Structural characterization of <Emphasis Type="Italic">Tobacco etch virus</Emphasis> coat protein mutants

Summary. The assembly of Tobacco etch potyvirus (TEV) coat protein (CP) and truncated mutants in Escherichia coli was studied. CP from which 28, 63 or 112 amino acids were deleted from the N-terminus polymerized into potyvirus-like particles (PVLPs). These structures were more rigid and progressively smaller in diameter than those produced by full length TEV-CP. CP from which 175 N-terminal amino acids were removed, failed to polymerize. A fragment containing amino acids 131 to 206 of TEV-CP is sufficient for PVLP assembly in E. coli.To determine the function of the highly conserved amino acids Ser152, Arg154, and Asp198 point mutants were generated. The mutant CP63(Asp198Glu) exhibited different spectral properties following circular dichroism analysis showing a lower amount of -helix compared to the wild type molecule. No differences were observed in spectra obtained from fluorescence spectroscopy. The point mutants bind RNA in vitro to the same degree as the wild type protein. However, while the wild type and the Arg154Gln mutant CP were each able to form PVLPs in E. coli, the Asp198Glu and the double mutant Ser152Pro/Arg154Gln mutants did not. These results suggest that the Asp198Glu mutation has an altered secondary structure which affects the capacity of the protein to polymerize but did not affect in vitro protein-RNA interactions.     

Diagnosis of heart failure in older adults: predictive value of dyspnea at rest

The objective of this study was to describe the predictive value of dyspnea at rest in the diagnosis of heart failure in older adults. We have conducted a retrospective chart review of older adults hospitalized with heart failure in 11 hospitals in Alabama, US. We defined dyspnea at rest as dyspnea not accompanied by activities, sleep, or lying down. We confirmed diagnosis of heart failure using modified Framingham criteria (based on available variables). We estimated values for sensitivity, specificity, predictive value positive and likelihood ratio positive of dyspnea at rest along with their 95% confidence intervals (95% CI). Patients had a mean (+/-S.D.) age 79 (+/-7.5) years, were 60% female and 18% African-American. The prevalence (pre-test probability) of heart failure was 77%. Dyspnea at rest was reported by 975 (89%) patients. Dyspnea on exertion, orthopnea and paroxysmal nocturnal dyspnea were, respectively, reported by 279 (26%), 356 (33%) and 230 (21%) patients. The sensitivity, specificity and positive predictive value (post-test probability) of dyspnea at rest were, respectively, 92% (95% CI = 90 - 94%), 19% (95% CI = 14 - 24%) and 79% (95% CI = 77 - 82%). Patients with dyspnea at rest were 13% (likelihood = 1.13; 95% CI = 1.06 - 1.20) more likely to have heart failure than those without. Presence of any three of the symptoms of dyspnea at rest, dyspnea on exertion, orthopnea, paroxysmal nocturnal dyspnea, fatigue and lower extremity edema increased the positive predictive value to 90% (95% CI = 87 - 93%). Dyspnea at rest was the commonest symptom among elderly hospitalized HF patients and had high sensitivity, but low specificity and positive predictive value, which increased in the presence of other symptoms.     

Prevalence of coronary artery disease, lower extremity peripheral arterial disease, and cerebrovascular disease in 110 men with an abdominal aortic aneurysm

Of 110 men (mean age 66 years) with an abdominal aortic aneurysm, 78 (71%) had coronary artery disease (CAD), 50 (46%) had lower extremity peripheral arterial disease (PAD), and 30 (27%) had cerebrovascular disease. Twenty-four percent of the patients with an abdominal aortic aneurysm had CAD plus lower extremity PAD plus cerebrovascular disease, 22% had CAD plus lower extremity PAD and no cerebrovascular disease, 22% had only CAD, 4% had CAD plus cerebrovascular disease and no lower extremity PAD, and 29% had no CAD, lower extremity PAD, or cerebrovascular disease.     

Mycobacterium marinum strains can be divided into two distinct types based on genetic diversity and virulence

Mycobacterium marinum causes a systemic tuberculosis-like disease in a large number of poikilothermic animals and is used as a model for mycobacterial pathogenesis. In the present study, we infected zebra fish (Danio rerio) with different strains of M. marinum to determine the variation in pathogenicity. Depending on the M. marinum isolate, the fish developed an acute or chronic disease. Acute disease was characterized by uncontrolled growth of the pathogen and death of all animals within 16 days, whereas chronic disease was characterized by granuloma formation in different organs and survival of the animals for at least 4 to 8 weeks. Genetic analysis of the isolates by amplified fragment length polymorphism showed that M. marinum strains could be divided in two clusters. Cluster I contained predominantly strains isolated from humans with fish tank granuloma, whereas the majority of the cluster II strains were isolated from poikilothermic species. Acute disease progression was noted only with strains belonging to cluster I, whereas all chronic-disease-causing isolates belonged to cluster II. This difference in virulence was also observed in vitro: cluster I isolate Mma20 was able to infect and survive more efficiently in the human macrophage THP-1 and the carp leukocyte CLC cell lines than was the cluster II isolate Mma11. We conclude that strain characteristics play an important role in the pathogenicity of M. marinum. In addition, the correlation between genetic variation and host origin suggests that cluster I isolates are more pathogenic for humans.     

Identification of deoxynivalenol, 3-acetyldeoxynivalenol,and zearalenone in galactose oxidase-producing isolates of Fusarium graminearum

Fusarium graminearum strains are well known for their role as plant pathogens and for their production of mycotoxins, and less known for their secretion of galactose oxidase, a well-studied and useful enzyme. Three galactose oxidase-producing isolates of F. graminearum were grown on rice to identify the production of zearalenone and trichothecenes through the use of thin layer chromatography and gas chromatography coupled to mass fragmentation. Detection and identification of deoxynivalenol, 3-acetyldeoxynivalenol, and zearalenone were accomplished.     

Glutathione and glutathione S-transferases A1-1 and P1-1 in seminal plasma may play a role in protecting against oxidative damage to spermatozoa

OBJECTIVE: To study the levels of glutathione, glutathione S-transferase A1-1, and glutathione S-transferase P1-1 in seminal fluid of fertile and subfertile men. DESIGN: Retrospective case-control study. SETTING: Departments of gastroenterology, obstetrics and gynecology, and epidemiology and biostatistics in a university medical center. PATIENT(S): Twenty-five subfertile men visiting the fertility clinic and 25 fertile men from midwife practices were recruited. INTERVENTION(S): Collection of semen of subfertile and fertile men. MAIN OUTCOME MEASURE(S): Plasma levels of glutathione and glutathione S-transferases A1-1 and P1-1 in relation to seminal characteristics. RESULT(S): Glutathione, glutathione S-transferase A1-1, as well as glutathione S-transferase P1-1 were found in considerable amounts in seminal fluid of subfertile and fertile men. No differences between groups were found for glutathione S-transferases A1-1 and P1-1. Also, no associations with sperm count, motility, or morphology could be detected. Fertile men had significantly higher glutathione levels as compared with the case of subfertile men. Associations of glutathione with sperm motility quality (r(s) = 0.321) and abnormal sperm morphology (r(s) = -0.496) were found. CONCLUSION(S): The presence of glutathione S-transferases A1-1 and P1-1 in seminal fluid suggests a role in the protection against (oxidative) damage of spermatozoa, whereas glutathione may play a role in male fertility.     

Umbilical glutathione levels are higher after vaginal birth than after cesarean section

Glutathione plays an important role in quenching reactive oxygen species, resulting in oxidation of glutathione, which in times of prolonged oxidative stress may be excreted from the erythrocyte. We investigated arterial and venous umbilical cord levels of glutathione in neonates born by vaginal delivery (n = 140) or cesarean section (n = 38). In a subset of neonates who were delivered vaginally maternal levels were assessed in parallel (n = 14). Median (5th-95th percentile) glutathione levels in venous and arterial umbilical samples were higher after vaginal delivery as compared to cesarean section, 2.7 (0.9-7.3) versus 2.0 (0.6-11.5; P < 0.03) and 3.5 (0.6-22.7) versus 2.3 (0.7-24.3) micromol/L (P < 0.02), respectively. Maternal glutathione levels were higher, 7.8 (4.3-10.6) micromol/L, than corresponding venous (P < 0.001) or arterial (P < 0.02) umbilical levels. These results suggest that vaginal delivery is associated with more oxidative stress than delivery by cesarean section.