Spontaneous apoptosis, oxidative status and immunophenotype markers in blood lymphocytes of AIDS patients.

Peripheral blood mononuclear cells (PBMC) from 251 HIV-positive drug abusers of known clinical stage and from 40 healthy donors were tested for conventional immunologic markers (CD3, CD4, CD8, CD19, CD14, CD16/CD56, CD45 and HLA-DR). Additional cell parameters and the occurrence of spontaneous apoptosis (programmed cell death) were investigated on freshly isolated PBMC by flow cytometric measurement of either annexin-V bound to plasma membrane phosphatidylserine or propidium iodide uptake. The activity of gamma-glutamyltransferase (gamma-GT), an ectoenzyme contributing to the synthesis of the intracellular antioxidant glutathione (GSH) and involved in early apoptosis, was also determined in these cells. Immunocompetent T-cell counts were lower in HIV+ patients, with the exception of CD8+ and HLA-DR+ lymphocytes. The external binding of annexin-V was significantly higher in HIV+ PBMC and occurred in both CD8+ and CD4+ T-lymphocyte subsets. The activity of gamma-GT, was significantly lower in the PBMC from HIV+ patients, indicating that the redox status of PBMC may be affected in HIV+ individuals. Finally, the most dominant features characterising patients receiving antiretroviral therapy were greater long-term stability in the distribution of various cell parameters excepted the level of apoptosis.

The aspartate aminotransferase-catalysed exchange of the alpha-protons of aspartate and glutamate: the effects of the R386A and R292V mutations on this exchange reaction.

1H-NMR was used to follow the aspartate aminotransferase-catalysed exchange of the alpha-protons of aspartate and glutamate. The effect of the concentrations of both the amino acids and the cognate keto acids on exchange rates was determined for wild-type and the R386A and R292V mutant forms of aspartate aminotransferase. The wild-type enzyme is found to be highly stereospecific for the exchange of the alpha-protons of L-aspartate and L-glutamate. The R386A mutation which removes the interaction of Arg-386 with the alpha-carboxylate group of aspartate causes an approximately 10,000-fold decrease in the first order exchange rate of the alpha-proton of L-aspartate. The R292V mutation which removes the interaction of Arg-292 with the beta-carboxylate group of L-aspartate and the gamma-carboxylate group of L-glutamate causes even larger decreases of 25,000- and 100,000-fold in the first order exchange rate of the alpha-proton of L-aspartate and L-glutamate respectively. Apparently both Arg-386 and Arg-292 must be present for optimal catalysis of the exchange of the alpha-protons of L-aspartate and L-glutamate, perhaps because the interaction of both these residues with the substrate is essential for inducing the closed conformation of the active site.

Effects of steroid hormones on nuclear membrane and membrane-bound heterochromatin from breast cancer cells evaluated by fractal morphometry.

OBJECTIVE: To evaluate the effect of steroid hormones on the ultrastructure of nuclear heterochromatin and perinuclear membranes in human MCF-7 breast cancer cells. STUDY DESIGN: MCF-7 cells were cultured briefly (five minutes) in the presence of 10(-9) M estrogen 17 beta-estradiol, a stimulator of cell proliferation and/or 10(-9) M glucocorticoid dexamethasone. Changes in the morphologic complexity of nuclear membrane-bound heterochromatin (NMBHC) and nuclear membranes (ENM) were assessed by means of the fractal capacity dimension, D, a noneuclidean geometric descriptor of complex, irregular bodies. RESULTS: 17 beta-estradiol (10(-9) M) enhanced the ultrastructural irregularity of NMBHC, as documented by the increased value of D, whereas dexamethasone (10(-9) M) reduced it when compared to NMBHC from untreated MCF-7 control cells. In contrast, neither steroid modified ENM ultrastructure. Changes in the nuclear heterochromatin complexity induced by estrogen 17 beta-estradiol occurred concomitantly with functional changes at the cell periphery, such as activation of the phospholipase C, a cell membrane-associated enzyme involved in signal transduction. Dexamethasone reduced the ultrastructural complexity of NMBHC without affecting functional processes. CONCLUSION: Fractal morphometry proved its usefulness in quantifying early ultrastructural changes in nuclear components induced in MCF-7 cells by steroid hormones, 17 beta-estradiol and dexamethasone.

Conversion of aspartate aminotransferase into an L-aspartate beta-decarboxylase by a triple active-site mutation.

The conjoint substitution of three active-site residues in aspartate aminotransferase (AspAT) of Escherichia coli (Y225R/R292K/R386A) increases the ratio of L-aspartate beta-decarboxylase activity to transaminase activity >25 million-fold. This result was achieved by combining an arginine shift mutation (Y225R/R386A) with a conservative substitution of a substrate-binding residue (R292K). In the wild-type enzyme, Arg(386) interacts with the alpha-carboxylate group of the substrate and is one of the four residues that are invariant in all aminotransferases; Tyr(225) is in its vicinity, forming a hydrogen bond with O-3' of the cofactor; and Arg(292) interacts with the distal carboxylate group of the substrate. In the triple-mutant enzyme, k(cat)' for beta-decarboxylation of L-aspartate was 0.08 s(-1), whereas k(cat)' for transamination was decreased to 0.01 s(-1). AspAT was thus converted into an L-aspartate beta-decarboxylase that catalyzes transamination as a side reaction. The major pathway of beta-decarboxylation directly produces L-alanine without intermediary formation of pyruvate. The various single- or double-mutant AspATs corresponding to the triple-mutant enzyme showed, with the exception of AspAT Y225R/R386A, no measurable or only very low beta-decarboxylase activity. The arginine shift mutation Y225R/R386A elicits beta-decarboxylase activity, whereas the R292K substitution suppresses transaminase activity. The reaction specificity of the triple-mutant enzyme is thus achieved in the same way as that of wild-type pyridoxal 5'-phosphate-dependent enzymes in general and possibly of many other enzymes, i.e. by accelerating the specific reaction and suppressing potential side reactions.

Prevention of Oxidation and Apoptosis in Human Peripheral Blood Mononuclear Cells Exposed to Calcium Dobesilate.

The antioxidant effects of calcium dobesilate (CD) (Doxium(R)) were investigated in relation to the oxidative status, apoptosis, and in vitro proliferation of human peripheral blood mononuclear cells (PBMC) isolated from healthy donors. Calcium dobesilate alone did not modify cell growth in vitro until it reached 10 µM. This molecule counteracted oxidative damage generated by the highly reducing sugar 2-deoxy-D-ribose (dR) and was shown to reduce apoptosis by delaying both membrane permeability changes and DNA fragmentation. Calcium dobesilate (10 µM) was effective in a time-dependent manner on several parameters, representative of the cellular oxidative status. In particular, CD significantly increased the activity of glutathione S-transferase (GST) after 3 days of treatment and also the activity of gamma-glutamyltransferase (gamma-GT). Both of these enzymes are known to be involved in the glutathione (GSH) metabolic cycle. This enzymatic behavior was reversed after 7 days of treatment, with a significant GST decrease and a gamma-GT activation. After 7 days of CD exposure, the intracellular GSH content was enhanced and this resulted in a dramatic decrease of lipid peroxidation, underlining the powerful antioxidant properties of CD in human PBMC.

Apoptosis and oxidative status in peripheral blood mononuclear cells of diabetic patients.

We have compared the concentrations of intracellular glutathione (GSH), glutathione-dependent antioxidative enzymes, the cell death rate and immunophenotype profile of peripheral blood mononuclear cells (PBMC) from healthy donors and from patients with insulin-dependent type II (NIDDM) diabetes mellitus. The IDDM and NIDDM patients had above-normal absolute lymphocyte counts, whereas the percentages of CD3, CD4 adn CD8 T lymphocytes were significantly reduced. In contrast, the absolute number and percentage of B lymphocytes was higher in diabetic patients than in healthy donors. The low intracellular reduced glutathione(GSH) and the unbalanced profile of key enzymes involved in GSH metabolism, gamma-glutamyltransferase (gamma-GT) and glutathione-S-transferase (GST), account for the increased oxidative status of PBMC from diabetic patients. The plasma membranes of PBMC for diabetic patients were less permeable to propidium iodide than those of PBMC from healthy donors, indicating that the apoptotic cell death rate was lower in the cells from diabetic patients. These differences are potentially useful markers of pathogenic metabolic changes which occur during clinical diabetes and if they are confirmed could be use dot identify the onset of diabetes.

Safety and efficacy of early extubation of elderly coronary artery bypass surgery patients.

OBJECTIVE: Early extubation and fast-track management protocols on younger, low-risk patients result in shorter hospital stays and decreased costs. The impact of such protocols on elderly patients undergoing coronary artery bypass graft (CABG) surgery is not presently known. DESIGN: A matched retrospective cohort study. SETTING: A university teaching hospital. PARTICIPANTS: Six hundred ninety-eight consecutive patients undergoing isolated CABG between January 1995 and September 1996. INTERVENTIONS: Three hundred seventy-seven patients underwent early extubation, defined as extubation within 8 hours of arrival in the intensive care unit. They were divided into groups of patients 70 years of age and younger (n = 263) and patients older than 70 years of age (n = 114). RESULTS: The mean length of stay (LOS) for all patients extubated within 8 hours or less was 5.5 days versus 8.4 days for patients who underwent later extubation (p < 0.0001). The percentage of patients undergoing early extubation was greater for the younger cohort (59% v 48%; p < 0.003) compared with the older cohort of patients. Analysis of demographics showed the older patients to have a greater incidence of peripheral vascular disease, congestive heart failure, and prior strokes (p < 0.05). Although the intensive care unit LOS was similar, postoperative LOS was 5.3 +/- 1.8 days for the younger patients versus 6.1 +/- 2.6 days for the older patients (p = 0.001). The overall surgical mortality rate was 2.6% (18/698), and there were no deaths among patients undergoing early extubation. Reintubation rate was negligible in both groups of patients. CONCLUSION: This study confirms the safety and efficacy of early extubation among elderly patients undergoing CABG. Elderly patients have more comorbid conditions, yet a significant number can be extubated early, with resultant shortened LOSs.

Steroid hormones modify nuclear heterochromatin structure and plasma membrane enzyme of MCF-7 cells. A combined fractal, electron microscopical and enzymatic analysis.

Ultrastructural features of the nuclear membrane envelope (ENM) and the nuclear membrane-bound heterochromatin (NMBHC) were investigated in MCF-7 human breast cancer cells by fractal morphometry. The fractal dimension D established by the box counting method proved to be effective for quantifying nuclear changes in MCF-7 cells treated with steroid hormones, namely the estrogen 17 beta-estradiol, which stimulates cell proliferation, and the glucocorticoid dexamethasone. When MCF-7 cells were briefly (5 min) cultured in the presence of 17 beta-estradiol (10(-9) M), the irregularity of the NMBHC outline was increased as documented by the increased fractal dimension D. Changes in the ultrastructural complexity of the nuclear heterochromatin were observed in concomitance with functional changes at the cell periphery, namely the modulation of the estrogen-induced activity of phospholipase C, a cell membrane-associated enzyme involved in the signal transduction pathway via phosphoinositides metabolism. Dexamethasone did not affect the in vitro proliferation, the phospholipase C activity nor the shape of the ENM of MCF-7 cells, but reduced the structural complexity of the nuclear membrane-bound heterochromatin.

Apoptotic cell death and the proliferative capacity of human breast cancers.

The proliferative capacity (%S-phase fraction), DNA ploidy, apoptosis frequency (DNA fragmentation) and steroid hormone receptor status (estrogen receptor, ER; progesterone receptor, PR) of 110 samples of human breast tissues with ductal invasive carcinoma were measured using biochemical and cytofluorimetric procedures. The DNA fragmentation had a left-skewed frequency distribution and an overall median value of 1.64%, whilst the median %S-phase fraction was 8%. The median %DNA fragmentation and %S-phase fraction were 1.96% and 16% in hyperdiploid tumours (n = 29; DNA index >1.1) higher than in hypodiploid tumors (n = 10; DNA index <0.96), 0.38% and 7.5%. DNA diploid tumours (n = 71) had median %DNA fragmentation and %S-phase values of 1.68% and 6%, consistently lower than the median values of DNA hyperdiploid tumours. The ER content of hypodiploid tumours was about one half (median: 5.9 fmol/mg) the median values in hyperdiploid (10.6 fmol/mg) and diploid tumours (14.6 fmol/mg). This may correlate with the lowest frequency of apoptosis in hypodiploid tumours, at least when measured by biochemical methods which only detect cells in the late phases of apoptosis. In contrast, the median PR was lowest in hyperdiploid tumours than in hypo and/or diploid tumours. The %S-phase/%fragmented DNA ratio for the hypodiploid tumours was 19.7, significantly higher than the ratios for hyperdiploid (8.2) and diploid tumours (3.6). These findings indicated that there is an imbalance between proliferative capacity and cell death or growth arrest in human breast tumours. This imbalance may well be linked to a loss of steroid hormone control.

Calcium Dobesilate protects human peripheral blood mononuclear cells from oxidation and apoptosis.

The antioxidant effects of Calcium Dobesilate (CD, Doxium) were investigated in relation to the oxidative status, apoptosis and in vitro proliferation of human peripheral blood mononuclear cells (PBMC) isolated from healthy donors. CD alone did not modify cell growth in vitrountil 10 microM. This molecule counteracted oxidative damages generated by the high reducing sugar dR and was shown to reduce apoptosis by delaying both membrane permeability changes and DNA fragmentation. CD 10 microM affected in a time-dependent dynamics several parameters representative of the cellular oxidative status. In particular, CD significantly increased the activity of glutathione S-transferase (GST) after three days of treatment and also, but to a lower extent, the activity of gamma-glutamyltransferase (gamma-GT). Both enzymes are known to be involved in the glutathione (GSH) metabolic cycle. This enzymatic behaviour was reversed at seven days of treatment, with a significant GST decrease and a gamma-GT activation. After seven days of CD exposure, the intracellular GSH content was enhanced and this resulted in a dramatic decrease in lipid peroxidation, underlining the powerful antioxidant properties of CD in human PBMC.

Active-site Arg --> Lys substitutions alter reaction and substrate specificity of aspartate aminotransferase.

Arg386 and Arg292 of aspartate aminotransferase bind the alpha and the distal carboxylate group, respectively, of dicarboxylic substrates. Their substitution with lysine residues markedly decreased aminotransferase activity. The kcat values with L-aspartate and 2-oxoglutarate as substrates under steady-state conditions at 25 degrees C were 0.5, 2.0, and 0.03 s-1 for the R292K, R386K, and R292K/R386K mutations, respectively, kcat of the wild-type enzyme being 220 s-1. Longer dicarboxylic substrates did not compensate for the shorter side chain of the lysine residues. Consistent with the different roles of Arg292 and Arg386 in substrate binding, the effects of their substitution on the activity toward long chain monocarboxylic (norleucine/2-oxocaproic acid) and aromatic substrates diverged. Whereas the R292K mutation did not impair the aminotransferase activity toward these substrates, the effect of the R386K substitution was similar to that on the activity toward dicarboxylic substrates. All three mutant enzymes catalyzed as side reactions the beta-decarboxylation of L-aspartate and the racemization of amino acids at faster rates than the wild-type enzyme. The changes in reaction specificity were most pronounced in aspartate aminotransferase R292K, which decarboxylated L-aspartate to L-alanine 15 times faster (kcat = 0.002 s-1) than the wild-type enzyme. The rates of racemization of L-aspartate, L-glutamate, and L-alanine were 3, 5, and 2 times, respectively, faster than with the wild-type enzyme. Thus, Arg --> Lys substitutions in the active site of aspartate aminotransferase decrease aminotransferase activity but increase other pyridoxal 5'-phosphate-dependent catalytic activities. Apparently, the reaction specificity of pyridoxal 5'-phosphate-dependent enzymes is not only achieved by accelerating the specific reaction but also by preventing potential side reactions of the coenzyme substrate adduct.

Cost analysis of early extubation after coronary bypass surgery.

BACKGROUND: Although early extubation after coronary bypass surgery has been shown to reduce length of stay, a systematic cost analysis of its economic benefit has not been reported, and previous studies have used hospital charges that are typically confused with actual costs. METHODS: A consecutive series of 690 patients undergoing coronary bypass surgery during a 24-month period were studied to determine the effect of early extubation, defined as removal of the endotracheal tube within 8 hours of arrival to the intensive care unit, on length of stay and hospital costs. Patients in group 2 (n = 362) who underwent coronary bypass surgery in 1995, subsequent to the initiation of an early extubation protocol, were compared with those in group 1 (n = 328) operated on in 1994, before implementation of early extubation. To reflect true hospital resource consumption, only costs (not charges) directly related to patient health core (variable direct cost) were analyzed. RESULTS: Baseline characteristics such as age, gender, previous myocardial infarctions, ejection fraction, reoperations, diabetes, and left main stenosis were similar in both groups. Operative mortality for the entire group was 3.3% and did not differ between the two groups; the incidence of serious morbidity was 10.9% for the entire group. Early extubation was accomplished in 38% of patients in group 2 versus 3% in group 1 (p < 0.001), and postoperative length of stay declined from 9.4 days to 7.7 days (p < 0.01). This was accompanied by a significant (p = 0.001) reduction in variable direct cost per case. CONCLUSIONS: Early extubation after coronary bypass surgery is an effective strategy of reducing length of stay and does not appear to impact on either morbidity or mortality. An additional benefit is significant cost savings realized through accelerated recovery and control of resource use.

Changing the reaction specificity of a pyridoxal-5'-phosphate-dependent enzyme.

The electron distribution in the coenzyme-substrate adduct of aspartate aminotransferase was changed by replacing active-site Arg386 with alanine and introducing a new arginine residue nearby. [Y225R, R386A]Aspartate aminotransferase decarboxylates L-aspartate to L-alanine (kcat = 0.04 s-1), while its transaminase activity towards dicarboxylic amino acids is decreased by three orders of magnitude (kcat = 0.19 s-1). Molecular-dynamics simulations based on the crystal structure of the mutant enzyme suggest that a new hydrogen bond to the imine N atom of the pyridoxal-5'-phosphate- aspartate adduct and an altered electrostatic potential around its beta-carboxylate group underlie the 650,000-fold increase in the ratio of beta-decarboxylase/transaminase activity.

Apoptosis in human lymphoblastoid cells induced by acivicin, a specific gamma-glutamyltransferase inhibitor.

We examined the effects of acivicin, a specific inhibitor of the ectoenzyme gamma-glutamyltransferase (gamma-GT), on gamma-GT activity and apoptosis in 2 human T-lymphoblastoid CEM cell lines, CCRF and VBL-100. In both cell lines, acivicin was found to cause morphological and biochemical changes of apoptosis in a dose-dependent manner. There was a close correlation between inhibition of gamma-GT activity and the emergence of apoptotic cells. However, VBL-100 cells had a 50% higher gamma-GT basal activity than CCRF-CEM cells, their enzyme activity was more inhibited, and, they had a greater apoptotic response to acivicin. The gamma-GT-specific activity in apoptotic/dead cells was also almost totally inhibited, while that of cells that remained alive after 5 days of acivicin treatment was not. These findings confirm that gamma-GT is implicated in the process of apoptosis of CEM cells.

Changes in the activities of signal transduction and transport membrane enzymes in CEM lymphoblastoid cells by glucocorticoid-induced apoptosis.

The behaviour of the membrane enzymes PIP2-phospholipase C (PLC), that modulates the extracellular signals, and gamma-glutamyltransferase (gamma-GT), involved in metabolite transport, was followed during the early and active phases of apoptosis induced in CCRF-CEM cells by glucocorticoid (10(-6) M dexamethasone, DEX). The activities of gamma-GT and PLC increased significantly at 15 and 30 s after dexamethasone addition. Both activities decreased to the control level after 2 min but increased again at 5 min. The enzyme activities were high in apoptotic cells. Apoptosis was seen after incubation with 10(-6)M dexamethasone for 48 h, with decreased cell number, cellular activation (MTT) and S-phase percentage; enhanced DNA fragmentation and propidium iodide uptake; and ultrastructural changes in the chromatin and cell membranes. The changes in enzyme activity are indicators which occur much earlier than the cellular events related to the apoptotic death in CD4(+)-T CEM lymphoblastoid cells.

Fatty acids and cell signal transduction.

Fatty acids released from membrane phospholipids by cellular phospholipases or available to the cell from the extracellular environment are important cell signalling molecules. Fatty acids can act as second messengers involved in the transduction of external signals because their concentrations are rapidly and transiently altered in response to the binding of specific agonists to plasma membrane receptors, and they substitute for the classical second messengers of the inositide phospholipid and the cyclic AMP signal transduction pathways. Fatty acids are also modulators because they act in a reversible manner at a precise intracellular location for a very short time to amplify, attenuate or deviate a signal. Fatty acids modify the activities of phospholipases, protein kinases, G-proteins, adenylate and guanylate cyclases as well as ion channels and other biochemical events involved in stimulus-response coupling mechanisms. The action of fatty acids on signal transduction pathways can be direct and/or indirect (by catabolic conversion of arachidonic acid to eicosanoids). However, a number of studies clearly show that fatty acids per se are messenger and modulator molecules mediating responses of the cell to extracellular signals.

Rapid and long-term effects of 17 beta-estradiol on PIP2-phospholipase C-specific activity of MCF-7 cells.

Activity of the enzyme phosphatidylinositol 4,5-bisphosphate phospholipase C (PIP2-PLC) was demonstrated in MCF-7 human breast cancer cell homogenate. The addition of 10(-9) M 17 beta-estradiol to the culture medium elicited in the cells two types of responses depending on the period of exposure. Enzyme activity was rapidly activated at 15 s of incubation. After 5 min, PIP2-PLC activity was inhibited, and this effect continued at least until 24 h of exposure to the hormone. When 17 beta-estradiol was added in vitro to the total homogenate of untreated cells, enzyme activity was stimulated in a dose-dependent manner. These findings indicate that 17 beta-estradiol induces early and long-term modifications of the phosphoinositide signal pathway in intact MCF-7 cells as well as in vitro. The rapidity of the early effect suggests a non-genomic action of estradiol.