RESUMO
We investigated the nitrogen and carbohydrate allocation patterns of trees under heterogeneous light environments using saplings of the devil maple tree (Acer diabolicum) with Y-shaped branches. Different branch groups were created: all branches of a sapling exposed to full light (L-branches), all branches exposed to full shade (S-branches), and half of the branches of a sapling exposed to light (HL-branches) and the other half exposed to shade (HS-branches). Throughout the growth period, nitrogen was preferentially allocated to HL-branches, whereas nitrogen allocation to HS-branches was suppressed compared to L- and S-branches. HL-branches with the highest leaf nitrogen content (N(area)) also had the highest rates of growth, and HS-branches with the lowest N(area) had the lowest observed growth rates. In addition, net nitrogen assimilation, estimated using a photosynthesis model, was strongly correlated with branch growth and whole-plant growth. In contrast, patterns of photosynthate allocation to branches and roots were not affected by the light conditions of the other branch. These observations suggest that tree canopies develop as a result of resource allocation patterns, where the growth of sun-lit branches is favoured over shaded branches, which leads to enhanced whole-plant growth in heterogeneous light environments. Our results indicate that whole-plant growth is enhanced by the resource allocation patterns created for saplings in heterogeneous light environments.
Assuntos
Acer/crescimento & desenvolvimento , Acer/metabolismo , Luz , Nitrogênio/metabolismo , Metabolismo dos Carboidratos , Alocação de RecursosRESUMO
Cocultivation of spleen cells, lymph node cells, and thymocytes of female Wistar-King-Aptekman rats with short-term cultured male adult T cell leukemia (ATL) cells in the presence of 5-bromo-2'-deoxyuridine (BrdUrd) resulted in the establishment of rat lymphoid cell lines, TARS-1, TARL-2, and TART-1. Cytogenetic analysis of the three cell lines showed a female rat karyotype with 42 chromosomes. The surface phenotypes of TARS-1 and TART-1 were those of rat T cells. TARL-2 was only positive for rat Ia and leukocyte common antigens. The cell lines continuously produced a type C retrovirus, human T cell leukemia virus (HTLV) and expressed ATL-associated antigens. TARS-1 and TART-1, but not TARL-2 were transplantable into newborn syngeneic rats and nude mice. These results strongly indicate that HTLV not only immortalizes, but also transforms rat T cells in vitro. Adult rats immunized with either TARS-1 or TARL-2 produced antibodies specific for HTLV. The biochemical analysis of the antigens that reacted with rat sera revealed that they are the two HTLV-specific polypeptides, p24 and p28.
Assuntos
Transformação Celular Viral , Deltaretrovirus/análise , Glicoproteínas de Membrana , Linfócitos T/imunologia , Idoso , Animais , Anticorpos Antivirais/biossíntese , Antígenos de Neoplasias/imunologia , Antígenos de Superfície/genética , Antígenos Virais/imunologia , Linhagem Celular , Deltaretrovirus/imunologia , Deltaretrovirus/ultraestrutura , Feminino , Humanos , Leucemia/etiologia , Leucemia/imunologia , Leucemia/patologia , Linfonodos/citologia , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Ratos , Ratos Endogâmicos , Linfócitos T/patologia , Linfócitos T/ultraestrutura , Timo/citologia , Proteínas Virais/isolamento & purificaçãoRESUMO
Human T lymphocyte virus type I (HTLV-I) can be transmitted into several inbred strains of newborn and adult rats by inoculating newly established HTLV-I-immortalized rat T cell lines or the human T cell line MT-2. The transmission efficiency exceeds 80%, regardless of strain differences or the age at transmission. The production of anti-HTLV-I antibodies significantly differs among the strains and depends on the age at the time of transmission. Rats neonatally inoculated with HTLV-I-positive rat or human cells generally become seronegative HTLV-I carriers throughout their lives, whereas adult rats inoculated with HTLV-I-positive cells at 16 wk of age become seropositive HTLV-I carriers. The HTLV-I provirus genome is present in almost all organs, regardless of whether the carriers are seronegative or seropositive. According to antibody titers to HTLV-I, there are three groups of inbred rat strains: ACI, F344, and SDJ (high responders); WKA, BUF, and LEJ (intermediate responders); and LEW (low responder). Three of three 16-mo-old seronegative HTLV-I carrier rats of the WKA strain developed spastic paraparesis of the hind legs. Neuropathological examinations revealed that the lesions were confined primarily to the lateral and anterior funiculi of the spinal cord. Both myelin and axons were extensively damaged in a symmetrical fashion, and infiltration with massive foamy macrophages was evident. The most severe lesions were at levels of the thoracic cord and continued from the cervical to the lumbar area. These histopathological features as well as clinical symptoms largely parallel findings in humans with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). These HTLV-I carrier rats, in particular the WKA rats described above, can serve as a useful animal model for investigating virus-host interactions in the etiopathogenesis of HTLV-I-related immunological diseases, particularly HAM/TSP.
Assuntos
Formação de Anticorpos , Portador Sadio , DNA Viral/isolamento & purificação , Infecções por HTLV-I/fisiopatologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Linfócitos T/imunologia , Integração Viral , Envelhecimento/fisiologia , Animais , Animais Recém-Nascidos , Antígenos de Diferenciação/análise , Sequência de Bases , Linhagem Celular , DNA Viral/genética , Modelos Animais de Doenças , Feminino , Genoma Viral , Infecções por HTLV-I/complicações , Infecções por HTLV-I/imunologia , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/isolamento & purificação , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Especificidade de Órgãos , Reação em Cadeia da Polimerase/métodos , Provírus/genética , Ratos , Ratos Endogâmicos , Especificidade da Espécie , Medula Espinal/microbiologia , Medula Espinal/patologiaRESUMO
Tyrosine kinase receptors for angiogenic factors vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) are expressed not only by endothelial cells but also by subsets of hematopoietic stem cells (HSCs). To further define their role in the regulation of postnatal hematopoiesis and vasculogenesis, VEGF and Ang-1 plasma levels were elevated by injecting recombinant protein or adenoviral vectors expressing soluble VEGF(165), matrix-bound VEGF(189), or Ang-1 into mice. VEGF(165), but not VEGF(189), induced a rapid mobilization of HSCs and VEGF receptor (VEGFR)2(+) circulating endothelial precursor cells (CEPs). In contrast, Ang-1 induced delayed mobilization of CEPs and HSCs. Combined sustained elevation of Ang-1 and VEGF(165) was associated with an induction of hematopoiesis and increased marrow cellularity followed by proliferation of capillaries and expansion of sinusoidal space. Concomitant to this vascular remodeling, there was a transient depletion of hematopoietic activity in the marrow, which was compensated by an increase in mobilization and recruitment of HSCs and CEPs to the spleen resulting in splenomegaly. Neutralizing monoclonal antibody to VEGFR2 completely inhibited VEGF(165), but not Ang-1-induced mobilization and splenomegaly. These data suggest that temporal and regional activation of VEGF/VEGFR2 and Ang-1/Tie-2 signaling pathways are critical for mobilization and recruitment of HSCs and CEPs and may play a role in the physiology of postnatal angiogenesis and hematopoiesis.
Assuntos
Fatores de Crescimento Endotelial/fisiologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Linfocinas/fisiologia , Glicoproteínas de Membrana/fisiologia , Proteínas Proto-Oncogênicas , Transdução de Sinais/fisiologia , Adenoviridae , Angiopoietina-1 , Animais , Células da Medula Óssea , Fatores de Crescimento Endotelial/administração & dosagem , Fatores de Crescimento Endotelial/sangue , Fatores de Crescimento Endotelial/metabolismo , Feminino , Vetores Genéticos , Mobilização de Células-Tronco Hematopoéticas , Leucócitos/fisiologia , Linfocinas/administração & dosagem , Linfocinas/sangue , Linfocinas/metabolismo , Masculino , Glicoproteínas de Membrana/administração & dosagem , Glicoproteínas de Membrana/sangue , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos SCID , Proteínas de Neoplasias/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular , Baço/citologia , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
In Drosophila, the Jun amino-terminal kinase (JNK) homolog Basket (Bsk) is required for epidermal closure. Mutants for Src42A, a Drosophila c-src protooncogene homolog, are described. Src42A functions in epidermal closure during both embryogenesis and metamorphosis. The severity of the epidermal closure defect in the Src42A mutant depended on the amount of Bsk activity, and the amount of Bsk activity depended on the amount of Src42A. Thus, activation of the Bsk pathway is required downstream of Src42A in epidermal closure. This work confirms mammalian studies that demonstrated a physiological link between Src and JNK.
Assuntos
Proteínas de Drosophila , Drosophila/crescimento & desenvolvimento , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Actinas/metabolismo , Animais , Drosophila/embriologia , Drosophila/genética , Drosophila/metabolismo , Ativação Enzimática , Epiderme/embriologia , Genes de Insetos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno , Metamorfose Biológica , Fenótipo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Mutação Puntual , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Transdução de SinaisRESUMO
Individuals infected with the human immunodeficiency virus type 1 (HIV-1) may be asymptomatic or have AIDS-related complex or the acquired immuno deficiency syndrome (AIDS). Little is known about the factors that influence progression of infection to AIDS. In this study of isolates of HIV-1 obtained at intervals during the infection of four individuals, the development of disease was found to be correlated with the emergence of HIV-1 variants that were more cytopathic in vitro as the disease progressed and that replicated more efficiently in a wide variety of different human cells. The biologic properties of HIV-1 in vitro thus appear to reflect its virulence in the host. Further studies of such sequentially isolated viruses may lead to the identification of viral genes that govern pathogenesis.
Assuntos
HIV/patogenicidade , Complexo Relacionado com a AIDS/etiologia , Complexo Relacionado com a AIDS/microbiologia , Síndrome da Imunodeficiência Adquirida/etiologia , Síndrome da Imunodeficiência Adquirida/microbiologia , Efeito Citopatogênico Viral , DNA/genética , Variação Genética , HIV/genética , HIV/fisiologia , Proteína gp120 do Envelope de HIV , Humanos , Testes de Neutralização , Proteínas dos Retroviridae/genéticaRESUMO
Antibodies that enhance human immunodeficiency virus (HIV) infectivity have been found in the blood of infected individuals and in infected or immunized animals. These findings raise serious concern for the development of a safe vaccine against acquired immunodeficiency syndrome. To address the in vivo relevance and mechanism of this phenomenon, antibody-dependent enhancement of HIV infectivity in peripheral blood macrophages, lymphocytes, and human fibroblastoid cells was studied. Neither Leu3a, a monoclonal antibody directed against the CD4 receptor, nor soluble recombinant CD4 even at high concentrations prevented this enhancement. The addition of monoclonal antibody to the Fc receptor III (anti-FcRIII), but not of antibodies that react with FcRI or FcRII, inhibited HIV type 1 and HIV type 2 enhancement in peripheral blood macrophages. Although enhancement of HIV infection in CD4+ lymphocytes could not be blocked by anti-FcRIII, it was inhibited by the addition of human immunoglobulin G aggregates. The results indicate that the FcRIII receptor on human macrophages and possibly another Fc receptor on human CD4+ lymphocytes mediate antibody-dependent enhancement of HIV infectivity and that this phenomenon proceeds through a mechanism independent of the CD4 protein.
Assuntos
Síndrome da Imunodeficiência Adquirida/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , HIV-2/imunologia , Receptores Fc/fisiologia , Receptores Virais/fisiologia , Animais , Anticorpos Monoclonais , Citotoxicidade Celular Dependente de Anticorpos , Cobaias , Anticorpos Anti-HIV/biossíntese , Humanos , Técnicas In Vitro , Pan troglodytes , Receptores de HIVRESUMO
High-fidelity transfers of genetic information in the central dogma can be achieved by a reaction called editing. The crystal structure of an enzyme with editing activity in translation is presented here at 2.5 angstroms resolution. The enzyme, isoleucyl-transfer RNA synthetase, activates not only the cognate substrate L-isoleucine but also the minimally distinct L-valine in the first, aminoacylation step. Then, in a second, "editing" step, the synthetase itself rapidly hydrolyzes only the valylated products. For this two-step substrate selection, a "double-sieve" mechanism has already been proposed. The present crystal structures of the synthetase in complexes with L-isoleucine and L-valine demonstrate that the first sieve is on the aminoacylation domain containing the Rossmann fold, whereas the second, editing sieve exists on a globular beta-barrel domain that protrudes from the aminoacylation domain.
Assuntos
Isoleucina-tRNA Ligase/química , Isoleucina/metabolismo , Valina/metabolismo , Monofosfato de Adenosina , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/enzimologia , Ligação de Hidrogênio , Hidrólise , Isoleucina-tRNA Ligase/metabolismo , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , RNA de Transferência de Isoleucina/metabolismo , Especificidade por Substrato , Thermus thermophilus/enzimologia , Aminoacilação de RNA de TransferênciaRESUMO
Mitogen-activated protein kinase (MAPK) is a conserved eukaryotic signaling factor that mediates various signals, cumulating in the activation of transcription factors. Extracellular signal-regulated kinase (ERK), a MAPK, is activated through phosphorylation by the kinase MAPK/ERK kinase (MEK). To elucidate the extent of the involvement of ERK in various aspects of animal development, we searched for a Drosophila mutant which responds to elevated MEK activity and herein identified a lace mutant. Mutants with mild lace alleles grow to become adults with multiple aberrant morphologies in the appendages, compound eye, and bristles. These aberrations were suppressed by elevated MEK activity. Structural and transgenic analyses of the lace cDNA have revealed that the lace gene product is a membrane protein similar to the yeast protein LCB2, a subunit of serine palmitoyltransferase (SPT), which catalyzes the first step of sphingolipid biosynthesis. In fact, SPT activity in the fly expressing epitope-tagged Lace was absorbed by epitope-specific antibody. The number of dead cells in various imaginal discs of a lace hypomorph was considerably increased, thereby ectopically activating c-Jun N-terminal kinase (JNK), another MAPK. These results account for the adult phenotypes of the lace mutant and suppression of the phenotypes by elevated MEK activity: we hypothesize that mutation of lace causes decreased de novo synthesis of sphingolipid metabolites, some of which are signaling molecules, and one or more of these changes activates JNK to elicit apoptosis. The ERK pathway may be antagonistic to the JNK pathway in the control of cell survival.
Assuntos
Aciltransferases/metabolismo , Drosophila/embriologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Processamento de Proteína Pós-Traducional , Esfingolipídeos/biossíntese , Aciltransferases/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sobrevivência Celular , Clonagem Molecular , Cruzamentos Genéticos , DNA Complementar/genética , Regulação para Baixo , Olho/embriologia , Feminino , Cabeça/embriologia , Proteínas Quinases JNK Ativadas por Mitógeno , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Serina C-Palmitoiltransferase , Asas de Animais/embriologiaRESUMO
BACKGROUND: The 20 aminoacyl-tRNA synthetases are divided into two classes, I and II. The 10 class I synthetases are considered to have in common the catalytic domain structure based on the Rossmann fold, which is totally different from the class II catalytic domain structure. The class I synthetases are further divided into three subclasses, a, b and c, according to sequence homology. No conserved structural features for tRNA recognition by class I synthetases have been established. RESULTS: We determined the crystal structure of the class Ia methionyl-tRNA synthetase (MetRS) at 2.0 A resolution, using MetRS from an extreme thermophile, Thermus thermophilus HB8. The T. thermophilus MetRS structure is in full agreement with the biochemical and genetic data from Escherichia coli MetRS. The conserved 'anticodon-binding' residues are spatially clustered on an alpha-helix-bundle domain. The Rossmann-fold and anticodon-binding domains are connected by a beta-alpha-alpha-beta-alpha topology ('SC fold') domain that contains the class I specific KMSKS motif. CONCLUSIONS: The alpha-helix-bundle domain identified in the MetRS structure is the signature of the class Ia enzymes, as it was also identified in the class Ia structures of the isoleucyl- and arginyl-tRNA synthetases. The beta-alpha-alpha-beta-alpha topology domain, which can now be identified in all known structures of the class Ia and Ib synthetases, is likely to dock with the inner side of the L-shaped tRNA, thereby positioning the anticodon stem.
Assuntos
Metionina tRNA Ligase/química , Proteínas de Ligação a RNA/química , Thermus thermophilus/química , Anticódon , Domínio Catalítico , Cristalografia por Raios X , Metionina tRNA Ligase/metabolismo , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Proteínas de Ligação a RNA/metabolismoRESUMO
Telomerase is a ribonucleoprotein that synthesizes telomeric DNA onto chromosomal ends. The expression of telomerase is thought to be required for cellular immortality and oncogenesis. Telomerase activity has been detected not only in most cancers but also in some types of premalignant lesions, such as squamous intraepithelial lesions (SILs). In the present study, we used the telomerase assay to detect uterine cervical lesions in cervical scraping samples. A total of 82 cervical scraping samples were obtained from women with or without cervical lesions and examined by nonradioisotope telomeric repeat amplification protocol assay. Fifteen of 17 (88%) cervical cancer specimens exhibited telomerase activity, whereas 5 of 8 (63%) and 14 of 24 (58%) specimens from low-grade and high-grade SILs, respectively, also exhibited telomerase activity. In contrast, 3 of 33 (9%) specimens from normal cervices exhibited telomerase activity. Dilution telomeric repeat amplification protocol assay was performed to estimate telomerase activity; it revealed that high levels of activity were often expressed in cervical cancer. Cytological examination was also performed by Pap smear test, and 4 of 8 (50%) low-grade SILs, 21 of 24 (88%) high-grade SILs, and 16 of 17 (94%) cervical cancers were found to have cytological abnormalities. There were discordances in some cases between findings of smear abnormality and telomerase positivity. In particular, we found five cases of SILs without smear abnormality but with telomerase activity, suggesting that some lesions with false negative cytology can be detected by telomerase assay. These findings suggest that telomerase assay using cervical scrapings might be a useful screening method for cervical lesions especially when combined with a Pap smear test.
Assuntos
Biomarcadores Tumorais/metabolismo , Telomerase/metabolismo , Neoplasias do Colo do Útero/enzimologia , Colo do Útero/citologia , Colo do Útero/enzimologia , Feminino , Humanos , Reação em Cadeia da Polimerase/métodos , Valores de Referência , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/patologiaRESUMO
DNA methylation is the only known mechanism for an epigenetic genomic DNA modification that is capable of altering gene expression. A recent study reveals that the pattern of CpG island methylation is largely characteristic of tumor type, suggesting that distinct sets of genes are inactivated by methylation during development of each tumor type. We compared previously the methylation status between normal liver and liver tumors in SV40 T/t antigen transgenic mice (MT-D2 mice) using Restriction Landmark Genomic Scanning for Methylation (RLGS-M) and identified several loci/spots that appeared to be methylated frequently in liver tumors. One of these spots, B236, identified a locus on chromosome 12 (D12Ncvs7) syntenic with human 14q12-q21 that is frequently lost in certain human cancers. Shotgun sequencing of a bacterial artificial chro mosome clone containing this spot/locus was performed to identify genes within this region. The Genescan program predicted an open reading frame of a novel, intron-less gene adjacent to the B236 spot that encodes a putative 493-amino acid protein containing the SNAG repressor motif in the NH2-terminal region and five C2H2-type zinc finger motifs in the COOH-terminal half. This putative gene, methylated in liver tumor (mlt 1), is a novel member of the SNAG transcriptional repressor family with 43% amino acid identity to insulinoma-associated protein 1. An open reading frame encoding a protein quite similar to mouse mlt 1 (56% amino acid identity) was located in the syntenic region of the human genome, indi cating that mlt 1 is evolutionarily conserved in human. Northern blot analysis revealed that mlt 1 is normally expressed in brain, spleen, stom ach, and liver. However, mlt 1 expression was silenced in the liver tumors of MT-D2 mice. The putative promoter region of mlt 1 is unmethylated in normal tissues but methylated in all liver tumors from 11 MT-D2 mice We also found that mlt 1 was methylated and not expressed in N18TG-22 cells, a mouse neuroblastoma cell line. Treatment of N18TG-2 cells with a demethylating agent, 5-aza-deoxycytidine, resulted in an expression of mlt 1, indicating that the repression of mlt 1 is attributable to methylation Thus, mlt 1 is a novel target gene that is silenced by methylation during liver tumorigenesis initiated by SV40 T antigen.
Assuntos
Antígenos Transformantes de Poliomavirus/genética , Azacitidina/análogos & derivados , Metilação de DNA , Inativação Gênica , Neoplasias Hepáticas Experimentais/genética , Proteínas Repressoras/genética , Dedos de Zinco/genética , Sequência de Aminoácidos , Animais , Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/farmacologia , Sequência de Bases , Proteínas de Ligação a DNA/genética , Decitabina , Feminino , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Humanos , Neoplasias Hepáticas Experimentais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Neuroblastoma/genética , Fases de Leitura Aberta , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Postgraduate medical trainees experience high rates of burnout, but evidence regarding psychiatric trainees is missing. We aim to determine burnout rates among psychiatric trainees, and identify individual, educational and work-related factors associated with severe burnout. METHODS: In an online survey psychiatric trainees from 22 countries were asked to complete the Maslach Burnout Inventory (MBI-GS) and provide information on individual, educational and work-related parameters. Linear mixed models were used to predict the MBI-GS scores, and a generalized linear mixed model to predict severe burnout. RESULTS: This is the largest study on burnout and training conditions among psychiatric trainees to date. Complete data were obtained from 1980 out of 7625 approached trainees (26%; range 17.8-65.6%). Participants were 31.9 (SD 5.3) years old with 2.8 (SD 1.9) years of training. Severe burnout was found in 726 (36.7%) trainees. The risk was higher for trainees who were younger (P<0.001), without children (P=0.010), and had not opted for psychiatry as a first career choice (P=0.043). After adjustment for socio-demographic characteristics, years in training and country differences in burnout, severe burnout remained associated with long working hours (P<0.001), lack of supervision (P<0.001), and not having regular time to rest (P=0.001). Main findings were replicated in a sensitivity analysis with countries with response rate above 50%. CONCLUSIONS: Besides previously described risk factors such as working hours and younger age, this is the first evidence of negative influence of lack of supervision and not opting for psychiatry as a first career choice on trainees' burnout.
Assuntos
Esgotamento Profissional , Psiquiatria/estatística & dados numéricos , Tolerância ao Trabalho Programado/psicologia , Adulto , Esgotamento Profissional/diagnóstico , Esgotamento Profissional/epidemiologia , Esgotamento Profissional/etiologia , Escolha da Profissão , Demografia , Educação Médica Continuada/métodos , Feminino , Humanos , Masculino , Serviços de Saúde Mental/organização & administração , Pessoa de Meia-Idade , Inventário de Personalidade , Fatores de Risco , Fatores Socioeconômicos , Inquéritos e QuestionáriosRESUMO
The roll-twist-slide correlation in the DNA crystal structures that are collected in the Nucleic Acid Data Base is analyzed in order to obtain a general understanding of the effects of the nucleotide sequence on the 3D structure of a dinucleotide step. It is concluded that the differences between the pyrimidine bases and the purine bases in terms of their physical shapes are the major factors that determine the stereochemical characteristics of the steps through base to backbone and base to base interactions. The characteristics are further modulated by the differences between the A:T and G:C base-pairs, which can be explained by enhancement of the purine-pyrimidine asymmetry in the A:T base-pair.
Assuntos
DNA/química , Bases de Dados Factuais , Modelos Moleculares , Conformação de Ácido Nucleico , Biologia Computacional/métodos , Biologia Molecular/métodosRESUMO
By a kinetic analysis of 59 variant transcripts of Escherichia coli tRNA(Glu) with glutamyl-tRNA synthetase (GluRS), the U11.A24 base-pair, the U13.G22..A46 base-triple, and the lack of residue 47 (delta47) were found to serve as major determinants for tRNA(Glu) identity. This is the first system for which major identity determinants are reported to be clustered in the "augmented D helix", consisting of the D stem with some neighboring residues and the variable loop. Other identity determinants are U34, U35, C36 and A37 in the anticodon loop, and G1.C72 and U2.A71 in the acceptor stem. Phosphate-group protection by GluRS from ethylnitrosourea was observed most strongly for the minor groove side of D-stem helix, indicating that GluRs tightly binds to the D stem for recognition, on the minor groove side, of the potent identity-determinant groups of the U11.A24 and U13.G22 base-pairs. A46 is not involved in direct recognition by GluRS; the U13.G22..A46 base-triple is required probably for formation of the structural features that are recognized by GluRS. In this context, the essential role of characteristic delta47 in tRNA(Glu) identity may be to maintain the U13.G22..A46 base-triple.
Assuntos
Escherichia coli/química , Conformação de Ácido Nucleico , RNA de Transferência de Ácido Glutâmico/química , Anticódon/genética , Composição de Bases , Sequência de Bases , Clonagem Molecular , Códon/genética , Eletroforese em Gel de Poliacrilamida , Etilnitrosoureia/metabolismo , Glutamato-tRNA Ligase/metabolismo , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Oligorribonucleotídeos/química , Ligação Proteica , RNA de Transferência de Glutamina/química , RNA de Transferência de Glutamina/metabolismo , RNA de Transferência de Ácido Glutâmico/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
The Ras protein and its homolog, Rap1A, have an identical "effector region" (residues 32-40) preceded by Asp30-Glu31 and Glu30-Lys31, respectively. In the complex of the "Ras-like" E30D/K31E mutant Rap1A with the Ras-binding domain (RBD), residues 51-131 of Raf-1, Glu31 in Rap1A forms a tight salt bridge with Lys84 in Raf-1. However, we have recently found that Raf-1 RBD binding of Ras is indeed reduced by the E31K mutation, but is not affected by the E31A mutation. Here, the "Rap1A-like" D30E/E31K mutant of Ras was prepared and shown to bind the Raf-1 RBD less strongly than wild-type Ras, but slightly more tightly than the E31K mutant. The backbone 1H, 13C, and 15N magnetic resonances of the Raf-1 RBD were assigned in complexes with the wild-type and D30E/E31K mutant Ras proteins in the guanosine 5'-O-(beta,gamma-imidotriphosphate)-bound form. The Lys84 residue in the Raf-1 RBD exhibited a large change in chemical shift upon binding wild-type Ras, suggesting that Lys84 interacts with wild-type Ras. The D30E/E31K mutant of Ras caused nearly the same perturbations in Raf-1 chemical shifts, including that of Lys84. We hypothesized that Glu31 in Ras may not be the major salt bridge partner of Lys84 in Raf-1. A molecular dynamics simulation of a model structure of the Raf-1 RBD.Ras.GTP complex suggested that Lys84 in Raf-1 might instead form a tight salt bridge with Asp33 in Ras. Consistent with this, the D33A mutation in Ras greatly reduced its Raf-I RBD binding activity. We conclude that the major salt bridge partner of Lys84 in Raf-1 may be Asp33 in Ras.
Assuntos
Proteínas Proto-Oncogênicas c-raf/química , Proteínas ras/química , Sequência de Aminoácidos , Ácido Aspártico/metabolismo , Simulação por Computador , Escherichia coli , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Recombinantes de Fusão/química , Homologia de Sequência de Aminoácidos , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Pharmacokinetic properties of a new angiotensin-converting enzyme inhibitor, delapril (CV-3317), which converts to two active metabolites (M-1 and M-2) and one inactive metabolite (M-3) after oral administration, were investigated in six subjects with normal, 10 subjects with slight (SRF), and six subjects with markedly (MRF) deteriorated kidney function. The elimination half-life of M-1 was prolonged significantly in subjects with MRF and that of M-3 was also prolonged in subjects with SRF or MRF. The peak plasma drug concentration, time to reach peak concentration (tmax), and AUC were significantly larger in subjects with SRF and MRF than in normal subjects, except for Tmax in subjects with SRF. In M-2 and unchanged delapril, no difference was observed. The 24-hour cumulative urinary excretion of those metabolites was significantly lower in subjects with MRF than in normal subjects. Plasma angiotensin-converting enzyme activity, suppressed at 4 hours in all subjects, remained significantly low in patients with MRF at 24 hours. Blood pressure was reduced more in subjects with chronic renal failure. It was concluded that delapril is excreted mainly through the kidney and its pharmacodynamics and biologic effects are affected by the renal dysfunction.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacocinética , Indanos/farmacocinética , Indenos/farmacocinética , Falência Renal Crônica/metabolismo , Administração Oral , Inibidores da Enzima Conversora de Angiotensina/administração & dosagem , Inibidores da Enzima Conversora de Angiotensina/sangue , Inibidores da Enzima Conversora de Angiotensina/urina , Fenômenos Químicos , Química , Humanos , Indanos/administração & dosagem , Indanos/sangue , Indanos/urina , Fatores de TempoRESUMO
A docking model of glutamyl-tRNA synthetase (GluRS) and tRNAGlu was constructed, on the basis of the distinguished similarity between the X-ray crystallographic three-dimensional structures of the N-terminal halves of the Thermus thermophilus GluRS in the free state and the Escherichia coli glutaminyl-tRNA synthetase in a complex with tRNAGln. The modeled structure is energetically favorable and is also well consistent with the results of site-directed mutagenesis studies. The model indicates that the GluRS-specific insertions 2 and 3 fit and bind to the acceptor stem and the D arm, respectively, of the cognate tRNA without affecting other contacts. In particular, insertion 3 strongly interacts with the two D-stem base pairs that are essential for the tRNA-GluRS recognition.
Assuntos
Glutamato-tRNA Ligase/química , Modelos Moleculares , RNA de Transferência de Ácido Glutâmico/química , Anticódon , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/enzimologia , Glutamato-tRNA Ligase/genética , Glutamato-tRNA Ligase/metabolismo , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , RNA de Transferência de Ácido Glutâmico/genética , RNA de Transferência de Ácido Glutâmico/metabolismo , Termodinâmica , Thermus thermophilus/enzimologiaRESUMO
UNLABELLED: Recently, troglitazone has emerged as an insulin sensitizer for the treatment of type II diabetes. However, its effect on skeletal muscle glucose use (SMGU) has not been studied. METHODS: To investigate the effect of troglitazone on SMGU in patients with type II diabetes, we undertook skeletal muscle (18)F-FDG PET dynamic imaging under insulin clamping before and after administration of SMGU to 20 patients with type II diabetes. Data were compared with those for 12 age-matched healthy volunteers. RESULTS: The whole-body glucose disposal rate (GDR) was significantly lower in patients (29.9 +/- 9.83 micromol/min/kg) than in control subjects (55.6 +/- 16.5 micromol/min/kg, P < 0.01), as was the SMGU (patients, 3.27 +/- 2.17 micromol/min/kg; control subjects, 10.9 +/- 6.4 micromol/min/kg; P < 0.01). After the therapy, GDR significantly improved in patients (29.3 +/- 14.6 micromol/min/kg, P < 0.05), as did SMGU (5.06 +/- 2.11 micromol/min/kg, P < 0.05). When results for patients with and without hypertension were separately analyzed, a significant improvement in SMGU after troglitazone was seen in both normotensive and hypertensive patients (normotensive [n = 10]: baseline, 3.67 +/- 2.89 micromol/min/kg; after therapy, 5.28 +/- 2.61 micromol/min/kg; P < 0.05; hypertensive [n = 10]: baseline, 2.89 +/- 1.22 micromol/min/kg; after therapy, 4.72 +/- 1.39 micromol/min/kg; P < 0.05). GDR in patients with and without hypertension was significantly improved by troglitazone (normotensive: baseline, 17.9 +/- 10.2 micromol/min/kg; after therapy, 31.9 +/- 15.9 micromol/min/kg; P < 0.01; hypertensive: baseline, 39.6 +/- 15.1 micromol/min/kg; after therapy, 47.7 +/- 23.8 micromol/min/kg; P < 0.05). The plasma free fatty acid concentration during insulin clamping was not changed by troglitazone (baseline, 1.1 +/- 0.86 mEq/L; after therapy, 0.93 +/- 0.65 mEq/L; P = not significant). CONCLUSION: Troglitazone can improve whole-body insulin resistance through the improvement of SMGU but not through a decline in plasma free fatty acid concentration in patients with type II diabetes with or without hypertension.
Assuntos
Cromanos/uso terapêutico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Fluordesoxiglucose F18 , Glucose/metabolismo , Hipoglicemiantes/uso terapêutico , Resistência à Insulina , Músculo Esquelético/metabolismo , Compostos Radiofarmacêuticos , Tiazóis/uso terapêutico , Tiazolidinedionas , Tomografia Computadorizada de Emissão , Glicemia/análise , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/diagnóstico por imagem , Diabetes Mellitus Tipo 2/metabolismo , Ácidos Graxos não Esterificados/sangue , Feminino , Humanos , Hipertensão/complicações , Hipertensão/metabolismo , Insulina/sangue , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/diagnóstico por imagem , TroglitazonaRESUMO
UNLABELLED: Dual-isotope scintigraphic studies with 201Tl and radioiodinated metaiodobenzylguanidine (MIBG) suggest that acute myocardial infarction causes extensive regional myocardial denervation beyond the infarcted area. We therefore investigated the histopathological and biochemical significance of the discrepancy between 201Tl and 125I-MIBG distribution determined by dual-tracer autoradiography in experimental myocardial infarction. METHODS: Left coronary arteries of 12 male Wistar rats were ligated for 30 min followed by reperfusion. Dual-tracer autoradiography of infarcted heart sections was performed with 201Tl and [125I]MIBG 4 hr or 2 days after coronary reperfusion, followed by immunohistochemical staining with myoglobin monoclonal antibody to determine the area of myocardial infarction. Ultrastructural alterations and myocardial norepinephrine (NE) content in the region determined by dual-tracer autoradiography and myglobin immunostaining were studied. RESULTS: Thirty-minute coronary ligation with 4-hr reperfusion produced myocardial infarction associated with discrepant region in the peri-infarcted myocardium characterized by decreased [125I]MIBG uptake and normal 201Tl distribution (discrepant region), as determined by dual-tracer autoradiography. In the discrepant region, which disappeared after 2 days, the nerve terminals showed loss of granular cores, with normal structures between normal myocytes. The mean myocardial NE level in the discrepant region was significantly lower than that in the nonischemic region (255.2 +/- 85.9 versus 549.5 +/- 82.5 ng/mg). CONCLUSION: The uptake discrepancy of 201Tl and [125I]MIBG observed in the infarcted heart represents a transient functional denervation of the regional cardiac sympathetic nerve terminals in the noninfarcted myocardium.