Assuntos
Artérias/patologia , Arterite de Células Gigantes/diagnóstico , Polimialgia Reumática/complicações , Pele/patologia , Idoso , Evolução Fatal , Feminino , Arterite de Células Gigantes/complicações , Arterite de Células Gigantes/tratamento farmacológico , Glucocorticoides/uso terapêutico , Humanos , Polimialgia Reumática/tratamento farmacológico , Prednisolona/uso terapêutico , Pele/irrigação sanguíneaRESUMO
BACKGROUND: O-linked ß-N-acetylglucosamine (O-GlcNAc) is a glycan essential for fundamental cellular processes such as transcription/translation, nuclear transport, protein stability and protein-protein interactions. However, the role of O-GlcNAc in prostate cancer progression of patients remains poorly unknown. Here we investigated the clinicopathological significance of O-GlcNAc expression level in prostate cancer. METHODS: O-GlcNAc expression level in prostate cancer cells was determined by immunohistochemistry of prostate biopsy specimens obtained from 56 patients later treated with hormone deprivation therapy comparing with adjacent normal prostate glands in the same sections. Overall survival was determined by the Kaplan-Meier and Cox proportional hazards methods with univariate and multivariate models. The effects of reduced O-GlcNAc expression level on proliferation and invasion of prostate cancer LNCaP cells were examined using small interfering RNA (siRNA) targeting O-GlcNAc transferase (OGT), the enzyme responsible for O-GlcNAc biosynthesis. RESULTS: Defining cancer cells showing stronger cytoplasmic staining than normal prostate glands as overexpression of O-GlcNAc, 39% of prostate cancer patients were categorized as overexpression. The Kaplan-Meier and Cox proportional hazards methods with univariate model analysis revealed that O-GlcNAc overexpression was associated with overall survival (P=0.0012 for the Kaplan-Meier and P=0.0021 for Cox univariate hazard model analysis). Furthermore, O-GlcNAc was the only item in which a significant difference was observed at overall survival by multivariate analysis (P=0.0475). Finally, siRNA-mediated OGT knockdown in LNCaP cells resulted in decreased expression of O-GlcNAc and promoted decreased proliferation and tumor cell invasion compared with control siRNA-transfected LNCaP cells. CONCLUSIONS: These results indicate that O-GlcNAc expression level in prostate cancer cells is associated with poor prognosis of prostate cancer patients and likely enhances tumor cell proliferation and invasion.
Assuntos
Acetilglucosamina/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/mortalidade , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Gradação de Tumores , Invasividade Neoplásica , Estadiamento de Neoplasias , Prognóstico , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologiaRESUMO
OBJECTIVES: Immunoglobulin (Ig)G4-related disease is a recently proposed systemic disorder that includes autoimmune pancreatitis (AIP), Mikulicz's disease, and various other organ lesions. In the present retrospective study, we examined whether thyroid lesions should also be included in IgG4-related disease (Ig4-RD) under the new term IgG4-related thyroiditis. METHOD: We enrolled 114 patients with Ig4-RD, including 92 patients with AIP, 15 patients with Mikulicz's disease, and seven patients with IgG4-related cholangitis, and analysed clinical findings, function, serum values of activity markers, computed tomography (CT) images, and histology of the thyroid gland. RESULTS: Among the 22 patients (19%) in our cohort who were found to have hypothyroidism [thyroid stimulating hormone (TSH) > 4 mIU/L], 11 patients had clinical hypothyroidism [free thyroxine (FT4) < 1 ng/dL] and 11 patients had subclinical hypothyroidism (FT4 ≥ 1 ng/dL). Serum concentrations of IgG, IgG4, circulating immune complex (CIC), and ß2-microglobulin (ß2-MG) were significantly higher in the hypothyroidism group compared with the remaining 92 euthyroid patients, and serum C3 concentration was significantly lower. After prednisolone treatment, TSH values had decreased significantly (p = 0.005) in this group and FT4 values had increased significantly (p = 0.047). CT images showed that the thyroid glands of patients with clinical hypothyroidism had a significantly greater volume than those of the euthyroid and other groups. Pathological analysis of one resected thyroid gland disclosed a focused lesion with infiltration of lymphocytes and IgG4-bearing plasma cells and loss of thyroid follicles. CONCLUSIONS: Thyroid lesions associated with hypothyroidism can be considered as a new disease termed IgG4-related thyroiditis. Awareness of this condition should lead to appropriate corticosteroid treatment that may prevent progression to a fibrous state.
Assuntos
Doenças Autoimunes/diagnóstico , Hipotireoidismo/diagnóstico , Hipotireoidismo/imunologia , Imunoglobulina G/imunologia , Doença de Mikulicz/diagnóstico , Pancreatite/diagnóstico , Tireoidite Autoimune/diagnóstico , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Doenças Autoimunes/complicações , Colangite/complicações , Colangite/diagnóstico , Colangite/imunologia , Estudos de Coortes , Progressão da Doença , Feminino , Humanos , Hipotireoidismo/complicações , Masculino , Pessoa de Meia-Idade , Doença de Mikulicz/complicações , Doença de Mikulicz/imunologia , Pancreatite/complicações , Pancreatite/imunologia , Prognóstico , Estudos Retrospectivos , Medição de Risco , Fatores Sexuais , Estatísticas não Paramétricas , Testes de Função Tireóidea , Tireoidite Autoimune/complicações , Tireoidite Autoimune/imunologiaRESUMO
The present study was carried out to determine whether myoglobin (Mb) concentration ([Mb]) in human skeletal muscle is influenced by 8 weeks of endurance training under normal conditions, and under hypoxic conditions equivalent to an altitude of 2500 m. Fourteen healthy but sedentary male adults who did not participate in any regular exercise program took part in this study. They were divided into two groups according to the training regime to which they were submitted: the N group, who exercised under normobaric conditions, and the H group, who exercised under hypobaric conditions. All subjects performed an incremental cycling exercise at sea level to evaluate their maximal O2 uptake (VO2max) before and after the 8-week endurance training course period. Muscle tissue samples were obtained by needle biopsy from the vastus lateralis muscle for histochemical and biochemical analysis. Training induced an increase in VO2max in both the N and H groups (P < 0.05), although there was no significant difference in these changes between groups. The 8-week training had no effect on [Mb] in either group. Muscle fiber composition was also unaffected by the training course. In contrast, citrate synthase activity in both groups increased by [mean (SD)] 28.2 (33.3)% (N: P < 0.01) and 32.0 (18.2)% (H: P < 0.05) after training, and the number of capillaries (capillary:fiber ratio) increased by 47.7 (33.8)% (N: P < 0.01) and 32.3 (20.6)% (H: P < 0.05). There were no significant differences in these parameters between the N and H groups. These results suggest that significant improvement of aerobic potential as a result of endurance training are not accompanied by increases in [Mb] in human skeletal muscle. In addition, a lower absolute workload may not be sufficient to stimulate Mb synthesis in humans, even where endurance training is carried out under hypoxia.
Assuntos
Hipóxia/metabolismo , Músculo Esquelético/metabolismo , Mioglobina/metabolismo , Resistência Física/fisiologia , Adulto , Pressão Atmosférica , Capilares/fisiologia , Citrato (si)-Sintase/metabolismo , Humanos , Masculino , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/citologia , Consumo de Oxigênio/fisiologiaRESUMO
A case of membranous nephropathy, preexisting in a donor kidney, will be reported. A 41-year-old man underwent a cadaver renal transplantation. An allograft biopsy specimen obtained during the operation showed spike formation on periodic acid-silver methenamine staining and deposition of IgG along the glomerular capillary loop on immunoperoxidase staining. Immunofluorescence staining for IgG remained in the specimens obtained on day 11 and after 4 weeks, but markedly decreased in the specimen obtained 7 weeks after transplantation. Electron-dense deposits also decreased in amount, but irregular thickening of the glomerular basement membrane with spikes, electron-lucent washout lesions, and small amounts of electron-dense deposits remained 20 months after the transplantation. These findings suggest that membranous nephropathy, as well as IgA nephritis and diabetic nephropathy, resolve after renal transplantation and that deposition of IgG markedly decreases within a few months after transplantation, but that complete histological restoration of the basement membrane needs at least a few years.
Assuntos
Glomerulonefrite Membranosa/patologia , Transplante de Rim/fisiologia , Doadores de Tecidos , Adulto , Humanos , Falência Renal Crônica/cirurgia , Glomérulos Renais/patologia , Glomérulos Renais/ultraestrutura , Masculino , Microscopia EletrônicaRESUMO
A rare case of a mature teratoma of the placenta is reported. The tumor lay between the amnion and the chorion and contained skin and its appendages, bone, cartilage, fat and ganglia, without organization. There was no evidence of a recognizable umbilical cord. We concluded that the tumor was a mature teratoma of the placenta. The distinction between a teratoma and a fetus acardius amorphus, and the possible origin of the tumor in this site, are discussed with a brief review of the literature.
Assuntos
Doenças Placentárias/patologia , Teratoma/patologia , Adulto , Feminino , Humanos , GravidezRESUMO
The purpose of this study was to examine the relationships between the relative contents of phosphocreatine (PCr), inorganic phosphate (Pi), beta-adenosine triphosphate (ATP), and transverse relaxation time (T2) with fiber composition, which determined histochemically in the human skeletal muscle. The vastus lateralis muscles of 28 volunteers were subjected to phosphorus nuclear magnetic resonance (31P NMR) spectroscopy, magnetic resonance imaging (MRI) and muscle biopsy. Muscle fibers were divided into type I and type II fibers using myosin ATPase stain. A wide range of fiber composition levels were observed in the subjects (27.3-74.6% type I fibers). The PCr/ATP, Pi/ATP and (PCr + Pi)/ATP ratios were positively related to the percentage of type II fibers (r = 0.695, p < 0.001, r = 0.429, p < 0.05 and r = 0.773, p < 0.001, respectively). There was no correlation between fiber composition and the PCr/Pi ratio (r 0.127, n.s.) or intracellular pH (r = 0.305, n.s.). Moreover, no correlation was found between T2 and fiber type (r = 0.144, n.s.). These results suggest that 31P NMR can detect the differences in relative content of phosphates between type I and type II fibers, thereby noninvasively evaluating fiber composition in human skeletal muscle.
Assuntos
Espectroscopia de Ressonância Magnética , Fibras Musculares Esqueléticas/química , Músculo Esquelético/química , Trifosfato de Adenosina/análise , Adolescente , Adulto , Biópsia por Agulha , Feminino , Histocitoquímica , Humanos , Imageamento por Ressonância Magnética , Masculino , Fibras Musculares Esqueléticas/classificação , Relaxamento Muscular , Músculo Esquelético/fisiologia , Fosfatos/análise , Fosfocreatina/análiseRESUMO
A cine magnetic resonance (MR) imaging technique, involving the acquisition of kinematic sagittal images during knee movement, was used to evaluate 52 symptomatic knee joints. Results were compared with those obtained by means of static three-dimensional (3D) MR imaging. Twenty-seven of the 28 anterior cruciate ligament (ACL) tears and 22 of 24 normal ligaments were correctly identified at cine MR imaging for a sensitivity of 96% and a specificity of 92%. Static 3D MR imaging yielded a sensitivity of 71% and a specificity of 88%. All four posterior cruciate ligament tears were identified at cine and 3D MR imaging. For meniscal tears, cine MR imaging yielded a sensitivity of 48% and a specificity of 96%; the sensitivity and specificity for 3D MR imaging were 71% and 96%, respectively. Cine MR imaging proved to be more useful than static MR imaging in assessing the tightness of cruciate ligaments, especially of those that were partially torn, and in assessing the movement of meniscal-free fragments. The increased information obtained with cine MR imaging may warrant continued investigation and clinical application.
Assuntos
Lesões do Ligamento Cruzado Anterior , Processamento de Imagem Assistida por Computador , Traumatismos do Joelho/diagnóstico , Articulação do Joelho/fisiologia , Imageamento por Ressonância Magnética/métodos , Ligamento Cruzado Posterior/lesões , Lesões do Menisco Tibial , Adulto , Artroscopia , Feminino , Humanos , Masculino , Amplitude de Movimento Articular/fisiologia , Sensibilidade e EspecificidadeRESUMO
By digestion with HindIII restriction enzyme, a human hepatocellular carcinoma was shown to contain only 2 hepatitis B virus (HBV) DNA inserts. Both HindIII fragments (8 and 16 kb) were molecularly cloned and the structures of HBV DNA and adjacent host sequences were analyzed. One clone (lambda YH 8) contained part of pre-S(I) through the entire S gene and the other (lambda YH 16) had the middle section of the S to the end of X gene of HBV DNA. No gross rearrangements were observed in either HBV or cellular DNA sequences in lambda YH 16 clone. However, the 8 kb HindIII fragment was considered to be amplified together with flanking cellular sequences. Furthermore, the HBV DNA was integrated into cellular genome at the Alu repeated sequences in lambda YH 8.
Assuntos
Carcinoma Hepatocelular/microbiologia , DNA Viral/análise , Vírus da Hepatite B/genética , Neoplasias Hepáticas/microbiologia , Bacteriófago lambda/genética , Sequência de Bases , Clonagem Molecular , Enzimas de Restrição do DNA , Humanos , Dados de Sequência Molecular , OncogenesRESUMO
Ad5-HBL is a type 5 adenovirus bearing the large BglII fragment (2.8 kilobases; 87% of the total genome) of hepatitis B virus (HBV), subtype adr. Eight HBV RNAs expressed in HeLa cells infected with Ad5-HBL were mapped by the nuclease S1 technique. Three major RNAs spanning 2.4, 2.0, and 0.7 kilobases of the HBV sequences cover the coding regions of "presurface" plus surface antigen, surface antigen alone, and "X" protein, respectively. The 5' segment of an RNA which could code for core antigen (HBcAg) was also detected. All major HBV RNAs initiate from mutually exclusive 5' ends, terminate at the unique 3' end within the HBcAg coding region (except readthrough species), and have no spliced deletion, forming a novel RNA family structure. No TATA box-like sequences were found near the 5' end of these RNAs, except in the case of the 2.4-kilobase RNA. About two thirds of total HBV RNA does not terminate at the mapped 3'-end position, suggesting the termination signal is functionally inefficient. Since the potential 5' end of HBcAg mRNA was mapped at the same position as the minus-strand nick of HBV DNA previously reported, we propose a model that requires inefficient poly(A) addition to produce an RNA which serves both as HBcAg mRNA and as the putative RNA template of minus-strand DNA synthesis in the HBV life cycle.
Assuntos
Vírus da Hepatite B/genética , RNA Viral/genética , Adenovírus Humanos/genética , DNA Viral/biossíntese , Endonucleases , Vetores Genéticos , Células HeLa , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos de Superfície da Hepatite B/genética , Humanos , Splicing de RNA , RNA Mensageiro/genética , RNA Viral/análise , Endonucleases Específicas para DNA e RNA de Cadeia Simples , Moldes Genéticos , Transcrição Gênica , Proteínas Virais/genéticaRESUMO
A novel helper-free adenovirus type 5 (Ad5) vector system, which utilizes a cloning site 0.2 kilobase (kb) from the right end of the genome, has been developed. To construct a nondefective Ad5 bearing the 2.8-kb DNA fragment of hepatitis B virus (HBV) at this site, we deleted the 2.1-kb nonessential E3 fragment from cloned DNA covering the right one-fourth of the Ad5 genome (76 to 100 map units), inserted the HBV DNA into this site, ligated the recombinant DNA to the rest of the Ad5 genome, and transfected the ligated DNA into human embryo kidney cells. Most of the recovered virus clones had only the E3 deletion and no HBV insertion, suggesting that a homologous recombination occurs between transfected DNAs in these cells. The isolated Ad5 virus bearing the HBV DNA (Ad5-HBL) grew without helper virus in HeLa cells as efficiently as wild-type Ad5, although the 1.9-kb major E4 transcript was detected only poorly in the early phase in the Ad5-HBL-infected cells, suggesting that the HBV DNA inserted upstream of the E4 promoter reduces the E4 transcript. HBV mRNAs transcribed from the inserted DNA were at least as abundant as Ad5 early mRNAs in the late phase of Ad5-HBL infection, but the HBV surface antigen was barely detectable in the infected-cell lysate and culture medium. This result suggests that HBV mRNAs can be transcribed from the inserted genes but no protein can be translated from the HBV mRNAs, presumably because of the translational suppression of cellular mRNAs caused by adenovirus in its late phase.
Assuntos
Adenoviridae/genética , DNA Viral/análise , Genes Virais , Vírus da Hepatite B/genética , Antígenos de Superfície da Hepatite B/análise , Humanos , RNA Mensageiro/metabolismo , Radioimunoensaio , Recombinação Genética , Transcrição GênicaRESUMO
Rat 3Y1 cells were transfected with recombinant gARC ( pSV2gpt carrying the adenovirus 12 early region 1 [E1] gene), and focus formation was observed in monolayer cultures after culture of cells in gpt-selective medium (Eagle medium containing 10% fetal calf serum, xanthine, thymidine, aminopterin, and mycophenolic acid) for 10 days, followed by focus formation. Transformed E1Y cell lines were then established from these foci. The E1Y cells were transformed morphologically similarly to cells transformed with intact adenovirus 12 DNA but formed no colonies in soft-agar culture and induced tumors in transplanted rats only after a long incubation period. For the establishment of completely transformed cells, 3Y1 cells were transformed with combinations of gARC , pE3 (pBR322 carrying the adenovirus 12 E3 gene), and gE4 ( pSV2gpt carrying the adenovirus 12 E4 gene) DNA. E1- 3Y cells (3Y1 cells transformed with gARC and pE3 DNA), E1- 4Y cells (3Y1 cells transformed with gARC and gE4 DNA), and E1-3- 4Y cells (3Y1 cells transformed with gARC , pE3 , and gE4 DNA) were established. These transformed cell lines were compared for growth in Eagle medium with 2 or 10% fetal calf serum, colony formation in soft-agar culture, and tumor growth in rats transplanted with the transformed cells. Several transformed cell lines of E1- 4Y and E1-3- 4Y cells showed colony formation in soft-agar culture and abundant expression of the E1B gene. T antigen f was seen by immunofluorescence as flecks in these cells, in which the E4 gene was transcribed, but was not seen in E1Y cells, suggesting that T antigen f was encoded by the E4 gene. The suggestion was confirmed by the observation that T antigen f was detected in COS-1 cells transfected singly with gE4 DNA by immunofluorescence with polyclonal and monoclonal antibodies. Transcription of the E4 gene was confirmed in gE4 -transfected COS-1 cells. T antigen f, one of the E4 gene products, was identified as a polypeptide of molecular weight 11,000 (E4- 11K ) by immunoprecipitation with monoclonal antibodies. The above results also suggest that expression of the E4 gene gives cells the advantage of forming colonies in soft-agar culture. A tendency was noticed for E1B gene expression to be enhanced by E4 gene expression. The relationship between enhancement of colony formation in soft-agar culture and enhancement of E1B gene expression is discussed.
Assuntos
Adenovírus Humanos/genética , Transformação Celular Neoplásica , Genes Virais , Ágar , Animais , Sequência de Bases , Linhagem Celular , Chlorocebus aethiops , Clonagem Molecular , Enzimas de Restrição do DNA , DNA Recombinante/metabolismo , Embrião de Mamíferos , Humanos , Ratos , Ratos Endogâmicos F344 , Especificidade da Espécie , TransfecçãoRESUMO
We isolated an adenovirus 12 early region 1B mutant (in206B) by ligation of the cleaved DNA-protein complex and transfection of human embryo kidney cells with the ligation products. By deduction from the DNA sequencing analysis, the two polypeptides (with molecular weights of 19,000 and 54,000) coded in the early region 1B were fused in this mutant to produce a large polypeptide. This mutant could replicate efficiently in 293 cells but not efficiently in KB or human embryo kidney cells. In KB cells, viral DNA replication could not be detected after infection with in206B. In human embryo kidney cells, viral DNA replication did occur, but the transition of viral mRNA patterns from early to late did not occur even after DNA replication, resulting in failure to produce the late polypeptides. These results indicate that the early region 1B products may be involved in both viral DNA replication and regulation of transcription.
Assuntos
Adenovírus Humanos/fisiologia , Replicação do DNA , Regulação da Expressão Gênica , Genes Virais , Proteínas Virais/fisiologia , Replicação Viral , Adenovírus Humanos/genética , Linhagem Celular , Humanos , Mutação , RNA Mensageiro/genética , RNA Viral/genética , Transcrição Gênica , Proteínas Virais/biossíntese , Proteínas Virais/genéticaRESUMO
We isolated three adenovirus 12 early region 1B mutants (in205B, in205C, and dl205) by ligation of the cleaved DNA-protein complex and transfection of human embryo kidney cells with the ligation products. These mutants could replicate efficiently in human embryo kidney or KB cells but showed markedly reduced transforming capacities both in vitro and in vivo. In cells infected with the mutants, the early region 1B gene was transcribed efficiently. In cells infected with in205B, the products corresponding to the early region 1B-coded 19,000-molecular-weight polypeptide was detected by in vitro translation but not immunoprecipitated extract of labeled cells. In cells infected with in205C or dl205, the products corresponding to the same polypeptide were not detected by either in vitro translation or immunoprecipitation of labeled cell extracts. The results suggest that the 19,000-molecular-weight polypeptide encoded by early region 1B is required for cell transformation but not for viral propagation.
Assuntos
Adenovírus Humanos/genética , Transformação Celular Viral , Genes Virais , Replicação Viral , Deleção Cromossômica , Mapeamento Cromossômico , Endonucleases , Mutação , Biossíntese de Proteínas , RNA Mensageiro/genética , Endonucleases Específicas para DNA e RNA de Cadeia Simples , Transcrição Gênica , Proteínas Virais/genéticaRESUMO
We have constructed recombinant plasmids that direct the synthesis of the Mr 19 000 protein encoded by the adenovirus type 12 (Ad12) E1b region as either a native protein or a protein fused to the amino-terminal portion of the elongation factor EF-TuB in Escherichia coli cells. Using these recombinants, we could synthesize a large amount of the fused protein, while only a small amount of the native Mr 19 000 protein was produced. The failure to synthesize the native Mr 19 000 protein in E. coli cells was ascribed to inefficient translation.
Assuntos
Adenoviridae/genética , Plasmídeos , Biossíntese de Proteínas , Proteínas Virais/genética , Escherichia coli/genética , Lisogenia , Recombinação GenéticaRESUMO
Analysis of cytoplasmic RNA in Ad12-infected and -transformed cells showed that more than 20 mRNA species are transcribed from regions 1A and 1B. These mRNA species could be classified into four classes according to the extent to which they were expressed in infected and transformed cells under various conditions. The proteins synthesized from the major species of each of these mRNAs were identified by in vitro translation. In region 1A, the 40K (and 38K) proteins were synthesized from three mRNA species that differed from one another in their 5' ends, and in region 1B three proteins with molecular weights of 19,000 (19K), 50K, and 17K were synthesized from a single species of 2.2 kb mRNA. Only the 19K protein was immunoprecipitated by anti-T serum (G serum) from rats bearing GY1 cell tumors, [GY1 cells are rat cells transformed by the Ad12 HindIII-G fragment (0-6.8 map units).] The 19K protein was also detected by immunoprecipitation in extracts from both transformed and infected cells. The 17K protein was immunoprecipitated by anti-T serum (C serum) derived from rats bearing CY1 cell tumors, but was not by G serum. [CY1 cells are rat cells transformed by the Ad12 EcoRI-C fragment (0-16.5 map units).] The 17K protein was also translated in vitro from the 0.5 kb region 1B mRNA. These results suggest that the 19K and 17K proteins correspond to Ad12 T antigen f and polypeptide IX, respectively, and that these two proteins are translated in vivo from different coding regions in 2.2 kb mRNA in CY1 cells.
Assuntos
Adenovírus Humanos/genética , Antígenos Virais/genética , RNA Mensageiro/genética , RNA Viral/genética , Proteínas Virais/genética , Adenovírus Humanos/metabolismo , Antígenos Virais/análise , Antígenos Virais de Tumores , Linhagem Celular , Transformação Celular Neoplásica , Transformação Celular Viral , Genes Virais , Humanos , Biossíntese de Proteínas , Proteínas Virais/análise , Proteínas Virais/biossínteseRESUMO
A nondefective recombinant between adenovirus type 5 (Ad5) and type 12 (Ad12), rc-1 (Ad5 dl312, carrying the Ad12 E1A gene), was isolated from hamster cell foci transformed by a defective recombinant, rcB-1 (dl312, carrying the Ad12 E1 gene). The recombinant rc-1 grew in human embryo kidney and KB cells in the absence of helper and synthesized Ad12 T antigen g, the product of the E1A gene. The genome of rc-1 has a deletion between 79.9 and 82.5 map units of Ad5 dl312 DNA with an insertion of 0.1 to 5.5 map units of Ad12 DNA at the deletion site. The mRNAs of Ad12 E1A were transcribed from the Ad12 E1A promoter, and unusual RNAs were abundantly transcribed from the Ad5 E3 promoter on the opposite strand. The frequency of cell transformation with rc-1 was lower than those with Ad5 and Ad12 wild types.
Assuntos
Adenovírus Humanos/genética , Transformação Celular Viral , Genes Virais , Recombinação Genética , Adenovírus Humanos/fisiologia , Linhagem Celular , Humanos , RNA Mensageiro/genética , RNA Viral/genética , Transcrição GênicaRESUMO
The adenovirus type 12 (Ad12) early region 1 (E1) gene was introduced into KB cells by using a dominant selection vector, pSV2-gpt, and over 80 Gpt+ KB cell clones were established. Three types of recombinant DNAs (gAE1A, gARC, and gABA) were constructed. They contained the AccI-H, EcoRI-C, and BamHI-A fragments, respectively, of Ad12 DNA in pSV2-gpt. Five of 50 (10%) gABA-transformed cell clones, 12 of 18 (67%) gAE1A-transformed cell clones, and 10 of 18 (56%) gARC-transformed cell clones complemented the growth of Ad5 dl312 (deletion in E1A) and were designated as Gpt+ Ad+ cell clones. In these cell clones at their early passages, recombinant genome sequences were detected in cellular DNA and were expressed. T antigen g (the E1A gene product) was detected by immunofluorescence. The Gpt+ Ad+ cell clones supported the growth of Ad5 deletion mutants in parallel with the expression of Ad12 E1A or E1A plus E1B genes. After infection of Gpt+ Ad+ cell clones with Ad5 dl312, the early genes of dl312 were efficiently transcribed, indicating the expression of the pre-early function of the Ad12 E1A gene. Two clones each from gAE1A-,gARC-, and gABA-transformed cells were subcultured for a long period to determine the stability of the transfecting DNAs. Subculture in a nonselective medium resulted in cells which lost the transfecting DNAs. Subculture in a selective medium resulted in the selection of cells which maintained the gpt gene expression but lost the Ad12 gene expression. These results indicate that the transfecting DNA is present in an unstable state in KB cells.
Assuntos
Adenovírus Humanos/genética , Antígenos Virais/genética , DNA Recombinante , DNA Viral/genética , Genes Virais , Adenovírus Humanos/imunologia , Antígenos Virais de Tumores , Linhagem Celular , Transformação Celular Viral , Células Clonais , Regulação da Expressão Gênica , Humanos , Transcrição GênicaRESUMO
Simian virus 40 recombinant DNAs carrying the adenovirus type 12 E1B gene were constructed, propagated, and packaged in monkey cells. Monkey cells infected with the resulting virus stocks hyperproduced the E1B gene products in more than 80% of the cells as revealed by immunofluorescence. The products were distributed in both the nuclei and the cytoplasm, and a condensed form of fleck structure was observed in the cytoplasm. Polyacrylamide gel electrophoresis of the cell extracts and their immunoprecipitates detected the E1B-coded 19,000-molecular-weight protein but not the 50,000-molecular-weight protein. The 19,000-molecular-weight protein and the simian virus 40 VP1 protein were synthesized in nearly equal amounts.