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Probing the active site of mitogillin, a fungal ribotoxin[The first ] (1998)

Kao, R. ; Shea, J. E. ; Davies, J. ; [et al.]

Oxford BSL : Blackwell Science Ltd

Molecular microbiology 29 (1998), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: Fungal ribotoxins, such as mitogillin and the related Aspergillus toxins restrictocin and α-sarcin, are highly specific ribonucleases, which inactivate the ribosome enzymatically by cleaving the eukaryotic 28S RNA of the large ribosomal subunit at a single phosphodiester bond. The site of cleavage occurs between G4325 and A4326, which are present in a 14-base sequence (the α-sarcin loop) conserved among the large subunit rRNAs of all living species. The amino acid residues involved in the cytotoxic activities of mitogillin were investigated by introducing point mutations using hydroxylamine into a recombinant Met-mature mitogillin (mitogillin with a Met codon at the N-terminus and no leader sequence) gene constructed from an Aspergillus fumigatus cDNA clone. These constructs were cloned into a yeast expression vector under the control of the GAL1 promoter and transformed into Saccharomyces cerevisiae. Upon induction of mitogillin expression, surviving transformants revealed that substitutions of certain amino acid residues on mitogillin abolished its cytotoxicity. Non-toxic mutant genes were cloned into an Escherichia coli expression vector, the proteins overexpressed and purified to homogeneity and their activities examined by in vitro ribonucleolytic assays. These studies identified the His-49Tyr, Glu-95Lys, Arg-120Lys and His-136Tyr mutations to have a profound impact on the ribonucleolytic activities of mitogillin. We conclude that these residues are key components of the active site contributing to the catalytic activities of mitogillin.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.1998.00987.x

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Performance of a new, HIV/AIDS-targeted quality of life (HAT-QoL) instrument in asymptomatic seropositive individuals (1997)

Holmes, W. C. ; Shea, J. A.

Springer

Quality of life research 6 (1997), S. 561-571

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ISSN: 1573-2649

Keywords: QoL ; HIV ; AIDS ; asymptomatic.

Source: Springer Online Journal Archives 1860-2000

Topics: Medicine

Notes: Abstract Objective: to evaluate the psychometric performance of a new human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS)-targeted quality of life (QoL) instrument in asymptomatic HIV-seropositive individuals. Methods: 201 urban and rural, HIV-seropositive subjects were recruited to complete a 76-item pilot QoL measure developed using content provided in group process by seropositive individuals. Questionnaire responses from the full sample (n=201) were used to identify dimensions and to reduce the number of items to 42. The responses to the retained items were then analysed for the asymptomatic subsample (n=106). Results: 9 multi-item dimensions were identified: overall function (OF), sexual function (SF), disclosure worries (DW), health worries (HW), financial worries (FW), HIV mastery (HM), life satisfaction (LS), medication concerns (MC) and provider trust (PT). The responses by asymptomatic subjects (74% male, 64% non-White and 63% homosexual/bisexual) revealed no substantial floor or ceiling effects, except for the PT dimension (where 44% were found to have scored the highest score). The internal consistency coefficients (Cronbach's alphas) were between 0.80 and 0.89 for six dimensions. The coefficients were lower for the SF (0.52), HM (0.67) and MC (0.48) dimensions. Construct validity assessments, using self-reported HIV disease-severity and sociodemographic variables, revealed some significant relationships (p≤0.05) for all dimensions except SF, MC and PT. Conclusions: the results suggested that five dimensions (OF, DW, HW, FW and LS) from the new instrument have good psychometric properties for asymptomatic HIV-seropositive individuals. These dimensions may be useful in the study of asymptomatic, seropositive individuals' QoL. Four dimensions (SF, HM, MC and PS) require additional refinement for this subpopulation.