Multiple enzymatic activities associated with recombinant NS3 protein of hepatitis C virus

The hepatitis C virus (HCV) nonstructural 3 protein (NS3) contains at least two domains associated with multiple enzymatic activities; a serine protease activity resides in the N-terminal one-third of the protein, whereas RNA helicase activity and RNA-stimulated nucleoside triphosphatase activity are associated with the C-terminal portion. To study the possible mutual influence of these enzymatic activities, a full-length NS3 polypeptide of 67 kDa was expressed as a nonfusion protein in Escherichia coli, purified to homogeneity, and shown to retain all three enzymatic activities. The protease activity of the full-length NS3 was strongly dependent on the activation by a synthetic peptide spanning the central hydrophobic core of the NS4A cofactor. Once complexed with the NS4A-derived peptide, the full-length NS3 protein and the isolated N-terminal protease domain cleaved synthetic peptide substrates with comparable efficiency. We show that, as in the case of the isolated protease domain, the protease activity of full-length NS3 undergoes inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A-NS4B and NS5A-NS5B. We have also characterized and quantified the NS3 ATPase, RNA helicase, and RNA-binding activities under optimized reaction conditions. Compared with the isolated N-terminal and C-terminal domains, recombinant full-length NS3 did not show significant differences in the three enzymatic activities analyzed in independent in vitro assays. We have further explored the possible interdependence of the NS3 N-terminal and C-terminal domains by analyzing the effect of polynucleotides on the modulation of all NS3 enzymatic functions. Our results demonstrated that the observed inhibition of the NS3 proteolytic activity by single-stranded RNA is mediated by direct interaction with the protease domain rather than with the helicase RNA-binding domain.     

Floquet's theorem in three dimensions and its application to the dispersion characteristics of circular waveguides with screw periodicity for use with gyrotron backward-wave oscillators

Floquet's theorem for three dimensions is proved for each of the two mathematical conditions of periodicity: the differential equation and the boundary condition. The theorem applied to circular waveguides with screw periodicity provides the dispersion characteristics of the uncoupled modes. These characteristics are compared to those of a normal circular waveguide to show how screw periodic RF structures shift in phase the waveguide cutoff point and how such a shift is advantageous to gyrotron backward-wave oscillators.

     

The future of clinical communication in an electronic environment

Computer technologies, particularly electronic computer networks, can enhance nurses' abilities to initiate, facilitate, and sustain interpersonal contact with patients. Computer networks are electronic links between remote sites and as such provide a pathway for communication between nurses and patients. In an innovative project known as the ComputerLink, a team of nurses used an electronic network to provide information, communication, and decision support to homebound persons and their caregivers. This experiment allowed exploration of the unspoken language of nursing and provides direction for considering how nursing therapeutics can capitalize on the benefits of the electronic network.     

Molecular organization of the alcohol dehydrogenase loci of Drosophila grimshawi and Drosophila hawaiiensis

A total of 71 Staphylococcus aureus strains isolated from bovine mammary glands were identified and subtyped. The methods used to differentiate between the S. aureus isolates were the DNA polymorphism pattern after amplification with a Polymerase Chain Reaction using several primer combinations and phage typing. The DNA fingerprinting technique using RAPD, ERIC1R and ERIC primers proved to be useful in differentiating isolates of S. aureus. Differentiation of isolates using phage typing gave no additional information compared to the DNA technique. The outbreak of S. aureus in the herd studied was mainly caused by one S. aureus strain. Other strains were only found on three occasions, twice in subclinical infections and once from a case of clinical mastitis. In the latter case the dominant strain was isolated from a different quarter of the same cow. Four of the ten cows studied suffered from clinical mastitis. From those four cows, three remained infected with the same S. aureus strain despite antibiotic treatment.     

The modified beta-ketoadipate pathway in Rhodococcus rhodochrous N75: enzymology of 3-methylmuconolactone metabolism

Rhodococcus rhodochrous N75 is able to metabolize 4-methylcatechol via a modified beta-ketoadipate pathway. This organism has been shown to activate 3-methylmuconolactone by the addition of coenzyme A (CoA) prior to hydrolysis of the butenolide ring. A lactone-CoA synthetase is induced by growth of R. rhodochrous N75 on p-toluate as a sole source of carbon. The enzyme has been purified 221-fold by ammonium sulfate fractionation, hydrophobic chromatography, gel filtration, and anion-exchange chromatography. The enzyme, termed 3-methylmuconolactone-CoA synthetase, has a pH optimum of 8.0, a native Mr of 128,000, and a subunit Mr of 62,000, suggesting that the enzyme is homodimeric. The enzyme is very specific for its 3-methylmuconolactone substrate and displays little or no activity with other monoene and diene lactone analogues. Equimolar amounts of these lactone analogues brought about less than 30% (most brought about less than 15%) inhibition of the CoA synthetase reaction with its natural substrate.     

Renal transplantation in adults with thrombotic thrombocytopenic purpura/haemolytic-uraemic syndrome

BACKGROUND: Thrombotic thrombocytopenic purpura/haemolytic-uraemic syndrome (TTP/HUS) is a rare cause of renal failure in adults. There is little data concerning the outcome of adult patients who receive a renal transplant for TTP/HUS: METHODS: We have carried out a survey of 22 transplant centres in the USA to determine the outcome of patients who developed ESRD from TTP/HUS and latter received a renal transplant. RESULTS: Twelve of the 22 centres responded to our inquiry. Seven centres had not transplanted any patients with TTP/HUS, and five centres had transplanted a total of 24 grafts in 17 patients with TTP/HUS: Thirty-three per cent of patients demonstrated definite clinical and pathological evidence of recurrence of TTP/HUS: An additional 16% of patients demonstrated pathological evidence of possible recurrence of TTP/HUS in the absence of clinical manifestations. The overall 1-year graft survival rate was 42% and the 2-year graft survival rate was 35%. In our experience recurrence TTP/HUS was associated with universal graft failure. Although cyclosporin A does occasionally cause a thrombotic angiopathy in patients with no history of TTP/HUS, we found no evidence that it should be avoided in patients with a previous history of ESRD from TTP/HUS who subsequently receive a renal transplant. CONCLUSIONS: TTP/HUS frequently recurres in adults who receive a renal transplant, with a 2-year graft survival rate of 35%.     

Attachment styles and personality disorders: their connections to each other and to parental divorce, parental death, and perceptions of parental caregiving

Attachment theory was explored as a means of understanding the origins of personality disorders. We investigated whether adult attachment styles and personality disorders share a common underlying structure, and how both kinds of variables relate to family background factors, including parental death, parental divorce, and current representations of childhood relationships with parents. A nonclinical group of 1407 individuals, mostly adolescents and young adults, were surveyed about their attachment styles, parental marital status, parental mortality status, perceptions of treatment by parents in childhood, and 13 personality disorders. Results indicated substantial overlap between attachment and personality-disorder measures. Two of the personality-disorder dimensions are related to the two dimensions of the attachment space; that is, there is a two-dimensional space in which both the attachment patterns and most of the personality disorders can be arrayed. The one personality-disorder factor that is unrelated to attachment appears akin to psychopathy. Both personality disorders and attachment styles were associated with family-of-origin variables. Results are discussed in terms of encouraging further research to test the idea that insecure attachment and most of the personality disorders share similar developmental antecedents.     

Risk factors for death in homeless adults in Boston

BACKGROUND: Homeless individuals experience high mortality rates. Males, whites, and substance abusers are more likely to die, but other high-risk characteristics are unknown. OBJECTIVE: To identify demographic and clinical factors associated with an increased risk of death in homeless individuals. METHODS: We conducted a case-control study of 558 adults who were seen by a health care program for the homeless in Boston, Mass, and who died in 1988 to 1993. Age-matched paired controls were selected from among individuals seen by the program who were alive at the end of 1993. Predictive data were obtained by blinded review of medical records. Odds ratios (ORs) for death were calculated using logistic regression analysis models. RESULTS: In a multivariate analysis, the strongest risk factors for death were acquired immunodeficiency syndrome (OR, 55.8), symptomatic human immunodeficiency virus infection (OR, 17.7), asymptomatic human immunodeficiency virus infection (OR, 4.1), renal disease (OR, 18.4), a history of cold-related injury (OR, 8.0), liver disease (OR, 3.8), and arrhythmia (OR, 3.3). A history of substance abuse involving injection drugs (OR, 1.6) or alcohol (OR, 1.5) also increased the risk of mortality. Nonfluency in English was associated with a decreased risk of death (OR, 0.4). CONCLUSIONS: In a group of adults seen by a health care program for the homeless, specific medical illnesses were associated with the greatest risk of death. Substance abuse alone was less strongly associated with death. Interventions to reduce mortality among the homeless should focus on individuals with high-risk characteristics.