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.

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)

Selenium effects on the carcinogenicity and metabolism of 2-acetylaminofluorene.

Addition of 4 ppm Se to the drinking water of male albino rats fed diets containing 0.03% 2-acetylaminofluorene (AAF) provided protection against hepatic damage and also resulted in at least 50% reduction in liver tumor incidence. An in vitro assay system utilizing microsomes from Se supplemented or non-supplemented 3-methylcholanthrene (MC) induced rats was used to determine the effect of oral Se intake on the metabolism of AAF. Oral Se administration led to an increase in ring hydroxylation and a decrease in N-hydroxylation. Addition of Se to the microsomal assay system increased 3-OH AAF formation and decreased N-OH AAF formation, thus shifting the balance of metabolism toward detoxification pathways.

Aryl hydrocarbon hydroxylase induction in mitogen-stimulated lymphocytes by benzanthracene or cigarette tars adsorbed to asbestos fibers.

Human mitogen-stimulated lymphocytes, cultured in the presence of amosite asbestos (AS), demonstrated a slight increase in aryl hydrocarbon hydroxylase (AHH) activity compared with non-induced (control) cultures (P = 0.005). A much greater increase in enzyme activity occurred following addition of the inducers benzanthracene (BA) or cigarette tars (CT) to cell cultures (P less than 0.001 in both instances). Significant enzyme induction also occurred when AS fibers were first preincubated with CT or BA, washed with acetone, then added to lymphocyte cultures (P less than 0.003 in all instances). This increase in AHH activity was not as great, however, as the induction observed when BA or CT was added to cell cultures. No further increase in enzyme activity was noted when AS and CT or AS and BA were simultaneously added to culture lymphocytes (P greater than 0.070 in all instances). The results demonstrate that polycyclic aromatic hydrocarbons (PAH), such as BA and other components of CT, are adsorbed and transported by amosite AS particles. These AS-PAH complexes are capable of inducing AHH in cultured human lymphocytes.

Induction of aryl hydrocarbon hydroxylase in human peripheral blood lymphocytes by chrysene.

Many of the polycyclic aromatic hydrocarbons (e.g., benzo[a]pyrene (B[a]P), benzanthracene (BA), 3-methylcholanthrene (3-MC)) are not only carcinogenic, but also induce AHH in human tissues. Recently, chrysene has been implicated as an etiologic determinant of chemical carcinogenesis. Here we describe the ability of chrysene to induce AHH in cultured human lymphocytes. Lymphocytes were obtained from 9 healthy subjects, divided into 2 sets, and cultured in duplicate, triplicate, or quadruplicate for 48 h. Chrysene (25 microM final concentration) in acetone was then added to the induced culture set and the control set received acetone alone. Lymphocytes were then cultured an additional 24 h before harvesting. AHH was quantitated by a fluorometric analysis of the phenolic metabolites produced by incubating the lymphocytes with B[a]P for 35 min. A significant increase in enzyme induction occurred in the chrysene-induced cultures compared with control (non-induced) cells (one-tailed student t-test; P less than 0.001). It was also observed that the interindividual variation in AHH inducibility seen with other PAHs is also observed with chrysene.

Phagocytosis of asbestos fibers by human pulmonary alveolar macrophages.

Human pulmonary alveolar macrophages (PAMs) were cultured for 24--72 h with varying concentrations (0--300 microgram/ml) of amosite asbestos (AS). At lower AS concentrations, (less than 100 microgram/ml) no decrease in cell viability occurred during the first 24 h of culture. Significant cytotoxicity (P less than 0.005 in all instances) was observed, however, following incubation for 24 h with higher AS concentrations (greater than 100 microgram/ml). Even following incubation with lower concentrations of AS, significant cytotoxicity (P less than 0.006 in all instances) was observed after 48 or 72 h of culture. Scanning electron microscopy (SEM) clearly illustrates the various stages of AS phagocytosis by PAMs. SEM also documented morphological changes in PAMs following AS exposure. These included increased zeiosis and the appearance of a fibrous-like material on the surface of AS fibers following initial contact with the PAM cytoplasmic membrane. Further study of the biological interactions between AS and human cells, such as PAMs, might provide valuable information regarding the etiology of AS-related lung disorders.

Patterns of benzo[alpha]pyrene metabolism in normal human pulmonary alveolar macrophages.

Pulmonary alveolar macrophages (PAMs) obtained by bronchopulmonary lavage from 6 normal non-smoking volunteers were incubated with [3H]-benzo[alpha]pyrene to ascertain the normal metabolism and conjugation of polycyclic aromatic hydrocarbons. Through the use of a crude glucuronidase preparation, both glucuronic acid and sulfate conjugates were examined. Phenols and quinones were identified by high-pressure liquid chromatography as the principal free metabolites formed during 1 h incubation with benzanthracene induced PAMs. In addition, phenols and quinones were major substrates utilized by these cells for conjugation during the incubation period. The ranges of benzo[alpha]pyrene metabolites produced by PAMs from non-smokers were compiled and the variation in production as well as detoxification of proximate carcinogenic benzo[alpha]pyrene metabolites are presented.

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.

Red cell and hemorheological changes in multiple sclerosis.

Blood rheology in multiple sclerosis (MS) was investigated in 15 subjects with varying degrees of locomotor difficulties who were members of the local MS Society. Control data were obtained from blood samples from 25 male and 25 female normal blood donors. Whole blood viscosity was measured and blood filterability was assessed. Six MS females provided blood samples for scanning electron microscopy. Erythrocyte membrane fatty acids and phospholipids were assayed. Whole blood viscosity in MS females was higher than controls at 3 of 4 shear rates (p less than 0.001) but in MS males blood viscosity was higher only at shear rate of 1.0 s-1 (p less than 0.05). MS erythrocyte filtration rates were significantly lower than controls (p less than 0.001). Leucocyte counts in MS were greater than controls both in males (p less than 0.01) and females (p less than 0.001). MS erythrocyte morphology was greatly different from controls (p less than 0.0001) and erythrocyte membranes contained less sphingomyelin than controls (p less than 0.01) but more phosphatidylinositol plus phosphatidylserine (p less than 0.02). We conclude that, because our findings indicate an identifiable and potentially correctable abnormality, it is possible to envisage an inhibition of the progressive nature of MS, with the hope of a better prognosis for patients.

Biologic markers in cancer diagnosis and treatment.

We have reviewed several tumor markers that our advocates feel are now clinically useful, involve current assay technology, and are based on already available information. These include, in selected instances, estrogen receptors for breast cancer, thyrocalcitonin for medullary cancer of the thyroid, prostatic acid phosphatase for cancer of the prostate, alpha-fetoprotein for hepatocellular cancer, and carcinoembryonic antigen for monitoring colon cancer. We have considered the potential use of measurement of serum proteases and protein degradation products due to their activity as possible future areas of development, and we have explored measurement of tissue aryl hydrocarbon hydroxylase to identify populations at risk of cancer resulting from chemical carcinogenesis. It is clear that the study of tumor markers is already improving patient care in some specific areas and offers exciting potential for the future.

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.

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.