Cloning and characterization of the full length cDNA and promoter of mouse tissue transglutaminase

Transglutaminases are a family of enzymes that catalyze the covalent cross-linking of proteins through the formation of $\varepsilon$-($\gamma$-glutaminyl)-lysyl isopeptide bonds. Tissue transglutaminase (Tgase) is an intracellular enzyme which is expressed in terminally differentiated and senescent cells and also in cells undergoing apoptotic cell death. To characterize this enzyme and examine its relationship with other members of the transglutaminase family, cDNAs, the first two exons of the gene and 2 kb of the 5$\sp\prime$ flanking region, including the promoter, were isolated. The full length Tgase transcript consists of 66 bp of 5$\sp\prime$-UTR (untranslated) sequence, an open reading frame which encodes 686 amino acids and 1400 bp of 3$\sp\prime$-UTR sequence. Alignment of the deduced Tgase protein sequence with that of other transglutaminases revealed regions of strong homology, particularly in the active site region.^ The Tgase cDNA was used to isolate and characterize a genomic clone encompassing the 5$\sp\prime$ end of the mouse Tgase gene. The transcription start site was defined using genomic and cDNA clones coupled with S1 protection analysis and anchored PCR. This clone includes 2.3 kb upstream of the transcription start site and two exons that contain the first 256 nucleotides of the mouse Tgase cDNA sequence. The exon intron boundaries have been mapped and compared with the exon intron boundaries of three members of the transglutaminase family: human factor XIIIa, the human keratinocyte transglutaminase and human erythrocyte band 4.1. Tissue Tgase exon II is similar to comparable exons of these genes. However, exon I bears no resemblance with any of the other transglutaminase amino terminus exons.^ Previous work in our laboratory has shown that the transcription of the Tgase gene is directly controlled by retinoic acid and retinoic acid receptors. To identify the region of the Tgase gene responsible for regulating its expression, fragments of the Tgase promoter and 5$\sp\prime$-flanking region were cloned into the chloramphenicol actetyl transferase (CAT) reporter constructs. Transient transfection experiments with these constructs demonstrated that the upstream region of Tgase is a functional promoter which contains a retinoid response element within a 1573 nucleotide region spanning nucleotides $-$252 to $-$1825. ^

Inhibitor of cytochrome c messenger RNA -protein interaction in stimulated rat skeletal muscle

The purpose of this work was to examine the possible mechanisms for the regulation of cytochrome c gene expression in response to increased contractile activity in rat skeletal muscle. The working hypothesis was that increased contractile activity enhances cytochrome c gene expression through a cis-element. A 110% increase in cytochrome c mRNA concentration was observed in tibialis anterior (TA) muscle after 9 days of chronic stimulation. Similar difference (120%) exists between soleus (SO) muscle of higher contractile activity and white vastus lateralis (WV) muscle of lower contractile activity. These results suggest that the endogenous cytochrome c gene expression is regulated by contractile activity. Cytochrome c-reporter genes were injected into skeletal muscles to identify the cis-element that is responsible for the regulation. Although the data was inconclusive, part of it suggested the importance of the 3$\sp\prime$-untranslated region (3$\sp\prime$-UTR) in mediating the response to increased contractile activity.^ RNA gel mobility shift (GMSA) and ultraviolet (UV) cross-linking assays revealed specific RNA-protein interaction in a 50-nucleotide region of the 3$\sp\prime$-UTR in unstimulated TA muscle. Computer analysis predicted a stem-loop structure of 17 nucleotides, which provides a structural basis for RNA-protein interaction. These 17 nucleotides are 100% conserved among rat, mouse and human cytochrome c genes and their 13 pseudogenes, suggesting a functional role for this region. The RNA-protein interaction was significantly less in highly active SO muscle than in inactive WV muscle and was dramatically decreased in stimulated TA muscle due to a protein inhibitor(s) associated with ribosome. It is possible that cytochrome c mRNAs undergoing translation are subject to a compartmentalized regulatory influence.^ The conclusion from these results is that increases in contractile activity induce or activate a protein inhibitor(s) associated with ribosome in rat skeletal muscle. The inhibitor decreases RNA-protein interaction in the 3$\sp\prime$-UTR of cytochrome c mRNA, which may result in increased mRNA stability and/or translation. ^

Breast cancer on the world wide web: cross sectional survey of quality of information and popularity of websites.

OBJECTIVES: To determine the characteristics of popular breast cancer related websites and whether more popular sites are of higher quality. DESIGN: The search engine Google was used to generate a list of websites about breast cancer. Google ranks search results by measures of link popularity---the number of links to a site from other sites. The top 200 sites returned in response to the query "breast cancer" were divided into "more popular" and "less popular" subgroups by three different measures of link popularity: Google rank and number of links reported independently by Google and by AltaVista (another search engine). MAIN OUTCOME MEASURES: Type and quality of content. RESULTS: More popular sites according to Google rank were more likely than less popular ones to contain information on ongoing clinical trials (27% v 12%, P=0.01 ), results of trials (12% v 3%, P=0.02), and opportunities for psychosocial adjustment (48% v 23%, P<0.01). These characteristics were also associated with higher number of links as reported by Google and AltaVista. More popular sites by number of linking sites were also more likely to provide updates on other breast cancer research, information on legislation and advocacy, and a message board service. Measures of quality such as display of authorship, attribution or references, currency of information, and disclosure did not differ between groups. CONCLUSIONS: Popularity of websites is associated with type rather than quality of content. Sites that include content correlated with popularity may best meet the public's desire for information about breast cancer.

The impact of traditional psychiatric services and case management services on the functioning of people with schizophrenia: A cross-national study in Nova Scotia and Texas

The purpose of this study was to determine the impact of traditional psychiatric services with case management services on the functioning of people with schizophrenia. Traditional services were defined as routine clinic services consisting of medication follow-along, psychotherapy, and support services. Case management consisted of activities involved in linking, planning, and monitoring services for the outpatient client who has schizophrenia. The target population was adult schizophrenics who had been receiving outpatient clinic services for a minimum of six months. Structured interviews were conducted using standardized scales (e.g., Quality of Life, Self-Efficacy, and Brief Symptom Inventory) with 78 outpatient client volunteers from two sites: Nova Scotia (Canada) and Texas (USA). The researcher tested for differences in psychiatric symptomatology, recidivism, and quality of life for persons with schizophrenia receiving traditional psychiatric services in Nova Scotia and traditional plus case management services in Texas. Data were collected from the structured interviews and medical records review forms. Types of services were blocked into low and high levels of Intensity (frequency x minutes) and compared to determine the relative contribution of each. Finally, the role of clients' self-efficacy was tested as an intervening variable. Although the findings did not support the hypotheses in the direction anticipated, there were some interesting and useful results. From the Nova Scotia site, clients who received low levels of services were hospitalized less compared to the Texas site. The more psychotic a patient was the higher their involvement in medication follow-along and the more monitoring they received. The more psychotherapy received, the lower the reported satisfaction with social relationships. Of particular interest is the role that self-efficacy played in improved client outcomes. Although self-efficacy scores were related to improved functioning, the mechanism for this still needs to be clarified through subsequent research. ^