preserved central stress fibers, associated with decreased myosin light chain 2 (MLC2) phosphorylation but preserved cofilin phosphorylation. These effects resulted in a significant reduction in cell shrinkage, detachment, and predetachment apoptosis. In contrast, ROCK2^−/− MEFs showed increased periphery membrane folding and impaired cell adhesion, associated with reduced phosphorylation of both MLC2 and cofilin. Treatment with inhibitor of myosin (blebbistatin), inhibitor of actin polymerization (cytochalasin D), and ROCK pan-inhibitor (Y27632) confirmed the contributions of actomyosin contraction and stress fiber instability to stress-induced actin cytoskeleton reorganization. These results support a novel concept that ROCK1 is involved in destabilizing actin cytoskeleton through regulating MLC2 phosphorylation and peripheral actomyosin contraction, whereas ROCK2 is required for stabilizing actin cytoskeleton through regulating cofilin phosphorylation. Consequently, ROCK1 and ROCK2 can be functional different in regulating stress-induced stress fiber disassembly and cell detachment.     

SUMO-targeted ubiquitin ligase (STUbL) Slx5 regulates proteolysis of centromeric histone H3 variant Cse4 and prevents its mislocalization to euchromatin

Centromeric histone H3, CENP-A^Cse4, is essential for faithful chromosome segregation. Stringent regulation of cellular levels of CENP-A^Cse4 restricts its localization to centromeres. Mislocalization of CENP-A^Cse4 is associated with aneuploidy in yeast and flies and tumorigenesis in human cells; thus defining pathways that regulate CENP-A levels is critical for understanding how mislocalization of CENP-A contributes to aneuploidy in human cancers. Previous work in budding yeast shows that ubiquitination of overexpressed Cse4 by Psh1, an E3 ligase, partially contributes to proteolysis of Cse4. Here we provide the first evidence that Cse4 is sumoylated by E3 ligases Siz1 and Siz2 in vivo and in vitro. Ubiquitination of Cse4 by the small ubiquitin-related modifier (SUMO)-targeted ubiquitin ligase (STUbL) Slx5 plays a critical role in proteolysis of Cse4 and prevents mislocalization of Cse4 to euchromatin under normal physiological conditions. Accumulation of sumoylated Cse4 species and increased stability of Cse4 in slx5∆ strains suggest that sumoylation precedes ubiquitin-mediated proteolysis of Cse4. Slx5-mediated Cse4 proteolysis is independent of Psh1, since slx5∆ psh1∆ strains exhibit higher levels of Cse4 stability and mislocalization than either slx5∆ or psh1∆ strains. Our results demonstrate a role for Slx5 in ubiquitin-mediated proteolysis of Cse4 to prevent its mislocalization and maintain genome stability.     

The restriction of zoonotic PERV transmission by human APOBEC3G

The human APOBEC3G protein is an innate anti-viral factor that can dominantly inhibit the replication of some endogenous and exogenous retroviruses. The prospects of purposefully harnessing such an anti-viral defense are under investigation. Here, long-term co-culture experiments were used to show that porcine endogenous retrovirus (PERV) transmission from pig to human cells is reduced to nearly undetectable levels by expressing human APOBEC3G in virus-producing pig kidney cells. Inhibition occurred by a deamination-independent mechanism, likely after particle production but before the virus could immortalize by integration into human genomic DNA. PERV inhibition did not require the DNA cytosine deaminase activity of APOBEC3G and, correspondingly, APOBEC3G-attributable hypermutations were not detected. In contrast, over-expression of the sole endogenous APOBEC3 protein of pigs failed to interfere significantly with PERV transmission. Together, these data constitute the first proof-of-principle demonstration that APOBEC3 proteins can be used to fortify the innate anti-viral defenses of cells to prevent the zoonotic transmission of an endogenous retrovirus. These studies suggest that human APOBEC3G-transgenic pigs will provide safer, PERV-less xenotransplantation resources and that analogous cross-species APOBEC3-dependent restriction strategies may be useful for thwarting other endogenous as well as exogenous retrovirus infections.     

p53 promotes the fidelity of DNA end-joining activity by, in part, enhancing the expression of heterogeneous nuclear ribonucleoprotein G

Many studies have suggested the involvement of wild-type (wt) p53 in the repair of DNA double-strand breaks (DSBs) via DNA end-joining (EJ) process. To investigate this possibility, we compared the capacity and fidelity of DNA EJ in RKO cells containing wt p53 and RKO cells containing no p53 (RKO cells with p53 knockdown). The p53 knockdown cells showed lower fidelity of DNA EJ compared to the control RKO cells. The DNA end-protection assay revealed the association of a protein complex including heterogeneous nuclear ribonucleoprotein G (hnRNP G) with the DNA ends in RKO cells containing wt p53, but not with the DNA ends in RKO cells with p53 knockdown. Depletion of endogenous hnRNP G notably diminished the fidelity of EJ in RKO cells expressing wt p53. Moreover, an ectopic expression of hnRNP G significantly enhanced the fidelity of DNA EJ and the protection of DNA ends in human cancer cells lacking hnRNP G protein or containing mutant hnRNP G. Finally, using recombinant hnRNP G proteins, we demonstrated the hnRNP G protein is able to bind to and protect DNA ends from degradation of nucleases. Our results suggest that wt p53 modulates DNA DSB repair by, in part, inducing hnRNP G, and the ability of hnRNP G to bind and protect DNA ends may contribute its ability to promote the fidelity of DNA EJ.     

Comprehensive analysis of genetic polymorphisms in the interleukin-10 promoter: implications for immune regulation in specific ethnic populations

The association of interleukin-10 (IL-10) promoter single-nucleotide polymorphisms (SNPs) as risk factors for certain inflammatory diseases, viral infections, cancers, and transplant rejection have been the subject of recent studies. The SNPs -1082 G --> A, -819 C --> T, and -592 C --> A, which have been associated with differential IL-10 production, are strongly linked with ethnicity. In this study, we determined the ethnic distribution of IL-10 promoter SNPs and their haplotype rates among Hispanics, African Americans, and Caucasians from Texas and Ashkenazi Jews from New York. Significant differences in prevalence rates of IL-10 SNPs (and their haplotype distribution) were found. African Americans and Hispanics have a lower rate of putative high-producer SNPs and a higher rate of low IL-10 producers when compared to Caucasians or Ashkenazi Jews. No statistically significant differences in allelic frequencies and haplotype rates were observed between Caucasians and Ashkenazi Jews. This study provides critical new information on the ethnic distribution of IL-10 promoter SNPs in a regional U. S. population and is the first to analyze the rate of SNPs in an unstudied ethnic population, Ashkenazi Jews. Knowledge of IL-10 promoter polymorphisms may prove useful in prediction of immunization responses, disease severity, and in the intelligent design of customized immunotherapy.     

A model-based optimization framework for the inference on gene regulatory networks from DNA array data

MOTIVATION: Identification of the regulatory structures in genetic networks and the formulation of mechanistic models in the form of wiring diagrams is one of the significant objectives of expression profiling using DNA microarray technologies and it requires the development and application of identification frameworks. RESULTS: We have developed a novel optimization framework for identifying regulation in a genetic network using the S-system modeling formalism. We show that balance equations on both mRNA and protein species led to a formulation suitable for analyzing DNA-microarray data whereby protein concentrations have been eliminated and only mRNA relative concentrations are retained. Using this formulation, we examined if it is possible to infer a set of possible genetic regulatory networks consistent with observed mRNA expression patterns. Two origins of changes in mRNA expression patterns were considered. One derives from changes in the biophysical properties of the system that alter the molecular-interaction kinetics and/or message stability. The second is due to gene knock-outs. We reduced the identification problem to an optimization problem (of the so-called mixed-integer non-linear programming class) and we developed an algorithmic procedure for solving this optimization problem. Using simulated data generated by our mathematical model, we show that our method can actually find the regulatory network from which the data were generated. We also show that the number of possible alternate genetic regulatory networks depends on the size of the dataset (i.e. number of experiments), but this dependence is different for each of the two types of problems considered, and that a unique solution requires fewer datasets than previously estimated in the literature. This is the first method that also allows the identification of every possible regulatory network that could explain the data, when the number of experiments does not allow identification of unique regulatory structure.