Complete Nucleotide Sequence of the Rice Tungro Spherical Virus Genome of the Highly Virulent Strain Vt6

The complete nucleotide sequence of rice tungro spherical virus (RTSV) strain Vt6, originally from Mindanao, the Philippines, with higher virulence to resistant rice cultivars, was determined and compared with the published sequence for the Philippine-type strain A (RTSV-A-Shen). It was reported that RTSV-A was not able to infect a rice resistant cultivar TKM 6 (10). RTSV-Vt6 and RTSV-A-Shen share 90% and 95% homology at nucleotide and amino-acid levels, respectively. The N-terminal leader sequence of RTSV-Vt6 contained a 39-amino acids-region (positions 65 to 103) which was totally different from that of RTSV-A-Shen; the difference resulted from frame shifting by nucleotide insertions and deletions. To confirm the amino-acid sequence differences of the leader polypeptide, the same region was cloned and sequenced using a newly obtained variant of RTSV-type 6, which had been collected in the field of IRRI, and seven field isolates from Mindanao, the Philippines. Since all the sequences of the target region are identical to that of the Vt6 leader polypeptide, the sequence difference in the leader region seems not to correlate with the virulence of Vt6.     

Three novel mutations of the fibrillin-1 gene and ten single nucleotide polymorphisms of the fibrillin-3 gene in Marfan syndrome patients

Marfan syndrome (MFS) is an autosomal dominant disorder of the extracellular matrix. Allelic variations in the gene for fibrillin-1 (FBN1) have been shown to cause MFS. To date, over 550 mutations have been identified in patients with MFS and related connective tissue diseases. However, about a half of MFS cases do not possess mutations in the FBN1 gene. These findings raise the possibility that variants located in other genes cause or modify MFS. To explore this possibility, firstly we analyzed FBN1 allelic variants in 12 Japanese patients with MFS, and secondly we analyzed fibrillin-3 gene (FBN3) in patients without FBN1 mutations using conformation sensitive gel electrophoresis (CSGE) and direct sequencing analysis. We identified three novel FBN1 mutations and ten FBN3 single nucleotide polymorphisms (SNPs). In this report, we could not detect a responsible mutation of the FBN3 gene for MFS. Although the number of the cases in this report is small, at least these results suggest that disease-causing mutations in exon regions of the FBN3 gene are very rare in MFS.Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers: AB177797, AB177798, AB177799, AB177800, AB177801, AB177802, AB177803     

Heterogeneity of rice ragged stunt oryzavirus genome segment 9 and its segregation by insect vector transmission

Summary Genomic heterogeneity of genome segment 9 (S9) of rice ragged stunt virus (RRSV) was investigated and a point mutation was found to be responsible for an electrophoretic mobility shift of S9 on polyacrylamide gel electrophoresis (PAGE). A new form of S9 (S9L) which migrated slightly faster than natural S9 (S9U) had the same length with AC transversion at nt 843. Synthetic S9 with a C:G pair at nt 843 migrated slightly faster than that with an A:U pair. Therefore, we conclude that the single point mutation shifts the electrophoretic mobility. Using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), we could detect S9U and S9L alone or mixture in insect vectors after acquisition as well as in infected rice plants.     

Topostatin, a novel inhibitor of topoisomerases I and II produced by Thermomonospora alba strain No. 1520. III. Inhibitory properties.

A novel inhibitor of topoisomerases designated as topostatin was isolated from the culture filtrate of Thermomonospora alba strain No. 1520. The inhibitory activity of topostatin was shown to be pH- and temperature-dependent with a maximum around at pH 6 and 28 degrees C. The stability of topostatin decreased with decreasing pH and rising temperature. Topostatin inhibited topoisomerases I and II in a competitive manner with respect to DNA. The inhibitor also inhibited some restriction endonucleases such as Sca I, Hind III and Pst I, but not Alu I, Bam HI, Eco RI, RNase A, DNase I, DNase II and DNA ligase. Topostatin did not induce the nuclear accumulation of p53 protein by DNA damage in the normal human cells.     

Bacteriologic and endotoxin analysis of salvaged blood used in autologous transfusions during cardiac operations.

Autologous blood transfusion is a common method of reducing the need for heterologous blood transfusion during cardiac operations. Recently we investigated an outbreak of severe, nonsurgical postoperative bleeding among patients undergoing heart operations and receiving intraoperative transfusion of blood from a cell conservation device (Cell Saver System, Haemonetics Corp., Braintree, Mass.). As a result of this investigation, we conducted a prospective study to determine if bacterial or endotoxin contamination of the blood collected in the Cell Saver System and used for reinfusion during heart operations contributes to postoperative bleeding complications. Patients' blood samples were collected immediately before operation, at the end of cardiopulmonary bypass, 1 hour postoperatively, and from the Cell Saver System. All blood samples were cultured for bacteria, and all plasma samples were assayed for endotoxin. Preoperatively all patients having heart operations were without signs of infection, 33 of 37 blood cultures taken were negative, and none of the plasma samples had detectable endotoxin. After discontinuance of cardiopulmonary bypass but before delivery of blood from the Cell Saver System, bacteria and endotoxin were detected in 11 of 36 (30.6%) and five of 35 (14.3%) of the patients' blood samples, respectively. The blood aspirated from the open chest and collected by the Cell Saver System was culture positive in 30 of 31 (96.8%) samples, and seven of 29 (24.1%) contained endotoxin. One of 28 blood samples collected 1 hour postoperatively was culture positive, and five of 25 samples contained endotoxin. Of 61 total microorganisms isolated, 50 (82%) were coagulase-negative staphylococci, four (6.6%) aerobic diphtheroids, five (8.2%) anaerobic "diphtheroids" (Propionibacterium acnes), and two (3.2%) gram-negative bacilli. Plasma endotoxin concentrations ranged from 10 to 765 pg/ml. No signs of endotoxemia or unusual bleeding were observed intraoperatively or postoperatively in any of the 38 patients. Although blood collected in the Cell Saver System and used for reinfusion during heart operations often was contaminated with gram-positive bacterial commensals of the skin and low concentrations of endotoxin, no adverse effects were noted in the patients.     

Preseismic anomalous telluric current signals observed in Kozu-shima Island, Japan

Monitoring of telluric current, which is practically a synonym for geoelectric potential difference, was conducted on Kozu-shima Island about 170 km south of Tokyo from May 14, 1997 to June 25, 2000. During the monitoring period, 19 anomalous telluric current changes (ATCs) were observed. Their possible correlation with nearby earthquakes was statistically examined by assuming various lead times for different ranges of magnitude and focal distance. The best correlation may be obtained for earthquakes with a magnitude greater than 3.0 occurring within 20 km of focal distance. There were 23 such earthquakes, of which 11 were preceded by ATCs within 30 d. Of these 11 earthquakes, preceding ATCs of 5 and 6 were positive and negative polarities of telluric current, respectively. Their epicenters were spatially well clustered in the east and west of the island. These facts were clearly beyond those expected by chance and led to a simple speculative model.     

Confirmation by FRET in individual living cells of the absence of significant amyloid beta -mediated caspase 8 activation

When cells are exposed to death-inducing molecules such as tumor necrosis factor-alpha or Fas, caspase 8 is activated and cleaves an apoptotic facilitator, Bid, that is a member of the Bcl-2 family. After additional modification, the C-terminal moiety of Bid is translocated to the mitochondria and induces the release of cytochrome c into the cytoplasm. In an attempt to directly observe the cleavage of Bid and the following events in living cells, we constructed a vector that encoded Bid fused with yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) (YFP-Bid-CFP). On expression of YFP-Bid-CFP in mammalian cells, we were able to observe the efficient transfer of energy from excited CFP to YFP within the YFP-Bid-CFP molecule and, importantly, the fusion protein YFP-Bid-CFP was fully functional in cells. When YFP-Bid-CFP was cleaved by caspase 8, on activation by anti-Fas Abs but not by Abeta or tunicamycin, no such transfer of energy was detected. To our knowledge, this is the first report of (i) visualization of the activation of Bid by proteolytic cleavage, with direct observation of the cleavage of YFP-Bid-CFP in the cytoplasm and subsequent translocation of the cleaved Bid to mitochondria and (ii) the absence of Abeta- or tunicamycin-mediated significant activation of caspase 8 in individual living cells.     

Nonoperative management of injuries of the spleen in adults

Seventeen adult patients with proved splenic injuries were managed nonoperatively without a single instance of morbidity or mortality. All patients had a clinical history and physical findings suggestive of splenic trauma; the diagnosis was confirmed by splenic scintigraphy and, in some patients, additionally by angiography. The selection of patients for nonoperative management was made on the basis of a stable clinical status and laboratory evidence of cessation of bleeding. Patients who had progressive hypovolemia, worsening abdominal signs and laboratory evidence of continued bleeding were subjected to laparotomy for the performance of total splenectomy, partial splenectomy or splenorrhaphy. An arbitrary limit of three transfusions was set beyond which laparotomy was mandated. Nonoperative management is clearly not indicated for all patients with splenic injuries. In selected patients, however, nonoperative management is feasible and safe, providing careful clinical and laboratory observations are available. The theoretical long term sequelae of splenosis and splenic cyst formation have not yet been observed.