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.     

Ultrastructure of a Hexagonal Array in Exosporium of a Highly Sporogenic Mutant of Clostridium botulinum Type A Revealed by Electron Microscopy Using Optical Diffraction and Filtration

The ultrastructure of a hexagonal array in the exosporium from spores of a highly sporogenic mutant of Clostridium botulinum type A strain 190L was studied by electron microscopy of negatively stained exosporium fragments using optical diffraction and filtration. The exosporium was composed of three or more lamellae showing an equilateral, hexagonal periodicity. Images of the single exosporium layer from which the noise had been filtered optically revealed that the hexagonally arranged, morphological unit of the exosporium was composed of three globular subunits about 2.1 nm in diameter which were arranged at the vertices of an equilateral triangle with sides of about 2.4 nm. The morphological units were arranged with a spacing of about 4.5 nm. The adjacent globular subunits appeared to be interconnected by delicate linkers.     

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)     

Retinal Attachment Instability Is Diversified among Mammalian Melanopsins

Melanopsins play a key role in non-visual photoreception in mammals. Their close phylogenetic relationship to the photopigments in invertebrate visual cells suggests they have evolved to acquire molecular characteristics that are more suited for their non-visual functions. Here we set out to identify such characteristics by comparing the molecular properties of mammalian melanopsin to those of invertebrate melanopsin and visual pigment. Our data show that the Schiff base linking the chromophore retinal to the protein is more susceptive to spontaneous cleavage in mammalian melanopsins. We also find this stability is highly diversified between mammalian species, being particularly unstable for human melanopsin. Through mutagenesis analyses, we find that this diversified stability is mainly due to parallel amino acid substitutions in extracellular regions. We propose that the different stability of the retinal attachment in melanopsins may contribute to functional tuning of non-visual photoreception in mammals.     

The APC/C activator Cdh1 regulates the G2/M transition during differentiation of placental trophoblast stem cells

Differentiation of placental trophoblast stem (TS) cells to trophoblast giant (TG) cells is accompanied by transition from a mitotic cell cycle to an endocycle. Here, we report that Cdh1, a regulator of the anaphase-promoting complex/cyclosome (APC/C), negatively regulates mitotic entry upon the mitotic/endocycle transition. TS cells derived from homozygous Cdh1 gene-trapped (Cdh1^GT/GT) murine embryos accumulated mitotic cyclins and precociously entered mitosis after induction of TS cell differentiation, indicating that Cdh1 is required for the switch from mitosis to the endocycle. Furthermore, the Cdh1^GT/GT TS cells and placenta showed aberrant expression of placental differentiation markers. These data highlight an important role of Cdh1 in the G2/M transition during placental differentiation.     

Polyclonal Antibody Production in Murine Spleen Cells Induced by Staphylococcus

Polyclonal plaque-forming cell (PFC) responses in murine spleen cells induced by Staphylococcus aureus and S. epidermidis were studied. Injection of Balb/c mice with S. aureus strain 248βH resulted in the generation of anti-trinitrophenyl (TNP) and anti-sheep red blood cell PFC in their spleens. Cultures of Balb/c spleen cells in the presence of S. aureus 248βH, Cowan I, or a protein A-deficient mutant yielded many anti-TNP PFC. The larger the number of organisms that were added to the cultures, the better was the PFC response. Both living and killed organisms, were capable of inducing the response, but an excess of living 248βH organisms in the cultures abrogated the response. All of the organisms (12 strains of S. aureus and 11 strains of S. epidermidis) freshly isolated from patients had the ability to induce the polyclonal PFC response in cell cultures. These organisms stimulated cultured C3H/HeJ mouse spleen cells, which were unresponsive to bacterial lipopolysaccharide (LPS). Cultured cells from the spleens of athymic nu/nu mice also responded to these organisms, and the number of PFC in nu/nu cell cultures was always greater than that in nu/+ cells prepared from a haired litter mate. Moreover, the responses of nu/nu spleen cell cultures to which nylon wool column-filtered splenic nu/+ T cells were added were lower than expected. These findings suggest that the polyclonal PFC response to staphylococci is thymus independent, but that the magnitude of the response is regulated by mature T cells. Cultures of macrophage-depleted spleen cells responded to the organisms to an extent similar to that of the control. The 248βH organisms were less capable of stimulating spleen cells of 2-week-old mice (i.e., early maturing B cells) than LPS. However, spleen cells from adult (7-week-old) and aged (9-month-old) mice responded well to both the organisms and LPS. Previous sensitization with the organisms in vivo did not affect any polyclonal responses of spleen cells in vitro to either the organisms or LPS. The role of staphylococcal protein A in the polyclonal PFC response to staphylococci is discussed.     

Tangential cell migration during layer formation of chick optic tectum

The laminated structure of the optic tectum is formed by radial and tangential cell migration during development. Studies of developing chick optic tectum have revealed two streams of tangential cell migration in the middle and superficial layers, which have distinctive origins, migratory paths, modes of migration, and destinations. We will review the process of the two types of tangential migrations, in order to elucidate their roles in the formation of the optic tectum layers.