RESUMO
Recent advancement in liquid-environment atomic force microscopy (AFM) has enabled us to visualize three-dimensional (3D) hydration structures as well as two-dimensional (2D) surface structures with subnanometer-scale resolution at solid-water interfaces. However, the influence of ions present in solution on the 2D- and 3D-AFM measurements has not been well understood. In this study, we perform atomic-scale 2D- and 3D-AFM measurements at fluorite-water interfaces in pure water and a supersaturated solution of fluorite. The images obtained in these two environments are compared to understand the influence of the ions in solution on these measurements. In the 2D images, we found clear difference in the nanoscale structures but no significant difference in the atomic-scale contrasts. However, the 3D force images show clear difference in the subnanometer-scale contrasts. The force contrasts measured in pure water largely agree with those expected from the molecular dynamics simulation and the solvent tip approximation model. In the supersaturated solution, an additional force peak is observed over the negatively charged fluorine ion site. This location suggests that the observed force peak may originate from cations adsorbed on the fluorite surface. These results demonstrate that the ions can significantly alter the subnanometer-scale force contrasts in the 3D-AFM images.
RESUMO
Recently, possibilities of improving operation speed and force sensitivity in atomic-scale atomic force microscopy (AFM) in liquid using a small cantilever with an electron beam deposited (EBD) tip have been intensively explored. However, the structure and properties of an EBD tip suitable for such an application have not been well-understood and hence its fabrication process has not been established. In this study, we perform atomic-scale AFM measurements with a small cantilever and clarify two major problems: contaminations from a cantilever and tip surface, and insufficient mechanical strength of an EBD tip having a high aspect ratio. To solve these problems, here we propose a fabrication process of an EBD tip, where we attach a 2 µm silica bead at the cantilever end and fabricate a 500-700 nm EBD tip on the bead. The bead height ensures sufficient cantilever-sample distance and enables to suppress long-range interaction between them even with a short EBD tip having high mechanical strength. After the tip fabrication, we coat the whole cantilever and tip surface with Si (30 nm) to prevent the generation of contamination. We perform atomic-scale AFM imaging and hydration force measurements at a mica-water interface using the fabricated tip and demonstrate its applicability to such an atomic-scale application. With a repeated use of the proposed process, we can reuse a small cantilever for atomic-scale measurements for several times. Therefore, the proposed method solves the two major problems and enables the practical use of a small cantilever in atomic-scale studies on various solid-liquid interfacial phenomena.
RESUMO
AIMS: To investigate the influence of heat-killed Lactobacillus gasseri TMC0356 on changes in respiratory immune function and intestinal microbiota in a diet-induced obese mouse model. METHODS AND RESULTS: Male C57BL/6J mice were fed a high-fat diet for 16 weeks. After 8 weeks, the high-fat-diet-induced obese mice (DIO mice) were randomly divided into two 0067roups, the DIO and DIO0356 groups. DIO0356 group mice were orally fed with heat-killed TMC0356 every day for 8 weeks, while DIO group mice were exposed to 0·85% NaCl over the same time period as controls. After intervention, the pulmonary mRNA expression of cytokines and other immune molecules in DIO0356 mice compared to those in DIO group mice was significantly increased (P < 0·05, P < 0·01). In faecal bacterial profiles, analysed using the terminal restriction fragment length polymorphism (T-RFLP) method, T-RFLP patterns in 75% of the DIO0356 group mice were apparently changed compared with those in control group mice. CONCLUSION: These results suggest that inactive lactobacilli may stimulate the respiratory immune responses of obese host animals to enhance their natural defences against respiratory infection, partially associating with their potent impact on intestinal microbiota. SIGNIFICANCE AND IMPACT OF THE STUDY: We have demonstrated that oral administration of inactive lactobacilli may protect host animals from the lung immune dysfunction caused by obesity.
Assuntos
Intestinos/microbiologia , Lactobacillus/imunologia , Pulmão/imunologia , Metagenoma , Obesidade/imunologia , Administração Oral , Animais , Citocinas/genética , Citocinas/metabolismo , Dieta Hiperlipídica/efeitos adversos , Fezes/microbiologia , Mucosa Intestinal/metabolismo , Células Matadoras Naturais/imunologia , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/microbiologia , Polimorfismo de Fragmento de Restrição , Probióticos/administração & dosagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Baço/citologia , Baço/imunologiaRESUMO
AIMS: The aim of this study was to investigate the influence of heat treatment and culture media on the immunoregulatory effects of a probiotic strain, Lactobacillus gasseri TMC0356 (TMC0356). METHODS AND RESULTS: TMC0356 cultured in deMan-Rogosa-Sharpe and same food grade (FG) media were inactivated with the heat treatment at 70 and 90°C. Viable and heat-killed TMC0356 were tested for their ability to induce interleukin (IL)-12 production in the murine macrophage cell line J774.1. These TMC0356 were examined for their resistance to N-acetylmuramidase. Their morphology was observed by scanning electron microscopy. The heat-killed TMC0356 significantly induced IL-12 production in J774.1 cells and exhibited enhanced resistance to N-acetylmuramidase compared with viable TMC0356. Morphological changes were observed in TMC0356 when cultured in FG medium. Cell morphology and induction of IL-12 production in J774.1 cells were also associated. CONCLUSIONS: These results suggest that heat treatment and culture medium composition modified the immunoregulatory effects of TMC0356 to induce IL-12 production in macrophages. SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrate that probiotic immunoregulatory effects may be modified by the processing technology of cell preparation.
Assuntos
Meios de Cultura/metabolismo , Lactobacillus/crescimento & desenvolvimento , Probióticos/farmacologia , Animais , Linhagem Celular , Glicosídeo Hidrolases/toxicidade , Temperatura Alta , Interleucina-12/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Viabilidade MicrobianaRESUMO
Estrogen deficiency causes bone loss, which can be prevented by estrogen replacement therapy. Using a recently developed technique for isolation of highly purified mammalian osteoclasts, we showed that 17 beta-estradiol (E2) was able to directly inhibit osteoclastic bone resorption. At concentrations effective for inhibiting bone resorption, E2 also directly induced osteoclast apoptosis in a dose- and time-dependent manner. ICI164,384 and tamoxifen, as pure and partial antagonists, respectively, completely or partially blocked the effect of E2 on both inhibition of osteoclastic bone resorption and induction of osteoclast apoptosis. These data suggest that the protective effects of estrogen against postmenopausal osteoporosis are mediated in part by the direct induction of apoptosis of the bone-resorbing osteoclasts by an estrogen receptor- mediated mechanism.
Assuntos
Apoptose/efeitos dos fármacos , Reabsorção Óssea/prevenção & controle , Estrogênios/farmacologia , Osteoclastos/efeitos dos fármacos , Animais , Osteoclastos/fisiologia , Coelhos , Receptores de Estrogênio/fisiologiaRESUMO
Articular cartilage repair remains a major obstacle in tissue engineering. In the present study, we investigated the potential for demineralized dentin matrix (DDM; organic material derived from dentin) obtained from extracted teeth to be used as bone graft material. To evaluate the extent to which DDM induces osteochondral regeneration, we implanted DDM from bovine teeth in rabbit knees with full-thickness articular cartilage defects. Thirty-three 13-week-old male rabbits weighing 2.5-3.0 kg were randomly assigned to a control group (n = 11) and two experimental groups (n = 11 for each group). The knees were divided into three groups according to the subsequent treatment: in group I (n = 22), the control group, the defect was left untreated; and in groups II (n = 22) and III (n = 22), 50 and 100 mg of DDM, respectively, was implanted. The rabbits were killed 1, 3, 6, or 9 weeks after the surgical procedure, and the knees were collected. The harvested tissues were examined radiographically and histologically. The 100-mg DDM group (group III) had significantly more new bone forming inside the defect (as measured using the BV/TV value) compared with the other two groups as early as at week 3 postoperatively, but thereafter, the difference gradually decreased. Cartilage repair in the surface region remained significantly better in group III because hyaline-like cartilage appeared in the peripheral area of the defect at week 6 and the surface was covered with hyaline-like cartilage with a thickness similar to that of normal cartilage at week 9. In conclusion, the results of this study suggest that DDM acts as a scaffold for osteochondral regeneration, yielding active new bone formation early in the postoperative period. Thus, DDM may represent an effective bone implant material.
Assuntos
Regeneração Óssea , Transplante Ósseo , Cartilagem Articular/fisiologia , Cartilagem Articular/cirurgia , Dentina/transplante , Animais , Bovinos , Dentina/fisiologia , Fêmur/cirurgia , Masculino , Coelhos , Engenharia Tecidual , Alicerces Teciduais , Dente/fisiologiaRESUMO
We characterized Apg8/Aut7p essential for autophagy in yeast. Apg8p was transcriptionally upregulated in response to starvation and mostly existed as a protein bound to membrane under both growing and starvation conditions. Immunofluorescence microscopy revealed that the intracellular localization of Apg8p changed drastically after shift to starvation. Apg8p resided on unidentified tiny dot structures dispersed in the cytoplasm at growing phase. During starvation, it was localized on large punctate structures, some of which were confirmed to be autophagosomes and autophagic bodies by immuno-EM. Besides these structures, we found that Apg8p was enriched on isolation membranes and in electron less-dense regions, which should contain Apg8p-localized membrane- or lipid-containing structures. These structures would represent intermediate structures during autophagosome formation. Here, we also showed that microtubule does not play an essential role in the autophagy in yeast. The result does not match with the previously proposed role of Apg8/Aut7p, delivery of autophagosome to the vacuole along microtubule. Moreover, it is revealed that autophagosome formation is severely impaired in the apg8 null mutant. Apg8p would play an important role in the autophagosome formation.
Assuntos
Proteínas Associadas aos Microtúbulos/fisiologia , Fagossomos/fisiologia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiologia , Família da Proteína 8 Relacionada à Autofagia , Proteínas Fúngicas/fisiologia , Microscopia Eletrônica , Fagocitose/fisiologia , Fagossomos/ultraestrutura , Saccharomyces cerevisiae/ultraestruturaRESUMO
p120 was originally identified as a substrate of pp60src and several receptor tyrosine kinases, but its function is not known. Recent studies revealed that this protein shows homology to a group of proteins, beta-catenin/Armadillo and plakoglobin (gamma-catenin), which are associated with the cell adhesion molecules cadherins. In this study, we examined whether p120 is associated with E-cadherin using the human carcinoma cell line HT29, as well as other cell lines, which express both of these proteins. When proteins that copurified with E-cadherin were analyzed, not only alpha-catenin, beta-catenin, and plakoglobin but also p120 were detected. Conversely, immunoprecipitates of p120 contained E-cadherin and all the catenins, although a large subpopulation of p120 was not associated with E-cadherin. Analysis of these immunoprecipitates suggests that 20% or less of the extractable E-cadherin is associated with p120. When p120 immunoprecipitation was performed with cell lysates depleted of E-cadherin, beta-catenin was no longer coprecipitated, and the amount of plakoglobin copurified was greatly reduced. This finding suggests that there are various forms of p120 complexes, including p120/E-cadherin/beta-catenin and p120/E-cadherin/plakoglobin complexes; this association profile contrasts with the mutually exclusive association of beta-catenin and plakoglobin with cadherins. When the COOH-terminal catenin binding site was truncated from E-cadherin, not only beta-catenin but also p120 did not coprecipitate with this mutated E-cadherin. Immunocytological studies showed that p120 colocalized with E-cadherin at cell-cell contact sites, even after non-ionic detergent extraction. Treatment of cells with hepatocyte growth factor/scatter factor altered the level of tyrosine phosphorylation of p120 as well as of beta-catenin and plakoglobin. These results suggest that p120 associates with E-cadherin at its COOH-terminal region, but the mechanism for this association differs from that for the association of beta-catenin and plakoglobin with E-cadherin, and thus, that p120, whose function could be modulated by growth factors, may play a unique role in regulation of the cadherin-catenin adhesion system.
Assuntos
Caderinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Fosfoproteínas/metabolismo , Transativadores , Sítios de Ligação , Caderinas/isolamento & purificação , Cateninas , Moléculas de Adesão Celular/isolamento & purificação , Compartimento Celular , Células Cultivadas , Detergentes , Imunofluorescência , Humanos , Fosfoproteínas/isolamento & purificação , Fosforilação , Testes de Precipitina , Ligação Proteica , Células Tumorais Cultivadas , Tirosina/metabolismo , beta Catenina , delta CateninaRESUMO
BACKGROUND: The technique of preserving the major tributaries of the middle hepatic vein (MHV) (V5 and V8) until just before graft retrieval is beneficial to minimize congestion time of the graft. However, it remains unclear whether this technique exerts a burden on donors in terms of operative time, blood loss, and postoperative hepatic dysfunction. In this study we investigated adverse effects of the MHV tributaries preserving technique until immediately before graft retrieval on donors' surgical outcomes. METHODS: Data from 71 donors who underwent right hepatectomy without MHV for a liver transplantation at our hospital from January 2002 to August 2016 were retrospectively reviewed. Donors were divided into 3 groups as follows: group 1 (n = 12), no MHV tributary reconstruction; group 2 (n = 33), single MHV tributary reconstruction; group 3 (n = 26), 2 or 3 MHV tributaries reconstruction. Donor operation time, blood loss, proportion of the remnant liver, maximum postoperative total bilirubin, aspartate aminotransferase, alanine transaminase, minimum platelets, prothrombin time, albumin level, number of days in hospital from surgery to discharge, and surgical complications were compared. RESULTS: Compared with groups 2 and 3, group 1 exhibited shorter average operational time and less average blood loss, but the difference was not significant. Comparisons of all other factors indicated no significant differences. CONCLUSION: The technique of preserving the major tributaries of the MHV until just immediately before graft retrieval does not appear to impose an apparent burden on donors.
Assuntos
Hepatectomia/métodos , Veias Hepáticas/cirurgia , Transplante de Fígado/métodos , Tratamentos com Preservação do Órgão/métodos , Complicações Pós-Operatórias/prevenção & controle , Coleta de Tecidos e Órgãos/métodos , Adulto , Feminino , Hepatectomia/efeitos adversos , Humanos , Fígado/irrigação sanguínea , Fígado/enzimologia , Fígado/cirurgia , Transplante de Fígado/efeitos adversos , Doadores Vivos , Masculino , Pessoa de Meia-Idade , Duração da Cirurgia , Estudos Retrospectivos , Coleta de Tecidos e Órgãos/efeitos adversos , Transplantes/irrigação sanguínea , Transplantes/cirurgia , Resultado do TratamentoRESUMO
OBJECTIVE: Graft injuries sometimes occur and may cause complications such as the leakage of pancreatic secretions, which is often lethal. We report our experience of a case of successful simultaneous pancreas-kidney transplantation using injured pancreas graft. PATIENTS AND METHODS: The recipient was a 57-year-old woman with type 1 diabetes mellitus, and the donor was a 30-year-old man with a brain injury. In the donation, the pancreas parenchyma, splenic artery, and gastroduodenal artery were injured iatrogenically. We therefore reconstructed these arteries using vessel grafts and then performed simultaneous pancreas-kidney transplantation. RESULTS: Five days after transplantation, we noted a high titer of amylase in the ascites; therefore, we performed an urgent laparotomy. The origin of the amylase was the injured pancreatic parenchyma, and continued washing and drainage were carried out. We reconstructed the duodenojejunostomy using the Roux-en-Y technique to separate the passage of food from the pancreas graft to prevent injury to other organs due to exposure to pancreatic secretions. Thereafter, we inserted a decompression tube into the anastomosis thorough the blind end of the jejunum. Finally, we inserted 3 drainage tubes for lavage. Following this procedure, the patient recovered gradually and no longer required hemodialysis and insulin therapy. She was discharged from our hospital 56 days after transplantation. CONCLUSION: The restoration of the injured graft was possible by management of pancreatic secretions and use of the donor's vessel grafts. Shortage of donors is a problem throughout the world; thus, it is important to use injured grafts for transplantation if possible.
Assuntos
Transplante de Rim/efeitos adversos , Transplante de Pâncreas/efeitos adversos , Pâncreas/lesões , Complicações Pós-Operatórias , Coleta de Tecidos e Órgãos/efeitos adversos , Transplantes/lesões , Adulto , Anastomose em-Y de Roux/métodos , Diabetes Mellitus Tipo 1/cirurgia , Drenagem/métodos , Duodenostomia/métodos , Duodeno/irrigação sanguínea , Duodeno/cirurgia , Feminino , Humanos , Jejuno/cirurgia , Transplante de Rim/métodos , Masculino , Pessoa de Meia-Idade , Pâncreas/cirurgia , Transplante de Pâncreas/métodos , Tecido Parenquimatoso/lesões , Artéria Esplênica/lesõesRESUMO
The Japanese black bear (Ursus thibetanus japonicus) is an endangered species in some areas of Japan, and semen collection and cryopreservation are important means to preserve genetic resources and prevent extinction. The aim of the present study was to improve the methods for electroejaculation and cryopreservation in these bears. We collected the semen from captive mature Japanese black bears during the mating season by the electroejaculation method using maximum stimulus voltages of 10 V (n=9) and 7 V (n=15), and compared the characteristics of fresh semen after collection. None of the variables examined (i.e. total sperm count, motility, viability, morphology, and rate of urine contamination) were statistically different between the two electro-stimulus voltages. After the semen obtained was cryopreserved, the effects of three different diluents (egg yolk-TRIS-citrate-glucose, egg yolk-TRIS-citrate-fructose, and egg yolk-TRIS-citrate-fructose-lactose-raffinose; n=10) and two different incubation times (1-1 and 3-0 h of cooling-glycerol equilibration times; n=12) on frozen-thawed spermatozoa. None of the variables examined (i.e. motility, viability and morphology) were statistically different among the three diluents and between the two incubation times. The results indicate that, in the collection and cryopreservation of Japanese black bear semen: (1) a maximum voltage of 7 V may be enough to obtain ejaculates; (2) the three diluents examined were useful; and (3) glycerol equilibration time may be omitted with prior cooling for 3h.
Assuntos
Criopreservação/veterinária , Preservação do Sêmen/veterinária , Sêmen , Ursidae/fisiologia , Acrossomo/fisiologia , Animais , Sobrevivência Celular/fisiologia , Conservação dos Recursos Naturais , Criopreservação/métodos , Ejaculação/fisiologia , Estimulação Elétrica , Japão , Masculino , Preservação do Sêmen/métodos , Contagem de Espermatozoides/veterinária , Motilidade dos Espermatozoides/fisiologiaRESUMO
The epithelial-mesenchymal transition (EMT) is a crucial morphological event that occurs during the progression of epithelial tumors. EMT can be induced by transforming growth factor ß (TGF-ß) in certain kinds of cancer cells through the induction of Snail, a key regulator of EMT. We have previously found that TGF-ß remarkably induces Snail expression in cooperation with Ras signals; however, the underlying mechanism of this synergism has not yet been determined. Here, we demonstrate that signal transducer and activator of transcription 3 (STAT3) acts as a mediator that synergizes TGF-ß and Ras signals. The overexpression of STAT3 enhanced Snail induction, whereas siRNA-mediated knockdown of STAT3 inhibited it. The STAT3-YF mutant, which has Tyr 705 substituted with Phe, did not enhance Snail induction. Several STAT3 mutants lacking transcriptional activity also failed to enhance it; however, the putative STAT3-binding elements in the Snail promoter regions were not required for STAT3-mediated Snail induction. Protein inhibitor of activated STAT3 (PIAS3) inhibited the enhanced Snail promoter activity induced by TGF-ß and Ras. The interaction between PIAS3 and STAT3 was reduced by TGF-ß in cells harboring oncogenic Ras, whereas TGF-ß promoted the binding of PIAS3 to Smad3, a crucial mediator of TGF-ß signaling. Therefore, these findings suggest that STAT3 enhances Snail induction when it is dissociated from PIAS3 by TGF-ß in cooperation with Ras signals.
Assuntos
Fator de Transcrição STAT3/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas ras/metabolismo , Células Cultivadas , Células HeLa , Humanos , Transdução de Sinais , Fatores de Transcrição da Família SnailRESUMO
BACKGROUND: Successful liver transplantation from non-heart-beating donors (NHBDs) might enlarge donor source. Some studies have reported that resveratrol (RES), an activator of sirtuins, has cytoprotective effects on ischemia-reperfusion (I/R) injury. The purpose of this study was to investigate the effects of RES on warm I/R injury in rats. METHODS: Male Wister rats were divided into 5 groups: (1) the heart-beating (HB) group, whose livers were retrieved from HB donors; (2) the NHB group, whose livers were retrieved under apnea-induced NHB conditions; (3) the ethanol group, retrieved in the same manner as the NHB group with ethanol (10 µL) as a solvent; (4) the RES-1 group, retrieved in the same manner as the NHB group and pretreated with RES (0.4 mg/kg, dissolved in 10 µL ethanol); and (5) the RES-2 group, retrieved in the same manner as the NHB group and pretreated with RES (2 mg/kg, dissolved in 10 µL ethanol). The resected livers were perfused for 60 minutes with Krebs-Henseleit bicarbonate buffer after 6 hours of cold preservation, after which the perfusate and liver tissues were investigated. RESULTS: The bile production, portal vein flow volume, tumor necrosis factor-α level, and adenosine triphosphate level in the RES-2 group were significantly improved compared with in the NHB group. Histology revealed numerous well-preserved sinusoidal endothelial cells in the RES-2 group. CONCLUSIONS: RES might reduce warm I/R injury and improve the viability of liver grafts from NHBDs. We considered that this method may represent a promising approach for clinical liver transplantation from NHBDs.
Assuntos
Transplante de Fígado/métodos , Substâncias Protetoras/uso terapêutico , Traumatismo por Reperfusão/prevenção & controle , Estilbenos/uso terapêutico , Isquemia Quente , Animais , Masculino , Ratos , Ratos Wistar , Traumatismo por Reperfusão/diagnóstico , Resveratrol , Resultado do TratamentoRESUMO
As the first examples among marine algal hemagglutinins, the primary structures of two hemagglutinins, named hypnin A-1 and A-2, from the red alga Hypnea japonica, were determined by Edman degradation. Both hemagglutinins were single-chain polypeptides composed of 90 amino acid residues including four half-cystines, all of which were involved in two intrachain disulfide bonds, Cys(5)-Cys(62) and Cys(12)-Cys(89). Hypnin A-1 and A-2 had calculated molecular masses of 9146.7 and 9109.7 Da which coincided with determined values, 9148 and 9109 Da, by electrospray ionization-mass spectrometry, respectively. Both hemagglutinins only differed from each other at three positions; Pro(19), Arg(31) and Phe(52) of hypnin A-1 as compared with Leu(19), Ser(31), and Tyr(52) of hypnin A-2. Approximately 43% of total residual numbers consisted of three kinds of amino acids: serine, glycine and proline. The hemagglutination activities were lost by reduction and alkylation of the disulfide bonds. The nature of the small-sized polypeptides, including disulfide bonds, may contribute to the extreme thermostability of the hemagglutinins. Sequence having overall similarity to hypnin A-1 or A-2 was not detected in databases. Unexpectedly, however, hypnins contained a motif similar to the alignment of the C-terminal conserved amino acids within carbohydrate-recognition domains of C-type animal lectins. Furthermore, interestingly, the hemagglutination activities were inhibited by a protein, phospholipase A-2 besides some glycoproteins, suggesting that hypnins may possess both a protein-recognition site(s) and a carbohydrate-recognition site(s).
Assuntos
Hemaglutininas/química , Rodófitas/química , Sequência de Aminoácidos , Dissulfetos/química , Dados de Sequência Molecular , Peso Molecular , Fosfolipases A/farmacologia , Homologia de Sequência de AminoácidosRESUMO
Four active forms of chymotrypsin C (C1, C2A, C2B, and C3) were isolated from the autolyzed porcine pancreas glands. Their molecular weights were estimated by SDS-polyacrylamide gel electrophoresis to be 29 100 for C1, 26 300 for C2A and C3, and 25 500 for C2B. The kinetic analyses of esterase activity of the enzymes toward Ac-LLeu-OEt and Ac-LPhe-OEt showed that chymotrypsin C1 hydrolyzed the two substrates more efficiently than did chymotrypsin C3. Chymotrypsin C1 consisted of chain A (H-Cys-...-Asn-OH, Mr 886) and chain BC (H-Val-...-Lys-OH, Mr 28 200). Chymotrypsin C3 consisted of the two components of C3L and C3S that could be dissociated in the presence of 2.3% SDS. C3L consisted of the chain A and the chain C (H-Ser-...-Lys-OH, Mr 13 600). C3S was the chain B (H-Val-...-Lys-OH, Mr 11 800). These kinetic and chemical analyses show that chymotrypsins C1 and C3 correspond to chymotrypsin A delta and A alpha, respectively.
Assuntos
Quimotripsina/isolamento & purificação , Isoenzimas/isolamento & purificação , Pâncreas/enzimologia , Aminoácidos/análise , Animais , Fenômenos Químicos , Química , Quimotripsina/metabolismo , Eletroforese em Gel de Poliacrilamida , Esterases/metabolismo , Isoenzimas/metabolismo , Cinética , Peso Molecular , SuínosRESUMO
By a PCR-based screen for cDNA clones of protein kinases, we have isolated a cDNA clone encoding a novel protein kinase (referred to as EDPK) of 305 amino acids. EDPK has a catalytic domain of 271 amino acids that contains all conserved subdomains characteristic of the protein kinase family. Only short sequences are present at the N- and C-terminal ends outside the catalytic domain. EDPK expressed in Escherichia coli and in mammalian cells phosphorylated serine and threonine, but not tyrosine, residues in an exogenous substrate. The amino acid sequence similarity between EDPK and known serine/threonine kinases was less than 35%. Thus, the newly isolated protein kinase EDPK is a novel member of the serine/threonine kinase family. Northern blot analysis showed that the EDPK mRNA was highly expressed in various stages of mouse embryo development. The expression of the mRNA was also found in a variety of mouse adult tissues. These results suggest that EDPK plays a crucial role in intracellular signaling not only during mouse development but also in adult tissues.
Assuntos
Embrião de Mamíferos/enzimologia , Proteínas Serina-Treonina Quinases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS/citologia , Células COS/metabolismo , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Embrião de Mamíferos/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Camundongos , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Distribuição TecidualRESUMO
A cDNA encoding mouse hepatocyte growth factor activator (HGFA) has been cloned by RT-PCR, based on the screening result from the database of expressed sequence tags. Subsequently, its gene was cloned from a mouse genomic bacterial artificial chromosome library using the cDNA as a probe. Sequencing analysis revealed that mouse HGFA protein deduced from the cDNA, similar to its human and rat counterparts, has two epidermal growth factor-like domains, type 1 and 2 fibronectin homology domains, a single kringle domain and a catalytic domain of serine proteinase, and the gene consists of 14 exon spanning approximately 7.5 kb. Interestingly, mouse HGFA mRNA was detected not only in the liver but also in the gastrointestinal tract by RNA blot analysis. Since hepatocyte growth factor (HGF) is up-regulated in the damaged gastrointestinal mucosa, our present data suggest that HGFA might activate proHGF directly in the gastrointestinal mucosa and play an important role in wound repair throughout the gastrointestinal tract.
Assuntos
Sistema Digestório/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Fígado/metabolismo , Serina Endopeptidases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Etiquetas de Sequências Expressas , Expressão Gênica , Biblioteca Gênica , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/análise , Mapeamento por Restrição , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de SequênciaRESUMO
A competitive enzyme-linked immunosorbent assay (ELISA) was developed to quantify endogenous acetaldehyde-protein adducts (APAs) produced in plant seeds at low acetaldehyde concentrations without exogenous reducing agents. The key point of this technique is the use of a gelatin-acetaldehyde adduct, which is synthesized under 1 mM acetaldehyde and 10 mM NaCNBH3, to pre-coat plate wells to obtain the proper binding parameters for the quantification of APA in seed proteins. Compared with the traditional, direct ELISA method, the competitive one has higher sensitivity and less background. Using competitive ELISA, we determined the accumulation of endogenous APAs in seeds in relation to the loss of seed viability. Lettuce seeds were exposed to 2 mM gaseous acetaldehyde during storage for 30 or 45 d; the relative humidity and temperature of storage were studied independently. Viability decreased only in acetaldehyde-treated seeds, as either the temperature or the relative humidity increased. A loss in viability was accompanied by an increase in the accumulation of APA. The APA content also increased as viability decreased in five species of seeds, which were aged naturally without exposure to acetaldehyde. It is suggested that the modification of functional seed proteins with endogenously evolved acetaldehyde may be an important cause of seed aging.
RESUMO
Constitutive activation of BCR-ABL tyrosine kinase fusion protein has been shown to be an essential step in the pathogenesis of Philadelphia chromosome (Ph)-positive leukemias. We studied the tyrosine phosphorylated proteins which might be involved in the signaling pathway p185BCR-ABL using a Ph-positive acute lymphoblastic leukemia cell line. p185BCR-ABL but not p145c-abl was constitutively phosphorylated on tyrosine in this cell line. p21ras GTPase-activating protein (GAP) was physically associated with p185BCR-ABL, but not with p145c-abl, and GAP-associated proteins p62/p190 were found to be tyrosine-phosphorylated. Furthermore, p185BCR-ABL was also physically associated with phospholipase C-gamma 1 (PLC-gamma) and phosphatidylinositol 3'-kinase (P13-kinase). Concomitantly, both PLC-gamma and p85 subunit of P13-kinase are tyrosine-phosphorylated in the cells with p185BCR-ABL. These data suggest that GAP, GAP-associated proteins, PLC-gamma, and P13-kinase may participate in downstream signaling for p185BCR-ABL tyrosine kinase.
Assuntos
Proteínas de Fusão bcr-abl/fisiologia , Cromossomo Filadélfia , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/fisiopatologia , Proteínas/fisiologia , Transdução de Sinais/fisiologia , Fosfolipases Tipo C/fisiologia , Animais , Proteínas de Fusão bcr-abl/metabolismo , Proteínas Ativadoras de GTPase , Humanos , Camundongos , Fosfatidilinositol 3-Quinases , Fosforilação , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Tirosina Quinases/metabolismo , Tirosina/metabolismoRESUMO
We originally reported that vitamin K(2) (VK2) effectively induces apoptosis in various types of primary cultured leukemia cells and leukemia cell lines in vitro. In addition, VK2 was shown to induce differentiation of leukemia cells when the cells were resistant against VK2-inducing apoptosis. A novel synthetic vitamin D(3)derivative, 22-oxa-1,25-dihydroxyvitamin D(3) (OCT: oxacarcitriol) shows a more potent differentiation-inducing ability among myeloid leukemia cells in vitro with much lesser extent of the induction of hypercalcemia in vivo as compared to the effects of 1alpha,25(OH)(2)D(3). In the present study, we focused on the effects of a combination of OCT plus VK2 on leukemia cells. Treatment of HL-60 cells with OCT for 72 h induces monocytic differentiation. A combination of OCT plus VK2 dramatically enhances monocytic differentiation as assessed by morphologic features, positivity for non-specific esterase staining, and cell surface antigen expressions. This combined effect far exceeds the maximum differentiation induction ability at the optimal concentrations of either OCT or VK2 alone. In addition, pronounced accumulation of the cells in the G0/G1 phase is observed by combined treatment with OCT plus VK2 as compared with each vitamin alone. In contrast to cell differentiation, caspase-3 activation and apoptosis induction in response to VK2 are significantly suppressed in the presence of OCT in HL-60 cells. These data suggest that monocytic differentiation and apoptosis induction of HL-60 cells are inversely regulated. Furthermore, pronounced induction of differentiation by combined treatment with VK2 plus OCT was also observed in four out of six cases of primary cultured acute myeloid leukemia cells in vitro, suggesting that VK2 plus OCT might be a potent combination for the differentiation-based therapy for acute myeloid leukemias.