Effects of the tom1 mutation of Arabidopsis thaliana on the multiplication of tobacco mosaic virus RNA in protoplasts.

For the multiplication of RNA viruses, specific host factors are considered essential, but as of yet little is known about this aspect of virus multiplication. To identify such host factors, we previously isolated PD114, a mutant of Arabidopsis thaliana, in which the accumulation of the coat protein of tobacco mosaic virus (TMV) in uninoculated leaves of an infected plant was reduced to low levels. The causal mutation, designated tom1, was single, nuclear, and recessive. Here, we demonstrate that the tom1 mutation affects the amplification of TMV-related RNAs in a single cell. When protoplasts were inoculated with TMV RNA by electroporation, the percentage of TMV-positive protoplasts (detected by indirect immunofluorescence staining with anti-TMV antibodies) was lower (about 1/5 to 1/10) among PD114 protoplasts than among wild-type protoplasts. In TMV-positive PD114 protoplasts, the amounts of the positive-strand RNAs (the genomic RNA and subgenomic mRNAs) and coat protein reached levels similar to, or slightly lower than, those reached in TMV-positive wild-type protoplasts, but the accumulation of the positive-strand RNAs and coat protein occurred more slowly than with the wild-type protoplasts. The parallel decrease in the amounts of the coat protein and its mRNA suggests that the coat protein is translated from its mRNA with normal efficiency. These observations support the idea that the TOM1 gene encodes a host factor necessary for the efficient amplification of TMV RNA in an infected cell. Furthermore, we show that TMV multiplication in PD114 protoplasts is severely affected by the coinoculation of cucumber mosaic virus (CMV) RNA. When PD114 protoplasts were inoculated with a mixture of TMV and CMV RNAs by electroporation, the accumulation of TMV-related molecules was approximately one-fifth of that in PD114 protoplasts inoculated with TMV RNA alone. No such reduction in the accumulation of TMV-related molecules was observed when wild-type protoplasts were inoculated with a mixture of TMV and CMV RNAs or when wild-type and PD114 protoplasts were inoculated with a mixture of TMV and turnip crinkle virus RNAs. These observations are compatible with a hypothetical model in which a gene(s) that is distinct from the TOM1 gene is involved in both TMV and CMV multiplication.     

Polymorphism of Crystals of Salmonella minnesota Re and Ra Lipopolysaccharides

Salmonella minnesota Re and Ra lipopolysaccharides (LPSs) formed three-dimensional crystals when they were precipitated by the addition of 2 volumes of 95% ethanol containing 375 mM MgCl₂ and incubated in 70% ethanol containing 250 mM MgCl₂ at 4 C. Besides typical shapes of crystals, hexagonal plates and solid columns, which were already reported (J. Bacteriol. 172: 1516–1528 (1990)), the LPSs thus treated formed crystals possessing various shapes such as square or rectangular plate, lozenge plate, discoid, and truncated hexangular pyramid forms. Electron diffraction patterns from all these crystals except square or rectangular plate crystals obtained by electron irradiation from the direction perpendicular to the basal plane were essentially the same as those from hexagonal plate crystals, indicating that they consist of hexagonal lattices with the lattice constant of 4.62 Å. From these results as well as the results of electron microscopic observations of these crystals, it was concluded that all these crystals except square or rectangular plate crystals are composed of hexagonal plate sheets as the basic structural units. Square or rectangular crystals were assumed to correspond to the {1011} planes of solid hexagonal column crystals.     

Diving pattern and performance in the macaroni penguin Eudptes chrysolophus

The pattern and characteristics of diving in two female macaroni penguins Eudyptes chrysolophus was studied, during the brooding period, using continuous-recording time-depth recorders, for a total of I8 days (15 consecutive days) during which the depth, duration and timing of 4876 dives were recorded. Diving in the first 11 days was exclusively diurnal, averaging 244 dives on trips lasting 12 hours. Near the end of the brooding period trips were longer and included diving at night. About half of all trips (except those involving continuous night-time diving) was spent in diving and dive rate averaged 14–25 dives per hour (42 per hour at night). The duration of day time dives varied between trips, and averaged 1.4–1.7 min, with a subsequent surface interval of 0.5–0.9 min. Dive duration was significantly directly related to depth, the latter accounting for 53% of the variation. The average depths of daytime dives were 20–35 m (maximum depth 11 5 m). Dives at night were shorter (average duration 0.9 min) and much shallower (maximum 11 m); depth accounted for only 6% of the variation in duration. Estimates of potential prey capture rates (3–5 krill per dive; one krill every 17–20 s) are made. Daily weight changes in chicks were directly related to number of dives, but not to foraging trip duration nor time spent diving. Of the other species at the same site which live by diving to catch krill, gentoo penguins forage exclusively diurnally, making longer. deeper dives; Antarctic fur seals, which dive to similar depths as macaroni penguins, do so mainly at night.     

Mapping of the human C3G gene coding a guanine nucleotide releasing protein for Ras family to 9q34.3 by fluorescence in situ hybridization

C3G, a human guanine nucleotide releasing protein for Ras protein, was mapped to human chromosome 9q34.3 by fluorescence in situ hybridization with Rbanded chromosomes. C3G was originally identified as one of the CRK-binding proteins, similar to c-ab1 (9q34.1). Our result suggests that the downstream factors of Crk are localized in close proximity on chromosome 9.     

Cytokine-mediated antitumor effect of bacillus Calmette-Guérin on tumor cells in vitro

Intravesical instillation therapy of bacillus Calmette-Guérin (BCG) is a useful modality for recurrent superficial transitional-cell carcinoma (TCC) of the urinary bladder. The mechanism of BCG effect has not yet been well characterized. BCG was tested in vitro for cytokine-mediated antiproliferative activity against T24 and KK47 cells (cell lines established from human TCC of the urinary bladder), and ACHN cells (cell line established from human renal cell carcinoma) using a modified human tumor clonogenic assay. Continuous exposure of cells to BCG at concentrations of more than 5 g/ml in the presence of peripheral blood mononuclear cells (PBMC) consisting of a mixture of 5×10⁴ monocytes/dish and 5×10⁵ lymphocytes/dish, obtained from healthy donors, significantly inhibited colony formation of T24 and ACHN cells in comparison with growth inhibition in the absence of PBMC (P<0.05). Slightly inhibited colony formation was observed with KK47 cells under the same conditions. At the same time various cytokines were measured in supernatants when BCG and the same conditioned PBMC were co-cultured. Tumor necrosis factor (TNF) and interleukin-1 (IL-1) were detected at markedly high levels at 24 h, and interferon (IFN) was detected at 120 h. IL-2 and macrophage-colony-stimulating factor were not detected. Neutralizing anti-TNF monoclonal antibody significantly reduced the anti-proliferative activity of ACHN cells, and anti-IFN antibody reduced that of T24 cells. The results obtained suggest that cytokines mediated by BCG play an important role in the antitumor activity of BCG and that the sensitivity of bladder cancer cells to the cytokines induced by BCG may differ considerably.     

Developmental ability of porcine ova matured in porcine follicular fluid in vitro and fertilized in vitro

Developmental ability of porcine ova matured in porcine follicular fluid (pFF) with FSH in vitro and fertilized in vitro was examined by culturing in BMOC-2. Forty-eight hours after insemination, 35.6% of ova cleaved normally, and this rate was significantly higher (13.0%) than that of the ova matured in a modified Krebs-Ringer bicarbonate solution. Twenty-four percent (29 120 ) of ova matured in pFF with FSH developed to the four-cell stage and two of them developed to the eight-cell stage 66 h after insemination. Most cleaved embryos stopped developing at the four-cell stage and neither the morula nor blastocyst stage was observed throughout the culture period as reported in the in vivo matured ova. In culture at 37 degrees C, the appearance of two-cell and four-cell embryos was delayed from that of in vivo embryos, but their development was significantly accelerated by culturing at 39 degrees C. These results show that pFF is an excellent maturation medium for porcine oocytes, and the developmental capacity of the ova matured in pFF seems to be similar to that of in vivo matured ova. Culturing at 39 degrees C was found to be more suit-able for the development of ova than 37 degrees C.     

Tryptophan 2,3-dioxygenase activity in rat skin

We have found an enzyme system that catalyzes the conversion of L-tryptophan to L-kynurenine, presumably via L-formylkynurenine, in soluble and insoluble fractions of rat skin. The enzymatic activity was stimulated by hematin, ascorbate, and catalase, but not by methylene blue. Highest activity was located in the skin of the dorsal posterior region and lowest activity in the abdominal region. The activity in plucked (depilated) skin was only about 25% of that obtained from unplucked (depilated) tissue of the same region. D-Tryptophan, 5-hydroxytryptophan, and tryptamine were not degraded by the skin enzyme and the Km for L-tryptophan determined with the crude enzyme was 1 microM. The decycling activity of rat skin and liver for L-tryptophan began to be stimulated after birth and reached the highest level at 6 weeks. But, 1 week later, most of the skin activity suddenly disappeared and the low level continued at least until 12 weeks. In contrast, the hepatic enzyme did not change so drastically. These findings suggest that an enzyme that catalyzes L-tryptophan to L-kynurenine via L-formylkynurenine is present in rat skin.     

Role of lipoprotein-copper complex in copper catalyzed-peroxidation of low-density lipoprotein.

The oxidative modification of low-density lipoprotein (LDL) is suggested to play an important role in the pathogenesis of atherosclerosis. The present study examined the role of the formation of LDL-copper (Cu) complex in the peroxidation of LDL. The amount of copper bound to LDL increased during incubation performed with increasing concentrations of CuSO4. More than 80% of the copper bound to the LDL particle was observed in the protein phase of LDL, suggesting that most of the copper ions formed complexes with the ligand-binding sites of apoprotein. The addition of histidine (1 mM), known to form a high affinity complex with copper, and EDTA (1 mM), a metal chelator, during the incubation of LDL with CuSO4 prevented the formation of both thiobarbituric acid-reactive substances (TBARS) and LDL-Cu complexes. EDTA inhibited the copper-catalyzed ascorbate oxidation whereas histidine had no effect, suggesting that the copper within the complex with histidine is available to catalyze the reaction, in contrast to EDTA. These observations indicate that the preventive effect of histidine on the copper-catalyzed peroxidation of LDL is not simply mediated by chelating free copper ions in aqueous phase. Evidence that copper bound to LDL particle still has a redox potential was provided by the observed increase in TBARS content during incubation of LDL-Cu complexes in the absence of free copper ions. The addition of either histidine or EDTA to LDL-Cu complexes inhibited the formation of TBARS by removing copper ions from the LDL forming the corresponding complexes. However, there still remained small amounts of copper in the LDL particles following the treatment of LDL-Cu complexes with histidine or EDTA. The copper ions remaining in the LDL particle lacked the ability to catalyze LDL peroxidation, suggesting that there may be two types of copper binding sites in LDL: tight-binding sites, from which the copper ions are not removed by chelation, and weak-binding sites, from which copper ions are easily removed by chelators. The formation of TBARS in the LDL preparation during incubation with CuSO4 was comparable to the incubation with FeSO4. In contrast, the formation of TBARS in the LDL-lipid micelles by CuSO4 was nearly eliminated even in the presence of ascorbate to promote metal-catalyzed lipid peroxidation, although a marked increase in TBARS content was observed in the LDL-lipid micelles with FeSO4, and with FeCl3 in the presence of ascorbate.(ABSTRACT TRUNCATED AT 400 WORDS)