Ibaraki Virus,an Agent of Epizootic Disease of Cattle Resembling Bluetongue

Replication of Ibaraki virus was not inhibited by 5-iodo-2′-deoxyuridine, indicating that the virus is an RNA virus. The virus was resistant to ether, chloroform and deoxycholate, sensitive to trypsin, very labile at acidic pH but stable at pH 6.4 or higher, and was resistant to repeated freezing and thawing. The virus was readily inactivated at 56 C or higher, was fairly stable at 37 C, and very stable at 4 C, while it rapidly lost infectivity when stored frozen at —20 C. The virus was readily sedimented by centrifugation at 40 000Xg for 60 min. It readily passed through membrane filters of 200 mμ pore size, passed through 100 μfilters but only with some titer loss and did not through 50 mμ filters. In these tests, the bluetongue virus used as a control behaved in the same manner as Ibaraki virus. These findings provide additional evidence for the similarity of Ibaraki virus to bluetongue virus which had been previously demonstrated on the basis of seasonal incidence, symptomatology and pathology of the diseases caused by these viruses and the behavior of the viruses in cell cultures, embryonated eggs and laboratory animals. The present study, however, provided no evidence for any serological relation between these two viruses. More Information is needed to reach a final decision on the classification of Ibaraki virus, particularly regarding the morphology of the virion, the doublestrandedness of the viral RNA and other basic features.     

Involvement of the Binuclear Copper Site in the Proteolytic Activity of Polyphenol Oxidase

Plant polyphenol oxidase (PPO) is apt to degrade during andeven after purification. We developed a method to stabilizePPO by 0.3 M NaCl, 0.1% (w/v) Tween 20, and 50% (w/v) ethyleneglycol at pH 6.5. The protein slowly degraded by itself whenthe stabilizing reagents were removed. Ascorbate and/or H₂O₂accelerated the degradation. The ascorbate-induced degradationwas inhibited by catalase, suggesting that H₂O₂ is generatedthrough reduction of PPO by ascorbate. It is likely that dissolvedoxygen is converted to peroxide through two-electron reductionby the reaction center of PPO, binuclear Cu site, and a Fenton-typereaction occurred on it. This understanding was supported bythe finding that the H₂O₂-induced degradation was inhibitedby metal-chelators as well as by polyphenolic substrate of PPO.Considering the postulated mechanism of the self-degradationof PPO, we re-examined the degradation of the 23-kDa proteinof PSII by PPO [Kuwabara et al. (1997) Plant Cell Physiol. 38:179]. The obtained results suggested that the 23-kDa proteintriggers the active oxygen production by the binuclear Cu site,probably as reductant, and receives the radical species preferentiallyto the polypeptide moiety of PPO. (Received April 15, 1999; Accepted July 21, 1999)     

Culture of newborn monkey liver epithelial progenitor cells in chemical defined serum-free medium

Studies with hepatic progenitor cells from non-human primates would allow better understanding of their human counterparts. In this study, rhesus monkey liver epithelial progenitor cells (mLEPCs) were derived from a small piece of newborn livers in chemical defined serum-free medium. Digested hepatic cells were treated in Ca²⁺-containing medium to form cell aggregates. Two types of cell aggregates were generated: elongated spindle cells and polygonal epithelial cells. Elongated spindle cells were expressed as vimentin and brachyury, and they were disappeared within 5 d in our cultures. The remaining type consisted of small polygonal epithelial cells that expressed cytokeratin 7 (CK7), CK8, CK18, nestin, CD49f, and E-cad, the markers of hepatic stem cells, but were negative for α-fetoprotein, albumin, and CK19. They can proliferate and be passaged, if on laminin or rat tail collagen gel, to initiate colonies. When cultured with dexamethasone and oncostatin M, the expression of mature hepatocyte markers, such as α-1-antitrypsin, intracytoplasmic glycogen storage, indocyanine green uptake, and lipid droplet generation, were induced in differentiated cells. If transferred onto mouse embryonic fibroblasts feeders, they gave rise to CK19-positive cholangiocytes with formation of doughnut-like structure. Thus, mLEPCs with bipotency were derived from newborn monkey liver and may serve as a preclinical model for assessment of cell therapy in humans.     

Pollen size-number trade-off and pollen-pistil relationships in Pedicularis (Orobanchaceae)

Current patterns of floral design in Pedicularis must have undergone an evolutionary process of interacting among components of floral traits, and then formed internal relationships among these traits. To detect such correlations, which may provide insight to understand flower evolution, 40 Pedicularis species representing all corolla types of the genus were studied. Results show that, interspecifically, pollen size correlates negatively with pollen number, but positively with pistil length. This suggests that plants evolve an optimal pollen size, which balances the advantages of large pollen size for gametophytic competition against the fecundity disadvantages of fewer pollen grains. In contrast to sex allocation theory, this study does not find a trade-off, but an interspecific positive correlation between pollen and ovule number. This is consistent with the hypothesis that genetic variation for resource acquisition may in part be responsible for the lack of negative correlation between male and female function.This work was supported by the State Key Basic Research and Development Plan, China (Grant No. G2000046804) to YHG. The authors would like to thank Peter K. Endress and two anonymous reviewers for providing critical comments and helpful suggestions, Qing-Feng Wang, Jing-Yuan Wang and Jin-Ming Chen for their helpful suggestions. Shi-Guo Sun, Jing Xia, and Qian Yu are thanked for their assistance in both the field work and laboratory phases of the project.     

Corolla wilting facilitates delayed autonomous self-pollination in Pedicularis dunniana (Orobanchaceae)

Structural changes associated with corolla wilting may serve as a mechanism for effecting self-pollination. Low pollinator visitation, high seed production and a corolla that persists after anthesis indicates that Pedicularis dunniana is autogamous. Delayed autonomous self-pollination is facilitated by corolla wilting. Wilting of the upper lip (galea) brought the pollen laden anthers into contact with the stigma resulting in the deposition of self pollen on the stigma. The seed set of flowers either emasculated, or with restrained galeae thus preventing anthers brushing against the stigma, was significantly lower than that of open-pollinated flowers. This demonstrates that autogamy occurs in this species through corolla wilting. Germination experiments indicated that outcross seedlings were more vigorous than selfed seedlings as a result of inbreeding depression. It is likely that autogamy provides reproductive assurance for P. dunniana under conditions of pollinator scarcity.     

A Novel Hyaluronidase Produced by Bacillus sp. A50

Hyaluronidases are a family of enzymes that degrade hyaluronic acid (hyaluronan, HA) and widely used in many fields. A hyaluronidase producing bacteria strain was screened from the air. 16S ribosomal DNA (16S rDNA) analysis indicated that the strain belonged to the genus Bacillus, and the strain was named as Bacillus sp. A50. This is the first report of a hyaluronidase from Bacillus, which yields unsaturated oligosaccharides as product like other microbial hyaluronate lyases. Under optimized conditions, the yield of hyaluronidase from Bacillus sp. A50 could reach up to 1.5×10⁴ U/mL, suggesting that strain A50 is a good producer of hyaluronidase. The hyaluronidase (HAase-B) was isolated and purified from the bacterial culture, with a specific activity of 1.02×10⁶ U/mg protein and a yield of 25.38%. The optimal temperature and pH of HAase-B were 44°C and pH 6.5, respectively. It was stable at pH 5–6 and at a temperature lower than 45°C. The enzymatic activity could be enhanced by Ca²⁺, Mg²⁺, or Ni²⁺, and inhibited by Zn²⁺, Cu²⁺, EDTA, ethylene glycol tetraacetic acid (EGTA), deferoxamine mesylate salt (DFO), triton X-100, Tween 80, or SDS at different levels. Kinetic measurements of HAase-B towards HA gave a Michaelis constant (K _m) of 0.02 mg/mL, and a maximum velocity (V _max) of 0.27 A ₂₃₂/min. HAase-B also showed activity towards chondroitin sulfate A (CSA) with the kinetic parameters, K _m and V _max, 12.30 mg/mL and 0.20 A ₂₃₂/min respectively. Meanwhile, according to the sequences of genomic DNA and HAase-B’s part peptides, a 3,324-bp gene encoding HAase-B was obtained.     

Enhanced viability of Lactobacillus reuteri for probiotics production in mixed solid-state fermentation in the presence of Bacillus subtilis

In order to develop a multi-microbe probiotic preparation of Lactobacillus reuteri G8-5 and Bacillus subtilis MA139 in solid-state fermentation, a series of parameters were optimized sequentially in shake flask culture. The effect of supplementation of B. subtilis MA139 as starters on the viability of L. reuteri G8-5 was also explored. The results showed that the optimized process was as follows: water content, 50 %; initial pH of diluted molasses, 6.5; inocula volume, 2 %; flask dry contents, 30～35 g/250 g without sterilization; and fermentation time, 2 days. The multi-microbial preparations finally provided the maximum concentration of Lactobacillus of about 9.01?±?0.15 log CFU/g and spores of Bacillus of about 10.30?±?0.08 log CFU/g. Compared with pure fermentation of L. reuteri G8-5, significantly high viable cells, low value of pH, and reducing sugar in solid substrates were achieved in mixed fermentation in the presence of B. subtilis MA139 (P?<?0.05). Meanwhile, the mixed fermentation showed the significantly higher antimicrobial activity against E. coli K88 (P?<?0.05). Based on the overall results, the optimized process enhanced the production of multi-microbe probiotics in solid-state fermentation with low cost. Moreover, the viability of L. reuteri G8-5 could be significantly enhanced in the presence of B. subtilis MA139 in solid-state fermentation, which favored the production of probiotics for animal use.