Apical membrane antigen-1 (AMA-1) gene sequences of re-emerging Plasmodium vivax in South Korea

Plasmodium vivax malaria re-emerged in South Korea in 1993, and epidemics continue since then. We examined genetic variation in the region encompassing the apical membrane antigen-1 (PvAMA-1) of the parasites by DNA sequencing of the 22 re-emerging P. vivax isolates. The genotype of the PvAMA-1, which was based on sequence data previously reported for the polymorphic regions, showed that two haplotypes were present at one polymorphic site. Compared with reported data, the two types, SKOR type I and type II, were similar to Chinese CH-10A and CH-05A isolates, respectively. Thus, the present study showed that two genotypes of AMA-1 genes coexist in the re-emerging Korean P. vivax.     

Nociceptin/orphanin FQ increases ANP secretion in neonatal cardiac myocytes

Nociceptin (N/OFQ) is a novel heptadecapeptide with an amino acid sequence similar to that of endogenous opioid peptide dynorphin A. Dynorphin have been reported to increase the secretion of atrial natriuretic peptide (ANP) via selective activation of kappa-opioid receptor in cultured atrial cardiocytes. The present study was designed to investigate the direct effect of N/OFQ on the ANP secretion in cultured neonatal rat cardiac myocytes via N/OFQ receptor (NOP) activation. The secretion of ANP from cultured neonatal cardiac myocytes was increased in terms of incubation time. N/OFQ, at a dose of 0.3, 1, 3, and 10 microM, caused increases in ANP secretion in a dose-dependent manner. The N/OFQ-induced ANP secretion was completely antagonized by antagonists of NOP, 1 microM each of [Phe1 (CH2-NH) Gly2] nociceptin (1-13)-NH2 ([FG]N/OFQ(1-13)NH2) or naloxone benzoylhydrazone. In contrast, naloxone (1 microM), the non-selective opioid receptor antagonist, did not alter ANP response to N/OFQ. N/OFQ at 3 microM inhibited basal and forskolin-stimulated cAMP production, which was partially antagonized with the pretreatment of [FG]N/OFQ(1-13)NH2. An increase in ANP secretion by N/OFQ was also partially blocked by the pretreatment of forskolin. Homologous competition studies in neonatal cardiomyocyte membranes revealed the presence of two distinct sites. The high affinity site (10.9 +/- 1.6 nM) was far less abundant than the low affinity site. Therefore, these results suggest that N/OFQ causes an increase in ANP secretion in cultured neonatal cardiac myocytes by decreasing cAMP through its binding sites.     

Flavonoid inhibition of sodium-dependent vitamin C transporter 1 (SVCT1) and glucose transporter isoform 2 (GLUT2), intestinal transporters for vitamin C and Glucose

Vitamin C and flavonoids, polyphenols with uncertain function, are abundant in fruits and vegetables. We postulated that flavonoids have a novel regulatory action of delaying or inhibiting absorption of vitamin C and glucose, which are structurally similar. From six structural classes of flavonoids, at least 12 compounds were chosen for studies. We investigated the effects of selected flavonoids on the intestinal vitamin C transporter SVCT1(h) by transfecting and overexpressing SVCT1(h) in Chinese hamster ovary cells. Flavonoids reversibly inhibited vitamin C transport in transfected cells with IC(50) values of 10-50 microm, concentrations expected to have physiologic consequences. The most potent inhibitor class was flavonols, of which quercetin is most abundant in foods. Because Chinese hamster ovary cells have endogenous vitamin C transport, we expressed SVCT1(h) in Xenopus laevis oocytes to study the mechanism of transport inhibition. Quercetin was a reversible and non-competitive inhibitor of ascorbate transport; K(i) 17.8 microm. Quercetin was a potent non-competitive inhibitor of GLUT2 expressed in Xenopus oocytes; K(i) 22.8 microm. When diabetic rats were administered glucose with quercetin, hyperglycemia was significantly decreased compared with administration of glucose alone. Quercetin also significantly decreased ascorbate absorption in normal rats given ascorbate plus quercetin compared with rats given ascorbate alone. Quercetin was a specific transport inhibitor, because it did not inhibit intestinal sugar transporters GLUT5 and SGLT1 that were injected and expressed in Xenopus oocytes. Quercetin inhibited but was not transported by SVCT1(h). Considered together, these data show that flavonoids modulate vitamin C and glucose transport by their respective intestinal transporters and suggest a new function for flavonoids.     

Phosphorylation of cucumber mosaic virus RNA polymerase 2a protein inhibits formation of replicase complex

The 2a (polymerase) protein of cucumber mosaic virus (CMV) was shown to be phosphorylated both in vivo and in vitro. In vitro assays using 2a protein mutants and tobacco protein kinases showed that the 2a protein has at least three phosphorylation sites, one of which is located within the N-terminal 126 amino acid region. This region is essential and sufficient for interaction with the CMV 1a protein. When phosphorylated in vitro, the 2a protein N-terminal region failed to interact with the 1a protein. Since the 1a-2a interaction is essential for the replication of CMV, this suggests that phosphorylation of the N-terminal region of the 2a protein negatively modulates the interaction in vivo, and may have a regulatory role acting directly in viral infection.     

Degradation of cholesterol by Bacillus subtilis SFF34 isolated from Korean traditional fermented flatfish

AIMS: To examine cholesterol degradation by Bacillus subtilis SFF34. METHODS AND RESULTS: Cholesterol degradation and cholesterol oxidase production by B. subtilis SFF34 were investigated in a medium containing 0.2% cholesterol. In addition, the oxidized product of cholesterol by the purified cholesterol oxidase was detected using a gas chromatograph. Cholesterol oxidase production reached its maximal level (3.14 U ml(-1) after 24 h of incubation in the cholesterol medium. The residual cholesterol content reduced to 0.98 mg g(-1) after 60 h of cultivation in the cholesterol medium. Two cholesterol oxidases were purified from the culture supernatant fluid and their reaction product against cholesterol was identified as 4-cholesten-3-one. CONCLUSIONS: B. subtilis SFF34 degraded cholesterol and produced a high level of extracellular cholesterol oxidase. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacillus subtilis will be very useful for the reduction of cholesterol in many fermented foods and as a source of cholesterol oxidase.     

Homogeneity of recombination rate within a conserved region on BTA3 that contains QTL

The D3S20-D3S34-D3S3 region on BTA3 contains quantitative trait loci (QTL) controlling milk production traits. This region also displays extensive conservation of synteny among several species including cattle, humans, mice and sheep. In this study, we evaluated the adjacent intervals D3S20-D3S34 and D3S34-D3S3 for differences in recombination rate (theta) among bulls in order to assess the suitability of population-based estimates of theta for marker assisted selection and to explore the relationship between variation in theta and chromosome breakpoints associated with mammalian evolution. Using sperm typing, thetaD3S20-D3S34 and theta D3S34-D3S3 were estimated for six triply heterozygous bulls. Recombination frequency ranged from 6.2 to 12.5% and from 9.7 to 19.2% for the D3S20-D3S34 and D3S34-D3S3 intervals, respectively. However, significant variation in theta was not detected between bulls for either interval (D3S20-D3S34 chi(2)5 d.f.=2.59, P < 0.90; D3S34-D3S3 chi(2)5 d.f.=3.72, P < 0.75). The observed differences in theta were most readily attributed to differences in allele-specific amplification efficiencies among bulls. Our results suggest that the positions of QTL in this region can be reliably determined from population data and therefore accurate marker-assisted selection can be performed for desirable alleles without concern for variation in theta. Furthermore, when considered with results of earlier studies, these findings support a correlation between the existence of evolutionary breakpoints or chromosome rearrangements and variation in theta.     

Role of the murein precursor UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-diaminopimelic acid-D-Ala-D-Ala in repression of beta-lactamase induction in cell division mutants

Certain beta-lactam antibiotics induce the chromosomal ampC beta-lactamase of many gram-negative bacteria. The natural inducer, though not yet unequivocally identified, is a cell wall breakdown product which enters the cell via the AmpG permease component of the murein recycling pathway. Surprisingly, it has been reported that beta-lactamase is not induced by cefoxitin in the absence of FtsZ, which is required for cell division, or in the absence of penicillin-binding protein 2 (PBP2), which is required for cell elongation. Since these results remain unexplained, we examined an ftsZ mutant and other cell division mutants (ftsA, ftsQ, and ftsI) and a PBP2 mutant for induction of beta-lactamase. In all mutants, beta-lactamase was not induced by cefoxitin, which confirms the initial reports. The murein precursor, UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-diaminopimelic acid-D-Ala-D-Ala (UDP-MurNAc-pentapeptide), has been shown to serve as a corepressor with AmpR to repress beta-lactamase expression in vitro. Our results suggest that beta-lactamase is not induced because the fts mutants contain a greatly increased amount of corepressor which the inducer cannot displace. In the PBP2(Ts) mutant, in addition to accumulation of corepressor, cell wall turnover and recycling were greatly reduced so that little or no inducer was available. Hence, in both cases, a high ratio of repressor to inducer presumably prevents induction.     

Long-term effect of interferon on keratinocytes that maintain human papillomavirus type 31

The long-term effects of interferon treatment on cell lines that maintain human papillomavirus type 31 (HPV-31) episomes have been examined. High doses and prolonged interferon treatment resulted in growth arrest of HPV-positive cells, with a high percentage of cells undergoing apoptosis. These effects were not seen with interferon treatment of either normal human keratinocytes or cells derived from HPV-negative squamous carcinomas, which exhibited only slight decreases in their rates of growth. Within 2 weeks of the initiation of treatment, a population of HPV-31-positive cells that were resistant to interferon appeared consistently and reproducibly. The resistant cells had growth and morphological characteristics similar to those of untreated cells. Long-term interferon treatment of HPV-positive cells also resulted in a reduction in HPV episome levels but did not significantly decrease the number of integrated copies of HPV. Cells that maintained HPV genomes lacking E5 were sensitive to interferon, while cells expressing only the E6/E7 genes were resistant. In contrast, cells that expressed E2 from a tetracycline-inducible promoter were found to be significantly more sensitive to interferon treatment than parental cells. This suggests that at least a portion of the sensitivity to interferon could be mediated through the E2 protein. These studies indicate that cells maintaining HPV episomes are highly sensitive to interferon treatment but that resistant populations arise quickly.     

Expression of exogenous Sam68, the 68-kilodalton SRC-associated protein in mitosis, is able to alleviate impaired Rev function in astrocytes

Human immunodeficiency virus type 1 (HIV-1) gene expression in astrocytes is restricted, resulting in a brief and limited synthesis of HIV-1 viral structural proteins. Impaired Rev function has been documented in these cells. However, the molecular mechanisms underlying the impaired Rev function are not fully understood. Using the astroglial cell line U87.MG as a model, we report here that HIV-1 gene expression down-regulated expression of Sam68, the 68-kDa Src-associated protein in mitosis, which was constitutively expressed at a lower level in astrocytes. Elevating the endogenous level of Sam68 expression considerably restored HIV-1 Rev function in astrocytes, as determined by a Rev-dependent reporter gene assay. However, elevation of Sam68 expression achieved only a modest increase in HIV-1 production, further supporting the notion that there are multiple cellular restrictions of HIV-1 gene expression in astrocytes. Mutagenesis analysis identified the region between amino acids 321 and 410 of Sam68 as being directly involved in the binding of Sam68 to Rev, while a double mutation in Rev, L78D and E79L, like those in the dominant-negative Rev mutant M10, eliminated Rev binding to Sam68. Moreover, subcellular fractionation and digital fluorescence microscopic imaging revealed that Sam68 expression promoted Rev nuclear export. Taken together, our studies demonstrate that a lower level of constitutive Sam68 expression, followed by further down-regulation by HIV-1 infection, contributes to impaired Rev function in astrocytes, and they suggest that Sam68 may play an important role in Rev nuclear export.