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Biomedical subjects

J L Hoffman

Publications and source records attributed to J L Hoffman.

At least 19 recordsLinked to original sources

S-phase modulation by irinotecan: pilot studies in advanced solid tumors.

Two studies of irinotecan (CPT-11) followed 24 h later by an antimetabolite were conducted. The objectives of the studies were: (1) to determine whether the increase in S-phase in tumor cells seen 24 h after CPT-11 administration in animal studies is seen in advanced solid tumors in patients, (2) to determine the dose of CPT-11 required to produce this effect, (3) to compare two methods (immunohistochemistry, IHC, for cyclin A, and DNA flow cytometry, FC) for evaluating S-phase in tumor biopsies from patients, and (4) to establish the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of CPT-11, given 24 h before gemcitabine (GEM, 1000 mg/m(2)). In one study CPT-11 was followed 24 h later by 5-fluorouracil (5-FU), 400 mg/m(2) per week for 4 weeks every 6 weeks. Tumor biopsies were obtained before and 24 h after CPT-11 administration before administration of 5-FU and assayed for S-phase by IHC for cyclin A and by FC. The starting dose of CPT-11 was 80 mg/m(2) per week with subsequent exploration of 40 and 60 mg/m(2) per week to establish the dose-effect relationship of the increase in tumor cells in S-phase. In the second study, CPT-11 was given 24 h before GEM 1000 mg/m(2) per week for 2 weeks every 3 weeks. Doses of 20-80 mg/m(2) were explored to establish the MTD and DLT and to study tumor cell S-phase in selected patients. CPT-11 80 mg/m(2) produced a mean increase in S-phase by IHC for cyclin A of 137%. Lesser increases were seen with 40 and 60 mg/m(2). CPT-11 followed 24 h later by 5-FU 400 mg/m(2) per week for 4 weeks was well tolerated. In the study of CPT-11 followed by GEM 1000 mg/m(2), 60 mg/m(2) of CPT-11 was the MTD.

Adenocarcinoma↗

Differential impairment of triazolam and zolpidem clearance by ritonavir.

BACKGROUND: The viral protease inhibitor ritonavir has the capacity to inhibit and induce the activity of cytochrome P450-3A (CYP3A) isoforms, leading to drug interactions that may influence the efficacy and toxicity of other antiretroviral therapies, as well as pharmacologic treatments of coincident or complicating diseases. METHODS: The inhibitory effect of ritonavir on the biotransformation of the hypnotic agents triazolam and zolpidem was tested in vitro using human liver microsomes. In a double-blind clinical study, volunteer study subjects received 0.125 mg triazolam or 5.0 mg zolpidem concurrent with low-dose ritonavir (four doses of 200 mg), or with placebo. RESULTS: Ritonavir was a potent in vitro inhibitor of triazolam hydroxylation but was less potent as an inhibitor of zolpidem hydroxylation. In the clinical study, ritonavir reduced triazolam clearance to < 4% of control values (p < .005), prolonged elimination half-life (41 versus 3 hours; p < .005), and magnified benzodiazepine agonist effects such as sedation and performance impairment. In contrast, ritonavir reduced zolpidem clearance to 78% of control values (p < .08), and slightly prolonged elimination half-life (2.4 versus 2.0 hours; NS). Benzodiazepine agonist effects of zolpidem were not altered by ritonavir. CONCLUSION: Short-term low-dose administration of ritonavir produces a large and significant impairment of triazolam clearance and enhancement of clinical effects. In contrast, ritonavir produced small and clinically unimportant reductions in zolpidem clearance. The findings are consistent with the complete dependence of triazolam clearance on CYP3A activity, compared with the partial dependence of zolpidem clearance on CYP3A.

Adult↗

Alprazolam-ritonavir interaction: implications for product labeling.

BACKGROUND: Pharmacokinetic interactions involving antiretroviral therapies may critically influence the efficacy and toxicity of these drugs, as well as pharmacologic treatments of coincident or complicating diseases. The viral protease inhibitor ritonavir is of particular concern since it both inhibits and induces the activity of cytochrome P450 3A (CYP3A) isoforms. METHODS: The inhibitory effect of ritonavir on the metabolism of alprazolam, a CYP3A-mediated reaction in humans, was tested in vitro using human liver microsomes. In a double-blind clinical study, volunteer subjects received 1.0 mg of alprazolam concurrent with low-dose ritonavir (four doses of 200 mg) or with placebo. RESULTS: Ritonavir was a potent in vitro inhibitor of alprazolam hydroxylation. The 50% inhibitory concentration was 0.11 micromol/L (0.08 microg/mL); this is below the usual therapeutic plasma concentration range (generally exceeding 2 microg/mL). In the clinical study, ritonavir reduced alprazolam clearance to 41% of control values (P < .001), prolonged elimination half-life (mean values, 30 versus 13 hours; P < .005), and magnified benzodiazepine agonist effects such as sedation and performance impairment. CONCLUSION: Consistent with in vitro results, administration of low doses of ritonavir for a short duration of time resulted in large impairment of alprazolam clearance and enhancement of clinical effects. Removal from product labeling of a warning against coadministration of ritonavir and alprazolam was based on a previous study only of extended exposure to ritonavir, in which CYP3A induction offset inhibition. Kinetic interactions involving antiretroviral therapies may be complex and time dependent. Product labeling should reflect this complexity.

Adult↗

Role of tyrosine kinase, ODC, and p34cdc2 kinase and cyclin B-associated cdc2 in jejunal enterocyte proliferation, maturation, and exfoliation in diabetic and DFMO-treated rats.

BACKGROUND: Several signal transduction pathways involved in rapidly proliferating cells of the intestine are currently not well understood. In the jejunum, crypt enterocytes are constantly replicating, lower villi are maturing, and upper villi are constantly shed. Type I diabetes is associated with jejunal mucosal hyperplasia, and administration of diflouromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), causes hypoplasia. Phosphorylation of proteins controls cellular proliferation and/or exfoliation. Cell division cycle kinase (p34cdc2) and cyclin B-associated p34cdc2 kinase regulate the cell cycle during the transition from G2-M phase. Our aims were to: 1) investigate the activities of tyrosine kinase, ODC, total p34cdc2 kinase, and cyclin B-associated p34cdc2 kinase and 2) phosphorylate proteins at tyrosine residues in jejunal upper villi, lower villi, and crypt enterocytes of DFMO-treated control and diabetic rats. METHODS: Diabetes was induced by streptozotocin. DFMO was administered in drinking water in both control and diabetic groups for 10 days after the induction of diabetes. Jejunal enterocytes were isolated and kept frozen at -70 degrees C until ready to process. RESULTS: Diabetic rats showed jejunal mucosal hyperplasia as indicated by increases in the jejunal mucosal weight/cm and DNA content compared to control rats. Diabetic crypt enterocytes showed significant increases in activities of tyrosine kinase, ODC, total p34cdc2 kinase, and cyclin B-associated cdc2 as well as increased phosphorylation of proteins at tyrosine residues compared to control rats. DFMO prevented diabetes-induced jejunal hyperplasia, and decreased the activities of these enzymes and phosphorylation of proteins at tyrosine residues in both diabetic and control rats. Phosphorylation of a 14 kd protein became prominent in crypt, upper villi, and lower villi enterocytes of DFMO-treated diabetic and control groups. CONCLUSION: Diabetic jejunal mucosal hyperplasia appears to involve activation of complex signal transduction pathways such as tyrosine kinase, ODC, p34cdc2 kinase, and cyclin B. These enzymes are involved in proliferation and/or exfoliation of jejunal enterocytes. Our results also suggest that tyrosine phosphorylation of a 14 kd protein may be involved in cell exfoliation and that jejunal mucosal hypoplasia may be a synergistic effect caused by down regulation of the above enzymes.

Animals↗

Effect of gamma subunit carboxyl methylation on the interaction of G protein alpha subunits with beta gamma subunits of defined composition.

A baculovirus expression system was used to determine the contribution of carboxyl methylation of specific G protein gamma subunits to the interaction between alpha and beta gamma subunits. beta gamma subunits were carboxyl methylated by a membrane bound methyltransferase in Sf9 cells, and periodate-oxidized adenosine inhibited this methylation by 90%. Carboxyl methylation of beta(1) gamma(2), beta(2) gamma(3), and beta(2) gamma(7) enhanced pertussis toxin-catalyzed ADP-ribosylation of alpha(i2) and alpha(i3) by about 2-fold. On the other hand, methylation did not enhance membrane attachment of beta gamma subunits. These results suggest that methylation of isoprenylated gamma subunits is required for optimal G protein-mediated signal transduction, but not membrane attachment.

Adenosine Diphosphate Ribose↗

Effects of diabetes and difluoromethylornithine treatment on hyperplasia, activity of MAP-kinase, and activity and association with cyclin B of p34cdc2 kinase in rat jejunal mucosa.

BACKGROUND: The different signal transduction pathways of rapidly proliferating cells of the intestine are not clearly understood. We report here a possible signaling pathway that involves regulation of activity of two closely related kinases, MAP-K (mitogen-activated protein kinase) and p34cdc2 kinase, during hyperplasia of diabetic jejunal mucosa. Our aim was to investigate the activity and phosphorylation of MAP-K and activity and association of p34cdc2 kinase with cyclin B during diabetes-induced jejunal mucosal hyperplasia in vivo. METHODS: We studied untreated and difluoromethylornithine (DFMO) treated control rats and rats with streptozotocin-induced type I diabetes. Assays were done 10 days after the induction of diabetes. In diabetic rats there was jejunal hyperplasia as indicated by increases in the jejunal mucosal weight/cm and DNA content as well as increased activities of MAP-K and p34cdc2 kinase and association of the latter with cyclin B as compared to corresponding values in control rats. Administration of DFMO, an irreversible inhibitor of the proliferation-associated enzyme ornithine decarboxylase (ODC), prevented diabetes-I induced jejunal hyperplasia and decreased all of the above enzymic parameters in both diabetic and control rats. In our previous in vivo study, DFMO administration also blocked diabetic jejunal hyperplasia and in addition decreased ornithine decarboxylase and tyrosine kinase activities jejunal and tyrosine phosphorylation of proteins. CONCLUSION: Thus the jejunal mucosal hyperplasia found in diabetes appears to involve activation of signal transduction pathways involving tyrosine kinases, MAP-K, p34cdc2 kinase, and cyclin B.

Animals↗

CNS sites involved in sympathetic and parasympathetic control of the pancreas: a viral tracing study.

The viral transneuronal tracing method was used to identify the CNS cell groups that regulate the parasympathetic and sympathetic outflow systems of the pancreas. Pseudorabies virus (PRV) was injected into the pancreas of vagotomized rats and after 6 days survival, the pattern of transneuronal labeling in the CNS sympathetic regulatory regions was determined. The converse experiment was performed in order to elucidate the central parasympathetic cell groups that regulate the pancreas. Immunohistochemical methods were used to identify putative neuropeptide- and catecholamine-containing CNS neurons involved in these regulatory circuits. The major finding of this study indicates that five brain regions, viz., paraventricular hypothalamic nucleus, perifornical hypothalamic region, A5 catecholamine cell group, rostral ventrolateral medulla, and lateral paragigantocellular reticular nucleus, contain a considerable amount of overlap in cell body labeling. In addition, the ventrolateral part of the periaqueductal gray matter and gigantocellular reticular nucleus, ventral part also showed a similar overlap, but the numbers of neurons found in these areas were considerably lower than the five major regions. These data suggest that these brain regions may provide parallel and possibly redundant, autonomic pathways affecting glucagon and adrenaline release.

Adrenal Medulla↗

Suicide inactivation of thioether S-methyltransferase by ethyl sulfide.

Thioether S-methyltransferase is an important enzyme in the metabolism of sulfur and selenium-containing compounds in animals. Ethyl vinyl sulfide was previously shown to be a substrate for this enzyme yielding methyl ethyl vinyl sulfonium ion (MEVS+) upon reaction with S-adenosylmethionine. Since vinyl sulfonium ions are reactive toward nucleophiles, the inactivation of thioether S-methyltransferase as a result of its methylation of ethyl vinyl sulfide was investigated. Ethyl vinyl sulfide was found to inactivate thioether S-methyltransferase in a time-dependent, pseudo-first-order process with k(inact) and KI values of 0.05 min(-1) and 0.275 mM, respectively. Calculation of the partition ratio revealed one inactivation event for every 100 turnovers. Dimethyl sulfide, an alternate substrate for thioether S-methyltransferase which yields the nonreactive product trimethyl sulfonium ion, protected the enzyme from inactivation by ethyl vinyl sulfide. The inactivation is a result of covalent reaction of methyl ethyl vinyl sulfonium ion with the enzyme as shown by comigration of radioactivity with the enzyme during denaturing gel filtration of reaction mixtures containing thioether S-methyltransferase, ethyl vinyl sulfide, and S-adenosyl[methyl-3H]methionine. Using this method the stoichiometry of inactivation was determined to be 1 mol of [3H]-methyl group/mol of thioether S-methyltransferase inactivated. Both the alternate substrate, dimethyl sulfide, and the competitive product inhibitor, S-adenosylhomocysteine, inhibited such covalent labeling of the enzyme by ethyl vinyl sulfide and S-adenosyl[methyl-3H]methionine. Chemically synthesized MEVS+ inactivated thioether S-methyltransferase, and [methyl-14C]MEVS+ covalently labeled the enzyme with 14C. These results reveal a previously unrecognized mechanism for biochemical activation of vinyl thioethers by methylation to form reactive vinyl sulfonium ions.

Animals↗

Relation of ornithine decarboxylase and tyrosine kinase activity in the jejunal mucosa in vivo.

Our aim was to study the relationship between jejunal mucosal activity of ornithine decarboxylase and tyrosine kinase during proliferation in adolescent rats in vivo. Their relationship in the proliferating intestinal mucosa under in vivo conditions has not been reported before. From the results of in vitro studies, it was speculated that tyrosine kinase activity modulated ornithine decarboxylase activity during colonic mucosal proliferation (Majumdar AP. Am J Physiol 259:G626-G630, 1990). Jejunal mucosal hyperplasia was induced by Type 1 diabetes and suppressed in both control and diabetic rats by administration of difluoromethylornithine. Jejunal mucosal weight and enzyme activity were determined after 3, 6, and 10 days, and tyrosine-specific phosphorylated proteins after 10 days of induction of diabetes. Difluoromethylornithine suppressed jejunal mucosal proliferation and tyrosine kinase activity after the 6- and 10- day study periods. After the 3-day study period although jejunal mucosal growth was suppressed, tyrosine kinase activity was not. Activity of tyrosine kinase and ornithine decarboxylase were highly significantly correlated at all time periods in both control and diabetic rats. Tyrosine-specific phosphorylated proteins of 34, 54, 80, and 200 kDa proteins were observed in jejunal mucosa of both control and diabetic rats. In the difluoromethylornithine-treated rats, phosphorylation of the above proteins was negligible while the phosphorylation of a 14-kDa protein was prominent. We speculate that in vivo ornithine decarboxylase activity may be modulating tyrosine kinase activity and that phosphorylation of a 14-kDa protein was associated with suppressed mucosal growth in difluoromethylornithine-treated rats.

Animals↗

Cloning and base sequence analysis of a cDNA encoding mouse lung thioether S-methyltransferase.

Thioether S-methyltransferase catalyzes transfer of the methyl group from S-adenosylmethionine to X in compounds of the structure R-X-R', where X may be sulfur, selenium, or tellurium, and R and R' may be various organic groups. To obtain a cDNA clone of thioether S-methyltransferase, a mouse lung cDNA library in lambda gt11 was screened with a 99 base-pair probe obtained by performing the polymerase chain reaction on oligo(dT) primed, reverse transcribed, mouse lung RNA using two degenerate primers designed from partial amino-acid sequences of the enzyme. The entire coding and 3'-untranslated regions were obtained and sequenced. The predicted protein contains 264 amino-acid residues and has a calculated M(r) of 29,460. The amino-acid sequence of thioether S-methyltransferase contains three motifs characteristic of many methyltransferases and has a high level of identity with the amino-acid sequences of nicotinamide N-methyltransferase and phenylethanolamine N-methyltransferase. However, in spite of the fact that they are both mammalian cytosolic sulfur methyltransferases, the sequences of thioether S-methyltransferase and thiopurine S-methyltransferase share little identity.

Amino Acid Sequence↗

Sequential methylation of 2-mercaptoethanol to the dimethyl sulfonium ion, 2-(dimethylthio)ethanol, in vivo and in vitro.

Thioether methyltransferase (S-adenosyl-L-methionine: thioether S-methyltransferase; EC 2.1.1.96) catalyzes the methylation of X in compounds of the type R-X-R'(X = S, Se, Te), yielding a methyl onium ion. Previous results using mice have demonstrated a role for thioether methyltransferase in the conversion and clearance of thioethers by methylation to more water-soluble methyl sulfonium ions suitable for excretion in the urine. A potential major physiological source of thiethers is reactions catalyzed by microsomal thiol methyltransferase (S-adenosyl-L-methionine: thiol S-methyltransferase; EC 2.1.1.9), which has been shown to methylate a diverse range of aliphatic sulfhydryl compounds. This study provides evidence for the sequential methylation of the aliphatic thiol, 2-mercaptoethanol, first to the methyl thioether, 2-(methylthio)ethanol, by thiol methyltransferase followed by methylation of this methyl thioeter to the dimethyl sulfonium ion, 2-(-dimethylthio)ethanol, by thioether methyltransferase. This sequence of reactions was demonstrated in vivo by injecting mice i.p. with radioactive 2-mercaptoethanol and analyzing the labeled methylated products, 2-(methylthio)ethanol and 2(dimethylthio)ethanol, in the urine by HPLC. In addition, the system converting 2-mercaptoethanol to 2-(dimethylthio)ethanol was reconstituted in vitro using solubilized mouse liver microsomes as a source of thiol methyltransferase and purified thioether methyltransferase from mouse lung. The results of these in vivo and in vitro studies established the sequential methylation of 2-mercaptoethanol by these two enzymes.

Animals↗

Role of carboxylmethylation in chemoattractant receptor-stimulated G protein activation and functional responses.

The role of G protein gamma subunit carboxylmethylation was examined in HL-60 granulocytes using an inhibitor of S-adenosylmethionine-dependent methylation, periodate-oxidized adenosine (Adox). A 40-60% reduction in gamma subunit carboxyl-methylation was associated with attenuation of fMet-Leu-Phe-stimulated GTP gamma S binding and GTP hydrolysis, while plasma membrane density of formyl peptide receptors, alpha i2, alpha i3, beta, gamma 5, and gamma 7 were not reduced. Reduced pertussis toxin-catalyzed ADP-ribosylation was re-established by in vitro methylation or addition of transducin beta gamma subunits. Superoxide release and inositol phosphate generation stimulated by fMet-Leu-Phe were significantly inhibited by Adox treatment. Carboxylmethylation contributes to transmembrane signalling and functional responses by enhancing association of alpha and beta gamma subunits.

Adenosine↗

Effect of prenylcysteine analogues on chemoattractant receptor-mediated G protein activation.

The hypothesis that carboxylmethylation of gamma subunits plays a role in G protein activation was tested by examining the ability of N-acetyl-S-farnesyl-L-cysteine (AFC) and its methyl ester (AFC-ME) to inhibit G protein-mediated signalling in intact HL-60 granulocytes and isolated HL-60 plasma membranes. Incubation of HL-60 granulocytes with AFC or AFC-ME inhibited superoxide release stimulated by fMet-Leu-Phe, but not by opsonized bacteria. AFC-ME, but not AFC, inhibited NaF- and PMA-stimulated superoxide release. Addition of AFC to HL-60 membranes inhibited fMet-Leu-Phe-, leukotriene B4- (LTB4) and C5a-stimulated GTP gamma S binding and GTP hydrolysis more potently than it inhibited basal guanine nucleotide exchange. AFC-ME inhibited basal- and ligand-stimulated G protein activation with equal potency, but less potently than AFC. AFC also inhibited mastoparan-stimulated GTP gamma S binding. Binding of fMet-Leu-Phe and LTB4 to HL-60 membranes was completely inhibited by AFC, while AFC-ME inhibited ligand binding by less than 50%. Neither AFC nor AFC-ME inhibited pertussis toxin or cholera toxin-catalysed ADP-ribosylation of alpha i. It was concluded that AFC interrupts signal propagation in G protein-dependent pathways by multiple mechanisms, including inhibition of ligand-receptor interactions, of receptor-G protein coupling and of guanine nucleotide binding to G proteins. Carboxylmethylation alters the specificity of AFC interruption of signal propagation in intact cells and isolated membranes.

Acetylcysteine↗

Polyamines and intestinal epithelial hyperplasia in streptozotocin-diabetic rats.

We measured specific activity of ornithine decarboxylase (ODC) and contents of putrescine and of the polyamines (spermidine and spermine) in isolated villus and crypt enterocytes from the jejunum of adolescent streptozotocin-diabetic and weight-matched control rats and diabetic and control rats treated with difluoromethyl ornithine (DFMO) 10 days after induction of diabetes. Consistent with previous observations by others of elevated ODC activity and contents of putrescine and of the polyamines in the intestinal epithelium undergoing hyperplasia, our studies showed elevated ODC activity and contents of putrescine and spermidine, but not of spermine, in the hyperplastic intestinal epithelium of diabetic rats. As in previous studies, suppression of ODC activity by DFMO prevented not only the jejunal epithelial hyperplasia in the diabetic rats, but also retarded jejunal epithelial growth in the control rats. DFMO administration lowered ODC activity by over 80% in both diabetic and control rat enterocytes and prevented the rise in enterocyte contents of putrescine and spermidine in the diabetic rat. The observation that, in both diabetic and control rats, treatment with DFMO lowered spermidine content in the crypt enterocytes but had no similar consistent effect on contents of putrescine or spermine suggested that spermidine could have been responsible for the intestinal epithelial hyperplasia in the diabetic rats and for the normal growth of the intestinal epithelium in control rats.

Animals↗

Ion chromatographic analysis of the purity and synthesis of sulfonium and selenonium ions.

The use of single-column ion chromatography with conductometric detection was shown to be useful for the analysis of sulfonium and selenonium ions. A Hamilton PRP X-200 cation column was eluted with either solvent A (5 mM nitric acid in 30% methanol) or solvent B (4 mM nitric acid). With solvent B, trimethylsulfonium ion was separated from trimethylselenonium ion. With solvent A, amounts of trimethylsulfonium ion from 2 to 250 nmol were detected with a linear response. The retention times and response factors for a series of sulfonium ions with various organic groups were determined. In general the ions with more hydrophobic groups eluted later, but all had similar response factors. The method was shown to be useful for optimizing conditions for the synthesis of methylsulfonium ions, specifically the reaction of methyl iodide with diallyl sulfide.

Allyl Compounds↗

Se-(8-azidoadenosyl)[75Se]selenomethionine as a photoaffinity label for S-adenosylmethionine binding proteins.

A method is described for the synthesis and purification of the photoaffinity label Se-(8-azidoadenosyl)[75Se]selenomethionine. This photoaffinity label can be used to specifically and covalently label the S-adenosylmethionine binding site of proteins that use this cofactor, as exemplified by labeling of thioether methyltransferase. By utilizing the gamma-emitting isotope of selenium, Se-(8-azidoadenosyl)[75Se]selenomethionine eliminates the need for the impregnation of acrylamide gels with fluorographic enhancers and dilution of liquid samples into scintillation cocktails, as is required with the commonly used methyl-3H-labeled and 35S-labeled S-(8-azidoadenosyl)methionine.

Affinity Labels↗