Transcriptional regulation and functional analysis of the human Wilms' tumor 1 gene

The Wilms' tumor 1 gene (WT1) encodes a zinc-finger transcription factor and is expressed in urogenital, hematopoietic and other tissues. It is expressed in a temporal and spatial manner in both embryonic and adult stages. To obtain a better understanding of the biological function of WT1, we studied two aspects of WT1 regulation: one is the identification of tissue-specific cis-regulatory elements that regulate its expression, the other is the downstream genes which are modulated by WT1.^ My studies indicate that in addition to the promoter, other regulatory elements are required for the tissue specific expression of this gene. A 259-bp hematopoietic specific enhancer in intron 3 of the WT1 gene increased the transcriptional activity of the WT1 promoter by 8- to 10-fold in K562 and HL60 cells. Sequence analysis revealed both GATA and c-Myb motifs in the enhancer fragment. Mutation of the GATA motif decreased the enhancer activity by 60% in K562 cells. Electrophoretic mobility shift assays showed that both GATA-1 and GATA-2 proteins in K562 nuclear extracts bind to this motif. Cotransfection of the enhancer containing reporter construct with a GATA-1 or GATA-2 expression vector showed that both GATA-1 and GATA-2 transactivated this enhancer, increasing the CAT reporter activity 10-15 fold and 5-fold respectively. Similar analysis of the c-Myb motif by cotransfection with the enhancer CAT reporter construct and a c-Myb expression vector showed that c-Myb transactivated the enhancer by 5-fold. A DNase I-hypersensitive site has been identified in the 258 bp enhancer region. These data suggest that GATA-1 and c-Myb are responsible for the activity of this enhancer in hematopoietic cells and may bind to the enhancer in vivo. In the process of searching for cis-regulatory elements in transgenic mice, we have identified a 1.0 kb fragment that is 50 kb downstream from the promoter and is required for the central nervous system expression of WT1.^ In the search for downstream target genes of WT1, we noted that the proto-oncogene N-myc is coexpressed with the tumor suppressor gene WT1 in the developing kidney and is overexpressed in many Wilms' tumors. Sequence analysis revealed eleven consensus WT1 binding sites located in the 1 kb mouse N-myc promoter. We further showed that the N-myc promoter was down-regulated by WT1 in transient transfection assays. Electrophoretic mobility shift assays showed that oligonucleotides containing the WT1 motifs could bind WT1 protein. Furthermore, a Denys-Drash syndrome mutant of WT1, R394W, that has a mutation in the DNA binding domain, failed to repress the N-myc promoter. This suggests that the repression of the N-myc promoter is mediated by DNA binding of WT1. This finding helps to elucidate the relationship of WT1 and N-myc in tumorigenesis and renal development. ^

Accuracy and self correction of information received from an internet breast cancer list: content analysis.

OBJECTIVES: To determine the prevalence of false or misleading statements in messages posted by internet cancer support groups and whether these statements were identified as false or misleading and corrected by other participants in subsequent postings. DESIGN: Analysis of content of postings. SETTING: Internet cancer support group Breast Cancer Mailing List. MAIN OUTCOME MEASURES: Number of false or misleading statements posted from 1 January to 23 April 2005 and whether these were identified and corrected by participants in subsequent postings. RESULTS: 10 of 4600 postings (0.22%) were found to be false or misleading. Of these, seven were identified as false or misleading by other participants and corrected within an average of four hours and 33 minutes (maximum, nine hours and nine minutes). CONCLUSIONS: Most posted information on breast cancer was accurate. Most false or misleading statements were rapidly corrected by participants in subsequent postings.

A comparison of healthcare access, healthcare utilization and asthma self-management for Hispanic children in border and non-border regions of Texas

The purpose of this culminating experience was to investigate the relationships between healthcare utilization, insurance coverage, and socioeconomic characteristics of children with asthma along the Texas-Mexico Border. A secondary data analysis was conducted on cross-sectional data from the Texas Child Asthma Call-back Survey, a follow-up survey to the random digit dialed Behavior Risk Factor Surveillance Study (BRFSS) conducted between 2006-2009 ( n = 556 adults living in households with a child with asthma).^ The proportion of Hispanic children with asthma in Border areas of Texas was more than twice that of non-Border areas (84.8% vs. 28.8%). Parents in Border areas were less likely to have their own health insurance (OR = 0.251, 95% C.I. = 0.117-0.540) and less likely to complete the survey in English than Spanish (OR = 0.251 95% C.I. = 0.117-0.540) than parents in non-Border areas. No significant socio-economic or health care utilization differences were noted between Hispanic children living in Border areas compared to Hispanic children living in non-Border areas. Children with asthma along the Texas-Mexico Border, regardless of ethnicity and language, have insurance coverage rates, reported cost barriers to care, symptom management, and medication usage patterns similar to those in non-Border areas. When compared to English-speakers, Spanish-speaking parents in Texas as a whole are far less likely to be taught what to do during an asthma attack (50.2% vs. 78.6%).^ Language preference, rather than ethnicity or geographical residence, played a larger role on childhood asthma-related health disparities for children in Texas. Spanish-speaking parents in are less likely to receive adequate asthma self-management education. Investigating the effects of Hispanic acculturation rates and incongruent parent-child health insurance coverage may provide better insight into the health disparities of children along the Texas-Mexico Border.^

The feasibility of self-report advance directives among elderly patients with cognitive impairment

The central objective of this dissertation was to determine the feasibility of self-completed advance directives (AD) in older persons suffering from mild and moderate stages of dementia. This was accomplished by identifying differences in ability to complete AD among elderly subjects with increasing degrees of dementia and cognitive incompetence. Secondary objectives were to describe and compare advance directives completed by elders and identified proxy decision makers. Secondary objectives were accomplished by measuring the agreement between advance directives completed by proxy and elder, and comparing that agreement across groups defined by the elder's cognitive status. This cross-sectional study employed a structured interview to elicit AD, followed by a similar interview with a proxy decision maker identified by the elder. A stratified sampling scheme recruited elders with normal cognition, mild, and moderate forms of dementia using the Mini Mental-State Exam (MMSE). The Hopkins Competency Assessment Test (HCAT) was used for evaluation of competency to make medical decisions. Analysis was conducted on "between group" (non-demented $\leftrightarrow$ mild dementia $\leftrightarrow$ moderate dementia, and competent $\leftrightarrow$ incompetent) and "within group" (elder $\leftrightarrow$ family member) variation.^ The 118 elderly subjects interviewed were generally male, Caucasian, and of low socioeconomic status. Mean age was 77. Overall, elders preferred a "trial of therapy" regarding AD rather than to "always receive the therapy". No intervention was refused outright more often than it was accepted. A test-retest of elders' AD revealed stable responses. Eleven logic checks measured appropriateness of AD responses independent of preference. No difference was found in logic error rates between elders grouped by MMSE or HCAT. Agreement between proxy and elder responses showed significant dissimilarity, indicating that proxies were not making the same medical decisions as the elders.^ Conclusions based on these data are: (1) Self reporting AD is feasible among elders showing signs of cognitive impairment and they should be given all opportunities to complete advance directives, (2) variation in preferences for advance directives in cognitively impaired elders should not be assumed to be the effects of their impairment alone, (3) proxies do not appear to forego life-prolonging interventions in the face of increasing impairment in their ward, however, their advance directives choices are frequently not those of the elder they represent. ^