RESUMEN
Analysis of SNPs for association, linkage, haplotype, and pharmacogenetic studies has led to a dramatic increase in the number and evolution of medium- to high-throughput genotyping technologies. This study introduces Plexor as a new method for medium-throughput (single SNP) genotyping. We compare this fluorescent-based chemistry for call rate, accuracy, affordability, throughput, and overall efficiency against two commonly used technologies. These include fluorescent-based TaqMan allelic discrimination for single SNP analysis (medium-throughput) and the homogenous MassEXTEND (hME) chemistry using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for multiple SNP analysis (high-throughput). Analysis of 11 SNPs, including all six possible nucleotide substitutions, showed Plexor to be highly comparable for both call rate (94.7%) and accuracy (99.2%) to the TaqMan (94.6% and 99.8%, respectively) and hME (91.9% and 98.1%, respectively) chemistries. We demonstrate that this novel method is an efficient, cost-effective alternative to TaqMan genotyping commonly used in diagnostic settings.
Asunto(s)
Análisis Mutacional de ADN/métodos , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Nucleótido Simple , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Genotipo , Humanos , Proteína Antagonista del Receptor de Interleucina 1/genética , Interleucina-2/genética , Interleucina-4/genética , Interleucina-6/genética , Interleucina-8/genética , Modelos Biológicos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Aromatic L-amino acid decarboxylase (AADC) is a relatively non specific enzyme involved in the biosynthesis of several classical neurotransmitters including dopamine and 5-hydroxytryptamine (5HT; serotonin). AADC does not catalyse the rate limiting step in either pathway, but is rate limiting in the synthesis of 2-phenylethylamine (2PE) which is a positive modulator of dopaminergic transmission and a candidate natural psychotogenic compound.1 We and others have proposed that polymorphism in AADC resulting in altered 2PE activity might contribute to the pathogenesis of psychosis. In order to test this hypothesis, we have used denaturing high performance liquid chromatography (DHPLC)3 to screen 3943 bases of the AADC gene and its promoter regions for variants that might affect protein structure or expression in 15 unrelated people with schizophrenia, and 15 unrelated people with bipolar disorder. Three polymorphisms were identified by DHPLC: a insertion/deletion polymorphism in the 5' UTR of the neuronal specific mRNA (g.-33-30delAGAG, bases 586-589 of GenBank M77828), a T>A variant in the non-neuronal exon 1 (g. -67T>A, GenBank M88070), and a G>A polymorphism within intron 8 (g. IVS8 +75G>A, GenBank M84598). Case-control analysis did not suggest that genetic polymorphism in the AADC gene is associated with liability for developing schizophrenia or bipolar disorder.
Asunto(s)
Descarboxilasas de Aminoácido-L-Aromático/genética , Trastorno Bipolar/enzimología , Trastorno Bipolar/genética , Esquizofrenia/enzimología , Esquizofrenia/genética , Secuencia de Bases , Humanos , Irlanda , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Valores de Referencia , Reino Unido , Población Blanca/genéticaRESUMEN
Neurotensin (NT) is an endogenous tridecapetide1 cleaved from a precursor proneurotensin/ proneuromedin protein. NT localises within dopaminergic neurones in the mesocortical, mesolimbic and nigrostriatal systems1-3 and it is now clear that NT can selectively modulate dopaminergic neurotransmission.2-9 These anatomical and functional connections have led to the hypothesis that NT dysfunction might contribute to the pathogenesis of neuropsychiatric disorders in which disordered dopaminergic neurotransmission is suspected, particularly schizophrenia.3 The latter hypothesis has been supported circumstantially by the observation that central administration of NT produces effects similar to those produced by the peripheral administration of atypical antipsychotics,10,11 and more directly by studies showing levels of NT in cerebral spinal fluid (CSF) is lower in schizophrenics than in controls.12,13 To allow such hypotheses to be tested, we used denaturing high performance liquid chromatography (DHPLC)14 to identify three sequence variants in the neurotensin gene (NTS) that might alter NT structure or expression. However, using a case-control study design and a novel genotyping system based upon a primer extension protocol and HPLC detection,15 we found no evidence to support the hypothesis that variation in the proneurotensin gene contributes to susceptibility to schizophrenia.
Asunto(s)
Variación Genética , Neurotensina/genética , Polimorfismo Genético , Precursores de Proteínas/genética , Esquizofrenia/genética , Alelos , Cartilla de ADN , Exones , Frecuencia de los Genes , Genotipo , Humanos , Neurotensina/líquido cefalorraquídeo , Reacción en Cadena de la Polimerasa , Esquizofrenia/líquido cefalorraquídeoRESUMEN
Denaturing high-performance liquid chromatography (DHPLC) is a novel high-capacity technique for detecting new mutations. We have evaluated the sensitivity and specificity of this method in a blind analysis of exon H of the factor IX gene and exon 16 of the neurofibromatosis type 1 gene. Under a single set of conditions for each exon, 55/55 individuals carrying 48 unique mutations were correctly identified as were 55/55 individuals with wildtype alleles. We conclude that DHPLC is a highly sensitive and specific method for mutation detection.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Análisis Mutacional de ADN/métodos , Desnaturalización de Ácido Nucleico , Exones/genética , Factor IX/genética , Tamización de Portadores Genéticos/métodos , Pruebas Genéticas/métodos , Humanos , Proteínas del Tejido Nervioso , Neurofibromina 1 , Proteínas/genética , Sensibilidad y EspecificidadRESUMEN
OBJECTIVE: The purpose of this study was to identify the specific expanded CAG/CTG trinucleotide repeat associated with bipolar disorder. METHOD: The study employed an efficient multistage approach for using a genomic CAG/CTG screening set. RESULTS: The authors found no evidence of expanded repeats at 43 polymorphic autosomal loci and seven X chromosomal loci. Secondary screening was pursued at the only locus that contained a large allele (37 repeats) in the primary screening. No association was found between allele size and diagnostic status. CONCLUSIONS: It is highly unlikely that expansions in repeat size at any of the 50 candidate trinucleotide repeat loci examined are responsible for the association between expanded CAG/ CTG repeats and bipolar disorder. However, although the authors prioritized the repeats that were a priori most likely to be involved, the study does not reject the more general hypothesis that expanded CAG/CTG repeats are implicated in the pathogenesis of bipolar disorder.
Asunto(s)
Trastorno Bipolar/genética , Repeticiones de Trinucleótidos/genética , Alelos , Trastorno Bipolar/etiología , ADN/genética , Marcadores Genéticos , Genotipo , Humanos , Modelos Genéticos , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Cromosoma XRESUMEN
The hypothesis that expanded trinucleotide repeats contribute to the pathogenesis of schizophrenia and bipolar disorder has been recently supported by three independent studies which have shown that patients with either disorder tend to have larger CAG/CTG repeat expansion detection products than controls. In an attempt to identify the specific expanded CAG/CTG locus or loci which are associated with schizophrenia and bipolar disorder, we determined the repeat size at CAG/CTG loci mapping to candidate regions for psychosis. In this study we report our findings from eight loci which map to chromosome 4. We conclude that these loci are unlikely candidates for CAG/CTG repeat expansion in schizophrenia and bipolar disorder.
Asunto(s)
Trastorno Bipolar/genética , Cromosomas Humanos Par 4 , Esquizofrenia/genética , Repeticiones de Trinucleótidos , Mapeo Cromosómico , HumanosRESUMEN
In a recent report, we demonstrated that intracerebral injections of the pleiotropic cytokine, ciliary neurotrophic factor (CNTF), into developing postnatal rats evoked a severe inflammatory response as determined by the appearance of reactive astrocytes and activated microglia. Considering the likely involvement of CNTF in the injury response, we felt it was important to further understand the role of CNTF in the developing rat CNS. In this study, we examined the responsiveness of other cell populations to intracerebral injections of CNTF. We report that CNTF increases glial fibrillary acidic protein (GFAP), while having no appreciable effect on the levels of other intermediate filaments including vimentin and neurofilament. Moreover, CNTF did not affect the expression of the mature oligodendrocyte gene, myelin basic protein. These results suggest that CNTF is highly specific in its regulation of GFAP. In our previous study, we showed CNTF to increase GFAP in a cell population that already exists in the CNS parenchyma. To determine the origin of the CNTF-induced reactive astrocytes, therefore, we have utilized a technique of combined in situ hybridization and immunocytochemistry. To examine the possibility that CNTF acts on oligodendrocyte precursors to give rise to reactive astrocytes, the platelet-derived growth factor alpha receptor (PDGF-alpha R) was utilized as a riboprobe in conjunction with an antibody to GFAP. Examination of CNTF-induced GFAP+ astrocytes revealed no colocalization with PDGF-alpha R mRNA. In contrast, when we utilized an S100 alpha antibody recognizing a calcium binding protein in immature astrocytes, we found colocalization of S100 alpha and GFAP mRNA. These data suggest that CNTF induces an upregulation of GFAP in immature S100 alpha + astrocytes. Examination of the CNTF-alpha receptor mRNA revealed no change in expression following CNTF treatment. Unexpectedly, however, the CNTF-induced astrogliotic response appears to be indirect since the CNTF-alpha receptor was solely expressed by neurons in the cytokine-treated animals.
Asunto(s)
Biomarcadores , Encéfalo/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/biosíntesis , Factores de Crecimiento Nervioso/farmacología , Proteínas del Tejido Nervioso/farmacología , Receptores de Factor de Crecimiento Nervioso/análisis , Proteínas S100/análisis , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Encéfalo/citología , Encéfalo/metabolismo , Factor Neurotrófico Ciliar , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Microglía/efectos de los fármacos , Microglía/metabolismo , Fenotipo , Ratas , Ratas Wistar , Receptor de Factor Neurotrófico Ciliar , Proteínas Recombinantes/farmacología , Células Madre/efectos de los fármacos , Células Madre/metabolismoRESUMEN
Studies of the transmission of schizophrenia in families with affected members in several generations have suggested that an expanded trinucleotide repeat mechanism may contribute to the genetic inheritance of this disorder. Using repeat expansion detection (RED), we and others have previously found that the distribution of CAG/CTG repeat size is larger in patients with schizophrenia than in controls. In an attempt to identify the specific expanded CAG/CTG locus or loci associated with schizophrenia, we have now used an approach based on a CAG/CTG PCR screening set combined with RED data. This has allowed us to minimize genotyping while excluding 43 polymorphic autosomal loci and 7 X-chromosomal loci from the screening set as candidates for expansion in schizophrenia with a very high degree of confidence.
Asunto(s)
Esquizofrenia/genética , Repeticiones de Trinucleótidos , Alelos , Femenino , Genotipo , Humanos , Masculino , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo Genético , Cromosoma XRESUMEN
In response to physical or chemical brain injury, the mammalian central nervous system (CNS) often reacts by evoking astrogliosis. The most prominent feature describing this state is an upregulation of glial fibrillary acidic protein (GFAP). The agent(s) responsible for inducing astrogliosis remains unclear; however, recent observations have shown cytokines may play a pivotal role. During CNS trauma, macrophages and lymphocytes infiltrate the CNS where they are thought to synthesize and secrete cytokines; moreover, activated microglia and reactive astrocytes are known to be capable of cytokine production. We are the first to report that an intracerebral injection of the pleiotropic cytokine, ciliary neurotrophic factor (CNTF), increases astrogliosis and the appearance of activated microglia in the neonatal rat. This response to CNTF was comparable to the response observed in animals receiving a well known pro-inflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha). Only a moderate increase was observed in the proliferative index of cytokine-injected animals; therefore, we conclude that GFAP is largely upregulated in a pre-existing GFAP negative cell population. Interestingly, coinjections of CNTF and TNF-alpha appeared to act synergistically. Coinjected animals displayed a wave of hypertrophied astrocytes reaching far into the contralateral hemisphere. No contralateral spreading of microglia was observed. This article clearly provides interesting information regarding the regulatory mechanisms that govern astrogliosis and discusses the probable relationship of reactive astrocytes to microglia.
Asunto(s)
Astrocitos/efectos de los fármacos , Sistema Nervioso Central/efectos de los fármacos , Gliosis/inducido químicamente , Microglía/efectos de los fármacos , Factores de Crecimiento Nervioso/farmacología , Proteínas del Tejido Nervioso/toxicidad , Animales , Astrocitos/patología , Sistema Nervioso Central/crecimiento & desarrollo , Factor Neurotrófico Ciliar , Femenino , Proteína Ácida Fibrilar de la Glía/análisis , Gliosis/patología , Masculino , Microglía/patología , Ratas , Ratas Wistar , Proteínas Recombinantes/toxicidad , Estimulación Química , Factor de Necrosis Tumoral alfa/toxicidad , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Nine proteins specified by Kunjin virus were labelled with [3H]lysine and digested with carboxypeptidase B which specifically cleaves carboxy-terminal Lys or Arg. The theoretical amount of [3H]lysine was released from the non-structural (ns) proteins NS2A, NS2B, NS3 and NS4B, which have a common carboxy terminus Lys-Arg deduced from cleavage sites established in the viral polyprotein by previous N-terminal amino acid analyses. This is a flavivirus consensus site, always followed by Gly, Ala or Ser. These results indicate that no truncations had occurred despite anomalous electrophoretic migrations of NS2A, NS2B and NS4B observed in some gel systems. No [3H]lysine was released from NS4A or NS5 which terminate in Ala and Leu, respectively, thus establishing that NS5 (observed Mr 98,000, theoretical Mr 103,600) was not cleaved post-translationally at any internal Lys-Arg site. Unexpectedly, [3H]lysine residues were apparently released from P14(C), the intracellular equivalent of virion C protein which terminates in Ala (adjacent to the established N-terminus of prM). However, a putative internal cleavage site (Lys-Arg decreases Gly) exists 18 residues upstream from the carboxy terminus of C, prior to the transmembrane spanning domain. Apparent internal cleavage in the cytosol at this site to produce P14(C) would expose [3H]lysine residues to carboxypeptidase B, and account for the previously observed differences in size and composition between C and P14(C).
Asunto(s)
Flavivirus/análisis , Proteínas del Núcleo Viral/metabolismo , Secuencia de Aminoácidos , Arginina/genética , Arginina/metabolismo , Carboxipeptidasa B , Carboxipeptidasas , Flavivirus/genética , Lisina/genética , Lisina/metabolismo , Datos de Secuencia Molecular , Conformación Proteica , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/aislamiento & purificaciónRESUMEN
A radiolabeled protein migrating in SDS-polyacrylamide gels near the core protein C of Kunjin virus-infected cells was isolated and subjected to N-terminal amino acid sequencing. Comparisons with the translation sequence deduced from the known nucleotide sequence identified a hydrophobic protein of 149 amino acids located in the polyprotein sequence between NS3 and NS4B, thus establishing its identity as NS4A with a calculated Mr 16,100. The cleavage sites identified at the N- and C-termini are KR decreases S....parallel....VAA decreases, both representing consensus sequences defined previously for Kunjin and other flaviviruses.
Asunto(s)
Cápside/análisis , Flavivirus/análisis , Proteínas del Núcleo Viral/análisis , Secuencia de Aminoácidos , Animales , ADN Viral/genética , Flavivirus/genética , Genes Virales , Datos de Secuencia Molecular , Peso Molecular , Células Vero , Proteínas no Estructurales ViralesRESUMEN
A Kunjin (KUN) virus cDNA sequence of 10664 nucleotides was obtained and it encoded a single open reading frame for 3433 amino acids. Partial N-terminal amino acid analyses of KUN virus-specified proteins identified the polyprotein cleavage sites and the definitive gene order. The gene order relative to that proposed for yellow fever (YF) virus is as follows: KUN 5'-C.GP20.E.GP44.P19.P10.P71.(?).P21.P98-3' YF 5'-C.prM.E.NS1.ns2a.ns2b.NS3.ns4a.ns4b. NS5-3'. The order of putative signal sequences and stop transfer sequences indicated that KUN NS1, NS2A and NS4B are probably cleaved in the lumen of the endoplasmic reticulum, at a consensus site Val-X-Ala decreases where X is an uncharged residue, and NS2B, NS3 and NS5 are cleaved in the cytosol at the site Lys-Arg decreases Gly. Comparisons with the complete amino acid sequences of YF and West Nile (WN) viruses showed that KUN virus shared 93% homology with WN virus, but only 46% homology with YF virus. Comparisons among individual gene products of six flaviviruses showed that E, NS1, NS3 and NS5 tended to be the most highly conserved, and C among the least conserved. Homologous cleavage sites were evident, and six domains in NS5, a total of over 170 residues, shared at least 85% homology. Comparisons with the KUN C to NS2B sequence defined a gradient of relationships of all gene products in decreasing order WN greater than Murray Valley greater than Japanese encephalitis greater than St Louis encephalitis viruses within this closely related serological complex. A non-coding 5' sequence (75 nucleotides) of KUN virus shared 95% homology with WN virus and a shorter imperfect match with Murray Valley encephalitis virus (15 of 18 nucleotides). The KUN non-coding 3' sequence of 290 nucleotides contained several short and imperfectly matched sequences, and shared 87% homology over the distal region of 191 nucleotides with the corresponding region of WN virus RNA.
Asunto(s)
Flavivirus/genética , Genes Virales , Proteínas Virales/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Codón/genética , Enzimas de Restricción del ADN , Virus de la Encefalitis Japonesa (Especie)/genética , Virus de la Encefalitis de San Luis/genética , Datos de Secuencia Molecular , ARN Viral/genética , Homología de Secuencia de Ácido Nucleico , Células Vero , Virus del Nilo Occidental/genética , Virus de la Fiebre Amarilla/genéticaRESUMEN
Partial N-terminal amino acid analyses of five radiolabelled non-structural (ns) proteins specified by Kunjin (KUN) virus provided positive identification of NS3, NS5 and three previously hypothetical ns proteins of flaviviruses, ns2a, ns2b and ns4b. Their correct gene order was obtained from their deduced amino acid sequences. Thus the gene order for KUN virus relative to that proposed for yellow fever (YF) virus was as follows: KUN 5'...GP44.P19.P10.P71.(?).P21.P98-3', YF 5'...NS1.ns2a.ns2b.NS3.ns4a.ns4b.NS5 -3'. The identity of GP44 as NS1 was assumed from the known nucleotide and deduced amino acid sequences; ns4a was not identified. The cleavage sites in the polyprotein for KUN NS2B, NS3 and NS5 were identical, Lys-Arg decreased Gly, similar in form to the sequence Arg-Arg decreased Ser defined at the cleavage sites of YF NS3 and NS5. A new consensus cleavage site for NS1, NS2A and NS4B in the form Val-X-Ala decreased, where X is any one of several uncharged amino acids, was found at corresponding sites homologous to those of KUN virus in all published flavivirus sequences (a total of 18 sites). NS1 and NS4B, but not NS2A, were preceded by a putative signal sequence.
Asunto(s)
Flavivirus/genética , Genes Virales , Proteínas Virales/genética , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Secuencia de Bases , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Señales de Clasificación de Proteína , Proteínas/análisis , Proteínas/genética , Proteínas/metabolismo , ARN Viral/genética , Células Vero , Proteínas Virales/análisisRESUMEN
The rates of inactivation of synthesis of individual virus-specified proteins by ultraviolet radiation provided an estimate of the target sizes of individual viral genes. In a control experiment with Semliki Forest virus, the genes for the structural proteins mapped in the known sequence 5' C-PE2-E1 3', and in accordance with initiation of translation from a single site on 26-S mRNA. Under the same conditions, the inactivation of synthesis of seven Kunjin virus-specified proteins also followed first-order kinetics, but the largest target size was equivalent to only about half the length of the genome. No gene sequence could be deduced using the premise that translation was initiated at a single site. However, genes for the major nonstructural proteins could be mapped in the order 5' P98-P71-P10 3' from a postulated ribosomal attachment site about midway along the RNA. The genes for the structural proteins C and E could then be mapped in a preceding sequence 5'...C-E-GP19-P21 3', with a vacant upstream region sufficient to code for at least one of two flavivirus genes not expressed in these experiments. The implications of the two postulated translational units are discussed in relation to previous data on translation strategy of flaviviruses.
Asunto(s)
Flavivirus/genética , Biosíntesis de Proteínas , Animales , Secuencia de Bases , Peso Molecular , Ribosomas/metabolismo , Rayos Ultravioleta , Proteínas Virales/biosíntesis , Proteínas Virales/efectos de la radiación , Proteínas Estructurales ViralesRESUMEN
The addition of a 1 mM N6, O2'-dibutyryl cyclic AMP to incubations of foetal calf articular cartilage for a 4 h period resulted in a 13 and a 15% decrease, respectively, in the rate of incorporation of [3H]glucosamine and [14C]glucose into glycosaminoglycans. Under the same conditions, a 41% increase in the rate of incorporation of [35S]sulphate into glycosaminoglycans was observed. Dibutyryl cyclic AMP had no effect on protein synthesis over a 4 h period or on the hydrodynamic size of the proteoglycan subunits or glycosaminoglycans synthesized by the cartilage. When the glycosaminoglycans were digested with chondroitin ABC lyase and the resulting disaccharides analysed on Dowex 1 (formate form), it was observed that dibutyryl cyclic AMP increased the degree of sulphation of the disaccharides. Furthermore, an over-sulphated disaccharide corresponding to chondroitin 4,6-disulphate was identified. It was concluded that dibutyryl cyclic AMP was stimulating the process of sulphation of glycosaminoglycans by cartilage.
Asunto(s)
Bucladesina/farmacología , Cartílago Articular/efectos de los fármacos , Proteoglicanos/metabolismo , Sulfatos/metabolismo , Animales , Cartílago Articular/embriología , Bovinos , Disacáridos/análisis , Glucosa/metabolismo , Glicina/metabolismo , Glicosaminoglicanos/metabolismoRESUMEN
The rate of proteoglycan synthesis by chondrocytes in vitro was depressed by either omitting L-glutamine from the incubation medium or by addition of proteoglycan subunit to the medium. The molecular size distribution on Sepharose 2B of the proteoglycan subunits synthesized by the chondrocytes under these conditions of reduced proteoglycan synthesis was found to be the same as those synthesized by the control cells. Likewise, the molecular size distribution on Sepharose 6B CL of the glycosaminoglycan chains synthesized by the depressed cells was found to be similar to that observed in untreated chondrocytes. This work demonstrates that, under conditions of reduced proteoglycan synthesis, fewer proteoglycan subunits are synthesized by chondrocytes and that the molecular size distribution of these macromolecules is similar to those synthesized by untreated cells.
Asunto(s)
Cartílago/metabolismo , Glicosaminoglicanos/análisis , Proteoglicanos/análisis , Animales , Células Cultivadas , Centrifugación por Gradiente de Densidad , Embrión de Pollo , Glutamina/farmacología , Glicosaminoglicanos/biosíntesis , Peso Molecular , Proteoglicanos/biosíntesisRESUMEN
The incorporation of [3H]glycine into acid-insoluble protein and of [3H]acetate into glycosaminoglycans by cultured chick chondrocytes was stimulated by the addition of L-glutamine to the incubation medium. The effect of exogenous L-glutamine on protein synthesis was studied further by examining changes in the sedimentation patterns on sucrose gradients of ribosomes isolated from chondrocytes incubated in presence and absence of L-glutamine. It was found that the absence of L-glutamine caused a disaggregation of polyribosomes that was revered by the addition of this amino acid to the culture medium. No detectable glutamine synthetase activity could be measured in avian articular cartilage. These results indicate that L-glutamine is an essential amino acid for cartilage in that an extracellular supply of this amino acid is required for the maintenance of protein and glycosaminoglycan synthesis. A dependence of L-glutamine was also demonstrated for other avain connective tissues.
Asunto(s)
Cartílago/metabolismo , Glutamina/metabolismo , Aminoácidos Esenciales/metabolismo , Animales , Células Cultivadas , Embrión de Pollo , Pollos , Tejido Conectivo/metabolismo , Glutamato-Amoníaco Ligasa/metabolismo , Glicosaminoglicanos/biosíntesis , Polirribosomas/metabolismo , Biosíntesis de ProteínasRESUMEN
The rate of synthesis of glycosaminoglycans by cartilage was shown to be dependent on an exogenous source of L-glutamine. In the absence of L-glutamine the tissue and cellular levels of this amino acid were rapidly depleted. The levels of nucleotide sugars and their precursors were measured after separation on Dowex 1 (formate form) in cartilage incubated with and without L-glutamine. It was found that the levels of N-acetylhexoamine 6-phosphate and UDP-N-acetylhexosamine were decreased by 27 and 40% respectively. This demonstrates that L-glutamine is required as the amido group donor in the synthesis of glucosamine 6-phosphate and that the decrease in glycosaminoglycan synthesis is due to the limitation in synthesis of UDP-N-acetylhexoamine.