RESUMO
Because of global mobility and migration resulting in a growing diversity of the donor pool, the risk for donor-derived tuberculosis in solid organ transplant recipients becomes more and more relevant, even in countries with a low overall tuberculosis incidence. Here, we describe a case series of donor-derived tuberculosis in 2 of 3 solid organ transplant recipients and one medical staff member in Germany resulting in the death of one recipient. This case series highlights the relevance of this topic to clinicians. It advocates for a better communication between organ procurement organizations and transplant centers regarding donor information and transplant recipient outcome. Furthermore, it underpins the necessity for a standardized critical incident reporting system in the german transplant system to improve short- and long-term recipient's safety, health and survival.
Assuntos
Transplante de Órgãos/efeitos adversos , Doadores de Tecidos , Transplantados , Transplantes/microbiologia , Tuberculose , Idoso , Antituberculosos/administração & dosagem , Antituberculosos/uso terapêutico , Feminino , Pessoal de Saúde , Humanos , Masculino , Pessoa de Meia-Idade , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia , Tuberculose/transmissãoRESUMO
We report a case of a 27-year-old anesthetist who acquired tuberculosis (TB) while performing general anesthesia in a renal transplant (RTX) patient who had donor-derived contagious TB. The anesthetist developed pleural TB 6 months after exposure. Contact investigations (CIs) did not include health care workers (HCWs) of the Department of Anesthesiology, thereby missing the opportunity for the early diagnosis and treatment of TB. Genomic fingerprinting revealed identical Mycobacterium tuberculosis (MT) isolates in the anesthetist and in the RTX patient. The recipient had acquired disseminated TB from the harvested renal graft. The donor (liver and kidneys), a 67-year-old immigrant, had died from brain death by cerebral herniation after a stroke. She had been treated for tuberculosis with a pneumectomy 40 years ago. Since that time, she had been suffering from latent tuberculous infection (TBI), but had been considered to have been cured.
Assuntos
Transmissão de Doença Infecciosa do Paciente para o Profissional , Transplante de Rim/efeitos adversos , Tuberculose Pulmonar/etiologia , Tuberculose Pulmonar/transmissão , Adulto , Idoso , Anestesistas , Antibióticos Antituberculose/uso terapêutico , Feminino , Humanos , Transplante de Fígado/efeitos adversos , Mycobacterium tuberculosis , Doadores de Tecidos , Tuberculose Pulmonar/terapiaRESUMO
BACKGROUND: Limited surgical procedures for benign cystic neoplasms and endocrine tumours of the pancreas have the potential advantage of pancreatic tissue sparing compared to standard oncological resections. METHODS: Searching PubMed/MedLine, Embase and Cochrane Library identified 86 full papers: 25 reporting on enucleation (EN), 38 on central pancreatectomy (CP) and 23 on duodenum-preserving total/partial pancreatic head resection (DPPHRt/p). The results are based on analysis of data of 838, 912 and 431 patients for EN, CP and DPPHRt/s, respectively. RESULTS: The indication for EN for cystic neoplasms and neuro-endocrine tumours to EN was 20.5 and 73 %; for CP 62.9 and 31 %; and for DPPHRt/p 69.6 and 10.2%, respectively. The estimated mean tumour sizes were in EN-group 2.4 cm, in CP-group 2.9 cm and in DPPHRt/p-group 3.1 cm (DPPHRt/p vs EN, p = 0.035). Postoperative severe complications developed after EN, CP and DPPHRt/p in 9.6, 16.8 and 11.5% of patients; pancreatic fistula in 36.7, 35.2 and 20.1%; and reoperation was required in 4.7, 6.5 and 1.8 %, respectively. Hospital mortality after EN was 0.95 %; after CP 0.72%; and after DPPHRt/p 0.49%. Compared to EN and CP, DPPHRt/p exhibited significant lower frequency of reoperation (p = 0.029, p < 0.001) and lower rate of fistula (p < 0.001; p = 0.001). CONCLUSION: EN, CP and DPPHRt/p applied for benign tumours are associated with low surgery-related early postoperative morbidity, a very low hospital mortality and the advantages of conservation of pancreatic functions. However, the level of evidence for EN and CP compared to standard oncological resections appears presently low. There is a high level of evidence from prospective controlled trials regarding the significant maintenance of exocrine and endocrine pancreatic functions after DPPHRt/p compared to pancreato-duodenectomy.
Assuntos
Neoplasias Císticas, Mucinosas e Serosas/cirurgia , Tumores Neuroendócrinos/cirurgia , Pancreatectomia/métodos , Fístula Pancreática/etiologia , Neoplasias Pancreáticas/cirurgia , Mortalidade Hospitalar , Humanos , Neoplasias Císticas, Mucinosas e Serosas/patologia , Tumores Neuroendócrinos/patologia , Tratamentos com Preservação do Órgão/métodos , Pancreatectomia/efeitos adversos , Neoplasias Pancreáticas/patologia , ReoperaçãoRESUMO
BACKGROUND: Experimental studies and small anecdotal reports have documented the potential and feasibility of transgastric appendicectomy. This paper reports the results of the new technique in a selected group of patients. METHODS: From April 2010 transgastric appendicectomy was offered to all patients with acute appendicitis, but without generalized peritonitis or local contraindications. RESULTS: Of 111 eligible patients 15 agreed to undergo the transgastric operation. After conversion of the first case to laparoscopy because of severe inflammation and adhesions, the following 14 consecutive transgastric procedures were completed. Two patients with initial peritonitis required laparoscopic lavage 4 days after transgastric appendicectomy, but no leaks were detected at the appendiceal stump or stomach. CONCLUSION: These preliminary results have shown the feasibility of this innovative procedure. Additional studies, however, are required to demonstrate the specific advantages and disadvantages of this approach, and define its role in clinical surgery.
Assuntos
Apendicectomia/métodos , Apendicite/cirurgia , Gastroscopia/métodos , Cirurgia Endoscópica por Orifício Natural/métodos , Doença Aguda , Adolescente , Adulto , Idoso , Estudos de Viabilidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
A reliable, noninvasive method for monitoring patients with transitional cell carcinoma (TCC) of the bladder would be of great clinical benefit. Cystoscopy is currently the "gold standard," but it is invasive, expensive and uncomfortable for the patient. Recently, we demonstrated a novel approach for the detection of primary bladder cancer based on microsatellite analysis of urine DNA. To determine the feasibility of this technique for following-up patients with TCC, we tested serial urine samples from 21 patients who had been treated for bladder cancer with 20 polymorphic microsatellite markers in a blinded fashion. We detected recurrent lesions in 10 out of 11 patients and correctly predicted the existence of a neoplastic cell population in the urine of two patients, 4 and 6 months before cystoscopic evidence of the tumor. The assay was negative in 10 of 10 patients who had no evident cancer. Microsatellite analysis of urine sediment represents a novel and potentially powerful clinical tool for the detection of recurrent bladder cancer.
Assuntos
Carcinoma de Células de Transição/urina , Repetições de Microssatélites , Recidiva Local de Neoplasia/diagnóstico , Recidiva Local de Neoplasia/urina , Neoplasias da Bexiga Urinária/urina , Idoso , Idoso de 80 Anos ou mais , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Método Simples-Cego , Neoplasias da Bexiga Urinária/genéticaRESUMO
Rabbits were immunized with diphtheria toxoid combined with complete Freund's adjuvant. Half of the animals were started on intramuscular injections of chloramphenicol 24 hr before the injection of the antigens. There was a general depression of protein synthesis in the immune system in the presence of chloramphenicol, but a greater effect on the synthesis of antibody than on the synthesis of proteins necessary for reproduction and maturation. In contrast to the finding of antibody in cells of the spleen and in the circulation of the control animals, those animals receiving chloramphenicol did not have measurable circulating antibody, and their spleens contained only a few cells with intracytoplasmic antibody late in the course of the experiment. Cytologically there was maturation of potential antibody-producing cells in the red pulp and nonfollicular white pulp of the spleen while the animals were receiving chloramphenicol. These cells developed more slowly, and were fewer and smaller than those of the control animals. They had numerous small, electron-opaque particles in their cytoplasm early in development. Ribosomes were synthesized, though fewer in number. The endoplasmic reticulum formed more slowly.
Assuntos
Cloranfenicol/farmacologia , Plasmócitos/citologia , Plasmócitos/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Feminino , Imunoglobulina G/imunologia , Masculino , Microscopia Eletrônica , Plasmócitos/ultraestrutura , Coelhos , Baço/citologiaRESUMO
Calcium activated skinned frog muscle fibers develop a large relative force at a sarcomere length of 1.0 micrometer. Since the normal myofilament lattice is perturbed at this length, regularity of the lattice does not appear to be an important factor in the contraction mechanism.
Assuntos
Fenômenos Biomecânicos , Cálcio/farmacologia , Contração Muscular , Miofibrilas/fisiologia , Animais , Anuros , Técnicas In Vitro , Contração Muscular/efeitos dos fármacos , Miofibrilas/efeitos dos fármacosRESUMO
Microsatellite DNA markers have been widely used as a tool for the detection of loss of heterozygosity and genomic instability in primary tumors. In a blinded study, urine samples from 25 patients with suspicious bladder lesions that had been identified cystoscopically were analyzed by this molecular method and by conventional cytology. Microsatellite changes matching those in the tumor were detected in the urine sediment of 19 of the 20 patients (95 percent) who were diagnosed with bladder cancer, whereas urine cytology detected cancer cells in 9 of 18 (50 percent) of the samples. These results suggest that microsatellite analysis, which in principle can be performed at about one-third the cost of cytology, may be a useful addition to current screening methods for detecting bladder cancer.
Assuntos
DNA de Neoplasias/urina , Repetições de Microssatélites , Neoplasias da Bexiga Urinária/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Deleção Cromossômica , Cromossomos Humanos Par 9 , DNA de Neoplasias/genética , Feminino , Marcadores Genéticos , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Projetos Piloto , Reação em Cadeia da Polimerase , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/urina , Urina/citologiaRESUMO
OBJECTIVE: Clinicians monitor cognitive effects of drugs primarily by asking patients to describe their side effects. We examined the relationship of subjective perception of cognition to mood and objective cognitive performance in healthy volunteers and neurological patients. METHODS: Three separate experiments used healthy adults treated with lamotrigine (LTG) and topiramate (TPM), adults with epilepsy on LTG or TPM, and patients with idiopathic Parkinson's disease. Correlations were calculated for change scores on and off drugs in the first two experiments and for the single assessment in Experiment 3. RESULTS: Across all three experiments, significant correlations were more frequent (chi(2)=259, P < or = 0.000) for mood versus subjective cognitive perception (59%) compared with subjective versus objective cognition (2%) and mood versus objective cognitive performance (2%). CONCLUSIONS: Subjective perception of cognitive effects is related more to mood than objective performance. Clinicians should be aware of this relationship when assessing patients' cognitive complaints.
Assuntos
Afeto/fisiologia , Anticonvulsivantes/farmacologia , Cognição/fisiologia , Epilepsias Parciais/psicologia , Doença de Parkinson/psicologia , Desempenho Psicomotor/fisiologia , Autoimagem , Adulto , Afeto/efeitos dos fármacos , Anticonvulsivantes/uso terapêutico , Cognição/efeitos dos fármacos , Estudos Cross-Over , Depressão/psicologia , Método Duplo-Cego , Epilepsias Parciais/tratamento farmacológico , Feminino , Frutose/análogos & derivados , Frutose/farmacologia , Frutose/uso terapêutico , Humanos , Lamotrigina , Masculino , Testes Neuropsicológicos , Doença de Parkinson/tratamento farmacológico , Desempenho Psicomotor/efeitos dos fármacos , Qualidade de Vida , Topiramato , Triazinas/farmacologia , Triazinas/uso terapêuticoRESUMO
The effect of sequential methotrexate and 5-fluorouracil on the clonal growth of the human colon adenocarcinoma cell, HCT-8, and the hormone-dependent human breast carcinoma cell, 47-DN, was examined. In both cell lines, when 5-fluorouracil was given during the last 6 h of a 24 h methotrexate exposure period, there was marked synergistic inhibition of clonal growth. Shorter intervals or the reverse sequence of drugs were either additive or antagonistic. These results indicate the importance of the drug sequence and time interval between drug administration for optimal cytotoxicity in these human cell lines. This information suggests that the administration of methotrexate 18 h before 5-fluorouracil may have potential application in the design of clinical trials for these malignancies.
Assuntos
Adenocarcinoma/tratamento farmacológico , Neoplasias da Mama/tratamento farmacológico , Carcinoma/tratamento farmacológico , Neoplasias do Colo/tratamento farmacológico , Fluoruracila/uso terapêutico , Metotrexato/uso terapêutico , Linhagem Celular , Esquema de Medicação , Fluoruracila/administração & dosagem , Humanos , Metotrexato/administração & dosagemRESUMO
BACKGROUND: Organ-confined renal malignancies can be cured in the majority of patients, whereas more extensive lesions have a poor prognosis. We sought to develop a noninvasive test for renal cancer detection based on a novel molecular approach. METHODS: Matched urine and serum DNA samples were obtained before surgery from 30 patients with clinically organ-confined solid renal masses (25 with malignant tumors and five with tumors of low malignant potential) and were subjected to microsatellite analysis. Serum samples and urine samples obtained from 16 individuals without clinical evidence of genitourinary malignancy served as controls. RESULTS: Nineteen (76%) of the 25 patients with malignant tumors were found to have one or more microsatellite DNA alterations in their urine specimen, and 15 (60%) were found to have alterations in their serum DNA by microsatellite analysis. In every case, the microsatellite changes in urine or serum were identical to those found in the primary tumor. Three of five patients with tumors of low malignant potential were found to have DNA alterations in their urine, but none displayed alterations in their serum. Moreover, microsatellite alterations were not identified in either the urine or the serum samples from normal control subjects and patients with hematuria due to nephrolithiasis (renal stones). CONCLUSION: These data suggest that microsatellite DNA analysis of urine specimens provides a potentially valuable tool for the early detection of resectable kidney cancer. Furthermore, microsatellite analysis of serum samples reveals evidence of circulating tumor-specific DNA in approximately half of these patients and may reflect the propensity of these tumors to spread to distant sites at an early stage.
Assuntos
DNA/urina , Neoplasias Renais/diagnóstico , Neoplasias Renais/genética , Repetições de Microssatélites , Idoso , Análise Química do Sangue , DNA/sangue , Humanos , Neoplasias Renais/sangue , Neoplasias Renais/urina , Perda de Heterozigosidade , Pessoa de Meia-Idade , Sensibilidade e Especificidade , UrináliseRESUMO
Collecting duct carcinoma (CDC) of the kidney is a rare malignant neoplasm of distal nephron origin. Previous studies of CDC have shown loss of heterozygosity on chromosomal arm 1q in 57% of the cases studied. To better characterize 1q loss in CDC, we performed high-density mapping of the entire long arm of chromosome 1 in 13 CDC tumor samples. We observed complete deletion of chromosomal arm 1q in 5 samples and partial deletion in 4 additional tumors. Our study further showed that the region of minimal deletion is located at 1q32.1-32.2. Sixty-nine percent (9 of 13) of the tumors showed loss of heterozygosity in this area. These data suggest that a gene or group of genes that contribute to the development of distal nephron tumors may be located within the 1q32.1-32.2 region.
Assuntos
Deleção Cromossômica , Mapeamento Cromossômico , Cromossomos Humanos Par 1 , Neoplasias Renais/genética , Túbulos Renais Coletores , HumanosRESUMO
Carcinoma in situ (CIS) of the urinary bladder is an aggressive lesion that frequently progresses to an invasive tumor, yet the underlying molecular changes in this lesion are largely unknown. In this study, we microdissected 31 cases of CIS and examined them for loss of heterozygosity (LOH) on 13 chromosomal arms. Twenty-nine microsatellite markers were chosen for this analysis based on their location in regions previously shown to be frequently lost in primary transitional cell carcinoma of the bladder. LOH of chromosome 9 was a frequent event in these samples, occurring in 77% of these lesions, with 19 of 31 cases showing deletion on the 9p arm (61%) and 17 of 28 cases displaying LOH on 9q (61%). Fine mapping at 9p21 demonstrated that CIS also displayed a high frequency of homozygous deletion surrounding the p16INK4A locus, like superficial papillary tumors, the other form of noninvasive lesion found in the bladder. However, loss of 14q (70%) was frequent in CIS yet extremely rare in papillary lesions (9%). Other chromosomal arms showing frequent LOH included 8p (65%), 17p (60%), 13q (56%), 11p (54%), and 4q (52%), whereas slightly lower frequencies of loss were observed for 11q (36%), 4p (32%), 3p (31%), 18q (29%), and 5q (20%). CIS lesions already possess many of the genetic alterations displayed by invasive transitional cell carcinomas, potentially accounting for the aggressive nature of these lesions.
Assuntos
Carcinoma in Situ/genética , Deleção Cromossômica , Neoplasias da Bexiga Urinária/genética , Alelos , Cromossomos Humanos Par 9 , HumanosRESUMO
Accumulating evidence implicates the presence of putative tumor suppressor genes on human chromosome 4 that are potentially inactivated in the genesis of several different neoplasms. To accurately determine the frequency of allelic loss on both arms of human chromosome 4, we screened 282 fresh-frozen human bladder carcinomas for allelic loss. Loss of heterozygosity of at least one marker for chromosome 4 was identified in 129 tumors (45.7%). Fine mapping was accomplished using up to 15 polymorphic markers on the p arm and 19 markers on the q arm. We identified a 3-cM minimal area of loss on the p arm between microsatellite markers D4S1608 and D4S404 deleted in 82 tumors (29%). A total of 68 tumors (24%) targeted a 14-cM critical region identified on the distal q arm between markers D4S426 and D4S408. Loss of these two regions correlated with advanced stage and grade of the lesions. These data identify distinct regions of loss on chromosome 4 potentially involved in the late progression of bladder carcinoma.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 4 , Neoplasias da Bexiga Urinária/genética , Mapeamento Cromossômico , HumanosRESUMO
Two hundred eighty-five primary human carcinomas of the urinary bladder were examined for allelic loss on chromosome 14q. Seventeen highly polymorphic dinucleotide markers spanning the long arm of this acrocentric chromosome were selected for fine PCR-based mapping. Loss of heterozygosity for at least one marker was observed in 72 (25.3%) tumors. Thirty-four of these 72 tumors (47.2%) lost the entire long arm (monosomy), as suggested by loss of heterozygosity at all informative sites. Allelic loss on 14q occurred in all grades and stages of bladder cancer but was more commonly associated with muscle-invasive tumors (Ta, 9.4%; T1, 24.1%; and > or = T2, 41%). A deletion map of 16 primary tumors with partial losses delineated two minimal regions of loss. One region (approximately 2 cM) was bounded by markers D14S75 and D14S288 and the other (approximately 3 cM) by D14S51 and D14S267. Our results demonstrate that 14q loss is common in invasive bladder cancer and suggest that two potential suppressor loci at 14q12 and 14q32.1-32.2 may contribute to the genetic progression of this common cancer.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 14/genética , Deleção de Genes , Genes Supressores , Neoplasias da Bexiga Urinária/genética , HumanosRESUMO
Tumors of varying malignant potential arise from the complex epithelial lining of the nephron. Although the molecular characteristics of renal clear cell carcinomas, which arise from the proximal tubule, have been studied, little is known about tumors that develop from other parts of the renal tubular system. To elucidate common molecular lesions that may contribute to the development or progression of nonproximal tubule renal tumors, we performed a detailed microsatellite allelotype of lesions thought to arise from the renal collecting duct. Eighteen collecting duct carcinomas (CDCs) and 13 renal oncocytomas were studied using highly informative microsatellite markers on all autosomal arms. Loss of heterozygosity (LOH) was identified on multiple chromosomal arms in CDCs and renal oncocytomas. Microsatellite analysis revealed LOH of 1q in 57% of informative CDCs. LOH was also observed on arms 6p (45%), 8p (40%), and 21q (40%). In renal oncocytomas, LOH of 1q occurred in approximately 30% of tumors, but 1p LOH was observed in 57% of informative cases analyzed. High levels of LOH were also observed on arms 8p, 14q, 19q, and 21q in the oncocytomas studied. Loss of chromosomal arm 3p was infrequent in both tumor types. Our results suggest that the molecular events that contribute to the development of distal nephron tumors are distinct from those associated with the etiology of proximal tubule renal cancers.
Assuntos
Deleção Cromossômica , Cromossomos Humanos , DNA Satélite/genética , Neoplasias Renais/genética , Repetições de Microssatélites/genética , Adenoma Oxífilo/genética , Alelos , Carcinoma/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 1 , Marcadores Genéticos , Humanos , Túbulos Renais Coletores , Néfrons , Reação em Cadeia da PolimeraseRESUMO
Mitochondrial DNA (mtDNA) mutations scattered through coding and noncoding regions have been reported in cancer. The mechanisms that generate such mutations and the importance of mtDNA mutations in tumor development are still not clear. Here we present the identification of a specific and highly polymorphic homopolymeric C stretch (D310), located within the displacement (D) loop, as a mutational hotspot in primary tumors. Twenty-two % of the 247 primary tumors analyzed harbored somatic deletions/insertions at this mononucleotide repeat. Moreover, these alterations were also present in head and neck preneoplastic lesions. We further characterized the D310 variants that appeared in the lung and head and neck tumors. Most of the somatic alterations found in tumors showed deletion/insertions of 1- or 2-bp generating D310 variants identical to constitutive polymorphisms described previously. Sequencing analysis of individual clones from lymphocytes revealed that patients with D310 mutations in the tumors had statistically significant higher levels of D310 heteroplasmy (more than one length variant) in the lymphocyte mtDNA as compared with the patients without D310 mutations in the tumor mtDNA. On the basis of our observations, we propose a model in which D310 alterations are already present in normal cells and achieve homoplasmy in the tumor through a restriction/amplification event attributable to random genetic drift and clonal expansion.
Assuntos
DNA Mitocondrial/genética , Repetições de Microssatélites/genética , Neoplasias/genética , Carcinoma de Células Escamosas/sangue , Carcinoma de Células Escamosas/genética , Feminino , Mutação em Linhagem Germinativa , Neoplasias de Cabeça e Pescoço/sangue , Neoplasias de Cabeça e Pescoço/genética , Humanos , Neoplasias Pulmonares/genética , Linfócitos/fisiologia , Masculino , Neoplasias/sangue , Polimorfismo Genético , Lesões Pré-Cancerosas/sangue , Lesões Pré-Cancerosas/genética , Análise de Sequência de DNARESUMO
The finding of Barnett et al. (Biophys. J. 61 (1992) 358) that NPM-reacted crossbridge heads do not bind strongly to actin in rigor solution is not easily interpreted in terms of the solution studies of Xie and Schoenberg (Biochemistry 37 (1998) 8048) who found strong binding of NPM-reacted myosin subfragment-1 to actin in solutions devoid of MgATP. For this reason, the current work uses stiffness measurement to re-investigate the binding of rabbit skeletal muscle crossbridges to actin in rigor solution. It is found that NPM-reacted crossbridge heads bind strongly to actin in rigor solution providing one is extremely careful to reduce MgATP contamination to levels well below those that would have a detectable effect on unmodified fibers. The reason for this is that NPM-reacted crossbridge heads, which hydrolyze MgATP extremely slowly, are especially susceptible to contaminant MgATP. The new fiber results show a strong correlation with the solution results. A further manifestation of this correlation is that pPDM-reacted crossbridge heads are different from NPM-reacted ones in that, like in solution, they remain weakly binding to actin even at extremely low MgATP levels. The findings suggest that the covalent crosslinking of SH1 and SH2 by pPDM is likely playing a significant role in locking pPDM-reacted crossbridge heads in a weakly binding conformation.
Assuntos
Músculo Esquelético/metabolismo , Actinas/química , Actinas/metabolismo , Actinas/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Fenômenos Biomecânicos , Fenômenos Biofísicos , Biofísica , Reagentes de Ligações Cruzadas , Técnicas In Vitro , Maleimidas , Contração Muscular/fisiologia , Músculo Esquelético/química , Músculo Esquelético/fisiologia , Subfragmentos de Miosina/química , Subfragmentos de Miosina/metabolismo , Subfragmentos de Miosina/fisiologia , CoelhosRESUMO
Treatment of relaxed skinned rabbit psoas muscle fibers with 0.1 mM N-phenylmaleimide (NPM) for 1 h locks all of the crossbridges in a weakly-binding state resembling that of the myosin.ATP crossbridge. Under these conditions, NPM reacts mainly with myosin heavy chain (Barnett et al. (1992) Biophys. J. 61, 358-367). Here the specific sites for that reaction are explored. Small bundles of rabbit psoas muscle fibers were treated with Triton X-100 to make the fiber sarcolemmas permeable. The bundles were treated with 0.1 mM [14C]NPM for 1 h, and homogenized for SDS-PAGE. 43 +/- 2.2% of the muscle fiber protein ran in the myosin heavy chain band, the same as for untreated fibers. An alkylating stoichiometry of 2.2 +/- 0.33 moles NPM per mole myosin heavy chain was determined. Exhaustive trypsin digestion followed by two-dimensional thin-layer chromatography and reverse-phase HPLC revealed two major sites on myosin heavy chain for NPM binding. The sites contained about the same amount of linked NPM, suggesting that the reaction stoichiometry of each site under the conditions studied is approx. 1 mol NPM/mol myosin heavy chain. Comparison of the labeled tryptic peptides with NPM-reacted synthetic SH1 and SH2 tryptic peptides and analysis of the treated fiber bundles' ATPase activity suggested that the sites for NPM reaction on myosin heavy chain when it locks crossbridges in a weakly-binding state are Cys-697 (SH2) and Cys-707 (SH1).