Tp53-dependent repression of cell cycle target genes: Global and mechanistic analysis

Most studies of p53 function have focused on genes transactivated by p53. It is less widely appreciated that p53 can repress target genes to affect a particular cellular response. There is evidence that repression is important for p53-induced apoptosis and cell cycle arrest. It is less clear if repression is important for other p53 functions. A comprehensive knowledge of the genes repressed by p53 and the cellular processes they affect is currently lacking. We used an expression profiling strategy to identify p53-responsive genes following adenoviral p53 gene transfer (Ad-p53) in PC3 prostate cancer cells. A total of 111 genes represented on the Affymetrix U133A microarray were repressed more than two fold (p ≤ 0.05) by p53. An objective assessment of array data quality was carried out using RT-PCR of 20 randomly selected genes. We estimate a confirmation rate of >95.5% for the complete data set. Functional over-representation analysis was used to identify cellular processes potentially affected by p53-mediated repression. Cell cycle regulatory genes exhibited significant enrichment (p ≤ 5E-28) within the repressed targets. Several of these genes are repressed in a p53-dependent manner following DNA damage, but preceding cell cycle arrest. These findings identify novel p53-repressed targets and indicate that p53-induced cell cycle arrest is a function of not only the transactivation of cell cycle inhibitors (e.g., p21), but also the repression of targets that act at each phase of the cell cycle. The mechanism of repression of this set of p53 targets was investigated. Most of the repressed genes identified here do not harbor consensus p53 DNA binding sites but do contain binding sites for E2F transcription factors. We demonstrate a role for E2F/RB repressor complexes in our system. Importantly, p53 is found at the promoter of CDC25A. CDC25A protein is rapidly degraded in response to DNA damage. Our group has demonstrated for the first time that CDC25A is also repressed at the transcript level by p53. This work has important implications for understanding the DNA damage cell cycle checkpoint response and the link between E2F/RB complexes and p53 in the repression of target genes. ^

Caenorhabditis elegans germinal center kinase (Gck-1) is a p-body component that inhibits precocious ectopic MAP kinase dependent meiotic maturation

The Caenorhabditis elegans germline is an excellent model system for studying meiosis, as the gonad contains germ cells in all stages of meiosis I prophase in a linear temporal and spatial pattern. To form healthy gametes, many events must be coordinated. Failure of any step in the process can reduce fertility. Here, we describe a C. elegans Germinal Center Kinase, GCK-1, that is essential for the accurate progression of germ cells through meiosis I prophase. In the absence of GCK-1, germ cells undergo precocious maturation due to the activation of a specific MAP kinase isoform. Furthermore, GCK-1 localizes to P-bodies, RNP particles that have been implicated in RNA degradation and translational control. Like two other components of C. elegans germline P-bodies, GCK-1 functions to limit physiological germ cell apoptosis. This is the first study to identify a role for a GCK-III kinase in metazoan germ cell development and to link P-body function with MAP kinase activation and germ cell maturation. ^

Blood-oxygen-level-dependent (BOLD) signal changes in total cortex and subcortex, pain, reward, and vigilance regions during mechanical pressure pain after morphine administration

Morphine is the most common clinical choice in the management of severe pain. Although the molecular mechanisms of morphine have already been characterized, the cerebral circuits by which it attenuates the sensation of pain have not yet been studied in humans. The objective of this two-arm (morphine versus placebo), between-subjects study was to examine whether morphine affects pain via pain-related cortical circuits, but also via reward regions that relate to the motivational state, as well as prefrontal regions that relate to vigilance as a result of morphine's sedative effects. Cortical activity was measured by the blood-oxygen-level-dependent (BOLD) signal changes using functional magnetic resonance imaging (fMRI). ^ The novelty of this study is at three levels: (i) to develop a methodology that will assess the average BOLD signal across subjects for the pain, reward, and vigilance cortical systems; (ii) to examine whether the reward and/or sedative effects of morphine are contributing factors to cortical regions associated with the motivational state and vigilance; and (iii) to propose a neuroanatomical model related to the opioid-sensitive effects of reward and sedation as a function of cortical activity related to pain in an effort to assess future analgesics. ^ Consistent with our hypotheses, our findings showed that the decrease in total pain-related volume activated between the post- and the pre-treatment morphine group was about 78%, while the post-treatment placebo group displayed only a 5% decrease when compared to pre-treatment levels of activation. The volume increase in reward regions was 451% in the post-treatment compared to the pre-treatment morphine condition. Finally, the volumetric decrease in vigilance regions was 63% in the posttreatment compared to the pre-treatment morphine condition. ^ These findings imply that changes in the blood flow of the reward and vigilance regions may be contributing factors in producing the analgesic effect under morphine administration. Future studies need to replicate this study in a higher resolution fMRI environment and to assess the proposed neuroanatomical model in patient populations. The necessity of pain research is apparent, since pain cuts across different diseases especially chronic ones, and thus, is recognized as a vital public health developing area. ^

The differential staurosporine mediated G1 arrest in normal versus tumor cells is dependent on the retinoblastoma and Chk1 proteins

The ability to regulate cell cycle progression is one of the differences that separates normal from tumor cells. A protein, which is frequently mutated or deleted in a majority of tumor cells, is the retinoblastoma protein (pRb). Previously, we reported that normal cells, which have a wild-type Rb pathway, can be reversibly arrested in the G1 phase of the cell cycle by staurosporine (ST), while tumor cells were unaffected by this treatment. As a result, ST may be used to protect normal cells against the toxic affects of chemotherapy. Here we set out to determine the mechanism(s) by which ST can mediate a reversible G1 arrest in pRb positive cells. To this end, we used an isogenic cell model system of normal human mammary epithelial cells (HMEC) with either intact pRb+ (p53-) or p53+ (pRb-) treated with ST. Our results show that pRb+ cells treated with low concentrations of ST, arrested in the G1 phase of the cell cycle; however, in pRb - cells there was no response. This was verified as a true G 1 arrest in pRb+ cells by two different methods for monitoring cell cycle kinetics and in two additional model systems for Rb (i.e. pRb -/- mouse embryo fibroblasts, and downregulation of RB with siRNA). Our results indicated that ST-mediated G1 arrest required pRb, which in turn initiated a cascade of events leading to inhibition of CDK4 and CDK2 activities and up-regulation of p21 protein. Further assessment of this pathway revealed the novel finding that Chk1 expression and activity were required for the Rb-dependent, ST-mediated G1 arrest. In fact, overexpression of Chk1 facilitated recovery from ST-mediated G1 arrest, an effect only observed in RB+ cells. Collectively, our data suggest pRb is able to cooperate with Chk1 to mediate a G1 arrest in pRb+ cells, but not in pRb- cells. The elucidation of this pathway can help identify novel agents that can be used to protect cancer patients against the debilitating affects of chemotherapy, by targeting only the normal proliferating cells in the body that are otherwise destroyed. ^

Reactive oxygen species-dependent mechanisms of N-(4-hydroxyphenyl)retinamide (4HPR)-induced apoptosis in human carcinoma cells

4HPR is a synthetic retinoid that has shown chemopreventive and therapeutic efficacy against premalignant and malignant lesions including oral leukoplakia, ovarian and breast cancer, and neuroblastoma. 4HPR induces apoptosis in various cancer cells and production of reactive oxygen species (ROS) has been suggested as a possible cause underlying these effects. However, the mechanisms governing these effects by 4HPR are not fully elucidated. In this study, we explored the mechanisms of 4HPR-induced ROS increase and apoptosis in human cancer cells. ^ First, we identified genes modulated by 4HPR using oligonucleotide gene expression arrays and found that they fall into specific functional canonical pathways and gene networks using Ingenuity Pathways Analysis®. Further analysis has shown that 4HPR induced up-regulation of Endoplasmic Reticulum (ER)-related genes such as Heat shock proteins 70 and 90 and the transcriptional factor, GADD153. These findings were validated using quantitative real-time PCR. ^ Second, we found that 4HPR induced extensive ER stress evidenced by dilation of the ER and endoribonuclease-mediated splicing and activation of the transcriptional factor, XBP-1. In addition, 4HPR induced the up-regulation of various ER stress-related genes and their protein products, as well as cleavage and activation of the ER specific Caspase-4. Concomitantly with XBP-1 splicing, all of these effects were dependent on ROS generation by 4HPR. Furthermore, chemical inhibition and RNA interference studies revealed a novel pro-apoptotic role for HSP70/A1A in 4HPR-mediated apoptosis. ^ Third, we observed rapid activation of the small GTPase Rac by 4HPR which was upstream of ROS generation. Inhibition of Rac activity or silencing of its expression by RNA interference inhibited ROS generation and apoptosis induction by 4HPR. siRNA targeting PAK1 and expression of a dominant negative Rac, decreased 4HPR-mediated ROS generation, while expression of a constitutive active Rac increased basal and 4HPR-induced ROS generation and PARP cleavage. Furthermore, metastatic cancer cells exhibited higher Rac activation, ROS generation, and cell growth inhibition due to 4HPR exposure compared to their primary cancer cell counterparts. ^ These findings provide novel insights into 4HPR-mediated ROS generation and apoptosis induction and support the use of ROS inducing agents such as 4HPR against metastatic cancer cells. ^

The connection between E2F and ATM in p53-dependent apoptosis

Programmed cell death is an anticancer mechanism utilized by p53 that when disrupted can accelerate tumor development in response to oncogenic stress. Defects in the RB tumor suppressor cause aberrant cell proliferation as well as apoptosis. The combinatorial loss of the p53 and RB pathways is observed in a large percentage of human tumors. The E2F family of transcription factors primarily mediates the phenotype of Rb loss, since RB is a negative regulator of E2F. Contrary to early expectations, it has now been shown that the ARF (alternative reading frame) tumor suppressor is not required for p53-dependent apoptosis in response to deregulation of the RB/E2F pathway. In this study, we demonstrate that ATM, known as a DNA double-strand break (DSB) sensor, is responsible for ARF-independent apoptosis and p53 activation induced by deregulated E2F1. Moreover, NBS1, a component of the MRN DNA repair complex, is also required for E2F1-induced apoptosis and apparently works in the same pathway as ATM. We further found that endogenous E2F1 and E2F3 both play a role in apoptosis and ATM activation in response to inhibition of RB by the adenoviral E1A oncoprotein. We demonstrate that, unlike deregulated E2F3 and Myc, ATM activation by deregulated E2F1 does not involve the induction of DNA damage, autophosphorylation of ATM on Ser 1981, a marker of ATM activation by DSB, but does depend on the presence of NBS1, suggesting that E2F1 activates ATM in a different manner from E2F3 and Myc. Results from domain mapping studies show that the DNA binding, dimerization, and marked box domains of E2F1 are required to activate ATM and stimulate apoptosis but the transactivation domain is not. This implies that E2F1's DNA binding and interaction with other proteins through the marked box domain are necessary to induce ATM activation leading to apoptosis but transcriptional activation by E2F1 is dispensable. Together these data suggest a model in which E2F1 activates ATM to phosphorylate p53 through a novel mechanism that is independent of DNA damage and transcriptional activation by E2F1.^

A systematic review of community-based participatory research interventions in pediatric and adolescent populations

Background. Community-based participatory research (CBPR) is a collaborative approach to research actively involving community members in all aspects of the research process. CBPR is not a new research method, but an approach that has gained increased attention in the field of public health over the last several years. Recognition of the inequalities in health status associated with social and environmental factors have led to calls for a renewed focus on ecological approaches to research. Ecological approaches acknowledge that the health of the community is dependent on an interaction between behavioral and environmental factors affecting the entire population. While many published studies document the benefits of CBPR in difficult-to-reach populations and describe successful implementation of this approach in adult populations, relatively few studies have been conducted in child and adolescent populations. Given that children and adolescents are particularly sensitive to the effects of their physical environments and may also be distrustful of outsiders, ecological approaches involving the community as partners, such as CBPR, may be especially useful in this population. ^ Objective. This thesis reviews published studies using a community-based participatory research approach in children and adolescents to assess the appropriateness of this approach in this population. ^ Method. Studies using CBPR in youth populations were identified using Medline and other Internet searches through both MeSH heading and text-word searches. ^ Results. A total of 16 studies were identified and analyzed for this review. Nine of the sixteen studies were experimental or quasi-experimental design, with Asthma being the most commonly studied disease. ^ Conclusions. While many studies using CBPR were not conducted with the level of scientific rigor typically found in clinical trial research, the studies reviewed each contributed to a greater understanding of the problems they investigated. Furthermore, interventional studies provided lasting benefits to communities under study above what would be found in studies using more traditional research approaches. While CBPR may not be appropriate for all research situations due to the time and resources required, we conclude that is a useful approach and should be considered when conducting community-based research for pediatric and adolescent populations.^

Assessment of US Air Force student pilots with intermittent monofixation syndrome on a non-stereoptic dependent flight maneuver in pilot training

The United States Air Force School of Aerospace Medicine (USAFSAM) and Aeromedical Consult Service (ACS) have developed waiver criteria for pilots with subtle substandard depth perception. This is to allow United States Air Force (USAF) pilots with mild depth perception deficiency to continue flying duties while limiting the risk to flight safety and ensuring the availability of costly human resources. From 1999 to 2005, 166 aviators were given waivers for intermittent monofixation syndrome (IMFS). Of these, 96 were student pilots who performed slightly worse at stereoptic dependent flight maneuvers than student pilots (8,907) with normal depth perception (Lowry, 2006).^ This study's purpose is to evaluate the performance of the extended-trail maneuver, a non-stereoptic dependent flying maneuver, as executed by a cohort of 12 United States Air Force student pilots with intermittent monofixation syndrome versus the cohort of 100 student pilots with normal depth perception. These subjects are extracted from the cohorts examined by Lowry (2006) and the null hypothesis predicts no statistical difference in the performance of the non-stereoptic dependant flight maneuver extended-trail between student pilots with intermittent monofixation syndrome and those without the condition. ^

Depolarization-dependent synaptic potentiation and hyperexcitability induced by a Ca2+-independent trigger

Despite vast research efforts since Cajal's seminal thoughts on the adaptation of the nervous system, researchers have only recently begun to understand the diversity of forms of neuronal plasticity and its mechanisms. All known forms of activity-dependent neuronal plasticity utilize alterations in [Ca 2+]i as a signal of changes in the membrane voltage. Ca 2+ sensors trigger modifications in excitability or synaptic strength that last from seconds to weeks and presumably years. Intriguingly, Kunjilwar et al., (unpublished observations) discovered in peripheral sensory axons of Aplysia that the induction of depolarization-dependent long-term axonal hyperexcitability does not require Ca2+ transients. Here we show that induction of depolarization-dependent intermediate-term and long-term synaptic potentiation in Aplysia occurs in conditions that prevent Ca2+ entry through voltage-gated channels and elevation of [Ca2+]i. We found that the intermediate-term synaptic potentiation induced under conditions expected to prevent Ca 2+ transients is associated with increased excitability of sensory neuron axons near presynaptic terminals, suggesting that the synaptic potentiation involves a presynaptic locus. The Ca2+-independent intermediate- and long-term synaptic potentiation appeared similar to previously reported Ca2+-dependent modifications in Aplysia. ^

Membrane trafficking from lysosomes to the plasma membrane regulates phosphatidylserine externalization during apoptosis

Programmed cell death is characterized by tightly controlled temporal and spatial intracellular Ca2+ responses that regulate the release of key proapoptotic proteins from mitochondria to the cytosol. Since apoptotic cells retain their ability to exclude membrane impermeable dyes, it is possible that the cells evoke repair mechanisms that, similar to those in normal cells, patch any damaged areas of the plasma membrane that preclude dye permeation. One critical distinction between plasma membrane repair in normal and apoptotic cells is the preservation of membrane lipid asymmetry. In normal cells, phosphatidylserine (PS) retains its normal asymmetric distribution in the inner membrane leaflet. In apoptotic cells, PS redistributes to the outer membrane leaflet by a Ca2+ dependent mechanism where it serves as a recognition ligand for phagocytes(1). In this study Ca 2+-specific fluorescent probes were employed to investigate the source of Ca2+ required for PS externalization. Experiments employing Rhod2-AM, calcium green 1, fura2-AM and the aqueous space marker FITC-dextran, demonstrated that exogenous Ca2+ imported with endocytotic vesicles into the cell was released into the cytosol in an apoptosis dependent manner. Labeling of the luminal side of the endocytotic vesicles with FITC-annexin 5, revealed that membrane lipid asymmetry was disrupted upon endosome formation. Specific labeling of the lysosomal luminal surface with the non-exchangeable membrane lipid probe, N-rhodamine-labeled-phosphatidylethanolamine (N-Rho-PE) and the lysosomal specific probe, lysotracker green, facilitated real-time monitoring of plasma membrane-to-endosome-to-lysosome transitions. Enforced elevation of cytosolic [Ca2+] with ionophore resulted in the redistribution of N-Rho-PE and PS from the inner membrane leaflet to the PM outer membrane leaflet. Identical results were obtained during apoptosis, however, the redistribution of both N-RhoPE and PS was dependent on the release of intra-lysosomal Ca2+ to the cytosol. Additional experiments suggested that lipid redistribution was dependent on the activity of lysosomal phospholipase A2 activity since lipid trafficking was abolished in the presence of chloroquine and lipase inhibitors. These data indicate that endosomal/lysosomal Ca2+ and the fusion of hybrid organelles to the plasma membrane regulates the externalization of PS during apoptosis. ^

The INO80 chromatin remodeling complex is involved in the nucleotide excision repair pathway

The genomic DNA of eukaryotic cells is well organized into chromatin structures. However, these repressed structures present barriers that block the access of regulatory factors to the genome during various nuclear events. To overcome the obstacle, two major cellular processes, post-modification of histone tails and ATP-dependent chromatin remodeling, are involved in reconfiguring chromatin structure and creating accessible DNA. Despite the current research progress, much remains to be explored concerning the relationship between chromatin remodeling and DNA repair. Recently, one member of the ATP-dependent chromatin remodeling complexes, INO80, has been found to play a crucial role in DNA damage repair. However, the functions of this complex in higher eukaryotes have yet to be determined. The goal of my study is to generate a human somatic INO80 conditional knockout model and investigate the functions of Ino80 in damage repair.^ By homologous targeting of the INO80 locus in human HCT116 colon epithelial cells, I established a human somatic INO80 conditional knockout model. I have demonstrated that the conditional INO80 cells exhibited a sufficiently viable period when the INO80 protein is removed. Moreover, I found that loss of INO80 resulted in deficient UV lesion repair in response to UV while the protein levels of the NER factors such as XPC, XPA, XPD were not affected. And in vitro repair synthesis assay showed that the NER incision and repair synthesis activities were intact in the absence of INO80. Examination on the damage recognition factor XPC showed its recruitment to damage sites was impaired in the INO80 mutant cells. Loss of INO80 also led to reduced enrichment of XPA at the site of UV lesions. Despite the reduced recruitment of XPC and XPA observed in INO80 mutants, no direct interaction was detected. Meanwhile, direct interaction between INO80 and DDB1, the initial UV lesion detector, was detected by coimmunoprecipitation. UV-induced chromosome relaxation was reduced in cells devoid of INO80. These results demonstrate the INO80 complex may participates in the NER by interacting with DDB1 and having a critical role of in creating DNA accessibility for the nucleotide excision pathway. ^

PREPARATION AND CHARACTERIZATION OF FAD DEPENDENT NADPH CYTOCHROME P-450 REDUCTASE (FLAVOPROTEINS, APOFLAVOPROTEIN)

NADPH cytochrome P-450 reductase releases FMN and FAD upon dilution into slightly acidic potassium bromide. The flavins are released with positive cooperativity. Dithiothreitol protects the FAD dependent cytochrome c reductase activity against inactivation by free radicals. Behavior in potassium bromide is sensitive to changes in the pH. High performance hydroxylapatite resolved the FAD dependent reductase from holoreductase. For 96% FAD dependent reductase, the overall yield was 12%.^ High FAD dependence was matched by a low FAD content, with FAD/FMN as low as 0.015. There were three molecules of FMN for every four molecules of reductase. The aporeductase had negligible activity towards cytochrome c, ferricyanide, menadione, dichlorophenolindophenol, nitro blue tetrazolium, oxygen and acetyl pyridine adenine dinucleotide phosphate. A four minute incubation in FAD reconstituted one half to all of the specific activity, per milligram protein, of untreated reductase, depending upon the substrate. After a two hour reconstitution, the reductase eluted from hydroxylapatite at the location of holoreductase. It had little flavin dependence, was equimolar in FMN and FAD, and had nearly the specific activity (per mole flavin) of untreated reductase.^ The lack of activity and the ability of FMN to also reconstitute suggest that the redox center of FAD is essential for catalysis, rather than for structure. Dependence upon FAD is consistent with existing hypotheses for the catalytic cycle of the reductase. ^

Antibody chemical reactivity: Beneficial and pathogenic roles

Antibodies (Abs) to autoantigens and foreign antigens (Ags) mediate, respectively, various pathogenic and beneficial effects. Abs express enzyme-like nucleophiles that react covalently with electrophiles. A subpopulation of nucleophilic Abs expresses proteolytic activity, which can inactivate the Ag permanently. This thesis shows how the nucleophilicity can be exploited to inhibit harmful Abs or potentially protect against a virus. ^ Inactivation of pathogenic Abs from Hemophilia A (HA) patients by means of nucleophile-electrophile pairing was studied. Deficient factor VIII (FVIII) in HA subjects impairs blood coagulation. FVIII replacement therapy fails in 20-30% of HA patients due to production of anti-FVIII Abs. FVIII analogs containing electrophilic phosphonate group (E-FVIII and E-C2) were hypothesized to inactivate the Abs by reacting specifically and covalently with nucleophilic sites. Anti-FVIII IgGs from HA patients formed immune complexes with E-FVIII and E-C2 that remained irreversibly associated under conditions that disrupt noncovalent Ab-Ag complexes. The reaction induced irreversible loss of Ab anti-coagulant activity. E-FVIII alone displayed limited interference with coagulation. E-FVIII is a prototype reagent suitable for further development as a selective inactivator of pathogenic anti-FVIII Abs. ^ The beneficial function of Abs to human immunodeficiency virus type 1 (HIV-1) was analyzed. HIV-1 eludes the immune system by rapidly changing its coat protein structure. IgAs from noninfected subjects hydrolyzed gp120 and neutralized HIV-1 with modest potency by recognizing the gp120 421-433 epitope, a conserved B cell superantigenic region that is also essential for HIV-1 attachment to host cell CD4 receptors. An adaptive immune response to superantigens is generally prohibited due to their ability to downregulate B cells. IgAs from subjects with prolonged HIV-1 infection displayed improved catalytic hydrolysis of gp120 and exceptionally potent and broad neutralization of diverse CCR5-dependent primary HIV isolates attributable to recognition of the 421-433 epitope. This indicates that slow immunological bypass of the superantigenic character of gp120 is possible, opening the path to effective HIV vaccination. ^ My research reveals a novel route to inactivate pathogenic nucleophilic Abs using electrophilic antigens. Conversely, naturally occurring nucleophilic Abs may help impede HIV infection, and the Abs could be developed for passive immunotherapy of HIV infected subjects. ^

Macular edema in Hispanics with non-insulin dependent diabetes mellitus in Starr County, Texas: Prevalence, incidence and risk factors

This project is based on secondary analyses of data collected in Starr County, Texas from 1981 till 1991 to determine the prevalence, incidence and risk factors for macular edema in Hispanics with non-insulin-dependent diabetes in Starr County, Texas. Two studies were conducted. The first study examined the prevalence of macular edema in this population. Of the 310 diabetics that were included in the study 22 had macular edema. Of these 22 individuals 9 had clinically significant macular edema. Fasting blood glucose was found to be significantly associated with macular edema. For each 10 mg/dl increase in fasting blood glucose there was a 1.07 probability of an increase in the risk of having macular edema. Individuals with fasting blood glucose $\ge$200 mg/dl were found to be more than three times at risk of having macular edema compared to those with fasting blood glucose $<$200 mg/dl.^ In the second study the incidence and the risk factors that could cause macular edema in this Hispanic population were examined. 240 Hispanics with non-insulin-dependent diabetes mellitus and without macular edema were followed for 1223 person-years. During the follow-up period 27 individuals developed macular edema (2.21/100 person-years). High fasting blood glucose and glycosylated hemoglobin were found to be strong and independent risk factors for macular edema. Participants taking insulin were 3.9 times more at risk of developing macular edema compared to those not taking insulin. Systolic blood pressure was significantly related to macular edema, where each 10 mmHg increase in systolic blood pressure was associated with a 1.3 increase in the risk of macular edema.^ In summary, this study suggests that hyperglycemia is the main underlying factor for retinal pathological changes in this diabetic population, and that macular edema probably is not the result of sudden change in the blood glucose level. It also determined that changes in blood pressure, particularly systolic blood pressure, could trigger the development of macular edema.^ Based on the prevalence reported in this study, it is estimated that 35,500 Hispanic diabetics in the US have macular edema. This imposes a major public health challenge particularly in areas with high concentration of Mexican Americans. It also highlights the importance of public health measures directed to Mexican Americans such as health education, improved access to medical care, and periodic and careful ophthalmologic examination by ophthalmologists knowledgeable and experienced in the management of diabetic macular edema. ^

A GENETIC MARKER STUDY OF NON - INSULIN DEPENDENT DIABETICS IN THE MEXICAN-AMERICAN COMMUNITY FROM STARR COUNTY, TEXAS (PLASMA GLUCOSE, HEMOGLOBIN ANALYSIS)

Diabetes mellitus occurs in two forms, insulin-dependent (IDDM, formerly called juvenile type) and non-insulin dependent (NIDDM, formerly called adult type). Prevalence figures from around the world for NIDDM, show that all societies and all races are affected; although uncommon in some populations (.4%), it is common (10%) or very common (40%) in others (Tables 1 and 2).^ In Mexican-Americans in particular, the prevalence rates (7-10%) are intermediate to those in Caucasians (1-2%) and Amerindians (35%). Information about the distribution of the disease and identification of high risk groups for developing glucose intolerance or its vascular manifestations by the study of genetic markers will help to clarify and solve some of the problems from the public health and the genetic point of view.^ This research was designed to examine two general areas in relation to NIDDM. The first aims to determine the prevalence of polymorphic genetic markers in two groups distinguished by the presence or absence of diabetes and to observe if there are any genetic marker-disease association (univariate analysis using two by two tables and logistic regression to study the individual and joint effects of the different variables). The second deals with the effect of genetic differences on the variation in fasting plasma glucose and percent glycosylated hemoglobin (HbAl) (analysis of Covariance for each marker, using age and sex as covariates).^ The results from the first analysis were not statistically significant at the corrected p value of 0.003 given the number of tests that were performed. From the analysis of covariance of all the markers studied, only Duffy and Phosphoglucomutase were statistically significant but poor predictors, given that the amount they explain in terms of variation in glycosylated hemoglobin is very small.^ Trying to determine the polygenic component of chronic disease is not an easy task. This study confirms the fact that a larger and random or representative sample is needed to be able to detect differences in the prevalence of a marker for association studies and in the genetic contribution to the variation in glucose and glycosylated hemoglobin. The importance that ethnic homogeneity in the groups studied and standardization in the methodology will have on the results has been stressed. ^