Nucleotide sequence and expression of the ncr nickel and cobalt resistance in Hafnia alvei 5-5

The structural genes for the nickel and cobalt resistance of the conjugative plasmid pEJH 501 of Hafnia alvei 5-5, contained on a SalI-EcoRI fragment of 4.8 kb, were cloned and sequenced. The DNA sequence included five genes in the following order: ncrA, ncrB, ncrC, ncrY, and ncrX. The predicted amino acid sequences of ncrA were homologous to the amino acid sequences of nreB of Achromobacter xylosoxidans 31A. Expression of ncr with the T7 RNA polymerase-promoter system allowed Escherichia coli BL21 (DE3) to overexpress NcrA, NcrB, and NcrC but not NcrY, and NcrX. The apparent molecular masses of NcrA, NcrB, and NcrC were 30, 33, and 17 kDa, respectively. Primer-extension analysis showed that ncr mRNA started at nucleotide position 23 upstream from ncrA. The promoter region of the ncr operon possessed a strong, putative –35 element of σ32-type promoter sequence, and transcriptional 'lacZ fusion studies indicated that the –35 element influenced σ32-specific transcription. [Int Microbiol 2004; 7(1):27–34

Conjugative plasmid mediated inducible nickel resistance in Hafnia alvei 5-5

Hafnia alvei 5-5, isolated from a soil-litter mixture underneath the canopy of the nickel-hyperaccumulating tree Sebertia acuminata (Sapotaceae) in New Caledonia, was found to be resistant to 30 mM Ni2+ or 2 mM Co2+. The 70-kb plasmid, pEJH 501, was transferred by conjugation to Escherichia coli, Serratia marcescens, and Klebsiella oxytoca. Transconjugant strains expressed inducible nickel resistance to between 5 and 17 mM Ni2+, and cobalt resistance to 2 mM Co2+. A 4.8-kb Sal–EcoRI fragment containing the nickel resistance determinant was subcloned, and the hybrid plasmid was found to confer a moderate level of resistance to nickel (7 mM Ni2+) even to E. coli. The expression of nickel resistance was inducible by exposure to nickel chloride at a concentration as low as 0.5 mM Ni2+. By random TnphoA´-1 insertion mutagenesis, the fragment was shown to have structural genes as well as regulatory regions for nickel resistance. Southern hybridization studies showed that the nickel-resistance determinant from pEJH501 of H. alvei 5-5 was homologous to that of pTOM9 from Alcaligenes xylosoxydans 31A

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

Seed Dormancy Class and Germination Characteristics of <i>Prunus spachiana</i> (Lavallée ex Ed.Otto) Kitam. f. <i>ascendens</i> (Makino) Kitam Native to the Korean Peninsula

Prunus spachiana (Lavallée ex Ed.Otto) Kitam. f. ascendens (Makino) Kitam leaves exert natural anti-inflammatory effects by inhibiting nitric oxide formation. P. spachiana flowers bloom earlier than other Prunus spp. and thus could serve as a valuable resource for the horticulture and pharmaceutical industries. However, its seed dormancy class and germination traits remain uncharacterized. Thus, this study aimed to characterize the seed dormancy and germination of P. spachiana. Imbibition, phenological, and move-along experiments were performed, and the effects of H2SO4 treatment, hormone soaking, warm/cold stratification, and endocarp removal on germination were explored. Observation revealed that ripe seeds of P. spachiana contain developed embryos and are water permeable. Radicle and shoot emergence began in March and April, respectively, under natural conditions in the year following production. No seed germination was observed after 30 days of incubation at 4, 15/6, 20/10, or 25/15 °C under light/dark conditions, indicating the physiological dormancy of the seeds. Germination increased with prolonged stratification and was affected by incubation temperature. Seed scarification by H2SO4 and soaking with gibberellic acid (GA3) and fluridone were ineffective in breaking dormancy. However, GA3 soaking of the seeds after endocarp removal effectively induced germination (100%). These results indicate that P. spachiana seeds exhibit intermediate physiological dormancy

Prevalence of Toxoplasma gondii in Stray Cats of Gyeonggi-do, Korea

Toxoplasma gondii is an obligate intracellular zoonotic protozoan with a worldwide distribution. It infects humans as well as a broad spectrum of vertebrate hosts. Cats and wild felidae play crucial roles in the epidemiology of toxoplasmosis. This study was performed to survey the prevalence of T. gondii infection among stray cats in the Gyeonggi-do, Republic of Korea. A total of 174 stray cat blood samples were collected from Gwacheon-si (n = 20), Bucheon-si (82), and Yangju-si (72). Positive sera for T. gondii were identified in 14 samples (8.1%) exclusively via the latex agglutination test, 28 (16.1%) via ELISA, and 23 (13.2%) via PCR analysis. The overall infection rate of female stray cats (29.2%) presented as higher than that of male cats (24.0%). This study suggests that T. gondii is widespread in the stray cat population of Gyeonggi-do, Korea. It is urgently needed to control urban stray cat population and to reduce the risk of zoonotic transmission of toxoplasmosis to other animal hosts and humans

Genotype of Toxoplasma gondii from Blood of Stray Cats in Gyeonggi-do, Korea

Genotyping of Toxoplasma gondii has been performed in 23 PCR positive blood samples from stray cats in Korea. We used 2 separate PCR-restriction fragment length polymorphism (RFLP) patterns of SAG2 gene, amplifying the 5'and 3'ends of the locus. The results revealed that all samples belonged to the type I clonal lineage. Although T. gondii organisms were not isolated from the samples, the results of the present study represent that stray cats with T. gondii infection should be seriously concerned in our environment. Adequate and continuous control programs of stray cats are needed to reduce the risk of transmission of T. gondii as a zoonotic infection threatening the public health

Rapid Dissemination of Newly Introduced Plasmodium vivax Genotypes in South Korea

Reemerged Plasmodium vivax malaria in South Korea has not yet been eradicated despite continuous governmental efforts. It has rather become an endemic disease. Our study aimed to determine the genetic diversity in P. vivax merozoite surface protein-1 (PvMSP-1) and circumsporozoite protein (PvCSP) genes over an extended period after its reemergence to its current status. Sequence analysis of PvMSP-1 gene sequences from the 632 P. vivax isolates during 1996–2007 indicates that most isolates recently obtained were different from isolates obtained in the initial reemergence period. There was initially only one subtype (recombinant) present but its subtypes have varied since 2000; six MSP-1 subtypes were recently found. A similar variation was observed by CSP gene analysis; a new CSP subtype was found. Understanding genetic variation patterns of the parasite may help to analyze trends and assess extent of endemic malaria in South Korea

Murine immune responses to a Plasmodium vivax-derived chimeric recombinant protein expressed in Brassica napus

<p>Abstract</p> <p>Background</p> <p>To develop a plant-based vaccine against <it>Plasmodium vivax</it>, two <it>P. vivax </it>candidate proteins were chosen. First, the merozoite surface protein-1 (MSP-1), a major asexual blood stage antigen that is currently considered a strong vaccine candidate. Second, the circumsporozoite protein (CSP), a component of sporozoites that contains a B-cell epitope.</p> <p>Methods</p> <p>A synthetic chimeric recombinant 516 bp gene encoding containing PvMSP-1, a Pro-Gly linker motif, and PvCSP was synthesized; the gene, named MLC, encoded a total of 172 amino acids. The recombinant gene was modified with regard to codon usage to optimize gene expression in <it>Brassica napus</it>. The Ti plasmid inducible gene transfer system was used for <it>MLC </it>chimeric recombinant gene expression in <it>B. napus</it>. Gene expression was confirmed by polymerase chain reaction (PCR), beta-glucuronidase reporter gene (GUS) assay, and Western blot.</p> <p>Results</p> <p>The MLC chimeric recombinant protein expressed in <it>B. napus </it>had a molecular weight of approximately 25 kDa. It exhibited a clinical sensitivity of 84.21% (n = 38) and a clinical specificity of 100% (n = 24) as assessed by enzyme-linked immunosorbent assay (ELISA). Oral immunization of BALB/c mice with MLC chimeric recombinant protein successfully induced antigen-specific IgG1 production. Additionally, the Th1-related cytokines IL-12 (p40), TNF, and IFN-γ were significantly increased in the spleens of the BALB/c mice.</p> <p>Conclusions</p> <p>The chimeric MLC recombinant protein produced in <it>B. napus </it>has potential as both as an antigen for diagnosis and as a valuable vaccine candidate for oral immunization against vivax malaria.</p