Zinc-induced sodium-dependent vitamin C transporter 2 expression: potent roles in osteoblast differentiation

Zinc is an essential trace element that increases osteoblast numbers and bone formation. However, the mechanisms involved in the Zn-induced differentiation of osteoblasts are poorly understood. We examined the roles of L-ascorbic acid (AA) and its transporter, sodium-dependent vitamin C transporter (SVCT) 2, in the Zn-induced expression of osteoblastic differentiation markers. Zinc time- and dose-dependently induced SVCT2 mRNA expression in the absence or presence of AA. Western blotting and kinetic assays showed that Zn increased functional SVCT2 protein levels and AA transport. In the presence of AA, 50 microM Zn enhanced mRNA expression of the osteoblastic differentiation markers alkaline phosphatase, alpha(1)(I) procollagen, osteopontin (OPN), and osteocalcin (OCN) by 3.9-, 3.8-, 3.3-, and 3.5-fold, respectively; in the absence of AA, the Zn-induced increase was 2.8-, 2.5-, 1.3-, and 1.1-fold, respectively. These findings suggest that AA and SVCT2 mediate Zn-induced OPN and OCN expression and partly regulate Zn-induced osteoblastic differentiation.     

Identification of cry1I-type genes from Bacillus thuringiensis strains and characterization of a novel cry1I-type gene

A PCR-restriction fragment length polymorphism method for identification of cry1I-type genes from Bacillus thuringiensis was established by designing a pair of universal primers based on the conserved regions of the genes to amplify 1,548-bp cry1I-type gene fragments. Amplification products were digested with the Bsp119I and BanI enzymes, and four kinds of known cry1I-type genes were successfully identified. The results showed that cry1I-type genes appeared in 95 of 115 B. thuringiensis isolates and 7 of 13 standard strains. A novel cry1I-type gene was found in one standard strain and six isolates. The novel cry1I gene was cloned from B. thuringiensis isolate Btc007 and subcloned into vector pET-21b. Then it was overexpressed in Escherichia coli BL21(DE3). The expressed product was shown to be toxic to the diamondback moth (Plutella xylostella), Asian corn borer (Ostrinia furnacalis), and soybean pod borer (Leguminivora glycinivorella). However, it was not toxic to the cotton bollworm (Helicoverpa armigera), beet armyworm (Spodoptera exigua), or elm leaf beetle (Pyrrhalta aenescens) in bioassays. Subsequently, the Cry protein encoded by this novel cry gene was designated Cry1Ie1 by the B. thuringiensis delta-endotoxin nomenclature committee.     

Structural Variation and Uniformity among Tetraloop-Receptor Interactions and Other Loop-Helix Interactions in RNA Crystal Structures

Tetraloop-receptor interactions are prevalent structural units in RNAs, and include the GAAA/11-nt and GNRA-minor groove interactions. In this study, we have compiled a set of 78 nonredundant loop-helix interactions from X-ray crystal structures, and examined them for the extent of their sequence and structural variation. Of the 78 interactions in the set, only four were classical GAAA/11-nt motifs, while over half (48) were GNRA-minor groove interactions. The GNRA-minor groove interactions were not a homogeneous set, but were divided into five subclasses. The most predominant subclass is characterized by two triple base pair interactions in the minor groove, flanked by two ribose zipper contacts. This geometry may be considered the “standard” GNRA-minor groove interaction, while the other four subclasses are alternative ways to form interfaces between a minor groove and tetraloop. The remaining 26 structures in the set of 78 have loops interacting with mostly idiosyncratic receptors. Among the entire set, a number of sequence-structure correlations can be identified, which may be used as initial hypotheses in predicting three-dimensional structures from primary sequences. Conversely, other sequence patterns are not predictive; for example, GAAA loop sequences and GG/CC receptors bind to each other with three distinct geometries. Finally, we observe an example of structural evolution in group II introns, in which loop-receptor motifs are substituted for each other while maintaining the larger three-dimensional geometry. Overall, the study gives a more complete view of RNA loop-helix interactions that exist in nature.     

Isolation and molecular characterization of herpesvirus from cultured European eels Anguilla anguilla in Taiwan

A herpesvirus has been isolated for the first time from a population of European eels Anguilla anguilla cultured in a recirculated system in Taiwan. Syncytia formation was detected in EP-1 (eel epidermis) cell cultures inoculated with cell-free homogenates prepared from both integument and visceral organs of moribund fish. Inoculation of homogenates onto EK (eel kidney) cell cultures induced giant cell formation. Subsequent passages produced a consistent and progressive cytopathic effect (CPE) in cell cultures. In this study, EP-1 cell cultures infected with EEHV (European eel herpesvirus) were examined using an electron microscope. Numerous nucleocapsids of about 100 nm in diameter were found within the nucleus of infected cells, whereas enveloped particles were observed within the cytoplasm. The mature viral particle, about 235 nm in diameter, had an electron-dense core with a hexagonal nucleocapsid surrounded by a coarse capsule. Histopathological examination of moribund fish showed epithelial hyperplasia with intracytoplasmic metabolic inclusions in the skin. Macrophage aggregates were found in liver, spleen, and kidney. A pair of primers designed from channel catfish virus and salmonid herpesvirus 1 was used in a polymerase chain reaction. A 402 bp fragment was amplified and cloned from genomic DNA of EEHV. The nucleotide homology was 99% (298 of 300) with DNA polymerase of eel herpesvirus (anguillid herpesvirus). EEHV nucleic acids were detected within melanomacrophages in the skin, liver, spleen and kidney by in situ hybridization (ISH).     

Role of the Rab GTP-binding protein Ypt3 in the fission yeast exocytic pathway and its connection to calcineurin function

A genetic screen for mutations synthetically lethal with fission yeast calcineurin deletion led to the identification of Ypt3, a homolog of mammalian Rab11 GTP-binding protein. A mutant with the temperature-sensitive ypt3-i5 allele showed pleiotropic phenotypes such as defects in cytokinesis, cell wall integrity, and vacuole fusion, and these were exacerbated by FK506-treatment, a specific inhibitor of calcineurin. Green fluorescent protein (GFP)-tagged Ypt3 showed cytoplasmic staining that was concentrated at growth sites, and this polarized localization required the actin cytoskeleton. It was also detected as a punctate staining in an actin-independent manner. Electron microscopy revealed that ypt3-i5 mutants accumulated aberrant Golgi-like structures and putative post-Golgi vesicles, which increased remarkably at the restrictive temperature. Consistently, the secretion of GFP fused with the pho1(+) leader peptide (SPL-GFP) was abolished at the restrictive temperature in ypt3-i5 mutants. FK506-treatment accentuated the accumulation of aberrant Golgi-like structures and caused a significant decrease of SPL-GFP secretion at a permissive temperature. These results suggest that Ypt3 is required at multiple steps of the exocytic pathway and its mutation affects diverse cellular processes and that calcineurin is functionally connected to these cellular processes.     

Myocardial protection of MCI-186 in rabbit ischemia-reperfusion

We observed that 3-methyl-1-1phenyl-2-pyrazolin-5-one (MCI-186), a newly-developed free radical scavenger, attenuated necrosis in the in vivo rabbit hearts upon reperfusion after prolonged ischemia. In rabbits undergoing 1 hour ligation of the anterior ventricular coronary artery, a single bolus injection of MCI-186 (1.5 mg/kg) was introduced into the post-ischemic heart immediately before 4 hour reperfusion. Compared to negligible necrosis in sham-operated control animals and 33.81 +/- 13.50% necrosis in the area at risk for the saline control group (n = 8), the MCI-186 - treated group (n = 8) had a necrosis of 13.27 +/- 4.60% (p < 0.05 vs saline control group). The pressure-rate index had a slight decrease in MCI-186 treated group compared to the control group (p > 0.05). However, the blood levels of malondialdehyde (MDA) in MCI-186 treated group (2.08 +/- 0.23 microM) was significantly smaller than that of 2.65 +/- 0.31 microM in control animals (p < 0.01), while sham control had an average MDA level of 1.91 +/- 0.40 microM, with p > 0.05 relative to that in the MCI-186 treated group. These data support our contention that MCI-186 reduces reperfusion injury in perfused hearts with prolonged ischemia and the mechanism for the in vivo efficacy of MCI-186 is predominantly related to its antioxidant activities.     

Evaluation of the natural refuge function for Helicoverpa arnigera (Lepidoptera: Noctuidae) within Bacillus thuringiensis transgenic cotton growing areas in north China

The density of Helicoverpa armigera (Hübner) populations on Bacillus thuringiensis Berliner (Bt) transgenic cotton, corn, peanut, and soybean; differences in its development on Bt cotton and common (nontransgenic) cotton; and the potential for mating among populations from Bt cotton fields and other crop fields were investigated in the suburbs of Xinxiang City (Henan Province) and Langfang City (Hebei Province) in the southern and northern parts of north China, respectively. Although development of H. armigera on Bt cotton was much slower than on common cotton, there was a still high probability of mating between populations from Bt cotton and other sources due to the scattered emergence pattern of H. armigera adults, and overlap of the second and third generations. In a cotton and corn growing region, early and late planted corn provided suitable refugia for the third and fourth generations of H. armigera, but not for the second generation. In a cotton and soybean/ peanut mix system, noncotton crops provided a natural refugia from the second- to fourth-generation H. armigera, but function of the refuge would closely depend on the proportion of Bt cotton. Consequently, it may be necessary to compensate the original mixed cropping patterns in different areas for delaying resistance development of H. armigera to Bt cotton.     

Side-chain metabolism of propranolol: involvement of monoamine oxidase and mitochondrial aldehyde dehydrogenase in the metabolism of N-desisopropylpropranolol to naphthoxylactic acid in rat liver

We examined the metabolism of N-desisopropylpropranolol (NDP), which is generated from propranolol (PL) by side-chain N-desisopropylation, to naphthoxylactic acid (NLA) in rat liver. S(-)-NDP (S-NDP) and R(+)-NDP (R-NDP) were enantioselectively metabolized to NLA in isolated rat hepatocytes and in an enzyme reaction system of rat liver mitochondria with cofactor NAD+. Furthermore, the clearance profiles of NDP enantiomers were examined in an enzyme reaction system of rat liver mitochondria without NAD+. The amounts of S-NDP remaining in the incubation medium were similar to those of R-NDP, suggesting that monoamine oxidase (MAO) catalyzes the deamination of NDP to the aldehyde intermediate, but fails to deaminate enantioselectively S-NDP or R-NDP. Cyanamide, a potent inhibitor of aldehyde dehydrogenase (ALDH), markedly decreased the formation of NLA from racemic NDP in the enzyme reaction system of rat liver mitochondria with NAD+. When rat liver cytosol and microsomes were added to this enzyme reaction system, no significant alterations were observed in the amount of NLA generated from racemic NDP. We concluded that MAO deaminates NDP to an aldehyde intermediate, and that mitochondrial ALDH subsequently catalyzes the enantioselective metabolism of the aldehyde intermediate to NLA in rat liver.