RESUMO
Objective: To explore the mechanisms of prickle planar cell polarity protein 1 (PRICKLE1) involved in the occurrence of skeletal Class â ¢ malocclusion. Methods: After extracting the genomic DNA of all family members of the skeletal Class â ¢ malocclusion pedigree with maxillary hypoplasia collected in the Department of Orthodontics at the Affiliated Stomatological Hospital of Nanjing Medical University in October 2021, whole exome sequencing and Sanger sequencing were performed to screen pathogenic genes/mutation sites and validate the mutations. Jaw tissue was collected during the operation of orthognathic patients who were treated in the Department of Oral and Maxillofacial Surgery at the same hospital from October 2021 to December 2022. Following the extraction of human jaw bone marrow mesenchymal stem cells and transfection with overexpressing lentivirus (lentiviruses overexpressing the gene of interest served as the wild group, lentiviruses overexpressing mutation site served as the mutant group) and knockdown lentivirus (divided into knockdown group 1 and 2, with transfection interference negative lentiviruses as the control group). Various assays including real-time fluorescence quantitative PCR (RT-qPCR), Western blotting, proliferation and Transwell assays, alkaline phosphatase staining and alizarin red staining were performed. Construction of zebrafish animal model, morpholino oligonucleotide (MO) were injected to knock down the expression of prickle1a and prickle1b in zebrafish (co-knocking group), and the control group was injected with standardized MO as a reference. Transcriptome sequencing, enrichment analysis and co-expression analysis were performed on the zebrafish craniofacial tissues of the two groups. Results: Two patients of this family carried this mutation PRICKLE1 c.113C>T. The transfection experiments showed that compared with the wild group (relative expression of PRICKLE1 was 21.97±0.60), the relative expression of mutant group (5.05±0.05) was significantly reduced (P<0.05), and cell proliferation and migration ability significantly enhanced (P<0.05), and osteogenic differentiation ability was significantly reduced (P<0.05). Compared with the control group, the proliferation and migration ability of cells in the two knockdown groups were significantly enhanced (P<0.05), and the osteogenic differentiation ability was significantly reduced (P<0.05). Zebrafish model experiments showed the width of the ethmoid plate was significantly reduced in the co-knocking group (282.50±61.77, t=5.29, P<0.001) compared with the control group (338.80±24.92). Transcriptome data and enrichment analysis showed that the differentially expressed genes were significantly enriched in the mitogen-activated protein kinase (MAPK) signaling pathway after the simultaneous knockdown of prickle1a and prickle1b in zebrafish. Conclusions: PRICKLE1 c.113C>T mutation might suppress the osteoblastic differentiation ability of jaw bone marrow mesenchymal stem cells by downregulating the MAPK signaling pathway, thereby involving the development of skeletal Class â ¢ malocclusion.
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Diabetic foot wound repair is a challenging issue in clinical practice. Due to the influence of multiple factors including the damage and regeneration failure of local tissue, the impaired pathways of wound repairing through blood vessels and nerve nutrition, and disorders of a variety of cellular factors, traditional treatment methods are often difficult to achieve good therapeutic effects. Stem cells are a type of cells with potentials of multidirectional differentiation, which also possess functions such as regulating immunity and paracrine to facilitate the comprehensive wound repair, so they have promising application prospect at present for the treatment of diabetic foot wounds. Because the relevant parameters of stem cell treatment are in the exploratory phase, there were no standardized data. This paper reviews the application of stem cells in the research of diabetic foot wound treatment over the past 6 years, analyzing and summarizing the contents in focused aspects including the types and sources of stem cells, effects of donor age and gender on stem cells, mode of administration, transplantation survival rate and safety, which may provide a reference for further application of stem cells in the clinical treatment of diabetic foot wound.
Assuntos
Diabetes Mellitus , Pé Diabético , Diferenciação Celular , Pé Diabético/terapia , Humanos , Transplante de Células-Tronco , Cicatrização/fisiologiaRESUMO
Hypoxia plays a critical role during the evolution of malignant cells and tumour microenvironment (TME).Tumour-derived exosomes contain informative microRNAs involved in the interaction of cancer and stromal cells, thus contributing to tissue remodelling of tumour microenvironment. This study aims to clarify how hypoxia affects tumour angiogenesis through exosomes shed from lung cancer cells. Lung cancer cells produce more exosomes under hypoxic conditions than do parental cells under normoxic conditions. miR-23a was significantly upregulated in exosomes from lung cancer under hypoxic conditions. Exosomal miR-23a directly suppressed its target prolyl hydroxylase 1 and 2 (PHD1 and 2), leading to the accumulation of hypoxia-inducible factor-1 α (HIF-1 α) in endothelial cells. Consequently, hypoxic lung cancer cells enhanced angiogenesis by exosomes derived from hypoxic cancer under both normoxic and hypoxic conditions. In addition, exosomal miR-23a also inhibits tight junction protein ZO-1, thereby increasing vascular permeability and cancer transendothelial migration. Inhibition of miR-23a by inhibitor administration decreased angiogenesis and tumour growth in a mouse model. Furthermore, elevated levels of circulating miR-23a are found in the sera of lung cancer patients, and miR-23a levels are positively correlated with proangiogenic activities. Taken together, our study reveals the clinical relevance and prognostic value of cancer-derived exosomal miR-23a under hypoxic conditions, and investigates a unique intercellular communication, mediated by cancer-derived exosomes, which modulates tumour vasculature.
Assuntos
Permeabilidade Capilar/fisiologia , Exossomos/metabolismo , Neoplasias Pulmonares/metabolismo , MicroRNAs/metabolismo , Neovascularização Patológica/metabolismo , Prolil Hidroxilases/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , Animais , Hipóxia Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Hipóxia/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas de Junções Íntimas/metabolismoRESUMO
As an essential trace element, copper can be toxic in mammalian cells when present in excess. Metallothioneins (MTs) are small, cysteine-rich proteins that avidly bind copper and thus play an important role in detoxification. Yeast CUP1 is a member of the MT gene family. The aim of this study was to determine whether yeast CUP1 could bind copper effectively and protect cells against copper stress. In this study, CUP1 expression was determined by quantitative real-time PCR, and copper content was detected by inductively coupled plasma mass spectrometry. Production of intracellular reactive oxygen species (ROS) was evaluated using the 2',7'-dichlorofluorescein-diacetate (DCFH-DA) assay. Cellular viability was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the cell cycle distribution of CUP1 was analyzed by fluorescence-activated cell sorting. The data indicated that overexpression of yeast CUP1 in HeLa cells played a protective role against copper-induced stress, leading to increased cellular viability (P<0.05) and decreased ROS production (P<0.05). It was also observed that overexpression of yeast CUP1 reduced the percentage of G1 cells and increased the percentage of S cells, which suggested that it contributed to cell viability. We found that overexpression of yeast CUP1 protected HeLa cells against copper stress. These results offer useful data to elucidate the mechanism of the MT gene on copper metabolism in mammalian cells.
Assuntos
Animais , Humanos , Mamíferos/fisiologia , Feromônios/fisiologia , Comportamento Animal/fisiologia , Comportamento/fisiologia , Odorantes , Bulbo Olfatório/fisiologia , Mucosa Olfatória/fisiologia , Condutos Olfatórios/anatomia & histologia , Condutos Olfatórios/fisiologia , Neurônios Receptores Olfatórios/fisiologia , Feromônios Humano/fisiologia , Olfato/fisiologiaRESUMO
As an essential trace element, copper can be toxic in mammalian cells when present in excess. Metallothioneins (MTs) are small, cysteine-rich proteins that avidly bind copper and thus play an important role in detoxification. Yeast CUP1 is a member of the MT gene family. The aim of this study was to determine whether yeast CUP1 could bind copper effectively and protect cells against copper stress. In this study, CUP1 expression was determined by quantitative real-time PCR, and copper content was detected by inductively coupled plasma mass spectrometry. Production of intracellular reactive oxygen species (ROS) was evaluated using the 2',7'-dichlorofluorescein-diacetate (DCFH-DA) assay. Cellular viability was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the cell cycle distribution of CUP1 was analyzed by fluorescence-activated cell sorting. The data indicated that overexpression of yeast CUP1 in HeLa cells played a protective role against copper-induced stress, leading to increased cellular viability (P<0.05) and decreased ROS production (P<0.05). It was also observed that overexpression of yeast CUP1 reduced the percentage of G1 cells and increased the percentage of S cells, which suggested that it contributed to cell viability. We found that overexpression of yeast CUP1 protected HeLa cells against copper stress. These results offer useful data to elucidate the mechanism of the MT gene on copper metabolism in mammalian cells.
Assuntos
Cobre/metabolismo , Metalotioneína/fisiologia , Estresse Oxidativo/fisiologia , Proteínas de Transporte/análise , Proteínas de Transporte/metabolismo , Ciclo Celular/fisiologia , Sobrevivência Celular/fisiologia , Cobre/análise , Formazans , Células HeLa , Humanos , Metalotioneína/análise , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Sais de Tetrazólio , Fatores de TempoRESUMO
The authors emphasize the possible pharmacological enhancement of axonal regeneration using a specific growth factor/ extracellular media incorporated in a biodegradable nonneural nerve conduit material. They investigated the early effects on nerve regeneration of continuous local delivery of nerve growth factor (NGF) and the local incorporation of hyaluronic acid (HA) inside a newly manufactured nerve conduit material from fresh human amnionic membrane. Human amnionic membrane contains important biochemical factors that play a major neurotrophic role in the nerve regeneration process. The process of manufacturing a nerve conduit from fresh human amnionic membrane is described. This nerve conduit system was used in rabbits to bridge a 25-mm nerve gap over 3 months. NGF was released locally, over 28 days, at the distal end of the tube via a system of slow release, and HA was incorporated inside the lumen of the tube at the time of surgery. NGF/HA treatment promoted axonal regeneration across the amnionic tube nerve conduit (8,962 +/- 383 myelinated axons) 45% better than the nontreated amnionic tube group (6,180 +/- 353 myelinated axons). The authors demonstrate that NGF/HA media enhances additional axonal regeneration in the amnionic tube nerve conduit. This result is secondary to the effect of the amnion promoting biochemical factors, in combination with the NGF/HA effect on facilitating early events in the nerve regeneration process.
Assuntos
Âmnio , Ácido Hialurônico/farmacologia , Fator de Crescimento Neural/farmacologia , Regeneração Nervosa , Nervo Isquiático/cirurgia , Animais , Axônios/efeitos dos fármacos , Biodegradação Ambiental , Feminino , Humanos , Masculino , Próteses e Implantes , Coelhos , Distribuição Aleatória , Estatísticas não ParamétricasRESUMO
OBJECTIVE: To improve the clinical result of repair on flexor tendon injury, and recover the defected finger function in children as far as possible. METHODS: From January 1990 to October 1997, 12 cases with flexor tendon injury were repaired by microsurgical technique, sutured by modified Kessler method with 3/0 or 5/0 nontraumatic thread and followed by invering suture of the gap edge with 7/0 or 8/0 nontraumatic thread after debridement. Appropriate functional practice was performed postoperatively. RESULTS: All the defected fingers were healed by first intention. Followed up 6 months to 1 year, there was excellent in 7 cases, better in 4 cases, moderate in 1 case and 91.67% in excellent rate according to the TAM standard of International Hand Committee. CONCLUSION: The important measures to improve the clinical result in children's flexor tendon injury are prompt and accurate diagnosis and repair of the injured tendon by microsurgical technique, and effective postoperative functional practice.
Assuntos
Traumatismos dos Dedos/cirurgia , Microcirurgia/métodos , Traumatismos dos Tendões/cirurgia , Criança , Pré-Escolar , Desbridamento , Feminino , Seguimentos , Humanos , MasculinoRESUMO
UNLABELLED: The use of microsurgical techniques (vasovasostomy and epididymovasostomy) for vasectomy reversal has now enabled surgeons to perform both procedures with certainly acceptable success rates. However, these operations are technically demanding and require special training in microsurgery. PURPOSE: A new method of performing these procedures using laser tissue soldering is described and results are evaluated. Laser tissue soldering is different from laser welding in that it involves the laser activation of a protein solder with a dye specific for the specific wavelength of laser light; therefore, surrounding tissue is not affected by the laser. MATERIALS AND METHODS: Ten rats underwent bilateral vasovasostomy and eleven underwent bilateral epididymovasostomy. In each rat, a sutured anastomosis was performed on one side while laser tissue soldering was performed on the other. Animals were sacrificed after one month and anastomoses were evaluated for patency and presence of sperm granulomas. Histologic analysis was also performed. RESULTS: Patency rates were 8/10 (80%) for sutured vasovasostomy versus 9/10 (90%) for the laser technique. Epididymovasostomy patency rates were 8/11 (73%) for sutured versus 9/11 (82%) for the laser technique. Mean operative times were significantly shorter for lasered anastomoses when compared to controls. The frequency of granuloma formation did not significantly differ between laser and control groups. CONCLUSIONS: Laser tissue soldering resulted in similar patency when compared to a conventional 2 layered sutured anastomosis while decreasing operative time. In addition, since fewer sutures are placed, the laser method is less technically demanding.
Assuntos
Terapia a Laser , Microcirurgia , Vasovasostomia/métodos , Animais , Epididimo/cirurgia , Estudos de Avaliação como Assunto , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
To demonstrate a dependence of spinal cord motoneurons on the communication with their targets, the expression of immediate early gene c-fos and neurotrophin genes in the lumbar (L3-L6) spinal cord neurons was examined in Sprague-Dawley rats (male > or = 9-weeks-old) with unilateral sciatic nerve transection. Using in situ hybridization, we detected the expression of c-fos mRNA in the motoneurons of the spinal cord segments within 45 min to 3 h of peripheral nerve transection (n = 4 in each time point). The expression of c-fos mRNA was also correlated positively with the expression of Fos antigen using immunohistochemistry, while no change in calbindin and parvalbumin antigens were noted. The expression of BDNF mRNA increased at 90 min after sciatic nerve transection. However, no detectable enhancement in the expression of NGF mRNA was observed. DNA fragmentation in neurons was observed using the incorporation of digoxigenin-dUTP by terminal transferase into 3'-OH terminals of DNA fragments in the ipsilateral section of the spinal cords 48h after nerve injury. Nuclei that exhibited DNA fragmentation were not observed in the spinal cord of the control animals. Lastly, we observed that the majority of astrocytes did not have DNA fragmentation. Because the detection of DNA fragmentation using this assay is one of early detections of apoptosis or programmed cell death, the result suggested we could detect early cell death in spinal cord, and indicated a target dependence of the neurons in the spinal cord after transection of sciatic nerve.
Assuntos
Apoptose/genética , Regulação da Expressão Gênica , Neurônios/citologia , Nervo Isquiático/lesões , Medula Espinal/citologia , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Fragmentação do DNA , Genes fos , Hibridização In Situ , Masculino , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
The purpose of this study was to investigate gait-pattern changes after complete tibial nerve lesion in the cat, and to observe whether nerve repair could reverse some of the changes. In six cats, a 5-cm segment of the tibial nerve was transected. The nerve gap was then repaired with nerve autograft in three animals and was unrepaired in three as controls. The walking patterns of the cats were videotaped, and the hip, knee, ankle, and metatarsophalangeal joint angles were measured at the beginnings of the F, E1, E2, and E3 phases of the step cycle. Two weeks after surgery, abnormal gait patterns were observed, and four gait parameters (E3.Hip, E3.Ankle, E3.M-P, and F.Ankle) were found to be statistically significantly different from normal. Six months after surgery, the nerve-graft group had gait-parameter values approaching normal, while the control group showed no measurable improvement. Correspondingly, electrophysiologic testing revealed considerable nerve regeneration in the nerve-graft group but not in the control group. It was concluded that these gait parameters can be used as valid functional indices to evaluate the degree of tibial nerve recovery in the cat model.
Assuntos
Marcha/fisiologia , Nervo Tibial/lesões , Nervo Tibial/cirurgia , Potenciais de Ação , Animais , Gatos , Eletromiografia , Masculino , Regeneração Nervosa , Condução Nervosa , Nervo Tibial/fisiopatologiaRESUMO
Tumor-elicited neutrophils (tcPMN) purified from 13762NF mammary adenocarcinoma tumor-bearing rats enhanced metastasis of syngeneic cells when co-injected intravenously; whereas, circulating (cPMN) and phorbol esteractivated (PMA-PMN) neutrophils did not [Welch et al. (1989) Proc. Natl. Acad. Sci. 86:5859-63]. We hypothesized that differential protein expression was responsible for functional differences between the neutrophil subtypes. Two-dimensional polyacrylamide gel electrophoresis was used to compare neutrophils (cPMN, PMA-PMN) purified from the peripheral blood of healthy, syngeneic nontumor-bearing rats, to tcPMN collected from rats with highly metastatic [clone MTLn3, subclone MTLn3(T44).5] or poorly metastatic [subclone MTLn3(T44).11] tumors growing in the mammary fat pads. Quantitative differences in polypeptide expression were observed between these functionally distinct PMN populations. Compared to cPMN, expression of a M(r) approximately 38.8 kDa (pl approximately 8) polypeptide was similar in tcPMN collected from poorly metastatic tumor-bearing rats, higher in PMA-PMN, and further increased in tcPMN from rats with highly metastatic tumors. Expression of two polypeptides, M(r) approximately 14.1 kDa (pl approximately 6) and M(r) approximately 43.3 kDa (pl approximately 5), was greater in tcPMN from rats with highly metastatic tumors compared to cPMN, PMA-PMN, or tcPMN from rats bearing poorly metastatic tumors. The latter two polypeptides thus appeared to be specifically increased in tcPMN from rats bearing highly metastatic tumors. Because it was most abundant and displayed the greatest differences between PMN subtypes, the M(r) approximately 14.1 kDa protein was further analyzed. Tryptic digests followed by internal sequence analyses of resulting peptide fragments revealed that the M(r) approximately 14.1 kDa contained amino acid sequences that were identical to those of MRP-14, a 14 kDa neutrophil calcium-binding protein belonging to the S-100 protein family of calcium-binding proteins. These results suggest a novel function for MRP-14 and suggest that MRP-14 may represent a marker for distinguishing phenotypically distinct subpopulations of neutrophils, particularly tcPMN with metastasis-enhancing abilities.
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Antígenos de Diferenciação/análise , Proteínas de Ligação ao Cálcio/análise , Metástase Neoplásica , Neutrófilos/química , Sequência de Aminoácidos , Animais , Antígenos de Diferenciação/fisiologia , Proteínas de Ligação ao Cálcio/fisiologia , Calgranulina B , Feminino , Neoplasias Mamárias Experimentais/sangue , Dados de Sequência Molecular , Neutrófilos/fisiologia , Ratos , Ratos Endogâmicos F344 , Células Tumorais CultivadasRESUMO
This study attempted to develop a motor functional index, ankle stance angle (ASA), to assess rat sciatic nerve regeneration subsequent to autografting. ASA, 50 degrees in normal rats, is the ankle joint angle at the mid-stance phase of the gait cycle. In a nerve graft group, a 1-cm segment of the right sciatic nerve was transected and then repaired with nerve autograft. In an ungrafted group, the nerve gap was left unrepaired. ASA measured 4 months after surgery was statistically significantly larger in the nerve graft group (36 degrees) than in the ungrafted group (22 degrees). The results suggest that ASA is more sensitive than sciatic function index in detecting functional recovery after a complete sciatic nerve lesion. ASA also showed a significant correlation with the passive range of ankle joint motion and gastrocnemius muscle weight. The study concluded that ASA is a reliable index for assessment of regeneration of rat sciatic nerve after a complete lesion. The intra-rater reliability (r = 0.97 and 0.90) and inter-rater reliability (r = 0.85) tests performed support the conclusions.
Assuntos
Marcha , Regeneração Nervosa , Nervo Isquiático/fisiologia , Nervo Isquiático/cirurgia , Tarso Animal , Animais , Membro Posterior , Masculino , Ratos , Ratos Endogâmicos Lew , Nervo Isquiático/lesões , Sensibilidade e EspecificidadeRESUMO
Vascularized skeletal tissue allografts would greatly expand the domain of reconstructive surgery. Few studies to date have examined the functional aspects of these allografts or their long-term fate. An orthotopic transplant model of rat distal femur and surrounding muscular cuff was developed to assess graft function in fracture healing and weight bearing. Isografts (RT1l to RT1l, n = 40), weak-barrier allografts (RT1l to RT1lv, n = 40), and strong-barrier allografts (RT1l to RT1n, n = 40) were transplanted. As the histocompatibility barrier increased between the donor and recipient animals, the graft viability and performance deteriorated according to radiographic, histologic, and immunologic analyses. Administration of cyclosporine led to survival of strong-barrier allografts similar to that of isografts. A long-term study of these allografts (RT1l to RT1n) was then performed on various immunosuppressive regimens. After an initial 10-week course of cyclosporine to achieve bony union and remodeling, subsequent cessation (n = 20) or intermittent "pulsing" (n = 20) of the immunosuppressant was insufficient in maintaining graft survival. However, graft viability and function were preserved through 1 year on continuous daily cyclosporine (n = 32). There was no evidence of host renal or hepatic toxicity by serum chemistry or histologic sections. Thus long-term survival of functional skeletal allografts was achieved in this orthotopic model without significant host toxicity from immunosuppression.
Assuntos
Transplante Ósseo/métodos , Consolidação da Fratura , Sobrevivência de Enxerto , Músculos/transplante , Animais , Transplante Ósseo/diagnóstico por imagem , Transplante Ósseo/imunologia , Transplante Ósseo/patologia , Osso e Ossos/irrigação sanguínea , Ciclosporina , Fêmur/irrigação sanguínea , Fêmur/transplante , Terapia de Imunossupressão , Microcirculação , Músculos/irrigação sanguínea , Radiografia , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew , Imunologia de Transplantes , Transplante Homólogo , Transplante Isogênico , Suporte de CargaRESUMO
Three subunits, Ac115, Ac39, and the proteolipid, were positively identified in the membrane sectors of V-ATPases from different sources. We searched for organelle-specific protein in purified preparations of V-ATPase from bovine chromaffin granules. A diffused protein band at a position of about 45 kDa was identified in SDS-polyacrylamide gels of the above preparation. Following digestion with endopeptidase Glu-C (V-8), a polypeptide of about 10 kDa was isolated and subjected to amino acid sequencing. Hence, the cDNA encoding the protein Ac45 was cloned from a bovine adrenal medulla library. The cDNA sequence contains an open reading frame encoding a protein of 468 amino acids with a calculated molecular mass of 51,786 daltons. A potential signal sequence comprised of the first 35 amino acids and a potential transmembrane domain at the C terminus of the protein were identified. There exist seven potential glycosylation sites between the aforementioned protein motifs. Experiments with a specific antibody against Ac45 demonstrated that it is copurifying with the V-ATPase from chromaffin granules. Immunological cross-reactivity was observed with purified V-ATPase from bovine kidney microsomes but not from plasma membranes of epithelial cells. Cell-free expression of the protein from synthetic mRNA produced a single protein band at about 50 kDa on SDS gels. Upon inclusion of dog pancreas microsomes in the reaction mixture, a slow migrating band sensitive to peptide:N-glycosidase F was observed.
Assuntos
Grânulos Cromafim/enzimologia , Proteínas de Membrana/genética , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/genética , ATPases Vacuolares Próton-Translocadoras , Vacúolos/enzimologia , Medula Suprarrenal/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , DNA Complementar , Cães , Humanos , Imuno-Histoquímica , Rim/enzimologia , Proteínas de Membrana/metabolismo , Microssomos/enzimologia , Dados de Sequência Molecular , Pâncreas/enzimologia , ATPases Translocadoras de Prótons/metabolismoRESUMO
Upon 2-3 hours cold treatment (0 degrees C), the keratin filaments of some PcaSE-1 cells and BEL-7404 cells are partly transformed into granular aggregates. But such structural transformation does not occur in HeLa cells and CNE cells. By rewarming (37 degrees C) cells within 15-30 minutes, this structural changes of keratin filaments in PcaSE-1 cells and BEL-7404 cells are readily reversed. In contrast, in HeLa cells and CNE cells, keratin filaments are transformed into granula aggregates during mitosis, but the keratin filament network in PcaSE-1 cells and BEL-7404 cells remained intact and encircled the developing mitotic spindle as the cells entered mitosis. Results suggest that the above two types of keratin filament structural transformation might be induced by different factors. Our results also indicate that: (1) PcaSE-1 cells treated with colchicine alone or with combination of colchicine and cytochalasin D does not cause granular aggregates of keratin filaments. However, after depolymerization of microtubules with colchicine, the response of the cells to cold treatment is intensified. (2) The aggregate formation during cold treatment is unrelated to whether epithelial cells contain two different type intermediate filaments or not. (3) Epithelial cells preextracted with Triton X-100 do not induce granular aggregate formation of keratin filaments upon cold treatment. (4) The structural transformation upon cold treatment may be a characteristic of keratin filaments of certain epithelial cell lines.
Assuntos
Filamentos Intermediários/ultraestrutura , Queratinas , Temperatura Baixa , Grânulos Citoplasmáticos/ultraestrutura , Epitélio/patologia , Células HeLa , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Nasofaríngeas/patologia , Neoplasias Cutâneas/patologia , Células Tumorais CultivadasRESUMO
A simple technique for harvesting skin grafts of predetermined size, shape, and thickness in rodents is described. In this technique, the donor skin is immobilized and tensed by means of a tongue depressor, or similar template, inserted into the loose areolar tissue below the panniculus, stretching the overlying skin to permit easy dermatome harvesting of a skin graft.
Assuntos
Transplante de Pele/instrumentação , Instrumentos Cirúrgicos , Animais , Cobaias , Ratos , Pele/patologia , Transplante de Pele/patologiaRESUMO
An important means by which tumor cells influence the vasculature is through the production of soluble mediators altering vascular properties. A approximately 22-kDa polypeptide was purified to homogeneity from conditioned medium of murine methylcholanthrene A (meth A) fibrosarcoma cells by ion-exchange chromatography and preparative sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE), based on its ability to induce tissue factor procoagulant activity in endothelial cells (ECs). The final product migrated as a broad band on reduced and nonreduced SDS-PAGE and had an unique amino-terminal sequence. This meth A-derived polypeptide modulated EC coagulant properties through the induction of tissue factor, induced monocyte migration and tissue factor expression, and was also chemotactic for granulocytes. Injection of the polypeptide into mouse footpads resulted in an inflammatory response with tissue swelling and polymorphonuclear leukocyte infiltration. The ability of this mediator to activate ECs and monocytes has led us to name it EMAP II (endothelial monocyte-activating polypeptide). EMAP II is distinct from a previously described approximately 40-kDa meth A-derived polypeptide termed EMAP I. Through its potential to activate host effector mechanisms, EMAP II could contribute to the biology of immunogenic tumors, such as the meth A fibrosarcoma.
Assuntos
Citocinas , Endotélio Vascular/metabolismo , Monócitos/metabolismo , Proteínas de Neoplasias/análise , Proteínas de Ligação a RNA , Sequência de Aminoácidos , Animais , Western Blotting , Movimento Celular , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Humanos , Camundongos , Dados de Sequência Molecular , Monócitos/efeitos dos fármacos , Proteínas de Neoplasias/imunologia , Testes de Precipitina , Homologia de Sequência de Aminoácidos , Células Tumorais CultivadasRESUMO
The DNA binding activity of Fos and Jun is regulated in vitro by a post-translational mechanism involving reduction-oxidation. Redox regulation occurs through a conserved cysteine residue located in the DNA binding domain of Fos and Jun. Reduction of this residue by chemical reducing agents or by a ubiquitous nuclear redox factor (Ref-1) recently purified from Hela cells, stimulates AP-1 DNA binding activity in vitro, whereas oxidation or chemical modification of the cysteine has an inhibitory effect on DNA binding activity. Here we demonstrate that the protein product of the ref-1 gene stimulates the DNA binding activity of Fos-Jun heterodimers, Jun-Jun homodimers and Hela cell AP-1 proteins as well as that of several other transcription factors including NF-kappa B, Myb and members of the ATF/CREB family. Furthermore, immunodepletion analysis indicates that Ref-1 is the major AP-1 redox activity in Hela nuclear extracts. Interestingly, Ref-1 is a bifunctional protein; it also possesses an apurinic/apyrimidinic (AP) endonuclease DNA repair activity. However, the redox and DNA repair activities of Ref-1 can, in part, be distinguished biochemically. This study suggests a novel link between transcription factor regulation, oxidative signalling and DNA repair processes in higher eukaryotes.
Assuntos
Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas de Escherichia coli , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas de Saccharomyces cerevisiae , Transativadores , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Desoxirribonuclease IV (Fago T4-Induzido) , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Escherichia coli , Imunofluorescência , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Oxirredução , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/metabolismoRESUMO
Advanced glycosylation end products of proteins (AGEs) are nonenzymatically glycosylated proteins which accumulate in vascular tissue in aging and at an accelerated rate in diabetes. A approximately 35-kDa polypeptide with a unique NH2-terminal sequence has been isolated from bovine lung and found to be present on the surface of endothelial cells where it mediates the binding of AGEs (receptor for advanced glycosylation end product or RAGE). Using an oligonucleotide probe based on the amino-terminal sequence of RAGE, an apparently full-length cDNA of 1.5 kilobases was isolated from a bovine lung cDNA library. This cDNA encoded a 394 amino acid mature protein comprised of the following putative domains: an extracellular domain of 332 amino acids, a single hydrophobic membrane spanning domain of 19 amino acids, and a carboxyl-terminal domain of 43 amino acids. A partial clone encoding the human counterpart of RAGE, isolated from a human lung library, was found to be approximately 90% homologous to the bovine molecule. Based on computer analysis of the amino acid sequence of RAGE and comparison with databases, RAGE is a new member of the immunoglobulin superfamily of cell surface molecules and shares significant homology with MUC 18, NCAM, and the cytoplasmic domain of CD20. Expression of the RAGE cDNA in 293 cells allowed them to bind 125I-AGE-albumin in a saturable and dose-dependent manner (Kd approximately 100 nM), blocked by antibody to RAGE. Western blots of 293 cells transfected with RAGE cDNA probed with anti-RAGE IgG demonstrated expression of immunoreactive protein compared to its absence in mock-transfected cells. These results suggest that RAGE functions as a cell surface receptor for AGEs, which could potentially mediate cellular effects of this class of glycosylated proteins.