Acute infection of encephalomyocarditis (EMC) virus in Syrian hamsters.

One-month-old Syrian hamsters of the APA and Std: golden strains were inoculated intraperitoneally with 10(5) PFU/head of the D variant of encephalomyocarditis (EMC) virus and examined virologically and pathologically up to 7 days after inoculation. APA hamsters developed apparent hyperglycemia due to pancreatic islet cell damage while Std:golden hamsters did not. Hamsters of both strains showed clear histopathologic changes in the testis with prominent viral replication as well as in the brain, heart and exocrine pancreas. The susceptibility to EMC virus-infection was higher in males than in females and in APA than in Std: golden hamsters.     

Spindle formation and microtubule organization during first division in reconstructed rat embryos produced by somatic cell nuclear transfer

The present study was conducted to demonstrate the spindle formation and behavior of chromosomes and microtubules during first division in reconstructed rat embryos produced by somatic cell nuclear transfer (SCNT) with cumulus cell nuclei. To demonstrate the effect of oocyte aging after ovulation on the cleavage of SCNT embryos, micromanipulation was carried out 11, 15 and 18 h after injection of hCG. SCNT oocytes were activated by incubation in culture medium supplemented with 5 microM ionomycin for 5 min followed by treatment with 2 mM 6-dimethylaminopurine (6-DMAP) in mR1ECM for 2-3 h. For immunocytochemical observation, the SCNT embryos were incubated with monoclonal anti-alpha-tubulin antibody and then fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG. Cleavage rates were significantly higher for oocytes collected after 15 and 18 h rather than for those collected 11 h after injection of hCG (56 and 53%, respectively vs. 28%; P<0.05). Premature chromosome condensation occurred before activation of the SCNT oocytes, but adequate spindle formation was only rarely observed. The distribution of microtubules in SCNT embryos after activation was different from those of fertilized and parthenogenic oocytes, i.e., a dense microtubule organization shaped like a ring was observed. Eighteen to 20 h post-activation, most SCNT embryos were in the 2-cell stage, but no nucleoli were clearly visible, which was quite different from the fertilized oocytes. In addition, first division with and without small cellular bodies containing DNA was observed in the rat SCNT embryos in some cases. The present study suggests that reorganization of transferred nuclei in rat SCNT embryos may be inadequate in terms of formation of the mitotic assembly and nucleolar reorganization.     

The incredible fungal genus —Drechslera — and its phytotoxic ophiobolins

ManyDrechslera species that haveCochliobolus sp. as a perfect stage produce a related group of ophiobolins, sesterterpenoids (C-25’s), which are phytotoxic to a wide range of plants. One of the most toxic ophiobolins is 6-epiophiobolin A and it is produced by all of theDrechslera species thus far studied. On the other hand, some novel ophiobolins have been found inD. oryzae: ophiobolin J, 6-epiophiobolin I, and 8-deoxyophiobolin J. Potentially, genetic crosses made between these fungi could yield novel organisms, producing novel combinations of the ophiobolins, and thus new pathotypes. Ophiobolins are being used in tissue culture systems to screen plants for sensitivity to these toxins. Paper presented at the Bat-Sheva Seminar on Host - Fungus Interaction, Jerusalem, Israel (March 14–25,1988).     

MytiLec,a Mussel R-Type Lectin,Interacts with Surface Glycan Gb3 on Burkitt’s Lymphoma Cells to Trigger Apoptosis through Multiple Pathways

MytiLec; a novel lectin isolated from the Mediterranean mussel (Mytilus galloprovincialis); shows strong binding affinity to globotriose (Gb3: Galα1-4Galβ1-4Glc). MytiLec revealed β-trefoil folding as also found in the ricin B-subunit type (R-type) lectin family, although the amino acid sequences were quite different. Classification of R-type lectin family members therefore needs to be based on conformation as well as on primary structure. MytiLec specifically killed Burkitt''s lymphoma Ramos cells, which express Gb3. Fluorescein-labeling assay revealed that MytiLec was incorporated inside the cells. MytiLec treatment of Ramos cells resulted in activation of both classical MAPK/ extracellular signal-regulated kinase and extracellular signal-regulated kinase (MEK-ERK) and stress-activated (p38 kinase and JNK) Mitogen-activated protein kinases (MAPK) pathways. In the cells, MytiLec treatment triggered expression of tumor necrosis factor (TNF)-α (a ligand of death receptor-dependent apoptosis) and activation of mitochondria-controlling caspase-9 (initiator caspase) and caspase-3 (activator caspase). Experiments using the specific MEK inhibitor U0126 showed that MytiLec-induced phosphorylation of the MEK-ERK pathway up-regulated expression of the cyclin-dependent kinase inhibitor p21, leading to cell cycle arrest and TNF-α production. Activation of caspase-3 by MytiLec appeared to be regulated by multiple different pathways. Our findings, taken together, indicate that the novel R-type lectin MytiLec initiates programmed cell death of Burkitt’s lymphoma cells through multiple pathways (MAPK cascade, death receptor signaling; caspase activation) based on interaction of the lectin with Gb3-containing glycosphingolipid-enriched microdomains on the cell surface.     

Physiological and Mineralogical Properties of Arsenic-Induced Chlorosis in Barley Seedlings Grown Hydroponically

The experiment was carried out to investigate the effects of arsenic (As) on the physiological and mineralogical properties of barley (Hordeum vulgare L. cv. ‘Minorimugi’). The plants were grown in nutrient solution treated with 0, 6.7, 33.5, and 67 μ M As (0, 0.5, 2.5, and 5 ppm As, respectively) in the phytotron. Dry matter yield of shoots and roots decreased significantly with the As treatments, indicating that barley plants are As-sensitive and As-toxicity depends on the As concentration in the rooting medium. Necrosis in older leaves and chlorosis symptoms (whitish color) in the fully developed young leaves were observed at the 33.5 and 67 μ M As treatments. Arsenic concentration, accumulation, and translocation increased with the increase of As concentration in the rooting medium. Arsenic was mostly concentrated in roots and a little amount was moved to shoots, indicating that As was not easily translocated to shoots of barley seedlings. Concentrations and accumulations of phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), manganese (Mn), zinc (Zn), and copper (Cu) decreased significantly in shoots for 33.5 and 67 μ M As treatments as compared to the 0 μ M As treatment. Concentrations of P, K, Ca, Mg, Mn, and Cu decreased in roots, but Zn concentration increased in roots at 67 μ M As treatment. Accumulations of P, K, Ca, Mg, Mn, Zn, and Cu in roots also decreased significantly at 67 μ M As treatment. Accumulation of P and the cations showed negative relationship with As. Concentration of Fe decreased in shoots at 33.5 and 67 μ M As treatments where chlorosis was induced in the young leaf but increased in roots at 33.5 and 67 μ M As treatments. It was suggested that As might induce iron (Fe)-chlorosis in the plants. Among the micronutrients, Fe translocation was more affected than others by As. Phytosiderophore (PS) accumulation in roots, which is a symptom of Fe-deficiency in grasses, did not change significantly between 0 and 33.5 μ M As treatments; indicating that As-induced chlorosis did not enhance PS accumulation in roots and decreased due to As-toxicity at 67 μ M As treatment.     

Evaluation of Intestinal Absorption of Dietary Halocynthiaxanthin,a Carotenoid from the Sea Squirt Halocynthia roretzi

Halocynthiaxanthin is an acetylenic carotenoid mainly found in Halocynthia roretzi. To date, several bioactivities of halocynthiaxanthin have been reported, but its mechanism of digestion and absorption in mammals has not been studied yet. In this study, we evaluated the intestinal absorption of halocynthiaxanthin in mice. The halocynthiaxanthin-rich fraction was prepared from the tunicate Halocynthia roretzi. Mice were orally administered the fraction at a dose of 5 mg/kg body weight. The halocynthiaxanthin levels in the plasma, liver, and small intestine, were quantified using HPLC-PDA, 1, 3, 6, and 9 h after ingestion. The halocynthiaxanthin-rich fraction mainly consisted of the all-trans form and a small amount of cis forms. These three isomers were detected in the plasma of mice 3 h after ingestion. Time-course changes after the ingestion of this fraction were found, with cis isomers being more abundant than the all-trans isomer in the mouse plasma and liver. In the small intestine, however, the all-trans isomer was primarily detected. The possibility that cis isomers might be absorbed rapidly from the small intestine cannot be denied, but our results suggest that dietary all-trans-halocynthiaxanthin might be isomerized to the cis isomer after intestinal absorption.     

Binding profiles and cytokine-inducing effects of fish rhamnose-binding lectins on Burkitt’s lymphoma Raji cells

Rhamnose-binding lectin (RBL) is one of the animal lectin categories which take part in the innate immune responses of fish. Osmerus lanceolatus lectin (OLL) from shishamo smelt eggs is an RBL composed of two tandem-repeated domains, both of which are considered to be a carbohydrate-recognition domain. SAL, catfish (Silurus asotus) egg RBL composed of three domains, binds to Burkitt’s lymphoma Raji cells through globotriaosylceramide (Gb3) carbohydrate chain and to reduce cell size and growth by altering membrane composition without causing cell death. In this experiment, we tried to compare the binding effects of these two RBLs on Raji cells. Flow cytometric and fluorescence microscopic analyses revealed that OLL also directly bound to and shrunk Raji cells with ten times less reactivity than SAL but reduced cell growth with decreasing cell viability. Anti-Gb3 antibody completely blocked the binding of SAL to Raji cells but not that of OLL. In addition, the direct bindings of OLL and SAL to Raji cells were comparably inhibited by melibiose, but lactose was more effective inhibitor for the binding of OLL than that of SAL. These results suggest that OLL has slightly different cell-binding property compared with SAL and binds not only to Gb3 but also to the other carbohydrate receptor-bearing β-galactoside chains. The quantitative RT-PCR analysis revealed that SAL induced the expression of TNF-α but not of IFN-γ, IL-1β, and IL-10. Thus, SAL-induced cytostatic effect on Raji cells might be partially caused by TNF-α-mediated signaling pathway.     

Induction of cellular senescence in immortalized cells by human chromosome 1

The control of cellular senescence by specific human chromosomes was examined in interspecies cell hybrids between diploid human fibroblasts and an immortal, Syrian hamster cell line. Most such hybrids exhibited a limited life span comparable to that of the human fibroblasts, indicating that cellular senescence is dominant in these hybrids. Karyotypic analyses of the hybrid clones that did not senesce revealed that all these clones had lost both copies of human chromosome 1, whereas all other human chromosomes were observed in at least some of the immortal hybrids. The application of selective pressure for retention of human chromosome 1 to the cell hybrids resulted in an increased percentage of hybrids that senesced. Further, the introduction of a single copy of human chromosome 1 to the hamster cells by microcell fusion caused typical signs of cellular senescence. Transfer of chromosome 11 had no effect on the growth of the cells. These findings indicate that human chromosome 1 may participate in the control of cellular senescence and further support a genetic basis for cellular senescence.     

Rapid and reliable detection of phytoplasma by loop-mediated isothermal amplification targeting a housekeeping gene

Phytoplasmas are plant pathogenic bacteria that infect more than 700 plant species. Because phytoplasma-resistant cultivars are not available for the vast majority of crops, the most common practice to prevent phytoplasma diseases is to remove infected plants. Therefore, developing a rapid, accurate diagnostic method to detect a phytoplasma infection is important. Here, we developed a phytoplasma detection assay based on loop-mediated isothermal amplification (LAMP) by targeting the groEL gene and 16S rDNA. We designed 19 primer sets for the LAMP assay and evaluated their amplification efficiency, sensitivity, and spectra to select the most suitable primer sets to detect Candidatus Phytoplasma asteris. As a result, DNA was efficiently amplified by one of the primer sets targeting the groEL gene, and LAMP assay sensitivity with this primer set was 10-fold higher than that of the polymerase chain reaction. Moreover, the groEL gene was successfully amplified from several strains of Ca. Phytoplasma asteris by this primer set, indicating that the groEL gene can be used as a LAMP assay target gene for a broad range of phytoplasma strains. Additionally, a simple DNA extraction method that omits the homogenizing and phenol extraction steps was combined with the LAMP assay to develop a simple, rapid, and convenient diagnostic method for detecting phytoplasma.     

Effect of selenium and vitamin E on oxidative stress in lambs experimentally infected with <Emphasis Type="Italic">Haemonchus contortus</Emphasis>

The objective of this study was to evaluate oxidative stress in lambs experimentally infected with Haemonchus contortus and supplemented with selenium and vitamin E. Twenty male Corriedale lambs were divided into four experimental groups with five animals each: G1 consisted of animals infected and supplemented with 0.2 mg/kg of live weight (LW) sodium selenite by intramuscular injection (IM); G2 consisted of animals infected with larvae and supplemented with 0.2 mg/kg LW sodium selenite IM and 2000 IU per animal of Vitamin E IM; G3 consisted of animals infected with larvae and supplemented with 2000 IU per animal of Vitamin E IM; and G4 consisted of animals infected with larvae. The animals were infected orally with 500 H. contortus larvae (L3) every 48 hours for 20 days. For biochemical analyses and eggs per grams of feces (EPG) evaluation, blood and feces were both collected at zero (T0), 20 (T1), 40 (T2) and 60 (T3) days. The weight of the animals was also measured at these times. Lower TBARS values were observed in the supplemented animals compared to the control group. The groups supplemented with Selenium exhibited blood GSH-Px activity higher than that of non-supplemented animals. Supplementation with selenium provided greater antioxidant protection against oxidative stress generated from experimental infection of lambs with H. contortus.