Molecular cloning of Plasmodium vivax calcium-dependent protein kinase 4

A family of calcium-dependent protein kinases (CDPKs) is a unique enzyme which plays crucial roles in intracellular calcium signaling in plants, algae, and protozoa. CDPKs of malaria parasites are known to be key regulators for stage-specific cellular responses to calcium, a widespread secondary messenger that controls the progression of the parasite. In our study, we identified a gene encoding Plasmodium vivax CDPK4 (PvCDPK4) and characterized its molecular property and cellular localization. PvCDPK4 was a typical CDPK which had well-conserved N-terminal kinase domain and C-terminal calmodulin-like structure with 4 EF hand motifs for calcium-binding. The recombinant protein of EF hand domain of PvCDPK4 was expressed in E. coli and a 34 kDa product was obtained. Immunofluorescence assay by confocal laser microscopy revealed that the protein was expressed at the mature schizont of P. vivax. The expression of PvCDPK4-EF in schizont suggests that it may participate in the proliferation or egress process in the life cycle of this parasite.     

Intestinal Helminthic Infections Diagnosed by Colonoscopy in a Regional Hospital during 2001-2008

The present study investigated characteristics of 24 parasite infection cases detected during colonoscopy in a regional hospital from January 2001 to December 2008. Sixteen patients were confirmed with Trichuris trichiura infection, 6 patients were with Ascaris lumbricoides infection, 1 patient with Enterobius vermicularis infection, and 1 patient with Anisakis infection. Among them, 7 patients (43.8%) were asymptomatic. Colonoscopy findings were normal in 18 patients (75.0%). Among the patients with T. trichiura infection, colonoscopy showed several erosions in 2 patients (8.3%) and non-specific inflammation of the affected segment of the colon in 3 patients (12.5%). In 1 patient with anisakiasis, colonoscopy revealed a markedly swollen colonic wall. Stool examinations were performed before treatment in 7 patients (29.2%) and were all negative for parasite eggs or worms. These results suggest that colonoscopy is a useful diagnostic approach for parasitic infections even for asymptomatic patients and for patients with negative stool examinations.     

Modulation of nonselective cation current by oxidized LDL and lysophosphatidylcholine and its inhibitory contribution to endothelial damage

AimsThis study examined the effects of oxidized low-density lipoprotein (LDL) and its major lipid constituent lysophosphatidylcholine (LPC) on nonselective cation (NSC) current and its inhibitory contribution to LPC-induced cytotoxicity in cultured human umbilical endothelial cells (HUVECs).Main methodsPatch-clamp technique and the resazurin-based cell viability assay were used.Key findingsIn voltage-clamped cells, oxidized LDL or LPC slowly activated NSC current. NSC current was also activated by loading cells with Ca²⁺ solution buffered at various concentrations using a patch pipette or by applying the sarcoplasmic reticulum Ca²⁺ pump blocker 2,5-di-t-butyl-1,4-benzohydroquinone (BHQ), the metabolic inhibitor CN^? or the hydroperoxide donor tert-butyl hydroperoxide (TBHP). On the contrary, when intracellular Ca²⁺ was strongly buffered with 12 mM BAPTA or cells were loaded with superoxide dismutase using a patch pipette, LPC or BHQ did not activate NSC current. Furthermore, NSC current activated by LPC, TBHP or CN^? was inhibited by the antioxidant tempol or extracellular Ca²⁺ depletion and NSC current activated by intracellular Ca²⁺ was further augmented by oxidized LDL or LPC. LPC or oxidized LDL released Ca²⁺ from intracellular stores and further enhanced store-operated Ca²⁺ entry. LPC-induced cytotoxicity was augmented by inhibiting Ca²⁺ influx and NO synthesis.SignificanceOxidized LDL or its main component LPC activated Ca²⁺-permeable NSC current via releasing Ca²⁺ from intracellular stores and producing ROS and thereby increased Ca²⁺ influx. Ca²⁺ influx through NSC channel might protect endothelial cells by producing NO.     

Metabolic classification of South American Ilex species by NMR-based metabolomics

The genus Ilex to which mate (Ilex paraguariensis) belongs, consists of more than 500 species. A wide range of metabolites including saponins and phenylpropanoids has been reported from Ilex species. However, despite the previous works on the Ilex metabolites, the metabolic similarities between species which can be used for chemotaxonomy of the species are not clear yet. In this study, nuclear magnetic resonance (NMR) spectroscopy-based metabolomics was applied to the classification of 11 South American Ilex species, namely, Ilex argentina, Ilex brasiliensis, Ilex brevicuspis, Ilex dumosa var. dumosa, I. dumosa var. guaranina, Ilex integerrima, Ilex microdonta, I. paraguariensis var. paraguariensis, Ilex pseudobuxus, Ilex taubertiana, and Ilex theezans. ¹H NMR combined with principal component analysis (PCA), partial least square-discriminant analysis (PLS-DA) and hierarchical cluster analysis (HCA) showed a clear separation between species and resulted in four groups based on metabolomic similarities. The signal congestion of ¹H NMR spectra was overcome by the implementation of two-dimensional (2D)-J-resolved and heteronuclear single quantum coherence (HSQC). From the results obtained by 1D- and 2D-NMR-based metabolomics it was concluded that species included in group A (I. paraguariensis) were metabolically characterized by a higher amount of xanthines, and phenolics including phenylpropanoids and flavonoids; group B (I. dumosa var. dumosa and I. dumosa var. guaranina) with oleanane type saponins; group C (I. brasiliensis, I. integerrima, I. pseudobuxus and I. theezans) with arbutin and dicaffeoylquinic acids, and group D (I. argentina, I. brevicuspis, I. microdonta and I. taubertiana) with the highest level of ursane-type saponins. Clear metabolomic discrimination of Ilex species and varieties in this study makes the chemotaxonomic classification of Ilex species possible.     

Production of functionalized polyhydroxyalkanoates by genetically modified <Emphasis Type="Italic">Methylobacterium extorquens</Emphasis> strains

Background  

Methylotrophic (methanol-utilizing) bacteria offer great potential as cell factories in the production of numerous products from biomass-derived methanol. Bio-methanol is essentially a non-food substrate, an advantage over sugar-utilizing cell factories. Low-value products as well as fine chemicals and advanced materials are envisageable from methanol. For example, several methylotrophic bacteria, including Methylobacterium extorquens, can produce large quantities of the biodegradable polyester polyhydroxybutyric acid (PHB), the best known polyhydroxyalkanoate (PHA). With the purpose of producing second-generation PHAs with increased value, we have explored the feasibility of using M. extorquens for producing functionalized PHAs containing C-C double bonds, thus, making them amenable to future chemical/biochemical modifications for high value applications.     

Peptidyl arginine deiminase type IV (<Emphasis Type="Italic">PADI4</Emphasis>) haplotypes interact with shared epitope regardless of anti-cyclic citrullinated peptide antibody or erosive joint status in rheumatoid arthritis: a case control study

Introduction  

Anti-cyclic citrullinated peptide autoantibodies (anti-CCP) are the most specific serologic marker for rheumatoid arthritis (RA). Genetic polymorphisms in a citrullinating (or deiminating) enzyme, peptidyl arginine deiminase type IV (PADI4) have been reproducibly associated with RA susceptibility in several populations. We investigated whether PADI4 polymorphisms contribute to anti-CCP-negative as well as -positive RA, whether they influence disease severity (erosive joint status), and whether they interact with two major risk factors for RA, Human Leukocyte Antigen-DRB1 (HLA-DRB1) shared epitope (SE) alleles and smoking, depending on anti-CCP and erosive joint status.     

Reorganization of horizontal cell processes in the developing FVB/N mouse retina

We investigated the morphological changes of horizontal cells after postnatal photoreceptor degeneration in the developing FVB/N mouse retina, using immunocytochemistry with anti-calbindin D-28K. From postnatal day 14 (P14) onwards, processes emerging from horizontal cells descend into the inner plexiform layer (IPL) and ramify mainly in stratum 1 of the IPL. Electron microscopy revealed that the descending processes make synaptic contacts with bipolar cells in the outer plexiform layer. Our results clearly demonstrate that loss of photoreceptor cells induces the reorganization of horizontal cell processes in the retinas of FVB/N mice as they mature.