RESUMO
Although a molecular diagnostic assay using clinically accessible tissue, such as blood, would facilitate evaluation of disease conditions in humans and animals, little information exists on microarray-based gene expression profiling of circulating leukocytes from clinically hypocalcemic cows. Therefore, peripheral blood mononuclear cells from dairy cows with experimentally induced hypocalcemia or spontaneous milk fever were subjected to oligo-microarray analysis to identify specific biomarker genes. In experimental hypocalcemia induced by a 4-h infusion of 10% disodium EDTA (n=4), 32 genes were significantly up- or downregulated compared with control treatment (4-h infusion of 11% calcium EDTA; n=4). In cows with milk fever (n=8), 98 genes were expressed differentially (either up- or downregulated) compared with healthy parturient cows (n=5). From these data, the following 5 genes were selected as being strongly related to both experimental hypocalcemia and milk fever: protein kinase (cAMP-dependent, catalytic) inhibitor ß (PKIB); DNA-damage-inducible transcript 4 (DDIT4); period homolog 1 (PER1); NUAK family, SNF1-like kinase, 1 (NUAK1); and expressed sequence tag (BI537947). Another gene (neuroendocrine secretory protein 55, NESP55) was also determined to be specific for milk fever, independently of hypocalcemia. The mRNA expression of these 6 genes in milk fever cases was verified by quantitative real-time reverse-transcription PCR and was significantly different compared with their expression in healthy parturient cows. In the present study, the selected genes appeared to be candidate biomarkers of milk fever because the continuous interactions between blood cells and the entire body suggest that subtle intracellular changes occur in association with disease. However, before any genomic biomarkers are incorporated into clinical evaluation of the disease, the effect of hypocalcemia on the mRNA expression of these genes in the tissues that regulate calcium homeostasis in dairy cows should be determined.
Assuntos
Doenças dos Bovinos/sangue , Perfilação da Expressão Gênica/veterinária , Hipocalcemia/veterinária , Leucócitos Mononucleares/metabolismo , Análise em Microsséries/métodos , Paresia Puerperal/sangue , Animais , Bovinos , Doenças dos Bovinos/genética , Feminino , Humanos , Hipocalcemia/sangue , Hipocalcemia/genética , Paresia Puerperal/genética , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
Field experiments were conducted to determine the effect of green manure as fallow on common scab of potato caused by Streptomyces turgidiscabies. Significantly fewer diseased tubers were harvested from soil incorporated with lopsided oat or woolly pod vetch compared with those from oat and continuous potato cultivation in a planter experiment. Each field experiment consisted of lopsided oat cultivated during the spring and summer prior to the potato planting. Comparisons were also made with several other treatments, including cultivation of woolly pod vetch, oat, soybean, sugar beet, and potato ('Yukirasya', which is resistant to potato common potato scab) and soil application of Ferosand (Fe, mainly FeSO4, to decrease the soil pH). In field experiments conducted during 1999-2000, treatment with lopsided oat followed by lopsided oat or woolly pod vetch was significantly more effective at suppressing the disease severity than oat and continuous potato cultivation (P < 0.001). An increase in the marketable tuber ratio was also more significant than for oat and continuous potato cultivation (P < 0.001). In field experiments conducted during 2000-01, lopsided oat cultivation alone and with the application of Ferosand (1.8 t/ha) or resistant potato cultivar treatment were significantly more effective at suppressing the disease severity and incidence than sugar beet cultivation (P < 0.001), even under high disease intensity in the field. However, potato yield had a tendency to reduce after lopsided oat treatment with an application of Ferosand (1.8 t/ha) compared with lopsided oat alone or the application of Ferosand at 600 kg/ha, due to low pH conditions. In field experiments conducted during 2001-02, the lowest severity and incidence of common scab of potato were observed in soil treated with lopsided oat than with other treatments (P < 0.05 and P < 0.001, respectively). These findings suggest that lopsided oat used as fallow green manure can reduce the severity of common scab and increase potato yield.
RESUMO
Ion microprobe analyses show that solar wind nitrogen associated with solar wind hydrogen implanted in the first tens of nanometers of lunar regolith grains is depleted in 15N by at least 24% relative to terrestrial atmosphere, whereas a nonsolar component associated with deuterium-rich hydrogen, detected in silicon-bearing coatings at the surface of some ilmenite grains, is enriched in 15N. Systematic enrichment of 15N in terrestrial planets and bulk meteorites relative to the protosolar gas cannot be explained by isotopic fractionation in nebular or planetary environments but requires the contribution of 15N-rich compounds to the total nitrogen in planetary materials. Most of these compounds are possibly of an interstellar origin and never equilibrated with the 15N-depleted protosolar nebula.
Assuntos
Lua , Nitrogênio , Argônio , Deutério , Meio Ambiente Extraterreno , Hidrogênio , Isótopos , Isótopos de Nitrogênio , Sistema SolarRESUMO
mu-Crystallin is an NADPH-dependent cytosolic T3-binding protein. A knockout study in mice showed that mu-crystallin has a physiological function as a reservoir of T3 in the cytoplasm in vivo. Patients with nonsyndromic deafness were reported to have point mutations in the mu-crystallin gene. The expression of mu-crystallin is regulated by multiple factors. The present study was performed to determine whether thyroid function is related to the expression of mu-crystallin mRNA in peripheral mononuclear cells. We examined 23 normal healthy male and female subjects and 15 patients with Graves' disease. mu-Crystallin protein expression was determined immunohistochemically in peripheral mononuclear cells. The expression of mu-crystallin mRNA was assessed by reverse transcription of total RNA from peripheral mononuclear cells followed by quantitative PCR. mu-Crystallin protein was detected in peripheral mononuclear cells. The mRNA expression was negatively correlated with age in normal female subjects. The values in female subjects were significantly higher than those in males. The values were positively correlated with serum TSH concentration. The values of the thyrotoxic patients with Graves' disease were lower than those in healthy subjects. A transient increase in mu-crystallin expression was observed within 14-42 days after the initial treatment with antithyroid medication. Thyroid hormone inversely relates to the expression of mu-crystallin mRNA in euthyroid mononuclear cells. Abrupt suppression of thyroid function leads to overexpression of mu-crystallin mRNA in thyrotoxic mononuclear cells. Thyroid hormone-regulated mu-crystallin expression may control thyroid hormone action via the intracytoplasmic T (3) capacity.
Assuntos
Antitireóideos/uso terapêutico , Cristalinas/genética , Expressão Gênica/efeitos dos fármacos , Doença de Graves/tratamento farmacológico , Metimazol/uso terapêutico , Adulto , Fatores Etários , Células Cultivadas , Cristalinas/metabolismo , Feminino , Doença de Graves/genética , Doença de Graves/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores Sexuais , Testes de Função Tireóidea , Hormônios Tireóideos/sangue , Cristalinas muRESUMO
In cattle, interferon-stimulated genes (ISGs) such as ISG15, MX1, MX2, and OAS1 are known as classic ISGs that are highly involved in the implantation process. Various molecules play a crucial role in the mechanisms underlying ISG effects. Although microarray analyses have highlighted the expression of various molecules during the implantation period, these molecules remain incompletely characterized. In the present study, various specifically expressed genes were selected and their characteristics were examined. The microarray data from peripheral blood leukocytes derived from artificially inseminated cows and granulocytes obtained from embryo-transferred cows, respectively, were used to identify new ISG candidates. Seven common genes, including ISG15 and OAS1, were confirmed, but only 4 of the 5 genes were amplified by reverse transcription quantitative polymerase chain reaction. In addition, 3 expressed sequence tags (ESTs) exhibited significantly greater expression in granulocytes from pregnant cows than that observed in bred nonpregnant cows, and the expression in granulocytes increased after interferon-tau stimulation. Sequence alignment revealed similar sequences within 2 ESTs on the Hairy and enhancer of split (Hes) family basic helix-loop-helix transcription factor 4 (HES4) gene. An additional EST was identified as cytidine/uridine monophosphate kinase 2 (CMPK2). In silico analysis facilitated the identification of transcription factor-binding sequences, including an interferon-stimulated response element and interferon regulatory factor-binding sites, within the promoter region of HES4 and CMPK2. These genes may function as new ISGs in the context of implantation and may participate in the coordination of the feto-maternal interface in cows.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Bovinos/genética , Granulócitos/efeitos dos fármacos , Interferon Tipo I/farmacologia , Núcleosídeo-Fosfato Quinase/metabolismo , Proteínas da Gravidez/farmacologia , Prenhez , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Bovinos/fisiologia , Células Cultivadas , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Granulócitos/metabolismo , Núcleosídeo-Fosfato Quinase/genética , Gravidez , Prenhez/fisiologia , TranscriptomaRESUMO
POU3F2/BRN-2 is a transcription factor that is mainly expressed in the central nervous system and plays an important role in brain development. The transactivation domain of POU3F2 includes multiple mammalian-characteristic tandem amino acid repeats (homopolymeric amino acid repeats). We previously generated knock-in mice (Pou3f2Δ/Δ mice) in which all three homopolymeric amino acid repeats were deleted from the Pou3f2 transactivation domain and identified phenotypic impairments in maternal behavior and pup recognition. Yet, the exact biological implications of homopolymeric repeats are not completely understood. In this study, we investigated cognitive function and hippocampal neurogenesis in Pou3f2Δ/Δ mice. Pou3f2Δ/Δ mice exhibited cognitive impairment in object recognition and object location tests. Immunohistochemistry for doublecortin, a marker of immature neurons, showed a lower number of newborn neurons in the dentate gyrus of adult Pou3f2Δ/Δ mice compared with wild-type mice. Consistent with this observation, adult Pou3f2Δ/Δ mice had lower numbers of 5-bromo-2'-deoxyuridine (BrdU) and NeuN double-positive cells at 4 weeks after BrdU injection compared with control mice, indicating the decreased generation of mature granule cells in Pou3f2Δ/Δ mice. Taken together, these results suggest that POU3F2 is involved in cognitive function as well as adult hippocampal neurogenesis, and that homopolymeric amino acid repeats in this gene play a functional role.
Assuntos
Aminoácidos/metabolismo , Cognição/fisiologia , Hipocampo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fatores do Domínio POU/metabolismo , Envelhecimento , Animais , Proliferação de Células/fisiologia , Giro Denteado/metabolismo , Hipocampo/patologia , Camundongos Transgênicos , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismoRESUMO
Aims: The aim of this study was to report the mid-term clinical outcome of cemented unlinked J-alumina ceramic elbow (JACE) arthroplasties when used in patients with rheumatoid arthritis (RA). Patients and Methods: We retrospectively reviewed 87 elbows, in 75 patients with RA, which was replaced using a cemented JACE total elbow arthroplasty (TEA) between August 2003 and December 2012, with a follow-up of 96%. There were 72 women and three men, with a mean age of 62 years (35 to 79). The mean follow-up was nine years (2 to 14). The clinical condition of each elbow before and after surgery was assessed using the Mayo Elbow Performance Index (MEPI, 0 to 100 points). Radiographic loosening was defined as a progressive radiolucent line of >1 mm that was completely circumferential around the prosthesis. Results: The mean MEPI scores significantly improved from 40 (10 to 75) points preoperatively to 95 (30 to 100) points at final follow-up (p < 0.0001). Complications were noted in ten elbows (ten patients; 11%). Two had an intraoperative humeral fracture which was treated by fixation and united. One had a postoperative fracture of the olecranon which united with conservative treatment and one had a radial neuropathy which resolved. Further surgery was required for one with a dislocation, three with an ulnar neuropathy and one with a postoperative humeral fracture. Revision with removal of the components was performed in one elbow due to deep infection. There was no radiographic evidence of loosening around the components. With any revision surgery or revision with implant removal as the endpoint, the rates of survival up to 14 years were 93% (95% confidence interval (CI), 83.9 to 96.6) and 99% (95% CI 91.9 to 99.8), respectively, as determined by Kaplan-Meier analysis. Conclusion: With the appropriate indications, the mid-term clinical performance of the cemented JACE TEA is reliable and comparable to other established TEAs in the management of the elbow in patients with RA. Cite this article: Bone Joint J 2018;100-B:1066-73.
Assuntos
Óxido de Alumínio/administração & dosagem , Artrite Reumatoide/cirurgia , Artroplastia de Substituição do Cotovelo/métodos , Cimentos Ósseos/efeitos adversos , Prótese de Cotovelo , Adulto , Idoso , Artrite Reumatoide/diagnóstico por imagem , Artroplastia de Substituição do Cotovelo/instrumentação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Desenho de Prótese , Radiografia , Sucção/métodos , Técnicas de Sutura , Sinovectomia/métodos , Resultado do TratamentoRESUMO
Mammalian poly(ADP-ribose) polymerase (PARP) is a nuclear chromatin-associated protein with a molecular mass of 114 kDa that catalyzes the transfer of ADP-ribose units from NAD+ to nuclear proteins that are located within chromatin. We report here the identification of a novel property of PARP as a modulator of nuclear receptor signalling. PARP bound directly to retinoid X receptors (RXR) and repressed ligand-dependent transcriptional activities mediated by heterodimers of RXR and thyroid hormone receptor (TR). The interacting surface is located in the DNA binding domain of RXRalpha. Gel shift assays demonstrated that PARP bound to TR-RXR heterodimers on the response element. Overexpression of wild-type PARP selectively blocked nuclear receptor function in transient transfection experiments, while enzyme-defective mutant PARP did not show significant inhibition, suggesting that the essential role of poly(ADP-ribosyl) enzymatic activity is in gene regulation by nuclear receptors. Furthermore, PARP fused to the Gal4 DNA binding domain suppressed the transcriptional activity of the promoter harboring the Gal4 binding site. Thus, PARP has transcriptional repressor activity when recruited to the promoter. These results indicates that poly(ADP-ribosyl)ation is a negative cofactor in gene transcription, regulating a member of the nuclear receptor superfamily.
Assuntos
Regulação da Expressão Gênica , Poli(ADP-Ribose) Polimerases/metabolismo , Receptores do Ácido Retinoico/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Humanos , Ligação Proteica , Processamento de Proteína Pós-Traducional , Ratos , Elementos de Resposta , Receptores X de Retinoides , Transdução de SinaisRESUMO
BACKGROUND: In a search for mutations of mu-crystallin (CRYM), a taxion specific crystalline which is also known as an NADP regulated thyroid hormone binding protein, two mutations were found at the C-terminus in patients with non-syndromic deafness. OBJECTIVE: To investigate the mechanism of hearing loss caused by CRYM mutations METHODS: T3 binding activity of mutant mu-crystallin was compared with that of wild-type mu-crystallin, because mu-crystallin is known to be identical to T3 binding protein. To explore the sites within the cochlea where mu-crystallin is functioning, its localisation in the mouse cochlea was investigated immunocytochemically using a specific antibody. RESULTS: One mutant was shown to have no binding capacity for T3, indicating that CRYM mutations cause auditory dysfunction through thyroid hormone binding properties. Immunocytochemical results indicated that mu-crystallin was distributed within type II fibrocytes of the lateral wall, which are known to contain Na,K-ATPase. CONCLUSIONS: CRYM mutations may cause auditory dysfunction through thyroid hormone binding effects on the fibrocytes of the cochlea. mu-Crystallin may be involved in the potassium ion recycling system together with Na,K-ATPase. Future animal experiments will be necessary to confirm a causal relation between Na,K-ATPase, T3, and deafness.
Assuntos
Cóclea/metabolismo , Cristalinas/genética , Surdez/genética , Surdez/metabolismo , Mutação de Sentido Incorreto , Tri-Iodotironina/metabolismo , Animais , Proteínas de Transporte/metabolismo , Cóclea/citologia , Cristalinas/análise , Cristalinas/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Modelos Biológicos , Subunidades Proteicas/análise , Subunidades Proteicas/metabolismo , Reticulócitos/metabolismo , ATPase Trocadora de Sódio-Potássio/análise , ATPase Trocadora de Sódio-Potássio/metabolismo , Hormônios Tireóideos/metabolismo , Cristalinas mu , Proteínas de Ligação a Hormônio da TireoideRESUMO
Early detection of gestation is important in the bovine industry. New methods have been developed to detect gene expression in leucocytes induced by interferon-tau (IFNT) as gestation biomarkers. However, it is debatable which blood cell is suitable for detecting gene expression. This study was aimed at confirming whether granulocytes respond to IFNT specifically. Granulocytes and mononuclear cells (MNCs) from cows, and several types of bovine cultured cells, were treated with recombinant (r) IFNT and gene expression was analysed by quantitative real-time reverse transcriptase (RT)-PCR and microarray analysis. Expression levels of IFN receptors (R1 and R2) were approximately 30- to 900-fold higher in granulocytes than in other cultured cells, and 1.5- to 2.5-fold higher in MNCs than in granulocytes. Microarray analysis following a 2h recombinant IFNT (rIFNT) treatment revealed expression changes for 900 genes in granulocytes. Genes with expression changes included known IFN-stimulated genes (ISGs; ISG15, OAS1, MX1, and MX2). Eighteen genes were selected following granulocyte microarray analysis and their expression changes were confirmed in early gestation, which revealed that nine genes had significantly higher expression levels in pregnant than in non-pregnant animals. In conclusion, granulocytes specifically responded to rIFNT treatment and the resulting gene expression changes correlated with those in vivo. Microarray analysis indicated that various genes showed expression changes in rIFNT-treated granulocytes, which may result in the identification of alternate candidate genes for the early detection of gestation. These results strongly indicate that gene expression in granulocytes is a suitable tool to determine pregnancy status.
Assuntos
Granulócitos/metabolismo , Inseminação Artificial/veterinária , Proteínas da Gravidez/genética , Testes de Gravidez/veterinária , Prenhez/genética , Animais , Bovinos , Feminino , Expressão Gênica , Granulócitos/efeitos dos fármacos , Interferon Tipo I/farmacologia , Valor Preditivo dos Testes , Gravidez , Testes de Gravidez/métodosRESUMO
A 64-year-old male received coronary angiography because of chest pain. Although coronary angiography showed total occlusion of right coronary artery (RCA) # 2 and left anterior descending branch (LAD) #6, and a significant stenosis of left circumflex (LCx) #11, it could not visualize LAD distal to LAD # 6. Since coronary multidetector-row computed tomography (MD CT) could visualize the distal LAD, coronary artery bypass grafting (CABG) was indicated for this patient. Left internal thoracic artery (LITA) was anastomosed to LAD and saphenous vein graft (SVG) was used for distal anastomoses to obtuse marginal branch (OM) and 4-posterior descending branch (# 4 PD). Postoperative course was uneventful. LITA anastomosed to LAD and SVG to OM and # 4 PD were visualized by postoperative coronary angiography. MD CT in addition to coronary angiography was demonstrated useful to assess precise lesions of the coronary artery disease in this case.
Assuntos
Ponte de Artéria Coronária , Doença das Coronárias/diagnóstico por imagem , Doença das Coronárias/cirurgia , Tomografia Computadorizada por Raios X/métodos , Angiografia Coronária , Humanos , Masculino , Artéria Torácica Interna/cirurgia , Pessoa de Meia-Idade , Veia Safena/transplanteRESUMO
The aim of this study is to clarify the relationship between CRP and postoperative infection after cardiovascular surgery. We had 5 cases of surgical site infection, and 3 cases of infective endocarditis (IE) among 57 patients selected for this study out of 405 patients who had undergone cardiovascular surgery from May 1995 to March 2005. CRP, WBC and body temperature (BT) were evaluated during 1 week after the operation. Our results showed not only that the mean value of CRP level in the 49 non-infection patients attained the peak on the 2nd or 3rd day after the operation (18.2 +/- 4.7 and 17.7 +/- 5.7 mg/dl), but also that each patient in this group showed the same pattern of CRP sequence. CRP in the 5 cases of postoperative infection showed different patterns from that in the non-infection group. CRP in 3 cases of valve replacement for IE showed significantly higher level than that in 16 cases of valve replacement without IE through 1 week after the surgery. WBC level in the non-infection group reached the peak just after the operation (11.3 +/- 4.4 x 10(3)/microl) and then decreased gradually during 1 week after the operation. WBC in the 3 cases of valve replacement for IE, did not show different sequence pattern from that in the 16 cases of valve replacement without IE. WBC in a case of postoperative mediastinal infection showed a similar pattern of sequence to that in the non-infection group although it showed a remarkably high level of CRP sequence through 1 week after the surgery. BT in the non-infection group became the lowest just after the operation and reached the peak 8 hours after the operation. It then decreased gradually during 1 week after the operation. Our study demonstrates that CRP sequence after the surgery might be useful to detect postoperative infection after cardiovascular surgery.
Assuntos
Temperatura Corporal , Proteína C-Reativa/análise , Procedimentos Cirúrgicos Cardiovasculares , Contagem de Leucócitos , Infecção da Ferida Cirúrgica/diagnóstico , Idoso , Biomarcadores , Diagnóstico Precoce , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos TestesRESUMO
Survivin (SVV) is a family member of inhibitor of apoptosis proteins (IAPs) and its expression is cell cycle regulated. The gene is mapped to chromosome 17q25, the region of which is frequently gained in advanced stages of neuroblastoma (NBL). However, the role of SVV in NBL is poorly understood. Here we studied the clinical and biological role of SVV in NBL. A 1.9 kb SVV transcript was expressed in all of 9 NBL cell lines at higher levels than those in adult cancer cell lines. In 34 primary NBLs, high levels of SVV expression was significantly associated with age greater than 12 months (two sample t-test: P= 0.0003), advanced stages (P = 0.0136), sporadic tumors (P= 0.0027) and low levels of TrkA expression (P = 0.0030). In NBL cell lines, SVV mRNA expression was dramatically down-regulated in CHP134 and IMR32 cells undergoing apoptosis after treatment with all-trans retinoic acid (RA) or serum deprivation. It was only moderately decreased in cells (SH-SY5Y and CHP901) undergoing RA-induced differentiation. On the other hand, in proliferating NBL cells or RA-treated SK-N-AS line which is refractory to RA, the SVV mRNA remained at steady state levels or rather up-regulated. Furthermore, transfection of SVV into CHP134 cells induced remarkable inhibition of the RA-induced apoptosis. Collectively, our results suggest that high expression of SVV is a strong prognostic indicator for the advanced stage neuroblastomas, and that it could be one of the candidate genes for the 17q gain.
Assuntos
Cromossomos Humanos Par 17/genética , Proteínas Associadas aos Microtúbulos , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Biossíntese de Proteínas , Proteínas/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Caspase 3 , Caspases/biossíntese , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Ativação Enzimática , Indução Enzimática , Humanos , Lactente , Recém-Nascido , Proteínas Inibidoras de Apoptose , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neuroblastoma/enzimologia , Neuroblastoma/genética , Prognóstico , Fatores de Risco , Survivina , Transfecção , Tretinoína/farmacologia , Células Tumorais CultivadasRESUMO
In order to clarify the pathological outcome of congenital diaphragmatic hernia (CDH), we devised an animal model of CDH by administration of 2,4-dichlorophenyl-p-nitrophenyl ether (nitrofen) to pregnant rats, and determined the level and distribution of lung surfactant using the monoclonal antibody toward sphingomyelin and disaturated phosphatidylcholine (disat-PC). In control rats, the concentration of disat-PC was found to increase greatly from 16 to 18 days of gestation. Intragastric administration of nitrofen to pregnant rats at day 9 of gestation resulted in CDH in 42.7% of fetuses delivered after 20 days of gestation. In nitrofen-treated fetuses, the concentration of disat-PC in the lungs was lower than those in control fetuses, and surfactant apoprotein SP-A was similarly reduced in nitrofen-treated fetuses. However, the concentration of disat-PC in nitrofen-treated fetuses was higher than that in control fetuses at 18 days of gestation, indicating a synthetic potential of surfactant in nitrofen-treated fetuses comparable to that at the late stage of normal gestation. Immunohistochemical study with the antibody revealed that surfactant phospholipid was mainly in the form of intracellular granules in nitrofen-treated fetuses, probably causing the hypoplastic lungs and then CDH, in contrast to the uniform distribution on the pulmonary alveolar surface in control fetuses.
Assuntos
Hérnias Diafragmáticas Congênitas , Pulmão/metabolismo , Surfactantes Pulmonares/metabolismo , Líquido Amniótico/metabolismo , Animais , Animais Recém-Nascidos , Western Blotting , Peso Corporal , Cromatografia em Camada Fina , Modelos Animais de Doenças , Eletroforese em Gel de Poliacrilamida , Herbicidas , Hérnia Diafragmática/induzido quimicamente , Imuno-Histoquímica , Pulmão/anormalidades , Pulmão/química , Tamanho do Órgão , Éteres Fenílicos , Fosfatidilcolinas/análise , Surfactantes Pulmonares/química , Ratos , Ratos Sprague-Dawley , Esfingomielinas/análiseRESUMO
Bovine zona pellucida (ZP) glycoproteins from ovarian egg emerged as three bands with molecular mass of 78 kDa, 64 kDa and 21 kDa in SDS-PAGE under reducing conditions. Endo-beta-galactosidase (E beta G) digestion of the glycoproteins yielded five products with molecular mass of 76 kDa (E beta G-76), 68 kDa (E beta G-68), 63 kDa (E beta G-63), 47 kDa (E beta G-47) and 21 kDa (E beta G-21) under the same conditions. The N-terminal amino acid sequences of E beta G-76 and E beta G-21 were identical. This fact together with the results of diagonal SDS-PAGE indicated that E beta G-21 (N-terminal region) is linked to E beta G-63 (C-terminal region) through disulfide bond to form E beta G-76. Immunoblot analysis using anti-pig ZP protein antibodies revealed that bovine E beta G-76, E beta G-68 and E beta G-47 correspond to pig PZP2, PZP3 alpha and PZP3 beta glycoproteins, respectively. The E beta G-76 and E beta G-68 components were shown to be specifically cleaved during fertilization.
Assuntos
Glicoproteínas/análise , Óvulo/química , Zona Pelúcida/química , Amidoidrolases , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Bovinos , Cromatografia em Gel , Fertilização , Dados de Sequência Molecular , Monossacarídeos/análise , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase , beta-GalactosidaseRESUMO
Cyclic AMP potentiates glucose-stimulated insulin release by actions predominantly at a site, or sites, distal to the elevation of the cytosolic free Ca2+ concentration ([Ca2+]i). Glucose also acts at a site, or sites, distal to the elevation of [Ca2+]i via the ATP-sensitive K+ channel (K+ATP channel)-independent signaling pathway. Accordingly, using rat pancreatic islets, we studied the location of the action of cAMP and its interaction with the glucose pathway. Forskolin, an activator of adenylyl cyclase, raised intracellular cAMP levels and enhanced KCl-induced (Ca2+ -stimulated) insulin release in the presence, but not in the absence, of glucose. Thus, cAMP has no direct effect on Ca2+ -stimulated insulin release. The interaction between cAMP and glucose occurs at a step distal to the elevation of [Ca2+]i because forskolin enhancement of KCl-induced insulin release, in the presence of glucose, was demonstrated in the islets treated with diazoxide, a K+ATP channel opener. The enhancement of insulin release was not associated with any increase in [Ca2+]i. Furthermore, the interaction between cAMP and glucose was unequivocally observed even under stringent Ca2+ -free conditions, indicating the Ca2+ -independent action of cAMP. This action of cAMP is physiologically relevant, because not only forskolin but also glucagon-like peptide 1, glucose-dependent insulinotropic polypeptide, and pituitary adenylyl cyclase activating polypeptide exerted similar actions. In conclusion, the cAMP/protein kinase A pathway has no direct effect on Ca2+ -stimulated insulin exocytosis. Rather, it strongly potentiates insulin release by increasing the effectiveness of the K+ATP channel-independent action of glucose.
Assuntos
AMP Cíclico/farmacologia , Glucose/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Canais de Potássio/fisiologia , Transdução de Sinais , Trifosfato de Adenosina/farmacologia , Animais , Cálcio/farmacologia , Colforsina/farmacologia , AMP Cíclico/metabolismo , Diazóxido/farmacologia , Ativação Enzimática/efeitos dos fármacos , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Cloreto de Potássio/farmacologia , Proteína Quinase C/metabolismo , Ratos , Ratos Wistar , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Glucose augments Ca2+-stimulated insulin release from the pancreatic beta-cell in an ATP-sensitive K+ channel (K(ATP) channel)-independent manner. In studying the mechanisms underlying this action, we used rat pancreatic islets and examined the effects of exogenous free fatty acids (FFAs), which are precursors of long-chain acyl-CoA (LC-CoA), on KCl-induced Ca2+-stimulated insulin release. Myristate, palmitate, and stearate augmented insulin release induced by 50 mmol/l KCl in the presence of 2.8 mmol/l glucose. Added acutely, their potency was weak compared with that of glucose-induced augmentation. The FFA-induced augmentation became much greater, however, when islets were preincubated with FFAs under stringent Ca2+-free conditions (with 1 mmol/l EGTA) before the KCl stimulation. Under these conditions, 16.7 mmol/l glucose augmented 13-fold insulin release induced by 50 mmol/l KCl, whereas palmitate or myristate (both at a free concentration of 10 micromol/l) produced 5.8- and 5.2-fold augmentations. Effects of FFAs and glucose were concentration-dependent. The temporal profiles of augmentation induced by 11.1 mmol/l glucose and 10 micromol/l palmitate were similar. Glucose and palmitate caused almost identical augmentation patterns for the initial 10 min of stimulation; subsequently, glucose augmentation was better sustained than palmitate augmentation. This suggests the existence of a longer-term glucose-specific signaling moiety that cannot be mimicked by FFAs. Our results provide direct evidence that FFAs can mimic the K(ATP) channel-independent action of glucose. Taking these results together with previous results, we conclude that glucose augments Ca2+-stimulated insulin release, at least in part, by increasing malonyl-CoA and cytosolic LC-CoA. However, one or more other glucose-specific signaling molecules are required for the full expression of augmentation.
Assuntos
Cálcio/farmacologia , Ácidos Graxos/farmacologia , Glucose/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Cálcio/metabolismo , Diazóxido/farmacologia , Combinação de Medicamentos , Espaço Extracelular/metabolismo , Ácidos Graxos/química , Ácidos Graxos não Esterificados/farmacologia , Secreção de Insulina , Masculino , Ácido Mirístico/farmacologia , Ácido Palmítico/farmacologia , Canais de Potássio/fisiologia , Cloreto de Potássio/farmacologia , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Nutrients such as glucose stimulate insulin release from pancreatic beta-cells through both ATP-sensitive K+ channel-independent and -dependent mechanisms, which are most likely interrelated. Although little is known of the molecular basis of ATP-sensitive K+ channel-independent insulinotropic nutrient actions, mediation by cytosolic long-chain acyl-CoA has been implicated. Because protein acylation might be a sequel of cytosolic long-chain acyl-CoA accumulation, we examined if this reaction is engaged in nutrient stimulation of insulin release, using cerulenin, an inhibitor of protein acylation. In isolated rat pancreatic islets, cerulenin inhibited the glucose augmentation of Ca2+-stimulated insulin release evoked by a depolarizing concentration of K+ in the presence of diazoxide and Ca2+-independent insulin release triggered by a combination of forskolin and phorbol ester under stringent Ca2+-free conditions. Cerulenin inhibition of glucose effects was concentration dependent, with a 50% inhibitory concentration (IC50) of 5 microg/ml and complete inhibition at 100 microg/ml. Cerulenin also inhibited augmentation of insulin release by alpha-ketoisocaproate, a mitochondrial fuel. Furthermore, cerulenin abolished augmentation of both Ca2+-stimulated and Ca2+-independent insulin release by 10 micromol/l palmitate, which causes palmitoylation of cellular proteins. In contrast, cerulenin did not attenuate insulin release elicited by nonnutrient secretagogues, such as a depolarizing concentration of K+, activators of protein kinases A and C, and mastoparan. Glucose oxidation, ATP content in islets, and palmitate oxidation were not affected by cerulenin. In conclusion, cerulenin inhibits nutrient augmentation of insulin release with a high selectivity. The finding is consistent with a prominent role of protein acylation in the process of beta-cell nutrient sensing.
Assuntos
Fenômenos Fisiológicos da Nutrição Animal , Cerulenina/farmacologia , Antagonistas da Insulina/farmacologia , Ilhotas Pancreáticas/metabolismo , Acilação/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/fisiologia , Sinergismo Farmacológico , Glucose/metabolismo , Glucose/farmacologia , Masculino , Concentração Osmolar , Oxirredução/efeitos dos fármacos , Ácido Palmítico/metabolismo , Ácido Palmítico/farmacologia , Cloreto de Potássio/farmacologia , Proteínas/metabolismo , Ratos , Ratos WistarRESUMO
Binucleate cells are endocrine cells generated by the acytokinesis and endoreduplication of the trophectoderm in the ruminant placenta. These cells are migratory and secrete hormones into the maternal circulation after fusing with uterine epithelial cells. In this study, we performed immunohistochemistry for E-cadherin and beta-catenin in bovine placenta and a bovine trophoblast cell line (BT-1). We found that E-cadherin and beta-catenin were distributed not only at the cell to cell boundary but throughout the cytoplasm in binucleate cells, although they were concentrated at the cell to cell boundary in epithelial cells in bovine placenta. Moreover, beta-catenin was detected in the nuclei of binucleate cells. Binucleate cells after fusion with uterine epithelial cells (feto-maternal hybrid cells) in the maternal side showed no intracellular expression of E-cadherin and beta-catenin. The transformation into binucleate cells in the BT-1 cell line was also accompanied by the cytoplasmic accumulation of E-cadherin and beta-catenin. We further demonstrated that levels of cytoplasmic beta-catenin were well correlated with the DNA content of binucleate cells in BT-1. The dynamic changes in the distribution of E-cadherin and beta-catenin suggest an important role in binucleate cells, including the rearrangement of cadherin-mediated cell adhesions during cell migration and the onset of endoreduplication probably via the nuclear transfer of beta-catenin.
Assuntos
Caderinas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Transativadores/metabolismo , Trofoblastos/citologia , Trofoblastos/metabolismo , Actinas/metabolismo , Animais , Bovinos , Diferenciação Celular , Linhagem Celular , Citoplasma/metabolismo , Feminino , Imuno-Histoquímica , Placenta/citologia , Placenta/metabolismo , Gravidez , beta CateninaRESUMO
PURPOSE: Activation of transcription factor nuclear factor-kappaB (NF-kappaB) has been shown to play a role in cell proliferation, apoptosis, cytokine production, and oncogenesis. The purpose of this study was to determine whether NF-kappaB is constitutively activated in human gastric carcinoma tissues and, if so, to determine any correlation between NF-kappaB activity and clinicopathological features of gastric carcinoma. EXPERIMENTAL DESIGN: NF-kappaB activation was determined by immunohistochemical analysis of formalin-fixed, paraffin-embedded specimens from 64 gastric carcinoma patients. We quantified nuclear staining of RelA as a marker of NF-kappaB activation. RESULTS: Nuclear translocation of RelA was significantly high in tumor cells in comparison to that in adjacent normal epithelial cells (22.5 +/- 2.4% versus 8.6 +/- 1.5%, P < 0.0001). There was a significant correlation between NF-kappaB activation (nuclear translocation of RelA) and expression of urokinase-type plasminogen activator, an invasion-related factor and target of NF-kappaB in tumor cells (rho = 0.393; P = 0.0013). NF-kappaB activation was correlated with tumor invasion-related clinicopathological features such as lymphatic invasion of tumor cells (P = 0.0126), depth of invasion (P = 0.0539), peritoneal metastases (P = 0.0538), and tumor size (P = 0.0164). CONCLUSIONS: Collectively, the data show that NF-kappaB is constitutively activated in human gastric carcinoma tissues and suggest that NF-kappaB activity is related to tumor progression due to its transcriptional regulation of invasion-related factors such as urokinase-type plasminogen activator.