Mapping and characterization of the Xlsirt P11 RNA cis-acting localization element

In many organisms, polarity of the oocyte is established post-transcriptionally via subcellular RNA localization. Many RNAs are localized during oogenesis in Xenopus laevis, including Xlsirts ( Xenopus laevis short interspersed repeat transcripts) [Kloc, 1993]. Xlsirts constitute a large family defined by highly homologous repeat units 79–81 nucleotides in length. Endogenous Xlsirt RNAs use the METRO (Message Transport Organizer) pathway of localization, where RNAs are transported from the nucleus to the mitochondrial cloud in stage I oocytes. Secondly, RNAs anchor at the vegetal pole in stage II oocytes. Exogenous Xlsirt RNAs can also utilize the Late pathway of localization, which involves localization to the vegetal cortex during stage III of oogenesis and results in RNAs anchored in the cortex of the entire vegetal hemisphere. ^ The Xlsirts localization signal is contained within the repeat region. This study was designed to test the hypothesis that there are cis -acting localization elements in Xlsirts, and that higher order structure plays a role. Results of experiments on Xlsirt P11, a 1700 basepair (bp) family member, led to the conclusion that a 137-bp fragment of the repetitive region is necessary and sufficient for METRO and Late pathway localization. This analysis definitively demonstrates that the Xlsirt localization signal for the METRO and Late pathways reside within the repetitive region and not within the flanking regions. Analysis of Xlsirt linker scanning mutations revealed two METRO-pathway specific subelements, and one Late-pathway specific subelement. Functional, computer, and biochemical evidence relates the higher order structure of this element to its ability to function as a localization element. ^ Xlsirt 137 is 99% identical to the Xlsirt consensus sequence identified in this study, suggesting that it is the localization element for all localized Xlsirt family members. The repeat unit was reframed based on function, rather than arbitrarily based on sequence. This work supports the hypothesis presented in 1981 by George Spohr, who originally isolated the Xlsirts, which stated that the highly conserved repetitive elements must be constrained from variability due to some unknown function of the repeats themselves. These studies shed light on the mechanism of RNA localization, linking structure and function. ^

Cardiolipin membrane domains in prokaryotes and eukaryotes.

Cardiolipin (CL) plays a key role in dynamic organization of bacterial and mitochondrial membranes. CL forms membrane domains in bacterial cells, and these domains appear to participate in binding and functional regulation of multi-protein complexes involved in diverse cellular functions including cell division, energy metabolism, and membrane transport. Visualization of CL domains in bacterial cells by the fluorescent dye 10-N-nonyl acridine orange is critically reviewed. Possible mechanisms proposed for CL dynamic localization in bacterial cells are discussed. In the mitochondrial membrane CL is involved in organization of multi-subunit oxidative phosphorylation complexes and in their association into higher order supercomplexes. Evidence suggesting a possible role for CL in concert with ATP synthase oligomers in establishing mitochondrial cristae morphology is presented. Hypotheses on CL-dependent dynamic re-organization of the respiratory chain in response to changes in metabolic states and CL dynamic re-localization in mitochondria during the apoptotic response are briefly addressed.

Somatic and cognitive domains of depression in an underserved region of Ecuador: some cultural considerations.

Not enough research efforts on depression have been carried out up to now in Latin America. The knowledge that has resulted from research activities in the United States or Europe offers limited generalizability to other regions of the world, including Latin America. In the Andean highlands of Ecuador, we found very high rates of moderate and severe depressive symptoms, a finding that must be interpreted within its cultural context. Somatic manifestations of depression predominated over cognitive manifestations, and higher education level was protective against depression. These findings call for an appreciation of culturally-specific manifestations of depression and the social factors that influence them. These factors must be further studied in order to give them the deserved priority, allocate resources appropriately, and formulate innovative psychosocial interventions.

Neural analogs of associative learning in {\it Aplysia\/}: Experimental and theoretical approaches

A change in synaptic strength arising from the activation of two neuronal pathways at approximately the same time is a form of associative plasticity and may underlie classical conditioning. Previously, a cellular analog of a classical conditioning protocol has been demonstrated to produce short-term associative plasticity at the connections between sensory and motor neurons in Aplysia. A similar training protocol produced long-term (24 hour) enhancement of excitatory postsynaptic potentials (EPSPs). EPSPs produced by sensory neurons in which activity was paired with a reinforcing stimulus were significantly larger than unpaired controls 24 hours after training. To examined whether the associative plasticity observed at these synapses may be involved in higher-order forms of classical conditioning, a neural analog of contingency was developed. In addition, computer simulations were used to analyze whether the associative plasticity observed in Aplysia could, in theory, account for second-order conditioning and blocking. ^

Design and analysis for three level hierarchical structures with count response

In numerous intervention studies and education field trials, random assignment to treatment occurs in clusters rather than at the level of observation. This departure of random assignment of units may be due to logistics, political feasibility, or ecological validity. Data within the same cluster or grouping are often correlated. Application of traditional regression techniques, which assume independence between observations, to clustered data produce consistent parameter estimates. However such estimators are often inefficient as compared to methods which incorporate the clustered nature of the data into the estimation procedure (Neuhaus 1993).1 Multilevel models, also known as random effects or random components models, can be used to account for the clustering of data by estimating higher level, or group, as well as lower level, or individual variation. Designing a study, in which the unit of observation is nested within higher level groupings, requires the determination of sample sizes at each level. This study investigates the design and analysis of various sampling strategies for a 3-level repeated measures design on the parameter estimates when the outcome variable of interest follows a Poisson distribution. ^ Results study suggest that second order PQL estimation produces the least biased estimates in the 3-level multilevel Poisson model followed by first order PQL and then second and first order MQL. The MQL estimates of both fixed and random parameters are generally satisfactory when the level 2 and level 3 variation is less than 0.10. However, as the higher level error variance increases, the MQL estimates become increasingly biased. If convergence of the estimation algorithm is not obtained by PQL procedure and higher level error variance is large, the estimates may be significantly biased. In this case bias correction techniques such as bootstrapping should be considered as an alternative procedure. For larger sample sizes, those structures with 20 or more units sampled at levels with normally distributed random errors produced more stable estimates with less sampling variance than structures with an increased number of level 1 units. For small sample sizes, sampling fewer units at the level with Poisson variation produces less sampling variation, however this criterion is no longer important when sample sizes are large. ^ 1Neuhaus J (1993). “Estimation efficiency and Tests of Covariate Effects with Clustered Binary Data”. Biometrics , 49, 989–996^

Studies of subcellular localization and complex formation of the Agrobacterium tumefaciens transport ATPase VirB11

The VirB11 ATPase is an essential component of an Agrobacterium tumefaciens type IV bacterial secretion system that transfers oncogenic nucleoprotein complexes to susceptible plant cells. This dissertation investigates the subcellular localization and homo-oligomeric state of the VirB11 ATPase in order to provide insights about the assembly of the protein as a subunit of this membrane-associated transfer system. Subcellular fractionation studies and quantitative immunoblot analysis demonstrated that $\sim$30% of VirB11 partitioned as soluble protein and $\sim$70% was tightly associated with the bacterial cytoplasmic membrane. No differences were detected in VirB11 subcellular localization and membrane association in the presence or absence of other transport system components. Mutations in virB11 affecting protein function were mapped near the amino terminus, just upstream of a region encoding a Walker 'A' nucleotide-binding site, and within the Walker 'A' motif partitioned almost exclusively with the cytoplasmic membrane, suggesting that an activity associated with nucleotide binding could modulate the affinity of VirB11 for the cytoplasmic membrane. Merodiploid analysis of VirB11 mutant and truncation derivatives provided strong evidence that VirB11 functions as a homo- or heteromultimer and that the C-terminal half of VirB11 contains a protein interaction domain. A combination of biochemical and molecular genetic approaches suggested that VirB11 and the green fluorescence protein (GFP) formed a mixed multimer as demonstrated by immunoprecipitation experiments with anti-GFP antibodies. Second, a hybrid protein composed of VirB11 fused to the N-terminal DNA-binding domain of bacteriophage $\lambda$ cI repressor conferred immunity to $\lambda$ superinfection, demonstrating that VirB11 self-association promotes dimerization of the chimeric repressor. A conserved Walker 'A' motif, though required for VirB11 function in T-complex export, was not necessary for VirB11 self-association. Sequences in both the N- and the C-terminal halves of the protein were found to contribute to self-association of the full length protein. Chemical cross-linking experiments with His$\sb6$ tagged VirB11 suggested that VirB11 probably assembles into a higher order homo-oligomeric complex. ^

Prediction of cardiovascular disease risk factors in Mexican-American children from parental risk factors, and social and demographic characteristics in the HHANES

Objectives. Cardiovascular disease (CVD) including CVD secondary to diabetes type II, a significant health problem among Mexican American populations, originates in early childhood. This study seeks to determine risk factors available to the health practitioner that can identify the child at potential risk of developing CVD, thereby enabling early intervention. ^ Design. This is a secondary analysis of cross-sectional data of matched Mexican American parents and children selected from the HHANES, 1982–1984. ^ Methods. Parents at high risk for CVD were identified based on medical history, and clinical and physical findings. Factor analysis was performed on children's skinfold thicknesses, height, weight, and systolic and diastolic blood pressures, in order to produce a limited number of uncorrelated child CVD risk factors. Multiple regression analyses were then performed to determine other CVD markers associated with these Factors, independently for mothers and fathers. ^ Results. Factor analysis of children's measurements revealed three uncorrelated latent variables summarizing the children's CVD risk: Factor1: ‘Fatness’, Factor2: ‘Size and Maturity’, and Factor3: ‘Blood Pressure’, together accounting for the bulk of variation in children's measurements (86–89%). Univariate analyses showed that children from high CVD risk families did not differ from children of low risk families in occurrence of high blood pressure, overweight, biological maturity, acculturation score, or social and economic indicators. However, multiple regression using the factor scores (from factor analysis) as dependent variables, revealed that higher CVD risk in parents, was significantly associated with increased fatness and increased blood pressure in the children. Father's CVD risk status was associated with higher levels of body fat in his children and higher levels of blood pressure in sons. Mother's CVD risk status was associated with higher blood pressure levels in children, and occurrence of obesity in the mother associated with higher fatness levels in her children. ^ Conclusion. Occurrence of cardiovascular disease and its risk factors in parents of Mexican American children, may be used to identify children at potentially higher risk for developing CV disease in the future. Obesity in mothers appears to be an important marker for the development of higher levels of body fatness in children. ^

Genetic variants in the mammalian target of rapamycin (mTOR) signaling pathway as predictors of survival and clinical response in women with ovarian cancer

Background. The mTOR pathway is commonly altered in human tumors and promotes cell survival and proliferation. Preliminary evidence suggests this pathway's involvement in chemoresistance to platinum and taxanes, first line therapy for epithelial ovarian cancer. A pathway-based approach was used to identify individual germline single nucleotide polymorphisms (SNPs) and cumulative effects of multiple genetic variants in mTOR pathway genes and their association with clinical outcome in women with ovarian cancer. ^ Methods. The case-series was restricted to 319 non-Hispanic white women with high grade ovarian cancer treated with surgery and platinum-based chemotherapy. 135 SNPs in 20 representative genes in the mTOR pathway were genotyped. Hazard ratios (HRs) for death and Odds ratios (ORs) for failure to respond to primary therapy were estimated for each SNP using the multivariate Cox proportional hazards model and multivariate logistic regression model, respectively, while adjusting for age, stage, histology and treatment sequence. A survival tree analysis of SNPs with a statistically significant association (p<0.05) was performed to identify higher order gene-gene interactions and their association with overall survival. ^ Results. There was no statistically significant difference in survival by tumor histology or treatment regimen. The median survival for the cohort was 48.3 months. Seven SNPs were significantly associated with decreased survival. Compared to those with no unfavorable genotypes, the HR for death increased significantly with the increasing number of unfavorable genotypes and women in the highest risk category had HR of 4.06 (95% CI 2.29–7.21). The survival tree analysis also identified patients with different survival patterns based on their genetic profiles. 13 SNPs on five different genes were found to be significantly associated with a treatment response, defined as no evidence of disease after completion of primary therapy. Rare homozygous genotype of SNP rs6973428 showed a 5.5-fold increased risk compared to the wild type carrying genotypes. In the cumulative effect analysis, the highest risk group (individuals with ≥8 unfavorable genotypes) was significantly less likely to respond to chemotherapy (OR=8.40, 95% CI 3.10–22.75) compared to the low risk group (≤4 unfavorable genotypes). ^ Conclusions. A pathway-based approach can demonstrate cumulative effects of multiple genetic variants on clinical response to chemotherapy and survival. Therapy targeting the mTOR pathway may modify outcome in select patients.^

L -Sox5 and Sox6: Architectural transcription factors in chondrogenesis

Transcription factors often determine cell fate and tissue development. Chondrogenesis is the developmental process by which cartilages form. Recently, gene targeting studies have shown that two transcription factors, L-Sox5 and Sox6, play essential and redundant roles in chondrogenesis in vivo by converting precartilaginous cell condensations into cartilages. Both are highly similar High-Mobility-Group (HMG)-domain proteins that bind and subsequently bend DNA containing the 7bp HMG site (A/T)(A/T)CAA(A/T)G. They have no transactivation domain, but homo- and hetero-dimerize and preferentially bind DNA containing two HMG sites. They are thought to play an architectural role in transactivation by facilitating long-range DNA and protein interactions. To understand their molecular mechanism of action, we investigated how phasing, orientation, and spacing between HMG sites affect L-Sox5 and Sox6 DNA-binding. We determined that L-Sox5 and Sox6 dimers bind with high affinity to paired HMG sites in DNA rather than a single HMG site. Binding of paired sites is independent of DNA helical phasing, orientation of paired HMG sites and independent of distance up to 255 base pairs between sites. Mutational analysis demonstrated that binding of L-Sox5 and Sox6, independent of orientation of the sites, is critically dependent on the presence of paired HMG sites rather than one HMG site alone. Our data support a unique and novel model whereby L-Sox5 and Sox6 dimerize and bind DNA with pronounced spatial flexibility, possibly by a flexible hinge, and act as architectural transcription factors that bring distant DNA sites and proteins together to form higher order transcriptional complexes that are essential for the activation of their target genes in chondrogenesis. ^

Molecular mechanism of the relaxin signaling pathways: Activation of cAMP, PI3K, and PKCzeta

Relaxin is a polypeptide hormone that has diverse effects on reproductive and non-reproductive tissues. Relaxin activates the G-protein coupled receptors, LGR7 and LRG8. Early studies described increased cAMP and protein kinase A activity upon relaxin treatment, but cAMP accumulation alone could not account for all of the relaxin-mediated effects. We utilized the human monocyte cell line THP-1 to study the mechanism of relaxin-stimulated CAMP production. ^ Relaxin treatment in THP-1 cells produces a biphasic time course in cAMP accumulation, where the first peak appears as early as 1–2 minutes with a second peak at 10–20 minutes. Selective inhibitors for phosphoinositide 3-kinase (P13K), such as wortmannin and LY294002, show a dose-dependent inhibition of relaxin-stimulated cAMP accumulation, specific for the second peak of the relaxin time course. Neither the effects of relaxin nor the inhibition of relaxin by LY294002 is mediated by the activity of phosphodiesterases. Furthermore, LY294002 blocks upregulation of vascular endothelial growth factor transcript levels by relaxin. ^ To further delineate relaxin signaling pathways, we searched for downstream targets of PI3K that could activate adenylyl cyclase (AC). Protein kinase C ζ (PKCζ) was a prime candidate because it activates types II and V AC. Chelerythrine chloride (a general PKC inhibitor) inhibits relaxin-induced cAMP production to the same degree as LY294002 (∼40%). Relaxin stimulates PKCζ translocation to the plasma membrane in THP-1, MCF-7, PHM1-31, and MMC cells, as shown by immunocytochemistry. PKCζ translocation is P13K-dependent and independent of cAMP production. Antisense PKCζ oligodeoxynucleotides (PKCζ-ODNs) deplete both PKCζ transcript and protein levels in THP-1 cells. PKCζ-ODNs abolish relaxin-mediated PKCζ translocation and inhibit relaxin stimulation of cAMP by 40%, as compared to mock and random ODN controls. Treatment with LY294002 in the presence of PKCζ-ODNs results in little further inhibition. Taken together, we present a novel role for PI3K and PKCζ in relaxin stimulation of cAMP and provide the first example of the PKCζ regulation of AC in an endogenous system. Furthermore, we have identified higher order complexes of AC isoforms and PKA anchoring proteins in attempts to explain the differential coupling of relaxin to cAMP and PI3K-signaling pathways in various cell types. ^