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Posttranslational modifications (PTM) including glycosylation, phosphorylation, acetylation, methylation and ubiquitination dynamically alter the proteome. The evolutionarily conserved enzymes O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and O-GlcNAcase are responsible for the addition and removal, respectively, of the nutrient-sensitive PTM of protein serine and threonine residues with O-GlcNAc. Indeed, the O-GlcNAc modification acts at every step in the "central dogma" of molecular biology and alters signaling pathways leading to amplified or blunted biological responses. The cellular roles of OGT and the dynamic PTM O-GlcNAc have been clarified with recently developed chemical tools including high-throughput assays, structural and mechanistic studies and potent enzyme inhibitors. These evolving chemical tools complement genetic and biochemical approaches for exposing the underlying biological information conferred by O-GlcNAc cycling.
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Acetilglucosamina/metabolismo , Pruebas de Enzimas/métodos , Inhibidores Enzimáticos/farmacología , N-Acetilglucosaminiltransferasas/antagonistas & inhibidores , N-Acetilglucosaminiltransferasas/metabolismo , Acetilglucosamina/análogos & derivados , Acetilglucosamina/química , Animales , Humanos , N-Acetilglucosaminiltransferasas/química , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy. Mutations in the pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) gene have been found to cause X-linked dominant CMT type 6 (CMTX6). This study identified the p.R158H PDK3 mutation after screening 67 probable X-linked CMT families. The mutation fully segregated with the phenotype, and genotyping the family indicated the mutation arose on a different haplotype compared with the original Australian CMTX6 family. Results of bisulphite sequencing suggest that methylated deamination of a CpG dinucleotide may cause the recurrent p.R158H mutation. The frequency of the p.R158H PDK3 mutation in Koreans is very rare. Magnetic resonance imaging revealed fatty infiltration involving distal muscles in the lower extremities. In addition, fatty infiltrations were predominantly observed in the soleus muscles, with a lesser extent in tibialis anterior muscles. This differs from demyelinating CMT1A patients and is similar to axonal CMT2A patients. The clinical, neuroimaging, and electrophysiological findings from a second CMTX6 family with the p.R158H PDK3 mutation were similar to the axonal neuropathy reported in the Australian family.
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Enfermedad de Charcot-Marie-Tooth/genética , Proteínas Serina-Treonina Quinasas/genética , Adulto , Femenino , Genotipo , Humanos , Masculino , Mutación , Linaje , Fenotipo , Piruvato Deshidrogenasa Quinasa Acetil-TransferidoraRESUMEN
The dynamic glycosylation of serine/threonine residues on nucleocytoplasmic proteins with a single N-acetylglucosamine (O-GlcNAcylation) is critical for many important cellular processes. Cellular O-GlcNAc levels are highly regulated by two enzymes: O-GlcNAc transferase (OGT) is responsible for GlcNAc addition and O-GlcNAcase (OGA) is responsible for removal of the sugar. The lack of a rapid and simple method for monitoring OGT activity has impeded the efficient discovery of potent OGT inhibitors. In this study we describe a novel, single-well OGT enzyme assay that utilizes 6 × His-tagged substrates, a chemoselective chemical reaction, and unpurified OGT. The high-throughput Ni-NTA Plate OGT Assay will facilitate discovery of potent OGT-specific inhibitors on versatile substrates and the characterization of new enzyme variants.
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Pruebas de Enzimas , Inhibidores Enzimáticos/farmacología , N-Acetilglucosaminiltransferasas/antagonistas & inhibidores , N-Acetilglucosaminiltransferasas/metabolismo , Compuestos Organometálicos/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Humanos , Estructura Molecular , Níquel/química , Compuestos Organometálicos/química , Relación Estructura-Actividad , Especificidad por Sustrato/efectos de los fármacos , beta-N-Acetilhexosaminidasas/antagonistas & inhibidores , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
Microbial biohydrogenation of dietary poly-unsaturated fatty acids (PUFA) to saturated fatty acids (SFA) in the rumen results in the high ratio of SFA/PUFA in ruminant products, such as meat and milk. In vitro, Butyrivibrio proteoclasticus-related bacteria extensively biohydrogenate PUFA to SFA, yet their contribution in the rumen has not been confirmed. The aim of this study was to evaluate the role of Butyrivibrio proteoclasticus group bacteria in ruminal biohydrogenation and to assess the possible role of other bacteria. Fish oil at 0%, 1.5% and 3% dry matter intake was fed to eight Holstein × Friesian steers, in order to elicit changes in the extent of PUFA biohydrogenation. Fatty acid and B. proteoclasticus group 16S rRNA concentrations in rumen digesta were determined. Correlation between digesta 18:0 concentration and B. proteoclasticus group 16S rRNA concentration was low. Terminal restriction fragment length polymorphism and denaturing gradient gel electrophoresis (DGGE) coupled with multivariate statistics revealed that many terminal restriction fragments (T-RFs) and DGGE bands were linked to cis-9, trans-11 conjugated linoleic acid (CLA), 18:1 trans-11 and 18:0 ruminal concentrations. MiCA T-RF predictive identification software showed that these linked T-RFs were likely to originate from as yet uncultured bacteria classified as Prevotella, Lachnospiraceae incertae sedis, and unclassified Bacteroidales, Clostridiales and Ruminococcaceae. Sequencing of linked DGGE bands also revealed that as yet uncultured bacteria classified as Prevotella, Anaerovoax (member of the Lachnospiraceae incertae sedis family), and unclassified Clostridiales and Ruminococcaceae may play a role in biohydrogenation.
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Bacteroidetes/genética , Butyrivibrio/genética , Prevotella/genética , Animales , Bacteroidetes/clasificación , Secuencia de Bases , Butyrivibrio/clasificación , Electroforesis en Gel de Gradiente Desnaturalizante , Ácidos Grasos/metabolismo , Ácidos Grasos Insaturados/metabolismo , Aceites de Pescado/metabolismo , Hidrogenación , Ácidos Linoleicos Conjugados/metabolismo , Datos de Secuencia Molecular , Filogenia , Prevotella/clasificación , Rumen/microbiologíaRESUMEN
The concepts of both protein glycosylation and cellular signaling have been influenced by O-linked-ß-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) on the hydroxyl group of serine or threonine residues. Unlike conventional protein glycosylation, O-GlcNAcylation is localized in the nucleocytoplasm and its cycling is a dynamic process that operates in a highly regulated manner in response to various cellular stimuli. These characteristics render O-GlcNAcylation similar to phosphorylation, which has long been considered a major regulatory mechanism in cellular processes. Various efficient chemical approaches and novel mass spectrometric (MS) techniques have uncovered numerous O-GlcNAcylated proteins that are involved in the regulation of many important cellular events. These discoveries imply that O-GlcNAcylation is another major regulator of cellular signaling. However, in contrast to phosphorylation, which is regulated by hundreds of kinases and phosphatases, dynamic O-GlcNAc cycling is catalyzed by only two enzymes: uridine diphospho-N-acetyl-glucosamine:polypeptide ß-N-acetylglucosaminyl transferase (OGT) and ß-D-N-acetylglucosaminidase (OGA). Many useful chemical tools have recently been used to greatly expand our understanding of the extensive crosstalk between O-GlcNAcylation and phosphorylation and hence of cellular signaling. This review article describes the various useful chemical tools that have been developed and discusses the considerable advances made in the O-GlcNAc field.
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Acetilglucosamina/química , Cromatografía de Afinidad , Espectrometría de Masas , ProteómicaRESUMEN
Lactate accumulation is a hallmark of solid cancers and is linked to the immune suppressive phenotypes of tumor-infiltrating immune cells. We report herein that interleukin-4 (IL-4)induced M0 â M2 macrophage polarization is accompanied by interchangeable glucose- or lactate-dependent tricarboxylic acid (TCA) cycle metabolism that directly drives histone acetylation, M2 gene transcription, and functional immune suppression. Lactate-dependent M0 â M2 polarization requires both mitochondrial pyruvate uptake and adenosine triphosphatecitrate lyase (ACLY) enzymatic activity. Notably, exogenous acetate rescues defective M2 polarization and histone acetylation following mitochondrial pyruvate carrier 1 (MPC1) inhibition or ACLY deficiency. Lastly, M2 macrophagedependent tumor progression is impaired by conditional macrophage ACLY deficiency, further supporting a dominant role for glucose/lactate mitochondrial metabolism and histone acetylation in driving immune evasion. This work adds to our understanding of how mitochondrial metabolism affects macrophage functional phenotypes and identifies a unique tumor microenvironment (TME)driven metabolic-epigenetic link in M2 macrophages.
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INTRODUCTION: While identifying Alzheimer's Disease (AD) in its early stages is crucial, traditional neuropsychological tests tend to lack sensitivity and specificity for its diagnosis. Neuropsychological studies have reported visual processing deficits of AD, and event-related potentials (ERPs) are suitable to investigate pre-attentive processing with superior temporal resolution. OBJECTIVE: This study aimed to investigate visual attentional characteristics of adults with AD, from pre-attentive to attentive processing, using a visual oddball task and ERPs. METHODS: Cognitively normal elderly controls (CN) and patients with probable AD (AD) were recruited. Participants performed a three-stimulus visual oddball task and were asked to press a designated button in response to the target stimuli. The amplitudes of 4 ERPs were analyzed. Mismatchnegativity (vMMN) was analyzed around the parieto-occipital and temporo-occipital regions. P3a was analyzed around the fronto-central regions, whereas P3b was analyzed around the centro-parietal regions. RESULTS: Late vMMN amplitudes of the AD group were significantly smaller than those of the CN group, while early vMMN amplitudes were comparable. Compared to the CN group, P3a amplitudes of the AD group were significantly smaller for the infrequent deviant stimuli, but the amplitudes for the standard stimuli were comparable. Lastly, the AD group had significantly smaller P3b amplitudes for the target stimuli compared to the CN group. CONCLUSION: Our findings imply that AD patients exhibit pre-attentive visual processing deficits, known to affect later higher-order brain functions. In a clinical setting, the visual oddball paradigm could be used to provide helpful diagnostic information since pre-attentive ERPs can be induced by passive exposure to infrequent stimuli.
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Enfermedad de Alzheimer/diagnóstico , Atención/fisiología , Potenciales Evocados Visuales/fisiología , Síntomas Prodrómicos , Percepción Visual/fisiología , Anciano , Cognición , Femenino , Humanos , Masculino , Pruebas Neuropsicológicas/estadística & datos numéricos , Lóbulo ParietalRESUMEN
BACKGROUND: Contusive spinal cord injury is complicated by a delayed loss of oligodendrocytes, resulting in chronic progressive demyelination. Therefore, transplantation strategies to provide oligodendrocyte lineage cells and to enhance the extent of myelination appear to be justified for spinal cord repair. The present study investigated whether transplantation of human neural stem cells (NSCs) genetically modified to express Olig2 transcription factor, an essential regulator of oligodendrocyte development, can improve locomotor recovery and enhance myelination in a rat contusive spinal cord injury model. RESULTS: HB1.F3 (F3) immortalized human NSC line was transduced with a retroviral vector encoding Olig2, an essential regulator of oligodendrocyte development. Overexpression of Olig2 in human NSCs (F3.Olig2) induced activation of NKX2.2 and directed differentiation of NSCs into oligodendrocyte lineage cells in vitro. Introduction of Olig2 conferred higher proliferative activity, and a much larger number of F3.Olig2 NSCs were detected by 7 weeks after transplantation into contused spinal cord than that of parental F3 NSCs. F3.Olig2 NSCs exhibited frequent migration towards the white matter, whereas F3 NSCs were mostly confined to the gray matter or around the lesion cavities. Most of F3.Olig2 NSCs occupying the spared white matter differentiated into mature oligodendrocytes. Transplantation of F3.Olig2 NSCs increased the volume of spared white matter and reduced the cavity volume. Moreover, F3.Olig2 grafts significantly increased the thickness of myelin sheath around the axons in the spared white matter. Finally, animals with F3.Olig2 grafts showed an improvement in the quality of hindlimbs locomotion. CONCLUSION: Transplantation of NSCs genetically modified to differentiate into an oligodendrocytic lineage may be an effective strategy to improve functional outcomes following spinal cord trauma. The present study suggests that molecular factors governing cell fate decisions can be manipulated to enhance reparative potential of the cell-based therapy.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Células Madre Fetales/trasplante , Vaina de Mielina/patología , Proteínas del Tejido Nervioso/genética , Traumatismos de la Médula Espinal/terapia , Médula Espinal/patología , Análisis de Varianza , Animales , Recuento de Células , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Femenino , Vectores Genéticos/genética , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Microscopía de Contraste de Fase , Actividad Motora , Fibras Nerviosas Mielínicas/patología , Neuronas/citología , Neuronas/trasplante , Proteínas Nucleares , Factor de Transcripción 2 de los Oligodendrocitos , Ratas , Ratas Sprague-Dawley , Recuperación de la Función , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatología , Telencéfalo/citología , Vértebras Torácicas , Factores de Transcripción , TransfecciónRESUMEN
Ruminant fat is rich in SFA, partly due to the biohydrogenation of dietary PUFA to SFA in the rumen. This process can be inhibited by the dietary inclusion of fish oil. The only bacteria isolated from the rumen capable of converting PUFA to SFA are closely related to Clostridium proteoclasticum. The aim of this study was to investigate if a correlation could be found in vivo between dietary fish oil inclusions and the composition of the ruminal bacterial community and specifically of C. proteoclasticum. Six Hereford x Friesian steers, prepared with ruminal and duodenal cannulae, received grass silage plus 1 of 3 concentrates resulting in total dietary fish oil contents of 0, 1, or 3% of dry matter. A dual flow marker technique was employed to estimate the relative flow of fatty acids. Steers fed the 3% fish oil diet had 100% increases in trans 18:1 flow, whereas 18:0 flow declined to 39% of steers fed the control diet. 16S ribosomal RNA-based denaturing gradient gel electrophoresis profiles obtained from ruminal digesta showed major changes in the bacterial community within steers fed the 3% fish oil diet. Quantitative PCR indicated only a weak relation between numbers of C. proteoclasticum and 18:0 flow between treatments and in individual steers (P < 0.05, but the percentage variance accounted for only 22.8) and did not provide unambiguous evidence that numbers of C. proteoclasticum in the rumen dictate the ratios of SFA:PUFA available for absorption by the animal. Understanding which microbes biohydrogenate PUFA in the rumen is key to developing novel strategies to improve the quality of ruminant products.
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Bacterias/metabolismo , Bovinos/metabolismo , Duodeno/metabolismo , Ácidos Grasos Insaturados/metabolismo , Aceites de Pescado/administración & dosificación , Rumen/microbiología , Animales , Bacterias/genética , Clostridium/genética , Clostridium/metabolismo , ADN Bacteriano/análisis , Dieta , Ácidos Grasos/administración & dosificación , Ácidos Grasos/metabolismo , Fermentación , Hidrogenación , Lolium , Masculino , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/genética , EnsilajeRESUMEN
Toxigenic Vibrio cholerae strains arise upon infection and integration of the lysogenic cholera toxin phage, the CTX phage, into bacterial chromosomes. The V. cholerae serogroup O1 strains identified to date can be broadly categorized into three main groups: the classical biotype strains, which harbor CTX-cla; the prototype El Tor strains (Wave 1 strains), which harbor CTX-1; and the atypical El Tor strains, which harbor CTX-2 (Wave 2 strains) or CTX-3~6 (Wave 3 strains). The efficiencies of replication and transmission of CTX phages are similar, suggesting the possibility of existence of more diverse bacterial strains harboring various CTX phages and their arrays in nature. In this study, a set of V. cholerae strains was constructed by the chromosomal integration of CTX phages into strains that already harbored CTX phages or those that did not harbor any CTX phage or RS1 element. Strains containing repeats of the same kind of CTX phage, strains containing the same kind of CTX phage in each chromosome, strains containing alternative CTX phages in one chromosome, or containing different CTX phages in each chromosome have been constructed. Thus, strains with any CTX array can be designed and constructed. Moreover, the strains described in this study contained the toxT-139F allele, which enhances the expression of TcpA and cholera toxin. These characteristics are considered to be important for cholera vaccine development. Once their capacity to provoke immunity in human against V. cholerae infection is evaluated, some of the generated strains could be developed further to yield cholera vaccine strains.
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O-GlcNAcase (OGA) promotes O-GlcNAc removal, and thereby plays a key role in O-GlcNAc metabolism, a feature of a variety of vital cellular processes. Two splice transcripts of human OGA encode "long OGA", which contains a distinct N-terminal O-GlcNAcase domain and a C-terminal histoneacetylferase (HAT) domain, and "short OGA", which lacks the HAT domain. The functional roles of long OGA are only beginning to be unraveled, and the characteristics of short OGA remain almost unknown. We find that short OGA, which possesses O-GlcNAcase catalysis machinery like that of long OGA, exhibits comparative resistance to previously described potent inhibitors of long OGA and lysosomal hexosaminidases, including PUGNAc and NAG-thiazoline, suggesting a role for the HAT domain in O-GlcNAcase catalysis. We also find that alpha-GlcNAc thiolsulfonate (2) is the most potent inhibitor of short OGA yet described (Ki = 10 microM), and exhibits some degree of selectivity versus long OGA and lysosomal hexosaminidases. In contrast to its mode of inhibition of short OGA, 2 acts as a irreversible inhibitor of long OGA by covalently modifying the enzyme as an S-GlcNAc derivative. Covalent attachment of GlcNAc to the HAT domain of long OGA dramatically changes its properties with respect to enzymatic activity and caspase-3 cleavage.
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Inhibidores Enzimáticos/farmacología , Tioglucósidos/farmacología , Compuestos de Tosilo/farmacología , beta-N-Acetilhexosaminidasas/antagonistas & inhibidores , beta-N-Acetilhexosaminidasas/metabolismo , Inhibidores Enzimáticos/química , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Estructura Molecular , Tioglucósidos/química , Compuestos de Tosilo/químicaRESUMEN
The pharmacokinetics (including distribution in the gastrointestinal tract) of 7-carboxymethyloxy-3',4',5-trimethoxy flavone (DA-6034) has been investigated in several mouse and rat models of chemically-induced inflammatory bowel disease (IBD). In the female ICR mouse model, IBD was induced by dextran sulfate and the mice administered 30 mg kg(-1) DA-6034 intravenously or orally. In the male SJL mouse model of IBD induced by oxazolone, 30 mg kg(-1) DA-6034 was administered orally. In the male Sprague-Dawley rat model of IBD induced by trinitrobenzene sulfonic acid (TNBS), 10 mg kg(-1) DA-6034 was administered intravenously and orally. After intravenous administration, the total area under the plasma concentration-time curve from time zero to the last measured time, t, in plasma (AUC(0-t)) values were comparable between control and dextran sulfate-induced IBD mice, and between control and TNBS-induced rats. This suggested that the disposition of DA-6034 was not affected considerably by dextran sulfate in mice and TNBS in rats. However, after oral administration in mice and rats with IBD, the AUC(0-t) values were greater compared with the respective controls. This could have been due to an increase (slow) in the gastrointestinal transit time (in IBD mice and rats, the percentages of the oral dose recovered from the rinsing fluid of the small intestine and large intestine as unchanged drug were greater and smaller, respectively), and an increase in intestinal permeability.
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Antiinflamatorios/farmacocinética , Flavonoides/farmacocinética , Enfermedades Inflamatorias del Intestino/metabolismo , Administración Oral , Animales , Antiinflamatorios/sangre , Sulfato de Dextran/farmacología , Femenino , Flavonoides/sangre , Enfermedades Inflamatorias del Intestino/inducido químicamente , Inyecciones Intravenosas , Intestino Delgado/efectos de los fármacos , Intestino Delgado/patología , Masculino , Ratones , Ratones Endogámicos ICR , Oxazolona/farmacología , Ratas , Ratas Sprague-Dawley , Ácido Trinitrobencenosulfónico/farmacologíaRESUMEN
BACKGROUND: Patients undergoing laparoscopic gynecological surgery are at high risk of postoperative nausea and vomiting (PONV). We compared the antiemetic efficacy of ondansetron plus betahistine with that of ondansetron alone in this patient population. METHODS: In this randomized, double-blinded study, 168 patients were randomly allocated to receive placebo (O group) or betahistine 18 mg (OB group) orally 3 hours before surgery and 24 hours thereafter. In both groups, ondansetron 4 mg was administered at the end of surgery and 8 mg were added to an intravenous patient-controlled analgesia (IV-PCA) fentanyl solution. The primary outcome was complete response (no PONV and no rescue antiemetics) during the first 48 hours after surgery. The severity of nausea, pain score, and adverse events were assessed. RESULTS: The incidence of complete response was significantly higher in OB group than in O group (69% vs. 46%, P=0.004). The severity of nausea was lower in OB group than in O group during 30 minutes to 6 hours and 6 to 24 hours after surgery (P=0.001 and P<0.001). Pain score was similar between the groups. The incidence of dizziness was lower in OB group than in O group (13% vs. 40%, P < 0.001). Six patients (7%) in OB group and 15 patients (18%) in O group required early IV-PCA discontinuation, primarily because of PONV and/or dizziness (P=0.038). CONCLUSIONS: Compared to ondansetron alone, ondansetron plus betahistine was more effective to prevent PONV and dizziness in high-risk patients undergoing laparoscopic gynecological surgery.
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Antieméticos/uso terapéutico , Betahistina/uso terapéutico , Laparoscopía/efectos adversos , Ondansetrón/uso terapéutico , Náusea y Vómito Posoperatorios/tratamiento farmacológico , Adulto , Anciano , Antieméticos/efectos adversos , Betahistina/efectos adversos , Método Doble Ciego , Esquema de Medicación , Quimioterapia Combinada/métodos , Femenino , Procedimientos Quirúrgicos Ginecológicos , Humanos , Persona de Mediana Edad , Ondansetrón/efectos adversos , Dimensión del Dolor , Estudios Prospectivos , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
Understanding the relationship between ingested plant material and the attached microbiome is essential for developing methodologies to improve ruminant nutrient use efficiency. We have previously shown that perennial ryegrass (PRG) rumen bacterial colonization events follow a primary (up to 4 h) and secondary (after 4 h) pattern based on the differences in diversity of the attached bacteria. In this study, we investigated temporal niche specialization of primary and secondary populations of attached rumen microbiota using metagenomic shotgun sequencing as well as monitoring changes in the plant chemistry using mid-infrared spectroscopy (FT-IR). Metagenomic Rapid Annotation using Subsystem Technology (MG-RAST) taxonomical analysis of shotgun metagenomic sequences showed that the genera Butyrivibrio, Clostridium, Eubacterium, Prevotella, and Selenomonas dominated the attached microbiome irrespective of time. MG-RAST also showed that Acidaminococcus, Bacillus, Butyrivibrio, and Prevotella rDNA increased in read abundance during secondary colonization, whilst Blautia decreased in read abundance. MG-RAST Clusters of Orthologous Groups (COG) functional analysis also showed that the primary function of the attached microbiome was categorized broadly within "metabolism;" predominantly amino acid, carbohydrate, and lipid metabolism and transport. Most sequence read abundances (51.6, 43.8, and 50.0% of COG families pertaining to amino acid, carbohydrate and lipid metabolism, respectively) within these categories were higher in abundance during secondary colonization. Kyoto encyclopedia of genes and genomes (KEGG) pathways analysis confirmed that the PRG-attached microbiota present at 1 and 4 h of rumen incubation possess a similar functional capacity, with only a few pathways being uniquely found in only one incubation time point only. FT-IR data for the plant residues also showed that the main changes in plant chemistry between primary and secondary colonization was due to increased carbohydrate, amino acid, and lipid metabolism. This study confirmed primary and secondary colonization events and supported the hypothesis that functional changes occurred as a consequence of taxonomical changes. Sequences within the carbohydrate metabolism COG families contained only 3.2% of cellulose activities, on average across both incubation times (1 and 4 h), suggesting that degradation of the plant cell walls may be a key rate-limiting factor in ensuring the bioavailability of intra-plant nutrients in a timely manner to the microbes and ultimately the animal. This suggests that a future focus for improving ruminant nutrient use efficiency should be altering the recalcitrant plant cell wall components and/or improving the cellulolytic capacity of the rumen microbiota.
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We isolated a novel cytokeratin gene of zebrafish (Danio rerio), DAPK-1 closely related to other vertebrate type I cytokeratin genes. Zygotic transcription starts at the sphere stage. After the mid-blastula stage, DAPK-1 is expressed in all surface cells, notably in those of the outer enveloping layer. DAPK-1 messages are also present specifically during the segmentation, pharyngula, and hatching periods. In particular, after 24 h post-fertilization, its expression is restricted to the developing eye region, otic vesicle, pectoral fin, dorsal aorta, and pronephric duct. In the mindbomb mutant embryo that has defects in the dorsal aorta development, DAPK-1 transcripts are not detected in the dorsal aorta and pronephric duct. The characteristic expression pattern of DAPK-1 may facilitate more detailed studies related to the morphogenesis of dorsal aorta and pronephric duct.
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Aorta/metabolismo , Embrión no Mamífero/metabolismo , Queratinas/genética , Riñón/metabolismo , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Aorta/embriología , ADN Complementario/química , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Riñón/embriología , Datos de Secuencia Molecular , Mutación , Filogenia , Alineación de Secuencia , Análisis de Secuencia de ADN , Pez Cebra/embriologíaRESUMEN
Effects of cysteine on the pharmacokinetics of clarithromycin were investigated after intravenous administration of the drug at a dose of 20 mg/kg to control rats (4-week fed on 23% casein diet) and rats with PCM (protein-calorie malnutrition, 4-week fed on 5% casein diet) and PCMC (PCM treated with 250 mg/kg for oral cysteine twice daily during the fourth week). Clarithromycin has been reported to be metabolized via hepatic microsomal cytochrome P450 (CYP) 3A4 to 14-hydroxyclarithromycin (primary metabolite of clarithromycin) in human subjects. It has also been reported that in rats with PCM, CYP3A23 level decreased to 40-50% of control level, but decreased CYP3A23 level in rats with PCM completely returned to control level by oral cysteine supplementation (rats with PCMC). Human CYP3A4 and rat CYP3A23 proteins have 73% homology. In rats with PCM, the area under the plasma concentration-time curve from time zero to time infinity, AUC (567, 853 and 558 microg min/ml for control rats and rats with PCM and PCMC, respectively) and percentage of clarithromycin remaining after incubation with liver homogenate (69.6, 83.9 and 71.7%) were significantly greater than those in control rats and rats with PCMC. Moreover, in rats with PCM, the total body clearance, CL (35.3, 23.4 and 35.8 ml/min/kg), nonrenal clearance, CL(NR) (21.3, 15.2 and 24.1 ml/min/kg) and maximum velocity for the disappearance of clarithromycin after incubation with hepatic microsomal fraction, V(max) (351, 211 and 372 pmol/min/mg protein) were significantly slower than those in control rats and rats with PCMC. However, above mentioned each parameter was not significantly different between control rats and rats with PCMC. The above data suggested that metabolism of clarithromycin decreased significantly in rats with PCM as compared to control due to significantly decreased level of CYP3A23 in the rats. By cysteine supplementation (rats with PCMC), some pharmacokinetic parameters of clarithromycin (AUC, CL, CL(NR) and V(max)) were restored fully to control levels because CYP3A23 level was completely returned to control level in rats with PCMC.
Asunto(s)
Claritromicina/farmacocinética , Cisteína/farmacología , Desnutrición Proteico-Calórica/metabolismo , Administración Oral , Animales , Área Bajo la Curva , Cromatografía Líquida de Alta Presión , Claritromicina/sangre , Claritromicina/orina , Cisteína/administración & dosificación , Modelos Animales de Enfermedad , Inyecciones Intravenosas , Masculino , Microsomas Hepáticos/metabolismo , Estado Nutricional/efectos de los fármacos , Desnutrición Proteico-Calórica/tratamiento farmacológico , Ratas , Ratas Sprague-Dawley , Distribución TisularRESUMEN
Thioacetamide has been known to cause immune suppression. The object of the present study is to investigate the role of metabolic activation by flavin-containing monooxygenases (FMO) in thioacetamide-induced immune response. To determine whether the metabolites of thioacetamide produced by FMO causes the immunosuppression, methimazole, an FMO inhibitor, was used to block the FMO pathway. Antibody-forming cell (AFC) response measured in BALB/c mice sensitized with sheep red blood cells (SRBCs) was compared between the groups treated with thioacetamide in the presence or absence of methimazole pretreatment. The pretreatment abolished the decrease in AFC number observed in the mice treated with thioacetamide alone. In addition, when spleen cells isolated from untreated mice were exposed to thioacetamide with a drug-metabolizing system, liver microsome and NADPH, for 4 h in vitro prior to the stimulation with mitogens, such as lipopolysaccharide (LPS) or concanavalin A (Con A), spleen cell proliferation was also decreased. The inhibitory effect of thioacetamide on cell growth was not detectable without the liver microsome. Moreover, the thioacetamide-suppressed proliferation of spleen cells in the presence of the metabolic activation system was prevented when coincubated with either SKF-525A, a cytochrome P450 (P450) inhibitor, or methimazole. We also found that the level of interleukin-2 (IL-2) in the culture supernatant was decreased by thioacetamide treatment and that the decrease of IL-2 level can be prevented by either SKF-525A or methimazole coincubation. Since IL-2 is one of the responsible factors that determine the proliferation level of lymphocytes, the change of IL-2 production was consistent with that of lymphoproliferation. In conclusion, thioacetamide-induced immunosuppression was, at least in part, due to the metabolites produced by FMO as well as by P450.
Asunto(s)
Inmunosupresores/toxicidad , Monoaminooxidasa/metabolismo , Tioacetamida/toxicidad , Animales , División Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Eritrocitos/inmunología , Femenino , Inmunoglobulina M/biosíntesis , Interleucina-2/biosíntesis , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Metimazol/farmacología , Ratones , Ratones Endogámicos BALB C , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , Proadifeno/farmacología , Ovinos/inmunología , Bazo/citología , Bazo/efectos de los fármacosRESUMEN
The effects of glucose on CYP2E1 expression in rats with acute renal failure induced by uranyl nitrate (U-ARF) have been reported. CYP2E1 was significantly induced (2.3-fold) in rats with U-ARF compared with that in control rats. In contrast, CYP2E1 expression was significantly decreased in rats with U-ARF supplied with glucose (dissolved in tap water to make 10%, w/v) in their drinking water for 5 days (U-ARFG) compared with that in rats with U-ARF. However, CYP2E1 in rats with U-ARFG was significantly greater than that in control rats. Chlorzoxazone (CZX) primarily undergoes hydroxylation, catalyzed mainly by CYP2E1, to form 6-hydroxychlorzoxazone (OH-CZX) rats. Hence, it could be expected that in rats with U-ARFG, formation of OH-CZX could significantly decrease and increase compared with those in rats with U-ARF and control rats, respectively. This expectation is proven by the following results of a study of intravenous administration of CZX at a dose 20 mg/kg to control rats and rats with U-ARF and U-ARFG. First, the total area under the plasma concentration-time curve from time zero to 8 h (AUC(0-8 h)) of OH-CZX in rats with U-ARFG (8730 microg x min/mL) was significantly greater than that in control rats (414 microg x min/mL) and significantly smaller than that in rats with U-ARF (11500 microg x min/mL). Second, the AUC(0-8 h, OH-CZX)/AUC(CZX) ratio in rats with U-ARFG (10.0) was significantly greater than that in control rats (0.252) and significantly smaller than that in rats with U-ARF (17.5). Finally, the in vitro intrinsic OH-CZX formation clearance (CL(int)) in rats with U-ARFG (27.9 mL/min/mg protein) was significantly slower than that in rats with U-ARF (36.7 mL/min/mg protein) and significantly faster than that in control rats (17.7 mL/min/mg protein).
Asunto(s)
Lesión Renal Aguda/metabolismo , Clorzoxazona/administración & dosificación , Clorzoxazona/farmacocinética , Glucosa/farmacología , Nitrato de Uranilo/toxicidad , Lesión Renal Aguda/inducido químicamente , Animales , Interacciones Farmacológicas/fisiología , Infusiones Intravenosas , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Dose-independent pharmacokinetic parameters of SR-4668 were observed after intravenous (i.v.) administrations at doses of 25, 50, and 75 mg/kg and oral administrations at doses of 50, 100, and 150 mg/kg to rats. The hepatic, gastric, and intestinal first-pass effects of SR-4668 were also measured after i.v., intraportal (i.p.), intraduodenal (i.d.), and intragastric (i.g.) administrations at a dose of 50 mg/kg to rats. Although a considerable amount of orally administered SR-4668 was absorbed, the F was low--only 33%. This indicates considerable first-pass (gastric, intestinal, and/or hepatic) effects of SR-4668 in rats. After i.v. administrations, the total body clearances of SR-4668 were considerably slower than the reported cardiac output in rats, suggesting that the first-pass effects of SR-4668 in the lung and heart could be negligible, if any, in rats. The AUCs of SR-4668 were comparable between i.v. and i.p. administrations, suggesting that the hepatic first-pass effect of SR-4668 was not considerable in rats. The AUCs were also comparable between i.d. and i.g. administrations, suggesting that gastric first-pass effect was almost negligible in rats. However, the AUC after an i.d. administration was significantly smaller (approximately 55% decrease) than that after an i.p. administration, suggesting that the intestinal first-pass effect was approximately 55% of oral dose. The rests of the orally administered dose could be mainly due to degradation of SR-4668 in gastric juices; 77.3-95.6% of the spiked amount of SR-4668 were recovered after 4-h incubation in five human gastric juices. The above data suggested that the low F of SR-4668 could be mainly due to considerable intestinal first-pass effect in rats.
Asunto(s)
Mucosa Intestinal/metabolismo , Fármacos del Sistema Nervioso Periférico/farmacocinética , Tiofenos/farmacocinética , Administración Oral , Animales , Área Bajo la Curva , Bilis/metabolismo , Cromatografía Líquida de Alta Presión , Neuropatías Diabéticas/tratamiento farmacológico , Relación Dosis-Respuesta a Droga , Estabilidad de Medicamentos , Jugo Gástrico/metabolismo , Mucosa Gástrica/metabolismo , Semivida , Inyecciones Intravenosas , Hígado/metabolismo , Masculino , Fármacos del Sistema Nervioso Periférico/administración & dosificación , Fármacos del Sistema Nervioso Periférico/farmacología , Ratas , Ratas Sprague-Dawley , Tiofenos/administración & dosificación , Tiofenos/farmacología , Factores de Tiempo , Distribución TisularRESUMEN
The purpose of this study was to report dose-independent pharmacokinetics of KR-31543, a new neuroprotective agent for ischemia-reperfusion damage, after intravenous (iv) and oral (po) administration and first-pass effects after iv, intraportal, intragastric, and intraduodenal administration in rats. After iv (10, 20, and 50 mg/kg) and oral (10, 20, and 50 mg/kg) administration, the pharmacokinetic parameters of KR-31543 were dose independent. The extent of absolute oral bioavailability (F) was 27.4% at 20 mg/kg. Considering the amount of unabsorbed KR-31543 from the gastrointestinal tract at 24 h (4.11%), the low F value could be due to the hepatic, gastric, and/or intestinal first-pass effects. After iv administration of three doses, the total body clearances were considerably slower than the reported cardiac output in rats, suggesting almost negligible first-pass effect in the heart and lung in rats. The areas under the plasma concentration-time curves from time zero to time infinity (AUCs) were not significantly different between intragastric and intraduodenal administration of KR-31543 (20 mg/kg), suggesting that the gastric first-pass effect of KR-31543 was almost negligible in rats. However, the values were significantly smaller (305 and 318 microg x min/mL) than that after intraportal administration (494 microg x min/mL), indicating a considerable intestinal first-pass effect of KR-31543 in rats; that is, approximately 40% of the oral dose. Approximately 50% of KR-31543 absorbed into the portal vein was eliminated by the liver (hepatic first-pass effect) based on iv and intraportal administration (the value, 50%, was equivalent to approximately 30% of the oral dose). The low F value of KR-31543 after oral administration of 20 mg/kg to rats was mainly due to considerable intestinal (approximately 40%) and hepatic (approximately 30%) first-pass effects.