3' Terminal putative stem-loop structure required for the accumulation of cymbidium ringspot viral RNA

Defective interfering (DI) RNA of cymbidium ringspot tombusvirus (CymRSV) was used to identify the cis-acting nature of the last 77-nt sequence of the viral genome which is required for DI RNA accumulation. The 3'-terminal cis-essential domain of both genomic and DI RNAs can be folded into a stable stem-loop structure composed of three hairpins and two short non-base-paired regions. None of the three conserved stem-loops can be deleted without abolishing the infectivity of DI RNA. Similarly, those mutants in which base-paired stem regions were disrupted by single-, double-, or triple-base substitutions were unable to replicate. However, when the original structures were reconstructed by compensatory mutations the viability of the molecules was also restored. Limited mutation (1 or 2 nt) in the non-base-paired region did not show any significant effect on viral replication. Our results strongly suggest that the proposed structure for the 3' terminus of the viral genome is very important for viral RNA replication. It is very likely that the function of this structure is to promote the minus-strand synthesis of CymRSV DI RNA. Evidence is provided that the proposed 3'-terminal structure is relevant not only for CymRSV DI but for genomic RNA as well.     

Increase of satellite tobacco ringspot virus RNA initiated by inoculating circular RNA

The present studies examined the actions of a series of novel arylpiperazine 5-hydroxytryptamine1A (5-HT1A) agonists, developed originally for anxiolytic efficacy, in several models of gastric secretion and experimental gastric mucosal injury. These models included conscious gastric acid secretion, pylorus ligation (gastric acid and pepsin secretion), stress-induced gastric mucosal injury, ethanol-induced gastric mucosal damage and gastric adherent mucus levels. 2-(4-[4-(4-Nitropyrazol-1- yl)butyl]-1-piperazinyl)pyrimidine (E4414) and 2-(4-[4-(4-chloropyrazol-1-yl)butyl]-1-piperazinyl)pyrimidine dihydrochloride (E4424) significantly inhibited gastric acid secretion in conscious as well as in pylorus-ligated rats. Both compounds also significantly reduced pepsin secretion in pylorus-ligated animals. E4414 and E4424 significantly reduced both stress-induced and ethanol-induced gastric mucosal injury, and both compounds significantly maintained gastric adherent mucus levels in rats subjected to stress. The antisecretory and gastroprotective actions of E4414 and E4424 were of significantly greater magnitude than those of the reference 5-HT1A agonists, buspirone and 8-hydroxy-2-(di-n- propylamino)tetralin. These results suggest that some novel 5-HT1A agonists exert gastroprotection not only through reduction of aggressive elements in the gut (acid and pepsin secretion) but also through enhancement of defensive gastrointestinal factors such as adherent mucus.     

Functional analysis of the herpes simplex virus UL42 protein

The herpes simplex virus UL42 gene encodes a multifunctional polypeptide (UL42) that is essential for virus DNA replication. To further understand the relationship between the structure of UL42 and the role that it plays during virus replication, we analyzed an extensive set of mutant UL42 proteins for the ability to perform the three major biochemical functions ascribed to the protein:binding to DNA, stably associating with the virus DNA polymerase (Pol), and acting to increase the length of DNA chains synthesized by Pol. Selected mutants were also assayed for their ability to complement the replication of a UL42 null virus. The results indicated that the N-terminal 340 amino acids of UL42 were sufficient for all three biochemical activities and could also support virus replication. Progressive C-terminal truncation resulted in the loss of detectable DNA-binding activity before Pol binding, while several mutations near the N terminus of the polypeptide resulted in an altered interaction with DNA but had no apparent affect on Pol binding. More dramatically, an insertion mutation at residue 160 destroyed the ability to bind Pol but had no effect on DNA binding. This altered polypeptide also failed to increase the length of DNA product synthesized by Pol, and the mutant gene could not complement the growth of a UL42 null virus, indicating that the specific interaction between Pol and UL42 is necessary for full Pol function and for virus replication. This study confirms the validity of the Pol-UL42 interaction as a target for the design of novel therapeutic agents.     

Formation of circular satellite tobacco ringspot virus RNA in protoplasts transiently expressing the linear RNA

The most abundant form of the satellite RNA of tobacco ringspot virus (sTRSV RNA) is a linear, unit length molecule of 359 nucleotide residues, designated L-(+)M. A postulated replication scheme for the satellite RNA has as its first, and apparently virus-independent, step the ligation of L-(+)M into the corresponding circular form C-(+)M. We transiently expressed L-(+)M wild type and L-(+)M mutants in tobacco protoplasts using an African cassava mosaic geminivirus vector. Measured extents of C-(+)M accumulation were correlated with computer-predicted folding to suggest wild-type secondary structure elements that might be deleted without reducing ligation. A 127-nucleotide residue mutant L-(+)M was created by replacing, with 7 and 3 residues, respectively, nucleotide residues 53-211 and 268-350, each of which was predicted to form a set of three adjacent imperfect stem-loops in wild-type L-(+)M. The mutant L-(+)M was found to be extensively ligated to C-(+)M in protoplasts and to retain a calculated helix of the wild-type molecule that incorporates the 3' terminal sequence. A trinucleotide in the 3' region was mutated so as to disrupt and restore, respectively, the calculated helix, reducing and restoring, respectively, C-(+)M formation. These results suggest that the 3' stem contributes to the suitability of the small L-(+)M molecules as a substrate for a protoplast RNA ligase and that computed folding of sTRSV RNA may be predictive of sTRSV RNA structure in vivo.     

Detection of antitumor promoting activity in Raji cells carrying Epstein-Barr virus genome by immunoblotting analysis

A survey of death investigation systems in Canada was conducted by questionnaire and included questions on the type of system used (coroner or medical examiner), budget, method of appointment and qualifications of coroners and medical examiners, responsible department of government, training requirements, laboratory facilities, and the utilization of investigators and pathologists by each jurisdiction. Of the population of Canada 81.5% reside in coroner jurisdictions, the primary death investigator in 81% of the population is a licensed physician. The majority of jurisdictions either require or provide training in death investigation. All death investigation systems are under the respective provincial or territorial department of Justice or Attorney General with the chief coroner or chief medical examiner appointed by their respective provincial cabinet. The qualifications of the chief coroner vary with jurisdiction, while in 75% of medical examiner jurisdictions the chief medical examiner must be a pathologist. On a per capita basis medical examiner systems are less expensive to operate than coroner systems.     

Evidence of transmission of hepatitis B virus to spouses from sequence analysis of the viral genome

Despite advances in postnatal care, patients born with a congenital diaphragmatic hernia (CDH) suffer substantial morbidity and mortality. The present study was undertaken to determine the prognostic influence of prenatally-diagnosed liver herniation in the hemithorax in fetuses with CDH. The medical records of 48 patients evaluated for a prenatally-diagnosed left CDH were retrospectively reviewed. Patients were analysed according to the position of the liver by prenatal ultrasound; 32 fetuses had a major portion of the liver herniated into the left hemithorax ('liver up') and 16 had an intra-abdominal liver ('liver down'). Liver position was determined using colour-flow Doppler ultrasonography. There were two fetal deaths in the liver-up group and one in the liver-down group. The liver-up group more frequently required extracorporeal membrane oxygenation (ECMO) support (53 per cent) compared with the liver-down group (19 per cent). Postnatal survival was significantly less in the liver-up group (43 per cent) vs. the liver-down group (93 per cent). Fetuses with congenital diaphragmatic hernia and liver herniated into the hemithorax have a much worse prognosis than similarly afflicted fetuses without liver herniation. Prenatal ultrasonographic diagnosis of congenital diaphragmatic hernia allows for preparation for a critically ill newborn and aids in prenatal family counselling.     

Segmented genome and nucleocapsid of La Crosse virus

La Crosse (LAC) virions purified by velocity and equilibrium gradient centrifugation contained three single-stranded RNA species. The three segments had sedimentation coefficients of 31S, 25S, and 12S by sodium dodecyl sulfate-sucrose gradient centrifugation. By comparison with other viral and cellular RNA species, the LAC viral RNAs had molecular weights of 2.9 x 10(6), 1.8 x 10(6), and 0.4 x 10(6). Phenol-sodium dodecyl sulfate-extracted LAC virion RNA was not infectious for BHK-21 cell cultures under conditions in which Sindbis viral RNA was infectious. Treatment of LAC virus with the nonionic detergent Triton X-100 and salt released three nucleocapsid structures, each containing one species of virion RNA. The nucleocapsids had sedimenation coefficients of 115S, 90S, and 65S. Negative-contrast electron microscopy of the nucleocapsids indicated that they were convoluted, supercoiled, and apparently circular. They had a mean diameter of 10 to 12 nm and modal lengths of 200, 510, and 700 nm (some were even longer). By chemical and enzymatic analysis of purified viral RNA, one type of 5' nucleotide (pppAp) present in the proportion of one per RNA segment was identified. After periodate oxidation, each virion RNA species was labeled by reduction with [3H]sodium borohydride. Taken together, these results suggest that although the nucleocapsids appear as closed loops, the viral RNA has free 5' and 3' ends and is, therefore, not circular.     

Functional analysis of vaccinia virus B5R protein: role of the cytoplasmic tail

Vaccinia extracellular enveloped virus (EEV) is important for cell-to-cell and long-range virus spread both in vitro and in vivo. Six genes have been identified that encode protein constituents of the EEV outer membrane, and some of these proteins are critical for EEV formation. The B5R gene encodes an EEV-specific type I membrane protein, and deletion of this gene markedly decreases EEV formation and results in a small plaque phenotype. Data suggest that the transmembrane domain, cytoplasmic tail, or both contain the EEV localization signals that are required for targeting of the B5R protein to EEV and for EEV formation. Here, we report the construction of mutant vaccinia viruses in which the wild-type B5R gene was replaced with a mutated one that encodes a protein with the putative cytoplasmic tail deleted. The mutated protein showed normal intracellular distribution and was properly incorporated into EEV. Vaccinia viruses expressing the B5R protein lacking the cytoplasmic tail formed plaques that were similar in type and size to those formed by wild-type viruses and produced equivalent amounts of infectious EEV. These results indicate that the B5R cytoplasmic tail is not necessary for EEV formation and points to the transmembrane domain as the major determinant for targeting the B5R protein to the outer membrane of EEV and for supporting EEV formation.     

Nucleotide sequence and genome structure of mink enteritis virus

Diabetes mellitus is a common disease. It affects multiple organ systems. Adverse effects of hyperglycemia on infection, fracture healing, and bone remodeling have been recently reported. This study was conducted to evaluate the clinical and radiographic results of 93 total hip arthroplasties in 78 consecutive patients with diabetes. All femoral components were cemented using contemporary cementing techniques. Prophylactic antibiotics were given in each case. The mean follow-up period was 4.1 years (range, 2-6.5 years). Ninety-six percent of the hips were rated excellent or good. Radiolucencies were observed in only 3.7% of the stems, while 22.2% of the cups showed radiolucencies. There was a 4% revision incidence. There was no postoperative infection in this series--a distinct improvement from previously reported series. However, complications remained high at 24.3%. The most frequent complication was urinary tract infection (14.2%). The most serious complication was myocardial infarction. The authors believe total hip arthroplasty can be safely performed in patients with diabetes, provided that adequate medical and follow-up evaluations are performed. The medium-term clinical and radiographic follow-up evaluations are not inferior to reported series in patients without diabetes.     

Sequence determination of hepatitis C virus genome isolated from Taiwan

The partial genome sequence of the hepatitis C virus (HCV) was determined in the serum of a Taiwanese patient with chronic community-acquired type C hepatitis. The cDNA fragments synthesized with the HCV RNA as a template were amplified by polymerase chain reaction using specific oligonucleotide primers. The amplified fragments represented the regions coding for the putative core, matrix and envelope proteins as well as the N-terminal amino acid sequence of the nonstructural protein NS1, the partial nonstructural NS3 and NS4 proteins and the region of the partial 5'-end noncoding sequence. The cDNA fragments were cloned and sequenced. Sequence analysis of these clones showed that they share 83.7%, 93.2% and 93.6% similarity at the nucleotide level, and 86.6%, 94.1% and 92.9% homology at the amino acid level, with the previously published American, Japanese and Taiwanese isolates, respectively. Accordingly, the RNA genome we obtained is HCV type II, probably, the predominant subtype in Taiwan.     

Systematic functional analysis of the yeast genome

The genome sequence of the yeast Saccharomyces cerevisiae has provided the first complete inventory of the working parts of a eukaryotic cell. The challenge is now to discover what each of the gene products does and how they interact in a living yeast cell. Systematic and comprehensive approaches to the elucidation of yeast gene function are discussed and the prospects for the functional genomics of eukaryotic organisms evaluated.     

The structure of tobacco ringspot virus: a link in the evolution of icosahedral capsids in the picornavirus superfamily

Electronic portal imaging devices use the high energy treatment beam to project the body interior of the patient during radiation onto a fluorescent screen that is scanned by a camera. Because of the imaging physics, the unprocessed images of very poor quality, but they are the only available information during treatment for observation of the patient's organs. This paper presents an approach that combines an associative restoration algorithm with a fuzzy image enhancement technique. By fusion of the electronic portal image (EPI) with a pre-treatment captured simulator image (SI) a higher image quality than by conventional techniques is achieved.