RESUMO
Pulsed-field gel electrophoresis was used to examine the distribution of telomere-associated sequences on individual chromosomes in four strains of Saccharomyces cerevisiae. The pattern of X and Y' distribution was different for each strain. At least one chromosome in each strain lacked Y', and in some strains, chromosome I, the smallest yeast chromosome, lacked detectable amounts of both X and Y'.
Assuntos
Cromossomos/ultraestrutura , Saccharomyces cerevisiae/genética , Sequência de Bases , Saccharomyces cerevisiae/ultraestruturaRESUMO
A 9-kilobase pair CEN4 linear minichromosome constructed in vitro transformed Saccharomyces cerevisiae with high frequency but duplicated or segregated inefficiently in most cells. Stable transformants were only produced by events which fundamentally altered the structure of the minichromosome: elimination of telomeres, alteration of the centromere, or an increase of fivefold or greater in its size. Half of the stable transformants arose via homologous recombination between an intact chromosome IV and the CEN4 minichromosome. This event generated a new chromosome from each arm of chromosome IV. The other "arm" of each new chromosome was identical to one "arm" of the unstable minichromosome. Unlike natural yeast chromosomes, these new chromosomes were telocentric: their centromeres were either 3.9 or 5.4 kilobases from one end of the chromosome. The mitotic stability of the telocentric chromosome derived from the right arm of chromosome IV was determined by a visual assay and found to be comparable to that of natural yeast chromosomes. Both new chromosomes duplicated, paired, and segregated properly in meiosis. Moreover, their structure, as deduced from mobilities in orthogonal field gels, did not change with continued mitotic growth or after passage through meiosis, indicating that they did not give rise to isochromosomes or suffer large deletions or additions. Thus, in S. cerevisiae the close spacing of centromeres and telomeres on a DNA molecule of chromosomal size does not markedly alter the efficiency with which it is maintained. Taken together these data suggest that there is a size threshold below which stable propagation of linear chromosomes is no longer possible.
Assuntos
Cromossomos/ultraestrutura , Saccharomyces cerevisiae/genética , Composição de Bases , Enzimas de Restrição do DNA , Meiose , Peso Molecular , Plasmídeos , Saccharomyces cerevisiae/citologia , Transformação GenéticaRESUMO
Fragments of chromosomal DNA from a variety of eucaryotes can act as ARSs (autonomously replicating sequence) in yeasts. ARSs enable plasmids to be maintained in extrachromosomal form, presumably because they function as initiation sites for DNA replication. We isolated eight different sequences from mouse chromosomal DNA which function as ARSs in Saccharomyces cerevisiae (bakers' yeast). Although the replication efficiency of the different mouse ARSs in yeasts appears to vary widely, about one-half of them functions as well as the yeast chromosomal sequence ARS1. Moreover, five of the ARSs also promote self replication of plasmids in Schizosaccharomyces pombe (fission yeast). Each of the ARSs was cloned into plasmids suitable for transformation of mouse tissue culture cells. Plasmids were introduced into thymidine kinase (TK)-deficient mouse L cells by the calcium phosphate precipitation technique in the absence of carrier DNA. In some experiments, the ARS plasmid contained the herpes simplex virus type 1 TK gene; in other experiments (cotransformations), the TK gene was carried on a separate plasmid used in the same transformation. In contrast to their behavior in yeasts, none of the ARS plasmids displayed a significant increase in transformation frequency in mouse cells compared with control plasmids. Moreover, only 1 of over 100 cell lines contained the original plasmid in extrachromosomal form. The majority of cell lines produced by transformation with an ARS TK plasmid contained multiple copies of plasmid integrated into chromosomal DNA. In most cases, results with plasmids used in cotransformations were similar to those for plasmids carrying TK. However, cell lines produced by cotransformations with plasmids containing any one of three of the ARSs (m24, m25, or m26) often contained extrachromosomal DNAs.
Assuntos
Replicação do DNA , DNA Recombinante/isolamento & purificação , Leveduras/genética , Animais , Cromossomos/metabolismo , Células L/metabolismo , Camundongos , Plasmídeos , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Timidina Quinase/genética , Transformação GenéticaRESUMO
Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) are potent immunomodulatory cytokines which are produced principally by cells of the macrophage-monocyte lineage. We conducted an investigation to assess the secretion of these cytokines by bronchoalveolar macrophages from patients with progressive stages of human immunodeficiency virus (HIV-1) infection. The mean level of TNF-alpha produced by macrophages from 9 patients with AIDS was significantly reduced compared with the responses of macrophages from 6 healthy HIV-1-seronegative persons, 6 patients with either asymptomatic HIV-1 infection or persistent generalized lymphadenopathy, and 6 patients with AIDS-related complex (ARC). The four study groups did not differ in their mean IL-1 beta responses. However, within the HIV-1-infected patient population, macrophages from 4 patients, 3 of whom had AIDS and 1 with ARC, failed to secrete detectable levels of IL-1 beta. All 4 patients were also nonresponsive in assays for TNF-alpha. These data establish that advanced HIV-1 infection may result in a pronounced dysfunction in the cytokine responses of alveolar macrophages.
Assuntos
Síndrome da Imunodeficiência Adquirida/imunologia , HIV-1 , Interleucina-1/biossíntese , Macrófagos/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Adulto , Líquido da Lavagem Broncoalveolar/citologia , Humanos , Lipopolissacarídeos/farmacologia , Alvéolos Pulmonares/metabolismoRESUMO
To evaluate bronchoalveolar lavage (BAL) findings in patients infected with human immunodeficiency virus (HIV), 39 patients seropositive for the virus but with no history of opportunistic infection were studied. Opportunistic organisms such as Pneumocystis carinii were not found in any of the 35 BAL fluids sent for special stains and cultures. Three of 16 (18 percent) BAL fluids sent for HIV culture were positive compared with a 60.9 percent blood HIV culture positivity in the same group. To evaluate cellular recovery, the patients were divided into Walter Reed (WR) groups 1 and 2 (blood CD4 greater than or equal to 400/cu mm) and WR3 to WR5 (blood CD4 less than 400/cu mm). Compared with ten nonsmoking healthy controls, the WR1 and WR2 group had a greater overall cellular recovery but this was not statistically significant when the smokers were excluded. There was no difference in macrophage or lymphocyte percentages in either patient group compared with controls. T-cell subset analysis of a small group of WR1 to WR5 patient BAL fluids revealed no difference in CD4 numbers or the CD4/CD8 rate between WR1 and WR2 and WR3 to WR5 patients. We conclude that opportunistic pulmonary infection is unlikely in HIV-seropositive patients with normal chest roentgenograms despite symptoms of dyspnea on exertion. Also, HIV can be isolated from BAL fluid from these patients although not as often as from blood. Finally, there appears to be no distinct progression in BAL cellular findings before the onset of acquired immunodeficiency syndrome.
Assuntos
Síndrome da Imunodeficiência Adquirida/patologia , Líquido da Lavagem Broncoalveolar , Soropositividade para HIV/patologia , Infecções Oportunistas/diagnóstico , Síndrome da Imunodeficiência Adquirida/complicações , Adulto , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/microbiologia , Eosinófilos , Feminino , HIV-1/isolamento & purificação , Humanos , Contagem de Leucócitos , Macrófagos , Masculino , Infecções Oportunistas/complicações , Pneumonia por Pneumocystis/complicações , Pneumonia por Pneumocystis/diagnóstico , Linfócitos TRESUMO
Lung cancer is the leading cause of cancer mortality in the United States in 1985. Early diagnosis and an aggressive surgical approach offer the best chance for a cure. Unfortunately, the majority of patients will be diagnosed after the disease has metastasized. Radiation therapy and chemotherapy, despite recent advances, are still basically palliative modalities. The most efficacious approach to the control of lung cancer is an effective anti-smoking campaign.
Assuntos
Carcinoma Broncogênico/mortalidade , Neoplasias Pulmonares/mortalidade , Carcinoma Broncogênico/diagnóstico , Carcinoma Broncogênico/patologia , Carcinoma Broncogênico/terapia , Diagnóstico Diferencial , Feminino , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Masculino , Estadiamento de Neoplasias , PrognósticoRESUMO
The use of gallium scanning in the treatment of patients with AIDS has been well described. In this study, 27 HIV-infected patients (Walter Reed Staging Classification I-V) with normal chest roentgenograms were evaluated to determine the occurrence of thoracic gallium abnormalities in early HIV infection. SPECT was used for gallium scanning. Patients received gallium injection on day 1 and bronchoalveolar lavage on day 2, and scanning was performed on day 3. Twenty-eight scans in 27 patients were performed. Mean nodal SPECT activity was compared with corresponding values for lung parenchyma, bronchoalveolar lavage cell counts, and peripheral blood T4 lymphocyte counts. No relationship between nodal and parenchymal activity and cell counts was observed. Although visual scan interpretation was unaffected, gallium activity was significantly increased in the region of bronchoalveolar lavage compared with uptake in other lung regions. It is concluded that SPECT gallium scanning demonstrates significant gallium avidity in recently lavaged lung areas, although no impact on visual scan interpretation was seen. Further, no correlation was seen between the degree of nodal uptake and the immunologic status of these patients with early HIV infection.
Assuntos
Radioisótopos de Gálio , Infecções por HIV/diagnóstico por imagem , Pulmão/diagnóstico por imagem , Linfonodos/diagnóstico por imagem , Adulto , Líquido da Lavagem Broncoalveolar/citologia , Infecções por HIV/imunologia , Infecções por HIV/patologia , Humanos , Masculino , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
The prevalence of sleep apnea-hypopnea syndrome (SAHS) was investigated in a selected group of veterans of the Persian Gulf War at Brooke Army Medical Center. One hundred ninety-two self-referred patients participated in the full evaluation of the Comprehensive Clinical Evaluation Program (CCEP) for veterans of the Persian Gulf War. After completing an initial survey, an interview and examination were performed by staff internists. Forty-six participants with histories suggestive of a sleep disorder were referred for further evaluation. Those patients suspected of SAHS then completed a sleep disorders questionnaire and underwent standard nocturnal polysomnography (PSG). SAHS was defined as a respiratory disturbance index > or = 15 in a symptomatic patient. Fifteen of 46 patients undergoing PSG at this institution met criteria for SAHS. The majority of these patients had symptoms of fatigue and memory loss. Overall, 16 of the 192 patients (8.3%) in the CCEP of our institution were diagnosed with SAHS. SAHS may play a significant role in the symptom complex presented by many veterans of the Persian Gulf War.
Assuntos
Militares , Síndromes da Apneia do Sono , Veteranos , Guerra , Adulto , Humanos , Kuweit , Pessoa de Meia-Idade , Arábia Saudita , Síndromes da Apneia do Sono/diagnósticoRESUMO
Natural termini from macronuclear DNA of the ciliated protozoans Tetrahymena thermophila and Oxytricha fallax can support telomere formation yeast. However, plasmids carrying these ciliate termini are modified by the addition of DNA which hybridizes to the synthetic oligonucleotide poly [d(C-A]), a sequence which also hybridizes to terminal restriction fragments from yeast chromosomes but not to Tetrahymena or Oxytricha macronuclear DNAs. Thus, in yeast, the creation of new telomeres on ciliate termini involves the acquisition of yeast-specific terminal sequences presumably by either recombination or non-templated DNA synthesis. The RAD52 gene is required for the majority of yeast mitotic and meiotic recombination events. Moreover, the absence of an active RAD52 gene product results in high rates of chromosome loss. Here we demonstrate that terminal restriction fragments from Tetrahymena macronuclear ribosomal DNA (rDNA) support the formation of modified telomeres in a yeast strain carrying a defect in the RAD52 gene. Moreover, linear plasmids bearing these modified ciliate termini are stably propagated in rad52- cells.
Assuntos
Mutação , Plasmídeos , Recombinação Genética , Saccharomyces cerevisiae/genética , Animais , Eucariotos/genética , Tetrahymena/genéticaRESUMO
Two middle repetitive DNA sequences called X and Y' are found near the telomeres of many chromosomes in Saccharomyces cerevisiae. Orthogonal field gel electrophoresis (OFAGE) was used to examine the distribution of X and Y' on different yeast chromosomes. Although the distribution of X and Y' varies among different laboratory strains of yeast, most yeast chromosomes in four different strains carry both X and Y'. However, at least one chromosome in each strain lacks the Y' element. This result indicates that Y' is not essential for replication or segregation of at least some yeast chromosomes.
Assuntos
Cromossomos/análise , Saccharomyces cerevisiae/genética , Sequência de Bases , DNA Fúngico/genéticaRESUMO
This study prospectively evaluated the alveolar content of transbronchial biopsy specimens based on whether or not they floated in formalin. One hundred biopsy specimens were obtained from 18 patients. Specimens with any alveoli were significantly more likely to float (45 of 61, 74%) than those with no alveolar tissue (15 of 39, 39%; p less than 0.001). The majority of diagnostic specimens were obtained in eight patients with sarcoidosis, where 47 biopsies were obtained. Fourteen of 19 specimens (74%) with noncaseating granulomas floated, whereas only 11 of 28 (39%; p less than 0.05) nondiagnostic specimens floated. The results of this study suggest that this simple bedside observation may serve to determine the adequacy of transbronchial biopsy specimens, particularly in sarcoidosis.
Assuntos
Biópsia/métodos , Pneumopatias/patologia , Alvéolos Pulmonares/patologia , Sarcoidose/patologia , Manejo de Espécimes , Broncoscopia , Estudos de Avaliação como Assunto , Tecnologia de Fibra Óptica/instrumentação , Formaldeído , HumanosRESUMO
In order to determine the effect of breathing high fractional concentrations of oxygen on forced expiratory flow in individuals with chronic obstructive pulmonary disease (COPD), we studied 18 patients with moderately severe disease. The patients were studied breathing air, 100% oxygen, or a four-gas mixture in a randomized double-blind study design. The four-gas mixture (oxygen 21.0%, argon 48.6%, nitrogen 19.3%, and helium 11.1%) was calculated to have a density and viscosity similar to oxygen. During spirometric testing, breathing oxygen produced a detectable reduction in timed volumes by 1 minute that was sustained at 5 minutes (FEV1 reduction 4.9% at 1 minute and 6.3% at 5 minutes). Breathing the gas mixture for 5 minutes resulted in similar reductions in flow. We conclude that high concentrations of oxygen reduce forced expiratory flow in patients with airflow obstruction, an effect probably related to the increased density and viscosity relative to air. This reduction in forced expiratory flow may contribute to the deterioration seen when COPD patients with acute respiratory failure are treated with 100% oxygen.
Assuntos
Fluxo Expiratório Forçado , Pneumopatias Obstrutivas/fisiopatologia , Oxigenoterapia , Oxigênio/análise , Idoso , Método Duplo-Cego , Humanos , Pneumopatias Obstrutivas/terapia , Masculino , Pessoa de Meia-Idade , Respiração , Insuficiência Respiratória/terapia , Capacidade VitalRESUMO
This study prospectively compared the diagnostic yield of transbronchial biopsies using large and small forceps (cup sizes, 3 x 2 x 0.9 versus 2 x 1.5 x 0.6 mm, respectively). Diagnostic yield was compared by a pathologist, blinded to the size of forceps used on the basis of the relative amount of tissue obtained, alveolar tissue obtained, and ability to ascertain a histopathologic diagnosis. Large forceps obtained significantly more tissue than did small forceps (20 of 27 patients [74%] versus five of 27 patients [19%], p < 0.005, with similar amounts obtained in two patients). Also, large forceps obtained significantly more alveolar tissue than did small forceps (16 of 22 patients [73%] versus six of 22 patients [27%], p < 0.05, with no alveolar tissue obtained in five patients). In 18 of the 27 patients, biopsies performed resulted in nonspecific diagnoses, including fibrosis or chronic inflammation. All nine of the patients with a specific diagnosis were ultimately proved to have sarcoidosis. There was a trend toward more of these patients having noncaseating granulomas obtained with the large forceps than with the small forceps (seven of nine patients versus four of nine patients). No difference was observed in the amount of postbiopsy bleeding with either forceps. We conclude that large forceps used for transbronchial biopsy yield more tissue and more alveolar tissue than do small forceps. These findings may have an impact on the diagnostic yield in some diseases such as sarcoidosis.
Assuntos
Biópsia/instrumentação , Brônquios/patologia , Adulto , Biópsia/métodos , Broncoscopia , Feminino , Humanos , Masculino , Estudos ProspectivosRESUMO
To confirm the presence of cardiac dysfunction in a group of patients seropositive for the human immunodeficiency virus with either dyspnea on exertion or a reduced anaerobic threshold, 9 patients with no history of opportunistic infection underwent exercise right-sided heart catheterization. When compared with 13 control patients previously exercised in the same manner, the patients showed elevated exercise pulmonary capillary wedge pressure (14.6 +/- 3.3 mm of mercury versus 9.9 +/- 3.3 mm of mercury; P less than .005) and right atrial pressure (10.1 +/- 2.1 mm of mercury versus 4.7 +/- 3.2 mm of mercury; P less than .001) at a similar exercise oxygen consumption and cardiac index. Of the 9 patients, 8 had at least 1 catheterization value outside the 95% confidence limits for the control group and 4 patients had multiple abnormalities. Values for blood CD4 lymphocytes were 0.2 x 10(9) per liter or more for 7 of the 9. One patient underwent endomyocardial biopsy with findings consistent with a cardiomyopathy. We conclude that cardiac disease may occur at any immunologic stage of human immunodeficiency virus infection. These observations suggest an effect of this disease on the heart.
Assuntos
Soropositividade para HIV/fisiopatologia , Coração/fisiopatologia , Adulto , Biópsia , Cateterismo Cardíaco , Eletrocardiografia , Teste de Esforço , Soropositividade para HIV/complicações , Soropositividade para HIV/patologia , Cardiopatias/complicações , Hemodinâmica , Humanos , Masculino , Miocárdio/patologiaRESUMO
To confirm the presence of exercise dysfunction in patients seropositive for the human immunodeficiency virus (HIV), 32 such patients without AIDS were evaluated with cardiopulmonary exercise testing, pulmonary function testing, bronchoalveolar lavage, chest roentgenography, and gallium scanning. No evidence of pulmonary opportunistic infection was found. When compared to an otherwise similar group of HIV-seronegative controls, the patients exercised to a significantly lower workload (195 +/- 30 versus 227 +/- 31 W, p less than 0.001). The ventilatory anaerobic threshold (VAT) values were also significantly lower for the patients (49.2 +/- 13.0 versus 61.9 +/- 9.1% of maximum predicted VO2, p less than 0.001). Nine of the patients had VAT values less than the 95% confidence interval for the controls. This subgroup exercised to a significantly lower maximum VO2 (69.9 +/- 11.2 versus 95.9 +/- 17.5% of maximum predicted VO2, p less than 0.001) and workload (165 +/- 21 versus 227 +/- 31 W) when compared to the control group. These patients demonstrated a mild tachypnea throughout exercise relative to the controls and had a significant increase in the slope of the heart rate to VO2 relationship. These findings are most consistent with a limitation of oxygen delivery to exercising muscles, which may represent occult cardiac disease in this group.