Activation of the anti-cancer drug ifosphamide by rat liver microsomal P450 enzymes

The NADPH-dependent metabolism of ifosphamide catalyzed by rat liver microsomes was investigated in order to identify individual P450 enzymes that activate this anti-cancer drug and to ascertain their relationship to the P450 enzymes that activate the isomeric drug cyclophosphamide. Pretreatment of rats with phenobarbital or clofibrate increased by up to 8-fold the activation of both ifosphamide and cyclophosphamide catalyzed by isolated liver microsomes. Studies using P450 form-selective inhibitory antibodies demonstrated that constitutively expressed P450s belonging to subfamily 2C (forms 2C11/2C6) make significant contributions to the activation of both oxazaphosphorines in uninduced male rat liver microsomes, while the phenobarbital-inducible P450 2B1 was shown to be a major catalyst of these activations in phenobarbital-induced microsomes. Pretreatment of rats with dexamethasone increased liver microsomal activation of ifosphamide approximately 6-fold without a corresponding effect on cyclophosphamide activation rates. Ifosphamide activation catalyzed by dexamethasone-induced liver microsomes was minimally inhibited by anti-P450 2B or anti-P450 2C antibodies, but was selectively inhibited by anti-P450 3A antibodies. Selective inhibition of liver microsomal ifosphamide activation was also effected by the macrolide antibiotic triacetyloleandomycin, an inhibitor of several dexamethasone-inducible 3A P450s. These studies establish that a dexamethasone-inducible family 3A P450 can make an important contribution to rat liver microsomal ifosphamide activation, and suggest that dexamethasone pretreatment might provide a useful approach for modulation of ifosphamide metabolism in order to improve its therapeutic efficacy in cancer patients.     

Role of cholesterol in regulating apolipoprotein B secretion by the liver

The review examines the evidence that the supply of cholesterol available for incorporation into nascent lipoprotein particles exerts a regulatory influence on apolipoprotein (apo) B secretion by the liver. Support for this hypothesis comes both from in vitro experiments and from recent observations in normal subjects and patients with dyslipidemia associated with familial hypercholesterolemia, obesity, noninsulin dependent diabetes mellitus, growth hormone deficiency and cholesteryl ester storage disease. The findings do not negate a role for triglyceride synthesis in determining apoB secretion in very low density lipoprotein, but the inhibitory effects on the latter process of pharmacological blockade of cholesterol synthesis or esterification suggest that it is conditional upon an adequate supply of cholesteryl ester.     

Homologous and heterologous desensitisation of somatostatin-induced increases in intracellular Ca2+ and inositol 1,4,5-trisphosphate in CHO-K1 cells expressing human recombinant somatostatin sst5 receptors

The mechanisms responsible for somatostatin (SRIF)-induced increases in intracellular Ca2+ concentration ([Ca2+]i) and subsequent desensitisation were studied in CHO-K1 cells expressing human sst5 receptors (CHOsst5 cells). To study the nature of the desensitisation, interactions with uridine triphosphate (UTP) were examined. SRIF (pEC50 7.10) and UTP (pEC50) 5.14) caused concentration-dependent increases in [Ca2+]i but the SRIF maximum was about 40% of that to UTP. SRIF-, but not UTP-, induced increases in [Ca2+]i were transient and abolished by pertussis toxin. SRIF and UTP caused sustained increases in Ins(1,4,5)P3 but the SRIF maximum was about 30% of that to UTP. Removal of [Ca2+]e attenuated the SRIF-induced peak rise in [Ca2+]i but had no effect on the peak increases in Ins(1,4,5)P3. UTP-induced increases in [Ca2+]i and Ins(1,4,5)P3 were attenuated in the absence of [Ca2+]e. Following pre-exposure to SRIF (1 microM) or UTP (100 microM) for 5 min, subsequent SRIF responses were desensitised. Similar results were obtained in the absence of [Ca2+]e. Pre-exposure to SRIF had no effect on subsequent responses to UTP but in the absence of [Ca2+]e, responses to UTP were attenuated. The results suggest that SRIF but not UTP-induced increases in [Ca2+]i in CHOsst5 cells are mediated by pertussis toxin sensitive G proteins and are caused by an entry of extracellular Ca2+ and release from an Ins(1,4,5)P3 sensitive Ca2+ store. Homologous or heterologous desensitisation of agonist-induced increases in [Ca2+]i could be demonstrated in the presence or absence of extracellular Ca2+ respectively, and the latter appeared to involve depletion of a common intracellular Ca2+ store.     

Use of a thick-film capillary column for the analysis of organic acids in body fluids

Contradictory evidence as to the effects of alcohol on early information processing stages has been obtained from behavioral and psychophysiological investigations. In the present study, choice reaction times, error rates, and event-related potentials (ERPs) were recorded in a task in which variations in stimulus discriminability and of the (task irrelevant) correspondence between stimulus location and response location were orthogonally combined. Both discriminability and stimulus-response correspondence affected reaction time and electrophysiological chronometric measures as expected. However, no behavioral effects of alcohol were observed, possibly because of strategic adjustments. Psychophysiological chronometric measures indicated that alcohol leaves the initial flow of perceptual evidence to motor stages unimpaired, whereas it appears to increase the duration of stimulus evaluation. Interestingly, a number of alcohol effects appeared in the ERP amplitudes. Decrements in early ERP components indicate alcohol-induced impairments of involuntary visual attention and/or the automatic stimulus location-dependent activation of response channels. In contrast, a strong enhancement of a late slow-wave component under alcohol may reflect the investment of processing resources in order to maintain normal performance levels.     

The predictive value of electrodiagnostic studies in carpal tunnel syndrome

In recent years, electrodiagnostic studies have become an expected component in the work up and evaluation of carpal tunnel syndrome. We conducted a retrospective review of 460 carpal tunnel decompressions to determine whether the accuracy of diagnosis and the prediction of therapeutic outcome could be related to the positivity and severity of findings on preoperative electrical studies. The 349 patients (460 hands) were divided into two groups: group 1 consisted of hands with the clinical diagnosis of carpal tunnel syndrome but with normal electrodiagnostic studies (n = 62); in group 2 the hands had a clinical diagnosis of carpal tunnel syndrome with confirmatory electrodiagnostic studies (n = 398). The number and distribution of signs and symptoms of carpal tunnel syndrome were not statistically different between these two groups. There was not a statistically significant difference in the success rate of surgery or the incidence of complications. The similarities between these two groups suggests that the distinction between them (the positivity of electrodiagnostic studies) is an artificial one and that the clinical diagnosis of carpal tunnel syndrome is sufficient to predict the presence of the disease, as well as outcome of surgery. On the basis of these data, strict adherence to electrodiagnostic studies to confirm the diagnosis will exclude 13 percent of the patients with legitimate carpal tunnel syndrome from receiving appropriate therapy.     

Characterization of a baculovirus-encoded ATP-dependent DNA ligase

Sequence analysis of the Lymantria dispar multicapsid nucleopolyhedrovirus (LdMNPV) genome identified an open reading frame (ORF) encoding a 548-amino-acid (62-kDa) protein that showed 35% amino acid sequence identity with vaccinia virus ATP-dependent DNA ligase. Ligase homologs have not been reported from other baculoviruses. The ligase ORF was cloned and expressed as an N-terminal histidine-tagged fusion protein. Incubation of the purified protein with [alpha-32P]ATP resulted in formation of a covalent enzyme-adenylate intermediate which ran as a 62-kDa labeled band on a sodium dodecyl sulfate-polyacrylamide gel. Loss of the radiolabeled band occurred upon incubation of the intermediate with pyrophosphate, poly(dA) . poly(dT)12-18, or poly(rA) . poly(dT)12-18, characteristics of a DNA ligase II or III. The protein was able to ligate a double-stranded synthetic DNA substrate containing a single nick and inefficiently ligated a 1-nucleotide (nt) gap but did not ligate a 2-nt gap. It was able to ligate short, complementary overhangs but not blunt-ended double-stranded DNA. In a transient DNA replication assay employing six plasmids containing the LdMNPV homologs of the essential baculovirus replication genes, a plasmid containing the DNA ligase gene was neither essential nor stimulatory. All of these results are consistent with the activity of type III DNA ligases, which have been implicated in DNA repair and recombination.