Mutant genes of cytochrome P-450IID6, glutathione S-transferase class Mu, and arylamine N-acetyltransferase in lung cancer patients. Epidemiological studies suggested a protective effect of certain phenotypes of polymorphic foreign-compound-metabolizing enzymes in some types of cancer. Poor metabolizers (PM) of debrisoquine 4-hydroxylase (cytochrome P-450IID6, CYP2D6) were found to be underrepresented among patients with lung cancer. Recent advances in molecular genetic characterization of CYP2D6, glutathione S-transferase (GST) class Mu, and arylamine N-acetyltransferase enabled genotypical determination of mutant alleles in lung cancer patients. Restriction fragment length polymorphism (RFLP) with a cDNA gene probe of CYP2D6 was analyzed in 79 lung cancer patients who were phenotyped with debrisoquine. Mutant alleles were detected by allele-specific polymerase chain reaction (PCR). In the same individuals, genotype of GST class Mu was analyzed by PCR and correlated with ex vivo activity of glutathione conjugation towards trans-stilbene oxide. RFLP patterns allowed discrimination between the slow and fast genotype of N-acetyltransferase as well as the heterozygotes. Three phenotypical PMs of debrisoquine (3.8%) were confirmed by PCR and RFLP. No PM could be unambiguously recognized only by RFLP patterns. The PMs were characterized by PCR and RFLP as carriers of the 29B/29B (n = 1), 29A/29B (n = 1), and 29A/44 (n = 1) mutant alleles. Higher debrisoquine hydroxylase activities were found in the homozygous EMs, who possess two active genes, as compared to heterozygous EMs, who have only one active gene. The patients with phenotypically impaired GST Mu activity were confirmed as such by PCR. A complete correspondence between phenotyping of N-acetyltransferase (with caffeine) and genotyping was found. The new genetic techniques proved to be powerful tools for molecular-epidemiological studies aimed at establishing host factors of cancer susceptibility.


Use of autotransplants in chronic myeloid leukaemia. Patients with newly diagnosed CML have Ph-negative stem cells in their marrow and peripheral blood. A minority can be restored to durable Ph-negative haemopoiesis by treatment with interferon-alpha but in general autografting with marrow collected after IFN treatment has not proved feasible. Some patients autografted with blood-derived stem cells collected in chronic phase have achieved durable Ph-negative haemopoiesis and selected patients autografted with 10-day incubated marrow cells have also achieved complete remission. An alternative approach involves collection and isolation of Ph-negative stem cells that may then be used for autografting. Different lines of evidence suggest that induction of remission for all patients with CML should be an attainable goal.


Drug treatment of the personality disorders. Many people with well defined borderline and schizotypal personality disorders may benefit considerably from small doses of neuroleptics. Depression that occurs with personality disorders, which is frequent, responds poorly to tricyclics but may respond better to neuroleptics, while the response to ECT is usually short lived. Selected borderline subjects may respond to MAOIs, particularly where there is a history of childhood hyperactivity. Carbamazepine and lithium may help some individuals with episodic behavioural dyscontrol and aggression, even in the absence of epileptic, affective or organic features. Drug treatments can be combined with psychotherapy, but further placebo-controlled trials are needed to clarify which drugs are most useful, and whether there are any useful clinical predictors of drug responsiveness.


Abnormal subjective time experience in depression. Abnormality of subjective time experience is well recognised in psychiatric illness. Earlier authors suggested that slowed time experience in depression is an aspect of psychomotor retardation, while more recently it has been argued that this disturbance is non-specifically linked to the global severity of the depressive syndrome. This study offers evidence that both views can be justified: slowed time awareness is a common symptom of depression, related particularly to retardation, and to the severity of the mood disturbance. Some of the experimental difficulties in this kind of research are illustrated.


Expressed emotion and schizophrenia in Italy. A study of an urban population. Forty-two schizophrenic patients and their close relatives took part in an Italian replication study of expressed emotion (EE). The patients were selected from the psychiatric ward of a general hospital in Milan and were subsequently followed up for nine months. All patients attended a community service clinic as out-patients, and all but one were prescribed neuroleptics for the duration of the study. Relatives were assigned to the high-EE group if they scored 4 or 5 on the emotional overinvolvement (EOI) scale, or showed hostility, or made six or more critical comments. On this basis, 18 (42%) families were rated as low EE and 24 (57%) as high EE. At follow-up, the admission rate for the 9-month period was significantly higher for the high-EE group (P less than 0.05). Furthermore, significantly fewer patients were readmitted from families showing high warmth (P less than 0.05). The presence of high warmth appeared to be associated with a lower admission rate, even in high-EE families.


Psychological well-being in families with a member suffering from schizophrenia. An investigation into long-standing problems. Levels of stress in carers of long-term schizophrenia sufferers attending a depot clinic were assessed. Nine out of 25 carers (36%) were identified as possible or definite cases on either the GHQ or the HAD. However, a substantial proportion of carers managed to cope with the difficulties without suffering psychologically.


Risk for definite neuroleptic malignant syndrome. A prospective study in 223 consecutive in-patients. The occurrence of neuroleptic malignant syndrome (NMS) was studied prospectively in two series of consecutive psychiatric in-patients (n = 223). The first group (n = 120) suffered from schizophrenia and was treated only with haloperidol. The second group (n = 103) was treated with diverse neuroleptics. All patients were on a single antipsychotic agent with no anticholinergic drug as prophylaxis. The incidence of full NMS per admission and first neuroleptic exposure was 5/223 (2.2%). Patients with bipolar affective disorder and those treated with injections were significantly over-represented in the NMS group.


Excitatory amino acid receptors appear to mediate paroxysmal depolarizing shifts in rat neocortical neurons in vitro. This study was designed to assess some of the contributions of excitatory amino acids to locally evoked responses in neurons in slices from frontal motor cortex in Sprague-Dawley rats. Intracellular recordings were obtained from 54 cortical neurons. Paroxysmal depolarization shifts (PDS) were evoked by local single pulse stimulation in cortex or in a small number of cases (n = 2) occurred spontaneously. These potentials could be abolished by application of kynurenic acid, a broad spectrum excitatory amino acid receptor antagonist. They were enhanced in Mg(2+)-free medium and could then be antagonized by application of D,L-2-amino-5-phosphonovalerate (AP5), a selective blocker of the N-methyl-D-aspartate (NMDA) receptors.


Domoic acid-containing toxic mussels produce neurotoxicity in neuronal cultures through a synergism between excitatory amino acids. In 1987, an intoxication by cultured mussels produced neurological problems, such as headache, confusion, and loss of memory, particularly severe at times. Neuronal damage was found in the hippocampus and amygdala of four patients. The intoxication was attributed to the presence in mussels of domoic acid, a rare excitatory amino acid acting at the non-NMDA receptor. We now report that a domoic acid-containing mussel extract is more neurotoxic for cultured neurons than purified domoic acid. Moreover, we show that this increase in neurotoxicity is selectively due to domoic acid potentiation of the excitotoxic effect of glutamic acid and aspartic acid present in high concentrations in mussel tissue. We also show that subtoxic concentrations of domoic acid are sufficient to potentiate glutamic acid and aspartic acid neurotoxicity, and we present evidence suggesting that the neurotoxic synergism may occur through a reduction of the voltage-dependent Mg2+ block at the NMDA receptor-associated channel, following activation of non-NMDA receptors by domoic acid. Thus, based on our results, we suggest that the contemporary presence in the brain of concentrations of domoic acid insufficient alone to be toxic, together with excitatory amino acids, of endogenous and eventually of diet-related origin, may have been relevant in the occurrence of the neurological problems reported.


In vivo voltammetric evidence for the detection of norepinephrine release in the thalamus of freely moving rats. The ventrobasal complex (VB) of the thalamus was monitored in awake rats for the presence of norepinephrine (NE) overflow following pharmacological manipulations and physiological stimulation. Overflow was detected using chronoamperometry with electrochemically pretreated, Nafion-coated carbon fiber microelectrodes. In vivo evaluation of the electrode responses to systemic drug administration showed that alpha-methyl-p-tyrosine (alpha-MPT) and FLA-63 caused decreases in baseline current. Increases in baseline current in the VB were observed in animals treated with pargyline, yohimbine and yohimbine injected 2 h postpargyline. The results suggest that an electrochemical signal primarily due to NE overflow can be monitored in thalamic regions. Vigorous somatosensory stimulation induced small, long-lasting (approximately 30 min), reproducible electrochemical signals in the VB which were suppressed by alpha-MPT or FLA-63. These studies provide in vivo evidence which suggests that stressful somatosensory input to the VB initiates the release of NE.


A high percentage yield of tyrosine hydroxylase-positive cells from rat E14 mesencephalic cell culture. In the ventral mesencephalon of the E14 rat fetus, 90% of the dopaminergic, tyrosine hydroxylase positive (TH+) cells are localized in 1.0 mm3 of tissue. This same ventral mesencephalic region also contains 90% of the dopamine content of the E14 ventral brainstem (2.2 +/- 0.3 nmol/mg protein). When cells were prepared for culturing from this localized area, and plated at a density of 2.5 x 10(5) cells/cm2, 17-21% of the cells were TH+, at 4 and 12 h, and at 1, 5, 7 and 10 days after plating. The percentage of TH+ cells was also 17-21% when examined at 4 h, 12 h or 5 days after plating at densities ranging from 7.8 x 10(3) to 2.5 x 10(5) cells/cm2. However, cell survival at a density of less than 6.2 x 10(4) cells/cm2 was poor after 5 days in culture. Based on the degree of neurite elongation and complexity, cell maturation appeared to be complete at 5 days in culture (DIV5), and appeared to be maintained at this level up to DIV10. By DIV14, neurite retraction was evident, and the cells were more rounded. These signs may indicate the inception of senescence in the cultures. A benztropine-sensitive, concentration-dependent dopamine uptake mechanism was demonstrated in the cultures at DIV7, and DA could be released from preloaded cells using 50 mM K+. Five morphological subtypes of TH+ cells were identified in the cultures. This primary culture of the ventral mesencephalic, dopaminergic area, with a high percentage of TH+ cells, is suitable for use in acute biochemical and cellular studies, between DIV 5 and DIV10.


Problems associated with the measurement of mean circulatory filling pressure by the atrial balloon technique in anaesthetized rats. To examine the existence of pressure equilibrium between tributary veins and the central vena cava during the mean circulatory filling pressure manoeuvre, pressures in the hepatic portal vein, renal vein, and inferior vena cava were determined at 4-s intervals over a 20-s period of circulatory arrest induced by inflating a right atrial balloon in normal blood volume, 10% volume depletion, and 10% volume expansion states in urethane-anaesthetized rats. Portal vein pressure determined 8 s after arrest during volume depletion and expansion was significantly higher than vena caval pressure (6.2 +/- 0.8 vs. 3.4 +/- 0.2 and 7.7 +/- 0.5 vs. 6.2 +/- 0.4 mmHg (1 mmHg = 133.32 Pa), respectively; p less than 0.01); this pressure disequilibrium continued for 16 s during volume expansion and for the entire 20 s during volume depletion. Renal vein pressure was equal to vena caval pressure during this manoeuvre. Portal vein pressure at normal blood volume was not significantly different from vena caval pressure following circulatory arrest (4.6 +/- 0.3 vs. 3.8 +/- 0.4 mmHg, respectively). Following ganglionic blockade, portal vein pressure was still significantly higher than vena caval pressure for 12 s during volume alterations. At the 8th s of the arrest the portal pressure determined in volume depletion was 3.6 +/- 0.3 mmHg and the inferior vena caval pressure was 2.6 +/- 0.4 mmHg (p less than 0.05). Under the volume expansion condition, the respective values were 6.5 +/- 0.3 and 5.3 +/- 0.4 mmHg (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)


Cocaine-enhanced arrhythmogenesis: neural and nonneural mechanisms. Cocaine abuse increases the susceptibility to cardiovascular complications and sudden cardiac death in man. We used programmed electrical stimulation of the heart to examine the arrhythmogenic influence of cocaine. Twenty-three pentobarbital-anesthetized adult dogs underwent programmed electrical stimulation using one to four extrastimuli before and during cocaine infusion. Autonomic decentralization was performed prior to the protocol in eight dogs. Induced ventricular arrhythmias included single premature ventricular depolarizations, doublets, triplets, ventricular tachycardia, and ventricular fibrillation. Intravenous cocaine, and subsequent adrenergic and muscarinic receptor blockade, or calcium channel blockade were evaluated for their influence on arrhythmogenesis. The incidence of induced ventricular arrhythmias was significantly elevated following cocaine and was reduced following propranolol and atropine. Verapamil, however, did not reduce the incidence of induced arrhythmias. In addition, cocaine significantly increased arrhythmia induction in decentralized animals, but propranolol, atropine, and phentolamine failed to reduce the proarrhythmic effects of cocaine in these animals. Thus, cocaine has a proarrhythmic effect on the heart with multiple mechanisms. The adrenergic mechanism appears to be a result of neurotransmitter uptake blockade, whereas the likely ionic mechanism is a neurally independent, direct effect on the heart.


A coupled assay detecting defects in fibroblast isoleucine degradation distal to enoyl-CoA hydratase: application to 3-oxothiolase deficiency. We developed a coupled NaH14CO3 fixation assay to detect 3-oxothiolase deficiency in extracts of cultured human fibroblasts. Cell extracts were incubated with tiglyl-CoA, NAD, CoASH, ATP and NaH14CO3. The enzymatic activities of tiglyl-CoA (enoyl-CoA) hydratase, 2-methyl-3-hydroxybutyryl-CoA dehydrogenase and 2-methylacetoacetyl-CoA thiolase (3-oxothiolase) were coupled to produce propionyl-CoA. Propionyl-CoA produced in the assay was estimated by fixation of NaH14CO3 into [14C]methylmalonyl-CoA employing endogenous propionyl-CoA carboxylase. The control activity was 32 +/- 23 pmol/min per mg protein (+/- 1 S.D., range 7-94; 28 cell lines). Five known cases of 3-oxothiolase deficiency had a mean activity of 2% of the control; a sixth case of 3-oxothiolase deficiency was significantly higher at 27% of the mean control value. Coupled assay activity was also low (3% of control) in the cells from a patient with propionyl-CoA carboxylase deficiency.


Polymorphisms of candidate genes in essential hypertension. 1. Family and population studies have reported that blood pressure has a heritability of 30-50%, but simple genetic models do not readily explain the patterns of inheritance of hypertension. 2. Restriction fragment length polymorphisms were used to study allele frequencies of a selection of candidate genes that may be important in determining the genetic component of hypertension. These included the genes for renin, haptoglobin, neuropeptide Y and cardiac myosin beta heavy chain. 3. There was no significant association between alleles at any of these loci and the presence of hypertension in this population, suggesting that the contribution of variation at these loci to the genetic component of the variance in hypertension may be quite small.


Tay-Sachs disease as a model for screening inborn errors. In the absence of treatments for most inborn errors of metabolism, the goal of both geneticists and health care providers has been the prevention of disease through identification of at-risk couples. When the enzyme deficiency responsible for a disorder is known, heterozygotes can frequently be identified by enzyme assay. The presence or absence of specific mutations in the genes coding for these enzymes may be determined directly if the gene of interest has been identified and characterized. Because the inherited metabolic disorders are rare, these approaches are useful only for individuals with a family history of a specific disease or for populations in which the gene frequency for a specific disease is increased. Tay-Sachs disease is a fatal, autosomal recessive, metabolic disease caused by deficient activity of the lysosomal enzyme Hex A. Although it is rare in the general population, in which the heterozygote frequency is approximately 1/167, it is elevated in a few populations, including the Ashkenazi Jewish community, in which the heterozygote frequency is 1/30. The ability to detect TSD heterozygotes reliably and to diagnose TSD prenatally using a simple and rapid enzyme assay has made prevention of this disorder possible through education and carrier screening. The identification of specific TSD mutations at the DNA level enables laboratories to provide more accurate screening and diagnosis in some families. The success of TSD screening in the Ashkenazi Jewish population has made it the prototype for screening among the inborn errors of metabolism. The TSD example becomes increasingly relevant as heterozygote detection becomes possible for other genetic disorders that are increased in well-defined populations. Cystic fibrosis is such a disease in the caucasian population.


Technical progress in parentage analysis. At the turn of the 20th century, Mendel's laws were found to be applicable to human blood groups. Within two decades, blood group genetics were applied to problems of parentage. Expansion of immunohematology into leukocyte antigen identification produced the single most informative, expressed polymorphism. About the same time, analysis of a great number of soluble protein polymorphisms followed advances in electric separation methods, enzymology, and immunochemistry. As new, independent loci were discovered, the power to exclude the falsely accused increased, and it became possible to apply Bayesian principles to determined probabilities of biologic relationships. The revolution in nucleic acid technology has dramatically improved analysis and statistical inferences. By the turn of the 21st century, laboratories should be able to determine biologic parentage with virtual certainty.


Hemodynamic effects of dobutamine in an intact animal model. Actions of dobutamine at the beta 1, beta 2, and alpha 1 adrenoreceptors were studied in anesthetized dogs. Six animals received dobutamine (at infusion rates of 0 to 160 micrograms/kg/min) with and without beta-adrenergic receptor blockade. Five animals received phenylephrine (0 to 16 micrograms/kg/min), with and without concurrent dobutamine (20 micrograms/kg/min); this procedure was repeated in five animals after beta-blockade. Dobutamine (10 to 160 micrograms/kg/min) increased heart rate (HR), cardiac output, and left ventricular change in pressure over time, and decreased systemic vascular resistance. beta-blockade prevented only dobutamine-induced changes in HR. Mean arterial pressure (MAP), unaffected by dobutamine alone, decreased with concurrent beta-blockade. Phenylephrine (1 to 16 micrograms/kg/min)-induced increases in MAP were unaffected by dobutamine; with beta-blockade, phenylephrine reduced MAP. Dobutamine prevented a phenylephrine-induced increase in systemic vascular resistance, an effect eliminated by beta-adrenergic receptor blockade. Dobutamine appeared to be an agonist at the beta 1- and beta 2-adrenoreceptors and at the myocardial alpha-adrenoreceptor. Dobutamine appeared to be an alpha-adrenergic receptor antagonist in the peripheral vasculature.


The effects of age on glutathione synthesis enzymes in lenses of Old World simians and prosimians. The activities of gamma-glutamylcysteine synthetase and glutathione synthetase, the two enzymes required for glutathione synthesis, were determined as a function of age in lenses of three species of Old World higher primates: orangutan, pigtail monkey and olive baboon. These were compared to enzyme activities in lenses of two prosimians: mouse lemur and galago. gamma-Glutamylcysteine synthetase activity decreased as a function of age in all three Old World simians. The rate of decrease was greatest in the juvenile lenses. In contrast, the enzyme activity increased continuously with age in the galago lens. In the mouse lemur the enzyme activity increased per lens, but was constant when expressed as specific activity or as units per gram of lens. The loss of enzyme activity with age was limited to Old World higher primates apparently representing genetic change. Glutathione synthetase activity decreased logarithmically with age in the lenses of all five species.


Stereoselective disposition of S-8666, a novel uricosuric antihypertensive diuretic, and its N-monodemethylated metabolite in a perfused rat liver preparation. Effect of protein binding on the kinetics of S-8666. S-8666 (5-dimethyl-sulfamoyl-6,7-dichloro-2,3-dihydrobenzofuran-2-carboxylic acid), a novel uricosuric antihypertensive diuretic, and its N-monodemethylated metabolite (M-I) were studied in a single pass perfused rat liver preparation under constant perfusate flow (ca. 16 ml/min). During perfusion with 100 nmol/ml of racemic S-8666 not containing bovine serum albumin (BSA), the steady-state hepatic extraction ratio of R(+)-S-8666 was two times higher (0.65 +/- 0.08) than that of S(-)-S-8666 (0.34 +/- 0.08). R(+)- and S(-)-M-I in the effluent perfusate plasma accounted for 64 and 18% of the influx rate of each enantiomeric S-8666, respectively. The N-monodemethylation was found to be responsible for the hepatic extraction of S-8666 enantiomers. S(-)-S-8666 was excreted into bile at a more rapid rate than the R(+)-enantiomer. Biliary excretion of R(+)-M-I was faster than S(-)-M-I, although the excretion rates of M-I were slower than those of S-8666 for both enantiomers. The steady-state extractions of preformed R(+)- and S(-)-M-I were low and a significant difference [S(-) greater than R(+)] was observed during the perfusion of 100 nmol/ml preformed racemic M-I without BSA. Increasing the concentration of BSA in the perfusate led to decreases in the extraction ratios of S-8666 enantiomers and biliary excretion rates of all chemicals, which was due to the decreases in the free fractions of S-8666 and M-I enantiomers. The binding of S-8666 and M-I enantiomers to BSA also showed stereoselectivity [R(+) less than S(-)].(ABSTRACT TRUNCATED AT 250 WORDS)


Diphenyl ether cleavage of 3-phenoxybenzoic acid by chicken kidney microsomal preparations. Incubation of 3-phenoxybenzoic acid[carbonyl-14C] with chicken kidney microsomal fraction produced two major radioactive compounds identified as 3-hydroxybenzoic acid and 4'-hydroxy-3-phenoxybenzoic acid. It was found that 4'-hydroxy-3-phenoxybenzoic acid was transitory in nature and was rapidly converted into 3-hydroxybenzoic acid. The conversion of 3-phenoxybenzoic acid to the products was NADPH dependent. This is a first example of metabolism of 4'-hydroxy-3-phenoxybenzoic acid to 3-hydroxybenzoic acid. Cytosol did not promote any cleavage. Similar activities were present in liver microsomes, but the activity was lower than in kidney. Intestinal contents, homogenates, or microsomal fractions did not metabolize 3-phenoxybenzoic acid.


Homeostasis of sulfate and 3'-phosphoadenosine 5'-phosphosulfate in rats with deficient dietary intake of sulfur. This study was designed to determine the role of dietary organic and inorganic sulfur on 3'-phosphoadenosine 5'-phosphosulfate (PAPS) homeostasis. Organic sulfur was altered by adding various amounts of methionine (0.15, 0.3, 0.6, or 1.2%) to a sulfhydryl-deficient diet. Inorganic sulfur was altered by providing rats with no sulfate or sulfate in their diets (0.12%) and distilled or tap water. Rats received these diets for 5 days. The two lowest methionine-containing diets produced a 60% reduction in liver glutathione concentrations, and the addition of sulfate to the diets did not restore hepatic glutathione levels. Urinary sulfate excretion was reduced by 95% in rats fed the three low-methionine diets. Addition of sulfate to these diets increased the urinary excretion of sulfate, but did not return sulfate levels to control values. The three low-methionine-containing diets decreased serum and liver sulfate concentrations about 50% and addition of sulfate to these diets largely restored them to control levels. Hepatic PAPS concentration was decreased (10%) only in the group receiving the lowest methionine content in their diet, and addition of sulfate had no effect on hepatic PAPS. In summary, dietary alterations of sulfur lowered the glutathione concentration in the liver as well as decreased sulfate levels in serum, liver, and urine, but had minimal effect on hepatic PAPS concentrations. Therefore, it appears that hepatic steady-state PAPS levels are not highly dependent on the sulfur content of the diet.


Purification and characterization of a cytochrome P-450 isozyme catalyzing bunitrolol 4-hydroxylation in liver microsomes of male rats. A cytochrome P-450 isozyme, P-450 bunitrolol (BTL), catalyzing bunitrolol 4-hydroxylation was partially purified from liver microsomes of adult male Sprague-Dawley rats by hydrophobic affinity chromatographic (omega-aminooctyl-Sepharose 4B) and high-performance liquid chromatographic (anion-exchange diethylaminoethyl-5PW) techniques. The specific content of the final preparation was 5.02 nmol/mg protein, which was 7.8-fold that of microsomes. It showed two protein bands of 49 and 32 kDa in sodium dodecylsulfate-polyacrylamide gel electrophoresis. N-Terminal 20 amino acid sequence of the protein of a higher molecular mass (49 kDa) isolated by an electroblotting technique is 94% homologous with that of CYP2D2. In a reconstituted system including NADPH-cytochrome P-450 reductase and an NADPH-generating system, the final preparation had the highest activity toward BTL and debrisoquine 4-hydroxylation among 12 isozymes of cytochrome P-450 examined. Kinetic parameters, KM and Vmax values, of P-450 BTL calculated for BTL 4-hydroxylation were 10.7 microM and 19.68 nmol/min/nmol P-450, respectively, whereas those values (mean +/- SE) of rat liver microsomes were 0.84 +/- 0.05 microM and 2.05 +/- 0.11 nmol/min/nmol P-450. When preincubated with rat liver microsomes, the antibody against the final P-450 BTL preparation suppressed bunitrolol and debrisoquine 4-hydroxylase activities dose-dependently and almost completely. These results suggest that cytochrome P-450 BTL and its immunochemically related P-450 isozyme(s) play a major role in debrisoquine 4-hydroxylation as well as in BTL 4-hydroxylation in rat liver microsomes.


Inhibition of debrisoquin clearance in perfused rat livers and inhibition of dextromethorphan metabolism in human liver microsomes by 4-hydroxydebrisoquin or other metabolites of debrisoquin. Debrisoquin undergoes oxidative metabolism to 4-hydroxydebrisoquin, catalyzed by cytochrome CYP2D1 in rats and CYP2D6 in humans. Cytochrome CYP2D6 also plays a major role in dextromethorphan O-demethylation. In preliminary studies in perfused Lewis rat livers, we observed a difference in repeat clearance experiments using debrisoquin, but not dextromethorphan. To determine whether this change in clearance with time was due to the accumulation of 4-hydroxydebrisoquin, we sequentially used a recirculating and nonrecirculating perfusion system in the same liver perfusion experiment. We also studied the kinetics of dextromethorphan O-demethylation in microsomes prepared from human and rat livers in the presence and absence of 4-hydroxydebrisoquin. Results from the perfused rat liver experiments showed a drop in clearance from 3.27 +/- 0.57 ml/min (clearance 1) to 1.61 +/- 0.27 ml/min (clearance 2) (p less than 0.05 vs. clearance 1) during recirculation, but clearance returned to 3.21 +/- 0.46 ml/min (clearance 3, no significance vs. clearance 1) after a 30-min period of liver perfusion using a nonrecirculating system. There was significant accumulation of 4-hydroxydebrisoquin in the liver perfusate during recirculation, and concentrations fell when the nonrecirculating system was used. In microsomal studies, 4-hydroxydebrisoquin competitively inhibited dextromethorphan metabolism in human microsomes was 600 microM. These data suggest that: (a) 4-hydroxydebrisoquin and/or other metabolites of debrisoquin have an inhibitory effect on CYP2D1 and CYP2D6; (b) the active site of human CYP2D6 has different substrate specificity than the rat isozyme (CYP2D1) and/or that the pathways of metabolism of dextromethorphan are different in the Lewis rat and not primarily dependent on the activity of CYP2D1.


In vitro and in vivo biotransformations of the potent leukotriene D4 antagonist verlukast in the rat. Verlukast, (S)3-((((3-(2-(7-chloroquinolin-2-yl)-(E)-ethenyl)phenyl)- 3-dimethylamino-3-oxopropylthio)methyl)thio)propionic acid, formerly known as MK-679, is a potent leukotriene D4 antagonist. Verlukast was incubated with rat liver microsomes under oxidative conditions to generate five metabolites, which were identified as the four possible isomeric monosulfoxides (M1-M4), and the N-hydroxymethyl amide (M5). This latter metabolite loses the elements of formaldehyde to yield the N-monomethyl amide (M6). These metabolites were isolated from a large microsomal incubation and were characterized by UV, 1H-NMR, and fast atom bombardment-MS. These data were identical to those obtained from synthetically prepared standards. Microsomal incubations of verlukast supplemented with UDP-glucuronic acid yielded the acyl glucuronide metabolite (M7), which was isolated and characterized by UV, 1H-NMR, and fast atom bombardment-M5. Verlukast was regenerated from M7 upon treatment with either beta-glucuronidase or strong aqueous base (pH greater than 11). The metabolites described above were all detected in bile collected from a rat dosed with verlukast.


Physiological disposition and metabolism of L-365,260, a potent antagonist of brain cholecystokinin receptor, in laboratory animals. L-365,260 [3R(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4- benzodiazepine-3-yl)-N'-(3-methylphenylurea)], a potent nonpeptide antagonist of the CCKB receptor, is currently under investigation to treat anxiety and panic disorders. This study describes absorption and disposition of the drug in rats, dogs, and monkeys. Following iv administration (5 mg/kg), L-365,260 was cleared very rapidly in rats, dogs, and monkeys. In all species, the concentrations of the drug in plasma declined in a polyphasic manner. There was no difference in total blood clearance among species, whereas considerable species differences were observed in volume of distribution and terminal half-lives. Binding of 14C-L-365,260 to plasma protein was extensive for all test species (greater than 96%). Interspecies differences in absorption were also observed. The bioavailability for rats, dogs, and monkeys was approximately 14%, 9%, and 2%, respectively. HPLC radiohistograms of urine and bile revealed that only trace amounts of intact drug were present; the drug was mainly eliminated by biotransformation. NMR and mass spectral analyses indicate that hydroxylation and glucuronide conjugation are the major biotransformation pathways.


Differential effects of human recombinant interleukin-1 beta on cytochrome P-450-dependent activities in cultured fetal rat hepatocytes. The immunomodulator interleukin-1 beta (IL-1) is one of the major inflammatory mediators. In vivo, it has been reported to depress some rat liver cytochromes P-450 (cytochrome P-450). Our aim was to study those effects in vitro, using cultured fetal rat hepatocytes as a model. Testosterone 6 beta-hydroxylase (cytochrome P-450 IIIA family activity) was not depressed by IL-1 treatments, but its induction by dexamethasone was prevented. The effect was time- and dose-dependent. Ethoxyresorufine-O-deethylase (cytochrome P-450 IA1 activity) decreased after IL-1 treatment, and dexamethasone partially prevented this inhibition. Acute phase effects of IL-1 were assayed by albumin and transferrin secretions. The cell's sensitivity to glucocorticoids was determined by tyrosine-aminotransferase activity. Our data demonstrate that IL-1 was able to prevent the glucocorticoid induction of cytochrome P-450 IIIA involving at least two different mechanisms. This is in agreement with the theory suggesting that the induction of CYPIIIA family by glucocorticoids requires the presence of the glucocorticoid receptor and some other regulatory elements. Other cytochrome P-450-dependent activities (IIA1, IIB1/2, and IIC11) were inhibited by IL-1 treatments, depending on dose and time, but some were also protected by dexamethasone.


Pharmacokinetic evaluation of drug interactions with anti-human immunodeficiency virus drugs. V. Effect of soluble CD4 on 2',3'-dideoxycytidine kinetics in monkeys. This study was conducted to determine if soluble CD4 (ST4) altered the pharmacokinetics of 2',3'-dideoxycytidine (ddC) in nonhuman primates. Each of six monkeys received 5 mg/kg of ddC iv in the absence and presence of two different iv regimens of ST4. The ST4 regimens produced steady-state plasma concentrations of 10.3 micrograms/ml (N = 3) and 22.2 micrograms/ml (N = 3) for 30 min following ddC administration. Pharmacokinetic parameters for ddC and ST4 were calculated based on plasma and urine concentrations of ddC and plasma concentrations of ST4. Following combined ddC and ST4 administration, in both the low- and high-dose ST4 groups, plasma concentration-time profile of ddC were similar for each monkey, and no statistical differences were observed in the pharmacokinetic parameters compared with those obtained when ddC was given alone. Complete urinary excretion data for ddC was obtained in 3 of the 6 animals studied. At the low ST4 dose, one animal had a reduced renal clearance of ddC, whereas at the high ST4 dose two animals recorded an increased renal clearance of ddC. ST4 plasma concentrations were comparable to in vitro concentrations of antiviral activity, with pharmacokinetic parameters similar to those reported previously. The kinetic information provides a basis for rational dosage design for combination chemotherapeutic regimens of ddC and ST4 in human immunodeficiency virus infection.


Disposition kinetics of amphotericin B in rats. The influence of dose. Amphotericin B (Am-B), an antifungal drug, has been used for the treatment of most disseminated fungal infections. The current therapeutic regimens for this drug are complex, at least in part as a result of limited pharmacokinetic information. In this study, we examined the disposition of Am-B as a function of dose in rats. Groups of male Sprague-Dawley rats were given Am-B by a 15-min iv infusion at three different doses. We observed no significant differences in systemic clearance among the Am-B doses studied (4.29 +/- 1.42, 3.83 +/- 0.29, and 3.92 +/- 0.68 ml/min.kg for doses of 0.28, 0.45, and 0.84 mg/kg, respectively). Similarly, small differences were seen in volumes of distribution as a function of dose. We conclude that the disposition kinetics of Am-B were linear over the dose range studied.


Sulfation of estrone and 17 beta-estradiol in human liver. Catalysis by thermostable phenol sulfotransferase and by dehydroepiandrosterone sulfotransferase. Sulfation is a major pathway in humans for the biotransformation of estrogens. However, the nature of the enzymes that catalyze the sulfation of estrone (E1) and 17 beta-estradiol (E2) in human liver is unclear. Human liver contains at least three well-characterized cytoplasmic sulfotransferases, the thermostable (TS) and thermolabile (TL) forms of phenol sulfotransferase (PST) and dehydroepiandrosterone sulfotransferase (DHEA ST). Therefore, we determined optimal conditions for the assay of E1 and E2 ST activities in human hepatic cytosol to compare their properties and regulation with those of the three well-characterized human liver ST activities. Thermal inactivation studies showed that human liver E2 ST and TS PST had very similar thermal stabilities. The thermal inactivation profile of E1 ST suggested that this activity might be related to both DHEA ST and TS PST. Inhibition studies performed with 2,6-dichloro-4-nitrophenol (DCNP) also showed similar inhibition profiles for E2 ST and TS PST. Neither thermal inactivation nor DCNP inhibition studies indicated a possible relationship between TL PST activity and E1 or E2 sulfation. Experiments performed with 20 individual human liver samples showed highly significant correlations between activity levels of E2 ST and TS PST (rs = 0.944, p less than 0.0001), E1 ST and DHEA ST (rs = 0.845, p less than 0.0001), and, to a lesser degree, E1 ST and TS PST (rs = 0.608, p less than 0.01). Ion exchange chromatography of a human liver preparation, followed by assay of all five ST activities, confirmed the important roles played by TS PST and DHEA ST in the sulfation of E2 and E1. Similar results were found by study of the elution patterns of ST activities after ion exchange chromatography of human jejunal mucosal preparations. Partially purified TL PST, however, was unable to catalyze the sulfate conjugation of either E1 or E2. All of these results were compatible with the conclusion that, in human liver, TS PST is the enzyme predominantly responsible for the sulfate conjugation of E2, DHEA ST is the major enzyme responsible for the sulfation of E1, and TL PST does not appear to catalyze the sulfation of either E1 or E2.


Obesity as a risk factor in drug-induced organ injury. V. Toxicokinetics of gentamicin in the obese overfed rat. Obese human patients and obese overfed rats treated chronically with gentamicin suffer greater renal injury than nonobese patients and control animals. To understand the mechanism of this heightened susceptibility to the nephrotoxic effects of gentamicin, this study examines the plasma-time course and renal uptake of gentamicin in control and obese overfed rats following a single bolus dose. Gentamicin was administered ip to control rats at 30 mg/kg total body mass and to obese rats at 30 mg/kg ideal body mass plus 40% of excess body mass. Following gentamicin dosing, only 1 of 11 concentration-time points taken over 6/hr postdosing was different between control and obese groups. In addition, the area under the plasma concentration curve extrapolated to infinite time was not different between obese and control rats (mean +/- SD of 4.47 +/- 0.85 vs. 4.13 +/- 0.35 mg.min.ml-1, p greater than 0.5). The gentamicin plasma concentrations after 6 hr were less than 1 microgram/ml and not different between the groups; however, the concentration of gentamicin in the kidneys was 33% greater in obese than control rats at this time (324 +/- 66.9 vs. 244 +/- 34.7 micrograms/g, p less than 0.05). The fraction of dose and the total amount of drug that accumulated in the kidneys were also greater in the obese rats (42 and 72% increases). Considered with the results of previous studies, it appears that obese overfed rats sustain more severe nephrotoxicity following comparable plasma gentamicin exposure because of increased renal uptake and/or retention of drug.


Physiological disposition of aerosolized MK-679 in rats. [14C]MK-679, a potent antagonist of leukotriene D4, was suspended in freon under pressure and sprayed into rat lungs through a tracheal cannula. The particle size of the drug was 1 to 5 microns, and the mean dose was 98.8 +/- 4.46 micrograms/rat. Time course studies indicate that MK-679 was slowly but efficiently absorbed from the lung, with only 6% of the dose remaining in the lung at 6 hr. Biliary excretion, the major route of elimination of aerosolized MK-679, accounted for 48% of the dose in 6 hr. Concentrations of the parent drug plateaued in plasma at 1 to 4 hr, and drug was not detectable in plasma at 6 hr. The parent drug accounted for 94% of the radioactivity in the lung, indicating no significant metabolism by lung tissue. The concentration of MK-679 after aerosol administration was higher in the lung and lower in plasma than after iv administration of the drug at 28 times the aerosol dose. The results of this study suggest that inhalation of MK-679 should be a considered route of administration for the treatment of asthma.


Tissue concentrations of coenzyme Q10 in the rat following its oral and intraperitoneal administration. Daily oral or ip administration of coenzyme Q10 to rats for time periods of 2 to 10 weeks leads to its accumulation in liver, concentrating in the soluble fraction of the liver cells. No uptake of coenzyme Q10 can be detected in the heart or kidney. Intraperitoneal administration also results in the accumulation of coenzyme Q10 in the spleen. It is concluded that the normal endogenous levels of quinone in the rat heart and kidney cannot be supplemented over the long term by administration of exogenous quinone.


Treatment of acromegaly with dopamine agonists. This article reviews the current understanding of how dopamine agonists stimulate growth hormone secretion in normal individuals, yet suppress growth hormone secretion in acromegaly patients. Although bromocriptine normalizes growth hormone or somatomedin C hypersecretion in a minority of patients, a significant number of subjects have a clinical response. Side effects of bromocriptine treatment and newer dopamine agonists are discussed. These drugs can be useful medical therapy, either used alone in selected patients or in combination with other therapeutic modalities.


Somatostatin analogs in the treatment of acromegaly. Medical therapy of acromegaly with the somatostatin analog octreotide is very successful. Both clinical symptomatology and hormonal hypersecretion by the growth hormone-secreting pituitary adenomas are controlled, and peripheral IGF-I levels also return to near normal levels. Tumor shrinkage is observed in most patients. Somatostatin analogs can be used after noncurative surgery and for a limited period after radiotherapy, and octreotide pretreatment might improve the outcome of surgery.


Cytochrome P-450 in the brain. Potential evolutionary and therapeutic relevance of localization of drug-metabolizing enzymes. The cytochrome P-4502D6 enzyme is reportedly expressed in the brain. It was hypothesized that brain P-450 may serve to diminish exposure and toxicity from exogenous substances by enhancing their elimination from the central nervous system (CNS). To test this, a physiologic-based kinetic model was developed to simulate drug concentrations in brain and blood in the presence and absence of CNS metabolism. The amount of cytochrome P-450 in the brain was set at 0.25% of hepatic levels to reflect the small amounts of enzyme reportedly present in the CNS. Simulations were performed for low, intermediate, and high clearance drugs, assuming the presence or absence of brain P-450 enzymes. Enzyme localization was simulated by systematically reducing the volume into which the enzyme was expressed. No difference could be detected in drug concentrations in the blood, regardless of whether enzyme was present and/or localized in brain tissue. Marked differences, however, were observed in steady-state tissue drug levels that were highly dependent on the presence or absence of the brain enzyme, its degree of localization, and its efficiency for its substrate. These results suggest that large intersubject variability in CNS response to some drugs could reflect interpatient differences in CNS drug metabolism.


Effect of diets rich in medium-chain and long-chain triglycerides on lipogenic-enzyme gene expression in liver and adipose tissue of the weaned rat. The activity and mRNA concentrations of two lipogenic enzymes, fatty-acid synthase and acetyl-CoA carboxylase were measured in the liver and white adipose tissue of rats weaned to a carbohydrate-rich diet containing either long-chain or medium-chain fatty acids, and compared to those of rats weaned on a diet containing less than 1% (total energy) fat (high-carbohydrate diet). In the liver, the diet containing long-chain fatty acids inhibited the increase of both lipogenic-enzyme mRNA concentrations and activities seen at weaning on the high-carbohydrate diet but did not prevent the decrease in phosphoenolpyruvate carboxykinase mRNA and activity. In contrast, the diet containing medium-chain fatty acids induced a slower but finally similar increase in lipogenic-enzyme mRNA concentrations and activities. In adipose tissue, a similar trend was observed, although the inhibitory effect of the diet containing long-chain fatty acids was considerably less marked than in liver. It is concluded that medium-chain and long-chain fatty acids have not the same inhibitory potency of the gene expression of lipogenic enzymes, and that long-chain fatty acids have a more marked effect in the liver.


Effect of dose on the percutaneous absorption of 2- and 4-chloronitrobenzene in rats. The effect of dose on the dermal absorption of 2- and 4-chloronitrobenzene (2- and 4-CNB) has been investigated in rats following nonocclusive protective dermal application on an area of 4 cm2 per animal at approximately 0.0325, 0.325, and 3.25 mg/cm2 (0.65, 6.5, and 65 mg/kg, respectively). At the three-dose levels, 33-40% and 51-62% of the dose of 2- and 4-CNB, respectively, was absorbed from the skin within 72 hr. The balance of the dose was recovered in the protective device and the organic trap (i.e. that portion unavailable for dermal absorption). The absorbed radioactivity was excreted in urine (21-28% of dose, 2-CNB; 43-45%, 4-CNB) and feces (11-15%, 2-CNB; 5-12%, 4-CNB). The extent and rate of dermal absorption and urinary and fecal excretion of 2-CNB were linear over the 0.65-65 mg/kg dose range; for 4-CNB they were linear over the 0.65-6.5 mg/kg dose, and nonlinear at the 65 mg/kg dose.


Changes in calpain and calpastatin mRNA induced by beta-adrenergic stimulation of bovine skeletal muscle. Administration of beta-adrenergic agonists to mammals can produce skeletal muscle hypertrophy in some species and muscle types. The growth-promoting effect appears to be due to suppression of protein breakdown rather than stimulation of synthesis, although evidence from turnover studies is equivocal. In ovine muscle, changes in the activity of the calcium dependent neutral proteinases (calpains I and II) and their specific inhibitor (calpastatin) accompany beta-agonist-induced hypertrophy. These observations suggest that the calpain system is involved in myofibrillar protein turnover in some way. Alternatively, the relationship with hypertrophy may be indirect, since the calpains also interact with hormone and growth-factor receptors, protein kinase C and transcription factors, in addition to a range of membrane, cytoskeletal and nuclear proteins. In the present study, attempts have been made to determine if the beta-agonist-induced effects on the calpain system are associated with corresponding changes in specific mRNA. The activity of both calpain isoforms and calpastatin was measured in bovine longissimus dorsi samples from trials in which test animals were treated with the beta agonist cimaterol. Total RNA was extracted from the muscle samples. A cDNA probe for calpastatin mRNA was generated from bovine RNA by the polymerase chain reaction. This cDNA and a human calpain-II large-subunit cDNA were used to detect specific mRNA by Northern-blot analysis. beta-agonist treatment of Friesian steers caused significant longissimus dorsi hypertrophy. Increases in muscle mass (+37%, P less than 0.005), calpain-II specific activity (+27%, P less than 0.05) and calpastatin-specific activity (+76%, P less than 0.05) were found in treated animals. Total RNA was unchanged, but there was a 96% overall increase in calpastatin mRNA and a 30% increase in calpain-II large-subunit mRNA in muscle from treated animals. The mRNA changes are similar in direction and degree to the activity changes. Both calpain-II large subunit and inhibitor expression may therefore be stimulated by agonist action at the level of transcription or mRNA stabilisation. Multiple calpastatin mRNA species were detected in steers, as reported for other species. Differential changes in these messages, induced by the beta agonist, suggest that expression or stability of alternative mRNA species may be a factor in calpastatin regulation.


Isolation and characterization of carbinolamide and phenolic glucuronide conjugates of (+-)-N-methyl-N-(1-methyl-3,3-diphenylpropyl) formamide and N-formylmethamphetamine by FAB/MS, LC/MS/MS, and NMR. The metabolic disposition of (+-)-N-methyl-N-(1-methyl-3,3- diphenyl-propyl)formamide, especially with regard to the formation of water soluble glucuronides, is described. The glucuronide conjugates, (+-)-N-hydroxymethyl-N-(1-methyl-3,3-diphenylpropyl)formamide glucuronide, (+-)-N-methyl-N-[1-methyl-3-(4'-hydroxyphenyl)-3-phenylpropyl]formamide glucuronide, and (+-)-N-methyl-N-[1-methyl-3-(4'-hydroxy-3'-methoxyphenyl)-3- phenylpropyl]formamide glucuronide were isolated from the bile of rats dosed with the parent compound. These conjugates were characterized spectroscopically by 1H-NMR, FAB/MS, and LC/MS/MS. Because it is becoming more common to isolate the intact glucuronide conjugates of xenobiotics, we investigated some common mass spectral fragmentation patterns of these conjugates, especially by LC/MS/MS. The fragmentation patterns for each of the conjugates were obtained under MS/MS conditions and compared. Specifically, the fragmentation patterns of phenolic glucuronide and an aliphatic O-glucuronide, in particular a carbinolamide glucuronide, were investigated. The data obtained from these studies was used to predict the nature of glucuronide conjugates obtained from rats dosed with the formamide analog, N-formylmethamphetamine. This is the first spectroscopic characterization of an intact carbinolamide glucuronide conjugate isolated from the bile of rats.


Interference of azelastine with anaphylaxis induced by ovalbumin challenge in actively sensitized rats. The inhibition of the haematological alterations and prevention of death due to systemic anaphylaxis after antigen challenge were investigated in rats after various drug treatments. The i.v. injection of ovalbumin (250 micrograms/kg) into actively sensitized rats induced marked thrombocytopenia and haemoconcentration within 5 min and significant leukocytosis within 30 min, lasting for 2 h after the challenge. Pretreatment with meclizine or terfenadine (15-30 mg/kg i.p.) inhibited antigen-induced haemoconcentration, whereas WEB 2086 (2-10 mg/kg i.p.) and PCA 4248 (5-10 mg/kg p.o.), two platelet-activating factor (PAF) antagonists, interfered with thrombocytopenia only. Azelastine (1-20 mg/kg p.o.) dose dependently inhibited antigen-induced haemoconcentration and thrombocytopenia but failed to block leukocytosis. Azelastine also inhibited the thrombocytopenia observed after the i.v. administration of PAF (4 micrograms/kg). Administration of ovalbumin at a dose of 1.5 mg/kg resulted in a lethal anaphylactic reaction in about 85% of the rats. Pretreatment with WEB 2086 (10 mg/kg i.p.), meclizine (30 mg/kg i.p.) or both increased the survival rate from 15 to 57, 68 and 87%, respectively. Azelastine alone (20 mg/kg p.o.) completely blocked the lethal reaction. It was concluded that the ability of azelastine to antagonize histamine and PAF is important for its effectiveness against anaphylactic shock.


Selectivity profile of the alpha 2-adrenoceptor antagonist efaroxan in relation to plasma glucose and insulin levels in the rat. The effects of efaroxan (RX 821037A; 2-[2-(2-ethyl-2,3-dihydrobenzofuranyl)]-2-imidazoline HCl) at alpha 1- and alpha 2-adrenoceptors were investigated in isolated tissues, pithed rats and conscious rats. In isolated tissues, efaroxan competitively antagonised the inhibitory effects of p-aminoclonidine in the electrically stimulated (0.1 Hz) rat vas deferens, (pA2 = 8.89) and the contractile effects of phenylephrine on the rat anococcygeus muscle (pA2 = 6.03). Efaroxan had a selectivity ratio (alpha 2/alpha 1) of 724 compared to a value of 182 for idazoxan. In pithed rats, the i.v. doses of efaroxan (mumol/kg) producing 2-fold shifts in dose-response curves for UK-14,304 at prejunctional cardiac alpha 2-adrenoceptors and postjunctional vascular alpha 2-adrenoceptors, and for cirazoline at postjunctional vascular alpha 1-adrenoceptors, were 0.05, 0.13 and 2.96, respectively. In conscious fasted rats, prazosin (5 mg/kg p.o.) increased resting glucose levels and exacerbated the hyperglycaemic effects of UK-14,304 and adrenaline. In contrast, efaroxan (1-5 mg/kg p.o.) had little effect on resting plasma glucose but markedly antagonised the hyperglycaemic actions of UK-14,304 and adrenaline. Efaroxan increased resting plasma insulin levels and markedly potentiated the rise in insulin levels produced by adrenaline; this latter effect was prevented by the co-administration of propranolol. These results demonstrate that efaroxan is a potent and selective alpha 2-adrenoceptor antagonist and provide further support for the involvement of alpha 2-adrenoceptors in glucose homeostasis.


Comparison of the effects of efaroxan and glibenclamide on plasma glucose and insulin levels in rats. The effect of efaroxan (1 and 5 mg/kg p.o.; a selective alpha 2-adrenoceptor antagonist) was compared to glibenclamide (1 and 5 mg/kg p.o.; a standard sulphonylurea) on basal plasma glucose levels of fed and fasted rats. In addition, the effect of efaroxan (5 mg/kg p.o.) and glibenclamide (2 or 5 mg/kg p.o.), alone and in combination, on the hyperglycaemia and hyperinsulinaemia induced by glucose challenges, were investigated. An intra-arterial (250 mg/kg i.a.) and a subcutaneous (1 g/kg s.c.) glucose challenge were used to stimulate the fast and slow release phases of insulin secretion. Efaroxan increased plasma insulin levels in both conscious fed and fasted rats without greatly affecting plasma glucose levels. Glibenclamide also elevated insulin levels, but was associated with marked hypoglycaemia. Efaroxan and glibenclamide potentiated the slow and fast release of insulin secretion, but glibenclamide had a tendency to produce hypoglycaemia in these test situations, a property not shared by efaroxan. A combination of efaroxan and glibenclamide produced a greater elevation in the slow and fast insulin release phases than either compound alone, but did not enhance the hypoglycaemia seen with glibenclamide alone. These results provide further evidence that pancreatic alpha 2-adrenoceptors are involved in the regulation of insulin secretion.


Pharmacological profiles of fentanyl analogs at mu, delta and kappa opiate receptors. Receptor binding assays using [3H]DAGO ([D-Ala2,MePhe4-Gly5-ol]enkephalin) (mu), [3H]DPDPE ([D-Pen2,D-Pen5]enkephalin) (delta) and [3H]U-69593 (kappa) were done in guinea pig whole brain membranes. Agonist activity was determined in norbinaltorphimine or beta-funaltrexamine (beta-FNA) treated guinea pig ileum (mu and kappa, respectively) and beta-FNA-treated mouse vas deferens (delta). The compounds with highest affinity were the most potent at the mu-receptor. The selectivity observed in the binding affinities was also found in in vitro activity. No correlation was found between mu-affinity and selectivity; the highest affinity analog, lofentanil, was found to be among the least selective, while another high affinity analog, R30490, was the most mu-selective. The results show that not all fentanyls are highly mu-selective, and could produce actions through delta- and kappa-opiate receptors.


Effects of dopamine receptor agonists on passive avoidance learning in mice: interaction of dopamine D1 and D2 receptors. The present study examined the effects of dopamine D1 and D2 receptor agonists on the acquisition stage of passive avoidance learning and on locomotor activity in mice. The D2 agonist, RU 24213 (1-10 mg/kg s.c.), and the non-selective agonist, apomorphine (0.3-3 mg/kg s.c.), but not the D1 agonist, SKF 38393 (1-10 mg/kg s.c.), impaired learning and activated locomotion. RU 24213 (1 mg/kg s.c.) was more effective in impairing learning than in activating locomotion. The concurrent administration of SKF 38393 (10 mg/kg i.p.) and RU 24213 (1 and 3 mg/kg s.c.) produced a synergistic effect in both behavioral situations. The D1 antagonist, SCH 23390 (0.025 mg/kg i.p.), slightly inhibited the effects of apomorphine and of the combination of SKF 38393 and RU 24213 on learning but not on locomotion. The D2 antagonist, (-)-sulpiride (40 mg/kg i.p.), completely blocked these effects in both situations. These results suggest that dopamine receptor agonists impair passive avoidance learning through the D2 receptor, and that D1 and D2 receptors act synergistically in this impairment, as they do in their effects on locomotion. The involvement of D1 and D2 receptors is qualitatively similar in each of these behaviors, although some small differences may exist.


Partial and full dopamine D1 receptor agonists in mice and rats: relation between behavioural effects and stimulation of adenylate cyclase activity in vitro. The dopamine (DA) D1 agonists, SK&F 83959, SK&F 75670, SK&F 38993, SK&F 81297 and SK&F 80723, had variable abilities to stimulate adenylate cyclase activity in rat striatal homogenates. Their efficacies, in relation to the effect of 100 microM DA were 0, 33, 69, 68 and 81%, respectively. In rats, all compounds induced (1) contralateral circling behaviour after unilateral 6-hydroxy-DA lesions, (2) ipsilateral circling behaviour after midbrain hemitransection after cotreatment with the D2 agonist quinpirole and (3) oral stereotypies after their combination with quinpirole. Maximum effects and rank order of potencies were similar in the three test models. In mice SK&F 83959, SK&F 75670 and SK&F 38393 inhibited methylphenidate-induced gnawing behaviour and induced no or only weak hypermotility. SK&F 81297 induced marked hypermotility which was partially inhibited by SK&F 83959 and SK&F 75670 and was completely blocked by the D1 antagonist, SCH 23390. It is concluded that no relation could be demonstrated between the efficacy to stimulate adenylate cyclase and to induce circling behaviours and stereotypies in rats. In contrast, a relation between biochemical and behavioural efficacies was found in the mouse models. The results suggest that different subtypes of D1 receptors mediate the behavioural effects reported in this study.


Competitive and non-competitive NMDA receptor antagonists in spatial learning tasks. The effects of the competitive N-methyl-D-aspartate (NMDA) receptor antagonists, CGP37849 (3 or 6 mg/kg i.p.) and its ethyl ester CGP39551 (5 or 15 mg/kg i.p.) and of the non-competitive NMDA receptor antagonist, dizocilpine (0.16 mg/kg; i.p.) on acquisition by rats of different spatial orientation tasks in an 8-arm radial maze were evaluated. Neither of the CGP compounds influenced locomotor activity during spontaneous alternation, only dizocilpine increased the number of arm entries (locomotion). Preferred angles between consecutive arm entries were changed by the high doses of CGP37849 (6 mg/kg), CGP39551 (15 mg/kg) and dizocilpine (0.16 mg/kg). The high doses of both CGP compounds as well as dizocilpine produced impairments in the acquisition of an egocentric orientation task and an allocentric reversal task indicated by an increased number of arm entries and re-entries. Such amnesic effects did not occur after administration of low doses of CGP37849 (3 mg/kg) and CGP39551 (5 mg/kg), doses which are sufficient to produce anticonvulsant and anticataleptic effects. In contrast, the non-competitive NMDA receptor antagonist, dizocilpine, produced amnesic effects over the entire behaviourally effective dose range.


Does rilmenidine act in vivo on central alpha 2-adrenoceptors modulating noradrenaline release? Noradrenaline release evoked by electrical stimulation was recorded in the rat hypothalamus by in vivo electrochemistry. Rilmenidine (0.3-10 mg kg-1 i.v.) did not diminish this evoked release while clonidine induced a dose-dependent decrease. These results further suggest that the antihypertensive action of rilmenidine is not mediated by central alpha 2-adrenoceptors and might explain why, unlike clonidine, rilmenidine does not have sedative effects.


Pharmacological and mechanical heterogeneity of cat isolated ophthalmociliary artery. The mechanical, histological and pharmacological properties of the isolated cat ophthalmociliary artery are reported and compared for passively stretched ring segments from three locations on the artery. Ring segments were mounted in a modified myograph system after the method of Hogestatt, Andersson and Edvinsson (1983, Acta Physiol Scand. 117, 49-61) and comparative length tension curves were measured. The most distal segment (DOA), nearest the eye, developed by far the smallest maximal tension compared to the middle segment (MOA) and the most proximal segment (POA), despite the fact that lumen diameters were similar. All segments displayed a phasic and tonic component in response to passive stretching and during activation with K(+)-Krebs. Cumulative contractile dose-response curves were measured for nine agonists: histamine (HIS), 5-hydroxytryptamine (5-HT), dopamine (DOPA), adrenaline (A), noradrenaline (NA), tyramine (TYR), phenylephrine (PHE), isoproterenol (ISOP) and xylazine (XYL) and the maximum active tension developed at a concentration of 10(-3) M was compared with that for 0.124 M K(+)-Krebs. The order of contraction magnitude in the three segments was: POA, HIS much greater than Kmax much greater than NA greater than A greater than DOPA greater than PH = TYR greater than 5-HT = ISOP greater than XYL; MOA, Kmax much greater than NA much greater than A = HIS much greater than DOPA greater than ISOP = PHE greater than TYR = 5-HT greater than XYL; DOA, Kmax much greater than NA greater than A greater than HIS = DOPA greater than ISOP greater than PHE = 5-HT greater than XYL = TYR. Pre-activation did not produce relaxation in response to histamine and isoproterenol. Testing with specific alpha 1 and beta antagonists supported the presence of alpha 1 receptors but not beta receptors. It is concluded that functional HIS, alpha 1 and 5-HT receptors are present and that the ophthalmociliary artery is heterogeneous in its response to HIS, with the POA segment producing the largest responses. The magnitude of the contractile responses to A and NA grew with increasing passive tension, whereas the response to histamine was independent of passive tension.


Reconstitution of the photoreceptor-pigment epithelium interface: L-glutamate stimulation of adhesive interactions and rod disc shedding after recombination of dissociated Xenopus laevis eyecups. In order to investigate adhesive interactions between photoreceptor and pigment epithelial cells, we have mechanically separated neural retinas from Xenopus laevis eyecups and then recombined the tissues in vitro. When tissue pairs are incubated in a defined medium, cell-cell contact is achieved within 3 hr. However, the average proportion of reassembled eyecups in which photoreceptor outer segments interdigitate with epithelial microvilli is limited. Furthermore, rod disc shedding does not take place in these cultures, even following a dark to light transition. When recombined tissues are placed in medium supplemented with 12 mM L-glutamate, retinal reattachment is enhanced and there is a four-fold increase in epithelial phagosome content. The positive effect of excitatory amino acid exposure on shedding, however, is restricted to regions where visual and epithelial cells interdigitate. These results indicate that re-establishment of cell contact may be necessary for shedding of apical disc membranes prior to their engulfment by the epithelium. While reattachment is not affected by pre-incubation of separated tissues in normal medium, rod photoreceptors fail to undergo membrane turnover in response to L-glutamate if a delay of 1 hr or more is interposed between isolation of the retina and its recombination with the pigment epithelium. This is probably due to a decline in retinal function in culture, since a similar preincubation of the pigment epithelium prior to reassembly with a freshly isolated retina does not inhibit the shedding response.(ABSTRACT TRUNCATED AT 250 WORDS)


Postnatal X-ray irradiation effects on glomerular layer of rat olfactory bulb: quantitative and immunocytochemical analysis. In the rat olfactory bulb, the majority of interneurons in the glomerular layer (GL) are supposed to be generated during first postnatal week. Low and repeated doses of X-rays (200 rad x 4 and 200 rad x 6) were used during this period to impair the development of interneurons. The resulting effects of olfactory bulb neurons were examined stereologically and immunocytochemically in animals of 4 and 12 weeks of age. Quantitative analysis showed that, 1) the volume of the GL decreased to 55% (1200 rad) - 70% (800 rad) of control, 2) numerical cell densities in GL decreased to 40% (1200 rad) - 60% (800 rad) of control, thus resulting in 3) a decrease of the total cell number in GL to 20% (1200 rad) - 40% (800 rad) of control in irradiated olfactory bulbs of animals 4 weeks old. In comparison, mitral cells, which are generated prenatally, were much less affected (total cell number: 70-80% of control), indicating a selective loss of cells generated during the first postnatal week in GL. Effects on somata and processes immunoreactive for GABA, tyrosine hydroxylase (TH), calbindin D-28K and parvalbumin (PV) were examined in irradiated bulbs of both 4 and 12 week-old rats. All of these immunoreactive elements showed a drastic decrease in all layers. Semiquantitative analysis showed that in the GL, calbindin D-28K immunoreactive (calbindin D-28K(+)) neurons decreased more extensively than TH immunoreactive (TH(+)) and GABA-like immunoreactive (GABA(+)) neurons; that is, TH(+) and GABA(+) neurons decreased to 20% (1200 rad) - 40% (800 rad) of control, whereas calbindin D-28K(+) neurons decreased to 10% (1200 rad) - 30% (800 rad) of control in the GL of irradiated bulbs. These findings indicated that larger proportions of calbindin D-28K(+) neurons might be generated during the first postnatal week than those of GABA(+) and TH(+) neurons. Furthermore, in irradiated bulbs the proportion of GABA(-)TH(+) cells in TH(+) cells increased to about twice of control, and the estimated total numbers of GABA(-)TH(+) cells in irradiated rats were 95% (800 rad) and 40% (1200 rad) of control. These observations suggest that the majority of GABA(-)TH(+) neurons were less affected by X-ray irradiation during the first postnatal week and thus that they might be generated in the prenatal period. Since during the first 2 postnatal weeks, neurons showing GABA(-)TH(+) were not seen in GL (Kosaka et al. 1987a), the majority of GABA(-)TH(+) neurons in adult olfactory bulb were assumed to change their phenotype at some postnatal developmental period.


MPTP induced hemiparkinsonism in monkeys: behavioral, mechanographic, electromyographic and immunohistochemical studies. A single infusion of 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP) into the right internal carotid artery of Macaca mulatta monkeys resulted in akinesia and rigidity of the contralateral limb. The immunohistochemical study revealed a dramatic reduction in the number of TH-immunoreactive cells in the substantia nigra of the infused side (70-81%). After unilateral MPTP-treatment movement parameters and EMG activity were altered; the agonist muscle developed increased EMG activity associated with a shift of antagonist muscle activity. These results confirm that hemiparkinsonian monkeys are a valuable model of parkinsonism which can be useful in studies of movement disorder physiology and therapy of Parkinson's disease.


Cryopreservation, survival and function of intrastriatal fetal mesencephalic grafts in a rat model of Parkinson's disease. In the present study we quantitatively assessed to what extent freeze-storage at liquid nitrogen temperature influences the survival and function of fetal mesencephalic grafts in the dopamine-depleted rat striatum. Ventral mesencephalic (VM) tissue was dissected from rat fetuses and stored overnight in a preservative medium at 4 degrees C (hibernation). It was grafted intrastriatally either as a fresh cell suspension or was frozen as tissue fragments or as a cell suspension after stepwise incubation in ascending concentrations of dimethyl-sulphoxide. Following a cryopreservation interval of 80 days in liquid nitrogen, the frozen samples were rapidly thawed, rinsed, and grafted. Cellular viabilities of graft cell suspensions, as assessed by ethidium bromide/acridine orange staining, were decreased from 90% in fresh tissue to 38-35% in frozen and thawed tissue. Amphetamine-induced turning behavior at 6 weeks post-grafting was significantly attenuated in hosts that had received fresh grafts or grafts that were frozen as tissue fragments. Tyrosine hydroxylase-(TH-) immunocytochemistry of recipient brains revealed significant decreases in TH-positive graft cell numbers in rats grafted with cryopreserved tissue (38-42% of fresh tissue). Moreover, the dye exclusion viability of thawed VM tissue was found to accurately predict the subsequent graft survival. There was no difference with respect to graft cell numbers between the two freezing methods employed, though block storage seems to be more simple from a practical point of view.(ABSTRACT TRUNCATED AT 250 WORDS)


An evaluation of the antihistamine activity of acrivastine and its onset in human skin. In a double-blind, two-period crossover study, 24 healthy volunteers were evaluated to establish the time of onset of action of activity of acrivastine in suppressing the weal and flare response to intradermally injected histamine. Volunteers received single doses of 8 mg acrivastine and placebo according to a fully randomized, balanced treatment plan. Acrivastine significantly (P less than 0.002) reduced the flare response induced by 0.4 micrograms histamine challenge 15 min after oral acrivastine dosing when compared with placebo. A significant (P less than 0.001) reduction of the weal response was noted at 25 min, although trends in this direction were already present at earlier time points.


Lansoprazole and omeprazole have similar effects on plasma gastrin levels, enterochromaffin-like cells, gastrin cells and somatostatin cells in the rat stomach. This study compares the effects of lansoprazole and omeprazole on the activation and proliferation of enterochromaffin-like (ECL) cells in the rat stomach. Lansoprazole was given orally once daily for 10 weeks in two doses, 135 and 200 mumol/kg. Omeprazole was given by the same regimen in a dose of 400 mumol/kg, which is equipotent in terms of acid inhibition to the higher lansoprazole dose. Lansoprazole (both doses) as well as omeprazole raised the plasma gastrin levels about 11-fold 2 h after dosing and 8-to 10-fold 24 h after dosing, reflecting complete (2 h) and 70-80% (24 h) reductions of gastric acid secretion. Administration of either drug for 10 weeks increased the weight of the stomach and the oxyntic mucosa. The oxyntic mucosal histidine decarboxylase activity, histamine concentration and ECL cell density were increased to the same extent in the rats given either of the two lansoprazole doses or omeprazole. The numbers of antral gastrin cells were doubled and the numbers of antral somatostatin cells half that in the controls. These results show that long-standing lansoprazole-evoked hypergastrinemia affects the ECL cell similarly to omeprazole, ranitidine and other acid secretion inhibitors.


Comparison between HLA-DRB and DQ DNA sequences and classic serological markers as type 1 (insulin-dependent) diabetes mellitus predictive risk markers in the Spanish population. The question of HLA susceptibility to Type 1 (insulin-dependent) diabetes mellitus remains unresolved. In the present study, 127 diabetic patients and 177 unrelated control subjects have been analysed for their class I and class II serological antigens, class II (DR, DQ) DNA restriction fragment length polymorphisms and DQA1 and B1 exon-2 nucleotide sequences and their corresponding amino acid residues. By using the aetiologic fraction (delta) as an almost absolute measure of the strongest linkage disequilibrium of an HLA marker to the putative Type 1 diabetes susceptibility locus, it has been found that the strength of association of the HLA markers may be quantified as follows: DR4 less than DR3 less than DR3 or DR4 less than non-Aspartate 57 beta DQ and Arginine 52 alpha DQ less than Arginine 52 alpha DQ. Thus, molecular HLA-DQ markers appear to be more accurate as susceptibility markers than the classic serologically defined ones (DR3 and DR4); however, any effect of DQ markers disappears when non-DR3/DR4 individuals are considered, suggesting that DR factors (or others in between DQ and DR) are also important. In addition, a dominant non-Aspartate 57 beta DQ susceptibility theory does not hold (but a recessive one does) in our diabetic population (probably due to the high frequency of the protective DR7-non-Aspartate 57 beta DQ haplotypes); Arginine 52 alpha DQ is the best single HLA marker found in our population, both as a recessive or as a dominant one. Also there are 13 patients in our sample who bear neither Arginine 52 alpha DQ nor non-Aspartate 57 beta DQ susceptibility factors.(ABSTRACT TRUNCATED AT 250 WORDS)


Comparison of Bacillus thuringiensis subsp. israelensis CryIVA and CryIVB cloned toxins reveals synergism in vivo. When the gene for the mosquitocidal protein CryIVA was expressed in two strains of Bacillus thuringiensis (Bt) cured of their resident delta-endotoxin genes, the protein accumulated as large inclusions. The inclusions produced in the Bt subsp. kurstaki recipient strain were twice as soluble at alkaline pH as the inclusions produced in Bt subsp. israelensis. Solubilized protoxins were activated by treatment with mosquito gut extracts or trypsin for varying lengths of time and tested for in vitro cytotoxicity on cell lines of three genera of mosquito. CryIVA treated with any of the mosquito gut extracts for 6 h showed significant toxicity against Anopheles gambiae cells and slight activity on Culex quinquefasciatus cells. For CryIVB, the only significant cytotoxicity observed was against Aedes aegypti cells after treatment with Aedes gut extract. In in vivo bioassays, both CryIVA, purified from either of the Bt recipient strains, and CryIVB inclusions were similarly toxic to A. aegypti and A. gambiae larvae but CryIVA was 25-fold more toxic to C. quinquefasciatus. Synergism in vivo between the two toxins was revealed when results from assaying single toxins and mixtures were compared. Mixtures of CryIVA and CryIVB proved to be 5-fold more toxic to Culex than either toxin used singly and showed a reduced but similar synergism when tested against Aedes and Anopheles larvae. The synergism was not duplicated in vitro using cell lines from these three insects.


Underrecognition of tardive dyskinesia and drug-induced parkinsonism by psychiatric residents. Recognition of tardive dyskinesia (TD) and other neuroleptic, drug-induced, extrapyramidal side effects presents a major challenge in modern clinical psychopharmacology. Failure to recognize these disorders can lead to poor patient care and may contribute to societal pressure for external control of psychiatric practice. This study reports the occurrence of tardive dyskinesia and drug-induced parkinsonism (DIP) in 101 inpatients, and documents underrecognition of both disorders by resident physicians. Researchers noted TD in 28% of cases and residents only described TD (or symptoms of TD) in 12%. The researcher determined DIP prevalence rate of 26% contrasted with an 11% rate found by residents. Patients with psychotic disorders were more likely than other patients to have researcher-identified TD, whereas DIP (researcher cases) occurred more often in patients with affective diagnoses. Residents tended to miss milder cases of TD, and to miss DIP in younger patients and in patients with affective disorders. Improved teaching and clinical exams are recommended to improve recognition.


The suppressor of forked locus in Drosophila melanogaster: genetic and molecular analyses. The suppressor of forked, su(f) locus is one of a class of loci in Drosophila whose mutant alleles are trans-acting allele-specific modifiers of transposable element-insertion mutations at other loci. Mutations of su(f) suppress gypsy insert alleles of forked and enhance the copia insert allele white apricot. Our investigations of su(f) include genetic and molecular analyses of 19 alleles to determine the numbers and types of genetic functions present at the locus. Our results suggest the su(f) locus contains multiple genetic functions. There are two distinct modifier functions and two vital functions. One modifier function is specific for enhancement and the other for suppression. One vital function is required for normal ecdysterone production in the third larval instar, the other is not. We present a restriction map of the su(f) genomic region and the results of an RFLP analysis of several su(f) alleles.


Intercellular adhesion molecule-1 (ICAM-1) and leukocyte function-associated antigen-1 (LFA-1) expression in human epiretinal membranes. Various histogenetically different cell types such as macrophages, retinal pigment epithelial cells, glial cells, and fibroblasts are involved in the formation of epiretinal membranes. In the development of such multicellular tissues, cellular adhesion molecules (CAMs) are necessary for cell migration, proliferation, and localization, and the transfer of information between the cells. We investigated the expression of the intercellular adhesion molecule 1 (ICAM-1) and the leukocyte function-associated antigen 1 (LFA-1) in frozen sections of epiretinal membranes in proliferative vitreoretinopathy (PVR), proliferative diabetic retinopathy (PDR), macular pucker, and recurrent membranes after intraocular silicone oil tamponade using the indirect immunoperoxidase method. ICAM-1 forms a receptor-ligand pair with LFA-1 and is involved in a number of significant cellular interactions, e.g. in providing dynamic position-specific information to guide lymphocyte and leukocyte localization in the immune response. ICAM-1 is a member of the immunoglobulin gene super-family of CAMs. LFA-1 is a member of the integrin family of cell membrane receptors. It mediates a wide range of lymphocyte, monocyte, natural killer cell, and granulocyte interactions with other cells in immunity and inflammation, and it is a receptor for ICAM-1. The LFA-1 interaction with its ligand ICAM-1 mediates not only cell adhesion but also signal transduction in immunologic and inflammatory cell responses. Basal ICAM-1 expression is normally low on nonhematopoietic cells, but it can be subject to an up-and-down regulation by various cytokines.(ABSTRACT TRUNCATED AT 250 WORDS)


Antihistaminic activity of quinethindole: a 2-substituted pyrazinopyridoindole derivative. Quinethindole, a 2-substituted pyrazinopyridoindole, showed specific antihistaminic (H1) activity in various in vivo and in vitro test models. It also inhibited antigen-induced contraction of ileum of sensitized guinea pig. The antihistaminic activity was of competitive nature.


Expression of HER-2/neu in renal-cell carcinoma. Correlation with histologic subtypes and differentiation. Twenty-four renal-cell carcinomas (RCC) and corresponding non-neoplastic kidney tissue were examined for amplification and expression of the HER-2/neu gene. Gene amplification was examined by slot-blot analysis, mRNA expression by in situ hybridization and Northern blot analysis, and protein expression by immunohistochemistry. Northern-blot analysis revealed lower expression of HER-2/neu mRNA in clear-cell (p less than 0.001) and compact (p less than 0.001) tumor subtypes, while chromophilic, chromophobic and tubulo-papillary subtypes did not show significant differences in HER-2/neu gene expression, as compared with non-neoplastic kidney tissues. HER-2/neu gene expression was not significantly associated with tumor stage. Low differentiation (G3) was associated with lower HER-2/neu gene expression, but the number of G3 cases was too small for statistical analysis. HER-2/neu gene amplification was not found in any of the tumors. The results of in situ hybridization and immunohistochemistry generally agreed with those of Northern-blot analysis. We conclude that HER-2/neu gene expression correlates with Thoenes' classification of RCC and may be inversely related to tumor differentiation; it is probably not involved in progression of RCC, in contrast to carcinomas of other locations (e.g. breast, ovary).


Over-expression of P-glycoprotein and glutathione S-transferase pi in MCF-7 cells selected for vincristine resistance in vitro. This study has provided evidence that exposure of the wild-type MCF-7 human breast carcinoma cell line to the mutagen ethyl methane sulphonate (EMS), followed by selection in vincristine (VCR), resulted in a stably-resistant subline, designated VCREMS, which expressed an approximately 14-fold level of resistance to VCR. This VCREMS subline showed cross-resistance (3-fold) to adriamycin (ADR) and to etoposide (3-fold), but not to cisplatin. The addition of a non-toxic concentration of verapamil (6.6 microM) significantly enhanced VCR cytotoxicity only in the resistant subline. This resistance was associated with over-expression of P-glycoprotein (Pgp), but without a concomitant increase in Pgp mRNA or gene amplification. In addition, activities of total glutathione S-transferases (GST) and glutathione peroxidase were elevated in this resistant subline, with over-expression of the GST-pi isozyme and its associated mRNA being identified, without gene amplification. This VCR-selected resistant MCF-7 cell line therefore provides another example of a breast carcinoma subline in which there is co-ordinate over-expression of both Pgp and GST-pi, without attributing a causal relationship to either event, and extends the range of anti-tumour drugs known to elicit modifications in glutathione metabolism.


Two new human cholangiocarcinoma cell lines and their cytogenetics and responses to growth factors, hormones, cytokines or immunologic effector cells. Two new human cholangiocarcinoma (CC) cell lines (CC-SW-I and CC-LP-I) were established and maintained in culture for 2 years. Histologically, both original liver tumors were adenocarcinomas, and the cell lines exhibited morphologic features of moderately differentiated adenocarcinoma. Immunohistochemistry showed that both cell lines were strongly positive for cytokeratin AEI but negative for carbohydrate tumor-associated antigen, CA19-9. Ultrastructural analysis of both cell lines showed the presence of tight junctional complexes and focally formed microvilli. Both CC cell lines were tumorigenic in nude mice. Cytogenetic analysis showed that both cell lines expressed highly aneuploid karyotypes with numerous structural and numerical deviations. CC-SW-I was hypodiploid with numerous chromosome losses and structural rearrangements, while CC-LP-I was hyperdiploid and displayed multiple additional chromosomes. Doubling times for the CC-SW-I and CC-LP-I cell lines in the presence of 15% fetal bovine serum were 72 hr and 180 hr, respectively. Growth of the CC-SW-I cell line was significantly stimulated in the presence of insulin, while that of the CC-LP-I cell line was significantly augmented by epidermal growth factor (EGF). In contrast, dexamethasone strongly inhibited proliferation of both cell lines in a dose-dependent manner. Among various recombinant cytokines examined for effects on growth or surface antigen expression on CC cell lines, only interleukin I-beta (ILI-beta) strongly inhibited growth of the CC-LP-I cell line, while interferons (IFNs) or tumor necrosis factor-alpha (TNF-alpha) were mildly inhibitory. Both tumor cell lines were resistant to natural killer (NK) cells but sensitive to lymphokine-activated killer (LAK) cells. Preincubation of tumor cells with IFN-gamma, IFN-alpha or TNF-alpha significantly decreased the susceptibility of each tumor cell line to lysis by LAK cells, and the change in sensitivity did not correlate with the expression of HLA antigens or intercellular adhesion molecule-I (ICAM-I) on the surface of tumor cells. These 2 CC cell lines are expected to provide valuable information about cell biology of human CC.


Epidermal growth factor reduces HER-2 protein level in human ovarian carcinoma cells. Over-expression of the proto-oncogene HER-2 (c-erbB-2/neu) in ovarian, endometrial, and mammary carcinoma is an indicator of poor prognosis. Interactions between the epidermal growth factor (EGF) receptor and the HER-2 protein have been described. The aim of this study was to elucidate the effects of EGF on HER-2 expression. In the human ovarian carcinoma cell lines HTB-77, OVCAR-3, 2780, SKOV-6, SKOV-8 and 2774, and the human mammary tumor cell line SKBR-3, total cellular p185HER-2 was determined by an ELISA, whereas the surface p185HER-2 was measured with a living-cell RIA. Stimulation of these cell lines with either EGF (0.1-30 nM) or TGF-alpha (0.1-30 nM) led to a significant reduction in p185HER-2 expression. The effect was more pronounced in cells with normal HER-2 expression. A reduction of mRNA levels for p185HER-2 by EGF was observed in OVCAR-3 cells but not in the over-expressing lines HTB-77 and SKBR-3. Interestingly, the EGF-induced effect was not always associated with growth stimulation and was not correlated with the number of EGF binding sites detected by a radioligand assay. Our data indicate that EGF treatment results in a down-regulation of p185HER-2.


Ovarian pregnancy in polycystic ovary syndrome: a case report. A case is reported of ectopic pregnancy occurring within an ovary with the morphologic appearance of polycystic ovary syndrome (PCOS). The hyperandrogenism and elevated LH/FSH ratio characteristic of PCOS were noted 2 months after removal of the ovarian gestation. The thickened ovarian cortex of the PCOS ovary and a defect in oocyte-cumulus complex detachment within the follicle are suggested as possible factors contributing to intraovarian fertilization in PCOS.


Identification of the Escherichia coli murI gene, which is required for the biosynthesis of D-glutamic acid, a specific component of bacterial peptidoglycan. The murI gene of Escherichia coli, whose inactivation results in the inability to form colonies in the absence of D-glutamic acid, was identified in the 90-min region of the chromosome. The complementation of an auxotrophic E. coli B/r strain by various DNA sources allowed us to clone a 2.5-kbp EcoRI chromosomal fragment carrying the murI gene into multicopy plasmids. The murI gene corresponds to a previously sequenced open reading frame, ORF1 (J. Brosius, T. J. Dull, D. D. Sleeter, and H. F. Noller. J. Bacteriol. 148:107-127, 1987), located between the btuB gene, encoding the vitamin B12 outer membrane receptor protein, and the rrnB operon, which contains the genes for 16S, 23S, and 5S rRNAs. The murI gene product is predicted to be a protein of 289 amino acids with a molecular weight of 31,500. Attempts to identify its enzymatic activity were unsuccessful. Cells altered in the murI gene accumulate UDP-N-acetylmuramyl-L-alanine to a high level when depleted of D-glutamic acid. Pools of precursors located downstream in the pathway are consequently depleted, and cell lysis finally occurs when the peptidoglycan content is 25% lower than that of normally growing cells.


Reference period analysis of vaginal bleeding with triphasic oral contraceptive agents containing norethindrone or levonorgestrel: a comparison study. The World Health Organization recommends the use of fixed reference periods for quantification of the incidence and severity of vaginal bleeding when patients use various forms of contraception. Ninety- and 110-day reference periods were used in the analysis of data from daily menstrual diaries kept by 72 healthy women in a one-year study of oral contraceptive agents containing ethinyl estradiol and either norethindrone or levonorgestrel. Analysis of bleeding patterns reported during both 90-day and 110-day periods revealed fewer days of bleeding and/or spotting overall with norethindrone than with levonorgestrel (e.g., a mean of 16.06 vs. 19.55 days, respectively, over the first 90-day period; P = .013) and significantly shorter bleeding and/or spotting episodes with the norethindrone preparation. This trend persisted when data were adjusted for a day-1 pill start. Using either method of analysis, duration of bleeding episodes was shorter among subjects taking norethindrone than levonorgestrel. Pills were missed in both study groups, but more women in the LNG/EE group missed from 1 to 3 pills in at least one cycle (31 vs. 21 in the NET/EE group). The between-group difference in bleeding events may be due to intrinsic hormonal differences in regimens or to the greater number of pills missed among levonorgestrel users.


Teratological evaluation of an injectable male antifertility agent, styrene maleic anhydride, in rats. Polymer styrene Maleic anhydride (SMA) dissolved in dimethylsulphoxide (DMSO) was injected into the lumen in each vas deferens of male rats at dose levels of 0.25 mg, 0.50 mg, 1.0 mg, 2.5 mg, and 5.0 mg, while control rats (groups 1) received 0.03 mL DMSO in each vas deferens. After 4 weeks, the lumen was flushed with 0.1 mL DMSO and the animals were left for another 6 weeks to regain fertility. These rats were mated with virgin and coeval females. No anomaly was observed which could be related to teratogenic action of the polymer in pregnant mothers or fetuses.


Ectopic pregnancy, the new gynecological epidemic disease: review of the modern work-up and the nonsurgical treatment option. Over the past few decades, the incidence of ectopic pregnancy has increased almost to the extent of an "epidemic disease." Early diagnosis of tubal pregnancy, with the aid of serum human chorionic gonadotropin, high-resolution ultrasound, and the more liberal use of laparoscopy, has dramatically reduced both maternal mortality and the need for radical surgery. Despite this, women with previous ectopic pregnancies still have reduced fertility potential. We report on some current aspects of the epidemiology, etiology, and work-up of ectopic pregnancy. In a review of 328 patients, gleaned from the literature, who were treated with various nonsurgical options, 283 (86%) were able to avoid surgery. The benefits, safety, and efficacy of the various treatment options are discussed, with appropriate recommendations for their use.


Lesions in two Escherichia coli type 1 pilus genes alter pilus number and length without affecting receptor binding. We describe the characterization of two genes, fimF and fimG (also called pilD), that encode two minor components of type 1 pili in Escherichia coli. Defined, in-frame deletion mutations were generated in vitro in each of these two genes. A double mutation that had deletions identical to both single lesions was also constructed. Examination of minicell transcription and translation products of parental and mutant plasmids revealed that, as predicted from the nucleotide sequence and previous reports, the fimF gene product was a protein of ca. 16 kDa and that the fimG gene product was a protein of ca. 14 kDa. Each of the constructions was introduced, via homologous recombination, into the E. coli chromosome. All three of the resulting mutants produced type 1 pili and exhibited hemagglutination of guinea pig erythrocytes. The latter property was also exhibited by partially purified pili isolated from each of the mutants. Electron microscopic examination revealed that the fimF mutant had markedly reduced numbers of pili per cell, whereas the fimG mutant had very long pili. The double mutant displayed the characteristics of both single mutants. However, pili in the double mutant were even longer than those seen in the fimG mutant, and the numbers of pili were even fewer than those displayed by the fimF mutant. All three mutants could be complemented in trans with a single-copy-number plasmid bearing the appropriate parental gene or genes to give near-normal parental piliation. On the basis of the phenotypes exhibited by the single and double mutants, we believe that the fimF gene product may aid in initiating pilus assembly and that the fimG product may act as an inhibitor of pilus polymerization. In contrast to previous studies, we found that neither gene product was required for type 1 pilus receptor binding.


Evidence that difference in size of fraternal twins may originate during early gestation: a case report. We describe a woman who conceived by in vitro fertilization (IVF) and embryo transfer (ET). Transvaginal ultrasound demonstrated at least 1 week's difference in size of twin gestations from 1 month post-transfer of embryos to delivery. Differences in sac size, crown-rump length, and gestational growth are discussed, as are implications of ultrasound in early pregnancy.


Incidence of congenital uterine anomalies in repeated reproductive wastage and prognosis for pregnancy after metroplasty. To discover the exact incidence of congenital uterine anomalies among infertile patients, hysterosalpingography was performed on 1,200 married women with a history of repeated reproductive wastage. Out of 1,200 hysterosalpingographies, 188 revealed congenital uterine anomaly (15.7%). The degree of uterine cavity deformity in the anomalies was evaluated during hysterosalpingography using the X/M ratio. This indicated that the incidence of repeated spontaneous abortion in cases with low-grade anomalies is as high as the incidence among cases with more severe anomalies. A significant improvement in maintaining pregnancy was observed after metroplasty; more than 84% of postoperative pregnancies were successfully maintained, whereas none of the 233 presurgical pregnancies had lasted full term. As a control group, 47 other women with anomalies were randomly chosen, and their subsequent pregnancies were monitored, without metroplasty. Of their pregnancies, 94.4% terminated spontaneously before 12 weeks of gestation.


Dexamethasone selectively regulates the activity of enzymatic markers of cerebral endothelial cell lines. Two endothelial cell lines were derived from grafts of the central nervous system using retrovirus mediated gene transfer to introduce the polyoma middle-T oncogene into fetal rat brain endothelial cells and transplantation of these cells into adult rat brain. In this report, we further characterize these cells and the effect of dexamethasone on the expression of specific enzymatic markers. These cells take up acetylated low density lipoprotein, leucine, and glucose, and express Factor VIII-related antigen, angiotensin converting enzyme, alkaline phosphatase, gamma-glutamyltranspeptidase, and as yet undescribed aminopeptidase A and B-like enzymes. When grown on semi-permeable membranes, these transformed cells do not spontaneously retain small hydrophilic molecules. In culture, one of the lines (EC 193) forms a confluent monolayer of spindle-shaped cells homogenously expressing gamma-glutamyltranspeptidase at a level comparable to primary cells. The other cell line (EC 219) grows as clusters of elongated cells, and gamma-glutamyltranspeptidase activity is expressed mainly in cells forming the clusters. This clustered pattern changes to a confluent one after culture on type-I collagen. Dexamethasone increases angiotensin-converting enzyme activity, and decreases the expression of gamma-glutamyltranspeptidase and aminopeptidase A, whereas the aminopeptidase B activity is little modified. Inhibition of aminopeptidase A activity by amastatin, potentiates angiotensin II effects on DNA synthesis. These results indicate that retrovirally transformed brain endothelial cells are a useful model for studying the blood-brain barrier in vitro and that dexamethasone, an agent with the potential to reduce brain edema, directly affects some blood-brain barrier properties in these endothelial cell lines.