RESUMO
Lupin cultivation worldwide is threatened by anthracnose, a destructive disease caused by the seed- and air-borne fungal pathogen Colletotrichum lupini. In this study we explored the intraspecific diversity of 39 C. lupini isolates collected from different lupin cultivating regions around the world, and representative isolates were screened for their pathogenicity and virulence on white and Andean lupin. Multi-locus phylogeny and morphological characterizations showed intraspecific diversity to be greater than previously shown, distinguishing a total of six genetic groups and ten distinct morphotypes. Highest diversity was found across South America, indicating it as the center of origin of C. lupini. The isolates that correspond to the current pandemic belong to a genetic and morphological uniform group, were globally widespread, and showed high virulence on tested white and Andean lupin accessions. Isolates belonging to the other five genetic groups were mostly found locally and showed distinct virulence patterns. Two highly virulent strains were shown to overcome resistance of advanced white lupin breeding material. This stresses the need to be careful with international seed transports in order to prevent spread of currently confined but potentially highly virulent strains. This study improves our understanding of the diversity, phylogeography and pathogenicity of a member of one of the world's top 10 plant pathogen genera, providing valuable information for breeding programs and future disease management.
Assuntos
Colletotrichum , Variação Genética , Lupinus/microbiologia , Doenças das Plantas , Fatores de Virulência/genética , Colletotrichum/genética , Colletotrichum/patogenicidade , Doenças das Plantas/genética , Doenças das Plantas/microbiologiaRESUMO
DNA motifs that encode for specific transcriptional regulatory sequences (TRS) when engineered adjacent to the structural protein coding domain of a suicide enzyme can provide cell-lineage specific protein expression. The disparate up-regulation of several genes in adult T-cell leukemia (ATL) versus HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP), seropositive carriers (SPC) and uninfected normals may reflect events at the molecular level related to leukemogenesis or to processes maintaining the heme-oncologic phenotype. Further, the genetic transduction of cytokine and receptor genes uniquely associated with ATL may provide targets for the development of leukemia-specific gene therapies aimed at exploiting differences in the production of certain growth factors and growth factor receptors. Comparisons of the transcriptional and translational levels of interleukin-2 receptor alpha chain (IL-2R alpha), transforming growth factor-beta 1 (TGF-beta 1) and intracellular adhesion molecule-1 (ICAM-1) in ATL, HAM/TSP, and SPC and in several control populations revealed selectively up-regulated expression in ATL. We evaluated the feasibility of using lymphoid-specific TRS to activate herpes simplex virus thymidine kinase (HSVtk) to achieve selective cytotoxicity in leukemias expressing terminal deoxynucleotidyl transferase (TdT). Selective and efficient leukemic cell killing was produced and suggests that similar chimeric gene constructs containing TRS elements for IL-2R alpha, TGF-beta 1, or ICAM-1 may prove useful in designing gene therapies to treat ATL.
Assuntos
Citocinas/biossíntese , Terapia Genética/métodos , Leucemia de Células T/terapia , Paraparesia Espástica Tropical/terapia , Receptores de Citocinas/biossíntese , Adulto , Portador Sadio/terapia , Citocinas/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Interleucinas/biossíntese , Engenharia de Proteínas , Receptores de Citocinas/genética , Receptores de Interleucina-2/biossíntese , Sequências Reguladoras de Ácido Nucleico , Simplexvirus/genética , Timidina Quinase/biossíntese , Timidina Quinase/genética , Fator de Crescimento Transformador beta/biossínteseRESUMO
CDC2 kinase activity was decreased by up to 75% when mitotic cell free extracts from mouse fibroblasts were incubated with cAMP and ATP. This effect was blocked by PKI, the heat stable inhibitor of cAMP-dependent protein kinase (PKA). An acidic, heat stable protein from G1 cells, consistent with inhibitor-1 of protein phosphatase 1, mimicked the effect of cAMP, but was not antagonized by PKI. Okadaic acid, another inhibitor of protein phosphatase 1, also downregulated CD2 activity, and the effect was independent of both cAMP and PKI. The evidence suggests that PKA exerts its effect by activating inhibitor-1 by phosphorylation, and that the next step in the regulatory pathway requires the inactivation of one or more protein phosphatase 1 isoenzymes. Non-denaturing gel electrophoresis suggested that the size and/or charge density of the CDC2 kinase complex was changed when the activity was downregulated by cAMP or G1 extracts.
Assuntos
Proteína Quinase CDC2/metabolismo , Mitose , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Sistema Livre de Células , AMP Cíclico/metabolismo , Fibroblastos/citologia , Fibroblastos/enzimologia , Fase G1 , Camundongos , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Especificidade por SubstratoRESUMO
A new method for the resolution of protein kinases in nondenaturing minigels was used to analyze the type of PKA induced by retinyl acetate in transformed mouse 10T1/2 cells. Protein kinases in 0.5 microliters aliquots of cell extracts were resolved on nondenaturing gradient Phastgels and assayed for PKA activity in situ using a specific peptide substrate and [32P]ATP. Retinyl acetate caused a decrease in type I and an increase in type II PKA isoforms after about 36 h in culture. The system provides a quick and sensitive means for analyzing the activity of PKA isoforms from small numbers of cells.
Assuntos
AMP Cíclico/farmacologia , Isoenzimas/biossíntese , Proteínas Quinases/biossíntese , Vitamina A/análogos & derivados , Animais , Linhagem Celular Transformada , Diterpenos , Eletroforese em Gel de Poliacrilamida , Indução Enzimática/efeitos dos fármacos , Isoenzimas/isolamento & purificação , Camundongos , Proteínas Quinases/isolamento & purificação , Ésteres de Retinil , Vitamina A/farmacologiaRESUMO
The synthesis and phosphorylation of H1 histones were studied in organ cultures of midpregnant mouse mammary glands exposed to various combinations of insulin, cortisol and prolactin over a 48-h period. The synthesis of specific H1 histone subtypes was changed only when all three hormones were present, and the effect was most pronounced during the first 24 h of culture, a period of cell replication. The 3-hormone combination also stimulated the phosphorylation of the N-terminal region of the H1 histone, and this also occurred maximally during the first 24 h of culture. The enhanced phosphorylation of the N-terminal region of the H1 histone included a site sensitive to phosphorylation by cyclic AMP-dependent protein kinase. Thus, hormones which stimulate mammary development in vitro influence the synthesis and specific phosphorylation of H1 histones during a period of cell replication preceding the expression of milk protein genes.
Assuntos
Histonas/biossíntese , Hidrocortisona/farmacologia , Insulina/farmacologia , Glândulas Mamárias Animais/metabolismo , Prolactina/farmacologia , Animais , Feminino , Glândulas Mamárias Animais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Técnicas de Cultura de Órgãos , Mapeamento de Peptídeos , Fosforilação , GravidezRESUMO
Twenty-six cases of ovarian carcinoma and six cases of other pelvic neoplasms were analyzed for microsatellite instability (MSI) using frozen specimens, fluorescence technology, and four selected markers (D2S123 on chromosome 2, D18S58 on chromosome 18, BAT26 on chromosome 2, and BAT40 on chromosome 1). This procedure also allowed the detection of loss of heterogeneity (LOH) at the four selected loci. One of the cases of ovarian carcinoma exhibited MSI and this was evident at three loci. Of 44 informative loci, 7 exhibited LOH representing 3 cases of ovarian carcinoma, 3 of 4 cases of primary peritoneal carcinoma, and one case of unknown primary. These data support other findings that MSI is not a frequent occurrence in ovarian cancer; however, LOH is a more frequent event and may be a target for the development of diagnostic/prognostic procedures for ovarian and primary peritoneal carcinoma.
Assuntos
Perda de Heterozigosidade , Repetições de Microssatélites , Neoplasias Ovarianas/genética , Neoplasias Pélvicas/genética , DNA de Neoplasias/análise , Feminino , HumanosRESUMO
The incidence of myofibrillar degeneration (MFD) was studied in the following different forensic-pathological diagnostic groups of 25 cases each: acute morphine intoxication, acute carbon monoxide intoxication, hanging, strangulation by hand/ligature, drowning, acute hemorrhagic shock, lethal acute brain injury, explainable death of babies or infants and sudden infant death syndrome, together with 18 cases of intoxication with various drugs. The MFD was demonstrated by the Luxol-fast-blue reaction, with two types of phenomena being differentiated, namely cross-band lesions and diffuse staining. All diagnostic groups included cases of MFD of differing degrees. Cross-band lesions were observed in practically all cases of hanging, strangulation and acute hemorrhagic shock. Diffuse stain was noted particularly in cases of drowning and acute brain injury. The diagnostic significance is discussed.
Assuntos
Medicina Legal , Miocárdio/patologia , Miofibrilas/patologia , Doença Aguda , Asfixia/patologia , Lesões Encefálicas/patologia , Intoxicação por Monóxido de Carbono/patologia , Afogamento/patologia , Humanos , Lactente , Morfina/intoxicação , Intoxicação/patologia , Choque Hemorrágico/patologia , Morte Súbita do Lactente/patologiaAssuntos
Histonas/análise , Fígado/análise , Glândulas Mamárias Animais/análise , Neoplasias Mamárias Experimentais/metabolismo , Proteínas de Neoplasias/análise , Baço/análise , Útero/análise , Animais , Isótopos de Carbono , Cromatografia por Troca Iônica , Feminino , Histonas/metabolismo , Lisina , Camundongos , Transplante de Neoplasias , TrítioAssuntos
Caseínas/biossíntese , Histonas/metabolismo , Hormônios/farmacologia , Neoplasias Mamárias Experimentais/metabolismo , Lesões Pré-Cancerosas/metabolismo , Animais , Feminino , Hidrocortisona/farmacologia , Hiperplasia/metabolismo , Insulina/farmacologia , Camundongos , Prolactina/farmacologiaAssuntos
Fosfatase Alcalina , Histonas/isolamento & purificação , Ribonucleases , Animais , Fenômenos Químicos , Química , Cromatografia por Troca Iônica , Lisina , Coelhos , TimoAssuntos
Regulação da Expressão Gênica , Genes , Histonas/genética , Animais , Ciclo Celular , Fenômenos Químicos , Química , Drosophila melanogaster/genética , Crescimento , Histonas/fisiologia , Células Híbridas , Modelos Genéticos , RNA Mensageiro/metabolismo , Ouriços-do-Mar/genética , Leveduras/genéticaAssuntos
Aminoácidos/metabolismo , Histonas/biossíntese , Hidrocortisona/farmacologia , Insulina/farmacologia , Glândulas Mamárias Animais/metabolismo , Prolactina/farmacologia , Animais , Isótopos de Carbono , Cromatografia , DNA/biossíntese , Eletroforese , Feminino , Lisina/metabolismo , Camundongos , Técnicas de Cultura de Órgãos , Gravidez , Timidina/metabolismo , Fatores de Tempo , TrítioAssuntos
Cromossomos/ultraestrutura , Histonas , Animais , Sítios de Ligação , Bovinos , Ciclo Celular , AMP Cíclico/farmacologia , DNA/biossíntese , Feminino , Genes , Histonas/análise , Histonas/metabolismo , Fígado , Glândulas Mamárias Animais/metabolismo , Modelos Biológicos , Conformação Molecular , Especificidade de Órgãos , Fosfatos/metabolismo , Gravidez , Prolactina/farmacologia , Ouriços-do-Mar , Especificidade da Espécie , Timo/ultraestruturaRESUMO
The effect of retinyl acetate (RAC) on the activity of cAMP-dependent protein kinase (PKA) was studied in mouse 10T1/2 cells. The studies revealed that normal 10T1/2 cells had about 13-fold more PKA activity than did methylcholanthrene-transformed cells (MCA cells). The addition of RAC to MCA cells increased the activity of PKA about 3-fold as measured by the in vitro phosphorylation of a specific site in H1 histone (site A) or Kemptide. The increased PKA activity coincided with a reduction in the rate of cell replication of MCA cells, about 24 hr after exposure to the retinoid. Addition of forskolin to RAC-treated MCA cells resulted in a further reduction in the rate of cell replication, and this suggested that the enhanced PKA activity was also capable of action in vivo. To test this notion, MCA cells were grown with and without RAC, and the phosphorylation of the H1 histone at site A, a site known to be phosphorylated by PKA in cells treated with hormones or other agonists which activate PKA, was studied in vivo. RAC, by itself, was capable of causing an increase in the phosphorylation of the H1 histone at site A, demonstrating that the retinoid-mediated increase in PKA activity was sufficient to cause the enhanced phosphorylation of a known substrate.
Assuntos
Transformação Celular Neoplásica , Proteínas Quinases/metabolismo , Vitamina A/análogos & derivados , Animais , Núcleo Celular/enzimologia , Células Cultivadas , Citosol/enzimologia , Diterpenos , Histonas/isolamento & purificação , Histonas/metabolismo , Camundongos , Mapeamento de Peptídeos , Fosfopeptídeos/isolamento & purificação , Proteínas Quinases/isolamento & purificação , Ésteres de Retinil , Vitamina A/farmacologiaRESUMO
The phosphorylation of H1 histones is reviewed. Consideration is given to phosphorylation reactions which occur in both replicating and nonreplicating cells. The available evidence suggests that H1 histones accept phosphate groups at different sites in response to different stimuli. The tentative location of the acceptor sites is summarized, and the effects of site-specific phosphorylation on the conformation of H1 histones in vitro is discussed. The phosphorylation of H1 histones which occurs during cell replication is reviewed in detail, and it is concluded that there is no clocklike mechanism which couples the phosphorylation of a particular site or region in H1 histones to a set point in the cell cycle. There is both species-and cell-specific variability in the phosphorylation of H1 histones during cell replication. Recent studies are discussed which show that an interspecific somatic cell hybrid of mouse and Chinese hamster can replicate the Chinese hamster genome in a stable manner using only mouse H1 histones and their phosphorylated forms. I speculate that H1 histone phosphorylation is a mechanism for the relaxation of long-term structures needed for differential gene activity in order to attain the short-term goal of genome replication.
Assuntos
Histonas/biossíntese , Histonas/metabolismo , Animais , Sítios de Ligação , Ciclo Celular , Divisão Celular , Linhagem Celular , Cromatina/metabolismo , Cricetinae , Cricetulus , Humanos , Células Híbridas/metabolismo , Camundongos , Fosforilação , Conformação Proteica , Ratos , Especificidade da EspécieRESUMO
The phosphorylation of the NH2- and COOH-terminal regions of H1 histones from normal mouse mammary glands, a mouse breast tumor (D1 tumor) and LM/TK- cells was compared. Chromatographic resolution of H1 subtypes revealed that the normal and transformed cells expressed the same H1 subtypes. Bisection of purified 32P-labeled H1 histones with N-bromosuccinimide, followed by fractionation of the NH2- and COOH-terminal regions of the molecules on Biogel P-60, demonstrated that explants of normal mouse breast tissue preferentially phosphorylated the NH2-terminal region (61% of the 32P) while the D1 tumor preferentially phosphorylated the COOH-terminal region (60% of the 32P). In keeping with numerous other examples, LM/TK- cells in culture also preferentially phosphorylated the COOH-terminal region of the H1 molecule (64% of the 32P).
Assuntos
Transformação Celular Neoplásica/metabolismo , Histonas/metabolismo , Sequência de Aminoácidos , Animais , Feminino , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação , GravidezRESUMO
Crude extracts from replicating normal and transformed cells were assayed for protein kinase activities specific for different sites in purified Hl histone in vitro. Extracts from normal cells favored the NH2-terminal region while extracts from transformed cells favored the COOH-terminal region. Analysis of phosphopeptides demonstrated that histone kinases from both normal and transformed cells catalyzed the phosphorylation of a number of sites in common, and these were typical of sites phosphorylated in replicating cells. The preference for the NH2-terminal region by extracts from normal cells was due to the extensive phosphorylation of a site previously shown to be phosphorylated by cyclic AMP-dependent protein kinase. This activity was very low in transformed cells.
Assuntos
Transformação Celular Neoplásica/metabolismo , Protamina Quinase/metabolismo , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Linhagem Celular , Histonas/metabolismo , CamundongosRESUMO
Information on the incidence of myofibrillary degeneration (MFD) was obtained by investigating the hearts of seven groups of cases with different forensic diagnosis using luxol fast blue as a staining method. The following diagnostic groups were examined: acute morphine intoxication, acute carbon monoxide intoxication, hanging, strangling by hand and/or ligature, drowning, acute hemorrhagic shock and sudden infant death syndrome. MFD positive cases were found in all diagnostic groups, but the morphologic expression of MFD as well the number of cases per group differed: The phenomenon of cytoplasmatic banding could be observed in nearly all cases of hanging, strangling and acute hemorrhagic shock; the phenomenon of diffuse cytoplasmatic staining in most cases of drowning. The diagnostic importance is discussed.
Assuntos
Cardiomiopatias/patologia , Causas de Morte , Miocárdio/patologia , Miofibrilas/ultraestrutura , Adulto , Homicídio/legislação & jurisprudência , Humanos , Lactente , Morte Súbita do Lactente/patologia , Suicídio/legislação & jurisprudênciaRESUMO
A new method of nondenaturing gel electrophoresis and a specific peptide based assay were used to study the histone kinases in mitotic and interphase mouse fibroblasts. The gels resolved four activities, one of which was shown to be the mitotic (CDC2) H1 kinase by virtue of its antigenicity. A new peptide substrate for the CDC2 kinase was phosphorylated by both S-phase and mitotic cell extracts and reacted with two protein kinases in the gels. Since the interphase enzyme did not react with the antibody, the results suggest that it is either a "masked" form of CDC2 or a second enzyme, functionally related to CDC2, which is responsible for the interphase phosphorylation of H1.
Assuntos
Histonas/análise , Interfase , Fosfoproteínas/análise , Proteínas Quinases/análise , Animais , Western Blotting , Proteína Quinase CDC2 , Eletroforese em Gel de Poliacrilamida , Fibroblastos/fisiologia , Técnicas In Vitro , Camundongos , Fosforilação , Especificidade por SubstratoRESUMO
The phosphorylation of H1 histone subtypes was studied in 3 Chinese hamster cell lines (CHO, V79, and CHW). Chromatographic resolution of H1 subtypes showed that all 3 cell lines contained 1 homologous (coeluting) H1 subtype (CHO-1, V79-1, and CHW-1) while V79 and CHW cells contained 2 additional H1 subtypes not found in CHO cells (V79-2,3 and CHW-2,3). N-Bromosuccinimide cleavage of 32P-labeled H1 subtypes demonstrated that all V79 subtypes were phosphorylated in both the NH2- and COOH-terminal regions during interphase while CHO-1 was phosphorylated only in the COOH-terminal region. Tryptic phosphopeptide fractionations, using 2 sequential electrophoretic steps on paper, demonstrated qualitative differences in the 32P-labeled peptides from the 7 H1 subtypes of the 3 cell lines. For example, CHO-1 differed from its V79-1 homologue by 1 phosphopeptide and from its CHW-1 homologue by 3 phosphopeptides. Phosphopeptide differences were also observed among the H1 subtypes of both V79 and CHW cells. The results demonstrate that Chinese hamster cell lines phosphorylated H1 histone subtypes differently during interphase and that there is no rigorous functional connection between the phosphorylation of the NH2-terminal region of 1 or all H1 histone subtypes and the initiation of mitosis in Chinese hamster cells.