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OBJECTIVE: Recently, we reported that angiopoietin-like protein 2 (Angptl2) functions in various chronic inflammatory diseases. In the present study, we asked whether Angptl2 and its associated chronic inflammation contribute to abdominal aortic aneurysm (AAA). METHODS AND RESULTS: Immunohistochemistry revealed that Angptl2 is abundantly expressed in infiltrating macrophages within the vessel wall of patients with AAA and in a CaCl(2)-induced AAA mouse model. When Angptl2-deficient mice were used in the mouse model, they showed decreased AAA development compared with wild-type mice, as evidenced by reduction in aneurysmal size, less severe destruction of vessel structure, and lower expression of proinflammatory cytokines and matrix metalloproteinase-9. However, no difference in the number of infiltrating macrophages within the aortic aneurysmal vessel wall was observed between genotypes. AAA development was also significantly suppressed in wild-type mice that underwent Angptl2-deficient bone marrow transplantation. Expression levels of proinflammatory cytokines and metalloproteinase-9 in Angptl2-deficient macrophages were significantly decreased, and those decreases were rescued by treatment of Angptl2 deficient macrophages with exogenous Angptl2. CONCLUSIONS: Macrophage-derived Angptl2 contributes to AAA development by inducing inflammation and degradation of extracellular matrix in the vessel wall, suggesting that targeting the Angptl2-induced inflammatory axis in macrophages could represent a new strategy for AAA therapy.
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Angiopoietinas/metabolismo , Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/metabolismo , Macrófagos/metabolismo , Proteína 2 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Angiopoietinas/deficiência , Angiopoietinas/genética , Animais , Aorta Abdominal/imunologia , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/imunologia , Aneurisma da Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/prevenção & controle , Transplante de Medula Óssea , Cloreto de Cálcio , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Genótipo , Humanos , Imuno-Histoquímica , Mediadores da Inflamação/metabolismo , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , FenótipoRESUMO
Cardiac manifestations are the major cause of mortality in patients with eosinophilic granulomatosis with polyangiitis (EGPA). Among these manifestations in EGPA patients, in the literature, there are fewer reports describing bradycardia in EGPA patients than those describing tachycardia. A 50-year-old woman with a history of childhood-onset asthma. At age 28, she was diagnosed with eosinophilic gastroenteritis without the diagnosis of EGPA and was started on a systemic steroid and had maintenance daily dose of 2.5 mg after gradually tapered. She had experiencing dizziness and palpitations 2 weeks after discontinuation of the steroid treatment. At emergency visit, electrocardiography revealed an advanced atrioventricular block of 3:1 or less. Forty-eight minutes after the start of electrocardiography, only a P wave was observed and cardiac arrest occurred for 9 s and temporary emergency pacing was performed immediately. She was diagnosed as EGPA presenting leukocyte count, 16,500/µL, 42.8% of which were eosinophils and sinusitis in computed-tomography. She could be survival by treatment of steroid, following the patient to withdraw from an external pacemaker. She received prednisolone of 60 mg, intravenous cyclophosphamide and intravenous immunoglobulin. She had relapsed presenting peripheral eosinophilia, abdominal and numbness in the toes of the left leg pain, but not arrythmia after tapered of prednisolone. Following additional steroid pulse, she had an increase of prednisolone and continued by intravenous cyclophosphamide, intravenous immunoglobulin and started mepolizumab. We presented a severe case of EGPA presenting an advanced atrioventricular block into cardiac arrest.
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Asma , Bloqueio Atrioventricular , Síndrome de Churg-Strauss , Granulomatose com Poliangiite , Parada Cardíaca , Feminino , Humanos , Adulto , Pessoa de Meia-Idade , Síndrome de Churg-Strauss/complicações , Síndrome de Churg-Strauss/diagnóstico , Síndrome de Churg-Strauss/tratamento farmacológico , Granulomatose com Poliangiite/complicações , Granulomatose com Poliangiite/diagnóstico , Granulomatose com Poliangiite/tratamento farmacológico , Imunoglobulinas Intravenosas/uso terapêutico , Bloqueio Atrioventricular/diagnóstico , Bloqueio Atrioventricular/etiologia , Bloqueio Atrioventricular/terapia , Prednisolona/uso terapêutico , Ciclofosfamida/uso terapêutico , Asma/tratamento farmacológico , Parada Cardíaca/tratamento farmacológicoRESUMO
Interparietal inguinal hernia, an exceedingly rare type of inguinal hernia in which the hernia sac anatomically lies between the tissue layers of the abdominal wall, is difficult to diagnose from physical findings. Given the few reports on interparietal inguinal hernias, this condition has remained fairly unrecognized. Herein, we report the successful imaging and laparoscopic diagnoses as well as repair of an interparietal inguinal hernia. Atypical physical findings and computed tomography data help in the diagnosis of an interparietal inguinal hernia. The laparoscopic approach is useful and feasible for both the diagnosis and treatment of interparietal inguinal hernia.
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BACKGROUND: Primary fascia closure is often difficult following an open abdomen (OA). While negative-pressure wound therapy (NPWT) is recommended to enhance successful primary fascia closure, the optimal methods and degree of negative pressure remain unclear. This study aimed to elucidate optimal methods of NPWT as a tentative abdominal closure for OA to achieve primary abdominal fascia closure. MATERIALS AND METHODS: A multicenter, retrospective, cohort study of adults who survived OA greater than 48 h was conducted in 12 institutions between 2010 and 2022. The achievement of primary fascia closure and incidence of enteroatmospheric fistula were examined based on methods (homemade, superficial NPWT kit, or open-abdomen kit) or degrees of negative pressure (<50, 50-100, or >100 mmHg). A generalized estimating equation was used to adjust for age, BMI, comorbidities, etiology for laparotomy requiring OA, vital signs, transfusion, severity of critical illness, and institutional characteristics. RESULTS: Of the 279 included patients, 252 achieved primary fascia closure. A higher degree of negative pressure (>100 mmHg) was associated with fewer primary fascia closures than less than 50 mmHg [OR, 0.18 (95% CI: 0.50-0.69), P =0.012] and with more frequent enteroatmospheric fistula [OR, 13.83 (95% CI: 2.30-82.93)]. The methods of NPWT were not associated with successful primary fascia closure. However, the use of the open-abdomen kit was related to a lower incidence of enteroatmospheric fistula [OR, 0.02 (95% CI: 0.00-0.50)]. CONCLUSION: High negative pressure (>100 mmHg) should be avoided in NPWT during tentative abdominal closure for OA.
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Técnicas de Fechamento de Ferimentos Abdominais , Fístula Intestinal , Tratamento de Ferimentos com Pressão Negativa , Adulto , Humanos , Estudos Retrospectivos , Estudos de Coortes , Resultado do Tratamento , Técnicas de Fechamento de Ferimentos Abdominais/efeitos adversos , Abdome , Fístula Intestinal/etiologia , Fístula Intestinal/cirurgia , Tratamento de Ferimentos com Pressão Negativa/métodosRESUMO
BACKGROUND: Definitive chemoradiotherapy (dCRT) using cisplatin plus 5fluorouracil (CF) with radiation is considered the standard treatment for unresectable locally advanced T4 esophageal squamous cell carcinoma (ESCC). Recently, induction chemotherapy has received attention as an effective treatment strategy. CASE SUMMARY: We report a successful case of a 59-year-old female with unresectable locally advanced T4 ESCC treated by two additional courses of chemotherapy with CF after induction chemotherapy with docetaxel, cisplatin and fluorouracil (DCF) followed by dCRT. Initial esophagogastroduodenoscopy (EGD) detected a type 2 advanced lesion located on the middle part of the esophagus, with stenosis. Computed tomography detected the primary tumor with suspected invasion of the left bronchus and 90° of direct contact with the aorta, and upper mediastinal lymph node metastasis. Pathological findings from biopsy revealed squamous cell carcinoma. We initially performed induction chemotherapy using three courses of DCF, but the lesion was still evaluated unresectable after DCF chemotherapy. Therefore, we subsequently performed dCRT treatment (CF and radiation). After dCRT, prominent reduction of the primary tumor was recognized but a residual tumor with ulceration was detected by EGD. Since the patient had some surgical risk, we performed two additional courses of CF and achieved a clinically complete response. After 14 mo from last administration of CF chemotherapy, recurrence has not been detected by computed tomography and EGD, and biopsy from the scar formation has revealed no cancer cells. CONCLUSION: We report successful case with tumor remnants even after DCF and subsequent dCRT, for whom a complete response was finally achieved with two additional courses of CF chemotherapy. Additional CF chemotherapy could be one radical treatment option for residual ESCC after treatment with induction DCF followed by dCRT to avoid salvage surgery, especially for high-risk patients.
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BACKGROUND: Pancreaticoduodenectomy (PD) for advanced gastric cancer is rarely performed because of the high morbidity and mortality rates and low survival rate. However, neoadjuvant chemotherapy for advanced gastric cancer has improved, and chemotherapy combined with trastuzumab may have a preoperative tumor-reducing effect, especially for human epidermal growth factor receptor 2 (HER2)-positive cases. CASE SUMMARY: We report a case of successful radical resection with PD after neoadjuvant S-1 plus oxaliplatin (SOX) and trastuzumab in a patient (66-year-old male) with advanced gastric cancer invading the pancreatic head. Initial esophagogastroduodenoscopy detected a type 3 advanced lesion located on the lower part of the stomach obstructing the pyloric ring. Computed tomography detected lymph node metastasis and tumor invasion to the pancreatic head without distant metastasis. Pathological findings revealed adenocarcinoma and HER2 positivity (immunohistochemical score of 3 +). We performed staging laparoscopy and confirmed no liver metastasis, no dissemination, negative lavage cytological findings, and immobility of the distal side of the stomach due to invasion to the pancreas. Laparoscopic gastrojejunostomy was performed at that time. One course of SOX and three courses of SOX plus trastuzumab were administered. Preoperative computed tomography showed partial response; therefore, PD was performed after neoadjuvant chemotherapy, and pathological radical resection was achieved. CONCLUSION: We suggest that radical resection with PD after neoadjuvant chemotherapy plus trastuzumab is an option for locally advanced HER2-positive gastric cancer invading the pancreatic head in the absence of non-curative factors.
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Terapia Neoadjuvante , Neoplasias Gástricas , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Gastrectomia , Humanos , Masculino , Pâncreas , Pancreaticoduodenectomia , Neoplasias Gástricas/diagnóstico por imagem , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/cirurgiaRESUMO
BACKGROUND: Inguinal hernia repair is one of the most common general surgical operations worldwide. We present a case of indirect inguinal hernia containing an expanded portosystemic shunt vessel. CASE SUMMARY: We report a 72-year-old man who had a 4 cm × 4 cm swelling in the right inguinal region, which disappeared with light manual pressure. Abdominal-pelvic computed tomography (CT) revealed a right inguinal hernia containing an expanded portosystemic shunt vessel, which had been noted for 7 years due to liver cirrhosis. We performed Lichtenstein's herniorrhaphy and identified the hernia sac as being indirect and the shunt vessel existing in the extraperitoneal cavity through the internal inguinal ring. Then, we found two short branches between the expanded shunt vessel and testicular vein in the middle part of the inguinal canal and cut these branches to allow the shunt vessel to return to the extraperitoneal cavity of the abdomen. The hernia sac was returned as well. We encountered no intraoperative complications. After discharge, groin seroma requiring puncture at the outpatient clinic was observed. CONCLUSION: If an inguinal hernia patient has portal hypertension, ultrasound should be used to determine the contents of the hernia. When atypical vessels are visualized, they may be shunt vessels and additional CT is recommended to ensure the selection of an adequate approach for safe hernia repair.
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BACKGROUND: Sclerodermataceae are known to be poisonous mushrooms; current published reports regarding the toxicity and treatment for Sclerodermataceae poisoning are limited. CASE PRESENTATION: A 66-year-old man was transferred to our hospital with complaints of visual disturbance, lightheadedness, bradycardia, and shock. The patient's medical history included cerebral hemorrhage and alcoholic hepatitis. He had eaten a mushroom growing in his garden, 30 min before arriving at our hospital. We carried out gastric lavage and gave the patient activated charcoal within an hour of mushroom ingestion. Particles of the mushroom were obtained during lavage, and most complaints were relieved immediately. However, the patient remained in shock for <2 h. He was admitted for observation and discharged 2 days later with no complications. The Public Health and Welfare Office later identified the mushroom as Scleroderma albidum. CONCLUSION: Scleroderma albidum caused muscarinic effects; features of central nervous system toxicity were also apparent.
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BACKGROUND: Survival rates in patients with esophageal cancer undergoing esophagectomy have improved, but the prevalence of gastric tube cancer (GTC) has also increased. Total resection of the gastric tube with lymph node dissection is considered a radical treatment, but GTC surgery is more invasive and involves a higher risk of severe complications or death, particularly in elderly patients. CASE SUMMARY: We report an elderly patient with early GTC that had invaded the duodenum who was successfully treated with resection of the distal gastric tube and Roux-en-Y (R-Y) reconstruction. The tumor was a type 0-IIc lesion with ulcer scars surrounding the pyloric ring. Endoscopic submucosal resection was not indicated because the primary lesion was submucosally invasive, was undifferentiated type, surrounded the pyloric ring, and had invaded the duodenum. Resection of distal gastric tube with R-Y reconstruction was safely performed, with preservation of the right gastroepiploic artery (RGEA) and right gastric artery (RGA). CONCLUSION: Distal resection of the gastric tube with preservation of the RGEA and RGA is a good treatment option for elderly patients with cT1bN0 GTC in the lower part of the gastric tube.
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We found 4-pyridylmethylthio derivative 1 to be very effective in using antiangiogenesis activity to prevent proliferation of HUVECs (Human Umbilical Vein Endothelial Cells), which was induced by vascular endothelial growth factor (VEGF). Compound 1 was equally effective in inhibiting VEGF receptor2 tyrosine kinase (KDR, IC(50)=26nM). We deduced that the inhibition was the result of binding the catalytic domain of VEGF receptor2 tyrosine kinase in a similar fashion to both phthalazine derivative PTK787 2 and anthranylamide derivative AAL993 3. In this report, we will describe the conformational analyses, from ab initio MO calculation and X-ray crystallographic analyses, of compound 1 and the analogs, which include non-active 9, all in comparison with 2 and 3. The conformation-activity relationships suggest that a nonbonded intramolecular interaction between the sulfur and the carbonyl oxygen of 1 was very important in inhibiting KDR.
Assuntos
Inibidores da Angiogênese/farmacologia , Endotélio Vascular/efeitos dos fármacos , Piridinas/farmacologia , Veias Umbilicais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Algoritmos , Inibidores da Angiogênese/síntese química , Domínio Catalítico , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Endotélio Vascular/citologia , Humanos , Concentração Inibidora 50 , Conformação Molecular , Ftalazinas/farmacologia , Piridinas/síntese química , Relação Estrutura-Atividade , Veias Umbilicais/citologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
BACKGROUND: Achondroplasia is an inherited disorder and the most common type of short-limbed dwarfism in human beings, affecting more than 250,000 individuals worldwide. To the best of our knowledge, no study has reported a correlation between achondroplasia and aortic dissection. Here, we report a rare case of acute type A aortic dissection repair in a patient with achondroplasia. CASE PRESENTATION: An 82-year-old Japanese female with achondroplasia was admitted to our hospital because of acute-onset severe chest pain migration to her back accompanied by numbness and pain in the right lower limb. A computed tomography scan revealed acute type A aortic dissection with right leg ischemia because of an occlusion of the right common iliac artery. We successfully performed hemiarch repair. CONCLUSIONS: This report presents the first case of a patient at such an advanced age with dwarfism and cardiac surgery and the second case to illustrate successful acute aortic dissection repair in achondroplasia. Of note, all procedures were performed without specialized equipment. Overall, this report adds to the experience of successful cardiac surgery in this unique patient population.
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AIM: Many studies have evaluated the risk factors for anastomotic leakage after laparoscopic anterior resection. In this study in order to increase the tightness of anastomoses and prevent bleeding from their staple lines, a linear stapler with pre-attached bioabsorbable polyglycolic acid (PGA) felt was used for rectal transection, and the short-term surgical outcomes were evaluated. PATIENTS AND METHODS: A prospective registry of 62 patients with rectosigmoidal or rectal carcinoma who initially underwent laparoscopic anterior resection using PGA felt for rectal transection was reviewed. RESULTS: The overall frequency of anastomotic leakage was 1.6% (1/62), and none of the patients developed postoperative staple line bleeding or other adverse events related to the use of PGA felt. CONCLUSION: The frequency of anastomotic leakage was relatively low, and therefore the use of a linear stapler with pre-attached bioabsorbable PGA felt might reduce the risk of adverse events related to anastomosis, especially anastomotic leakage.
Assuntos
Anastomose Cirúrgica/métodos , Fístula Anastomótica/prevenção & controle , Laparoscopia/métodos , Ácido Poliglicólico/química , Neoplasias Retais/cirurgia , Grampeamento Cirúrgico , Adulto , Idoso , Idoso de 80 Anos ou mais , Anastomose Cirúrgica/efeitos adversos , Fístula Anastomótica/etiologia , Feminino , Humanos , Laparoscopia/efeitos adversos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Reto/patologia , Reto/cirurgia , Fatores de Risco , Resultado do TratamentoRESUMO
Case: An 89-year-old man fell from stairs and sustained head trauma. He was taking warfarin and aspirin. Upon arrival at our hospital, his Glasgow Coma Scale score was 14. Initial head computed tomography showed small acute subdural hematoma. We immediately administered vitamin K and ordered fresh-frozen plasma. Repeat computed tomography 3 and 6 h after trauma revealed the acute subdural hematoma had increased to 14 and 20 mm, respectively, and there were several new intracranial hemorrhages. Fresh-frozen plasma and platelet transfusion were initiated. Outcome: Follow-up computed tomography revealed no further progression of intracranial hemorrhages, and the patient's consciousness did not deteriorate further. Conclusion: Appropriate administration of vitamin K, fresh-frozen plasma, and platelets successfully arrested progression of traumatic intracranial hemorrhages in this patient taking anticoagulant/antiplatelet agents and may have averted brain surgery.
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Four murine monoclonal anti-human deoxyribonuclease II (DNase II) antibodies were obtained from BALB/c mice immunized with human DNase II purified from human liver. Both single radial enzyme diffusion (SRED) and DNA-cast polyacrylamide gel electrophoresis (DNA-cast PAGE) were very useful for obtaining the DNase II-specific antibodies. All of the antibodies showed specific inhibition of human DNase II enzyme activity and specific immunostaining of the 32-kDa enzyme band, which is one of the three non-identical subunits of human DNase II molecule separated by sodium dodecyl sulfate (SDS)-PAGE followed by blotting on a transfer membrane. A formyl-cellulofine resin conjugated with each antibody specifically adsorbed and efficiently desorbed the active DNase II enzyme. Insertion of the immunoaffinity step in our purification procedure made the purification of human DNase II easier, faster and more effective than the conventional procedure.
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Anticorpos Monoclonais , Endodesoxirribonucleases/imunologia , Fígado/química , Animais , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Endodesoxirribonucleases/química , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , CoelhosRESUMO
Deoxyribonuclease I (DNase I) was confirmed to be expressed in the human pituitary gland, particularly the anterior lobe, at levels comparable to those in the pancreas. The DNase I activity and the amount of gene transcript present in the pituitary glands of individuals aged from 1 month to 89 years was significantly age-dependent, with an abrupt elevation after the neonatal and prepubertal periods irrespective of gender, followed by a gradual age-dependent decline in males and a marked reduction in females in their postreproductive period. This DNase I age dependence in the pituitary gland was not present in the pancreas and serum. These observations suggest that tissue-specific up-regulation of DNase I gene expression in the pituitary gland occur, possibly at the onset of puberty.
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Envelhecimento/metabolismo , Desoxirribonuclease I/genética , Expressão Gênica , Hipófise/enzimologia , Puberdade/fisiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Desoxirribonuclease I/metabolismo , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Pâncreas/enzimologia , RNA MensageiroRESUMO
A survey of DNase I in nine different carp tissues showed that the hepatopancreas has the highest levels of both DNase I enzyme activity and gene expression. Carp hepatopancreatic DNase I was purified 17,000-fold, with a yield of 29%, to electrophoretic homogeneity using three-step column chromatography. The purified enzyme activity was inhibited completely by 20 mM EDTA and a specific anti-carp DNase I antibody and slightly by G-actin. Histochemical analysis using this antibody revealed the strongest immunoreactivity in the cytoplasm of pancreatic tissue, but not in that of hepatic tissue in the carp hepatopancreas. A 995-bp cDNA encoding carp DNase I was constructed from total RNA from carp hepatopancreas. The mature carp DNase I protein comprises 260 amino acids, the same number as the human enzyme, however, the carp enzyme has an insertion of Ser59 and a deletion of Ala225 in comparison with the human enzyme. These alterations have no influence on the enzyme activity and stability. Three amino acid residues, Tyr65, Val67, and Ala114, of human DNase I are involved in actin binding, whereas those of carp DNase I are shifted to Tyr66, Val68, and Phe115, respectively, by the insertion of Ser59: the decrease in affinity to actin is due to one amino acid substitution, Ala114Phe. The results of our phylogenetic and immunological analyses indicate that carp DNase I is not closely related to the mammalian, avian or amphibian enzymes, and forms a relatively tight piscine cluster with the tilapia enzyme.
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Desoxirribonuclease I/química , Proteínas de Peixes/química , Fígado/enzimologia , Pâncreas/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Carpas , Desoxirribonuclease I/biossíntese , Desoxirribonuclease I/genética , Desoxirribonuclease I/imunologia , Proteínas de Peixes/biossíntese , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Regulação Enzimológica da Expressão Gênica , Fígado/imunologia , Dados de Sequência Molecular , Pâncreas/imunologia , FilogeniaRESUMO
The DNase I from canine pancreas was purified 260-fold to electrophoretic homogeneity with a 35% yield using three-step column chromatography. The activity of the purified enzyme was completely inhibited by 20 mM EDTA, an antibody specific to the purified enzyme and G-actin. A 1,373-bp cDNA encoding canine DNase I was constructed from the total canine pancreatic RNA using a rapid amplification of cDNA ends method, followed by sequencing. The mature canine DNase I protein was found to consist of 262 amino acids. A survey of DNase I in 13 different canine tissues revealed the highest levels of both DNase I enzyme activity and gene expression in the pancreas; therefore, the canine DNase I is of the pancreatic type. Phylogenetic and sequence identity analyses, studies of immunological properties and the tissue-distribution patterns of DNase I indicated that the canine enzyme is more closely related to the human DNase I than to other mammalian DNases I. Therefore, canine DNase I is found to be one of the best substitutes in studies of human DNase I.
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Desoxirribonuclease I , Pâncreas/enzimologia , Sequência de Aminoácidos , Animais , Células COS , Clonagem Molecular , DNA Complementar/genética , Desoxirribonuclease I/química , Desoxirribonuclease I/genética , Desoxirribonuclease I/imunologia , Desoxirribonuclease I/isolamento & purificação , Cães , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
We devised a procedure that combines a simple extraction method, isoelectric focusing and activity staining using the dried agarose film overlay method, for deoxyribonuclease I (DNase I) typing from aged urine stains. DNase I types were determined without difficulty from urine stains kept at room temperature for 3 months or more in all of the samples tested. The amounts of urine stains required for typing after 3 months of storage were estimated to be equivalent to 60-120 microl of liquid urine. Therefore, considering that useful PCR-based DNA typing has not yet been developed for urine stains, DNase I polymorphism could be considered the first biochemical marker found to be well suited for individualization from small aged urine stains.
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Desoxirribonuclease I/genética , Desoxirribonuclease I/urina , Cromatografia em Agarose , Impressões Digitais de DNA/métodos , Marcadores Genéticos , Humanos , Focalização Isoelétrica , Fenótipo , Reação em Cadeia da Polimerase/métodos , Polimorfismo Genético , Fatores de TempoRESUMO
Murine monoclonal anti-human deoxyribonuclease II (DNase II) antibodies were obtained from BALB/c mice immunized with human DNase II purified from human liver. Both single radial enzyme diffusion and DNA-cast polyacrylamide gel electrophoresis were very useful screening methods for obtaining the DNase II-specific antibodies. All of the antibodies showed specific inhibition of human DNase II enzyme activity and specific immunostaining. Insertion of the immunoaffinity step in our purification procedure made the purification of human DNase II easier, faster and more effective than the conventional procedure.
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Anticorpos Monoclonais , Endodesoxirribonucleases/imunologia , Fígado/enzimologia , Urina/química , Animais , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Endodesoxirribonucleases/isolamento & purificação , Humanos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
We devised a simple DNA extraction procedure suitable for STR typing of urine sample. Use of a commercially available DNA/RNA extraction kit equipped with a silica-gel-based membrane made it possible to omit the recovery of urinary nucleated cells by sedimentation before the extraction. Successful genotyping of the TH01, HumTPO and multiplex STRs was achieved using aliquots of urine as small as 100 microL. Furthermore, application of this DNA extraction procedure to frozen urine samples provided STR allele results comparable to results obtained from fresh samples. Therefore, this extraction procedure is considered to be effective for STR typing of urine samples in both the frozen and aqueous state. Furthermore, addition of sodium azide to fresh urine samples prolonged their storage duration even at room temperature.