RESUMO
The gene defect for hereditary papillary renal carcinoma (HPRC) has recently been mapped to chromosome 7q, and germline mutations of MET (also known as c-met) at 7q31 have been detected in patients with HPRC (ref. 2). Tumours from these patients commonly show trisomy of chromosome 7 when analysed by cytogenetic studies and comparative genomic hybridization (CGH). However, the relationship between trisomy 7 and MET germline mutations is not clear. We studied 16 renal tumours from two patients with documented germline mutations in exon 16 of MET. Fluorescent in situ hybridization (FISH) analysis showed trisomy 7 in all tumours. To determine whether the chromosome bearing the mutant or wild-type MET gene was duplicated, we performed duplex PCR and phosphoimage densitometry using polymorphic microsatellite markers D7S1801 and D7S1822, which were linked to the disease gene locus, and D1S1646 as an internal control. We determined the parental origin of chromosome alleles by genotyping parental DNA. In all 16 tumours there was an increased signal intensity (2:1 ratio) of the microsatellite allele from the chromosome bearing the mutant MET compared with the allele from the chromosome bearing the wild-type MET. Our study demonstrates a non-random duplication of the chromosome bearing the mutated MET in HPRC and implicates this event in tumorigenesis.
Assuntos
Carcinoma Papilar/genética , Cromossomos Humanos Par 7 , Neoplasias Renais/genética , Proteínas Proto-Oncogênicas c-met/genética , Trissomia , Adulto , Feminino , Marcadores Genéticos , Humanos , Hibridização in Situ Fluorescente , Masculino , Mutação , Linhagem , Reação em Cadeia da PolimeraseRESUMO
Normal diploid somatic mammalian cell division generates 2 daughter cells as a result of a strict and well-controlled mitotic process. However, some defects during the progression of that process could generate an unbalanced distribution of chromosomes, aneuploidy and eventually, a malignant phenotype. Previous observations using a transgenic mouse model with diminished DNA repair capacity revealed the presence of nuclear buds (NBs) induced in vitro by the nucleoside analog zidovudine (Retrovir(R), 3'-azido-3'-deoxythymidine, AZT). Here we used bone marrow mesenchymal cells, taken from mice with the Xpa(-/-)Trp53(+/-) genotype, that were cultured and exposed to 0 and 100 muM AZT for 24 hours. Fixed and denatured cells were processed by fluorescence in situ hybridization (FISH) with whole chromosome painting probes used to identify chromosomes in cells growing on glass chamber slides (2 probes/slide). A variety of sizes and shapes of NBs were observed. Some NBs had a large connection with the main nucleus (>(1/4) of the NB diameter), others hada smaller connection (<(1/4) of the NB diameter), some were circular and positioned close to the nucleus, while some resided in the cytoplasm separated from the nucleus or connected by a thin chromatin strand. We had hypothesized that NBs would progress in the process of budding until separation occurred, but this was not proven by time-lapse photography studies performed for 20 hours. From 1,126 cells scored in the unexposed cultures, 10.39 % of cells carried NBs, while from 1,108 cells scored in the AZT-exposed cultures 29.16% of cells carried NBs (p = 0.001). In AZT-exposed cells there were a total of 322 NBs scored; 46.6% or 150 NBs contained positive signals for one or both probes used, while 53% or 172 NBs had no probe signal. In addition, FISH analysis showed no preferential localization of any chromosome within the NBs. Among the NBs that carried no probe signal, the presence of positive signals with inversion of DAPI imaging demonstrated centromeric content. It has been hypothesized that NBs occur as a result of expulsion of amplified DNA from the main nucleus; however, this data demonstrates that NBs may contain any chromosome, suggesting that NBs do not consist of just amplified DNA.
Assuntos
Fármacos Anti-HIV/toxicidade , Núcleo Celular/efeitos dos fármacos , Zidovudina/toxicidade , Animais , Camundongos , Proteína Supressora de Tumor p53/genética , Proteína de Xeroderma Pigmentoso Grupo A/genéticaRESUMO
Embryonic stem (ES) cells have been investigated in many animal models of injury and disease. However, few studies have examined the ability of pre-differentiated ES cells to improve functional outcome following traumatic brain injury (TBI). The purpose of the present study was to compare the effect of murine ES cells that were pre-differentiated into GABAergic neurons or astrocytes on functional recovery following TBI. Neural and astrocyte induction was achieved by co-culturing ES cells on a bone marrow stromal fibroblast (M2-10B4) feeder layer and incubating them with various mitogenic factors. Rats were initially prepared with a unilateral controlled cortical contusion injury of the sensorimotor cortex or sham procedure. Rats were transplanted 7 days following injury with approximately 100K GABAergic neurons, astrocytes, fibroblasts, or media. Animals were assessed on a battery of sensorimotor tasks following transplantation. The stromal fibroblast cells (M2-10B4), as a control cell line, did not differ significantly from media infusions. Transplantation of GABAergic neurons facilitated complete and total recovery on the vibrissae-forelimb placing test as opposed to all other groups, which failed to show any recovery. It was also found that GABAergic neurons reduced the magnitude of the initial impairment on the limb use test. Histological analysis revealed infiltration of host brain with transplanted neurons and astrocytes. The results of the present study suggest that transplantation of pre-differentiated GABAergic neurons significantly induces recovery of sensorimotor function; whereas, astrocytes do not.
Assuntos
Astrócitos/transplante , Lesões Encefálicas/terapia , Células-Tronco Embrionárias/transplante , Neurônios/transplante , Recuperação de Função Fisiológica/fisiologia , Córtex Somatossensorial/citologia , Análise de Variância , Animais , Lesões Encefálicas/patologia , Transplante de Tecido Encefálico/fisiologia , Diferenciação Celular/fisiologia , Sobrevivência Celular , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Masculino , Camundongos , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Córtex Somatossensorial/lesões , Córtex Somatossensorial/fisiologia , Transfecção , Ácido gama-Aminobutírico/metabolismoRESUMO
Genetically altered mouse models constitute unique systems to delineate the role of adrenergic receptor (AR) signaling mechanisms as modulators of cardiomyocyte function. The interpretation of results from these models depends on knowledge of the signaling properties of endogenous ARs in mouse cardiomyocytes. In the present study, we identify for the first time several defects in AR signaling in cardiomyocytes cultured from mouse ventricles. beta(1)-ARs induce robust increases in cAMP accumulation and the amplitude of the calcium and cell motion transients in mouse cardiomyocytes. Selective beta(2)-AR stimulation increases the amplitude of calcium and motion transients, with only a trivial rise in cAMP accumulation in comparison. beta(2)-AR responses are not influenced by pertussis toxin in cultured mouse cardiomyocytes. alpha(1)-ARs fail to activate phospholipase C, the extracellular signal-regulated protein kinase, p38-MAPK, or stimulate hypertrophy in mouse cardiomyocytes. Control experiments establish that this is not due to a lesion in distal elements in the signaling machinery, because these responses are induced by protease-activated receptor-1 agonists and phospholipase C is activated by Pasteurella multocida toxin (a G(q) alpha-subunit agonist). Surprisingly, norepinephrine activates p38-MAPK via beta-ARs in mouse cardiomyocytes, but beta-AR activation of p38-MAPK alone is not sufficient to induce cardiomyocyte hypertrophy. Collectively, these results identify a generalized defect in alpha(1)-AR signaling and a defect in beta(2)-AR linkage to cAMP (although not to an inotropic response) in cultured mouse cardiomyocytes. These naturally occurring vagaries in AR signaling in mouse cardiomyocytes provide informative insights into the requirements for hypertrophic signaling and impact on the value of mouse cardiomyocytes as a reconstitution system to investigate AR signaling in the heart.
Assuntos
Miocárdio/metabolismo , Receptores Adrenérgicos alfa/fisiologia , Receptores Adrenérgicos beta/fisiologia , Animais , Cálcio/metabolismo , Cardiomegalia/etiologia , Células Cultivadas , AMP Cíclico/metabolismo , Ativação Enzimática , Camundongos , Camundongos Endogâmicos ICR , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Contração Miocárdica/fisiologia , Miocárdio/citologia , Miocárdio/patologia , Norepinefrina/fisiologia , Ratos , Ratos Wistar , Fosfolipases Tipo C/metabolismo , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Previous studies have established that cardiomyocytes express protease-activated receptor (PAR)-1, a high-affinity receptor for thrombin, which is also activated by the tethered-ligand domain sequence (SFLLRN) and which promotes inositol trisphosphate accumulation, stimulates extracellular signal-regulated protein kinase, and modulates contractile function. A single previous report identified PAR-1 as a hypertrophic stimulus, but there have been no subsequent investigations of the mechanism. This study reveals the coexpression of PAR-1 and PAR-2 (a second PAR, which is activated by trypsin/tryptase but not thrombin) by Northern blot analysis and compares their signaling properties in neonatal rat ventricular cardiomyocytes. SFLLRN and SLIGRL (an agonist peptide for PAR-2) promote inositol trisphosphate accumulation, stimulate mitogen-activated protein kinases (extracellular signal-regulated protein kinase and p38-mitogen-activated protein kinase), elevate calcium concentration, and increase spontaneous automaticity. SFLLRN (but not SLIGRL) also activates c-Jun NH(2)-terminal kinase and AKT. In keeping with their linkage to pathways that have been associated with growth and/or survival, SFLLRN and SLIGRL both induce hypertrophy. However, PAR agonists promote cell elongation, a morphology that is distinct from the uniform increase in cell dimension induced by alpha(1)-adrenergic receptor activation. These studies provide novel evidence that cardiomyocytes coexpress 2 functional PARs, which link to a common set of signals that culminate in changes in contractile function and hypertrophic growth. PAR actions may assume clinical importance in the border zone surrounding an infarction, where local proteolysis of PARs by serine proteases generated during inflammatory or thrombogenic pathways would elevate calcium concentration (setting the stage for arrhythmias), promote hypertrophic growth, and/or influence cardiomyocyte survival.
Assuntos
Miocárdio/metabolismo , Proteínas Serina-Treonina Quinases , Receptores de Trombina/metabolismo , Transdução de Sinais , Animais , Cardiomegalia/etiologia , Cardiomegalia/patologia , Células Cultivadas , Ativação Enzimática/fisiologia , Ventrículos do Coração , Hidrólise , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Miocárdio/citologia , Fosfatidilinositóis/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-akt , Ratos , Receptor PAR-1 , Receptor PAR-2 , Receptores de Trombina/fisiologiaRESUMO
Human centromeres remain poorly characterized regions of the human genome despite their importance for the maintenance of chromosomes. In part this is due to the difficulty of cloning of highly repetitive DNA fragments and distinguishing chromosome-specific clones in a genomic library. In this work we report the highly selective isolation of human centromeric DNA using transformation-associated recombination (TAR) cloning. A TAR vector with alphoid DNA monomers as targeting sequences was used to isolate large centromeric regions of human chromosomes 2, 5, 8, 11, 15, 19, 21 and 22 from human cells as well as monochromosomal hybrid cells. The alphoid DNA array was also isolated from the 12 Mb human mini-chromosome DeltaYq74 that contained the minimum amount of alphoid DNA required for proper chromosome segregation. Preliminary results of the structural analyses of different centromeres are reported in this paper. The ability of the cloned human centromeric regions to support human artificial chromosome (HAC) formation was assessed by transfection into human HT1080 cells. Centromeric clones from DeltaYq74 did not support the formation of HACs, indicating that the requirements for the existence of a functional centromere on an endogenous chromosome and those for forming a de novo centromere may be distinct. A construct with an alphoid DNA array from chromosome 22 with no detectable CENP-B motifs formed mitotically stable HACs in the absence of drug selection without detectable acquisition of host DNAs. In summary, our results demonstrated that TAR cloning is a useful tool for investigating human centromere organization and the structural requirements for formation of HAC vectors that might have a potential for therapeutic applications.
Assuntos
Centrômero/genética , Cromossomos Artificiais Humanos , Clonagem Molecular/métodos , Recombinação Genética , Saccharomyces cerevisiae/genética , Sequência de Bases , Linhagem Celular , Centrômero/química , Humanos , Cinetocoros/química , Modelos Genéticos , Dados de Sequência Molecular , Análise de Sequência de DNA , Transformação GenéticaRESUMO
von Hippel-Lindau (VHL) disease is an autosomal dominantly inherited cancer syndrome predisposing to a variety of tumor types that include retinal hemangioblastomas, hemangioblastomas of the central nervous system, renal cell carcinomas, pancreatic cysts and tumors, pheochromocytomas, endolymphatic sac tumors, and epididymal cystadenomas [W. M. Linehan et al., J. Am. Med. Assoc., 273: 564-570, 1995; E. A. Maher and W. G. Kaelin, Jr., Medicine (Baltimore), 76: 381-391, 1997; W. M. Linehan and R. D. Klausner, In: B. Vogelstein and K. Kinzler (eds.), The Genetic Basis of Human Cancer, pp. 455-473, McGraw-Hill, 1998]. The VHL gene was localized to chromosome 3p25-26 and cloned [F. Latif et al., Science (Washington DC), 260: 1317-1320, 1993]. Germline mutations in the VHL gene have been detected in the majority of VHL kindreds. The reported frequency of detection of VHL germline mutations has varied from 39 to 80% (J. M. Whaley et al., Am. J. Hum. Genet., 55: 1092-1102, 1994; Clinical Research Group for Japan, Hum. Mol. Genet., 4: 2233-2237, 1995; F. Chen et al., Hum. Mutat., 5: 66-75, 1995; E. R. Maher et al., J. Med. Genet., 33: 328-332, 1996; B. Zbar, Cancer Surv., 25: 219-232, 1995). Recently a quantitative Southern blotting procedure was found to improve this frequency (C. Stolle et al., Hum. Mutat., 12: 417-423, 1998). In the present study, we report the use of fluorescence in situ hybridization (FISH) as a method to detect and characterize VHL germline deletions. We reexamined a group of VHL patients shown previously by single-strand conformation and sequencing analysis not to harbor point mutations in the VHL locus. We found constitutional deletions in 29 of 30 VHL patients in this group using cosmid and P1 probes that cover the VHL locus. We then tested six phenotypically normal offspring from four of these VHL families: two were found to carry the deletion and the other four were deletion-free. In addition, germline mosaicism of the VHL gene was identified in one family. In sum, FISH was found to be a simple and reliable method to detect VHL germline deletions and practically useful in cases where other methods of screening have failed to detect a VHL gene abnormality.
Assuntos
Deleção de Genes , Hibridização in Situ Fluorescente/métodos , Doença de von Hippel-Lindau/genética , Cromossomos Humanos Par 3 , Saúde da Família , Feminino , Testes Genéticos/métodos , Humanos , Modelos Genéticos , Mutação PuntualRESUMO
Therapeutic irradiation for head and neck cancer, and the autoimmune disease Sjogren's syndrome, lead to loss of salivary parenchyma. They are the two main causes of irreversible salivary gland hypofunction. Such patients cannot produce adequate levels of saliva, leading to considerable morbidity. We are working to develop an artificial salivary gland for such patients. A major problem in this endeavor has been the difficulty in obtaining a suitable autologous cellular component. This article describes a method of culturing and expanding primary salivary cells obtained from human submandibular glands (huSMGs) that is serum free and yields cells that are epithelial in nature. These include morphological (light and transmission electron microscopy [TEM]), protein expression (immunologically positive for ZO-1, claudin-1, and E-cadherin), and functional evidence. Under confocal microscopy, huSMG cells show polarization and appropriately localize tight junction proteins. TEM micrographs show an absence of dense core granules, but confirm the presence of tight and intermediate junctions and desmosomes between the cells. Functional assays showed that huSMG cells have high transepithelial electrical resistance and low rates of paracellular fluid movement. Additionally, huSMG cells show a normal karyotype without any morphological or numerical abnormalities, and most closely resemble striated and excretory duct cells in appearance. We conclude that this culture method for obtaining autologous human salivary cells should be useful in developing an artificial salivary gland.
Assuntos
Órgãos Artificiais , Técnicas de Cultura de Células/métodos , Polaridade Celular , Células Epiteliais/citologia , Glândulas Salivares/citologia , Engenharia Tecidual/métodos , Células Cultivadas , Meios de Cultura Livres de Soro , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Humanos , Glândulas Salivares/metabolismo , Glândula Submandibular/citologia , Glândula Submandibular/metabolismo , Junções Íntimas/metabolismo , Junções Íntimas/ultraestruturaRESUMO
Deletions and rearrangements involving the long arm of chromosome 11 are not infrequent occurrences in the non-Hodgkin's lymphomas. Recently, a tumor suppressor gene, the multiple endocrine neoplasia type 1 gene (MEN1) was cloned and mapped to chromosome 11q13. To assess the potential involvement of this gene in lymphomagenesis, we examined 94 primary cases of lymphoma and 12 cell lines by a combination of fluorescent in situ hybridization and PCR-SSCP analysis. In our initial analysis of 41 primary B or T lymphomas, MEN1 FISH analysis revealed allelic deletions in 15 cases (three of four B cell chronic lymphocytic leukemias, six of 15 follicular lymphomas, three of nine diffuse large B cell lymphomas, two of five mantle cell lymphomas, one of four Burkitt's lymphoma). To discern whether the MEN1 gene was in fact the target of the deletions, we assessed 20 of these 41 cases and an additional 74 primary lymphomas and 12 cell lines for MEN1 gene mutations using PCR-SSCP analysis. Abnormal SSCP patterns were found in exon 2 in two of the primary lymphoma cases and in one of the cell lines, but not in any of the original cases that showed MEN1 deletions by FISH. Furthermore, sequencing analysis revealed that the abnormal SSCP patterns in exon 2 were the result of a previously described genetic polymorphism (S145S: AGC --> ACT), and in one sample, the result of this S145S polymorphism associated with a second nucleotide substitution at position 498 which left the encoded amino acid unchanged. Our study indicates that the 11q13 locus is a frequent target of deletion in lymphoid neoplasms, but that there are no associated mutations of the MEN1 gene. This suggests that the 11q deletions either target another gene in lymphomas, or that the MEN1 gene is inactivated through means other than mutation.
Assuntos
Cromossomos Humanos Par 11 , Deleção de Genes , Leucemia Linfocítica Crônica de Células B/genética , Linfoma/genética , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas , Substituição de Aminoácidos , Linfoma de Burkitt/genética , Mapeamento Cromossômico , Éxons , Humanos , Hibridização in Situ Fluorescente , Leucemia Linfocítica Crônica de Células B/patologia , Linfonodos/patologia , Linfoma/sangue , Linfoma/patologia , Linfoma de Células B/genética , Linfoma de Células T/genética , Neoplasia Endócrina Múltipla Tipo 1/genética , Mutação Puntual , Reação em Cadeia da Polimerase , Polimorfismo Genético , Polimorfismo Conformacional de Fita Simples , Células Tumorais CultivadasRESUMO
Carney complex (CNC) is an autosomal dominant multiple endocrine neoplasia and lentiginosis syndrome characterised by spotty skin pigmentation, cardiac, skin, and breast myxomas, and a variety of endocrine and other tumours. The disease is genetically heterogeneous; two loci have been mapped to chromosomes 17q22-24 (the CNC1 locus) and 2p16 (CNC2). Mutations in the PRKAR1A tumour suppressor gene were recently found in CNC1 mapping kindreds, while the CNC2 and perhaps other genes remain unidentified. Analysis of tumour chromosome rearrangements is a useful tool for uncovering genes with a role in tumorigenesis and/or tumour progression. CGH analysis showed a low level 2p amplification recurrently in four of eight CNC tumours; one tumour showed specific amplification of the 2p16-p23 region only. To define more precisely the 2p amplicon in these and other tumours, we completed the genomic mapping of the CNC2 region, and analysed 46 tumour samples from CNC patients with and without PRKAR1A mutations by fluorescence in situ hybridisation (FISH) using bacterial artificial chromosomes (BACs). Consistent cytogenetic changes of the region were detected in 40 (87%) of the samples analysed. Twenty-four samples (60%) showed amplification of the region represented as homogeneously stained regions (HSRs). The size of the amplicon varied from case to case, and frequently from cell to cell in the same tumour. Three tumours (8%) showed both amplification and deletion of the region in their cells. Thirteen tumours (32%) showed deletions only. These molecular cytogenetic changes included the region that is covered by BACs 400-P-14 and 514-O-11 and, in the genetic map, corresponds to an area flanked by polymorphic markers D2S2251 and D2S2292; other BACs on the centromeric and telomeric end of this region were included in varying degrees. We conclude that cytogenetic changes of the 2p16 chromosomal region that harbours the CNC2 locus are frequently observed in tumours from CNC patients, including those with germline, inactivating PRKAR1A mutations. These changes are mostly amplifications of the 2p16 region, that overlap with a previously identified amplicon in sporadic thyroid cancer, and an area often deleted in sporadic adrenal tumours. Both thyroid and adrenal tumours constitute part of CNC indicating that the responsible gene(s) in this area may indeed be involved in both inherited and sporadic endocrine tumour pathogenesis and/or progression.
Assuntos
Anormalidades Múltiplas/genética , Aberrações Cromossômicas , Cromossomos Humanos Par 2/genética , Neoplasia Endócrina Múltipla/patologia , Mixoma/patologia , Transtornos da Pigmentação/patologia , Anormalidades Múltiplas/patologia , Deleção Cromossômica , Cromossomos Artificiais Bacterianos/genética , Mapeamento de Sequências Contíguas , Feminino , Humanos , Hibridização in Situ Fluorescente , Masculino , Repetições de Microssatélites , Hibridização de Ácido Nucleico/métodos , Transtornos da Pigmentação/genética , Síndrome , Células Tumorais CultivadasRESUMO
The vitamin D receptor (VDR) is a member of the steroid hormone receptor superfamily of ligand-activated transcription factors. The VDR gene was previously mapped to human chromosome 12q13-12q14, but its precise physical and genetic localization are unknown. The present study reports the mapping of the human VDR gene by radiation hybrid (RH) analysis, the isolation of a bacterial artificial chromosome (BAC) containing this gene, and physical mapping of the VDR gene by fluorescent in situ hybridization (FISH). RH analysis placed the VDR gene locus at chromosome 12cen-q12, flanked by Stanford Human Genome Center (SHGC) 30216 and SHGC 9798 (D12S1892) markers. FISH analysis of a BAC containing the VDR gene confirmed its centromeric location. Thus, we have identified a BAC and genetic markers which can be used in the genetic analysis of the VDR gene and investigation of its involvement in osteoporosis and related disorders. We conclude that the VDR gene is centromeric to its previously reported locus on chromosome 12.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 12/genética , Receptores de Calcitriol/genética , Centrômero/genética , Cromossomos Bacterianos/genética , Biblioteca Gênica , Marcadores Genéticos , Humanos , Células Híbridas , Hibridização in Situ FluorescenteRESUMO
Carney complex (CNC) is a familial multiple neoplasia and lentiginosis syndrome with features overlapping those of McCune-Albright syndrome (MAS) and other multiple endocrine neoplasia (MEN) syndromes, MEN type 1 (MEN 1), in particular. GH-producing pituitary tumors have been described in individual reports and in at least two large CNC patient series. It has been suggested that the evolution of acromegaly in CNC resembles that of the other endocrine manifestations of CNC in its chronic, often indolent, progressive nature. However, histologic and molecular evidence has not been presented in support of this hypothesis. In this investigation, the pituitary glands of eight patients with CNC and acromegaly [age, 22.9+/-11.6 yr (mean +/- SD)] were studied histologically. Tumor DNA was used for comparative genomic hybridization (CGH) (four tumors). All tumors stained for both GH and prolactin PRL (eight of eight), and some for other hormones, including alpha-subunit. Evidence for somatomammotroph hyperplasia was present in five of the eight patients in proximity to adenoma tissue; in the remaining three only adenoma tissue was available for study. CGH showed multiple changes involving losses of chromosomal regions 6q, 7q, 11p, and 11q, and gains of 1pter-p32, 2q35-qter, 9q33-qter, 12q24-qter, 16, 17, 19p, 20p, 20q, 22p and 22q in the most aggressive tumor, an invasive macroadenoma; no chromosomal changes were seen in the microadenomas diagnosed prospectively (3 tumors). We conclude that, in at least some patients with CNC, the pituitary gland is characterized by somatotroph hyperplasia, which precedes GH-producing tumor formation, in a pathway similar to that suggested for MAS-related pituitary tumors. Three GH-producing microadenomas showed no genetic changes by CGH, whereas a macroadenoma in a patient, whose advanced acromegaly was not cured by surgery, showed extensive CGH changes. We speculate that these changes represent secondary and tertiary genetic "hits" involved in pituitary oncogenesis. The data (1) underline the need for early investigation for acromegaly in patients with CNC; (2) provide a molecular hypothesis for its clinical progression; and (3) suggest a model for MAS- and, perhaps, MEN 1-related GH-producing tumor formation.
Assuntos
Adenoma/metabolismo , Adenoma/patologia , Hormônio do Crescimento Humano/metabolismo , Neoplasias Hipofisárias/metabolismo , Neoplasias Hipofisárias/patologia , Acromegalia/genética , Acromegalia/metabolismo , Acromegalia/cirurgia , Adenoma/genética , Adolescente , Adulto , DNA de Neoplasias/genética , Doenças do Sistema Endócrino/genética , Doenças do Sistema Endócrino/metabolismo , Doenças do Sistema Endócrino/patologia , Feminino , Humanos , Hibridização In Situ , Masculino , Mixoma/genética , Mixoma/metabolismo , Mixoma/patologia , Neurilemoma/genética , Neurilemoma/metabolismo , Neurilemoma/patologia , Transtornos da Pigmentação/genética , Transtornos da Pigmentação/metabolismo , Transtornos da Pigmentação/patologia , Hormônios Hipofisários/sangue , Neoplasias Hipofisárias/genética , SíndromeRESUMO
Aldosterone synthase (AS) is encoded by the CYP11B2 gene, a candidate for familial hypertension. CYP11B2 was previously mapped to chromosome 8q but its precise localization is necessary for genetic studies of hypertension. The present study reports the genetic mapping of the human CYP11B2 gene by radiation hybrid (RH) analysis, the isolation of a bacterial artificial chromosome (BAC) containing this gene and its physical mapping by fluorescent in situ hybridization (FISH). The CYP11B2 locus is on the most distal segment of the long arm of chromosome 8, proximal to the microsatellite polymorphic marker D8S1704. This location, which was confirmed by FISH, is approximately 60cM telomeric to the currently listed human gene locus (chromosome 8q21-22) and corresponds to cytogenetic band 8q24.3. The BACs containing the gene and a high-resolution map of the CYP11B2 locus are useful for genetic studies of hypertension and other endocrine disorders.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 8 , Citocromo P-450 CYP11B2/genética , Mapeamento Cromossômico/métodos , Cromossomos Bacterianos/genética , Cromossomos Humanos Par 8/genética , Humanos , Hibridização in Situ FluorescenteRESUMO
BACKGROUND: Use of stainless steel wires in median sternotomy closure is at times associated with serious complications. In view of this, the efficacy and safety of a stainless steel band designed for fixation and approximation of the sternum in cardiothoracic procedures was evaluated in a prospective, randomized study. METHODS: Forty-eight patients undergoing open heart operations that involved a median sternotomy were studied. Group I (n = 21) was closed with four to six steel bands, and group II (n = 27) with six to eight standard stainless steel wires. The average age of the patients and the risk factors predisposing to dehiscence were similar in both groups. RESULTS: One postoperative death occurred in each group due to cardiac failure. In group I, the mean length of the postoperative hospital stay was 10.2 +/- 1.76 days (+/- 2 standard errors), whereas in group II the mean was 13.9 +/- 3.4 days (+/- 2 standard errors). Banded patients complained less of postoperative pain, although statistical significance was not achieved. No problems arose in either group during the 3-year follow-up. CONCLUSIONS: The steel bands, compared with wires, provided not only effective fixation, but a reduction in both postoperative pain and postoperative hospital stay. The band is now being studied in a larger group of patients to evaluate the incidence and type of complications associated with its use, as well as length of postoperative hospital stay.
Assuntos
Esterno/cirurgia , Suturas , Idoso , Fios Ortopédicos/estatística & dados numéricos , Procedimentos Cirúrgicos Cardíacos/métodos , Desenho de Equipamento , Seguimentos , Humanos , Pessoa de Meia-Idade , Aço Inoxidável , Suturas/estatística & dados numéricosRESUMO
PURPOSE: To describe a patient with metastasis of Ewing sarcoma to the choroid and the molecular genetics of the tumor. METHODS: A 26-year-old woman with metastatic Ewing sarcoma developed large choroidal masses in the left eye and died 2 months later. Autopsy of the eyes was performed. Dual-color fluorescent in situ hybridization was used to detect genetic alteration in the ocular tumor with EWS and FLI-1 probes. RESULTS: Histopathology confirmed choroidal metastatic Ewing sarcoma. Molecular analysis showed chromosomal translocation t(11;22)(q24;q12) or EWS/FLI-1 rearrangement in the malignant cells of the eye. CONCLUSIONS: Ewing sarcoma can rarely metastasize to the uvea. Molecular detection of the t(11;22)(q24;q12) translocation in Ewing sarcoma is valuable in the differential diagnosis of small round cell tumors.
Assuntos
Neoplasias Ósseas/genética , Neoplasias da Coroide/genética , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 22/genética , Sarcoma de Ewing/genética , Translocação Genética , Adulto , Neoplasias Ósseas/patologia , Neoplasias da Coroide/secundário , DNA de Neoplasias/análise , Evolução Fatal , Feminino , Humanos , Hibridização in Situ Fluorescente , Proteínas de Fusão Oncogênica/genética , Ossos Pélvicos , Proteína Proto-Oncogênica c-fli-1 , Proteína EWS de Ligação a RNA , Sarcoma de Ewing/secundário , Fatores de Transcrição/genéticaRESUMO
Allelic variation at a total of 20 nuclear-encoded microsatellites was examined among adult red snapper (Lutjanus campechanus) sampled from 4 offshore localities in the Gulf of Mexico. The number of alleles at the 20 microsatellites ranged from 5 to 20; average (+/- SE) direct count heterozygosity values ranged from 0.148 +/- 0.025 to 0.902 +/- 0.008. No significant departures from expectations of Hardy-Weinberg equilibrium were found for any locus within samples, and genotypes at pairs of microsatellites appeared to be randomly associated, i.e., in genotypic equilibrium. Tests of homogeneity in allele distributions among the 4 localities were nonsignificant for 19 of the microsatellites. Allele distribution at microsatellite Lca 43 was heterogeneous among localities before (but not after) Bonferroni corrections for multiple tests executed simultaneously. Tests of homogeneity in the distribution of individual alleles at Lca 43 gave similar results: one low frequency allele was distributed heterogeneously among samples before, but not after, Bonferroni correction. Molecular analysis of variance indicated that more than 99% of variation at each microsatellite was distributed within sample localities. These results generally are consistent with the hypothesis of a single population (stock) of red snapper in the northern Gulf of Mexico.
RESUMO
Homologous 1-naphtoxyalcanthiols of the type 1-C10H7O(CH2)nSH (n = 2-7) are used for structural studies of the microsomal cytochrome P450 active centre. It was found that the strongest complex of thiol with P450 is formed for n = 3. Microsomal oxidation of P450 substrates aminopyrine and benz(a) pyrene is inhibited by the 1-naphtoxyalcanthiols studied. A non-monotonous dependence of pI50 on n was found, the compound with a chain length n = 3 appeared to be the most effective inhibitor. The interaction of this thiol (n = 3) with both the heme group of P450 and the hydrophobic substrate zone is supposed and the distance between these points was estimated. It is possible to employ this approach for structural studies on the active centers of different isoforms of P450.
Assuntos
Sistema Enzimático do Citocromo P-450/análise , Membranas Intracelulares/enzimologia , Microssomos Hepáticos/enzimologia , Naftalenos , Compostos de Sulfidrila , Animais , Sítios de Ligação , Isoenzimas/análise , Cinética , Masculino , Oxirredução , Ratos , Ratos Endogâmicos , Especificidade por SubstratoRESUMO
Operations were performed on 192 patients with reflux esophagitis, 23 of them had peptic stricture of the esophagus. Esophago-fundoplication was the main operation. Nissen's (106), Tupe (47), Belsi's (3), Dor's (5), and atypical methods were applied. Whenever indicated it was supplemented by crurorhaphy, SPV, pylorotomy, correction of the duodenal junction, etc. Resection of the esophagus (19) was performed with one-stage esophagoplasty by means of the stomach through a left thoracoabdominal approach (14), the whole stomach passed through the posterior mediastinum from an abdomino-cervical approach (2) and the whole stomach with Lewis' intrathoracic anastomosis (3). Distal gastric resection was carried out in 6 and other operations in 3 patients. The mortality was 1%. Reoperations were performed in 5 patients. The results were good in 81.2% of cases. The tactics is individualized according to the presence or absence of a stricture, its length, and localization of the upper border.
Assuntos
Esofagite Péptica/cirurgia , Adolescente , Adulto , Idoso , Criança , Esofagite Péptica/diagnóstico , Esofagoplastia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , ReoperaçãoRESUMO
Examination of workers servicing radio location, radio navigation and communication means proved electromagnetic radiofrequency waves to induce some peripheral blood changes: cytopenia, Hb decrease, lower RBC and WBC counts, increased RBC with basophilic granularity, WBC metabolism alteration (higher acid phosphatase and myeloperoxidase activity), disordered lymphocytes subunits (T-helpers, T-suppressors) ratio and T- and B-cells numbers.