Robert F. O'Dea

Familial pyrimidinemia and pyrimidinuria associated with severe fluorouracil toxicity.

RAPIDLY growing tumor cells depend on a high rate of pyrimidine synthesis for the generation of RNA and DNA. Fluorouracil is a pyrimidine-base analogue that acts as an antimetabolite to block the s...

Antihypertensive effect and elimination kinetics of captopril in hypertensive children with renal disease

The efficacy and safety of captopril were studied in 10 patients with secondary hypertension (renal parenchymal disease, four patients; renal artery stenosis, two; and renal transplant rejection, four). Captopril was administered according to a dose titration protocol that randomized the initial three doses (0.5, 1.0, and 2.0 mg/kg) of drug to one of six possible sequences. All patients received diuretics prior to and during captopril therapy. A significant reduction in mean blood pressure was observed in all 10 patients during the initial dose titration. No correlation was observed between captopril dose and magnitude of the blood pressure reduction. The onset of antihypertensive action began approximately 15 minutes after each orally administered dose and reached the nadir approximately 1 1/2 hours later. Blood pressure returned to predrug levels between 6 and 10 hours after the dose. A significant reduction in systolic and diastolic blood pressure was noted in all subjects after 1 week of captopril treatment and was maintained during the course of continuous therapy in nine of 10 patients. Captopril combined with hydrochlorothiazide produced a satisfactory therapeutic response in five patients; in four others, additional antihypertensive drugs were required. No significant adverse effects were observed. Plasma renin activity determined prior to initiation of captopril was not predictive of blood pressure response to the drug. The clearance of captopril from patients with kidney failure ranged from 14.1 to 18.8 ml/min/kg in five subjects with creatine clearance between 10 and 21 ml/min/1.73 m2.

CYP3A5 Genotype has a Dose-dependent Effect on ABT-773 Plasma Levels

The metabolizing enzyme cytochrome P450 (CYP) 3A5 is polymorphically expressed as a result of genetic variants that do not encode functional protein. Because of overlapping substrate specificity with CYP3A4 and the multidrug efflux pump P‐glycoprotein, the importance of CYP3A5 genetic polymorphism for pharmacokinetics is controversial.

Protein methylesterase and leukocyte chemotaxis.

Abstract The involvement of protein carboxylmethylesterase in leukotaxis has been studied. The enzyme is localized in the cytosolic fraction of neutrophils. In intact cells the presence of a chemoattractant induces a rapid stimulation of enzymatic activity. An half-maximal stimulation of this activity is induced by a concentration (3 n M ) of peptide attractant that is close to that (1 n M ) producing an half-maximal chemotactic response. In addition, the presence of a specific antagonist of peptide attractants lowers the stimulated esterase response of neutrophils to these chemotactic factors. The role of another enzyme, protein carboxylmethylase, has been re-examined. In the initial stages (1 min) of chemotactic stimulation, a highly variable increase in the activity of this enzyme occurs, while over a longer period (30 min) there is an apparent decrease. The results are consistent with a role for an increased turnover of protein methylesters in leukotaxis. The reversible neutralization of charge that would accompany this turnover process may contribute to alterations of membrane events that occur in the earliest stages of leukotaxis.

REGULATION OF GUANOSINE 3′,5′ CYCLIC MONOPHOSPHATE IN THE RAT PINEAL AND POSTERIOR PITUITARY GLANDS

Potassium and norepinephrine stimulate the accumulation of cyclic AMP and cyclic GMP in rat pineal glands and their efflux into the medium. The efflux of both cyclic nucleotides was blocked by probenecid. The accumulation and efflux of cyclic GMP, but not of cyclic AMP, depends upon the presence of intact nerve endings and extracellular calcium. The calcium‐dependent release of norepinephrine caused by veratridine was accompanied by the efflux of both cyclic AMP and cyclic GMP. In contrast, the calcium‐independent release of norepinephrine caused by tyramine was accompanied by the efflux of cyclic AMP but not cyclic GMP. Changes in cyclic GMP therefore, may be related to exocytosis from the sympathetic nerve endings in the gland. High concentrations of potassium also increased tissue levels of cyclic GMP in the posterior pituitary gland. Veratridine and potassium, but not norepinephrine, stimulated the efflux of cyclic GMP from this neurosecretory gland. Thus, the relationship between cyclic GMP and exocytosis may extend beyond sympathetic nerve endings. The enhanced accumulation of cyclic GMP in the pineal gland after potassium does not appear to be mediated by extracellular (released) norepinephrine. Desmethylimipramine blocked the norepinephrine‐stimulated changes in cyclic GMP, but not those caused by potassium. Investigation of the possible relationship between cyclic GMP and release of neurotransmitters is complicated by the apparent seasonal variation in the response of pineal cyclic GMP to potassium or norepinephrine.

Dihydropyrimidine dehydrogenase activity in human blood mononuclear cells.

Dihydropyrimidine dehydrogenase (DPD; EC 1.3.1.2) catalyzes the rate-limiting reaction in the catabolism of endogenous uracil and thymine and exogenous fluoropyrimidines. DPD activity was studied in human blood mononuclear cell supernatants utilizing a new and sensitive radiochromatographic assay. Total DPD activity showed a linear correlation with supernatant protein concentration. The affinity constants (Km) for NADPH and thymine were approximately 10 and 1 mumol/l, respectively. Maximal activity (Vmax) was observed at 0.25 mmol/l NADPH and 10 mumol/l thymine, respectively. DPD activity in normal individuals was 8.0 +/- (SD) 2.2 nmol/mg protein/h, and ranged from 4.4 to 12.3 nmol/mg/h (n = 25). This activity range was quite similar to values obtained in patients with metastatic solid tumors treated with fluorodeoxyuridine (FUdR; n = 33, p = 0.57). No correlation was found to exist between mononuclear leucocyte DPD activity and the observed toxicity of FUdR in the tested patients. A bimodal distribution of DPD activity was observed in the patients and in normal individuals. The entire study population tested could be divided into two groups with respect to DPD activity; one group with high (greater than 8 nmol/mg/h) activity and another with low (less than 8 nmol/mg/h) activity. The possibility that sex differences may have been responsible for this distribution of DPD activity could not be excluded. The findings of this study are relevant to the pharmacogenetics of fluoropyrimidines in humans.

Protein carboxymethylase and methyl-acceptor proteins in human platelets and erythrocytes

Abstract The subcellular distribution and characteristics of protein carboxymethylase (protein methylase II) and its methyl-acceptor proteins have been studied in human platelets and erythrocytes. Human platelets constitute a rich source of protein carboxymethylase with the majority of the active enzyme localized to the cytosol. In contrast, the highest specific activity of protein substrate(s) for the enzyme, i.e. methyl-acceptor proteins, appears to be associated with the particulate fraction containing indoleamine storage vesicles. Thrombin promotes a 60 per cent increase in the carboxymethylation of platelet protein after pulse labeling of the cells with [ 3 H]methionine. Human erythrocytes also contain protein carboxymethylase which is localized almost entirely to the cytosol. Unlike platelet cytosol, erythrocyte cytosol appears to be devoid of methyl-acceptor proteins. Erythrocyte membrane ghosts, however, can be readily methylated and probably constitute the sole source of methyl-acceptor proteins in these cells.

The function of protein carboxylmethyltransferase in eucaryotic cells

Protein carboxylmethyltransferase (PCM) is an enzyme whose function in eucaryotic cells remains controversial. Early studies suggested that protein carboxylmethylation subserved a regulatory, post-translational role in such diverse processes as secretion, neuronal receptor function, chemotaxis, and cellular differentiation. Later work strongly supported a totally unrelated role for this enzyme, i.e., the repair of spontaneously altered aspartate residues in cellular proteins. More recent evidence, however, suggests that a distinct, membrane-associated PCM catalyzes the methylation of alpha-carboxyl groups of C-terminal cysteines on discrete proteins. In view of these recent investigations, the data supporting a regulatory role for PCM are critically discussed and re-evaluated. There now appears to be compelling evidence that PCM(s) subserves both repair and regulatory functions in eucaryotic cells, catalyzing post-translational modifications of proteins involved in cell division, hormonal secretion, calmodulin-associated events and the interaction of guanyl nucleotide-linked proteins with the cell membrane.

Protein carboxymethylation during in vitro culture of human peripheral blood monocytes and pulmonary alveolar macrophages

Protein carboxymethylase (PCM) activity was evaluated for long-term in vitro cultures of human peripheral blood monocytes and pulmonary alveolar macrophages. Both cell types exhibited increases in endogenous (without addition of the exogenous substrate, gelatin) and total (with gelatin) enzyme activity with increased time in culture. Monocytes developed increased activity after a 5-day lag period; three-to four-fold increases over day 1 values in both total and endogenous specific activity occurred. In contrast, PCM activity increased for pulmonary alveolar macrophage (PAM) without a detectable lag period. Although the increase in endogenous activity of 10--14-day PAM culture was similar to comparable age monocyte cultures, total enzyme activity increased only two-fold above day 1 values. The observation of changes in PCM endogenous specific activity in monocyte cultures may reflect alterations in enzyme activity and/or levels of endogenous methyl-acceptor proteins.

Protein Carboxylmethyltransferase Activity in Intact, Differentiated Neuroblastoma Cells: Quantitation by S[3H]Adlenosylmethionine Prelabeling

Abstract: Protein carboxylmethyltransferase has been proposed to play a role in the regulation of neuroblastoma differentiation (Kloog et al., 1983). When we investigated this hypothesis further, different results for methyl ester formation were obtained when measured in acid‐precipitated proteins and in proteins separated by acidic polyacrylamide gel electrophoresis, following the incubation of intact neuroblastoma cells with [3H]methionine. These unexpected findings led to the development of a modified assay using S‐[3H]‐adenosylmethionine as the radiolabeled precursor for quan‐titating carboxyl methylation in intact cells. Data obtained from either acid‐precipitated proteins or those separated on an electrophoresis gel following S‐[3H]adenosylmethionine incubation directly correlated with data obtained from proteins separated by electrophoresis following [3H]methionine incubation. Using each of the three methods, an approxi‐mately twofold increase in the carboxyl methylation of cellular proteins was detected in neuroblastoma cells differentiated by reducing the serum concentration from 10 to 0.5%, but not in those cells differentiated with either 5 mMhexa‐methylene bisacetamide or 2% dimethyl sulfoxide. The finding that all detectable methyl acceptor proteins are increasingly methylated following 0.5% serum treatment and that this modification is substoichiometric suggests that protein carboxyl methylation is not an essential component of the differentiation process in neuroblastoma cells.