Cardiolipin biosynthesis and remodeling enzymes are altered during development of heart failure.

Cardiolipin (CL) is responsible for modulation of activities of various enzymes involved in oxidative phosphorylation. Although energy production decreases in heart failure (HF), regulation of cardiolipin during HF development is unknown. Enzymes involved in cardiac cardiolipin synthesis and remodeling were studied in spontaneously hypertensive HF (SHHF) rats, explanted hearts from human HF patients, and nonfailing Sprague Dawley (SD) rats. The biosynthetic enzymes cytidinediphosphatediacylglycerol synthetase (CDS), phosphatidylglycerolphosphate synthase (PGPS) and cardiolipin synthase (CLS) were investigated. Mitochondrial CDS activity and CDS-1 mRNA increased in HF whereas CDS-2 mRNA in SHHF and humans, not in SD rats, decreased. PGPS activity, but not mRNA, increased in SHHF. CLS activity and mRNA decreased in SHHF, but mRNA was not significantly altered in humans. Cardiolipin remodeling enzymes, monolysocardiolipin acyltransferase (MLCL AT) and tafazzin, showed variable changes during HF. MLCL AT activity increased in SHHF. Tafazzin mRNA decreased in SHHF and human HF, but not in SD rats. The gene expression of acyl-CoA: lysocardiolipin acyltransferase-1, an endoplasmic reticulum MLCL AT, remained unaltered in SHHF rats. The results provide mechanisms whereby both cardiolipin biosynthesis and remodeling are altered during HF. Increases in CDS-1, PGPS, and MLCL AT suggest compensatory mechanisms during the development of HF. Human and SD data imply that similar trends may occur in human HF, but not during nonpathological aging, consistent with previous cardiolipin studies.

The cloning and expression of myogenin, a gene regulating myogenesis

Expression of the differentiated skeletal muscle phenotype is a process that appears to occur in at least two stages. First, pluripotent stem cells become committed to the myogenic lineage. Although undifferentiated and capable of continued proliferation, determined myoblasts are restricted to a single developmental fate. Upon receiving the appropriate environmental signals, these determined myoblasts withdraw from the cell cycle, fuse to form multi-nucleated myotubes, and begin to express a battery of muscle-specific gene products that make up the functional and contractile apparatus of the muscle. This project is aimed at the identification and characterization of factors that control the determination and differentiation of myogenic cells. We have cloned a cDNA, called myogenin, that plays an important role in these processes. Myogenin is expressed exclusively in skeletal muscle in vivo and myogenic cell lines in vitro. Its expression is sharply upregulated during differentiation. When constitutively expressed in fibroblasts, myogenin converts these cells to the myogenic lineage. Transfected cells behave as myogenic tissue culture cells with respect to the genes they express, the way they respond to environmental cues, and are capable of fusing to form multinucleated myotubes. Sequence analysis showed that this cDNA has homology to a family of transcription factors in a region of 72 amino acids known as the basic helix-loop-helix motif. This domain appears to mediate binding to a DNA sequence element known as an E-box (CANNTG) essential for the activity of the enhancers of many muscle-specific genes.^ Analysis of myogenin in tissue culture cells showed that its expression is responsive to many of the environmental cues, such as the presence of growth factors and oncogenes, that modulate myogenesis. In an attempt to identify the cis- and trans-elements that control myogenin expression and thereby understand what factors are responsible for the establishment of the myogenic lineage, we have cloned the myogenin gene. After analysis of the gene structure, we constructed a series of reporter constructs from the 5$\prime$ upstream sequence of the myogenin gene to determine which cis-acting sequences might be important in myogenin regulation. We found that 184 nucleotides of the 5$\prime$ sequence was sufficient to direct high-level muscle-specific expression of the reporter gene. Two sequence elements present in the 184 fragment, an E-box and a MEF-2 site, have been shown previously to be important in muscle-specific transcription. Mutagenesis of these sites revealed that both sites are necessary for full activity of the myogenin promoter, and suggests that a complex hierarchy of transcription factors control myogenic differentiation. ^

Mechanism of dendritic epidermal T cell-mediated tolerance induction and inhibition of proliferation

Dendritic epidermal T cells (DETC) comprise a unique population of T cells that reside in mouse epidermis and whose function remains unclear. Most DETC express a $\gamma\delta$ TCR, although some, including our DETC line, AU16, express an $\alpha\beta$ TCR. Additionally, AU16 cells express CD3, Thy-1, CD45, CD28, B7, and AsGM-1. Previous studies in our laboratory demonstrated that hapten-conjugated AU16 could induce specific immunologic tolerance in vivo and inhibit T cell proliferation in vitro. Both these activities are antigen-specific, and the induction of tolerance is non-MHC-restricted. In addition, AU16 cells are cytotoxic to a number of tumor cell lines in vitro. These studies suggested a role for these cells in immune surveillance. The purpose of my studies was to test the hypothesis that these functions of DETC (tolerance induction, inhibition of T cell proliferation, and tumor cell killing) were mediated by a cytotoxic mechanism. My specific aims were (1) to determine whether AU16 could prevent or delay tumor growth in vivo; and (2) to determine the mechanism whereby AU16 induce tolerance, using an in vitro proliferation assay. I first showed that AU16 cells killed a variety of skin tumor cell lines in vitro. I then demonstrated that they prevented melanoma growth in C3H mice when both cell types were mixed immediately prior to intradermal (i.d.) injection. Studies using the in vitro proliferation assay confirmed that DETC inhibit proliferation of T cells stimulated by hapten-bearing, antigen-presenting cells (FITC-APC). To determine which cell was the target, $\gamma$-irradiated, hapten-conjugated AU16 were added to the proliferation assay on d 4. They profoundly inhibited the proliferation of naive T cells to $\gamma$-irradiated, FITC-APC, as measured by ($\sp3$H) TdR uptake. This result strongly suggested that the T cell was the target of the AU16 activity because no APC were present by d 4 of the in vitro culture. In contrast, the addition of FITC-conjugated splenic T cells (SP-T) or lymph node T cells (LN-T) was less inhibitory. Preincubation of the T cells with FITC-AU16 cells for 24 h, followed by removal of the AU16 cells, completely inhibited the ability of the T cells to proliferate in response to FITC-APC, further supporting the conclusion that the T cell was the target of the AU16. Finally, AU16 cells were capable of killing a variety of activated T cells and T cell lines, arguing that the mechanism of proliferation inhibition, and possibly tolerance induction is one of cytotoxicity. Importantly, $\gamma\delta$ TCR$\sp+$ DETC behaved, both in vivo and in vitro like AU16, whereas other T cells did not. Therefore, these results are consistent with the hypothesis that AU16 cells are true DETC and that they induce tolerance by killing T cells that are antigen-activated in vivo. ^

Regulation of angiogenesis by interferon-beta

The purpose of these studies was to investigate the role of interferon-beta (IFN-$\beta$) in angiogenesis. IFN-$\alpha/\beta$ have been implicated in inhibiting a number of steps in the angiogenic pathway. We examined the balance of angiogenesis-regulating molecules in several systems including human infantile hemangiomas, UV-B irradiated mice, and dorsal incisional wound healing in mice. In each system, epidermal hyperplasia and cutaneous angiogenesis were directly related to the expression of positive angiogenic factors (bFGF and VEGF) and inversely related to the expression of endogenous IFN-$\beta.$ The re-expression of IFN-$\beta$ correlated with tumor regression and/or resolution of wound healing. In contrast to control mice, UV-B-induced cutaneous angiogenesis and hyperplasia persisted in IFN-$\alpha/\beta$ receptor knock-out mice. In normal mice, endogenous IFN-$\beta$ was expressed by all differentiated epithelial cells exposed to environmental stimuli. The expression of endogenous IFN-$\beta$ was necessary but insufficient for complete differentiation of epidermal keratinocytes.^ The tumor organ microenvironment can regulate angiogenesis. Human bladder carcinoma cells growing in the bladder wall of nude mice express high levels of bFGF, VEGF, and MMP-9, have higher vascular densities, and produce metastases to lymph nodes and lungs, whereas the same cells growing subcutaneously express less bFGF, VEGF, and MMP-9, have lower vascular densities, and do not metastasize. IFN-$\alpha/\beta$ was found to inhibit bFGF and MMP-9 expression both in vitro and in vivo in human bladder carcinoma cells. Systemic therapy with human IFN-$\alpha$ of human bladder cancer cells growing orthotopically in nude mice, resulted in decreased vascularity, tumorigenicity, and metastasis as compared to saline treated mice. Human bladder cancer cells resistant to the antiproliferative effects of IFN were transfected with the human IFN-$\beta$ gene. Hu-IFN-$\beta$ transfected cells expressed significantly less bFGF protein and gelatinase activity than parental or control-transfected cells and did not grow at ectopic or orthotopic sites. Collectively the data provide direct evidence that IFN-$\alpha/\beta$ can inhibit angiogenesis via down-regulation of angiogenesis-stimulating cytokines. ^

The pain experience of elderly hospice patients with cancer

Purpose/objectives. A grounded theory design was used to identify, describe, and generate a theoretical analysis of the pain experience of elderly hospice patients with cancer. ^ Sample. Eleven participants over the age of 65, receiving services from a for-profit hospice were interviewed in their homes. ^ Methods. Broad unstructured face to face audio-taped interviews were transcribed verbatim and analyzed using constant-comparative method of analysis. ^ Findings. Pain was described as a hierarchy of chronic, acute, and psychological pain with psychological pain as the worst. Suffering was the basic social problem of pain. Participants dealt with suffering by the basic social process of enduring. Enduring had two sub-processes, maintaining hope and adjusting. Trusting in a higher being and finding meaning were mechanisms of maintaining hope. Mechanisms of adjusting were dealing with uncertainty, accepting, and minimizing pain. ^ Implications for nursing practice. Nurses need to recognize and value the hard work of enduring to deal with suffering. Assisting elderly hospice patients with cancer to address the sub-processes of enduring and their mechanisms can foster enduring. ^

AN EPIDEMIOLOGIC CASE-CONTROL ANALYSIS OF CHILDHOOD INTRACRANIAL AND SPINAL CORD TUMORS IN RELATION TO PATERNAL OCCUPATION AT BIRTH (BRAIN, CANCER)

Epidemiologic case-control studies of small groups of childhood nervous system tumor patients have suggested that parental employment in occupations with exposure to hydrocarbons is a risk factor for disease. The main focus of this case-control study was to assess the paternal occupation at the time of birth of offspring who later developed childhood intracranial and spinal tumors. All children under 15 years of age dying of such tumors in Texas, during the period 1964-1980, were selected as cases. Disease and demographic data were abstracted from death certificates. The birth certificate for each child of the final group of 499 cases was located and parental occupation information, as well as demographic and obstetric data, were collected. The comparison group consisted of a random sample from all Texas live births with the same birth year, race and sex distribution as the cases.^ The paternal occupations were categorized into broad classifications of those involving hydrocarbon exposure versus those that did not, based on the occupation criteria used in the previous studies. Odds ratios did not indicate any increased risk associated with general paternal hydrocarbon exposure in the workplace. In prior studies, increased risk estimates were detected with narrower groups of occupations involving exposure to hydrocarbon materials. The data from this study were classified according to these groups, and again, no increased risks were indicated except for a statistically insignificant but elevated odds ratio for fathers who were paper and pulp mill workers.^ Odds ratios were calculated for specific occupations and industries previously implicated as risk factors. Significantly associated odds ratios (OR) were detected for electricians (OR = 3.5), especially those working for construction companies (OR = 10.0), for employment in the printing occupations (OR = 4.5), particularly graphic arts workers (OR = 21.9), and in the electronics and electronic machinery industries (OR = 3.5). Analysis of the petroleum refining and chemical industries, which were not found in previous study populations, revealed significantly elevated odds ratios of 3.0 for occupations with probable heavy exposure to chemicals and petroleum compounds and 10.0 for salesmen of chemical products. ^

THE TRANSLATION PRODUCTS OF MOLONEY MURINE SARCOMA VIRUS 124 GENOMIC RNA

Murine sarcoma viruses constitute a class of replication-defective retroviruses. Cellular transformation may be induced by these viruses in vitro; whereas, fibrosarcomas may result in animals infected with them in vivo (Tooze, 1973; Bishop, 1978). Hybridization studies suggest that murine sarcoma viruses arose by recombination between nondefective murine leukemia virus sequences and certain cellular sequences present in uninfected mouse cells (Hu et al., 1977). A specific gene product, however, has not been implicated in murine sarcoma virus transformation.^ One line of murine sarcoma virus-producing cells, Mo-MuSV-clone 124, (Ball et al., 1973), was studied biochemically because it mainly produces the sarcoma virus as a pseudotype packaged with helper murine leukemia virus proteins. The sarcoma viral RNA was translated in a sophisticated cell-free protein synthesizing system (Murphy and Arlinghaus, 1978). The translation products were analyzed by a number of techniques, including electrophoresis in denaturing gels of SDS polyacrylamide, immunoprecipitation, and peptide mapping. The major products of the total RNA purified from the virus preparation were shown to have molecular weights of about 63,000 (P63('gag)), 42,000 (P42), 40,000 (P40), 38,000 (P38), and 23,000 (P23). The size class of mRNA coding for each of the cell-free products was estimated using a poly(A) selection technique and sucrose gradient fractionation. These analyses were used to localize the coding information related to each of the in vitro synthesized cell-free products within the sarcoma virus genome.^ The major findings of these studies were: (1) the 5' half of the sarcoma viral RNA codes for the 63,000 dalton polypeptide and 42,000 - 38,000 dalton polypeptides derived from the "gag" gene; and (2) the 3' half of the sarcoma viral RNA codes for a 38,000 dalton polypeptide and possibly derived from the cellular acquired sequences. ^

A novel myeloid leukemia suppressor locus at human chromosome 5q13.3

Molecular mechanisms that underlie preleukemic myelodysplasia (MDS) and acute myelogenous leukemia (AML) are poorly understood. In MDS or AML with a refractory clinical course, more than 30% of patients have acquired interstitial or complete deletions of chromosome 5. The 5q13.3 chromosomal segment is commonly lost as the result of 5q deletion. Reciprocal and unbalanced translocations of 5q13.3 can also occur as sole anomalies associated with refractory AML or MDS. This study addresses the hypothesis that a critical gene at 5q13.3 functions either as a classical tumor suppressor or as a chromosomal translocation partner and contributes to leukemogenesis. ^ Previous studies from our laboratory delineated a critical region of loss to a 2.5–3.0Mb interval at 5q13.3 between microsatellite markers D5S672 and GATA-P18104. The critical region of loss was later resolved to an interval of approximately 2Mb between the markers D5S672 and D5S2029. I, then generated a long range physical map of yeast artificial chromosomes (YACs) and developed novel sequence tagged sites (STS). To enhance the resolution of this map, bacterial artificial chromosomes (BACs) were used to construct a triply linked contig across a 1 Mb interval. These BACs were used as probes for fluorescent in situ hybridization (FISH) on an AML cell line to define the 5q13.3 critical region. A 200kb BAC, 484a9, spans the translocation breakpoint in this cell line. A novel gene, SSDP2 (single stranded DNA binding protein), is disrupted at the breakpoint because its first four exons are encoded within 140kb of BAC 484a9. This finding suggests that SSDP2 is the critical gene at 5q13.3. ^ In addition, I made an observation that deletions of chromosome 5q13 co-segregate with loss of the chromosome 17p. In some cases the deletions result from unbalanced translocations between 5q13 and 17p13. It was confirmed that the TP53 gene is deleted in patients with 17p loss, and the remaining allele harbors somatic mutation. Thus, the genetic basis for the aggressive clinical course in AML and MDS may be caused by functional cooperation between deletion or disruption of the 5q13.3 critical gene and inactivation of TP53. ^

Health promotion attitudes and practices of Texas nurse practitioners

The purpose of this descriptive cross-sectional survey was to examine the health promotion attitudes and practices of Texas nurse practitioners and to evaluate the applicability of the Theory of Reasoned Action and the Theory of Planned Behavior as a theoretical model to guide nurse practitioner health promotion research. A questionnaire developed to elicit responses regarding demographic information, practice characteristics, behavior, behavior intention, attitudes toward health promotion, subjective norm and perceived behavioral control for health promotion practices was mailed to the home address of 727 Texas nurse practitioners. The majority of the 442 respondents reported positive attitudes toward health promotion. Texas nurse practitioners provide health promotion for more than 50% of their patients. Significant barriers to the provision of health promotion cited by Texas nurse practitioners were lack of time, lack of reimbursement and lack of patient desire to change behavior. The findings of this study support the use of the Theory of Reasoned Action and the Theory of Planned Behavior in nurse practitioner research. ^

New insights into the role of the duodenal vitamin D receptor in calcium homeostasis

It is generally believed that 1,25(OH)2D3, bound to its receptor (VDR) contributes to calcium homeostasis by regulating active calcium absorption in the proximal small intestine. However, studying patients with hereditary vitamin D-resistant rickets (HVDRR) provided investigators with a better understanding of VDR's role in calcium homeostasis. HVDRR patients have inactivating mutations in the VDR, and as a consequence they develop hypocalcemia, hyperparathyroidism and severe rickets. However, these phenotypes can be corrected if the patients are given IV infusions of calcium or dietary calcium. This raises the question of what is the physiological significance of VDR-regulated active calcium absorption if calcium homeostasis can be restored independently of the VDR. ^ In order to distinguish the contribution of VDR in the proximal small intestine to overall calcium homeostasis, I generated transgenic mice expressing the human VDR (hVDR) exclusively in the proximal small intestine of mVDR-/- mice by using an hVDR-expressing transgene driven by the duodenal-specific adenosine deaminase enhancer (hVDR+/mVDR-/-). hVDR+/mVDR-/- mice expressed transcriptionally active hVDR only in the proximal small intestine and responded to 1,25(OH)2D3 by up-regulating expression of TRPV6 and calbindin D9K, genes involved in calcium absorption. Furthermore, ligated duodenal loop assays determined that calcium absorption in hVDR+/mVDR-/- mice was as responsive to 1,25(OH)2D3 as in WT mice. Despite having a functional hVDR in the proximal small intestine, hVDR+/mVDR-/- mice were hypocalcemic, had hyperparathyroidism, and were rachitic when fed a normal rodent diet at weaning, as were the mVDR-/- mice. However, when fed a high calcium, phosphorus, and lactose diet (rescue diet), the hVDR+/mVDR-/- mice responded more effectively than the mVDR-/- mice by down-regulation of parathyroid hormone production and by a greater increase in bone mineralization. Furthermore, when three-month-old rachitic mice were fed a rescue diet for 3 weeks, serum calcium and bone mineral content were normalized in hVDR+/mVDR-/- mice, but not in mVDR-/- mice. ^ In conclusion, hVDR expression enabled young mice to better use the rescue diet than mVDR-/- mice. Expression of transgenic hVDR also protected the ability of older mice to respond to the rescue diet despite the absence of the VDR elsewhere in the intestinal tract. I propose that because hVDR+/mVDR-/- mice responded better than mVDR-/- mice to the rescue diet, it is likely that VDR expression in the proximal small intestine is necessary in nutritional (insufficient dietary calcium) and physiological (age) conditions when passive calcium absorption is inadequate. ^

Evaluation of web-based resources for pregnant and parenting teens

Current teen pregnancy and repeat pregnancy rates reveal that there is a pressing need for comprehensive care for pregnant and parenting teens to address their unique needs. The Internet has become a source of various types of information and as a result, several efforts have begun to assess the quality of health information provided on websites. The objective of this study was to assess the functionality and quality of websites containing health information and resources for pregnant and parenting teens. The three most widely used search engines currently: Google, MSN, and Yahoo were searched using three general search terms “teen pregnancy”, “pregnant teen”, and “teen parent”. The first 5 pages of each search were reviewed and categorized to yield 12 websites which met inclusion criteria for content evaluation. The 12 websites were rated using a pre-existing instrument encompassing two domains: functionality and content analysis. Within the functionality domain, this sample highlighted the need to improve accessibility and credibility for the target population. The content analysis revealed that among the topics which are recommended for pregnant and parenting teens, the topics most commonly covered were mental health and primary and preventive health care. The majority of websites neglected sexual health topics including STI’s and family planning. This study provides the first glimpse into health information and resources for pregnant and parenting teens on the Internet. Researchers, health care providers, social workers, health educators, and website sponsors can use these results to maintain and recommend websites which offer easily accessible, accurate, and practical information for pregnant and parenting teens.^

Validity of using item response theory to analyze data from the Work Limitations Questionnaire

The Work Limitations Questionnaire (WLQ) is used to determine the amount of work loss and productivity which stem from certain health conditions, including rheumatoid arthritis and cancer. The questionnaire is currently scored using methodology from Classical Test Theory. Item Response Theory, on the other hand, is a theory based on analyzing item responses. This study wanted to determine the validity of using Item Response Theory (IRT), to analyze data from the WLQ. Item responses from 572 employed adults with dysthymia, major depressive disorder (MDD), double depressive disorder (both dysthymia and MDD), rheumatoid arthritis and healthy individuals were used to determine the validity of IRT (Adler et al., 2006).^ PARSCALE, which is IRT software from Scientific Software International, Inc., was used to calculate estimates of the work limitations based on item responses from the WLQ. These estimates, also known as ability estimates, were then correlated with the raw score estimates calculated from the sum of all the items responses. Concurrent validity, which claims a measurement is valid if the correlation between the new measurement and the valid measurement is greater or equal to .90, was used to determine the validity of IRT methodology for the WLQ. Ability estimates from IRT were found to be somewhat highly correlated with the raw scores from the WLQ (above .80). However, the only subscale which had a high enough correlation for IRT to be considered valid was the time management subscale (r = .90). All other subscales, mental/interpersonal, physical, and output, did not produce valid IRT ability estimates.^ An explanation for these lower than expected correlations can be explained by the outliers found in the sample. Also, acquiescent responding (AR) bias, which is caused by the tendency for people to respond the same way to every question on a questionnaire, and the multidimensionality of the questionnaire (the WLQ is composed of four dimensions and thus four different latent variables) probably had a major impact on the IRT estimates. Furthermore, it is possible that the mental/interpersonal dimension violated the monotonocity assumption of IRT causing PARSCALE to fail to run for these estimates. The monotonicity assumption needs to be checked for the mental/interpersonal dimension. Furthermore, the use of multidimensional IRT methods would most likely remove the AR bias and increase the validity of using IRT to analyze data from the WLQ.^

Applying the Strengths Perspective to Increase Safety and Weil-Being: Views from Families and Providers

Consensus about the value of the strengths perspective is developing among child welfare and family service practitioners. Yet, few first-hand reports are available from the perspectives of family members and interdisciplinary service providers about the principles most important for engaging and supporting family members to achieve needed outcomes. This paper briefly highlights principles most often cited as key to application of the strengths perspective and compares first-hand accounts from family members and service providers. These views were elicited through focus groups facilitated by a community-based family support program.Implications for strengths-based practice with families are discussed.