RESUMO
A recombinant bacterial plasmid, pMS1, was constructed that contains 318 nucleotides complementary to a portion of pro-opiolipomelanocortin (proOLMC) messenger RNA from an ectopic adrenocorticotropin-producing tumor. The cloned complementary DNA insert, which contains the sequence that codes for all of the beta-melanocyte-stimulating hormone and beta-endorphin portions of proOLMC, as well as the 3' nontranslated section, is identical to the genomic sequence. Hybridization of tumor proOLMC complementary DNA to RNA subjected to electrophoresis and transferred to a nitrocellulose filter revealed two proOLMC messenger RNA species in the tumor polyadenylated RNA, but only one in pituitary polyadenylated RNA. At least one of the tumor proOLMC messenger RNA's is similar, if not identical, to human pituitary proOLMC messenger RNA.
Assuntos
DNA Recombinante/metabolismo , Endorfinas/genética , Hormônios Ectópicos/genética , Hormônios Estimuladores de Melanócitos/genética , Hormônios Adeno-Hipofisários/genética , Precursores de Proteínas/genética , Sequência de Aminoácidos , Sequência de Bases , Tumor Carcinoide/fisiopatologia , Clonagem Molecular , DNA de Neoplasias/genética , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/fisiopatologia , Pró-Opiomelanocortina , RNA Mensageiro/genética , beta-EndorfinaRESUMO
Single-chain antigen-binding proteins are novel recombinant polypeptides, composed of an antibody variable light-chain amino acid sequence (VL) tethered to a variable heavy-chain sequence (VH) by a designed peptide that links the carboxyl terminus of the VL sequence to the amino terminus of the VH sequence. These proteins have the same specificities and affinities for their antigens as the monoclonal antibodies whose VL and VH sequences were used to construct the recombinant genes that were expressed in Escherichia coli. Three of these proteins, one derived from the sequence for a monoclonal antibody to growth hormone and two derived from the sequences of two different monoclonal antibodies to fluorescein, were designed, constructed, synthesized, purified, and assayed. These proteins are expected to have significant advantages over monoclonal antibodies in a number of applications.
Assuntos
Cadeias Pesadas de Imunoglobulinas , Cadeias Leves de Imunoglobulina , Região Variável de Imunoglobulina , Proteínas Recombinantes , Sequência de Aminoácidos , Escherichia coli/genética , Genes , Vetores Genéticos , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Difração de Raios XRESUMO
The use of antibodies or antibody fragments for targeting tumors (either for tumor imaging or as carriers for drugs or toxins), has encountered problems of clearance, and non-specific or inefficient binding in clinical trials. A novel approach, linking two antibody variable fragments (Fvs), with a short peptide to generate a continuous polypeptide chain, may be able to overcome some of these problems. Since these single chain antibody variable regions (scFvs), are transcribed from constructed 'genes', large-scale production in, for example, E. coli, should be straightforward.
Assuntos
Cadeias Pesadas de Imunoglobulinas/química , Cadeias Leves de Imunoglobulina/química , Região Variável de Imunoglobulina/química , Engenharia de Proteínas/métodos , Animais , Humanos , Região Variável de Imunoglobulina/uso terapêutico , Imunotoxinas/síntese químicaRESUMO
A PCR based reverse transcriptase (RT) assay was developed that has 10(4)-fold higher sensitivity than conventional nucleotide incorporation assays and allows discrimination between false positive results generated by cellular polymerases and positives resulting from authentic RT activity. Recently, several reverse transcriptase (RT) assays have been developed where a reverse transcriptase reaction is performed on an RNA template/DNA primer combination. A specific region of the cDNA product is then amplified by the polymerase chain reaction to increase the sensitivity of cDNA detection. These reverse transcriptase assays up to 10(6)-fold more sensitive at detecting retroviruses than conventional methods. The drawback to these assays with increased sensitivity is the increased incidence of false positive results generated by cellular polymerases that can reverse transcribe. The MS2 bacteriophage RNA template and primers from one of the recently developed assays were used as the basis to develop the assay. A simple high resolution agarose gel was used as the endpoint for the assay without compromising sensitivity. In addition, the pH of the RT reaction was lowered to pH 5.5, the RT incubation was 1 h, and protease inhibitors were added to the RT reaction components. These modifications yield an assay that can discriminate between authentic RT activity and contaminating cellular polymerases.
Assuntos
Reação em Cadeia da Polimerase/métodos , DNA Polimerase Dirigida por RNA/metabolismo , Células 3T3 , Animais , Sistema Livre de Células/química , Células Cultivadas/virologia , Primers do DNA/genética , Reações Falso-Positivas , Concentração de Íons de Hidrogênio , Levivirus/química , Levivirus/genética , Camundongos , Dados de Sequência Molecular , RNA Viral/química , RNA Viral/genética , DNA Polimerase Dirigida por RNA/genética , Vírus Rauscher/química , Vírus Rauscher/enzimologia , Infecções por Retroviridae , Sensibilidade e Especificidade , Moldes GenéticosRESUMO
OLX-209 has readily measurable activity, is safe in experimental animals, and is efficacious in model systems. These results support the concept of OLX-209 and provide groundwork for further development of this oncoprotein targeted agent.
Assuntos
ADP Ribose Transferases , Toxinas Bacterianas , Exotoxinas/administração & dosagem , Imunotoxinas/administração & dosagem , Receptor ErbB-2/efeitos dos fármacos , Proteínas Recombinantes de Fusão/administração & dosagem , Fatores de Virulência , Animais , Anticorpos , Neoplasias da Mama/química , Desenho de Fármacos , Estudos de Avaliação como Assunto , Exotoxinas/genética , Exotoxinas/farmacologia , Humanos , Imunotoxinas/farmacologia , Imunotoxinas/toxicidade , Camundongos , Camundongos Nus , Neoplasias Experimentais/tratamento farmacológico , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/toxicidade , Proteínas Recombinantes/farmacologia , Anticorpos de Cadeia Única , Neoplasias Gástricas/tratamento farmacológico , Células Tumorais Cultivadas , Exotoxina A de Pseudomonas aeruginosaRESUMO
Image-guided core biopsy (IGCB) of nonpalpable mammographic abnormalities has gained attention as an alternative to needle-localized breast biopsy (NLB). This study evaluated IGCB in the diagnostic workup of patients with nonpalpable mammographic lesions suspicious for cancer. Eighty-six patients who underwent IGCB were compared to 85 patients who underwent NLB for the diagnosis of mammographic lesions suspicious for cancer. The incidence of positive margins was less in patients who subsequently underwent needle-localized resection in the IGCB group than in the NLB group (29 and 65%; P < 0.0001). The volume of excision was greater for patients in the IGCB group than for the NLB group (106 cm3 and 52 cm3; P < 0.0001). Patients in the IGCB group averaged 1.1 operative procedures compared with patients in the NLB group, who required an average of 1.9 operative procedures. The mean charge for an IGCB was $1011 compared to $2975 for a NLB. Subset analysis of 32 spiculated masses from the IGCB group and 21 from the NLB group showed similar advantages of IGCB over NLB. The preoperative use of IGCB for mammographically suspicious lesions can reduce the incidence of positive surgical margins and the number of surgical procedures required. The use of IGCB allows for a more efficient diagnostic workup and less expense to the patient.
Assuntos
Biópsia por Agulha/métodos , Neoplasias da Mama/diagnóstico , Diagnóstico por Imagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia/economia , Biópsia/métodos , Biópsia por Agulha/economia , Feminino , Humanos , Mamografia , Pessoa de Meia-Idade , Palpação , Estudos RetrospectivosAssuntos
Cromossomos Bacterianos , Escherichia coli/crescimento & desenvolvimento , Ácido Aspártico/metabolismo , Contagem de Células , Centrifugação com Gradiente de Concentração , Replicação do DNA , DNA Bacteriano/análise , Glucose/metabolismo , Peso Molecular , Succinatos/metabolismo , Fatores de Tempo , Trítio , Uracila/metabolismoAssuntos
Cromossomos Bacterianos , Replicação do DNA , Escherichia coli , Aminoácidos/metabolismo , Autorradiografia , Isótopos de Carbono , Centrifugação com Gradiente de Concentração , Mapeamento Cromossômico , Colífagos , DNA Bacteriano , DNA Viral , Escherichia coli/metabolismo , Lisogenia , Modelos Biológicos , Biologia Molecular , Mutação , Hibridização de Ácido Nucleico , Timina/metabolismo , TrítioAssuntos
Anticorpos Monoclonais/genética , Escherichia coli/genética , Genes de Imunoglobulinas , Região Variável de Imunoglobulina/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular/métodos , Rearranjo Gênico , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Plasmídeos , Reação em Cadeia da Polimerase/métodos , Conformação Proteica , Difração de Raios X/métodosRESUMO
A review of the results of 21,716 mammograms obtained at a low-cost screening center is presented, along with a report on the finances of that center. A total of 142 cancers were discovered, 12 of which gave false-negative results at mammography. The sensitivity was 91.5% and the specificity 90%. The positive predictive value for lesions categorized as "suspicious for malignancy" was 54%. Thirty-one percent of the cancers were "minimal," in other words, in situ or less than 1 cm in diameter and with no tumor-positive lymph nodes. An average of 42 examinations were performed each day at a cost of +28 each. Nonphysician expenses were +16 for each examination, leaving +12 per examination as professional revenue. This project demonstrates that high-quality, low-cost screening mammography can be provided if the volume is adequate and if there is sufficient attention to detail.
Assuntos
Neoplasias da Mama/prevenção & controle , Mamografia/economia , Programas de Rastreamento/economia , Análise Custo-Benefício , Coleta de Dados , Honorários Médicos , Feminino , Humanos , Mamografia/estatística & dados numéricos , Programas de Rastreamento/estatística & dados numéricos , North Carolina , Estatística como AssuntoRESUMO
A private, low-cost mammography screening program in Charlotte, North Carolina, has grown from one to four fixed sites in the past 6 years and now screens more than 25,000 women annually. An ongoing medical audit of the program shows that the percentage of patients with breast cancer with positive lymph nodes has decreased from 29% to 13%, and the percentage of minimal cancers found has increased from 31% to 52%. Compliance for regular screening has remained at approximately 50% for the past 2 years. Means of increasing compliance to desired levels are discussed.
Assuntos
Neoplasias da Mama/prevenção & controle , Programas de Rastreamento/organização & administração , Feminino , Humanos , Mamografia , North CarolinaRESUMO
The location and extent of the homology between plasmids ColE1 and p15A were determined by analysis of heteroduplexes formed between them as well as with a related plasmid, pBR322, and by hybridization of radioactive deoxyribonucleic acids to restriction fragments of p15A and ColE1. The homology between the plasmids contained the entire region of ColE1 required for its replication as well as an additional 400 base pairs downstream from the origin of replication. This region on p15A, which was 980 +/- 43 base pairs, started at 0.1 of the molecular length from one end formed by cleavage with the restriction endonuclease BglI and extended to 0.54 of the molecular length from the same end. Restriction cleavage maps for the enzymes BglI, HpaI, HaeII, HaeIII, and HincII are also presented.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II , Escherichia coli/genética , Plasmídeos , Sequência de Bases , Enzimas de Restrição do DNA , Ácidos Nucleicos Heteroduplexes , Hibridização de Ácido NucleicoRESUMO
Screening-detected microcalcifications are responsible for more benign biopsy results than any other mammographic lesion. The management of these lesions comes at a large cost in terms of morbidity and dollars spent. Both costs and morbidity could be reduced by decreasing the number of surgical biopsies. This could be accomplished by increasing the positive biopsy rate and by substituting core needle biopsy for surgical biopsy when appropriate. To increase the positive biopsy rate, we need to improve the preoperative evaluation of microcalcifications. A scheme is presented for the mammographic evaluation of these microcalcifications and for the appropriate use of core biopsy in the management of these lesions.
Assuntos
Doenças Mamárias/diagnóstico , Neoplasias da Mama/diagnóstico , Mama/patologia , Calcinose/diagnóstico , Carcinoma in Situ/diagnóstico , Carcinoma Ductal de Mama/diagnóstico , Procedimentos Clínicos , Biópsia , Diagnóstico Diferencial , Feminino , Humanos , MamografiaRESUMO
Newly synthesized DNA, in E. coli lysogenic for the phage lambda, was labeled by short pulses of [(3)H]-thymidine, isolated, and separated on the basis of size by alkaline sucrose density gradient centrifugation. The molecular polarity of this DNA was determined by hybridization with each of the separated strands of lambda DNA. The results show that, in the 3' to 5' direction, replication proceeds by synthesis of short chains that are subsequently joined to long DNA. This is true for both a polA(+) and a polA(-) strain. (The polA locus codes for DNA polymerase I.) In the 5' to 3' direction, replication proceeds continuously, by addition of nucleotides to long DNA, for the polA(+) strain. In the polA(-) strain, however, replication in the 5' to 3' direction is also discontinuous, but the discontinuities are 1-40 times less frequent than in the other direction.
Assuntos
Replicação do DNA , DNA Bacteriano/análise , Escherichia coli/análise , Centrifugação com Gradiente de Concentração , Colífagos , DNA Bacteriano/isolamento & purificação , DNA de Cadeia Simples/metabolismo , DNA Viral/isolamento & purificação , DNA Viral/metabolismo , Escherichia coli/metabolismo , Cinética , Lisogenia , Peso Molecular , Mutação , Hibridização de Ácido Nucleico , Especificidade da Espécie , Timidina/metabolismo , TrítioRESUMO
To investigate the events occurring at the replication forks during DNA synthesis, we studied the replication of plasmid ColE1 DNA in vivo and in vitro, using strains of Escherichia coli carrying either the dnaG3(Ts) or dnaG308(Ts) mutation. Extracts of both mutant strains supported in vitro DNA synthesis, but the amount of [3H]TMP incorporated into DNA was always less for mutant extracts than for extracts of revertant strains, which were able to grow at 42 degrees C. Sucrose gradient analysis, Southern blot analysis, and electron microscopy showed that mutant extracts synthesize a large number of early replicative intermediates containing one or two (one on each template strand) fragments at the origin of replication and some completed molecules, either open circles or covalently closed circles. The revertant extracts synthesized more completed molecules although the fraction of templates used was about the same, 0.27 for mutant extracts and 0.21 for revertant extracts. Our results show that a mutation in dnaG causes a block in the synthesis of both leading and lagging strands after initiation, which results in the accumulation of early replicative intermediates. The average size of the newly replicated region in the early replicative intermediates is 730 bases as measured from electron micrographs of early replicative intermediates. We conclude that the DnaG protein functions in lagging strand synthesis and may be necessary for the continuation of leading strand synthesis as well.
Assuntos
Proteínas de Bactérias/fisiologia , Plasmídeos de Bacteriocinas , Replicação do DNA , Escherichia coli/metabolismo , Plasmídeos , RNA Nucleotidiltransferases/fisiologia , Proteínas de Bactérias/genética , Centrifugação com Gradiente de Concentração , Cromossomos Bacterianos/metabolismo , DNA Primase , DNA Bacteriano/biossíntese , DNA Circular/biossíntese , Escherichia coli/genética , Mutação , RNA Nucleotidiltransferases/genéticaRESUMO
An in-depth "how-to" report on initiating and operating a low-cost screening mammography facility is presented. It is based on a successful program of this type in Charlotte, North Carolina, and includes information on feasibility determination, financial analysis, site selection and preparation, equipment, public relations, promotion, and operational details. The authors believe that use of such a model, with modification as necessary, should result in a successful program and a valuable community service.
Assuntos
Instituições de Assistência Ambulatorial , Mamografia , Instituições de Assistência Ambulatorial/economia , Instituições de Assistência Ambulatorial/métodos , Instituições de Assistência Ambulatorial/organização & administração , Feminino , Humanos , Mamografia/métodos , North CarolinaRESUMO
We have followed, by deoxyribonucleic acid-deoxyribonucleic acid hybridization, the order of replication of three chromosomal markers during a synchronous round of replication in three strains of Escherichia coli carrying a dnaAts mutation: one strain in which the F-like R factor R.100.1 was established as a plasmid and two strains in which the dnaA mutation was suppressed by the integration of R.100.1 into the chromosome. In the R+ strain at 30C, replication of the plasmid took place simultaneously with the initiation of chromosome replication at the normal origin. In the integratively suppressed Hfr strains, at 42.5 C, chromosome replication was initiated preferentially from the integrated plasmid; little or no initiation occurred at the normal origin. Similar results were obtained for the one strain tested at 30 C. For both Hfr strains at 42.5 C, the data suggest that at least part of the population replicated bidirectionally. This conclusion had been confirmed using an autoradiographic procedure. Both types of experiment indicate a wide variation in the rate of travel of individual replication forks within the population.
Assuntos
Cromossomos Bacterianos/metabolismo , Replicação do DNA , DNA Bacteriano/biossíntese , Resistência Microbiana a Medicamentos , Escherichia coli/metabolismo , Mutação , Fatores R , Divisão Celular , Escherichia coli/crescimento & desenvolvimento , Temperatura , Fatores de TempoRESUMO
The origin of replication in Escherichia coli K-12 was mapped by determining the rate of marker replication during a synchronous round of replication. Four isogenic strains were made lysogenic for lambdaind(-) and for phage Mu-1, with Mu-1 integrated into a different chromosomal location in each strain. Cultures were starved for amino acids to allow completion of chromosome replication cycles and then starved for thymine in the presence of amino acids, and a synchronous cycle of replication was initiated by the addition of thymine. Samples were exposed to radioactive thymidine at intervals, deoxyribonucleic acid was extracted, and the rate of marker replication was determined by deoxyribonucleic acid-deoxyribonucleic acid hybridization to filters containing Mu-1, lambda, and E. coli deoxyribonucleic acid. The results confirm that the origin of replication is near ilv. The travel times of the replication forks, calculated from the data obtained for cultures with doubling times of approximately 40 and 61 min, are 40 and 52 min, respectively.