In vitro metabolism of tegaserod in human liver and intestine: assessment of drug interactions.

Tegaserod is a selective 5-HT(4) receptor partial agonist with promotile activity in the gastrointestinal tract. This study was designed to describe the metabolic pathways of tegaserod in the human liver and small intestine in vitro, to identify the enzymes involved in tegaserod metabolism, and to investigate the effect of tegaserod on CYP-catalyzed reactions involving other compounds. Tegaserod was metabolized in human liver microsomes to O-desmethyl tegaserod at a low rate. This metabolite was also formed by cDNA expressed CYP2D6, and the reaction in human liver microsomes was inhibited by quinidine. In human liver slices, direct N-glucuronidation of tegaserod at the guanidine nitrogens (M43.2, M43.8, and M45.3) was found, with M43.8 being the major metabolite. Human small intestine slices also metabolized tegaserod to the N-glucuronides, suggesting a contribution of the small intestine to the presystemic metabolism. 5-Methoxyindole-3-carboxylic acid (M29.0), the main metabolite in human plasma, was generated in vitro by a sequence of reactions starting with nonenzymatic acid-catalyzed hydrolysis, followed by enzymatic oxidation and conjugation with glucuronic acid. Tegaserod inhibited CYP2C8, CYP2C9, CYP2C19, CYP2E1, and CYP3A only to a small extent with IC(50) values >30 microM. Tegaserod more effectively inhibited CYP1A2 and CYP2D6 with K(i) values of 0.84 and 0.85 microM, respectively. However, these K(i) values are approximately 140-fold greater than the maximal tegaserod plasma concentrations following the clinically relevant 6-mg oral dose given to healthy volunteers. M29.0, the main circulating metabolite, did not demonstrate any inhibitory potential toward cytochrome P450 enzymes in vitro. Therefore, clinically relevant metabolic drug interactions with tegaserod seem unlikely.

Gene products from LUQ neurons in the abdominal ganglion are present at the renal pore of Aplysia californica.

The L2-4,6 and L5 cells located in the left upper quadrant of the abdominal ganglion of Aplysia californica express the L5-67 and LUQ-1 genes, respectively, in a nonoverlapping manner. These cells send major neurites to the kidney and at least some of them were shown to innervate the renal pore closer muscle, and thereby control its function. By using in-situ hybridization and immunofluorescence, the presence of L5-67 and LUQ-1 mRNAs and peptides was studied in the kidney, with emphasis on the region of the renal pore. We detected immunoreactive materials in many small varicose nerve fibers running along the central epithelium in the inner parts of the kidney, and in neurites located within a large nerve associated with muscles inside the renal pore. Our observations represent the first direct evidence of the presence of gene products from LUQ cells at the renal pore, suggesting that they may be responsible for mediating LUQ cell signals. Furthermore, mRNAs coding for the L5-67 and LUQ-1 peptides were also found in the nerve structure inside the renal pore. Our report documents a striking example of neuropeptide mRNA targeting nerve terminals that are very distant from their cell bodies.

Multiple cytochrome P-450s involved in the metabolism of terbinafine suggest a limited potential for drug-drug interactions.

Biotransformation pathways and the potential for drug-drug interactions of the orally active antifungal terbinafine were characterized using human liver microsomes and recombinant human cytochrome P-450s (CYPs). The terbinafine metabolites represented four major pathways: 1) N-demethylation, 2) deamination, 3) alkyl side chain oxidation, and 4) dihydrodiol formation. Michaelis-Menten kinetics for the pathways revealed mean K(m) values ranging from 4.4 to 27.8 microM, and V(max) values of 9.8 to 82 nmol/h/mg protein. At least seven CYP enzymes are involved in terbinafine metabolism. Recombinant human CYPs predict that CYP2C9, CYP1A2, and CYP3A4 are the most important for total metabolism. N-demethylation is primarily mediated by CYP2C9, CYP2C8, and CYP1A2; dihydrodiol formation by CYP2C9 and CYP1A2; deamination by CYP3A4; and side chain oxidation equally by CYP1A2, CYP2C8, CYP2C9, and CYP2C19. Additionally, characteristic CYP substrates inhibited pathways of terbinafine metabolite formation, confirming the involvement of multiple enzymes. The deamination pathway was mainly inhibited by CYP3A inhibitors, including troleandomycin and azole antifungals. Dihydrodiol formation was inhibited by the CYP1A2 inhibitor furafylline. Terbinafine had little or no effect on the metabolism of many characteristic CYP substrates. Terbinafine, however, is a competitive inhibitor of the CYP2D6 reaction, dextromethorphan O-demethylation (K(i) = 0.03 microM). In summary, terbinafine is metabolized by at least seven CYPs. The potential for terbinafine interaction with other drugs is predicted to be insignificant with the exception that it may inhibit the metabolism of CYP2D6 substrates. Clinical trials are needed to assess the relevance of these findings.

S-nitrosoglutathione in rat cerebellum: identification and quantification by liquid chromatography-mass spectrometry.

Given the extreme lability and the facile inactivation of the messenger nitric oxide (NO) by many reactive biochemical species, it has been suggested that some intermediate compounds, for example, S-nitrosothiols, may act to stabilize NO and at the same time to preserve its biological activity. To test this hypothesis, we investigated if the S-nitrosothiol of glutathione, which is the predominant low molecular weight thiol in CNS, is present in the rat brain. The HPLC analysis of cerebellar extract from [35S]cysteine-prelabeled slices suggested that S-nitrosoglutathione (GSNO) was indeed present in rat brain. To detect endogenous GSNO, a methodology based on liquid chromatography-mass spectrometry was developed. Besides an unequivocal identification of the endogenous GSNO, this method also permitted its precise quantification using 15N-labeled GSNO ([15N]GSNO) as internal standard. GSNO level in adult cerebellum amounts to 15.4 +/- 1.4 pmol/mg of protein. This is the first direct demonstration of the presence of endogenous GSNO in CNS. The packaging of NO in the form of GSNO might serve to facilitate its transport, prolong its life, and target its delivery to specific effectors.

L5-67 and LUQ-1 peptide precursors of Aplysia californica: distribution and localization of immunoreactivity in the central nervous system and in peripheral tissues.

Two genes (L5-67 and LUQ-1) that encode neuropeptide precursors have recently been shown to be expressed in a distinct and non-overlapping manner in the five left upper quadrant (LUQ) cells of the abdominal ganglion of Aplysia (Landry et al. [1992]. J. Neurobiol 23:89-101). By using wholemount immunohistochemistry and radioimmunoassay (RIA), the pattern of expression of these two genes was assessed at the protein level throughout the central nervous system (CNS) and in peripheral tissues of Aplysia californica. The distribution of LUQ-1 precursor-like immunoreactivity was fairly limited, occurring in the ventral LUQ cell (L5) and in a total of approximately 20 additional neurons in the abdominal and cerebral ganglia. L5-67 precursor-like immunoreactive material was more prevalent, appearing in a total of approximately 100 neurons distributed among each of the central ganglia. Identified L5-67-immunoreactive neurons included the four dorsal LUQ cells (L2-4 and L6) and two giant neurons (R2 and LPI1). In one group of cells, the H cluster of the cerebral ganglion, L5-67 immunofluorescence was substantially more intense in larger versus smaller animals, suggesting that this peptide precursor is subject to developmental regulation in certain neurons. Immunoelectron microscopic examination of the subcellular localization of L5-67 immunoreactivity in LUQ cell somata and axons revealed its association with dense-core vesicles (approximately 114 nm in diameter). In the periphery, L5-67-immunoreactive fibers were detected in specific regions of the circulatory system (auricle, ventricle, cristae aorta, anterior aorta) and the reproductive system (genital ganglion, large hermaphroditie duct, small hermaphroditie duct, ovotestis). The kidney and the intestine, two tissues in which considerable secretion and absorption occur, contained material immunoreactive to both L5-67 and LUQ-1 antisera. The localization of the two peptide precursors in these tissues differed substantially, with L5-67 occurring in widely ramifying varicose fibers, whereas LUQ-1 was found in restricted foci of fibers and in small spherical cells that appeared to lack processes. These results support previous findings concerning the heterogeneity of neurotransmitter phenotypes in the LUQ cells. Furthermore, they are indicative of a fairly broad role for the L5-67-derived neuropeptides, and a more limited role for the LUQ-1-derived neuropeptides, in the regulation of the visceral organ systems of Aplysia.

gamma-Glutamylglutamine and taurine concentrations are decreased in the cerebrospinal fluid of drug-naive patients with schizophrenic disorders.

HPLC and gas chromatography-mass spectrometry analyses of 18 amino acids, N-acetylaspartate, N-acetylaspartylglutamate, and 5-hydroxyindoleacetic acid, derived from serotonin, and homovanillic acid, derived from dopamine, were performed in CSF collected from a group of patients with schizophrenia who either had been drug free for at least 1 year (n = 5) or were drug naive for psychotropic drugs (n = 21) and in 15 control subjects. Significant differences were found only for taurine (15% lower in the patients) and isoleucine (7% higher). A number of unidentified substances were detected, one of which proved to be markedly reduced (16%) among the schizophrenic patients. Liquid chromatography-mass spectrometry with continuous flow-fast atom bombardment interface allowed us to identify this substance as gamma-glutamylglutamine. The decreased level of gamma-glutamylglutamine may reflect a deficiency in the gamma-glutamyltransferase system, a system probably involved in glutamate uptake, or a deficiency in glutamine, an important precursor of releasable glutamate. Although glutamate was nonsignificantly reduced in the patients, it was one of the five substances (including gamma-glutamylglutamine) that were necessary for the best discrimination between the schizophrenic patients and the controls. These findings support the notion that the glutamatergic system is affected in schizophrenic disorders. In addition, they underscore the need to apply rigid bioanalytical techniques and use drug-naive patients to gain in-depth information on the pathophysiology of brain disorders such as schizophrenia.

Release of N-acetylaspartylglutamate from slices of rat cerebellum, striatum, and spinal cord, and the effect of climbing fiber deprivation.

The release of endogenous N-acetylaspartylglutamate (NAAG) from slices of rat cerebellum, striatum, and spinal cord upon depolarization with 50 mM K+ was investigated. NAAG in superfusates was prepurified using an ion exchanger, esterified, and then quantified by gas chromatography-mass spectrometry. Deuterated NAAG was used as internal standard. A depolarization-induced release of NAAG was found in all three regions. The release was Ca2+ dependent to over 85% in cerebellum and striatum, but only to approximately 70% in spinal cord. In addition, the effect of lesions of the olivocerebellar pathway on the K(+)-induced release of NAAG was studied: Treatment of the animals with 3-acetylpyridine reduced the release of NAAG from cerebellar hemispheres significantly, by about 40% compared with controls. These results suggest that part of the NAAG released from cerebellar slices on depolarization is related to climbing fibers. Implications of these findings concerning possible physiological roles of NAAG in the three CNS regions are discussed.

Tumor-forming ability in athymic nude mice of human cell lines devoid of mitochondrial DNA.

We have examined the contribution of the mitochondrial genome to the tumorigenic phenotype expressed by human cell lines derived from an ovarian and a cervical carcinoma and from an osteogenic sarcoma. All these continuous cell lines are anchorage-independent in soft agar and form tumors in athymic nude mice. Long-term exposure of the cells to ethidium bromide, an intercalating agent which inhibits mitochondrial DNA replication, gave rise to subclones depleted of mitochondrial DNA and RNA molecules and displaying either anchorage independence or dependence. These respiratory-deficient subclones contain disorganized and enlarged mitochondria, are auxotrophic for uridine and pyruvate, and grow in vitro at a rate nearly identical or moderately slower than their respective parent. The tumor-forming ability of both anchorage-independent and -dependent cell lines was tested by s.c. and intramuscular implantation of the cells in nude mice. We found that the tumorigenic capacity was influenced by the route of inoculation. Subcutaneously, mitochondrial DNA-less cell lines are either poorly or nontumorigenic, while all but one cell line form tumors when implanted into the hind leg muscle. The relative in vivo growth rate of the parent and the mitochondrial DNA-less subclones reflects their respective in vitro rate of growth. All intramuscular tumors introduced into culture mimic the molecular and phenotypic traits of the injected cells, with the exception of the anchorage-dependent cell lines which give rise to anchorage-independent tumor cell lines. The present observations indicate that human cells without mitochondrial DNA have the capacity to proliferate and form tumors in vivo.

Characterization of the multiple forms of mast cell degranulating peptide by NMR spectroscopy.

MCD peptide is a toxic component of bee venom that contains 22 amino acid residues and two disulfide bridges. The peptide was prepared synthetically and purified by HPLC. The proton NMR spectrum of aqueous MCD peptide was assigned at 300 MHz. The NMR and HPLC results showed that the peptide is an approximately 2:1 mixture of two slowly interconverting species, most likely conformers. Proline-12 is proposed as the locus of the conformational equilibrium. Measurements of the proton-proton nuclear Overhauser effect confirmed that the connectivity of the major form is C3-C15 and C5-C19.

Targeting of pro-opiomelanocortin to the regulated secretory pathway may involve cooperation between different protein domains.

The structure of pro-opiomelanocortin (POMC) can be divided into three main domains: an NH2-terminal domain formed by the NH2-terminal glycopeptide and the joining peptide, a central domain corresponding to the adrenocorticotropin sequences and a COOH-terminal domain containing the beta-lipotropin sequences. Expression of POMC in neuroendocrine cell lines such as the mouse neuroblastoma Neuro2A cells results in its targeting to the regulated secretory pathway of these cells. Intracellular targeting of proteins along non default pathways are widely believed to involve the recognition of specific structural features by a sorting machinery. To understand the nature of the signal involved in targeting prohormone to the regulated secretory pathway, we have constructed mutants of POMC in which sequences from the NH2-terminal, the central and the COOH-terminal domains were deleted and examined the sorting of these mutant POMC molecules in Neuro2A cells by immunofluorescence and immunoelectron microscopy. Our results indicate that POMC NH2-terminal glycopeptide or beta-LPH domain do not contain sufficient information for targeting to the regulated pathway since these peptides are not sorted to secretory vesicles when expressed in Neuro2A cells: Similarly, the ACTH domain does not contain essential targeting information since POMC mutants lacking these sequences were sorted to secretory vesicles. Mutant POMCs containing the sequences of more than one of the main protein domains were, however, correctly targeted to the regulated secretory pathway. Our results indicate that POMC is not targeted to the regulated secretory pathway through recognition of a unique continuous 'molecular address'.(ABSTRACT TRUNCATED AT 250 WORDS)

The biotransformation of the ergot derivative CQA 206-291 in human, dog, and rat liver slice cultures and prediction of in vivo plasma clearance.

Liver slice cultures from humans, dogs, and rats were used to investigate the biotransformation of the dopaminergic ergot agonist CQA 206-291 and to predict pharmacokinetic values for hepatic intrinsic clearance and plasma clearance. CQA 206-291 was extensively metabolized in the liver slice cultures and in vivo. The HPLC metabolite patterns from the liver slice cultures were similar for all three species, indicating the occurrence of the same metabolic pathways for CQA 206-291 biotransformation. The rate of formation of CQ 32-084, a pharmacologically active N-deethylated metabolite, exceeded that of metabolite d, a primary metabolite, by 1.4 fold in human liver slices, and by 1.7 fold in rat liver slices. In dog liver slice cultures, metabolite d formation exceeded CQ 32-084 formation by 1.3 fold and was formed at a statistically significantly greater rate (3 fold) than in either human or rat liver slices. The metabolism of ergots like CQA 206-291 by human fetal liver was also demonstrated in this study. However, the prominent metabolite from fetal and adult human liver microsomes was metabolite d with minor amounts of CQ 32-089 being formed. A major route of excretion for the metabolites of CQA 206-291 is the kidney, yet the kidney does not contribute to the metabolism of CQA 206-291. Kidney slices derived from humans, rats, and dogs did not metabolize CQA 206-291 within 24 hr. CQA 206-291 intrinsic clearance was derived from the half-life of parent drug disappearance in the liver slice and hepatocyte cultures, and from the ratio of Vmax/Km of human and rat liver microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkylacyl glycerophosphoinositol in human and bovine erythrocytes. Molecular species composition and comparison with glycosyl-inositolphospholipid anchors of erythrocyte acetylcholinesterases.

Glycosyl-inositolphospholipid (glycosyl-PtdIns) anchors of proteins in mammalian cells which have been analyzed so far are exclusively of the alkylacyl type. However, little is known about the putative precursor of glycosyl-PtdIns, the alkylacyl derivative of glycerophosphoinositol (GroPIns), in these cells since it is generally believed that cellular GroPIns consists of diacyl-type molecular species only. In this report, we describe the isolation and identification of alkylacyl GroPIns molecular species in both human and bovine erythrocytes, and compare it with the molecular species compositions of the glycosyl-PtdIns anchors of human and bovine erythrocyte acetylcholinesterase. Diradyl GroPIns was isolated from lipid extracts of ghost membranes and treated with phospholipase C. Diradylglycerols of the glycosyl-PtdIns anchors of affinity-purified human and bovine erythrocyte acetylcholinesterase were generated by sequential treatment with glycoprotein phospholipase D and acidic phosphatase and by PtdIns-specific phospholipase C, respectively. Diradylglycerols were subsequently converted into benzoate derivatives and separated into diacyl, alkylacyl, and alkenylacylglycerol subclasses. The molecular species compositions were quantitated and determined by combined HPLC/mass spectrometry. We found that human and bovine erythrocyte membrane diradyl GroPIns consist of 1.5-4.8% alkylacyl GroPIns. Molecular species analysis showed a heterogeneous species composition for both human and bovine erythrocyte alkylacyl GroPIns. Their compositions are distinctly different from those of human and bovine erythrocyte acetylcholinesterase glycosyl-PtdIns anchors. The number of alkylacyl GroPIns molecules/cell is roughly equal with the number of glycosyl-PtdIns-anchored proteins in human erythrocytes.

Characterization of the cytochrome P-450 gene family responsible for the N-dealkylation of the ergot alkaloid CQA 206-291 in humans.

The ergot alkaloid CQA 206-291 (CQA) was converted by human liver microsomes (n = 16) almost exclusively to the N-deethylated metabolite (I), as identified by the on-line coupling of liquid chromatography and mass spectroscopy. Metabolite I formation exhibited monophasic and linear enzyme kinetics (2.9-300 microM), and a 5.6-fold interindividual variability (7.2-40.2 nmol/mg/hr). Chemical inhibition experiments revealed that imidazole antimycotic agents (ketoconazole, miconazole, and clotrimazole) were potent inhibitors of this N-deethylation. Polymorphically metabolized substrates (sparteine and phenytoin), well-established cytochrome P-450 probe substrates (antipyrine and tolbutamide), and steroid hormones (estradiol and testosterone) were noninhibitory, indicating that their metabolism is catalyzed by forms of cytochrome P-450 that do not catalyze this route of CQA biotransformation. The ergot alkaloids--dihydroergotamine, bromocriptine, and SDZ 208-911--were competitive inhibitors of metabolite I formation, suggesting that these compounds are metabolized by similar enzymes. Cyclosporine A was a potent competitive inhibitor of CQA metabolism, providing initial evidence that formation of metabolite I was catalyzed by proteins of the CYP3 gene family. This was substantiated by the finding that CQA metabolism was completely inhibited by a polyclonal antibody directed against a pregnenolone 16 alpha-carbonitrile-inducible cytochrome P-450 of rat liver. The rate of CQA metabolism correlated significantly to the level of CYP3A4 expression, the rate of cyclosporine A metabolism to each of the primary metabolites (M-1, M-17, and M-21), and the rate of midazolam 4-hydroxylation. COS 1 cells transfected with human CYP3A4 and CYP3A5 provided direct evidence that these enzymes catalyze the metabolism of CQA.(ABSTRACT TRUNCATED AT 250 WORDS)

Investigation of a possible role of the amino-terminal pro-region of proopiomelanocortin in its processing and targeting to secretory granules.

Proopiomelanocortin (POMC) is a polyprotein which is targeted to the regulated secretory pathway of neuroendocrine cells where it undergoes tissue-specific proteolysis to yield peptides such as adrenocorticotropic hormone, beta-lipotropin and beta-endorphin. The pro-region of POMC is 49 amino acid long with two disulfide bonds between cysteine residues 2 and 24 and 8 and 20. These cysteine residues are conserved across the species. The pro-region contains no known hormonal sequence. Sorting to the regulated secretory pathway is thought to involve targeting signals encoded in the structure of secretory proteins. In the present study, we have examined the possibility that the disulfide bridges located in the NH2-terminal portion of the pro-region of POMC are essential for maintaining a determinant involved in the sorting of POMC to the regulated secretory pathway. Using site-directed and deletion mutagenesis of the porcine POMC cDNA, we created mutants in which one or both disulfide bridges were disrupted or in which the first 26 amino acid residues of the pro-region were deleted. Recombinant retroviruses carrying the mutated POMC cDNAs were used to infect Neuro2A cells. Immunofluorescence and immunoelectron microscopy studies performed on infected cells revealed that the unmutated and mutated POMC-immunoreactive peptides were localized in dense-core vesicles at the tips of cellular extensions. Analysis of the POMC-immunoreactive peptides extracted from the infected Neuro2A cells indicated that the mutated precursors in which one disulfide bridge was disrupted (POMC-S2 or POMC-S8) were stored and processed as efficiently as the unmutated POMC. By contrast, the mutated precursor in which both disulfide bridges were disrupted (POMC-S2,8) did not accumulate in intracellular compartments to the same extent as unmutated POMC. Moreover, this mutant was very inefficiently processed and no release could be observed upon stimulation of the cells with K+/Ca2+. These results suggest that POMC-S2,8 entered the regulated secretory pathway less efficiently than the unmutated precursor. However, when both disulfide bridges were removed from the precursor from the precursor by deletion of the first 26 amino acid residues of POMC, the truncated precursor (POMC delta 1-26) behaved as the unmutated POMC. Taken together our results indicate that the NH2-terminal portion of the pro-region including both disulfide bridges can be deleted without affecting the targeting of the molecule to secretory granules. However, when the entire POMC sequence is expressed in Neuro2A cells, the proper folding of the NH2-terminal region might be important for efficient processing and targeting.

Neutral endopeptidase, a major brush border protein of the kidney proximal nephron, is directly targeted to the apical domain when expressed in Madin-Darby canine kidney cells.

We have used a retroviral vector containing both the cDNA for rabbit neutral endopeptidase (EC 3.4.24.11; NEP) and the neomycin resistance gene to promote the expression of NEP in a polarized Madin-Darby canine kidney (MDCK) cell line. Cells resistant to G418 (a neomycin synthetic analog) were analyzed with a fluorescence-activated cell sorter to isolate a homogeneous population of cells which stably expressed NEP at their surface. When cells grown in Petri dishes were labeled with an antibody to NEP coupled to colloidal gold and examined under the electron microscope, a strong labeling of microvilli was observed, whereas very few particles were present on the basolateral domain, suggesting that the polarized distribution of this enzyme typical of proximal tubule cells is maintained in this MDCK cell population. To study more accurately the mechanism by which MDCK cells target NEP to the apical surface, cultures were grown to confluence on Costar Transwell chambers and used for pulse-chase experiments with [35S]methionine. Immunoprecipitation of recombinant NEP was then performed by adding an anti-NEP polyclonal antibody to the apical or basolateral surface of intact monolayers and by analyzing immunoprecipitates by gel electrophoresis and fluorography. Our results suggest that NEP is delivered directly to the apical domain and does not transit through the basolateral domain of the plasma membrane. This NEP-expressing MDCK cell line therefore constitutes a new model for investigating the molecular basis of apical membrane targeting in polarized epithelial cells.

[Sterilization of mentally retarded women]. / Zur Sterilisation geistig behinderter Patientinnen.

The sterilization of mentally retarded women raises gynecologic, psychiatric and legal questions. Gynecologic considerations include the experience of the reliability of reversible contraceptive methods with mentally retarded patients and questions concerning the type of operation. From the psychiatric point of view the prognoses of mentally retarded patients and the expected psychic coping with the operation are essential aspects in the indication for sterilization. Last but not least, the Swiss legal framework for sterilization must be considered. This aspect is illustrated on the basis of the results of a catamnestic analysis of 21 patients operated on between 1980 and 1987.

Expression of porcine pro-opiomelanocortin in mouse neuroblastoma (Neuro2A) cells: targeting of the foreign neuropeptide to dense-core vesicles.

Pro-opiomelanocortin (POMC) is the precursor to several pituitary hormones and neuropeptides including adrenocorticotropic hormone (ACTH) and beta-endorphin (beta-END). In neuroendocrine cells, peptide hormones and neuropeptides are targeted to the dense-core vesicles of the regulated secretory pathway. These vesicles are transported to the ends of cellular extensions where they are stored until they release their content upon external stimulation of the cell. In order to study the cellular mechanisms involved in targeting of neuropeptides, we have expressed POMC in Neuro2A cells, a cell line of neural origin. Using immunofluorescence labeling and immunoelectron microscopy we show that in Neuro2A cells POMC is packaged in dense-core vesicles which accumulate at the tips of cellular processes. Intracellular accumulation of POMC was not observed in NIH 3T3 fibroblasts. When a soluble form of an integral membrane protein, neutral endopeptidase (E.C. 3.4.24.11) (secNEP), was expressed in Neuro2A cells, the protein was found to be constitutively secreted without prior accumulation in dense-core vesicles. Our results suggest that in Neuro2A cells, targeting to the regulated secretory pathway is restricted to peptide hormones and neuropeptides and establish this cell line as a valid model for studying the molecular events involved in neuropeptide sorting into the regulated secretory pathway.

Características ultraestructural del reconocimiento específico de un anticuerpo monoclonal anti P24 del VIH-1 en células H9 y E. coli / Ultrastructural characterization of especific recognition of an Mab agains HIV-1 P24 in H9 and E. coli cells

Se realiza la caracterización del reconocimiento específico de un anticuerpo monoclonal contra la proteina P24 del VIH-1 mediante la combinación de métodos inmunológicos y ultraestructurales a través de la inmunomicroscopia electrónica de transmisión con el uso de oro coloidal como marcador en secciones ultrafinas de linfocitos de la linea celular H9 y células de E. coli transformadas para la expresión de las proteinas P24, GP41, GP160 y GP36. Se utilizó para el marcaje post-inclusión una sonda de IgH de cabra antiratón-oro (8 mn). Los resultados demostraron la especificidad del anticuerpo monoclonal utilizado para la proteina P24 en ambos tipos de células, así como la posibilidad de utilizar estos tipos de células, así como la posibilidad de utilizar estos sustratos antigénicos con vistas a la evaluación de otros anticuerpos monoclonales y policlonales relacionados al VIH

Características ultraestructural del reconocimiento específico de un anticuerpo monoclonal anti P24 del VIH-1 en células H9 y E. coli / Ultrastructural characterization of especific recognition of an Mab agains HIV-1 P24 in H9 and E. coli cells

Se realiza la caracterización del reconocimiento específico de un anticuerpo monoclonal contra la proteina P24 del VIH-1 mediante la combinación de métodos inmunológicos y ultraestructurales a través de la inmunomicroscopia electrónica de transmisión con el uso de oro coloidal como marcador en secciones ultrafinas de linfocitos de la linea celular H9 y células de E. coli transformadas para la expresión de las proteinas P24, GP41, GP160 y GP36. Se utilizó para el marcaje post-inclusión una sonda de IgH de cabra antiratón-oro (8 mn). Los resultados demostraron la especificidad del anticuerpo monoclonal utilizado para la proteina P24 en ambos tipos de células, así como la posibilidad de utilizar estos tipos de células, así como la posibilidad de utilizar estos sustratos antigénicos con vistas a la evaluación de otros anticuerpos monoclonales y policlonales relacionados al VIH (AU)

Production of monoclonal antibodies against the N-terminal glycopeptide of porcine pro-opiomelanocortin: their use for solid-phase radioimmunoassay, western blotting, and immunogold cytochemistry.

We have prepared two monoclonal antibodies for the N-terminal glycopeptide of pro-opiomelanocortin 1-77 (N-POMC(1-77)) purified from porcine pituitaries. Antibody 1-244 recognizes an epitope located within the gamma 3-melanotropin (gamma 3-MSH or POMC(51-77)) sequence, whereas antibody 2-197 binds specifically to a determinant in the 1-49 region of N-POMC. These monoclonal antibodies were used to construct a two-site solid-phase radioimmunoassay that can detect as little as 50 pg of N-POMC(1-77). The assay is linear between 0.5 and 5 ng of porcine peptide and recognizes equally well the homologous peptides purified from human and bovine pituitaries. The assay has been used to analyze reversed-phase high pressure liquid chromatography fractions of crude bovine pituitary extracts and detected a peptide with chromatographic properties identical to those of N-POMC(1-77). When used to stain immunoblots of bovine intermediate pituitary extracts, both the 2-197 and 1-244 antibodies could recognize a major peptide comigrating with purified N-POMC(1-77). In addition, antibody 2-197 also detected a peptide with a mobility similar to that of standard N-POMC(1-49). When used in conjunction with a second anti-mouse antibody coupled to colloidal gold particles, antibody 2-197 stained N-POMC immunoreactive material located in granules in thin sections of pituitary.