Molecular architecture of basement membranes

Basement membranes are specialized extracellular matrices with support, sieving, and cell regulatory functions. The molecular architectures of these matrices are created through specific binding interactions between unique glycoprotein and proteoglycan protomers. Type IV collagen chains, using NH2-terminal, COOH-terminal, and lateral association, form a covalently stabilized polygonal framework. Laminin, a four-armed glycoprotein, self-assembles through terminal-domain interactions to form a second polymer network, Entactin/nidogen, a dumbbell-shaped sulfated glycoprotein, binds laminin near its center and interacts with type IV collagen, bridging the two. A large heparan sulfate proteoglycan, important for charge-dependent molecular sieving, is firmly anchored in the basement membrane and can bind itself through a core-protein interaction to form dimers and oligomers and bind laminin and type IV collagen through its glycosaminoglycan chains. Heterogeneity of structure and function occur in different tissues, in development, and in response to different physiological needs. The molecular architecture of these matrices may be regulated during or after primary assembly through variations in compositions, isoform substitutions, and the modifying influence of exogenous macromolecules such as heparin and heparan sulfate.

Molecular characterization and chromosomal assignment of the bovine glycinamide ribonucleotide formyltransferase (GART) gene on cattle chromosome 1q12.1-q12.2

The mammalian glycinamide ribonucleotide formyltransferase (GART) genes encode a trifunctional polypeptide involved in the de novo purine biosynthesis. We isolated a bacterial artificial chromosome (BAC) clone containing the bovine GART gene and determined the complete DNA sequence of the BAC clone. Cloning and characterization of the bovine GART gene revealed that the bovine gene consists of 23 exons spanning approximately 27 kb. RT-PCR amplification of bovine GART in different organs showed the expression of two GART transcripts in cattle similar to human and mouse. The GART transcripts encode two proteins of 1010 and 433 amino acids, respectively. Eleven single nucleotide polymorphisms (SNPs) were detected in a mutation scan of 24 unrelated animals of three different cattle breeds, including one SNP that affects the amino acid sequence of GART. The chromosomal localization of the gene was determined by fluorescence in situ hybridization. Comparative genome analysis between cattle, human and mouse indicates that the chromosomal location of the bovine GART gene is in agreement with a previously published mapping report.

COMMUNITY-FOCUSED FUNCTIONS IN PUBLIC HEALTH NURSING: A COMPARISON OF PERCEPTIONS

This descriptive cross-sectional survey compared the perceptions of public health nursing practitioners, educators and administrators along two dimensions: the importance of community-focused functions in public health nursing and which occupational categories in public health are responsible for those functions. More than 50 percent of the mailed questionnaires that were sent to a systematic stratified nationwide sample of public health nurses were returned. In general, respondents: were female, were in their 40s, received their basic nursing education in baccalaureate programs, had either a baccalaureate or a master's degree, worked in official agencies or schools, and had approximately 14 years of experience in public health with six in their present position.^ Significant differences between practitioners, educators and administrators were found in their perceptions of both the importance of community-focused functions in public health nursing and in which occupational category they indicated as having the major responsibility to perform those functions. Educators and administrators perceived community-focused functions as more important than did practitioners. Overall the occupational category of administrator was indicated as having the major responsibility for performing community-focused functions.^

STUDIES OF THE MOLECULAR MECHANISMS OF CHROMOSOME ABERRATION FORMATION (DNA REPAIR, CROSSLINKS, MITOMYCIN C)

The objective of this research has been to study the molecular basis for chromosome aberration formation. Predicated on a variety of data, Mitomycin C (MMC)-induced DNA damage has been postulated to cause the formation of chromatid breaks (and gaps) by preventing the replication of regions of the genome prior to mitosis. The basic protocol for these experiments involved treating synchronized Hela cells in G(,1)-phase with a 1 (mu)g/ml dose of MMC for one hour. After removing the drug, cells were then allowed to progress to mitosis and were harvested for analysis by selective detachment. Utilizing the alkaline elution assay for DNA damage, evidence was obtained to support the conclusion that Hela cells can progress through S-phase into mitosis with intact DNA-DNA interstrand crosslinks. A higher level of crosslinking was observed in those cells remaining in interphase compared to those able to reach mitosis at the time of analysis. Dual radioisotope labeling experiments revealed that, at this dose, these crosslinks were associated to the same extent with both parental and newly replicated DNA. This finding was shown not to be the result of a two-step crosslink formation mechanism in which crosslink levels increase with time after drug treatment. It was also shown not to be an artefact of the double-labeling protocol. Using neutral CsCl density gradient ultracentrifugation of mitotic cells containing BrdU-labeled newly replicated DNA, control cells exhibited one major peak at a heavy/light density. However, MMC-treated cells had this same major peak at the heavy/light density, in addition to another minor peak at a density characteristic for light/light DNA. This was interpreted as indicating either: (1) that some parental DNA had not been replicated in the MMC treated sample or; (2) that a recombination repair mechanism was operational. To distinguish between these two possibilities, flow cytometric DNA fluorescence (i.e., DNA content) measurements of MMC-treated and control cells were made. These studies revealed that the mitotic cells that had been treated with MMC while in G(,1)-phase displayed a 10-20% lower DNA content than untreated control cells when measured under conditions that neutralize chromosome condensation effects (i.e., hypotonic treatment). These measurements were made under conditions in which the binding of the drug, MMC, was shown not to interfere with the stoichiometry of the ethidium bromide-mithramycin stain. At the chromosome level, differential staining techniques were used in an attempt to visualize unreplicated regions of the genome, but staining indicative of large unreplicated regions was not observed. These results are best explained by a recombinogenic mechanism. A model consistent with these results has been proposed.^

GENERAL WELL-BEING AND USE OF SOCIAL SUPPORT

Research interest on well-being and social support has focused largely on social factors as related to attaining and maintaining well-being, self-perceptions of well-being and to a lesser extent the relationship of current level of self-perceived well-being to use of formal or informal sources of social support. This study analyzed responses to the General Well-Being Schedule of 6,913 subjects (25-74 years) interviewed during the National Health and Nutrition Examination Survey (1971-1975). The purpose of this analysis was to relate the level of GWBS scores to the use of social support, both informal (family and friends) and formal (community professionals).^ Study questions addressed were whether well-being level was related to selection of a specific social support resource and/or rate of use of resources and whether gender differences were apparent in level of well-being and social support use. Because age, sex, race, socioeconomic status (income and education) and marital status may confound the relation between level of GWB and type of social support chosen, the association between these variables with GWB and use of social support were considered. For analysis, test scores were grouped into four categories and for detailed analysis, two categories: low (0-70) and high (71-110). Cross tabulations and percentages were computed and the chi-square test of significance was used.^ Although 16 to 25 percent of the sample population reported low well-being, less than 10 percent used formal resources to discuss emotional, mental or behavior problems. Medical resources, mostly physicians, were the most used formal social supports. Informal social support was important for all well-being levels where 65-77% of each category reported using this resource.^ While well-being level does not appear to serve as a screener/selector of type of formal social support used, it is related to rates of use. Females reported slightly lower well-being than males, and except in the lowest well-being group, had higher rates of social support use. Findings support the conclusion that perceived well-being is related to use of social support such that the lower the well-being, the greater tendency to use formal and/or informal social support. ^

Molecular regulatory mechanisms and embryonic tissue specification in sea urchin development

To understand how a eukaryote achieves differential transcription of genes in precise spatial patterns, the molecular details of tissue specific expression of the Strongylocentrotus purpuratus Spec2a gene were investigated by functional studies of the cis-regulatory components in the upstream enhancer. Regional activation of Spec2a in the aboral ectoderm is conferred by a combination of activators and repressors. The positive regulators include previously identified SpOtx and a trans-regulatory factor binding at the CCAAT site in the Spec2a enhancer. The nuclear protein binding to the CCAAT box was determined to be the heterotrimeric CCAAT binding factor (SpCBF). SpCBF also mediates general activation in the ectoderm. The negative regulators consist of an oral ectoderm repressor (OER), an endoderm repressor (ENR), and an S. Purpuratus goosecoid homologue (SpGsc). OER functions to prevent expression in the oral ectoderm, while ENR is required to repress endoderm expression. SpGsc antagonizes the SpOtx function by competing for binding at SpOtx target genes in oral ectoderm, where it functions as an active repressor. Thus, SpOtx and SpGsc perform collectively to establish and maintain the oral-aboral axis. Finally, purification of ENR and OER proteins from sea urchin blastula stage nuclear extracts was performed using site-specific DNA-affmity chromatography. ^

Alteration of CEACAM1 gene transcription in prostate cancer cells: The role of AR, Sp2, and HDAC

Cell to cell adhesion molecule (CEACAM1), a type II tumor suppressor, has been found to be down-regulated in prostate cancer cells. The mechanism that causes CEACAM1's down-regulation in tumorigenesis is unknown. Here we show that the transcriptional activity of CEACAM1 is decreased in prostate cancer cells. This decrease is not due to methylation of the CEACAM1's promoter, but rather to the alteration of transcription factors regulating CEACAM1 expression. ^ Since androgen/androgen receptors (AR) are potent regulators of prostate growth and differentiation, their role on CEACAM1 gene transcription was examined. The androgen receptor could directly increase CEACAM1 transcriptional activity in a ligand dependent manner by interacting with an AR consensus element that resides in the CEACAM1 promoter. However, AR binding to the CEACAM1 promoter is not related to the loss of CEACAM1 during prostate cancer progression. ^ Further analysis enabled us to determine the particular region in the CEACAM1 promoter that mediates a decrease in CEACAM1 transcriptional activity in prostate cancer cells. Upon further examination, we found that this CEACAM1 promoter region interacts with the Sp1, Sp2, and Sp3 transcription factors. However, only Sp2 expression was found to increase in prostate cancer cells. Inhibiting Sp2 from binding to the CEACAM1 promoter caused an increase in CEACAM1 transcriptional activity in prostate cancer cells. In addition, over-expressing Sp2 in normal prostate cells resulted in a decrease in CEACAM1 transcriptional activity and endogenous protein expression. These observations suggest that Sp2 is a transcription repressor of CEACAM1. Furthermore, prostate cancer cells treated with trichostatin A (TSA), a specific histone deacetylase (HDAC) inhibitor, activated CEACAM1 transcriptional activity. This implies that HDACs are involved in CEACAM1 transcriptional activity. Mutation of the Sp2 DNA binding region on the CEACAM1 promoter inhibited TSA activation of CEACAM1 transcriptional activity. This indicates that HDACs inhibit CEACAM1 transcriptional activity through Sp2. Base on these results, we propose that Sp2 is critical for down-regulating CEACAM1 expression, and one mechanism by which Sp2 represses CEACAM1 expression is by recruiting HDAC to the CEACAM1 promoter in prostate cancer cells. Collectively, these findings provide novel insights into mechanisms that cause the down-regulation of CEACAM1 expression in prostate cancer cells. ^

4HPR-X radiation mechanisms of interactions in non-small cell lung cancer (NSCLC) cells in vitro

Lung cancer is the leading cause of cancer death. However, poor survival using conventional therapies fuel the search for more rational interventions. The objective of this study was to design and implement a 4HPR-radiation interaction model in NSCLC, employing a traditional clinical modality (radiation), a relatively new, therapeutically unexplored agent (4HPR) and rationally combining them based on molecular mechanistic findings pertaining to their interactions. To test the hypothesis that 4HPR sensitizes cells to radiation-induced cell death via G2+M accumulation, we designed a working model consisting of H522 adenocarcinoma cells (p53, K-ras mutated) derived from an NSCLC patient; 4HPR at concentrations up to 10 μM; and X radiation up to 6 Gy generated by a patient-dedicated Phillips RT-250 X ray unit at 250 KV, 15 mA, 1.85 Gy/min. We found that 4HPR produced time- and dose-dependent morphological changes, growth inhibition, and DNA damage-inducing enhancement of reactive oxygen species. A transient G2+M accumulation of cells maximal at 24 h of continuous 4HPR exposure was used for irradiation time scheduling. Our data demonstrated enhanced cell death (both apoptotic and necrotic) in irradiated cells pre-treated with 4HPR versus those with either stressor alone. 4HPR's effect of increased NSCLC cells' radioresponse was confirmed by clonogenic assay. To explore these practical findings from a molecular mechanistic perspective, we further investigated and showed that levels of cyclin B1 and p34cdc2 kinase—both components of the mitosis promoting factor (MPF) regulating the G2/M transition—did not change following 4HPR treatment. Likewise, cdc25C phosphatase was not altered. However, enhanced p34cdc2 phosphorylation on its Thr14Tyr15 residues—indicative of its inactivation and increased expression of MPF negative regulators chk1 and wee1 kinases—were supportive of explaining 4HPR-treated cells' accumulation. Hence, p34cdc2 phosphorylation, chk1, and wee1 warrant further evaluation as potential molecular targets for 4HPR-X radiation combination. In summary, we (1) demonstrated that 4HPR not only induces cell death by itself, but also increases NSCLC cells' subsequent radioresponse, indicative of potential clinical applicability, and (2) for the first time, shed light on deciphering 4HPR-X radiation molecular mechanisms of interaction, including the finding of 4HPR's role as a p34cdc2 inactivator via Thr14Tyr15 phosphorylation. ^

Community preferences for cancer pain health states

The study's objective was to assess the reliability, acceptability, and concordance of cancer pain health states when using two utility assessment methods—simple rank order (RO) and numerical analogue scale (NAS). Additional aims were to describe the preferences of Hispanic and non-Hispanic community members toward cancer pain health states and identify predictors affecting these preferences. In this descriptive, cross-sectional study, telephone calls were made to a quota sample of 1,387 households that had telephone numbers listed for the Houston and surrounding Harris County area. Subjects (n = 302) within the general population completed a 20 minute telephone interview in their preferred language—English or Spanish. Study respondents assessed six cancer pain health states consisting of three attributes, pain intensity, presence of side effects, and interference with daily function. ^ Overall, the numerical analogue scale (NAS) had better test-retest reliability. Respondents were able to clearly distinguish the worst health state using both methods, but were not able to do so as clearly for less severe health states. Acceptability and subjects' ability to answer questions and complete the survey was high. Missing responses were low across methods for all health states. Concordance in the health state rankings was higher for the most severe health state in the non-Hispanic group, those in fair to poor health, males, and those $30,000 or greater income. Preferences for the less severe health states did not show much variation across methods. No significant predictors for health states were found except for ethnicity for a less severe health state when using the rank order method. ^ We found that the rank order (RO) and numerical analogue scale (NAS) are both robust in ranking the more severe cancer pain health states, e.g., moderate pain with three side effects. This study documents that RO and NAS methods to assess cancer pain preferences through a telephone-based approach among a relative diverse community dwelling, non-patient population for cancer pain health states represented a relatively valid and acceptable approach. ^

The dynamics of calmodulin signaling revealed using optical methods

The Ca2+-binding protein calmodulin (CaM) is a key transducer of Ca2+ oscillations by virtue of its ability to bind Ca 2+ selectively and then interact specifically with a large number of downstream enzymes and proteins. It remains unclear whether Ca2+ -dependent signaling alone can activate the full range of Ca 2+/CaM regulated processes or whether other regulatory schemes in the cell exist that allow specific targeting of CaM to subsets of Ca 2+/CaM binding sites or regions of the cell. Here we investigate the possibility that alterations of the availability of CaM may serve as a potential cellular mechanism for regulating the activation of CaM-dependent targets. By utilizing sensitive optical techniques with high spatial and temporal resolution, we examine the intracellular dynamics of CaM signaling at a resolution previously unattainable. After optimizing and characterizing both the optical methods and fluorescently labeled probes for intracellular measurements, the diffusion of CaM in the cytoplasm of HEK293 cells was analyzed. It was discovered that the diffusion characteristics of CaM are similar to that of a comparably sized inert molecule. Independent manipulation of experimental parameters, including increases in total concentrations of CaM and intracellular Ca2+ levels, did not change the diffusion of CaM in the cytoplasm. However, changes in diffusion were seen when the concentration of Ca2+/CaM-binding targets was increased in conjunction with elevated Ca2+. This indicates that CaM is not normally limiting for the activation of Ca 2+/CaM-dependent enzymes in HEK293 cells but reveals that the ratio of CaM to CaM-dependent targets is a potential mechanism for changing CaM availability. Next we considered whether cellular compartmentalization may act to regulate concentrations of available Ca2+/CaM in hippocampal neurons. We discovered changes in diffusion parameters of CaM under elevated Ca2+ conditions in the soma, neurite and nucleus which suggest that either the composition of cytoplasm is different in these compartments and/or they are composed of unique families of CaM-binding proteins. Finally, we return to the HEK293 cell and for the first time directly show the intracellular binding of CaM and CaMKII, an important target for CaM critical for neuronal function and plasticity. Furthermore, we analyzed the complex binding stoichiometry of this molecular interaction in the basal, activated and autophosphorylated states of CaMKII and determined the impact of this binding on CaM availability in the cell. Overall these results demonstrate that regulation of CaM availability is a viable cellular mechanism for regulating the output of CaM-dependent processes and that this process is tuned to the specific functional needs of a particular cell type and subcellular compartment. ^

RNA polymerase II large subunit degradation induced by ecteinascidin 743: Molecular characterization and subsequent rational investigation of antitumor mechanisms in cancers with clinical response

Ecteinascidin 743 (Et-743), which is a novel DNA minor groove alkylator with a unique spectrum of antitumor activity, is currently being evaluated in phase II/III clinical trials. Although the precise molecular mechanisms responsible for the observed antitumor activity are poorly understood, recent data suggests that post-translational modifications of RNA polymerase II Large Subunit (RNAPII LS) may play a central role in the cellular response to this promising anticancer agent. The stalling of an actively transcribing RNAPII LS at Et-743-DNA adducts is the initial cellular signal for transcription-coupled nucleotide excision repair (TC-NER). In this manner, Et-743 poisons TC-NER and produces DNA single strand breaks. Et-743 also inhibits the transcription and RNAPII LS-mediated expression of selected genes. Because the poisoning of TC-NER and transcription inhibition are critical components of the molecular response to Et-743 treatment, we have investigated if changes in RNAPII LS contribute to the disruption of these two cellular pathways. In addition, we have studied changes in RNAPII LS in two tumors for which clinical responses were reported in phase I/II clinical trials: renal cell carcinoma and Ewing's sarcoma. Our results demonstrate that Et-743 induces degradation of the RNAPII LS that is dependent on active transcription, a functional 26S proteasome, and requires functional TC-NER, but not global genome repair. Additionally, we have provided the first experimental data indicating that degradation of RNAPII LS might lead to the inhibition of activated gene transcription. A set of studies performed in isogenic renal carcinoma cells deficient in von Hippel-Lindau protein, which is a ubiquitin-E3-ligase for RNAPII LS, confirmed the central role of RNAPII LS degradation in the sensitivity to Et-743. Finally, we have shown that RNAPII LS is also degraded in Ewing's sarcoma tumors following Et-743 treatment and provide data to suggest that this event plays a role in decreased expression of the Ewing's sarcoma oncoprotein, EWS-Fli1. Altogether, these data implicate degradation of RNAPII LS as a critical event following Et-743 exposure and suggest that the clinical activity observed in renal carcinoma and Ewing's sarcoma may be mediated by disruption of molecular pathways requiring a fully functional RNAPII LS. ^

Zoning and the built environment: A public health analysis of zoning in Austin, Texas

The built environment is part of the physical environment made by people and for people. Because the built environment is such a ubiquitous component of the environment, it acts as an important pathway in determining health outcomes. Zoning, a type of urban planning policy, is one of the most important mechanisms connecting the built environment to public health. This policy analysis research paper explores how zoning regulations in Austin, Texas promote or prohibit the development of a healthy built environment. A systematic literature review was obtained from Active Living Research, which contained literature published about the relationships between the built environment, physical activity, and health. The results of these studies identified the following four components of the built environment that were associated to health: access to recreational facilities, sprawl and residential density, land use mix, and sidewalks and their walkability. A hierarchy analysis was then performed to demonstrate the association between these aspects of the built environment and health outcomes such as obesity, cardiovascular disease, and general health. Once these associations had been established, the components of the built environment were adapted into the evaluation criteria used to conduct a public health analysis of Austin's zoning ordinance. A total of eighty-eight regulations were identified to be related to these components and their varying associations to human health. Eight regulations were projected to have a negative association to health, three would have both a positive and negative association simultaneously, and nine were indeterminable with the information obtained through the literature review. The remaining sixty-eight regulations were projected to be associated in a beneficial manner to human health. Therefore, it was concluded that Austin's zoning ordinance would have an overwhelmingly positive impact on the public's health based on identified associations between the built environment and health outcomes.^

The construction of aging and health in older African American women

Persistence of racial and ethnic health disparities and governmental policies based on outdated ideas of aging call for inclusive approaches to the study of elder African Americans. The lived experiences of aging among urban, poor African American women, who comprise a vulnerable population, are not well known, as most studies focus on mainstream populations. Gerontological studies have tended to employ methods that collapse contextual information for ease of analysis, thus failing to capture nuanced information critically relevant to health of marginalized groups. Few researchers have been successful highlighting the importance of local knowledge, resilience, and resources for health by using participatory methods with older Black women. This study utilizes participatory principles to gather discursive data from nine older African American women, engaged in three generational cohorts: those born around World War II, women born after the great depression, and those born before the great depression. Videotaped and transcribed conversations of cohorts were analyzed in search of contextual factors that influence their experience of aging and health. As women responded to general themes that provoked their talk about their lives, they helped answer the study's questions: How do older African American women make sense of their aging experience? What are some of the important social and cultural influences that shape the construction of aging and health by these women? Are generational discourse groups an effective tool for exploring changes in the experiences of aging? A key finding demonstrated rich heterogeneity of experiences with strong generational influences on the construction of aging and health. The participants' moral orders comprised of traditional values of family, reinforced by personal experiences and the church, have guided their lives through oppression and stress but appear to be failing younger women who have greater exposure to new environmental pressures. Limited time and the size of the study were weaknesses although the women's interest in the study and their participation were gratifying. The participants served to highlight the importance of recognizing generational and other contextual factors in formation of ideas of aging and likelihood of additional challenges to the experience of old age among older, poorer, African Americans. ^

Molecular interactions and signal transfer between sensory rhodopsin-I and its cognate transducer

SRI is unique among known photoreceptors in that it produces opposite signals depending on the color of light stimuli. Absorption of orange light (587 nm) triggers an attractant response by the cell, whereas absorption of orange light followed by near-UV light (373 run) triggers a repellent response. Using behavioral mutants that exhibit aberrant color-sensing ability, we tested a two-conformation equilibrium model, using FRET and EPR spectroscopy. The essence of the model applied to SRI-HtrI is that the complex exists in a metastable two-conformer equilibrium which is shifted in one direction by orange light absorption (producing an attractant signal) and in the opposite direction by a second UV-violet photon (producing a repellent signal). First, by FRET we found that the E-F cytoplasmic loop of SRI moves toward the RAMP domain of the HtrI transducer during the formation of the orange-light activated signaling state of the complex. This is the first localization of a change in the physical relationship between the receptor and transducer subunits of the complex and provides a structural property of the two proposed conformers that we can monitor. Second, EPR spectra of a spin label probe at this cytoplasmic position showed shifts in the dark in the mutants toward shorter or longer EF loop-RAMP distances, explaining their behavior in terms of their mutations causing pre-stimulus shifts into one or the other conformer. ^ Next, we applied a novel electrophysiological method for monitoring the directionality of proton movement during photoactivation of SRI, to investigate the process of proton transfer in the photoactive site from the chromophore to proton acceptors on both the wildtype and aberrant color-response mutants. We observed an unexpected and critical difference in the two signaling conformations of the SRI-HtrI complex. The finding is that the vectoriality (i.e. movement away or toward the cytoplasm) of the light-induced proton transfer from the chromophore to the protein is opposite in formation of the two conformations. Retinylidene proton transfer is a common critical process in rhodopsins and these results are the first to show differences in vectoriality in a rhodopsin receptor, and to demonstrate functional importance of the direction of proton transfer. ^