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.

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.

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.

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.

Cloning and expression of a Porphyromonas (Bacteroides) gingivalis protease gene in Escherichia coli.

A genomic library of Bacteroides gingivalis W83 chromosomal DNA was constructed in the Escherichia coli lambda (lambda) vector EMBL 4. Three recombinant lambda phages expressing a cloned protease were identified in the library. All three lambda phages contained cloned overlapping DNA fragments from the same region of the chromosome and encoded the same cloned protease. The cloned protease was expressed poorly using its own promoter in E. coli.

The effect of pentamidine salts on the respiratory burst of human neutrophilic granulocytes.

Therapeutic concentrations (0.3-1.5 mg/l) of pentamidine isethionate and pentamidine mesylate, obtained after parenteral administration of either drug, did not affect oxygen consumption in the stimulated neutrophilic granulocyte. At concentrations of 0.7, 1.1 and 1.5 mg/l, superoxide production, hydrogen peroxide production, myeloperoxidase (MPO)-mediated iodination and hexose monophosphate shunt activity were suppressed relative to untreated cells (P less than 0.001 in each case). The depression in each activity was dose-related. There was no significant difference between the drugs with regard to these impairments in neutrophilic granulocyte function. This lowered respiratory burst activity, which would lead to a depression of MPO-dependent and MPO-independent processes in stimulated neutrophilic granulocytes, may be due to drug induced dysfunction of NADPH-oxidase.

The effect of pentamidine salts on the in-vitro responses of neutrophilic granulocytes.

Therapeutic concentrations (0.3-1.5 mg/l) of pentamidine isethionate and pentamidine mesylate, obtained after parenteral administration of the drugs, did not influence neutrophilic granulocyte adherence, random and chemotactic migration or phagocytosis of Candida albicans spores in vitro. At concentrations of 0.7, 1.1 and 1.5 mg/l, the ability of neutrophilic granulocytes to kill C. albicans spores was depressed (P less than 0.001); at all concentrations used, their ability to reduce nitroblue tetrazolium was decreased (P less than 0.001). There was no significant difference between the drugs with regard to these impairments in neutrophilic granulocyte function. It is likely that pentamidine salts inhibit superoxide radical formation in the stimulated neutrophilic granulocyte and that this dysfunction leads to depressed intracellular killing of C. albicans spores.