RESUMEN
Over the past 20 years, laparoscopic colectomy has become a well-established technique in the surgical armamentarium of colorectal operations, with proven reductions in postoperative pain, time to return of bowel function, and length of hospital stay. After early concerns over its oncologic effects, large prospective, multicenter trials have proven its safety in colorectal adenocarcinoma, with equivalence in nodal harvest, recurrence rates, disease-free survival, and overall survival. Laparoscopic right hemicolectomy in particular is a relatively accessible technique which may be performed by a single surgeon and an assistant/camera operator; this operation serves as an excellent method to develop laparoscopic skills for more complicated colorectal procedures. In this article, we describe the technical aspects of our approach to laparoscopic right hemicolectomy, which utilizes a medial-to-lateral, no-touch technique and either an intracorporeal or extracorporeal anastomosis.
Asunto(s)
Colectomía/métodos , Laparoscopía/métodos , Anastomosis Quirúrgica , Colectomía/instrumentación , Humanos , Laparoscopía/instrumentación , Posicionamiento del PacienteRESUMEN
Transmembrane and secreted glycoproteins of the semaphorin family are typically classified as inhibitory neuronal guidance molecules. However, although chemorepulsive activity has been demonstrated for several semaphorin family members, little is known about the function of the numerous transmembrane semaphorins identified to date. Here we demonstrated that the extracellular semaphorin domain of a transmembrane semaphorin, semaphorin-1a, could actively perturb axon pathfinding in vivo when presented homogenously as a recombinant freely soluble factor. When ectopic overexpression was limited to defined epithelial regions, semaphorin-1a could directly steer axons by acting as an attractive guidance molecule.
Asunto(s)
Axones/fisiología , Moléculas de Adhesión Celular Neuronal/farmacología , Moléculas de Adhesión Celular Neuronal/fisiología , Neuronas/fisiología , Semaforinas , Secuencia de Aminoácidos , Animales , Axones/efectos de los fármacos , Células COS , Moléculas de Adhesión Celular Neuronal/química , Línea Celular , Señales (Psicología) , Drosophila melanogaster , Embrión no Mamífero/fisiología , Saltamontes , Datos de Secuencia Molecular , Neuronas/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , TransfecciónRESUMEN
The mutant trpS gene of Bacillus subtilis encoding a thermosensitive tryptophanyl-tRNA synthetase that also confers resistance to growth inhibition by 5-fluorotryptophan at permissive temperature has been isolated and characterized. A point mutation at codon 82 of the gene from a wild-type TCA codon for Ser to a TTA codon for Leu has been identified.
Asunto(s)
Bacillus subtilis/genética , Genes Bacterianos/genética , Triptófano-ARNt Ligasa/genética , Triptófano/análogos & derivados , Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/enzimología , Clonación Molecular , Análisis Mutacional de ADN , Farmacorresistencia Microbiana/genética , Datos de Secuencia Molecular , Mutación Puntual/genética , Conformación Proteica , Temperatura , Triptófano/farmacología , Triptófano-ARNt Ligasa/químicaRESUMEN
A semi-conserved tryptophan residue of Bacillus subtilis tryptophanyl-tRNA synthetase (TrpRS) was previously asserted to be an essential residue and directly involved in tRNATrp binding and recognition. The crystal structure of the Bacillus stearothermophilus TrpRS tryptophanyl-5'-adenylate complex (Trp-AMP) shows that the corresponding Trp91 is buried and in the dimer interface, contrary to the expectations of the earlier assertation. Here we examine the role of this semi-conserved tryptophan residue using fluorescence spectroscopy. B. subtilis TrpRS has a single tryptophan residue, Trp92. 4-Fluorotryptophan (4FW) is used as a non-fluorescent substrate analog, allowing characterization of Trp92 fluorescence in the 4-fluorotryptophanyl-5'-adenylate (4FW-AMP) TrpRS complex. Complexation causes the Trp92 fluorescence to become quenched by 70%. Titrations, forming this complex under irreversible conditions, show that this quenching is essentially complete after half of the sites are filled. This indicates that a substrate-dependent mechanism exists for the inter-subunit communication of conformational changes. Trp92 fluorescence is not efficiently quenched by small solutes in either the apo- or complexed form. From this we conclude that this tryptophan residue is not solvent exposed and that binding of the Trp92 to tRNATrp is unlikely. Time-resolved fluorescence indicates conformational heterogeneity of B. subtilis Trp92 with the fluorescence decay being best described by three discrete exponential decay times. The decay-associated spectra (DAS) of the apo- and complexed-TrpRS show large variations of the concentration of individual fluorescence decay components. Based on recent correlations of these data with changes in the local secondary structure of the backbone containing the fluorescent tryptophan residue, we conclude that changes observed in Trp92 time-resolved fluorescence originate primarily from large perturbations of its local secondary structure. The quenching of Trp92 in the 4FW-AMP complex is best explained by the crystal structure conformation, in which the tryptophan residue is found in an alpha-helix. The amino acid residue cysteine is observed clearly within the quenching radius (3.6 angstroms) of the conserved tryptophan residue. These tryptophan and cysteine residues are neighbors, one helical turn apart. If this local alpha-helix was disrupted in the apo-TrpRS, this disruption would concomitantly relieve the putative cysteine quenching by separating the two residues. Hence we propose a substrate-dependent local helix-coil transition to explain both the observed time-resolved and steady-state fluorescence of Trp92. A mechanism can be further inferred for the inter-subunit communication involving the substrate ligand Asp132 and a small alpha-helix bridging the substrate tryptophan residue and the conserved tryptophan residue of the opposite subunit. This putative mechanism is also consistent with the observed pH dependence of TrpRS crystal growth and substrate binding. We observe that the mechanism of TrpRS has a dynamic component, and contend that conformational dynamics of aminoacyl-tRNA synthetases must be considered as part of the molecular basis for the recognition of cognate tRNA.
Asunto(s)
Bacillus subtilis/enzimología , Triptófano-ARNt Ligasa/química , Triptófano/química , Adenosina Monofosfato/química , Cisteína/química , Modelos Moleculares , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Espectrometría de Fluorescencia , Especificidad por Sustrato , Compuestos de Sulfhidrilo/química , Factores de Tiempo , Triptófano-ARNt Ligasa/genética , Triptófano-ARNt Ligasa/aislamiento & purificaciónRESUMEN
Tryptophanyl-tRNA synthetase from Bacillus subtilis was overexpressed in Escherichia coli and was purified using column chromatography on DEAE-Sephacel and hydroxyapatite columns. Single crystals of the synthetase were grown by vapor diffusion at 4 degrees C from pH 5.5 solutions of polyethylene glycol 8000 containing magnesium ATP and L-tryptophan. The crystals diffracted to about 4.0 A resolution at -150 degrees C and appeared to belong to the orthorhombic space group P2(1)2(1)2 with unit cell dimensions: a = 143.6 A, b = 111.6 A, c = 50.6 A with one dimer in the asymmetric unit.
Asunto(s)
Bacillus subtilis/enzimología , Triptófano-ARNt Ligasa/química , Clonación Molecular , Cristalización , Escherichia coli , Proteínas Recombinantes/química , Difracción de Rayos XRESUMEN
A 1.47-kb DNA fragment that carries the tryptophanyl-tRNA synthetase (TrpRS) gene of Bacillus subtilis has been cloned into the pUC8 plasmid. The recombinant plasmid, pTSQ2, conferred temperature-resistance to the temperature-sensitive trpS ts mutant of B. subtilis through chromosomal transformation, and to that of Escherichia coli through complementation. The pTSQ2 could be stably maintained in E. coli DH5 alpha, causing in the host cell a 200-fold amplification of TrpRS activity. The complete nucleotide sequence of the cloned fragment has been determined. A putative transcriptional promoter, a Shine-Dalgarno sequence, the 990-bp trpS gene proper, as well as a transcriptional terminator have been identified.
Asunto(s)
Aminoacil-ARNt Sintetasas/genética , Bacillus subtilis/genética , Genes Bacterianos , Genes , Triptófano-ARNt Ligasa/genética , Secuencia de Aminoácidos , Bacillus subtilis/enzimología , Secuencia de Bases , Clonación Molecular , ADN Recombinante/metabolismo , Datos de Secuencia Molecular , Plásmidos , Mapeo Restrictivo , Homología de Secuencia de Ácido NucleicoRESUMEN
Ever since the discovery that the human tryptophanyl-tRNA synthetase (TrpRS)-encoding gene is induced by interferon (IFN) [J. Fleckner et al., Proc. Natl. Acad. Sci. USA 88 (1991) 11520-11524] and contains IFN-response regulatory elements [Frolova et al., Gene 128 (1993) 237-245], the biological rationale for this induction has remained unresolved. A survey of immune system proteins in this study reveals that the human major histocompatibility complex (MHC) antigens, beta-2-microglobulin (beta MG) and complement factor B, which are known to be induced by IFN, together with immunoglobulins (Ig) are all exceptionally enriched in Trp residues, as compared to human proteins in general. It also reveals the conservation of a sequence motif, CX10-17 WX26-62C, in Ig domains. The conservation of this sequence motif and the utility of Trp residues within antigen-binding sites clearly contribute to the Trp enrichment in Ig. These observations suggest a biological rationale for the induction of TrpRS by IFN in safeguarding Trp incorporation for the IFN-enhanced synthesis of immunological molecules.
Asunto(s)
Interferones/farmacología , Proteínas/química , Triptófano-ARNt Ligasa/fisiología , Triptófano/análisis , Secuencia de Aminoácidos , Enfermedades Transmisibles/metabolismo , Secuencia Conservada , Epítopos/química , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Cirrosis Hepática/metabolismo , Datos de Secuencia Molecular , Páncreas/química , Biosíntesis de Proteínas , Proteínas/metabolismoRESUMEN
Antigen-specific lymphocytes are important in the immune response to viral infection. Peripheral blood mononuclear cells (PBMC) are traditionally used as a source of effector cells in most immunological studies. We described here the use of the bispecific monoclonal antibodies (BSMAB) anti CD3:CD8 (CD3,8) and anti CD3:CD4 (CD3,4B) to expand and selectively enrich CD4+ and CD8+ T cells populations, respectively. The expanded cells demonstrated >90% CD3+CD4+ or CD3+CD8+ by 14 days. We measured HIV- and CMV-specific responses of these subset-enriched T cell and found that sensitivity and specificity is similar or higher when compared to PBMC in various cellular immunology assays (CMI). Vbeta analysis of BSMAB-enriched cells demonstrated comparable repertoire to the parent PBMC. Although both CD45RA(hi) and CD45RO(hi) cell populations were expanded with the BSMAB, selective subset depletion demonstrated that the antigen-specific T cell responses were restricted to the initial CD45RO(hi) memory effector subgroup. In conclusion, BSMAB in vitro enrichment of T cells allows significant expansion of the cell population without loss of specificity. This technique of cell expansion permits studies of T cell subset function in situations where the initial cell source is scarce, and presents an alternative for viable and functional T cells in immunological assays.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Anticuerpos Monoclonales/inmunología , Antígenos Virales/inmunología , Activación de Linfocitos , Subgrupos de Linfocitos T/inmunología , Complejo CD3/inmunología , Antígenos CD4/inmunología , Linfocitos T CD4-Positivos/inmunología , Antígenos CD8/inmunología , Linfocitos T CD8-positivos/inmunología , Línea Celular , Citomegalovirus/inmunología , VIH/inmunología , Infecciones por VIH/inmunología , Humanos , Inmunofenotipificación , Cinética , Subgrupos de Linfocitos T/clasificaciónRESUMEN
Perfusion with deoxygenated dextran-hemoglobin provides an effective method for inducing hypoxic radioprotection of normal tissues during radiation treatment of tumors. In this study, the dependence of P50, the half-saturation pressure of oxygen binding to dextran-hemoglobin, was analyzed as a function of solution temperature and pH. The variation of attainable radioprotection with P50, and with the amount of collateral blood entering into the perfused region, was calculated. Upon perfusion of canine gracilis muscle with deoxygenated dextran-hemoglobin, a rapid onset of extensive venous hypoxia was observed.
Asunto(s)
Dextranos/farmacología , Hemoglobinas/farmacología , Protectores contra Radiación/farmacología , Animales , Perros , Músculos/efectos de los fármacos , Músculos/metabolismo , Oxígeno/sangreRESUMEN
Initial studies were performed to examine the potential of perfused dextran-hemoglobin to protect pig skin or mouse bone marrow cells against radiation damage. Some protection was indicated in both systems. In the pig skin a protection factor of 1.5 was observed for moist desquamation, and 2.0 for necrosis. These results suggest the possibility of using blood substitutes to induce tissue hypoxia for therapeutic purposes.
Asunto(s)
Médula Ósea/efectos de la radiación , Dextranos/farmacología , Hemoglobinas/farmacología , Traumatismos Experimentales por Radiación/prevención & control , Protectores contra Radiación/farmacología , Piel/efectos de la radiación , Animales , Médula Ósea/patología , Ensayo de Unidades Formadoras de Colonias , Relación Dosis-Respuesta en la Radiación , Femenino , Masculino , Ratones , Ratones Endogámicos C3H , Necrosis , Oxígeno/fisiología , Perfusión , Traumatismos Experimentales por Radiación/patología , Piel/patología , PorcinosRESUMEN
With flavone as a structural template, three-dimensional quantitative structure-activity relationship (3D-QSAR) studies and ab initio calculations were performed on a series of flavonoids. A reasonable pharmacophore model was built through CoMFA, CoMSIA, and HQSAR analyses and electrostatic potential calculations. A plausible binding mode for flavonoids with GABA(A) receptors was rationalized. On the basis of the commonly recognized binding site, the specific S1 and S2 subsites relating to substituent positions were proposed. The different binding affinities could be explained according to the frontier orbitals and electrostatic potential (ESP) maps. The ESP could be used as a novel starting point for designing more selective BZ-binding-site ligands.
Asunto(s)
Benzodiazepinas/metabolismo , Flavonoides/química , Flavonoides/metabolismo , Receptores de GABA-A/química , Receptores de GABA-A/metabolismo , Benzodiazepinas/química , Sitios de Unión , Calorimetría , Ligandos , Modelos Moleculares , Conformación Molecular , Mutagénesis Sitio-Dirigida , Estructura Secundaria de Proteína , Subunidades de Proteína , Relación Estructura-Actividad Cuantitativa , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Electricidad EstáticaRESUMEN
OKT3, an anti-CD3 MAB, depletes T cells in vivo and is among the most potent inhibitors of acute allograft rejection. The mechanism of this inhibition is unknown. The present studies investigate whether anti-CD3 antibodies have the ability to crosslink CD3 on two different cells and induce TCR-dependent antibody-bridged cell-mediated cytolysis (TCR-ABCMC) between T cells. Two different anti-CD3 antibodies (OKT3 and CD3,3) and OKT3 F(ab')2 were all highly effective in inducing cytolysis of CD8+ and CD4+ T cells by CD8+ T cells, and CD8+ T cells by CD4+ T cells. Monovalent OKT3 Fab was 25-125-fold less potent than OKT3 F(ab')2. Monovalent CD3,X was totally ineffective. The necessity for intercellular bridging was evidenced by the observation that an anti-CD3:anti-CD4 (CD3,4) bispecific MAB (BSMAB) was effective in mediating lysis of CD4+ but not CD8+ T cells by CD8+ T cells. These studies indicate that neither FcR-mediated ADCC nor complement fixation is necessary for bivalent anti-CD3 MAB to lyse T cells. Inter-T cell TCR-ABCMC may be particularly effective in inflammatory tissues, such as rejecting allografts and autoimmune diseases, in which numerous cytolytic T lymphocytes are present in close association with other T cells.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Citotoxicidad Inmunológica , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Antígenos de Diferenciación de Linfocitos T/análisis , Complejo CD3 , Antígenos CD4/análisis , Antígenos CD8 , Fragmentos Fab de Inmunoglobulinas/inmunología , RatonesRESUMEN
Monoclonal antibodies were used to determine the relationships between epitopes on the p55 chain of the IL-2 receptor and high-affinity IL-2 binding. Five monoclonal antibodies to the human P55 chain of the IL-2 receptor were induced by immunizing mice with murine L cells that were transfected with human p55 cDNA. Since the p55 chain is the only human antigen expressed on these cells, all antihuman MABs thus generated were directed against this molecule. These antibodies were used to map epitopes on the p55 chain and determine their relationship to high-affinity IL-2 binding. Extensive flow cytometric studies with these MABs and a large panel of other anti-p55 MABs revealed three major patterns of competition. Type I MABs compete with anti-Tac extensively but not with antibodies of other groups. Type II MABs do not block anti-Tac but do block 7E11. Type III MABs do not block either type I or type II antibodies. 125I-IL2 competition studies under high-affinity conditions revealed that types I and II MABs inhibit IL-2 binding. Type III MABs can be resolved into two subgroups, one that inhibits IL-2 binding and one that does not. Together these data suggest that there are at least four distinct immunogenic epitopes on the human p55 chain, with three epitopes related to IL-2 binding. The competitive component evident by a change in Kd on the Scatchard plots suggests that all three epitopes are close to or part of the IL-2-binding site of the p55 chain. The noncompetitive component, as evidenced by the lower number of high-affinity IL-2 receptors induced by these antibodies, suggests that the same epitopes are also close to the site(s) of interaction between the p55 and p70 chains to form the high-affinity receptor. These studies indicated that the IL-2-binding site and site of interaction between the p55 and p70 chains are close together or identical. Modulation studies revealed that one type II antibody (7E11) modulates the p55 chain in the absence of IL2 and the p70 chain, thus revealing that modulation of the p55 chain can occur by an active process, and not merely passively comodulate by the p70 chain upon IL-2 binding. Modulation of the p55 chain alone has no proliferative effect on IL-2-responsive T lymphoblasts. Potentially this antibody-dependent modulation may be used to deliver toxin to activated lymphocytes.
Asunto(s)
Receptores de Interleucina-2/inmunología , Linfocitos T/inmunología , Anticuerpos Monoclonales/inmunología , Unión Competitiva , Endocitosis , Epítopos , Citometría de Flujo , Humanos , Interleucina-2/metabolismo , Cinética , Activación de Linfocitos , Sustancias Macromoleculares , Receptores de Interleucina-2/metabolismo , Receptores de Interleucina-2/ultraestructura , Linfocitos T/metabolismoRESUMEN
The TCR and CD8 complexes of CD8+ T cells bind to different regions of MHC class I molecules and both play important roles in the response of the CD8+ T cells to Ag/MHC on APCs. In this report, we mimicked common MHC binding with an anti-CD3:anti-CD8 (CD3,8) BSMAB to isolate the effect of CD3: CD8 pairing, compared this with the effect of CD3: CD3 pairing by the parental bivalent anti-CD3 MAB, and with monovalent anti-CD3 binding by an anti-CD3: anti-CD4 (CD3,4) BSMAB. CD3: CD8 pairing induced an increase in cytosolic free [Ca2+] 1.5 to 3.0-fold greater than the increase induced by CD3: CD3 pairing whereas monovalent CD3 binding induced only 20%-30% of the increase. Postbinding receptor migration studies suggested that microaggregation increased from monovalent CD3 binding to CD3: CD3 pairing to CD3: CD8 pairing. Further studies revealed that progressively higher concentrations of antibodies were needed from CD3,8 to CD3,3 to CD3,4 to initiate the same degree of DNA synthesis. These results demonstrated that Ti/CD3 and CD8 can indeed be bridged by a single molecule. A model of direct CD8: CD3 synergism was raised as a possible explanation for the enhanced activation induced by CD3: CD8 pairing. The observed parallel between all three parameters and the number of TCRs that can be directly linked by the Abs raised a nonmutually exclusive model whereby CD3 binding induces activated TCR intermediaries (aTCRi) that progressively synergize with other adjacent aTCRis. In this model, this dominant inter-aTCRi synergism may be enhanced by the di- and multimeric CD8 alpha chains serving as aTCRi-aggregation foci.
Asunto(s)
Complejo CD3/inmunología , Antígenos CD8/inmunología , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Reacciones Antígeno-Anticuerpo/inmunología , Células Presentadoras de Antígenos/inmunología , Calcio/metabolismo , ADN/biosíntesis , Relación Dosis-Respuesta Inmunológica , HumanosRESUMEN
The bacterial formyl peptide N-formylmethionine-leucine-phenylalanine (fMLP) is a potent chemoattractant for mammalian neutrophils. In this study, we demonstrated the binding of fluorescent dye-conjugated-fMLP to haemocytes of the penaeid shrimp Penaeus penicillatus (Alcock), through the use of flow cytometry. Fluorescence microscopy with rhodamine-fMLP suggested that fMLP receptors are present only in sub-populations of the haemocytes: granulocytes and the semi-granular cells. In addition, fMLP dose-dependently mediated chemotaxis in sub-populations of haemocytes. Microphysiometry experiments demonstrated rapid extracellular acidification upon addition of fMLP, which is in agreement with the observation in neutrophils. t-BOC, the specific fMLP receptor antagonist, was able to block the binding, chemotaxis and extracellular acidification induced by the peptide. The ability of shrimp haemocytes to migrate toward fMLP in vitro suggests that this mechanism may be important for the accumulation of these cells in infected tissues of the shrimps.
Asunto(s)
Quimiotaxis/efectos de los fármacos , Decápodos/inmunología , Hemocitos/efectos de los fármacos , N-Formilmetionina Leucil-Fenilalanina/farmacología , Animales , Espacio Extracelular/química , Citometría de Flujo , Colorantes Fluorescentes/análisis , Granulocitos/efectos de los fármacos , Granulocitos/fisiología , Hemocitos/clasificación , Hemocitos/fisiología , Concentración de Iones de Hidrógeno , Microscopía Fluorescente , Oligopéptidos/farmacología , ATPasas de Translocación de Protón/metabolismo , Receptores de Formil Péptido , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/efectos de los fármacos , Receptores Inmunológicos/fisiología , Receptores de Péptidos/antagonistas & inhibidores , Receptores de Péptidos/efectos de los fármacos , Receptores de Péptidos/fisiología , Rodaminas/análisisRESUMEN
Virus-specific cytotoxic T lymphocytes (CTL) are frequently of the CD8+ surface phenotype, although CTL of the CD4+ surface phenotype have also been described. Published reports of CTL derived from peripheral blood mononuclear cells (PBMC) of individuals infected with human immunodeficiency virus type 1 (HIV-1) have described primarily cells of the CD8+ surface phenotype. However, CD4+ HIV-1 envelope-specific CTL have been reported after in vitro stimulation with HIV-1 envelope protein of peripheral blood cells obtained from HIV-1-seronegative donors, in peripheral blood cells after vaccination of HIV-1-seronegative persons with HIV-1 envelope proteins, and in cerebrospinal fluid cells of HIV-1-infected individuals. Recently, CD4+ HIV-1 gag-specific CTL were also reported. We now report a patient from whom we derived HIV-1 envelope-specific CTL cell lines of the CD4+ surface phenotype. Our cell culture technique did not employ exogenous viral antigenic stimulation, and may therefore yield cells that more closely reflect those in the underlying populations from which they were derived. These CTL did not appear to have the clear human leukocyte antigen (HLA) class II restriction pattern typically seen in CD4-expressing cells and were not functionally inhibited by anti-CD3 antibody. Further work will be required to define the role of CD4+ CTL in the pathogenesis of HIV-1 disease.
Asunto(s)
Antígenos CD4/análisis , Productos del Gen env/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Linfocitos T Citotóxicos/inmunología , Línea Celular , Epítopos , Antígenos HLA/inmunología , Humanos , Masculino , Fenotipo , Subgrupos de Linfocitos TRESUMEN
A rapid procedure has been developed for the analysis of phenylalanine in brain tissue. Perchloric acid extracts of brain tissue were made basic, and benzoyl chloride was added to derivatize the amine function. The aqueous layer was retained and made slightly acidic. To derivatize the carboxylic acid group, a solution of pentafluorobenzyl alcohol was added in the presence of the coupling agent dicyclohexylcarbodiimide and chloroform. After shaking for 15 min, the organic phase was retained and taken to dryness. The residue was taken up in toluene, washed, and an aliquot used for analysis on a gas chromatograph equipped with an automatic injector, a capillary column and an electron-capture detector. The procedure has been utilized for analysis of phenylalanine in brains of rats treated with vehicle or phenylalanine.
Asunto(s)
Química Encefálica , Encéfalo/metabolismo , Cromatografía de Gases/métodos , Fenilalanina/análisis , Animales , Masculino , Ratas , Ratas EndogámicasRESUMEN
Time- and dose-response studies were conducted to determine the effects of the antidepressant and antipanic drug phenelzine (a monoamine oxidase inhibitor) on whole brain levels of the aliphatic amino acid alanine. At a dose of phenelzine of 15 mg/kg IP, there was a significant increase in brain levels of alanine up to 16 hr after drug administration. Dose studies at 4 hr indicated that the alanine elevation after phenelzine was a dose-dependent effect.
Asunto(s)
Alanina/metabolismo , Química Encefálica/efectos de los fármacos , Fenelzina/farmacología , Aminoácidos/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Masculino , Ratas , Ratas EndogámicasRESUMEN
It has been suggested that inhibition of tryptophan (Trp) pyrrolase and a subsequent elevation of brain Trp may contribute to the actions of antidepressant drugs. In our laboratories, we have conducted a series of experiments measuring brain Trp levels in the rat after both acute and chronic administration of several monoamine oxidase (MAO) inhibitors. The drugs studied during the course of the long-term (28 day) experiments were phenelzine, N2-acetylphenelzine, tranylcypromine, 4-fluorotranylcypromine, 4-methoxytranylcypromine and (-)-deprenyl. High-pressure liquid chromatography with electrochemical detection was employed to measure Trp levels in brains of both MAO inhibitor- and vehicle-treated animals. No significant increases in brain Trp levels were observed as a consequence of MAO inhibitor treatment. Acute time-response (up to 24 h) and dose-response studies were conducted following the administration of phenelzine and tranylcypromine. Only after administration of high doses of these drugs was an elevation in brain Trp observed and the increase was relatively short-lived. These results suggest that elevation of brain Trp may be an important factor in the actions of MAO inhibitors only at high doses of these drugs.
Asunto(s)
Encéfalo/metabolismo , Inhibidores de la Monoaminooxidasa/farmacología , Triptófano/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Masculino , Concentración Osmolar , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Time- and dose-response studies were carried out on the effects of the monoamine oxidase-inhibiting antidepressant and antipanic drug phenelzine on GABA levels in rat whole brain. At a dose of 15 mg/kg i.p., phenelzine significantly elevated GABA levels at all time intervals studied (1, 2, 4, 8, 16, and 24 h). A further investigation indicated that this was a dose-dependent effect. The possible importance of GABA in the etiology and pharmacotherapy of depression and panic disorder is discussed.