Structural equation modeling of the medical outcome study: Short Form 36 (SF-36)

The factorial validity of the SF-36 was evaluated using confirmatory factor analysis (CFA) methods, structural equation modeling (SEM), and multigroup structural equation modeling (MSEM). First, the measurement and structural model of the hypothesized SF-36 was explicated. Second, the model was tested for the validity of a second-order factorial structure, upon evidence of model misfit, determined the best-fitting model, and tested the validity of the best-fitting model on a second random sample from the same population. Third, the best-fitting model was tested for invariance of the factorial structure across race, age, and educational subgroups using MSEM.^ The findings support the second-order factorial structure of the SF-36 as proposed by Ware and Sherbourne (1992). However, the results suggest that: (a) Mental Health and Physical Health covary; (b) general mental health cross-loads onto Physical Health; (c) general health perception loads onto Mental Health instead of Physical Health; (d) many of the error terms are correlated; and (e) the physical function scale is not reliable across these two samples. This hierarchical factor pattern was replicated across both samples of health care workers, suggesting that the post hoc model fitting was not data specific. Subgroup analysis suggests that the physical function scale is not reliable across the "age" or "education" subgroups and that the general mental health scale path from Mental Health is not reliable across the "white/nonwhite" or "education" subgroups.^ The importance of this study is in the use of SEM and MSEM in evaluating sample data from the use of the SF-36. These methods are uniquely suited to the analysis of latent variable structures and are widely used in other fields. The use of latent variable models for self reported outcome measures has become widespread, and should now be applied to medical outcomes research. Invariance testing is superior to mean scores or summary scores when evaluating differences between groups. From a practical, as well as, psychometric perspective, it seems imperative that construct validity research related to the SF-36 establish whether this same hierarchical structure and invariance holds for other populations.^ This project is presented as three articles to be submitted for publication. ^

Social support, health status and health services utilization among the noninstitutionalized elderly: An analysis of the Supplement on Aging to the 1984 national health interview survey

The association between Social Support, Health Status, and Health Services Utilization of the elderly, was explored based on the analysis of data from the Supplement on Aging to the National Health Interview Survey, 1984 (N = 11,497) using a modified framework of Aday and Andersen's Expanded Behavioral Model. The results suggested that Social Support as operationalized in this study was an independent determinant of the use of health services. The quantity of social activities and the use of community services were the two most consistent determinants across different types of health services use.^ The effects of social support on the use of health services were broken down into three components to facilitate explanations of the mechanisms through which social support operated. The Predisposing and Enabling component of Social Support had independent, although not uniform, effects on the use of health services. Only slight substitute effects of social support were detected. These included the substitution of the use of senior centers for longer stay in the hospital and the substitution of help with IADL problems for the use of formal home care services.^ The effect of financial support on the use of health services was found to be different for middle and low income populations. This differential effect was also found for the presence of intimate networks, the frequencies of interaction with children and the perceived availability of support among urban/rural, male/female and white/non-white subgroups.^ The study also suggested that the selection of appropriate Health Status measures should be based on the type of Health Services Utilization in which a researcher is interested. The level of physical function limitation and role activity limitation were the two most consistent predictors of the volume of physician visits, number of hospital days, and average length of stay in the hospital during the past year.^ Some alternative hypotheses were also raised and evaluated, when possible. The impacts of the complex sample design, the reliability and validity of the measures and other limitations of this analysis were also discussed. Finally, a revised framework was proposed and discussed based on the analysis. Some policy implications and suggestions for future study were also presented. ^

Validity and reliability of using MVP 4 Function Walk4Life digital pedometers to assess physical activity levels among preschool-aged Head Start children

The Surgeon General recommends preschoolers 3-5 years old accumulate 60 minutes of moderate-to-vigorous physical activity (MVPA) per day. However, there is limited data measuring physical activity (PA) and MVPA amongst this population. The purpose of this cross-sectional study is to determine the validity, reliability, and feasibility of using MVP 4 Function Walk4Life digital pedometers (MVP-4) in measuring MVPA among preschoolers using the newly modified direct observational technique, System for Observing Fitness Instruction Time-Preschool Version (SOFIT-P) as the gold standard. An ethnically diverse population of 3-5 year old underserved children were recruited from two Harris County Department of Education (HCDE) Head Start centers. For 2 days at baseline and 2 days at post-test, 75 children enrolled wore MVP-4 pedometers for approximately 6-hours per observation day and were observed using SOFIT-P during predominantly active times. Statistical analyses used Pearson "r" correlation coefficients to determine mean minutes of PA and MVPA, convergent and criterion validity, and reliability. Significance was set at p = <0.05. Feasibility was determined through process evaluation information collected during this study via observations from data collectors and teacher input. Results show mean minutes of PA and MVPA ranged between 30-42 and 11-14 minutes, respectively. Convergent validity comparing BMI percentiles with MVP-4 PA outcomes show no significance at pre-test; however, each measurement at post-test showed significance for MVPA (p = 0.0247, p = 0.0056), respectively. Criterion validity comparing percent MVPA time between SOFIT-P and MVP-4 pedometers was determined; however, results deemed insufficient due to inconsistency in observation times while using the newly developed SOFIT-P. Reliability measures show no significance at pre-test, yet show significant results for all PA outcomes at post-test (p = 0.001, p = 0.001, p = 0.0010, p = 0.003), respectively. Finally, MVP-4 pedometers lacked feasibility due to logistical barriers in design. Researchers feel the significant results at post-test are secondary to increased familiarity and more accurate placement of pedometers across time. Researchers suggest manufacturers of MVP-4 pedometers further modify the instrument for ease of use with this population, following which future studies ought to determine validity using objective measures or all-day direct observation techniques.^

Plasticity of lipid-protein interactions in the function and topogenesis of the membrane protein lactose permease from Escherichia coli.

Phosphatidylcholine (PC) has been widely used in place of naturally occurring phosphatidylethanolamine (PE) in reconstitution of bacterial membrane proteins. However, PC does not support native structure or function for several reconstituted transport proteins. Lactose permease (LacY) of Escherichia coli, when reconstituted in E. coli phospholipids, exhibits energy-dependent uphill and energy-independent downhill transport function and proper conformation of periplasmic domain P7, which is tightly linked to uphill transport function. LacY expressed in cells lacking PE and containing only anionic phospholipids exhibits only downhill transport and lacks native P7 conformation. Reconstitution of LacY in the presence of E. coli-derived PE, but not dioleoyl-PC, results in uphill transport. We now show that LacY exhibits uphill transport and native conformation of P7 when expressed in a mutant of E. coli in which PC completely replaces PE even though the structure is not completely native. E. coli-derived PC and synthetic PC species containing at least one saturated fatty acid also support the native conformation of P7 dependent on the presence of anionic phospholipids. Our results demonstrate that the different effects of PE and PC species on LacY structure and function cannot be explained by differences in the direct interaction of the lipid head groups with specific amino acid residues alone but are due to more complex effects of the physical and chemical properties of the lipid environment on protein structure. This conclusion is supported by the effect of different lipids on the proper folding of domain P7, which indirectly influences uphill transport function.

Long range physical map of the Wilms' tumor - aniridia region on human chromosome 11

Nephroblastoma or Wilms' tumor is a pediatric renal malignancy that is the most frequently occurring childhood solid tumor. Approximately 1-2% of children with Wilms' tumor also present with aniridia, a congenital absence of all or part of the iris of the eye. These children also have high rates of genitourinary anomalies and mental retardation resulting in what is called the WAGR (Wilms' tumor, aniridia, genitourinary anomaly, mental retardation) syndrome. Cytogenetic analysis of metaphase chromosomes from these patients revealed a consistent deletion of band P13 on chromosome 11. These observations suggest close physical linkage between the disease-related loci, and further imply that development of each phenotype results from the loss of normal gene function.^ The objective of this work is to understand the molecular events at chromosome band 11p13 that are essential to the development of sporadic Wilms' tumor and sporadic aniridia. Two human/hamster somatic cell hybrids have been used to identify sixteen independent DNA probes that map to this segment of the human genome. These newly identified DNA probes and four previously reported probes (CAT, FSHB, D11S16, and HBVIS) have been used to subdivide 11p13 into five intervals defined by overlapping constitutional deletions from several WAGR patients. A long-range physical map of 11p13 has been constructed using each of these probes in Southern blot analysis of genomic DNA after digestion with infrequently cutting restriction enzymes and pulse-field gel electrophoresis. This map, established primarily with MluI and NotI, spans approximately 13 $\times$ 10$\sp{6}$ bp and encompasses deletion and translocation breakpoints associated with genitourinary anomalies, aniridia, and sporadic Wilms' tumor. This complete physical map of human chromosome band 11p13 enables us to localize the genes for sporadic Wilms' tumor and sporadic aniridia to a small number of specific NotI fragments. ^

Insulin effects and receptor function in bovine aorta endothelial cells

The interaction of insulin with bovine aorta endothelial (BAE) cells has been studied to determine the effect of insulin on endothelial cells, and investigate the function of the insulin receptor in this cell type. BAE cell insulin receptor is similiar to insulin receptor in other cell types in the time to attain equilibrium binding, its physical properties in a solubilized assay system and affinity for insulin in the low nanomolar range. However, BAE cell insulin receptor has unusual properties in its interaction with insulin at 4$\sp\circ$C that include: (1) the inability to completely dissociate prebound $\sp{125}$I-insulin by dilution with excess insulin or acid rinse treatment, indicating that binding is not completely reversible (2) the inability to remove prebound insulin with trypsin and other proteases (3) the implication of disulfide complex formation during binding (4) the inability of pretreatment with trypsin to lower cell surface binding capacity and (5) the suppression of insulin binding by bacitracin. Interactions of insulin with the receptor at 37$\sp\circ$C showed that (1) BAE cells degrade insulin, but not as extensively as other cell types, and (2) an unusual biphasic interaction of insulin with the BAE cells is observed which is indicative of some regulatory mechanism which modulates binding affinity. Functional characterization of the BAE cell insulin receptor revealed that insulin-induced downregulation and phosphorylation of the receptor was observed, and the extent of these processes were comparable to that demonstrated in non-endothelial cell types. However, in contrast to other cell types, insulin did not stimulate deoxyglucose uptake in BAE cells. We were unable to confirm the receptor-mediated transport of insulin by the receptor across the endothelial cell monolayer as reported by a previous investigator. We could not demonstrate a role for the receptor to promote acute intracellular accumulation of insulin as postulated by several investigators. Thus, while BAE cell insulin receptor has many properties that are similiar to those in other cell types, it is distinctly different in its nondissociable binding at 4$\sp\circ$C, its interaction with insulin at 37$\sp\circ$C, and its functional role in the BAE cell. ^

STUDY OF THE EFFECTS OF TOTAL MODULATION TRANSFER FUNCTION CHANGES ON OBSERVER PERFORMANCE USING CLINICAL MAMMOGRAMS

The main goal of this study was to relate physical changes in image quality measured by Modulation Transfer Function (MTF) to diagnostic accuracy.^ One Hundred and Fifty Kodak Min-R screen/film combination conventional craniocaudal mammograms obtained with the Pfizer Microfocus Mammographic system were selected from the files of the Department of Radiology, at M.D. Anderson Hospital and Tumor Institute.^ The mammograms included 88 cases with a variety of benign diagnosis and 62 cases with a variety of malignant biopsy diagnosis. The average age of the patient population was 55 years old. 70 cases presented calcifications with 30 cases having calcifications smaller than 0.5mm. 46 cases presented irregular bordered masses larger than 1 cm. 30 cases presented smooth bordered masses with 20 larger than 1 cm.^ Four separated copies of the original images were made each having a different change in the MTF using a defocusing technique whereby copies of the original were obtained by light exposure through different thicknesses (spacing) of transparent film base.^ The mammograms were randomized, and evaluated by three experienced mammographers for the degree of visibility of various anatomical breast structures and pathological lesions (masses and calicifications), subjective image quality, and mammographic interpretation.^ 3,000 separate evaluations were anayzed by several statistical techniques including Receiver Operating Characteristic curve analysis, McNemar test for differences between proportions and the Landis et al. method of agreement weighted kappa for ordinal categorical data.^ Results from the statistical analysis show: (1) There were no statistical significant differences in the diagnostic accuracy of the observers when diagnosing from mammograms with the same MTF. (2) There were no statistically significant differences in diagnostic accuracy for each observer when diagnosing from mammograms with the different MTF's used in the study. (3) There statistical significant differences in detail visibility between the copies and the originals. Detail visibility was better in the originals. (4) Feature interpretations were not significantly different between the originals and the copies. (5) Perception of image quality did not affect image interpretation.^ Continuation and improvement of this research ca be accomplished by: using a case population more sensitive to MTF changes, i.e., asymptomatic women with minimum breast cancer, more observers (including less experienced radiologists and experienced technologists) must collaborate in the study, and using a minimum of 200 benign and 200 malignant cases.^

Measurement and correlates of physical activity behavior among older minority women

Research has shown that physical activity serves a preventive function against the development of several major chronic diseases. However, studying physical activity and its health benefits is difficult due to the complexity of measuring physical activity. The overall aim of this research is to contribute to the knowledge of both correlates and measurement of physical activity. Data from the Women On The Move study were used for this study (n = 260), and the results are presented in three papers. The first paper focuses on the measurement of physical activity and compares an alternate coding method with the standard coding method for calculating energy expenditure from a 7-day activity diary. Results indicate that the alternative coding scheme could produce similar results to the standard coding in terms of total activity expenditure. Even though agreement could not be achieved by dimension, the study lays the groundwork for a coding system that saves considerable amount of time in coding activity and has the ability to estimate expenditure more accurately for activities that can be performed at varying intensity levels. The second paper investigates intra-day variability in physical activity by estimating the variation in energy expenditure for workers and non-workers and identifying the number of days of diary self-report necessary to reliably estimate activity. The results indicate that 8 days of activity are needed to reliably estimate total activity for individuals who don't work and 12 days of activity are needed to reliably estimate total activity for those who work. Days of diary self-report required by dimension for those who don't work range from 6 to 16 and for those who work from 6 to 113. The final paper presents findings on the relationship between daily living activity and Type A behavior pattern. Significant findings are observed for total activity and leisure activity with the Temperament Scale summary score. Significant findings are also observed for total activity, household chores, work, leisure activity, exercise, and inactivity with one or more of the individual items on the Temperament Scale. However, even though some significant findings were observed, the overall models did not reveal meaningful associations. ^

Functional and physical associations of beta1 integrins in the activation of human T lymphocytes

Adhesion involves interactions between cells or cells with extracellular matrix components and is a fundamental process for all multicellular organisms as well as many pathogenic microbes. Integrins are heterodimeric transmembrane proteins that function as adhesion molecules and transduce signals between the extracellular environment and the intracellular cytoskeletal machinery. β1 integrin subfamily is highly expressed on T lymphocytes and mediates cell spreading, adhesion and coactivation. T lymphocytes have an important role in the regulation and homeostasis of the immune system therefore, the goals of this study were to first to investigate β1 integrin interaction with fibronectin binding protein A (FnbpA), a surface protein expressed on gram-negative bacteria Staphylococcus aureus. Second, characterize the association and function of a non-integrin surface protein, CD98, with β1 integrins on T lymphocytes. ^ FnbpA binds to fibronectin (FN), also a ligand for α5β1 and α4β1 integrins on T lymphocytes. Since both bacterial proteins FnbpA and T cell integrins utilize FN, it was of interest to determine the effects FnbpA on T cell activation. Results demonstrated that recombinant FnbpA (rFnbpA) coimmobilized with OKT3 mediated T cell coactivation in a soluble FN-dependent manner. Integrin α5β1 was identified as the main integrin utilized by Staphylococcus aureus FnbpA from studies using soluble antibodies to inhibit T cell proliferation and parallel plate flow chamber assays. The mechanism of rFnbpA-mediated coactivation was one that used soluble FN as a bridge between rFnbpA and integrin α5β1 on the T lymphocyte. ^ Since integrins are utilized by T lymphocytes and bacterial proteins, it was of interest to identify proteins involved in integrin regulation. Anti-CD98 mAb 80A10 was identified and characterized from a screen to identify surface proteins involved in integrin signaling and functions. CD98 is a non-integrin protein that was sensitive to integrin inhibition in human T lymphocyte aggregation and activation, thus suggested that CD98 shared a common signaling pathway with integrins. These results led to the question of whether CD98 physically associates with β1 integrins. Fluorescence microscopy and biochemical analysis determined that CD98 is specifically associated with β1 integrin on human T lymphocytes and may be part of a larger multimolecular signaling complex. ^

Lipid-dependent topogenesis and function of membrane proteins

Membranes are essential for the integrity and function of the cell. The collective property of the lipid bilayer is critical in providing an optimal functioning environment for membrane proteins. The simple yet well-characterized bacterium Escherichia coli serves an ideal model system to study the function of specific lipids since its lipid content can be easily manipulated. The most abundant lipid in E. coli membrane is phosphatidylethanolamine (PE, 70-80%). A PE-lacking E. coli mutant displays a complex mixture of deficient phenotypes, suggesting a profound role for PE in different aspects of cell function. A novel role of PE as a topological and functional determinant for membrane proteins has been established using lactose permease (LacY) as a model protein. PE is found to be required for energy-dependent uphill transport process of LacY. In PE-lacking membranes, LacY undergoes a dramatic conformational change, and the first half of the protein adopts an inverted topology with respect to the bilayer plane. ^ The work reported here was initiated to understand the molecular properties of lipids that enable their function as topological and functional determinants for membrane proteins. A glycolipid, monoglucosyldiacylglycerol (MGlcDAG) which shares physicochemical similarities with PE, was introduced to PE-lacking E. coli membranes. The introduction of MGlcDAG suppresses many of the PE-deficient phenotypes, and in particular supports the function and native topology of LacY. ^ The lipid-sensitive topogenic signals encoded in the amino acid sequence of LacY were also identified. Native LacY adopts an inverted topology when synthesized without PE, but mutation of specific acidic residues in the cytoplasmic extra-membrane domains can prevent this inversion and supports a native topological organization of LacY in PE-lacking membranes. These results suggest that it is the interplay between the collective charge properties of the lipid bilayer and extra-membrane loops of protein that determines the final orientation of transmembrane domains. By comparing the similarities as well as differences between these two lipids, we established how specific physical and chemical properties of lipids influence various cell functions and elucidated the molecular basis for the novel role of lipids in determining membrane protein topology. ^

Ambulatory blood pressure and physical activity in heart failure

Background. Ambulatory blood pressure (ABP) measurement is a means of monitoring cardiac function in a noninvasive way, but little is known about ABP in heart failure (HF) patients. Blood pressure (BP) declines during sleep as protection from consistent BP load, a phenomenon termed "dipping." The aims of this study were (1) to compare BP dipping and physical activity between two groups of HF patients with different functional statuses and (2) to determine whether the strength of the association between ambulatory BP and PA is different between these two different functional statuses of HF. ^ Methods. This observational study used repeated measures of ABP and PA over a 24-hour period to investigate the profiles of BP and PA in community-based individuals with HF. ABP was measured every 30 minutes by using a SpaceLabs 90207, and a Basic Motionlogger actigraph was used to measure PA minute by minute. Fifty-six participants completed both BP and physical activity for a 24-hour monitoring period. Functional status was based on New York Heart Association (NYHA) ratings. There were 27 patients with no limitation of PA (NYHA class I HF) and 29 with some limitation of PA but no discomfort at rest (NYHA class II or III HF). The sample consisted of 26 men and 30 women, aged 45 to 91 years (66.96 ± 12.35). ^ Results. Patients with NYHA class I HF had significantly greater dipping percent than those with NYHA class II/III HF after controlling their left ventricular ejection fraction (LVEF). In a mixed model analysis (PROC MIXED, SAS Institute, v 9.1), PA was significantly related to ambulatory systolic and diastolic BP and mean arterial pressure. The strength of the association between PA and ABP readings was not significantly different for the two groups of patients. ^ Conclusions. These preliminary findings demonstrate differences between NYHA class I and class II/III of HF in BP dipping status and ABP but not PA. Longitudinal research is recommended to improve understanding of the influence of disease progression on changes in 24-hour physical activity and BP profiles of this patient population. ^ Key Words. Ambulatory Blood Pressure; Blood Pressure Dipping; Heart Failure; Physical Activity. ^

Joint effects of pulmonary function and aerobic power on survival in a cohort of healthy adults

Objective measurements of physical fitness and pulmonary function are related individually to long-term survival, both in healthy people and in those who are ill. These factors are furthermore known to be related to one another physiologically in people with pulmonary disease, because advanced pulmonary disease causes ventilatory limitation to exercise. Healthy people do not have ventilatory limitation to exercise, but rather have ventilatory reserve. The relationship between pulmonary function and exercise performance in healthy people is minimal. Exercise performance has been shown to modify the effect of pulmonary function on mortality in people with chronic obstructive pulmonary disease, but the relationship between these factors in healthy people has not been studied and is not known. The purpose of this study is to quantify the joint effects of pulmonary function and exercise performance as these bear on mortality in a cohort of healthy adults. This investigation is an historical cohort study over 20 years of follow-up of 29,624 adults who had complete preventive medicine, spirometry and treadmill stress examinations at the Cooper Clinic in Dallas, Texas.^ In 20 years of follow-up, there were 738 evaluable deaths. Forced expiratory volume in one second (FEV$\sb1$) percent of predicted, treadmill time in minutes percent of predicted, age, gender, body mass index, baseline smoking status, serum glucose and serum total cholesterol were all significant, independent predictors of mortality risk. There were no frank interactions, although age had an important increasing effect on the risk associated with smoking when other covariates were controlled for in a proportional-hazards model. There was no confounding effect of exercise performance on pulmonary function. In agreement with the pertinent literature on independent effects, each unit increase in FEV$\sb1$ percent predicted was associated with about eight tenths of a percent reduction in adjusted mortality rate. The concept of physiologic reserve is useful in interpretation of the findings. Since pulmonary function does not limit exercise tolerance in healthy adults, it is reasonable to expect that exercise tolerance would not modify the effect of pulmonary function on mortality. Epidemiologic techniques are useful for elucidating physiological correlates of mortality risk. ^

The Role of the Arched Helicases in Exosome-Mediated Function

RNA processing and degradation are two important functions that control gene expression and promote RNA fidelity in the cell. A major ribonuclease complex, called the exosome, is involved in both of these processes. The exosome is composed of ten essential proteins with only one catalytically active subunit, called Rrp44. While the same ten essential subunits make up both the nuclear and cytoplasmic exosome, there are nuclear and cytoplasmic exosome cofactors that promote specific exosome functions in each of the cell compartments. To date, it is unclear how the exosome distinguishes between RNA substrates. We hypothesize that compartment specific cofactors may promote the substrate specificity of the exosome. In this work, I characterize several cofactors of the exosome, both nuclear and cytoplasmic. First, I describe the arch domain, which is a unique domain in a nuclear and a cytoplasmic cofactor of the exosome. Specifically, I show that the arch domain of the nuclear exosome cofactor, Mtr4, is required for specific exosome-mediated activities and overlaps functionally with the exosome-associated exonuclease, Rrp6. Further, I show that the arch domain of Ski2 is required for the degradation of normal and aberrant mRNAs. Additionally, this work describes in detail the Mtr4 domains involved in the physical association with other RNA processing proteins. Further, I characterize the minimal Mtr4-binding region in a third exosome cofactor, Trf5. Understanding how exosome cofactors synergistically promote exosome function will provide us a better understanding of how the exosome complex precisely regulates its catalytic activities. As described here, cofactors play a major role in determining the substrate specificity of the nuclear and cytoplasmic exosome. Moreover, specific accessory domains, which are not involved in the catalytic function of the cofactor, are required for substrate targeting of the eukaryotic RNA exosome.

Evidence that lymphokine -activated killer (LAK) cell function is regulated by both serine and tyrosine kinase pathways

Signal transduction pathways operative in lymphokine activated killer (LAK) cells during execution of cytolytic function have never been characterized. Based on ubiquitous involvement of protein phosphorylation in activation of cytolytic mechanisms used by CTL and NK cells, it was hypothesized that changes in protein phosphorylation should occur when LAK encounter tumor targets. It was further hypothesized that protein kinases would regulate LAK-mediated cytotoxicity. Exposure to either SK-Mel-1 (melanoma) or Raji (lymphoma) targets consistently led to increased phosphorylation of two 65-kD LAK proteins pp65a and -b, with isoelectric points (pI) of 5.1 and 5.2 respectively. Increased p65 phosphorylation was initiated between 1 and 5 min after tumor coincubation, occurred on Ser residues, required physical contact between LAK and tumors, correlated with target recognition, and also occurred after crosslinking Fc$\gamma$RIIIA in the absence of tumors. Both pp65a and -b were tentatively identified as phosphorylated forms of the actin-bundling protein L-plastin, based on pI, molecular weight, and cross-reactivity with specific antiserum. The known biochemical properties of L-plastin suggest it may be involved in regulating adhesion of LAK to tumor targets. The protein tyrosine kinase-specific inhibitor Herb A did not block p65 phosphorylation, but blocked LAK killing of multiple tumor targets at a post-binding stage. Greater than 50% inhibition of cytotoxicity was observed after a 2.5-h pretreatment with 0.125 $\mu$g/ml Herb A. Inhibition occurred over a period in pretreatment which LAK were not dependent upon IL-2 for maintenance of killing activity, supporting the conclusion that the drug interfered with mobilization of cytotoxic function. Granule exocytosis measured by BLT-esterase release from LAK occurred after coincubation with tumors, and was inhibited by Herb A LAK cytotoxicity was dependent upon extracellular calcium, suggesting that granule exocytosis rather than Fas ligand was the principal pathway leading to target cell death. The data indicate that protein tyrosine kinases play a pivotal role in LAK cytolytic function by regulating granule exocytosis, and that tumor targets can activate an adhesion dependent Ser kinase pathway in LAK resulting in phosphorylation of L-plastin. ^

Genetic integration of a nuclear -encoded mitochondrial gene with cardiac function

Cardiovascular disease (CVD) is the leading cause of death in the United States. One manifestation of CVD known to increase mortality is an enlarged, or hypertrophic heart. Hypertrophic cardiomyocytes adapt to increased contractile demand at the genetic level with a re-emergence of the fetal gene program and a downregulation of fatty acid oxidation genes with concomitant increased reliance on glucose-based metabolism. To understand the transcriptional regulatory pathways that implement hypertrophic directives we analyzed the upstream promoter region of the muscle specific isoform of the nuclear-encoded mitochondrial gene, carnitine palmitoyltransferase-1β (CPT-1β) in cultured rat neonatal cardiac myocytes. This enzyme catalyzes the rate-limiting step of fatty acid entry into β-oxidation and is downregulated in cardiac hypertrophy and failure, making it an attractive model for the study of hypertrophic gene regulation and metabolic adaptations. We demonstrate that the muscle-enriched transcription factors GATA-4 and SRF synergistically activate CPT-1β; moreover, DNA binding to cognate sites and intact protein structure are required. This mechanism coordinates upregulation of energy generating processes with activation of the energy consuming contractile promoter for cardiac α-actin. We hypothesized that fatty acid or glucose responsive transcription factors may also regulate CPT-1β. Oleate weakly stimulates CPT-1β activity; in contrast, the glucose responsive Upstream Stimulatory Factors (USF) dramatically depresses the CPT-1β reporter. USF regulates CPT-1β through a novel physical interaction with the cofactor PGC-1 and abrogation of MEF2A/PGC-1 synergistic stimulation. In this way, USF can inversely regulate metabolic gene programs and may play a role in the shift of metabolic substrate preference seen in hypertrophy. Failing hearts have elevated expression of the nuclear hormone receptor COUP-TF. We report that COUP-TF significantly suppresses reporter transcription independent of DNA binding and specific interactions with GATA-4, Nkx2.5 or USF. In summary, CPT-1β transcriptional regulation integrates mitochondrial gene expression with two essential cardiac functions: contraction and metabolic substrate oxidation. ^