Thermostability and thermoactivity of citrate synthases from the thermophilic and hyperthermophilic archaea, Thermoplasma acidophilum and Pyrococcus furiosus.

Citrate synthases from Thermoplasma acidophilum (optimal growth at 55 degrees C) and Pyrococcus furiosus (100 degrees C) are homo-dimeric enzymes that show a high degree of structural homology with each other, and thermostabilities commensurate with the environmental temperatures in which their host cells are found. A comparison of their atomic structures with citrate synthases from mesophilic and psychrophilic organisms has indicated the potential importance of inter-subunit contacts for thermostability, and here we report the construction and analysis of site-directed mutants of the two citrate synthases to investigate the contribution of these interactions. Three sets of mutants were made: (a) chimeric mutants where the large (inter-subunit contact) and small (catalytic) domains of the T. acidophilum and P. furiosus enzymes were swapped; (b) mutants of the P. furiosus citrate synthase where the inter-subunit ionic network is disrupted; and (c) P. furiosus citrate synthase mutants in which the C-terminal arms that wrap around their partner subunits have been deleted. All three sets of mutant enzymes were expressed as recombinant proteins in Escherichia coli and were found to be catalytically active. Kinetic parameters and the dependence of catalytic activity on temperature were determined, and the stability of each enzyme was analysed by irreversible thermal inactivation experiments. The chimeric mutants indicate that the thermostability of the whole enzyme is largely determined by the origin of the large, inter-subunit domain, whereas the dependence of catalytic activity on temperature is a function of the small domain. Disruption of the inter-subunit ionic network and prevention of the C-terminal interactions both generated enzymes that were substantially less thermostable. Taken together, these data demonstrate the crucial importance of the subunit contacts to the stability of these oligomeric enzymes. Additionally, they also provide a clear distinction between thermostability and thermoactivity, showing that stability is necessary for, but does not guarantee, catalytic activity at elevated temperatures.

In vitro measurement of cytotoxic T cell activity does not predict clinical progression in paediatric HIV disease--two case studies.

Cytotoxic T cells are believed to be an important immune response in HIV infection, both in the initial response to viraemia, and in controlling HIV replication and maintaining clinical stability. We report here the detailed findings in two vertically infected children, from the Edinburgh perinatal cohort. Both were clinically stable for the first 7 years of life. One had vigorous HIV-specific cytotoxic T lymphocyte (CTL) responses, and non-lytic suppression, measured in vitro, while the second had no CTL activity against HIV. Despite her HIV-specific immunity, the first child had a declining CD4 count, and a high and fluctuating viral load, whereas the second child maintained a stable CD4 count, a low viral load and had a virus which could not be cultured in peripheral blood mononuclear cells (PBMC) in vitro. The first child subsequently progressed to AIDS and has now died, while the second remains clinically well. More detailed investigations showed the clinically stable child to be heterozygous for the CCR5 receptor, and to be HLA-B49--both of which markers have been associated with slow HIV disease progression. These findings question the role of CTL in maintaining stable HIV disease, and stress the need for immunological investigations to be considered in the light of the genetic make-up of the patient. They may also reflect a different immunopathogenesis of HIV disease in children compared with adults.

Text and illustration: the digitisation of a medieval manuscript.

This paper considers the choice of the medieval Aberdeen Bestiary as the first project in Aberdeen University Library's digitisation programme, and discusses some of the unusual features of the manuscript itself. Attention is given to the content and depth of the accompanying commentaries, and particular notice is paid to the nature and extent of the textual apparatus (translation and transcription). The factors influencing the choice of (a) PhotoCD as the image capture method, and (b) JPEG as the image format for transmission of the page images across the World Wide Web are examined. The importance of the Web design to the effectiveness of the overall resource is emphasised.

HIV-induced cytopathology and viral load in a pentamidine-treated lymphocytic cell line.

Preincubation of CD4 lymphocytes with pentamidine isethionate at a concentration of 1.5 mg/L then removal from incubation medium prior to addition of HIV-1, or incubation of cells with the drug and virus simultaneously, increased HIV-1 DNA load but reduced p24 antigen release. The number of syncytia generated was not affected by the presence of pentamidine. The extent of balloon degeneration of cells was greater, however, although this was not associated with a discernable increase in cellular necrosis or reduction in cell viability. This suggests that drug-treatment resulted in an increased load of intracytoplasmic (but not necessarily integrated) forms of HIV- 1; this may explain the lower levels of antigen produced and also the balloon degeneration of treated cells, a phenomenon previously observed with other retroviruses.

Effects of adding spironolactone to an angiotensin-converting enzyme inhibitor in chronic congestive heart failure secondary to coronary artery disease.

In chronic heart failure, a diuretic plus an angiotensin-converting enzyme (ACE) inhibitor only partially suppresses aldosterone despite the fact that aldosterone has many harmful effects independent of angiotensin II. These possible harmful effects of aldosterone are magnesium loss, increased cardiac sympathetic activity, and increased ventricular arrhythmias. We have therefore assessed whether adding the aldosterone antagonist, spironolactone, to a loop diuretic and ACE inhibitor reverses any of these potentially harmful effects of residual aldosterone. In a preliminary animal study, we found that exogenous aldosterone reduced myocardial norepinephrine uptake by 24% in anesthetized rats in vivo. In our main study, 42 patients with New York Heart Association II to III congestive heart failure were randomized to spironolactone (50 to 100 mg/day, titrated to blood pressure and plasma potassium) or placebo in a double-blind fashion. Our principal finding is that cardiac norepinephrine uptake as assessed by 123I-metaiodobenzylguanidine scintigraphy increased with spironolactone (p < 0.01). Spironolactone also elevated plasma magnesium (p < 0.05), reduced urinary magnesium excretion (p < 0.05), and caused a reduction in ventricular arrhythmias on 24-hour ambulatory electrocardiography (p < 0.05). Spironolactone increased plasma renin activity, plasma aldosterone (p < 0.01), 24-hour urinary sodium excretion (p < 0.05), and urinary sodium/potassium ratio (p < 0.01). Echocardiographic-determined measurements of left ventricular systolic and diastolic function were unaltered by spironolactone.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective effects of pentamidine on cytosolic and granule-associated enzyme release from zymosan-activated human neutrophilic granulocytes.

Therapeutic concentrations (0.3-1.5 mgL-1) of pentamidine isethionate, normally obtainable in-vivo after parenteral administration of the drug, did not affect the in-vitro activity of the enzymes lysozyme, beta-glucuronidase or myeloperoxidase released from zymosan-activated human neutrophilic granulocytes. At concentrations of 0.7, 1.1 and 1.5 mgL-1, activity of cytosolic enzymes lactate dehydrogenase and glucose-6-phosphate dehydrogenase were reduced relative to untreated cells (P < 0.001 and P < 0.01, respectively), but not in a dose-dependent fashion. Cell viability, as determined by dye-exclusion remained unaffected.

Cognitive assessment of the medical patient.

Cognitive assessment is the clinical examination of higher mental functioning. A structured and logical approach leads the clinician to identify specific functional deficits and patterns of dysfunction. This allows accurate diagnosis, which is vital to identify treatable disorders and quantify the nature of decline in irreversible conditions. This article defines common terms in use, suggests a logical deductive procedure and discusses screening tools.

Cytotoxic T lymphocyte activity in children infected with HIV.

Of the Edinburgh cohort of approximately 130 children born to HIV-infected women, 9 are infected and alive. This article describes results from the first 18 months of a natural history study of seven of these, and two adopted children, studying the CD8 T cell-mediated cytotoxicity against HIV proteins (Gag, Tat, Pol, and Env), over time, and relating it to clinical progression and viral activity. Autologous EBV cell lines infected with vaccinia-HIV constructs were used as target cells, and bulk-cultured peripheral blood mononuclear cells as effector cells. The children ranged in age from 0 to 93 months, with six of the nine showing CTL activity to one or more HIV proteins. The specificity of the response was directed against Tat in the younger children, switching to Pol, then Gag or Env. Preliminary analysis of virological data showed no association between CTL and virus activity. The children with CTLs tended to be well clinically, but the cohort needs to be studied longer before conclusions can be made about CTL activity and HIV disease progression. Cytotoxic T lymphocyte activity has also been observed in two children diagnosed as HIV uninfected. These results show the importance of looking at CTL specificity, and may have implications in vaccine design.

A survey of the communication-impaired population of Tayside.

There are few published surveys that are designed specifically to describe the total population of clients with communication disorders who might potentially benefit from the use of a communication aid. Such data, however, would be useful in planning the funding and staffing of rehabilitation services in a region. Figures are also needed to identify populations whose needs are currently not being met and thus to establish areas of future developments both for service and research purposes. The survey reported here was carried out as part of a project concerned with the provision of augmentative communication to the neurologically impaired child and young adult. It was intended to produce a broad description of the communication-impaired population in Tayside. The survey was then used to identify detailed information about clients who may benefit from using a communication aid, and it also highlighted some provision and research issues. Access to communication aids for the non-speaking population was found to have been restricted by lack of funding, resources and opportunities for training for therapists in the use of augmentative and alternative communication. Future research and development should particularly address the needs of the aphasic population whose needs were not found to be met by currently available communication aids.

Ivermectin and coagulation: an in vitro study.

The prothrombin time, partial thromboplastin time with kaolin, and thrombin clotting time of plasma derived from healthy human volunteers were unaltered after in vitro addition of therapeutic concentrations (20-90 ng ml-1) of ivermectin. No difference in these coagulation tests, relative to untreated controls, was observed after 12 hours' incubation with the drug.

Social interactions of mice with congenital Toxoplasma infection.

The social interactions of male mice with subclinical congenital Toxoplasma infection towards their uninfected counterparts was assessed using a procedure based on the subdivision of behaviour into element groups. Infection had no effect on behavioural elements not directly associated with interaction. However infection was found to increase companion investigation as well as a number of elements directly associated with aggression. There was a complementary decrease in elements associated with flight behaviour. These findings suggest that congenital infection with Toxoplasma renders mice less cautious towards uninfected conspecifics and increases the tendency of adult male mice towards territorial aggression.

The effect of pentamidine salts on the NADPH-dependent oxidase of stimulated neutrophilic granulocytes.

Both pentamidine isethionate and pentamidine mesylate induced a depression in activity of the NADPH-dependent oxidase system of stimulated human neutrophilic granulocytes. This drug-induced effect occurred at concentrations of 0.7, 1.1 and 1.5 mg/l, values within the therapeutic range after parenteral administration of a standard dose of either pentamidine salt, and was dose-related. There was no significant difference between the two salts with regard to this suppression in neutrophilic granulocyte function. The reduced activity of the NADPH-dependent oxidase system, after incubation with pentamidine salts, may be associated with the previously observed depression in candidacidal capacity of human neutrophilic granulocytes treated with these drugs.