RESUMO
Protein adsorption on solid state media is important for the industrial affinity chromatography of biotherapeutics and for preparing materials for self-interaction chromatography where fundamental protein solution thermodynamic properties are measured. The adsorption of three model proteins (lysozyme, catalase and BSA) and two antibodies (a monoclonal and a polyclonal antibody) have been investigated on commercial affinity chromatography media with different surface functionalities (Formyl, Tresyl and Amino). Both the extent of protein immobilised (mg protein/ml media) and the reaction kinetics are reported for a range of reaction conditions, including pH, differing buffers as well as the presence of secondary reactants (glutaraldehyde, sodium cyanoborohydride, EDC and NHS). Compared to the reaction conditions recommended by manufacturers as well as those reported in previous published work, significant increases in the extent of protein immobilisation and reaction kinetics are reported here. The addition of glutaraldehyde or sodium cyanoborohydride was found to be especially effective even when not directly needed for the adsorption to happen. For mAb and pIgG, immobilisation levels of 50 and 31 mg of protein/ml of resin respectively were achieved, which are 100% or more than previously reported. Enhanced levels were achieved for lysozyme of 120 mg/ml with very rapid reaction kinetics (< 1 h) with sodium cyanoborohydride. It can be concluded that specific chromatography resins with Tresyl activated support offered enhanced levels of protein immobilisation due to their ability to react to form amine or thio-ether linkages with proteins. Additionally, glutaraldehyde can result in higher immobilisation levels whilst it can also accelerate immobilisation reaction kinetics.
RESUMO
Information on the factors that cause or amplify foodborne illness outbreaks (contributing factors), such as ill workers or cross-contamination of food by workers, is critical to outbreak prevention. However, only about half of foodborne illness outbreaks reported to the United States' Centers for Disease Control and Prevention (CDC) have an identified contributing factor, and data on outbreak characteristics that promote contributing factor identification are limited. To address these gaps, we analyzed data from 297 single-setting outbreaks reported to CDC's new outbreak surveillance system, which collects data from the environmental health component of outbreak investigations (often called environmental assessments), to identify outbreak characteristics associated with contributing factor identification. These analyses showed that outbreak contributing factors were more often identified when an outbreak etiologic agent had been identified, when the outbreak establishment prepared all meals on location and served more than 150 meals a day, when investigators contacted the establishment to schedule the environmental assessment within a day of the establishment being linked with an outbreak, and when multiple establishment visits were made to complete the environmental assessment. These findings suggest that contributing factor identification is influenced by multiple outbreak characteristics, and that timely and comprehensive environmental assessments are important to contributing factor identification. They also highlight the need for strong environmental health and food safety programs that have the capacity to complete such environmental assessments during outbreak investigations.
Assuntos
Surtos de Doenças , Contaminação de Alimentos , Doenças Transmitidas por Alimentos/epidemiologia , Vigilância da População , Centers for Disease Control and Prevention, U.S. , Doenças Transmitidas por Alimentos/microbiologia , Humanos , Estados Unidos/epidemiologiaRESUMO
Although contamination of food can occur at any point from farm to table, restaurant food workers are a common source of foodborne illness. We describe the characteristics of restaurant-associated foodborne disease outbreaks and explore the role of food workers by analysing outbreaks associated with restaurants from 1998 to 2013 reported to the Centers for Disease Control and Prevention's Foodborne Disease Outbreak Surveillance System. We identified 9788 restaurant-associated outbreaks. The median annual number of outbreaks was 620 (interquartile range 618-629). In 3072 outbreaks with a single confirmed aetiology reported, norovirus caused the largest number of outbreaks (1425, 46%). Of outbreaks with a single food reported and a confirmed aetiology, fish (254 outbreaks, 34%) was most commonly implicated, and these outbreaks were commonly caused by scombroid toxin (219 outbreaks, 86% of fish outbreaks). Most outbreaks (79%) occurred at sit-down establishments. The most commonly reported contributing factors were those related to food handling and preparation practices in the restaurant (2955 outbreaks, 61%). Food workers contributed to 2415 (25%) outbreaks. Knowledge of the foods, aetiologies, and contributing factors that result in foodborne disease restaurant outbreaks can help guide efforts to prevent foodborne illness.
Assuntos
Surtos de Doenças , Manipulação de Alimentos , Doenças Transmitidas por Alimentos/epidemiologia , Restaurantes , Contaminação de Alimentos , Doenças Transmitidas por Alimentos/etiologia , Humanos , Estados Unidos/epidemiologiaRESUMO
Surveillance data indicate that handling of food by an ill worker is a cause of almost half of all restaurant-related outbreaks. The U.S. Food and Drug Administration (FDA) Food Code contains recommendations for food service establishments, including restaurants, aimed at reducing the frequency with which food workers work while ill. However, few data exist on the extent to which restaurants have implemented FDA recommendations. The Centers for Disease Control and Prevention's Environmental Health Specialists Network (EHS-Net) conducted a study on the topic of ill food workers in restaurants. We interviewed restaurant managers (n = 426) in nine EHS-Net sites. We found that many restaurant policies concerning ill food workers do not follow FDA recommendations. For example, one-third of the restaurants' policies did not specifically address the circumstances under which ill food workers should be excluded from work (i.e., not be allowed to work). We also found that, in many restaurants, managers are not actively involved in decisions about whether ill food workers should work. Additionally, almost 70% of managers said they had worked while ill; 10% said they had worked while having nausea or "stomach flu," possible symptoms of foodborne illness. When asked why they had worked when ill, a third of the managers said they felt obligated to work or their strong work ethic compelled them to work. Other reasons cited were that the restaurant was understaffed or no one was available to replace them (26%), they felt that their symptoms were mild or not contagious (19%), they had special managerial responsibilities that no one else could fulfill (11%), there was non-food handling work they could do (7%), and they would not get paid if they did not work or the restaurant had no sick leave policy (5%). Data from this study can inform future research and help policy makers target interventions designed to reduce the frequency with which food workers work while ill.
Assuntos
Manipulação de Alimentos/normas , Saúde Ocupacional/normas , Gestão de Recursos Humanos/normas , Restaurantes/normas , Adulto , Centers for Disease Control and Prevention, U.S. , Surtos de Doenças , Contaminação de Alimentos/análise , Doenças Transmitidas por Alimentos/epidemiologia , Doenças Transmitidas por Alimentos/etiologia , Humanos , Pessoa de Meia-Idade , Licença Médica , Estados Unidos , United States Food and Drug Administration , Recursos HumanosRESUMO
BACKGROUND: Dasatinib is a small molecule kinase inhibitor that has recently been shown to inhibit Src family kinases (SFK) and also has activity against CaP. Of importance to metastatic CaP, which frequently metastasises to bone, SFK are also vital to the regulation of bone remodelling. We sought to determine the ability of dasatinib to inhibit growth of CaP in bone. METHODS: C4-2B CaP cells were injected into tibiae of SCID mice and treated with dasatinib, alone or in combination with docetaxel. Serum prostate-specific antigen levels, bone mineral density, radiographs and histology were analysed. RESULTS: Treatment with dasatinib alone significantly lowered sacrifice serum prostate-specific antigen levels compared to control, 2.3+/-0.4 vs 9.2+/-2.1 (P=0.004). Combination therapy improved efficacy over dasatinib alone (P=0.010). Dasatinib increased bone mineral density in tumoured tibiae by 25% over control tumoured tibiae (P<0.001). CONCLUSION: Dasatinib inhibits growth of C4-2B cells in bone with improved efficacy when combined with docetaxel. Additionally, dasatinib inhibits osteolysis associated with CaP. These data support further study of dasatinib in clinical trials for men with CaP bone metastases.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/secundário , Osteólise/tratamento farmacológico , Neoplasias da Próstata/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Tiazóis/farmacologia , Animais , Densidade Óssea/efeitos dos fármacos , Neoplasias Ósseas/sangue , Linhagem Celular Tumoral , Dasatinibe , Docetaxel , Humanos , Masculino , Camundongos , Camundongos SCID , Osteólise/sangue , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/sangue , Neoplasias da Próstata/patologia , Inibidores de Proteínas Quinases/efeitos adversos , Pirimidinas/efeitos adversos , Taxoides/administração & dosagem , Tiazóis/efeitos adversos , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases da Família src/antagonistas & inibidoresRESUMO
Zoledronic acid (ZA) has been shown to inhibit prostate tumor growth in vitro and have beneficial effects in patients with advanced prostate cancer (CaP). The aim of this study was to determine whether ZA exhibits direct anti-tumor effects on CaP cells in vivo. To distinguish the effects of inhibition of osteolysis and direct anti-tumor activity of ZA in vivo, we compared the results of treatment with ZA and osteoprotegerin (Fc-OPG), which inhibits osteolysis, but without significant direct anti-tumor effects. In vitro Fc-OPG had no significant effects on C4-2 proliferation, whereas ZA decreased proliferation. However, both agents decreased tumor growth in bone. Moreover, both increased bone volume and prevented the overall decreases in BMD associated with growth of C4-2 cells in bone. Our study provides novel and significant observations that the in vivo effects of ZA are consistent with indirect effects mediated by osteoclasts.
Assuntos
Anticorpos/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/secundário , Difosfonatos/farmacologia , Glicoproteínas/química , Glicoproteínas/imunologia , Imidazóis/farmacologia , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/imunologia , Receptores do Fator de Necrose Tumoral/química , Receptores do Fator de Necrose Tumoral/imunologia , Animais , Apoptose , Densidade Óssea , Conservadores da Densidade Óssea/farmacologia , Proliferação de Células , Densitometria , Difosfonatos/química , Glicoproteínas/farmacologia , Humanos , Imidazóis/química , Técnicas In Vitro , Masculino , Camundongos , Camundongos SCID , Metástase Neoplásica , Osteoclastos/metabolismo , Osteólise , Osteoprotegerina , Tíbia/patologia , Fatores de Tempo , Ácido ZoledrônicoRESUMO
Prostate cancer is the most commonly diagnosed malignancy in men and is often associated with bone metastases. Prostate cancer bone lesions can be lytic or schlerotic, with the latter predominating. Bone morphogenetic proteins (BMPs) are a family of growth factors, which may play a role in the formation of prostate cancer osteoblastic bone metastases. This study evaluated the effects of BMPs on prostate cancer cell lines. We observed growth inhibitory effects of BMP-2 and -4 on LNCaP, while PC-3 was unaffected. Flow cytometric analysis determined that LNCaP cell growth was arrested in G(1) after bone morphogenetic protein-2 treatment. Treatment of LNCaP and PC-3 with BMP-2 and -4 activated downstream signaling pathways involving SMAD-1, up-regulation of p21(CIP1/WAF1) and changes in retinoblastoma (Rb) phosphorylation. Interestingly, bone morphogenetic protein-2 treatment stimulated a 2.7-fold increase in osteoprotegerin (OPG), a molecule, which inhibits osteoclastogenesis, production in PC-3.
Assuntos
Proteínas Morfogenéticas Ósseas/farmacologia , Neoplasias Ósseas/metabolismo , Neoplasias da Próstata/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta , Proteína Morfogenética Óssea 2 , Proteína Morfogenética Óssea 4 , Neoplasias Ósseas/secundário , Divisão Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Citometria de Fluxo , Fase G1/fisiologia , Glicoproteínas/metabolismo , Humanos , Masculino , Osteoprotegerina , Fosforilação/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Fator de Necrose Tumoral , Proteína do Retinoblastoma/metabolismo , Proteínas Smad , Proteína Smad1 , Transativadores/metabolismo , Células Tumorais Cultivadas , Regulação para Cima/efeitos dos fármacosRESUMO
Deletions of the AZFc (azoospermia factor c) region of the Y chromosome are the most common known cause of spermatogenic failure. We determined the complete nucleotide sequence of AZFc by identifying and distinguishing between near-identical amplicons (massive repeat units) using an iterative mapping-sequencing process. A complex of three palindromes, the largest spanning 3 Mb with 99.97% identity between its arms, encompasses the AZFc region. The palindromes are constructed from six distinct families of amplicons, with unit lengths of 115-678 kb, and may have resulted from tandem duplication and inversion during primate evolution. The palindromic complex contains 11 families of transcription units, all expressed in testis. Deletions of AZFc that cause infertility are remarkably uniform, spanning a 3.5-Mb segment and bounded by 229-kb direct repeats that probably served as substrates for homologous recombination.
Assuntos
Deleção Cromossômica , Infertilidade Masculina/genética , Cromossomo Y/genética , Sequência de Bases , Inversão Cromossômica , Cromossomos Humanos Par 3/genética , Proteína 1 Suprimida em Azoospermia , Evolução Molecular , Duplicação Gênica , Humanos , Masculino , Modelos Genéticos , Dados de Sequência Molecular , Oligospermia/genética , Especificidade de Órgãos , Mapeamento Físico do Cromossomo , Proteínas de Ligação a RNA/genética , Recombinação Genética/genética , Análise de Sequência de DNA , Deleção de Sequência/genética , Homologia de Sequência do Ácido Nucleico , Espermatozoides/metabolismo , Sequências de Repetição em Tandem/genética , Testículo/metabolismo , Transcrição Gênica/genéticaRESUMO
The non-recombining region of the human Y chromosome (NRY), which comprises 95% of the chromosome, does not undergo sexual recombination and is present only in males. An understanding of its biological functions has begun to emerge from DNA studies of individuals with partial Y chromosomes, coupled with molecular characterization of genes implicated in gonadal sex reversal, Turner syndrome, graft rejection and spermatogenic failure. But mapping strategies applied successfully elsewhere in the genome have faltered in the NRY, where there is no meiotic recombination map and intrachromosomal repetitive sequences are abundant. Here we report a high-resolution physical map of the euchromatic, centromeric and heterochromatic regions of the NRY and its construction by unusual methods, including genomic clone subtraction and dissection of sequence family variants. Of the map's 758 DNA markers, 136 have multiple locations in the NRY, reflecting its unusually repetitive sequence composition. The markers anchor 1,038 bacterial artificial chromosome clones, 199 of which form a tiling path for sequencing.
Assuntos
Mapeamento Físico do Cromossomo , Cromossomo Y , Cromossomos Artificiais Bacterianos , Eucromatina , Amplificação de Genes , Genoma Humano , Heterocromatina , Humanos , Masculino , Mapeamento Físico do Cromossomo/métodos , Mapeamento de Híbridos Radioativos , Sitios de Sequências RotuladasRESUMO
The accurate mapping of clones derived from genomic regions containing complex arrangements of repeated elements presents special problems for DNA sequencers. Recent advances in the automation of optical mapping have enabled us to map a set of 16 BAC clones derived from the DAZ locus of the human Y chromosome long arm, a locus in which the entire DAZ gene as well as subsections within the gene copies have been duplicated. High-resolution optical mapping employing seven enzymes places these clones into two contigs representing four distinct copies of the DAZ gene and highlights a number of differences between individual copies of DAZ.
Assuntos
Mapeamento Cromossômico/métodos , Cromossomos Bacterianos/genética , Proteínas de Ligação a RNA/genética , Cromossomo Y/genética , Mapeamento Cromossômico/instrumentação , Clonagem Molecular/métodos , Mapeamento de Sequências Contíguas , Proteína 1 Suprimida em Azoospermia , Desoxirribonucleases de Sítio Específico do Tipo II/análise , Marcadores Genéticos/genética , Humanos , Mutagênese Insercional , Proteínas de Ligação a RNA/análise , Mapeamento por RestriçãoRESUMO
The DAZ genes are candidate fertility factors that lie within the human Y chromosome's AZFc region, whose deletion is a common cause of spermatogenic failure. The number of DAZ genes has been difficult to determine, in part because the nucleotide sequences of the DAZ genes are nearly identical. Here, fluorescence in situ hybridization and characterization of BAC clones revealed four full-length DAZ genes on the human Y chromosome. They exist in two clusters, each comprising an inverted pair of DAZ genes (3' <-- 5'::5' --> 3'). Analysis of genomic sequences and testicular transcripts suggested that three or four DAZ genes are translated. Each gene contains at least seven tandem copies of a previously described, 2.4-kb repeat unit that encodes 24 amino acids. In addition, two DAZ genes contain tandem copies of a 10.8-kb repeat unit that encodes the RNA-binding domain, which appears to be multimerized in some DAZ proteins. Combining our present results with previous studies, we can reconstruct several steps in the evolution of the DAZ genes on the Y chromosome. In the ancestral Y-chromosomal DAZ gene, amplification of both intragenic repeats began before the human and cynomolgus (Old World) monkey lineages diverged. During subsequent evolution, an inverted duplication of this modified gene occurred. Finally, the resulting two-gene cluster was duplicated, generating the two-cluster/four-gene arrangement found on modern human Y chromosomes.
Assuntos
Família Multigênica , Proteínas de Ligação a RNA/genética , Cromossomo Y/genética , Sequência de Bases , Southern Blotting , Cromossomos Artificiais de Levedura , Primers do DNA/química , Proteína 1 Suprimida em Azoospermia , Eletroforese em Gel de Campo Pulsado , Fibroblastos , Duplicação Gênica , Biblioteca Genômica , Humanos , Hibridização in Situ Fluorescente , Interfase , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas de Ligação a RNA/química , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Sequências de Repetição em TandemRESUMO
We have recently reported that complement factor H, a negative regulator of complement-mediated cytotoxicity, is produced and secreted by most bladder cancers. This observation was exploited in the development of the BTA stat and BTA TRAK diagnostic assays, both of which make use of two factor H-specific monoclonal antibodies in sandwich format. Here we show that both antibodies exert interesting effects on the biochemistry of complement activation in in vitro systems. Antibody X13.2 competes with C3b for association with factor H and strongly inhibits factor H/factor I-mediated cleavage of C3b, thereby evidently inactivating a negative regulator of complement; yet, the antibody strongly inhibits complement-mediated lysis as well. Conversely, antibody X52. 1, which does not compete with C3b and has no effect on solution-phase cleavage of C3b, is capable of enhancing complement-mediated lysis of various cell types, including cancer cells, by over 10-fold. Our observations indicate that it is possible to deconvolute the biochemical roles of factor H in complement by means of appropriate inhibitors, a finding with potentially valuable implications for both basic research and cancer therapy.
Assuntos
Anticorpos Monoclonais/imunologia , Ativação do Complemento/efeitos dos fármacos , Fator H do Complemento/imunologia , Animais , Especificidade de Anticorpos , Western Blotting , Complemento C3b/metabolismo , Relação Dose-Resposta a Droga , Eritrócitos/imunologia , Células HL-60 , Hemólise/imunologia , Humanos , Hibridomas/imunologia , Cinética , Modelos Biológicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ovinos , Fatores de Tempo , Células Tumorais Cultivadas , Regulação para Cima , Zimosan/metabolismoRESUMO
Deletion of the AZFc region of the Y chromosome is the most frequent molecularly defined cause of spermatogenic failure. We report three unrelated men in whom azoospermia or severe oligozoospermia was caused by de-novo AZFc deletions, and who produced sons by intracytoplasmic sperm injection (ICSI). We employed polymerase chain reaction (PCR) assays to examine the Y chromosomes of their four infant sons. All four sons were found to have inherited the Y chromosome deletions. Such sons are likely to be infertile as adults. This likelihood should be taken into account when counselling couples considering ICSI to circumvent infertility due to severe oligozoospermia or non-obstructive azoospermia.
Assuntos
Deleção Cromossômica , Fertilização in vitro , Infertilidade Masculina/genética , Infertilidade Masculina/terapia , Cromossomo Y/genética , Adulto , Citoplasma , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Microinjeções , Oligospermia/genética , Oligospermia/terapia , Reação em Cadeia da Polimerase , Gravidez , Sitios de Sequências Rotuladas , EspermatozoidesRESUMO
Deletion of the distal short arm of chromosome 9 (9p) has been reported in a number of cases to be associated with gonadal dysgenesis and XY sex reversal, suggesting that this region contains one or more genes required in two copies for normal testis development. Recent studies have greatly narrowed the interval containing this putative autosomal testis-determining gene(s) to the distal portion of 9p24.3. We previously identified DMRT1, a human gene with sequence similarity to genes that regulate the sexual development of nematodes and insects. These genes contain a novel DNA-binding domain, which we named the DM domain. DMRT1 maps to 9p24. 3 and in adults is expressed specifically in the testis. We have investigated the possible role of DM domain genes in 9p sex reversal. We identified a second DM domain gene, DMRT2, which also maps to 9p24.3. We found that point mutations in the coding region of DMRT1 and the DM domain of DMRT2 are not frequent in XY females. We showed by fluorescence in situ hybridization analysis that both genes are deleted in the smallest reported sex-reversing 9p deletion, suggesting that gonadal dysgenesis in 9p-deleted individuals might be due to combined hemizygosity of DMRT1 and DMRT2.
Assuntos
Cromossomos Humanos Par 9/genética , Genes/genética , Processos de Determinação Sexual , Testículo/crescimento & desenvolvimento , Adulto , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Pré-Escolar , Mapeamento Cromossômico , DNA Complementar/química , DNA Complementar/genética , Proteínas de Ligação a DNA/metabolismo , Transtornos do Desenvolvimento Sexual , Feminino , Disgenesia Gonadal 46 XY/genética , Humanos , Hibridização in Situ Fluorescente , Masculino , Dados de Sequência Molecular , Mutação Puntual , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Análise de Sequência de DNA , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Diferenciação Sexual/genética , Testículo/embriologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Phenylbutyrate (PB) is a potent differentiating agent and currently under investigation for the treatment of prostate cancer (CaP) and other malignancies. We have studied the impact of PB in vitro and in vivo on differentiation, proliferation and apoptosis in the LNCaP and LuCaP 23.1 prostate cancer xenograft models. In vitro we found that i) PB increased PSA secretion/cell, ii) inhibited cell proliferation in a time- and dose-dependent manner resulting in a cell cycle arrest in G1-phase and iii) induced apoptosis at concentrations of 2.5 mM after 3 days of treatment. In PB treated animals tumor growth stabilized or regressed. Combination of castration and PB treatment had a synergistic antiproliferative effect. The growth-inhibitory and differentiating properties and a low toxicity profile of PB provide rationale for further clinical studies in patients with CaP.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Fenilbutiratos/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Androgênios/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
In July 1997, symptoms characteristic of tomato yellow leaf curl virus (TYLCV-Is) were observed on one tomato plant in a field in Collier County, Florida, and on several tomato plants in a retail garden center in Sarasota, Florida. Amplification with three sets of primers, analysis of amplified fragments using restriction enzyme digestion, and hybridization with a clone of TYLCV-Is indicated that TYLCV-Is was present in symptomatic plants. The sequence of a 1,300-bp amplified fragment was 99% identical to TYLCV-Is from the Dominican Republic and 98% identical to an isolate from Israel. It appears that TYLCV-Is entered the United States in Dade County, Florida, in late 1996 or early 1997. Subsequently, infected tomato transplants produced for retail sale at two Dade County facilities were rapidly distributed via retail garden centers throughout the state. Infected plants purchased by homeowners and placed in and around homes appeared to be the source of TYLCV-Is for nearby commercial nurseries and production fields. It appears that transplants have played a role in the movement of this and probably other geminiviruses. A number of regulatory procedures, as well as field management practices, were implemented in the 1997-98 production season to minimize the movement of TYLCV-Is within and out of the state.
RESUMO
For small, brief targets incremental threshold is known to obey the de Vries-Rose law: threshold rises in direct proportion to the square-root of background intensity. We present data demonstrating a square-root law for brightness matching as well. The square-root law for brightness is obtained over the full range of scotopic vision, and the low intensity end of photopic vision. The classic theory of de Vries and Rose explains the square-root law on the basis of increased variability of the photon count as the background increases. Our brightness matching data instead indicates that the mean signal level is reduced by a factor which is inversely proportional to the standard deviation of the photon count. This result is consistent with the idea that in the retina there exists a gain control mechanism that is sensitive to the variance in the photon input, rather than to the mean illuminance. The importance of this idea to the modelling of retinal gain controls is discussed.
Assuntos
Retina/fisiologia , Percepção Visual/fisiologia , Adolescente , Adulto , Limiar Diferencial , Feminino , Humanos , Masculino , Matemática , Modelos Neurológicos , Estimulação Luminosa , Fotometria , Limiar Sensorial/fisiologiaRESUMO
BACKGROUND: Our objective was to investigate the presence of prostate specific antigen (PSA) and alpha-1-antichymotrypsin (ACT) mRNA and protein in prostate cancer cell lines, and the complexing characteristics of expressed PSA. METHODS: RT-PCR, immunohistochemistry, and Western blots were employed. Trypsin treatment of PSA was performed to establish the possible presence of an activatable form of PSA. RESULTS: ACT mRNA and protein were detected in LNCaP, PC-3, and DU 145 by RT-PCR and by immunohistochemistry, respectively. Only LNCaP cells were positive for PSA mRNA and protein. LNCaP expressed approximately 30% active PSA, approximately 40% putative zymogen form of PSA, and approximately 30% stably inactive PSA. CONCLUSIONS: We have shown that the majority of PSA expressed by LNCaP cells is present in free, noncomplexed forms in the conditioned media. A portion (40%) can be activated by trypsin, while the rest is stably inactive PSA. LNCaP cells may serve as a source of the "unreactive" PSA present in prostate cancer patients' serum.
Assuntos
Biomarcadores Tumorais/análise , Precursores Enzimáticos/análise , Peptídeos/análise , Antígeno Prostático Específico/análise , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/imunologia , Proteínas Secretadas pela Próstata , alfa 1-Antiquimotripsina/análise , Western Blotting , Primers do DNA , Humanos , Imuno-Histoquímica , Masculino , Reação em Cadeia da Polimerase/métodos , Antígeno Prostático Específico/genética , RNA Mensageiro/análise , RNA Neoplásico/análise , DNA Polimerase Dirigida por RNA , Células Tumorais Cultivadas , alfa 1-Antiquimotripsina/genéticaRESUMO
The human X and Y chromosomes share many blocks of similar DNA sequence. We conducted mapping and nucleotide sequencing studies of extensive, multi-megabase homologies between Yp and Xq21, which do not recombine during male meiosis. We confirmed and built upon previous evidence that a Yp inversion had occurred during evolution: a single contiguous segment of Xq21 is homologous to two non-contiguous segments of Yp. We precisely defined and sequenced the inversion breakpoints, obtaining evidence that the inversion was mediated by recombination between LINE-1 elements in otherwise non-homologous regions. This inversion appears to have followed a single transposition of an approximately 4 Mb segment from the X to the Y chromosome. These events jointly account for the present arrangement of Yp-Xq21 homologous sequences. Based on Southern blotting studies of primates and of humans drawn from diverse populations, we conclude that both the X-Y transposition and the subsequent, LINE-mediated Yp inversion occurred after the divergence of hominid and chimp lineages but before the radiation of extant human populations. This evolutionary scenario is consistent with our finding of 99.3 +/- 0.2% nucleotide identity between the X and Y chromosomes within the transposed region, which suggests that the transposition occurred approximately 3-4 million years ago, near the time of emergence of Homo . Comparative sequencing of the entire human X and Y chromosomes may reveal a succession of transpositions, inversions and other rearrangements underlying the complex pattern of sequence similarities between the present-day sex chromosomes. With the possible exception of cubitus valgus, phenotypic features of Turner syndrome are absent in individuals monosomic for Yp-Xq21 homologous sequences, suggesting that most of the critical 'Turner genes' are found elsewhere on the X and Y chromosomes.
Assuntos
Mapeamento Cromossômico , Elementos de DNA Transponíveis/genética , Proteínas de Ligação a DNA/genética , Recombinação Genética , Cromossomo X , Cromossomo Y , Sequência de Bases , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Análise de Sequência , Deleção de Sequência , Homologia de Sequência do Ácido NucleicoRESUMO
Y chromosome deletions encompassing the AZFc region have been reported in 13% of azoospermic men and 7% of severely oligozoospermic men. We examined the impact of these Y deletions on the severity of testicular defects in 51 azoospermic men undergoing intracytoplasmic sperm injection (ICSI) after testicular sperm extraction (TESE) and 30 men with severe oligozoospermia undergoing ICSI after ejaculation of spermatozoa. In addition, five azoospermic patients shown previously to have Y chromosome deletions underwent histological evaluation of their previously obtained testis biopsy specimens. A further 27 azoospermic men underwent TESE-ICSI, but not Y chromosome DNA testing. Ten of 51 azoospermic men (20%) who underwent TESE-ICSI and Y-DNA testing were found to be deleted for portions of the Y chromosome AZFc region. Of these 10, five had spermatozoa retrievable from the testis, and in two cases the wives became pregnant. Of the 41 azoospermic men with no Y chromosome deletion, 22 (54%) had spermatozoa retrievable from the testis, and in 12 cases (29%) the wives became pregnant. Four of 30 (13%) severely oligozoospermic patients were found to be deleted for AZFc and in three (75%) of these pregnancy was achieved. The other 26 severely oligozoospermic couples who had no AZFc deletions underwent ICSI, and 12 (46%) have an ongoing or delivered pregnancy. The embryo implantation rate was not significantly different for azoospermic (22%), oligozoospermic (16%), Y-deleted (14%) or Y-intact (18%) men. Of the total of 19 infertile men who had Y chromosome deletions, 14 had deletions within Y chromosome intervals 6D-6F, in the AZFc region. Twelve of those 14 had some spermatozoa (however few in number) in the ejaculate or testis. Five of the Y-deleted men had deletions that extended more proximally on the Y chromosome, and in none of these could any spermatozoa be observed in either ejaculate or testis. These results support the concept that, in azoospermic or oligozoospermic men with Y chromosome deletions limited to intervals 6D-6F (AZFc), there are generally very small numbers of testicular or ejaculated spermatozoa. Larger Y deletions, including and extending beyond the AZFc region and encompassing more Y genes, tend to be associated with a total absence of testicular spermatozoa. In those cases where spermatozoa were retrieved, the presence of Y deletions had no obvious impact on fertilization or pregnancy rate.