RESUMO
Organic materials, including carbon, exist inside the transmission electron microscope (TEM) chamber and are adsorbed onto samples under observation during TEM. When these adsorbed organic materials are irradiated by an electron beam, the adsorbed gas is decomposed. Carbon atoms remain on the sample and bond with each other forming a material with an amorphous structure. Due to the carbon deposition on the observation area of the sample, it is contaminated and the TEM image quality is decreased. Ar was introduced into environmental TEM (ETEM) to purge organic material from the sample chamber to reduce contamination growth. After Ar gas was introduced, the contamination was gradually removed. The contamination removal rate was dependent on the Ar pressure. Moreover, it was clear that Ar was ionised by electron beam irradiation and the Ar ions were produced in the ETEM during electron beam irradiation. It is proposed that the Ar ions removed the carbon contamination. LAY DESCRIPTION: Organic materials, including carbon, exist inside the transmission electron microscope (TEM) chamber and are adsorbed onto samples under observation during TEM. When these adsorbed organic materials are irradiated by an electron beam, the adsorbed gas is decomposed. Carbon atoms remain on the sample and bond with each other forming a material with an amorphous structure. Due to the carbon deposition on the observation area of the sample, it is contaminated and the TEM image quality is decreased. Ar was introduced into environmental TEM (ETEM) to purge organic material from the sample chamber to reduce contamination growth. After Ar gas was introduced, the contamination was gradually removed. The contamination removal rate was dependent on the Ar pressure. Moreover, it was clear that Ar was ionised by electron beam irradiation and the Ar ions were produced in the ETEM during electron beam irradiation. It is proposed that the Ar ions removed the carbon contamination.
RESUMO
Flow cytometric and immunocytochemical analyses of murine fetal thymus (FT) cells with antibodies to various surface markers and transcription factors reveal that the synthesis of TCF-1 and GATA-3 protein begins simultaneously in a fraction of the most immature population of FT cells, which have the phenotype of CD4-CD8-CD44+CD25-. No TCF-1-producing cells is found in the fetal liver (FL). In CD44+CD25- FT cells, the production of TCF-1 is immediately followed by intracellular expression of CD3 epsilon. It is also found that the T cell development from FL, but not FT, progenitors in the FT organ culture system is severely inhibited by the addition of antisense oligonucleotides for either TCF-1 or GATA-3. These results strongly suggest that TCF-1 and GATA-3 play essential roles in the initiation of the earliest steps of T cell development in the thymus.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Linfócitos T/citologia , Timo/citologia , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Dedos de Zinco , Células 3T3 , Animais , Anticorpos , Northern Blotting , Fator de Transcrição GATA3 , Haplorrinos , Fator 1-alfa Nuclear de Hepatócito , Fator de Transcrição Ikaros , Fator 1 de Ligação ao Facilitador Linfoide , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Coelhos , Fator 1 de Transcrição de Linfócitos T , Células Tumorais CultivadasRESUMO
A disintegrin and metalloproteinase (ADAM) represents a protein family possessing both metalloproteinase and disintegrin domains. ADAMTS-1, an ADAM family member cloned from cachexigenic colon adenocarcinoma, is unusual in that it contains thrombospondin type I motifs and anchors to the extracellular matrix. To elucidate the biological role of ADAMTS-1, we developed ADAMTS-1-null mice by gene targeting. Targeted disruption of the mouse ADAMTS-1 gene resulted in growth retardation with adipose tissue malformation. Impaired female fertilization accompanied by histological changes in the uterus and ovaries also resulted. Furthermore, ADAMTS-1(-/-) mice demonstrated enlarged renal calices with fibrotic changes from the ureteropelvic junction through the ureter, and abnormal adrenal medullary architecture without capillary formation. ADAMTS-1 thus appears necessary for normal growth, fertility, and organ morphology and function. Moreover, the resemblance of the renal phenotype to human ureteropelvic junction obstruction may provide a clue to the pathogenesis of this common congenital disease.
Assuntos
Desintegrinas/fisiologia , Fertilidade/fisiologia , Crescimento/fisiologia , Metaloendopeptidases/fisiologia , Proteínas ADAM , Proteína ADAMTS1 , Glândulas Suprarrenais/anormalidades , Animais , Desintegrinas/química , Desintegrinas/genética , Feminino , Fertilidade/genética , Crescimento/genética , Humanos , Infertilidade Feminina/etiologia , Rim/anormalidades , Masculino , Metaloendopeptidases/química , Metaloendopeptidases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovário/anormalidades , Fenótipo , Gravidez , Útero/anormalidadesRESUMO
The physical and reproductive traits known to be risk factors for breast cancer were correlated with familial predisposition for this disease by the use of hospital records of 3,867 patients with breast cancer, of whom 186 had one or more relatives with breast cancer and were called "familial patients." Various characteristics in the total series of patients were compared with familial patients matched for year of birth. The mean age at menarche was significantly less in familial compared with the total series of patients, especially for premenopausal patients; however, no difference was observed in the mean age at marriage between the two groups. Familial patients were slightly taller as compared to the total series of patients, but no difference was detected in body weight between the two groups. Because age at menarche and the stature are highly correlated, they may be influenced by an underlying common hormone effect that may influence the familial nature of breast cancer.
Assuntos
Neoplasias da Mama/genética , Adulto , Fatores Etários , Idoso , Estatura , Peso Corporal , Neoplasias da Mama/epidemiologia , Feminino , Humanos , Japão , Estilo de Vida , Casamento , Menarca , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
The basal plasma concentration of prolactin was determined by radioimmunoassay in Caucasian women at different risk for breast cancer and in three cases of women at risk for breast cancer. Samples taken under comparable conditions showed similar basal levels at birth and in prepubertal Bantu and Caucasian girls, or in healthy pre- and postmenopausal Bantu, Caucasian, or Japanese women. Elevated plasma prolactin levels were found in women castrated prior to 35 years of age and in women whose first pregnancy occured after 35 years. Evidence indicates that the prolactin/estrogen relationship may not be similar in women 35 to 45 years of age as compared to young women and suggests that elevation of serum prolactin per se does not appear to be related to an increased risk of breast cancer. The prolactin level was increased only in Caucasian women with breast cancer. If a high prolactin/estradiol ratio increases the susceptibility of the mammary epithelium to neoplastic growth, the lack of changes in prolactin levels in premenopausal Japanese patients and in postmenopausal patients of the three ethnic groups indicates that other factors are involved. Further study of the effects of life-style and diet on the basal level and stimulated release of prolactin is required to resolve the relationship of prolactin to breast cancer.
Assuntos
Neoplasias da Mama/sangue , Prolactina/sangue , Adulto , Fatores Etários , Envelhecimento , Povo Asiático , População Negra , Castração , Criança , Ritmo Circadiano , Climatério , Estradiol/sangue , Feminino , Sangue Fetal/análise , Humanos , Recém-Nascido , Menopausa , Menstruação , Pessoa de Meia-Idade , Gravidez , Radioimunoensaio , Risco , População BrancaRESUMO
It has been suggested that the urinary hormone profile is subject to environmental changes, such as urbanization and Westernization, and that the composition of the hormones can be used as a discriminate in determining the patient at risk for breast cancer. In this study, a comparison of the plasma hormone levels in Bantu and Japanese women, low-risk populations, and Caucasian women, a high-risk population, showed a higher level of 17 beta-estradiol in prepubertal girls and young Bantu and Japanese women. The higher estrogen level in the Bantu was evident in the early luteal and late follicular phases of the menstrual cycle. The difference in the dehydroepiandrosterone and testosterone levels in Bantu young women and prepubertal Bantu girls, suggests differences in adrenal activity between Bantu and Caucasian women. A fall in the plasma androstenedione was evident in postmenopausal Bantu and Japanese but not Caucasian women. Data suggest that the hormone profile is different among the 3 ethnic groups in both the pre- and postmenopausal women. Since the daily life-style of the women is comparable, it is suggested that the composition of the hormone profile is partially dependent on dietary on dietary factors.
Assuntos
Hormônios Esteroides Gonadais/sangue , Grupos Raciais , Glândulas Suprarrenais/fisiologia , Adulto , África , Androstenodiona/sangue , Animais , Povo Asiático , População Negra , Neoplasias da Mama/epidemiologia , Neoplasias da Mama/etiologia , Embrião de Galinha , Criança , Desidroepiandrosterona/sangue , Dieta , Estradiol/sangue , Estrogênios/sangue , Estrona/sangue , Feminino , Hormônio Foliculoestimulante/sangue , Humanos , Recém-Nascido , Japão , Hormônio Luteinizante/sangue , Menopausa , Menstruação , Testosterona/sangue , População BrancaRESUMO
Two clones, one cachexigenic (clone 20) and the other noncachexigenic (clone 5), from a murine colon adenocarcinoma, colon 26 cells, were used to analyze the involvement of immune reactions as well as the cytokine network in cachexia. Clone 20 induced cachexia in nude and SCID mice as well as in normal BALB/c mice, suggesting that lymphocytes played little, if any, role in the process. Both clones failed to express mRNA of interleukin (IL) 1 alpha, IL-1 beta, IL-6, and tumor necrosis factor alpha in vitro with or without the coculture of NIH3T3 cells or spleen cells. However, IL-6 mRNA was selectively detected at the tumor site of clone 20 but not at that of clone 5-bearing mice. In contrast, tumor necrosis factor alpha mRNA was detected at tumor sites and in spleens of only clone 5-bearing mice, suggesting a potential role of IL-6, but not tumor necrosis factor alpha, in inducing cachexia. Anti-IL-6 antibody partially reversed the weight loss induced by clone 20, whereas the continuous infusion of IL-6 failed to cause weight loss, despite being associated with an elevation of a serum acute phase protein. These results suggest that IL-6 is necessary but not sufficient for the induction of cachexia. Both clones expressed IL-6 mRNA in the presence of IL-1 in vitro, and mice bearing either clone expressed IL-1 beta mRNA at the tumor site. Moreover, IL-1 receptor antagonist (IL-1Ra) mRNA was detected at the tumor site of clone 5-bearing mice but not at that of clone 20-bearing mice, suggesting that IL-1Ra might block IL-1 activity to reduce IL-6 production in clone 5-bearing mice. However, the transfection of clone 20 with IL-1Ra cDNA failed to abolish its capacity to produce IL-6 and to cause cachexia. Collectively, additional factor(s) besides IL-1Ra and IL-1 beta may control IL-6 and some other cachexigenic factor production, thereby causing cachexia in this model.
Assuntos
Adenocarcinoma/complicações , Caquexia/etiologia , Neoplasias do Colo/complicações , Citocinas/fisiologia , Adenocarcinoma/imunologia , Animais , Anticorpos/farmacologia , Sequência de Bases , Peso Corporal/fisiologia , Caquexia/imunologia , Caquexia/prevenção & controle , Células Clonais , Neoplasias do Colo/imunologia , Citocinas/genética , Feminino , Interleucina-6/imunologia , Linfócitos/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Interleucina-1/genética , Transfecção , Células Tumorais CultivadasRESUMO
Exogenous ubiquinone-10 was efficiently reduced by rat liver microsomes in the presence of NADH and NADPH under anaerobic conditions. Ubiquinone-10 reduced under anaerobic conditions was rapidly re-oxidized by the re-aeration. The reduction and re-oxidation were not observed when the reactions were carried out with the boiled microsomes or without microsomes, suggesting that the reactions were enzymatically catalyzed by the electron transport system(s) from NAD(P)H to O2 through the ubiquinone. The Km and Vmax of the reductase activity for NADH were 0.4 mM and 1.7 nmol/min per mg of protein, and those for NADPH were 19 microM and 2.1 nmol/min per mg of protein, respectively. The NADH-dependent oxidoreduction system was different from the NADPH-dependent system because of the following observations; (1) rotenone inhibited only the NADH-dependent ubiquinone-10 reductase, (2) dicoumarol inhibited the NADPH-dependent ubiquinone-10 reduction more potently than the NADH-dependent reduction and (3) the activity oxidizing the reduced ubiquinone-10 in the presence of NADH was less than that in the presence of NADPH. Endogenous ubiquinone-9 was also reduced and re-oxidized in essentially the same manner as exogenous ubiquinone-10. Thus, ubiquinone-10 oxidoreductase in rat liver microsomes acts on endogenous ubiquinone-9.
Assuntos
Microssomos Hepáticos/enzimologia , NAD(P)H Desidrogenase (Quinona)/metabolismo , NADP/metabolismo , Animais , Concentração de Íons de Hidrogênio , Cinética , Magnésio/farmacologia , Masculino , Oxirredução , Ratos , Ratos Wistar , Rotenona/farmacologia , Ubiquinona/metabolismoRESUMO
A group of 243 patients with gastric cancer was subjected to a prospective randomized trial of adjuvant chemotherapy after curative gastrectomy. One of the three arms (regimen A) is induction therapy with a three-drug combination of mitomycin C (MMC), 5-fluorouracil (5-FU), and cytosine arabinoside (ara-C) followed by intermittent oral administration of 5-FU for two years (MFC + F). In regimen B, 5-FU in regimen A is replaced by ftorafur, administered on the same schedule as regimen A (MF'C + F'). Regimen C is the control, in which patients are treated by surgery alone. Regimen A was proven effective for the suppression of relapse in the limited groups of moderately locally advanced cancer; five-year survival rates of the subset of stages I, II, and III were 72.1% with regimen A and 53.1% with regimen C (P less than .05). Regimen B yielded a better survival than the control, although the difference did not reach statistical significance. The results seem to favor 5-FU as adjuvant chemotherapy for gastric cancer.
Assuntos
Fluoruracila/análogos & derivados , Fluoruracila/administração & dosagem , Neoplasias Gástricas/terapia , Tegafur/administração & dosagem , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Terapia Combinada , Feminino , Fluoruracila/efeitos adversos , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/cirurgia , Tegafur/efeitos adversosRESUMO
To clarify the role of disintegrin-like and metalloproteinase with thrombospondin type I motifs-1 (ADAMTS-1) in ovarian function, we examined abnormalities in ovulatory processes, folliculogenesis and the vascular system of ADAMTS-1 null ovaries. First, when immature female mice were treated with pregnant mare serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG), the number of ovulated oocytes was markedly decreased in ADAMTS-1 null mice in comparison to ADAMTS-1 (+/-) controls. The proportion of anovulated follicles to total mature follicles was significantly higher in ADAMTS-1 null females when compared with controls. The numbers of growing follicles at each stage were counted. The number of follicles at type 5b (late preantral) and later stages was markedly reduced in ADAMTS-1 null mice, irrespective of gonadotropin treatment (no gonadotropins, PMSG alone or PMSG/hCG). These data demonstrate that impairment of ovarian function to ovulate oocytes in ADAMTS-1 null mice occurs at two different levels: in the development of growing follicles and ovulatory processes. Furthermore, ADAMTS-1 null ovaries included a number of unusual atretic follicles that showed no sign of oocyte degeneration but lost the surrounding granulosa cell layers and were considered to be derived from type 4 or 5a follicles. These results suggest that ADAMTS-1 is important for follicular development beyond the type 4 and/or 5a and for maintaining normal granulosa cell layers in follicles. Finally, the number of large blood vessels in the medullar zone was significantly decreased in ADAMTS-1 null mice ovaries, suggesting that ADAMTS-1 is also involved in the organization of the medullary vascular network.
Assuntos
Proteínas ADAM/metabolismo , Folículo Ovariano/crescimento & desenvolvimento , Ovário/irrigação sanguínea , Ovulação/fisiologia , Proteínas ADAM/genética , Proteína ADAMTS1 , Animais , Gonadotropina Coriônica/metabolismo , Feminino , Atresia Folicular/metabolismo , Gonadotropinas Equinas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oócitos/fisiologia , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Folículo Ovariano/patologia , Ovário/metabolismo , GravidezRESUMO
Interleukin-1 (IL-1) exerts pleiotropic effects on a variety of tissues through binding to its receptor. Two distinct types of receptors for IL-1 have been characterized in mouse and human. Most of the IL-1 signal has been shown to be transmitted through type I IL-1R (80 kDa) in T lymphocytes as well as B lymphocytes and monocytes. Type II receptor may act as a suppressor of IL-1 biological activities by competing in binding with type I receptors on the cell surface. Functional studies of the type I IL-1R demonstrated that the cytoplasmic segments, possessing a sequence similarity with the Drosophila Toll gene product or IL-6R beta chain, gp130, are important for transmitting activity that induces cytokine genes. In the past three years, several groups reported that IL-1 and tumor necrosis factor (TNF) rapidly induce sphingomyelin turnover in various types of cells, producing ceramide, which may act as a second messenger molecule in an intracellular signaling cascade. Activation of both acid and neutral sphingomyelinases (SMases) has been suggested, and Schutz et al. proposed that the phosphatidylcholine-phospholipase C/acid SMase pathway is involved in TNF-induced NF-kappa B activation. However, our recent study showed that the NF-kappa B activation is induced by IL-1/TNF in fibroblasts from patients with type A Niemann-Pick disease, with acid SMase deficiency. This finding implies that acid SMase activity is not essential for the activation of NF-kappa B by IL-1/TNF at least in fibroblasts. Other signaling pathways including neutral SMase and unidentified protein kinases may be important for NF-kappa B-mediated cytokine gene activation.
Assuntos
Interleucina-1/metabolismo , Receptores de Interleucina-1/fisiologia , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Linhagem Celular , Ceramidas/metabolismo , Sequência Conservada , Proteína Quinase Tipo II Dependente de AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática , Fibroblastos/metabolismo , Humanos , Dados de Sequência Molecular , NF-kappa B/metabolismo , Proteína Quinase C/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Interleucina-1/química , Receptores de Interleucina-1/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Esfingomielinas/metabolismo , Relação Estrutura-AtividadeRESUMO
Lipopolysaccharide (LPS), a constituent of gram-negative bacteria cell wall, plays an essential role in the pathogenesis of septic shock by generating endogenous mediators such as cytokines, nitrous oxide, superoxide anions, and lipid mediators. In vitro, LPS induces the transcription of a set of genes involved in inflammatory reactions by activating several types of transcription factors, particularly nuclear factor-kappaB (NF-kappaB). An analysis of NF-kappaB activation using a cell-free system demonstated that two pathways converge to activate NF-kappaB; one is staurosporine-sensitive, the other is staurosporine-insensitive and tyrosine kinase-dependent. Furthermore, the latter pathway culminates in IkappaBalpha phosphorylation at serine/threonine residues in its carboxyl-terminal acidic region with dissociation of IkappaBalpha from NF-kappaB, thereby activating NF-kappaB. The requirement for the phosphorylation at this site was confirmed by the specific inhibition of NF-kappaB activation in a cell-free system by the synthetic peptide corresponding to this site. The in vivo administration of an anti-CD18 antibody prevented elevation of plasma tumor necrosis factor (TNF) levels and acute lethality induced by injection of a low dose of LPS into Propionibacterium acnes-primed rabbits or by the administration of a single high dose of LPS into animals. Anti-CD18 also prevented acute lethality induced by one of the main mediators of endotoxin shock, TNF-alpha. Furthermore, an antibody to a ligand for CD18, intercellular adhesion molecule-1, also prevented TNF-induced shock as well as endotoxin shock in rabbits. These observations suggest that the interaction between leukoytes and endothelium through beta2- integrin adhesion molecules may be of primary importance in mediating LPS signals in vivo.
Assuntos
Antígenos CD18/fisiologia , Citocinas/biossíntese , Receptores de Lipopolissacarídeos/fisiologia , NF-kappa B/fisiologia , Choque Séptico/etiologia , Transdução de Sinais/fisiologia , Animais , Sistema Livre de Células , Citocinas/fisiologia , Humanos , Choque Séptico/fisiopatologiaRESUMO
cDNA cloning revealed the presence of two related but distinct types of human interleukin-8 (IL-8) receptors, type I (type A) and type II (type B). By immunizing rabbits with glutathione-S-transferase fused with the NH2-terminal domain of each type of IL-8 receptor, we prepared polyclonal antibodies that specifically recognized the NH2-terminal domain of each type of IL-8 receptor. Immunofluorescence analysis of human peripheral blood leukocytes demonstrated that mature granulocytes except eosinophils express both types of IL-8 receptors. A majority of monocytes and CD16+ natural killer (NK) cells in peripheral blood were stained with both antibodies, whereas CD3+ T or CD20+ B lymphocytes in peripheral blood or CD34+ cells in cord blood were not stained with either antibody. These results suggest that both types of human IL-8 receptors were coordinately and selectively expressed in mature granulocytes, monocytes, and CD16+ NK cells.
Assuntos
Células Matadoras Naturais/química , Monócitos/química , Neutrófilos/química , Receptores de Interleucina/análise , Sequência de Aminoácidos , Sequência de Bases , Sequência Conservada , Sangue Fetal/citologia , Citometria de Fluxo , Imunofluorescência , Glutationa Transferase/imunologia , Glutationa Transferase/metabolismo , Granulócitos/química , Granulócitos/citologia , Granulócitos/ultraestrutura , Humanos , Células Matadoras Naturais/citologia , Células Matadoras Naturais/ultraestrutura , Leucócitos/química , Leucócitos/citologia , Leucócitos/ultraestrutura , Dados de Sequência Molecular , Monócitos/citologia , Monócitos/ultraestrutura , Neutrófilos/citologia , Neutrófilos/ultraestrutura , Receptores de Interleucina/química , Receptores de Interleucina/imunologia , Receptores de Interleucina-8A , Alinhamento de SequênciaRESUMO
Blocking monoclonal antibodies (mAbs) specific to mouse interleukin-1 receptor antagonist (IL-1ra) were prepared by immunizing Armenian hamsters with recombinant mouse IL-1ra. A sensitive and specific ELISA against mouse IL-1ra was also established. In Propionibacterium acnes-induced liver injury, P. acnes induced transient increase of serum tumor necrosis factor-alpha levels but not those of IL-1ra, IL-1, and IL-6. However, subsequent lipopolysaccharide (LPS) challenge induced the increase of serum levels of all these cytokines and the peak serum IL-1ra level was more than 20 times as high as serum IL-1 levels. Immunohistochemical analysis demonstrated that IL-1ra was predominantly produced by hepatocytes during the course of the priming phase by P. acnes and eliciting phase by LPS challenge. Furthermore, the administration of a mAb to mouse IL-1ra exacerbates the liver injury induced by P. acnes and sublethal dose of LPS, suggesting a protective role of endogenous IL-1ra in this liver injury model.
Assuntos
Citocinas/fisiologia , Hepatite/fisiopatologia , Sialoglicoproteínas/fisiologia , Animais , Anticorpos Monoclonais/imunologia , Reações Antígeno-Anticorpo , Cricetinae , Cricetulus , Modelos Animais de Doenças , Hepatite/patologia , Proteína Antagonista do Receptor de Interleucina 1 , Interleucina-1/metabolismo , Masculino , Camundongos , Propionibacterium acnes/patogenicidade , Coelhos , Sialoglicoproteínas/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Since plasmids containing autonomously replicating sequence(s) (ARS) can transform Saccharomyces cerevisiae cells at high frequency, ARS are considered to be the replication origins of chromosomes. To study the mechanism of initiation of eukaryotic chromosomal replication, we examined protein factors which interact with the ARS1 region located near the centromere of chromosome IV in S. cerevisiae. Using the gel-shift assay, we found protein factors which bound to a single-stranded, 97-bp fragment of the ARS1 region containing the core consensus. Competition experiments with various oligodeoxyribonucleotides (oligos) suggest that a site recognized by the factor(s) was within the element containing the core consensus and adjacent close matches to the core consensus of the minus strand. Indeed, when the oligo containing the minus strand of this element was used as a probe, two oligo-protein complexes were detected. Mutations in the core consensus reduced these binding activities. When the plus-strand oligo of the same region was used as a probe, a retarded band was also detected, but with less specific binding. Considering the fact that the core consensus and close matches to the core consensus are important for ARS function, these results imply that the protein factors detected in this experiment may participate in DNA replication.
Assuntos
Replicação do DNA , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Fatores de Transcrição , Sequência de Bases , Ligação Competitiva , Sondas de DNA , Eletroforese em Gel de Poliacrilamida , Dados de Sequência MolecularRESUMO
A mouse cDNA library was screened using a DNA fragment generated by polymerase chain reaction (PCR) with oligodeoxyribonucleotide primers which were derived from the conserved sequences in cDNAs encoding the human and rabbit interleukin-8 receptors (hIL-8R and rIL-8R). A novel cDNA was obtained encoding 359 amino acids (aa) with seven putative transmembrane portions similar to hIL-8R and rIL-8R. Its aa sequence shows 64 and 69% homology to those of type-1 and type-2 hIL-8R, respectively. COS-7 cells transfected with the isolated cDNA in a mammalian expression vector bind IL-8, but do not bind a related protein, monocyte chemotactic and activating factor, suggesting that the isolated cDNA encodes the mouse homolog of IL-8R. Northern blot analysis showed that mRNA of this clone was highly expressed in mouse peritoneal neutrophils, and the single band was observed in Southern blotting analysis on mouse genomic DNA digested with HindIII or KpnI, suggesting that this is a single-copy gene.
Assuntos
Clonagem Molecular , Interleucina-8/metabolismo , Receptores de Interleucina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , DNA Complementar/química , DNA Complementar/genética , Humanos , Camundongos , Dados de Sequência Molecular , Neutrófilos/química , RNA Mensageiro/análise , Coelhos , Receptores de Interleucina/metabolismo , Receptores de Interleucina-8A , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
A disintegrin-like and metalloproteinase with thrombospondin type I motifs-1 (ADAMTS-1) is an extracellular matrix-anchored metalloproteinase. In this study we have demonstrated that ADAMTS-1 is able to cleave a major cartilage proteoglycan, aggrecan. N-terminal sequencing analysis of the cleavage product revealed that ADAMTS-1 cleaves the Glu(1871)-Leu(1872) bond within the chondroitin sulfate attachment domain of aggrecan. In addition, deletional analysis demonstrated that the C-terminal spacer region of ADAMTS-1 is necessary to degrade aggrecan. These results suggest that ADAMTS-1 may be involved in the turnover of aggrecan in vivo.
Assuntos
Cartilagem/química , Desintegrinas/metabolismo , Proteínas da Matriz Extracelular , Metaloendopeptidases/metabolismo , Proteoglicanas/metabolismo , Proteínas ADAM , Proteína ADAMTS1 , Agrecanas , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Bovinos , Sulfatos de Condroitina/metabolismo , Desintegrinas/genética , Lectinas Tipo C , Metaloendopeptidases/genética , Camundongos , Dados de Sequência Molecular , Peso Molecular , Mutação/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteoglicanas/química , Proteoglicanas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de Proteína , Especificidade por SubstratoRESUMO
It was recently found that certain cells in the alveolar septum of the bovine lung are enriched in prostaglandin F (PGF) synthase. In this study we used immunohistochemical techniques at both light and electron microscopic levels to further characterize the PGF synthase-positive cells. By double immunofluorescence staining of bovine lung cryostat sections, the alveolar septal cells labeled by anti-PGF synthase antibody were also intensely labeled for cytoplasmic actin but not for alpha-smooth muscle actin. This labeling pattern suggests that the PGF synthase-positive cells in the septum are "contractile interstitial cells," which resemble conventional fibroblasts but characteristically contain prominent bundles of actin filaments. Immunogold electron microscopy of ultra-thin frozen sections of bovine lung showed that alveolar interstitial cells extending long cytoplasmic processes and closely associated with alveolar capillaries were intensely labeled for PGF synthase. Capillary endothelial cells, alveolar epithelial cells, and some fibroblastic cells were devoid of labeling. On the basis of these findings, we conclude that PGF synthase is specifically expressed in contractile interstitial cells within the alveolar septum. The protein may be a useful marker for contractile interstitial cells, whose physiological function and role in various pathological conditions have not been characterized in detail.
Assuntos
Hidroxiprostaglandina Desidrogenases/análise , Pulmão/enzimologia , Animais , Bovinos , Imuno-Histoquímica , Pulmão/citologia , Pulmão/ultraestrutura , Microscopia ImunoeletrônicaRESUMO
OBJECTIVE: The aim of this study was to determine the timing of brain injury in infants with periventricular leukomalacia (PVL) with serial electroencephalography (EEG) recordings during the neonatal period. PATIENTS AND METHODS: We evaluated 172 preterm infants having a gestational age <33 weeks and weighing <2000 g. Initial EEG was recorded within 72 hours of life and then recorded once every 1 to 4 weeks. Serial cranial ultrasonography was performed and cystic PVL was diagnosed when multiple cystic formations of >3 mm in diameter were observed. RESULTS: Of the 172 infants studied, 26 were diagnosed as having cystic PVL by ultrasonography. EEG abnormalities were observed in 25 of 26 infants with PVL, although EEG abnormalities were seen in 20 of 146 infants without PVL. The initial EEG recordings were normal in 7 infants, but EEG abnormalities were observed later in 6 of these infants. In these 6 infants, the timing of injury was presumed to be postpartum. Only acute stage abnormalities were observed on initial EEG recording in 14 infants, and the timing of injury was presumed to be just before or around birth. Chronic stage abnormalities were recognized already on initial EEG recordings in the other 5 infants, and the timing of injury was presumed to be some time before birth. CONCLUSIONS: Our study indicates that it may be possible to determine the timing of injury in infants with PVL by serial EEG recordings.
Assuntos
Eletroencefalografia , Doenças do Prematuro/diagnóstico , Leucomalácia Periventricular/diagnóstico , Paralisia Cerebral/diagnóstico , Paralisia Cerebral/etiologia , Desenvolvimento Infantil , Ecoencefalografia , Humanos , Recém-Nascido , Doenças do Prematuro/diagnóstico por imagem , Leucomalácia Periventricular/complicações , Fatores de TempoRESUMO
UNLABELLED: Iodine-123-MIBG imaging has been used to evaluate myocardial sympathetic function in various cardiac diseases. In patients with obstructive sleep apnea syndrome (OSAS), increased sympathetic activity has been widely recognized, as assessed by measuring the plasma concentration and urinary excretion of catecholamines and by measuring muscle sympathetic nerve activity. However, these measurements are not specific indices of cardiac sympathetic function. Therefore, this study was undertaken to assess cardiac sympathetic function in patients with OSAS using MIBG cardiac scintigraphy. METHODS: This study consisted of 11 patients (10 men, 1 woman; mean age 43 +/- 16 yr) with a diagnosis of OSAS established by polysomnography, and 8 age-matched normal control subjects (7 men, 1 woman; mean age 45 +/- 18 yr). Early (15 min) and delayed (3 hr) planar images were taken after the injection of 111 MBq of [123I]MIBG. The mean counts of the whole heart and the mediastinum were obtained to calculate heart-to-mediastinum count ratios from the early images (H/Me) and from the delayed images (H/Md) and the myocardial washout rate (WR). Eight patients were restudied after 1 mo of nasal continuous positive airway pressure treatment. RESULTS: The H/Me and H/Md ratios were significantly lower in the patients than in the control subjects (H/Me, 2.49 +/- 0.32 versus 2.84 +/- 0.34, p = 0.0207; and H/Md, 2.33 +/- 0.30 versus 3.02 +/- 0.36, p = 0.0013). The WR was higher in the patients than in the control subjects (36.2 +/- 9.0% versus 23.6 +/- 4.9%, p = 0.0022). The H/Me and H/Md ratios in the patients were significantly correlated with the apnea-hypopnea index and the degree of hypoxemia during sleep. After treatment, H/Me and H/Md remained unchanged, but WR significantly recovered (from 34.9 +/- 10.4% to 26.3 +/- 7.7%, p = 0.0357). CONCLUSION: Cardiac sympathetic function and integrity are impaired in subjects with OSAS when compared with age-matched control subjects. MIBG cardiac imaging can be helpful in evaluating cardiac involvement and efficacy of therapy in OSAS.