Characterization and epitope mapping of monoclonal antibodies to the nucleocapsid protein of severe acute respiratory syndrome coronavirus

The sudden emergence of severe acute respiratory syndrome (SARS) at the end of 2002 resulted in 774 reported deaths from more than 8000 cases worldwide. As no effective vaccines or antiviral agents are available, the most effective measure to prevent the expansion of a SARS epidemic is the rapid diagnosis and isolation of SARS patients. To establish specific diagnostic methods, we generated nine clones of monoclonal antibodies to nucleocapsid protein (NP) of SARS-coronavirus (SARS-CoV). On immunofluorescent antibody assay and Western blotting analysis, none of the monoclonal antibodies showed cross-reactivity to authentic and recombinant NPs of human coronavirus (HCoV) 229E strain. To determine the region on the NP molecule where the monoclonal antibodies bind, we generated four truncated recombinant NPs and analyzed the reactivity between monoclonal antibodies and truncated NPs. Two monoclonal antibodies reacted with a truncated NP covering from amino acid residues 111 to 230, and seven reacted with another truncated NP covering from amino acid residues 221 to 340. Epitope mapping analysis indicated that monoclonal antibody SN5-25 recognized the amino acid sequence Q(245)TVTKK(250) On SARS-NP. Within the epitope, Q245, T246, V247, K249, and K250 appeared to form an essential motif for monoclonal antibody SN5-25 to bind. The information about binding sites and epitopes of monoclonal antibodies may be useful for the development of new diagnostic methods for SARS and for analyzing the function of N protein of SARS-CoV.     

Search for low-mass exoplanets by gravitational microlensing at high magnification

Observations of the gravitational microlensing event MOA 2003-BLG-32/OGLE 2003-BLG-219 are presented, for which the peak magnification was over 500, the highest yet reported. Continuous observations around the peak enabled a sensitive search for planets orbiting the lens star. No planets were detected. Planets 1.3 times heavier than Earth were excluded from more than 50% of the projected annular region from approximately 2.3 to 3.6 astronomical units surrounding the lens star, Uranus-mass planets were excluded from 0.9 to 8.7 astronomical units, and planets 1.3 times heavier than Saturn were excluded from 0.2 to 60 astronomical units. These are the largest regions of sensitivity yet achieved in searches for extrasolar planets orbiting any star.     

Relationship between the chemiluminescent response of bovine neutrophils and changes in intracellular free calcium concentration.

The relationship between luminol dependent chemiluminescent (LDCL) response and changes in intracellular free Ca2+ concentrations in the bovine neutrophils was evaluated. LDCL responses and changes in intracellular Ca2+ concentrations of neutrophils were clearly detected by the stimulation with opsonized zymosan (OPZ), concanavalin A(ConA), heat-aggregated IgG (H-agg.IgG) and phorbol myristate acetate (PMA). Patterns of LDCL responses and intracellular Ca2+ of neutrophils showed characteristic features for each stimulant. PMA was a weak stimulant of the intracellular Ca2+ concentration, whereas it was a strong stimulant of LDCL response. Con A strongly stimulated an increase in the intracellular Ca2+ concentration, but was a weak stimulant of LDCL response. LDCL response of intracellular Ca(2+)-depleted neutrophils treated with ionomycin, stimulated with each stimulant was inhibited markedly without extracellular Ca2+. The sustained phase of intracellular Ca2+ concentrations stimulated with OPZ was inhibited significantly (P < 0.05) by the preincubation with anti-CD18 antibody, whereas the transient phase of intracellular Ca2+ concentrations was not inhibited. These results indicate that LDCL response is regulated at least in part by the elevation of the intracellular Ca2+, and a rise in intracellular Ca2+ concentration, which may be mediated by specific receptors appears to be essential in the LDCL response of bovine neutrophils.     

云南省水稻白叶枯病菌致病力差异的地理分布

使用12个分别持有不同抗病基因（Xa1,Xa2,Xa3,Xa4,Xa5,Xa7,Xa8,Xa10,Xa11,Xa13,Xa14和Xa21）的近等基因系水稻白叶枯病鉴别品种及3个对照品种（IR24，Toyonishiki和Sigadagabo），鉴别了从云南省收集的138个菌株。结果根据致病谱可以将这些菌株分成A－J共10组。A组菌株仅对Toyonishiki和Sigadagabo两个对照品种致病，J组菌株则能对9个鉴别品种和3个对照品种致病。对不同海拔采集的菌株进行致病力分析的结果表明，从大理、武定等较高海拔粳稻种植区采集的菌株致病力较弱，在鉴别品种上病斑的平均长度不到50mm；而元江、景洪、潞西等低海拔杂交稻种植区采集的菌株致病力较强，在鉴别品种上的病斑平均长度超过100mm。从高海拔稻区到低海拔稻区，病菌致病力有逐步增强的趋势。     

Antioxidant activities of pomegranate fruit extract and its anthocyanidins: delphinidin, cyanidin, and pelargonidin.

Antioxidant activities of freeze-dried preparations of a 70% acetone extract of pomegranate (Punica granatum L.) and its three major anthocyanidins (delphinidin, cyanidin, and pelargonidin) were evaluated. Free radical scavenging activities were examined using an ESR technique with spin trapping; DMPO for hydroxyl (.OH) and superoxide (O(2)(.-) ) radicals; and [(MGD)(2)Fe(2+)] for nitric oxide (NO). Inhibitory effects on lipid peroxidation were estimated by the levels of malonaldehyde and 4-hydroxyalkenals in rat brain homogenates. Pomegranate extract exhibited scavenging activity against.OH and O(2)(.-). Anthocyanidins inhibited a Fenton reagent.OH generating system possibly by chelating with ferrous ion. Anthocyanidins scavenged O(2)(.)- in a dose-dependent manner. The ID(50) values of delphinidin, cyanidin, and pelargonidin were 2.4, 22, and 456 microM, respectively. In contrast, anthocyanidins did not effectively scavenge NO. Anthocyanidins inhibited H(2)O(2)-induced lipid peroxidation in the rat brain homogenates. The ID(50) values of delphinidin, cyanidin, and pelargonidin for them were 0.7, 3.5, and 85 microM, respectively. These findings suggest that the above anthocyanidins contribute to the antioxidant activity of pomegranate fruits.     

Response of Wheat Grain to ABA and Imbibition at Low Temperature

During after-ripening of wheat grains, embryo responsiveness to abscisic acid (ABA) decreases in parallel with the loss of grain dormancy. Dormant grains of Triticum aestivum L. line ‘Kitakei-1354’ that had lost almost half their dormancy due to chilling still respond to ABA like fully dormant grains. Imbibition a: low temperature appears to break the dormancy without change of embryo responsiveness to ABA. Gibberellin (GA) and ABA-regulated α-amylase synthesis of the embryos. This is amplified when the grains are imbibed at low temperature. Imbibition at low temperature appears to condition cells and tissues of the embryo for germination and α-amylase synthesis.     

Breeding of ‘Manten-Kirari’, a non-bitter and trace-rutinosidase variety of Tartary buckwheat (Fagopyrum tataricum Gaertn.)

Here, we developed a new Tartary buckwheat cultivar ‘Manten-Kirari’, whose flour contains only trace amounts of rutinosidase and lacked bitterness. The trace-rutinosidase breeding line ‘f3g-162’ (seed parent), which was obtained from a Nepalese genetic resource, was crossed with ‘Hokkai T8’ (pollen parent), the leading variety in Japan, to improve its agronomic characteristics. The obtained progeny were subjected to performance test. ‘Manten-Kirari’ had no detectable rutinosidase isozymes in an in-gel detection assay and only 1/266 of the rutinosidase activity of ‘Hokkai T8’. Dough prepared from ‘Manten-Kirari’ flour contained almost no hydrolyzed rutin, even 6 h after the addition of water, whereas the rutin in ‘Hokkai T8’ dough was completely hydrolyzed within 10 min. In a sensory evaluation of the flour from the two varieties, nearly all panelists detected strong bitterness in ‘Hokkai T8’, whereas no panelists reported bitterness in ‘Manten-Kirari’. This is the first report to describe the breeding of a Tartary buckwheat cultivar with reduced rutin hydrolysis and no bitterness in the prepared flour. Notably, the agronomic characteristics of ‘Manten-Kirari’ were similar to those of ‘Hokkai T8’, which is the leading variety in Japan. Based on these characteristics, ‘Manten-Kirari’ is a promising for preparing non-bitter, rutin-rich foods.     

Demonstration of Akabane virus antigen using immunohistochemistry in naturally infected newborn calves

Eight newborn calves showing ataxia were necropsied and examined histologically. Six of seven cerebrospinal fluid samples collected from these animals had neutralizing antibody for Akabane virus (AKV). All examined calves had nonsuppurative encephalomyelitis, localized mainly in the midbrain and spinal cord. Corresponding to the encephalitic lesion, AKV antigen was demonstrated in neuroglial cells in the brain stem and neuronal cells in the ventral horn of the spinal cord. This is the first study to demonstrate AKV antigen by immunohistochemistry in naturally infected newborn calves.     

Homogeneity of <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> from farmed amberjack <Emphasis Type="Italic">Seriola dumerili</Emphasis> in Japan

Streptococcus dysgalactiae strains have been isolated from cultured amberjack Seriola dumerili and yellowtail Seriola quinqueradiata in Japan. To characterize the fish isolates, we performed genetic analysis and compared the biochemical properties of these isolates with those of the S. dysgalactiae subsp. dysgalactiae and S. dysgalactiae subsp. equisimilis strains isolated from mammals. The genetic analysis revealed that the fish isolates were genetically very similar to each other with high DNA–DNA relatedness (>95.4%) and sequence homology. Meanwhile, the DNA relatedness between mammalian isolates and the fish isolates was 73.4–82.6%. In biased sinusoidal gel electrophoresis (BSFGE) analysis, the restriction patterns of mammalian isolates were different from those of fish isolates. The fish isolates did not show streptokinase activity in plasminogen obtained from mammals. These characteristics enabled us to distinguish between the fish isolates and the Sdd and Sde strains isolated from mammals. In order to obtain epidemiological information on the fish isolates, BSFGE patterns from 284 S. dysgalactiae strains from fish in Japan were examined. Based on the results of BSFGE analysis, the fish isolates were classified into 16 groups (AP1–AP16) with restriction enzyme ApaI. The dendrogram based on BSFGE analysis indicated that all fish isolates using in this study were closely related.     

IP3 receptor types 2 and 3 mediate exocrine secretion underlying energy metabolism

Type 2 and type 3 inositol 1,4,5-trisphosphate receptors (IP3R2 and IP3R3) are intracellular calcium-release channels whose physiological roles are unknown. We show exocrine dysfunction in IP3R2 and IP3R3 double knock-out mice, which caused difficulties in nutrient digestion. Severely impaired calcium signaling in acinar cells of the salivary glands and the pancreas in the double mutants ascribed the secretion deficits to a lack of intracellular calcium release. Despite a normal caloric intake, the double mutants were hypoglycemic and lean. These results reveal IP3R2 and IP3R3 as key molecules in exocrine physiology underlying energy metabolism and animal growth.