RESUMO
The hypothesis that levonorgestrel (LNG) used as an emergency contraceptive interferes with endometrial receptivity remains unproven. We compared the endometrial gene expression profile during the receptive period after administering a single dose of LNG 1.5âmg or placebo on day 1 of the luteal phase. An endometrial biopsy was done on day LH+7 or LH+8 and samples were taken from seven volunteers, each one contributing with one cycle treated with placebo and another with LNG. The expression of 20â383 genes was determined using cDNA microarrays. Real-time RT-PCR was used 1) to confirm the differences found in DNA microarray analysis and 2) to determine the effect of LNG on transcript levels of C3, C4BPα, COX2, MAOA, S100A4, and SERPINB9, known to be upregulated during receptivity, and on cPLA2α, JAK1, JNK1, CTSL1, and GSTP1, known to respond to mifepristone. Additional endometrial biopsies were done during the pre-receptive (LH+3) and receptive (LH+7) period and samples were taken from eight untreated volunteers in order to determine the changes associated with acquisition of receptivity of 14 genes. Mean levels of PAEP, TGM2, CLU, IGF2, and IL6ST mRNAs increased after administering LNG while those of HGD, SAT1, EVA1, LOC90133, ANXA1, SLC25A29, CYB5A, CRIP1, and SLC39A14 decreased. Except for the level of ANXA1 transcript, all changes remained within the range observed in untreated controls, and none of the transcripts responding to mifepristone changed in response to LNG. Post-ovulatory administration of LNG caused minimal changes in gene expression profiling during the receptive period. Neither the magnitude nor the nature or direction of the changes endorses the hypothesis that LNG interferes with endometrial receptivity.
Assuntos
Anticoncepcionais Femininos/farmacologia , Endométrio/efeitos dos fármacos , Endométrio/metabolismo , Perfilação da Expressão Gênica , Levanogestrel/farmacologia , Fase Luteal/efeitos dos fármacos , Fase Luteal/genética , Anticoncepcionais Femininos/administração & dosagem , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Levanogestrel/administração & dosagem , Ciclo Menstrual/efeitos dos fármacos , Ciclo Menstrual/fisiologia , Mifepristona/farmacologia , Progesterona/metabolismo , Reprodutibilidade dos Testes , Transcriptoma/efeitos dos fármacosRESUMO
Successful implantation depends both on the quality of the embryo and on the endometrial receptivity. The latter depends on progesterone-induced changes in gene expression, a process that has been characterized by microarray analysis. One of the genes whose transcription appears to be enhanced during the receptive period is monoamine oxidase A (MAO-A). Our first objective was to confirm the increased expression of MAO-A in the endometrium during the receptive phase of spontaneous normal cycles using real time PCR and immunofluorescence. The second objective was to examine the endometrial expression of MAO-A during the receptive phase induced by exogenous estradiol (E(2)) and progesterone in patients whose endometrium was shown to have been either receptive or non-receptive to embryo implantation in repeated cycles of oocyte donation. Results showed that MAO-A transcript levels increased between the pre-receptive (LH+3) and receptive phase (LH+7) in all spontaneous cycles examined, with a median increase of 25-fold. Immunofluorescent labelling demonstrated MAO-A localization to the glandular and luminal epithelium with an increasing positive score between LH+3 and LH+7. Conversely, prior failure of embryo implantation was associated with a 29-fold decrease in MAO-A mRNA levels and a substantial reduction in MAO-A protein immunofluorescent label score. These results show a strong association between endometrial receptivity and MAO-A expression in the endometrial epithelium, suggesting an important role for this enzyme in normal implantation.
Assuntos
Implantação do Embrião/fisiologia , Perda do Embrião/etiologia , Endométrio/enzimologia , Infertilidade Feminina/enzimologia , Monoaminoxidase/deficiência , Doação de Oócitos , Adulto , Indução Enzimática , Células Epiteliais/enzimologia , Estradiol/farmacologia , Feminino , Humanos , Infertilidade Feminina/fisiopatologia , Fase Luteal , Monoaminoxidase/biossíntese , Monoaminoxidase/genética , Monoaminoxidase/fisiologia , Indução da Ovulação , Progesterona/farmacologia , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais/enzimologiaRESUMO
This unit describes three approaches that are widely used to define alignments between overlapping clones bearing large-insert genomic DNA and to generate extensive contiguous overlapping sets of clones (contigs). The three approaches are sequence-tagged site (STS) content mapping, repetitive-element hybridization fingerprinting, and Alu-PCR fingerprinting. Methods for isolating the necessary BAC DNA suitable for automated fluorescent sequencing and generating new STS markers are discussed in support protocols. An alternate protocol presents repetitive-element hybridization fingerprinting to detect overlaps and build contigs with full-genomic YAC libraries.
Assuntos
Mapeamento de Sequências Contíguas/métodos , Cromossomos Artificiais Bacterianos/genética , Cromossomos Artificiais de Levedura/genética , DNA/genética , DNA/isolamento & purificação , Impressões Digitais de DNA , Genética Médica , Biblioteca Genômica , Humanos , Reação em Cadeia da Polimerase , Sitios de Sequências RotuladasRESUMO
This unit describes several polymerase chain reaction (PCR)-based methods to obtain DNA fragments from clones with large inserts without prior knowledge of the insert DNA sequence. The protocols can be categorized into three groups: (1) methods to generate DNA fragments at random representing the entire length of the cloned insert, (2) methods to generate DNA fragments representing the extremities of an insert, and (3) methods to generate complex probes suitable for fluorescence in situ hybridization. Support protocols describe direct cloning of these PCR products and the isolation of total yeast DNA from yeast artificial chromosome (YAC) clones.
Assuntos
Técnicas de Sonda Molecular , Reação em Cadeia da Polimerase/métodos , Elementos Alu , Cromossomos Artificiais de Bacteriófago P1/genética , Cromossomos Artificiais de Levedura/genética , Clonagem Molecular , Sondas de DNA/genética , Sondas de DNA/isolamento & purificação , Vetores Genéticos , Genética Médica , Humanos , Hibridização in Situ FluorescenteRESUMO
OBJECTIVE: To examine the extent to which Illinois nursing facilities have developed relationships with other healthcare providers, particularly managed care organizations (MCOs). STUDY DESIGN: A cross-sectional survey of nursing facilities designed to determine: 1) relationship objectives; 2) obstacles to developing relationships; 3) currently available services; 4) staffing for these services and; 5) nursing facility approaches to networking. The survey was sent to a census sample of 867 nursing facilities serving the elderly in Illinois. Descriptive and multivariate logistic regression analyses of relationships determined to be formal/risk-sharing were performed. STUDY POPULATION: The sample included 523 Illinois nursing facilities. A total response rate of 60% was achieved (523/867). RESULTS: Higher strategic goals, urban location, nonprofit ownership status, higher percentages of private pay and/or Medicare clients (vs Medicaid), and provision of home care and subacute services were all significant predictors of formal or risk-sharing relationships with MCOs. CONCLUSIONS: Facilities with more relationships and higher goals have more formal/risk-sharing relationships with MCOs. Facilities in urban areas have more relationships, likely due to the fact that rural facilities have fewer options and operate in different markets. In addition, nursing facilities rely on Medicare referrals from hospitals, and these Medicare patients, especially those in urban areas, are increasingly controlled by MCOs.
Assuntos
Programas de Assistência Gerenciada/organização & administração , Casas de Saúde/organização & administração , Afiliação Institucional/estatística & dados numéricos , Participação no Risco Financeiro , Idoso , Comportamento Cooperativo , Estudos Transversais , Coleta de Dados , Eficiência Organizacional/estatística & dados numéricos , Pesquisa sobre Serviços de Saúde , Humanos , Illinois , Casas de Saúde/economia , Casas de Saúde/estatística & dados numéricos , Afiliação Institucional/economia , Objetivos Organizacionais , Propriedade , Análise de RegressãoRESUMO
We have constructed YAC, PAC, and cosmid contigs in the ataxia-telangiectasia gene region and used the assembled clones to isolate expressed sequences by exon trapping and hybridization selection. In the interval between D11S1819 and D11S2029, exons and cDNAs for potentially 13 different genes were identified. Three of these genes, F37, K28, and 6.82, are large novel genes expressed in a variety of different tissues. K28 shows sequence homology to the Rab GTP binding protein family and gene 6.82 homology to the rabbit vasopressin activated calcium mobilizing receptor, while gene F37 has no homology to any known sequence in the database. Three further clones, exon 6.41 and cDNAs K22 and E74, from the interval between D11S1819 and D11S2029, appear to be expressed endogenous retrovirus sequences. The fourth large novel genes, E14, together with two further possible novel genes, E13 and E3, was identified from exons and cDNAs in the more telomeric 300-kb interval between markers D11S2029 and D11S2179. These are in addition to the genes for mitochondrial acetoacetyl-CoA-acetyltransferase (ACAT) and the ATM gene in the same region. Genes E3, E13, and E14 do not show homology to any known genes. K28, 6.82, ACAT, and ATM all appear to have the same transcriptional orientation toward the telomere.
Assuntos
Ataxia Telangiectasia/genética , Mapeamento Cromossômico/métodos , Cromossomos Humanos Par 11/genética , Proteínas Serina-Treonina Quinases , Proteínas/genética , Transcrição Gênica/genética , Acetil-CoA C-Acetiltransferase/genética , Sequência de Aminoácidos , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular , DNA Complementar/genética , Proteínas de Ligação a DNA , Éxons/genética , Proteínas de Ligação ao GTP/genética , Humanos , Dados de Sequência Molecular , Especificidade de Órgãos , RNA Mensageiro/análise , RNA Mensageiro/genética , Retroviridae/genética , Homologia de Sequência de Aminoácidos , Proteínas Supressoras de TumorRESUMO
Several human Mendelian diseases, including the long-QT syndrome, malignant hyperthermia, and episodic ataxia/myokymia syndrome, have recently been demonstrated to be due to mutations in ion channel genes. Systematic mapping of ion channel genes may therefore reveal candidates for other heritable disorders. In this study, the GenBank and dbEST databases were used to identify members of several ion channel families (voltage-gated calcium and sodium, cardiac chloride, and all classes of potassium channels). Genes and ESTs without prior map localization were identified based on GDB and OWL database information and 15 genes and ESTs were selected for mapping. Of these 15, only the serotonin receptor 5HT3R had been previously mapped to a chromosome. A somatic cell hybrid panel (SCH) was screened with an STS from each gene and, if necessary the results verified by a second SCH panel. For three ESTs, rodent derived PCR products of the same size as the human STS precluded SCH mapping. For these three, human P1 clones were isolated and the genomic location was determined by metaphase FISH. These genes and ESTs can now be further evaluated as candidate genes for inherited cardiac, neuromuscular and psychiatric disorders mapped to these chromosomes. Furthermore, the ESTs developed in this study can be used to isolate genomic clones, enabling the determination of each transcript's genomic structure and physical map location. This approach may also be applicable to other gene families and may aid in the identification of candidate genes for groups of related heritable disorders.
Assuntos
Canais de Cloreto/genética , Mapeamento Cromossômico , Canais de Potássio/genética , Animais , Cromossomos Humanos , Humanos , Células Híbridas , Dados de Sequência Molecular , Receptores de Serotonina/genética , Sitios de Sequências RotuladasRESUMO
A contiguous physical map was constructed from the Harvey ras-1 (HRAS1) gene to the 11p telomere. The contig spans approximately 500 kb and is minimally composed of a telomere-containing YAC and P1 and cosmid clones. Included in the contig are 11 sequence-tagged sites derived from P1 and cosmid ends. Three genes were placed on the contig in the following order: telomere-ribonuclease/angiogenin inhibitor (RNH)-Harvey ras-1 (HRAS1)-HRAS1-related complex (HRC). Two novel tetranucleotide repeats (heterozygosity of 66 and 68%) and a complex CA repeat (heterozygosity of 78%) were isolated and characterized.
Assuntos
Cromossomos Humanos Par 11 , Genes ras , Telômero , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Artificiais de Levedura , Repetições de Dinucleotídeos , Triagem de Portadores Genéticos , Marcadores Genéticos , Humanos , Dados de Sequência Molecular , Polimorfismo Genético , Técnica de Amplificação ao Acaso de DNA PolimórficoRESUMO
The development of a highly reliable physical map with landmark sites spaced an average of 100 kbp apart has been a central goal of the Human Genome Project. We have approached the physical mapping of human chromosome 11 with this goal as a primary target. We have focused on strategies that would utilize yeast artificial chromosome (YAC) technology, thus permitting long-range coverage of hundreds of kilobases of genomic DNA, yet we sought to minimize the ambiguities inherent in the use of this technology, particularly the occurrence of chimeric genomic DNA clones. This was achieved through the development of a chromosome 11-specific YAC library from a human somatic cell hybrid line that has retained chromosome 11 as its sole human component. To maximize the efficiency of YAC contig assembly and extension, we have employed an Alu-PCR-based hybridization screening system. This system eliminates many of the more costly and time-consuming steps associated with sequence tagged site content mapping such as sequencing, primer production, and hierarchical screening, resulting in greater efficiency with increased throughput and reduced cost. Using these approaches, we have achieved YAC coverage for >90% of human chromosome 11, with an average intermarker distance of <100 kbp. Cytogenetic localization has been determined for each contig by fluorescent in situ hybridization and/or sequence tagged site content. The YAC contigs that we have generated should provide a robust framework to move forward to sequence-ready templates for the sequencing efforts of the Human Genome Project as well as more focused positional cloning on chromosome 11.
Assuntos
Cromossomos Humanos Par 11 , Quimera , Mapeamento Cromossômico , Cromossomos Artificiais de Levedura , Clonagem Molecular , DNA/química , Projeto Genoma Humano , Humanos , Hibridização in Situ Fluorescente , Reação em Cadeia da Polimerase , Mapeamento por RestriçãoRESUMO
We have developed a PCR-based method for rapid and effective screening of arrayed cDNA libraries. This strategy directly addresses the limitations of conventional hybridization-based schemes and provides a more rapid, cost-effective, and sensitive method compatible with large-scale and routine cDNA clone recovery. To prepare arrayed libraries, 1-2 x 10(6) cDNA clones were propagated as individual plaques on solid medium in 24-well culture dishes at approximately 250 plaque-forming units per well. Phage suspensions were prepared from each well and transferred to a 96-well format. To screen the library, pools were generated that correspond to each individual 96-well plate and to each row and column within "blocks" of six plates each. Library screening for specific cDNA clones was conducted in a systematic and hierarchical fashion beginning with the plate pools. Next, the row/column pools corresponding to each positive plate pool were screened. Finally, isolated clones from within each positive well were identified by hybridization. We have applied this approach to the screening of an arrayed human brain cDNA library resulting in the recovery of cDNAs corresponding to > 25 genes and expressed sequence tags.
Assuntos
Cromossomos Humanos Par 11 , DNA Complementar , Lobo Frontal/metabolismo , Biblioteca Gênica , Reação em Cadeia da Polimerase/métodos , Adulto , Animais , Sequência de Bases , Linhagem Celular , Mapeamento Cromossômico , Clonagem Molecular/métodos , Cricetinae , Humanos , Células Híbridas , Rim/metabolismo , Dados de Sequência MolecularRESUMO
The mammalian ribosome is a massive structure composed of 4 RNA species and about 80 different proteins. One of these ribosomal proteins, S3, appears to function not only in translation but also as an endonuclease in repair of UV-induced DNA damage. Moreover, the first intron of human RPS3 transcripts is processed to generate U15A, a small nucleolar RNA. We localized the nested RPS3/U15A genes to the immediate vicinity of D11S356 and D11S533 on human chromosome 11q13.3-q13.5 using a combination of somatic cell hybrid analysis, fluorescence in situ hybridization, and YAC/STS content mapping. These findings add to the evidence that genes encoding ribosomal proteins are scattered about the human genome.
Assuntos
Cromossomos Humanos Par 11 , Genes , RNA Nuclear Pequeno/genética , Proteínas Ribossômicas/genética , Animais , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Artificiais de Levedura , Cricetinae , Reparo do DNA/genética , Humanos , Células Híbridas , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
A significant issue in the analysis of any genomic DNA segment is the generation of a unique set of short single-copy sequences that are representative of that region. In this report we describe a novel technique, IRE-bubble PCR, which was designed to amplify the human DNA content of somatic cell hybrids, YACs, cosmids, and lambda phage and result in greater complexity and representation than standard inter-IRE PCR. Here we demonstrate that IRE-bubble PCR is species specific and that it results in the generation of a product that is at least 10-fold more complex and representative than that produced by standard inter-IRE PCR. In addition, we have addressed the factors that contribute to the representation of the IRE-bubble PCR product and show how they may be used to further increase the complexity of this reaction. Finally, we have illustrated how the complexity and distribution of products generated by IRE-bubble PCR can be exploited and applied to FISH mapping and "chromosome painting" as well as to the generation of STSs targeted to specific chromosomal or subchromosomal regions.
Assuntos
DNA/genética , Genoma Humano , Reação em Cadeia da Polimerase/métodos , Sequências Repetitivas de Ácido Nucleico , Animais , Sequência de Bases , Cricetinae , Primers do DNA , Humanos , Células Híbridas , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Moldes GenéticosRESUMO
This article is based upon research in Florida of the older population and its projected need for services. It includes discussion of the method and findings of the study. The focus, however, is on the policy implications for state government that emerged from the research. The data has enabled Florida to examine the experience of its own community care programs in order to help develop a strategic plan for expanding resources, reforming its organization, and improving its performance. In addition to funding and organization of the state's long-term care system, the issue of targeting limited community resources to severely frail elders emerged as a major concern of this research. Although the various states differ widely in history, demographics, and structure, these policy issues should be of immediate concern to each of them.
Assuntos
Idoso/estatística & dados numéricos , Necessidades e Demandas de Serviços de Saúde/tendências , Pesquisa sobre Serviços de Saúde/métodos , Serviços de Saúde para Idosos/estatística & dados numéricos , Serviço Social/tendências , Idoso de 80 Anos ou mais , Florida , Previsões , Serviços de Saúde para Idosos/organização & administração , Humanos , Pessoa de Meia-IdadeRESUMO
1. To be successful, an organizational career development program must include a differentiation of the responsibilities for which the various parties (employer, employee, career counselor) will be held accountable. 2. Project outcomes revealed that the career mobility program was attractive to nursing personnel and facility management personnel alike. However, it was more attractive to nonlicensed than licensed personnel. 3. Of the staff who participated and were promoted, the majority remained in their jobs. The program was most successful in enhancing retention with personnel who received within-level promotions. 4. The process of career development requires collaboration and support from all levels of leadership and staff throughout the organization. A career development program, including a career mobility program with a strong career counseling component, can serve as a catalyst in professionalizing the long-term care work environment.
Assuntos
Mobilidade Ocupacional , Ocupações em Saúde , Assistência de Longa Duração , Aconselhamento , Humanos , Modelos de Enfermagem , Recursos HumanosRESUMO
The purpose of this study was to determine the extent to which registered nurse (RN) staffing patterns influenced nursing home quality. Based upon extensive literature review, a model for nursing home quality was developed. Using data from reports of 455 Medicare-certified skilled nursing facilities, structural, process, and outcome factors thought to influence quality were entered into the model and analyzed using ordinary least squares regression. A small, though statistically significant, proportion of the variance in quality nursing home care was explained by the equation. A positive, significant relationship existed between nursing home quality and the ratio of RN hours to licensed vocational nurse (LVN) hours per resident day.
Assuntos
Casas de Saúde , Recursos Humanos de Enfermagem/normas , Gestão de Recursos Humanos , Admissão e Escalonamento de Pessoal , Qualidade da Assistência à Saúde/estatística & dados numéricos , Atividades Cotidianas , California , Humanos , Casas de Saúde/normas , Enfermagem Prática/organização & administração , Propriedade , Reorganização de Recursos Humanos , Fatores de Tempo , Recursos HumanosRESUMO
This article reports the results of a 3-year demonstration project in career mobility for nursing personnel in skilled nursing facilities. The goal of the project was to demonstrate the effectiveness of a career mobility program in addressing service delivery challenges such as recruitment, retention, and satisfaction of nursing personnel in skilled nursing facilities. The project was a joint effort between an educational department of a large university and a major long-term care corporation. The project evaluation addressed the effectiveness of this career mobility program in attracting and retaining nursing personnel, the satisfaction of nursing personnel with their jobs, and whether the additional training affected the quality of care. Five pilot and three comparison sites were designated in Southern California. Survey, self-report, and interview data were collected at yearly intervals and at the completion of the project. Descriptive statistics and analysis of variance formed the basis for data analysis and interpretation. Overall, the findings suggested that the career mobility program has the potential to retain staff who benefit from a career development program. The career mobility program was a valuable strategy, but its success was only as good as the management team's authority to support the program and emphasize its goals.