Modeling and E-M estimation of haplotype-specific relative risks from genotype data for a case-control study of unrelated individuals

The US National Cancer Institute has recently sponsored the formation of a Cohort Consortium (http://2002.cancer.gov/scpgenes.htm) to facilitate the pooling of data on very large numbers of people, concerning the effects of genes and environment on cancer incidence. One likely goal of these efforts will be generate a large population-based case-control series for which a number of candidate genes will be investigated using SNP haplotype as well as genotype analysis. The goal of this paper is to outline the issues involved in choosing a method of estimating haplotype-specific risk estimates for such data that is technically appropriate and yet attractive to epidemiologists who are already comfortable with odds ratios and logistic regression. Our interest is to develop and evaluate extensions of methods, based on haplotype imputation, that have been recently described (Schaid et al., Am J Hum Genet, 2002, and Zaykin et al., Hum Hered, 2002) as providing score tests of the null hypothesis of no effect of SNP haplotypes upon risk, which may be used for more complex tasks, such as providing confidence intervals, and tests of equivalence of haplotype-specific risks in two or more separate populations. In order to do so we (1) develop a cohort approach towards odds ratio analysis by expanding the E-M algorithm to provide maximum likelihood estimates of haplotype-specific odds ratios as well as genotype frequencies; (2) show how to correct the cohort approach, to give essentially unbiased estimates for population-based or nested case-control studies by incorporating the probability of selection as a case or control into the likelihood, based on a simplified model of case and control selection, and (3) finally, in an example data set (CYP17 and breast cancer, from the Multiethnic Cohort Study) we compare likelihood-based confidence interval estimates from the two methods with each other, and with the use of the single-imputation approach of Zaykin et al. applied under both null and alternative hypotheses. We conclude that so long as haplotypes are well predicted by SNP genotypes (we use the Rh2 criteria of Stram et al. [1]) the differences between the three methods are very small and in particular that the single imputation method may be expected to work extremely well.     

A chlorophyll a/b-binding protein homolog that is induced by iron deficiency is associated with enlarged photosystem I units in the eucaryotic alga Dunaliella salina

Adaptation of the halotolerant alga Dunaliella salina to iron deprivation involves extensive changes of chloroplast morphology, photosynthetic activities, and induction of a major 45-kDa chloroplast protein termed Tidi. Partial amino acid sequencing of proteolytic peptides suggested that Tidi resembles chlorophyll a/b-binding proteins which compose light-harvesting antenna complexes (LHC) (Varsano, T., Kaftan, D., and Pick, U. (2003) J. Plant Nutr. 26, 2197-2210). Here we show that Tidi shares the highest amino acid sequence similarity with light-harvesting I chlorophyll a/b-binding proteins from higher plants but has an extended proline-rich N-terminal domain. The accumulation of Tidi is reversed by iron supplementation, and its level is inversely correlated with photosystem I (PS-I) reaction center proteins. In native gel electrophoresis, Tidi co-migrates with enlarged PS-I-LHC-I super-complexes. Single particle electron microscopy analysis revealed that PS-I units from iron-deficient cells are larger (31 and 37 nm in diameter) than PS-I units from control cells (22 nm). The 77 K chlorophyll fluorescence emission spectra of isolated complexes suggest that the Tidi-LHC-I antenna are functionally coupled to the reaction centers of PS-I. These findings indicate that Tidi acts as an accessory antenna of PS-I. The enlargement of PS-I antenna in algae and in cyanobacteria under iron deprivation suggests a common limitation that requires rebalancing of the energy distribution between the two photosystems.     

Anaerobic Ammonium-Oxidizing (Anammox) Bacteria and Associated Activity in Fixed-Film Biofilters of a Marine Recirculating Aquaculture System

Microbial communities in the biological filter and waste sludge compartments of a marine recirculating aquaculture system were examined to determine the presence and activity of anaerobic ammonium-oxidizing (anammox) bacteria. Community DNA was extracted from aerobic and anaerobic fixed-film biofilters and the anaerobic sludge waste collection tank and was analyzed by amplifying 16S rRNA genes by PCR using anammox-selective and universal GC-clamped primers. Separation of amplified PCR products by denaturing gradient gel electrophoresis and sequencing of the different phylotypes revealed a diverse biofilter microbial community. While Planctomycetales were found in all three communities, the anaerobic denitrifying biofilters contained one clone that exhibited high levels of sequence similarity to known anammox bacteria. Fluorescence in situ hybridization studies using an anammox-specific probe confirmed the presence of anammox Planctomycetales in the microbial biofilm from the denitrifying biofilters, and anammox activity was observed in these biofilters, as detected by the ability to simultaneously consume ammonia and nitrite. To our knowledge, this is the first identification of anammox-related sequences in a marine recirculating aquaculture filtration system, and our findings provide a foundation for incorporating this important pathway for complete nitrogen removal in such systems.     

A Quantitative Model of the GIRK1/2 Channel Reveals That Its Basal and Evoked Activities Are Controlled by Unequal Stoichiometry of Gα and Gβγ

G protein-gated K⁺ channels (GIRK; Kir3), activated by Gβγ subunits derived from G_i/o proteins, regulate heartbeat and neuronal excitability and plasticity. Both neurotransmitter-evoked (I_evoked) and neurotransmitter-independent basal (I_basal) GIRK activities are physiologically important, but mechanisms of I_basal and its relation to I_evoked are unclear. We have previously shown for heterologously expressed neuronal GIRK1/2, and now show for native GIRK in hippocampal neurons, that I_basal and I_evoked are interrelated: the extent of activation by neurotransmitter (activation index, R_a) is inversely related to I_basal. To unveil the underlying mechanisms, we have developed a quantitative model of GIRK1/2 function. We characterized single-channel and macroscopic GIRK1/2 currents, and surface densities of GIRK1/2 and Gβγ expressed in Xenopus oocytes. Based on experimental results, we constructed a mathematical model of GIRK1/2 activity under steady-state conditions before and after activation by neurotransmitter. Our model accurately recapitulates I_basal and I_evoked in Xenopus oocytes, HEK293 cells and hippocampal neurons; correctly predicts the dose-dependent activation of GIRK1/2 by coexpressed Gβγ and fully accounts for the inverse I_basal-R_a correlation. Modeling indicates that, under all conditions and at different channel expression levels, between 3 and 4 Gβγ dimers are available for each GIRK1/2 channel. In contrast, available Gα_i/o decreases from ~2 to less than one Gα per channel as GIRK1/2''s density increases. The persistent Gβγ/channel (but not Gα/channel) ratio support a strong association of GIRK1/2 with Gβγ, consistent with recruitment to the cell surface of Gβγ, but not Gα, by GIRK1/2. Our analysis suggests a maximal stoichiometry of 4 Gβγ but only 2 Gα_i/o per one GIRK1/2 channel. The unique, unequal association of GIRK1/2 with G protein subunits, and the cooperative nature of GIRK gating by Gβγ, underlie the complex pattern of basal and agonist-evoked activities and allow GIRK1/2 to act as a sensitive bidirectional detector of both Gβγ and Gα.     

Linkage of acid α-glucosidase (Gaa) and Thymidine kinase (Tk-1) to Esterase-3 (Es-3) on mouse Chromosome 11

Inheritance in recombinant inbred (RI) strains of restriction fragment length variants (RFLVs) detected by probes specific for Gaa and Tk-1 showed tight linkage of both to Es-3 on mouse Chromosome (Chr) 11. This result extends the region of homology between mouse Chr 11 and human chr 17q.     

Activities of SOD and the ascorbate-glutathione cycle enzymes in subcellular compartments in leaves and roots of the cultivated tomato and its wild salt-tolerant relative Lycopersicon pennellii

The activities of the ascorbate-glutathione cycle enzymes ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) and SOD were studied in cell organelles of the cultivated tomato Lycopersicon esculentum (M82) and its wild salt-tolerant related species Lycopersicon pennellii (Lpa). All four enzymes of the ascorbate-glutathione cycle were present in chloroplasts/plastids, mitochondria and peroxisomes of leaf and root cells of both tomato species. In all leaf and root organelles of both species, the activity of MDHAR was similar to, or higher than, that of APX, while the activity of DHAR was one order of magnitude lower than that of MDHAR. Based on these results, it is suggested that in the organelles of both tomato species, ascorbate is regenerated mainly by MDHAR. In both tomato species, GR activity, and to a lesser extent DHAR activity, was found to reside in the soluble fraction of all leaf and root cell organelles, while APX and MDHAR activities were distributed between the membrane and soluble fractions. A higher SOD to APX activity ratio in all Lpa organelles was the major difference between the two tomato species. It is possible that this higher ratio contributes to the inherently better protection of Lpa from salt stress, as was previously reported.     

Patient-specific MRI-based 3D FSI RV/LV/patch models for pulmonary valve replacement surgery and patch optimization

A patient-specific right/left ventricle and patch (RV/LV/patch) combination model with fluid-structure interactions (FSIs) was introduced to evaluate and optimize human pulmonary valve replacement/insertion (PVR) surgical procedure and patch design. Cardiac magnetic resonance (CMR) imaging studies were performed to acquire ventricle geometry, flow velocity, and flow rate for healthy volunteers and patients needing RV remodeling and PVR before and after scheduled surgeries. CMR-based RV/LV/patch FSI models were constructed to perform mechanical analysis and assess RV cardiac functions. Both pre- and postoperation CMR data were used to adjust and validate the model so that predicted RV volumes reached good agreement with CMR measurements (error <3%). Two RV/LV/patch models were made based on preoperation data to evaluate and compare two PVR surgical procedures: (i) conventional patch with little or no scar tissue trimming, and (ii) small patch with aggressive scar trimming and RV volume reduction. Our modeling results indicated that (a) patient-specific CMR-based computational modeling can provide accurate assessment of RV cardiac functions, and (b) PVR with a smaller patch and more aggressive scar removal led to reduced stress/strain conditions in the patch area and may lead to improved recovery of RV functions. More patient studies are needed to validate our findings.     

Rat liver acyl-CoA synthetase 4 is a peripheral-membrane protein located in two distinct subcellular organelles,peroxisomes, and mitochondrial-associated membrane

Obesity and non-insulin-dependent diabetes favor storage of fatty acids in triacylglycerol over oxidation. Recently, individual acyl-CoA synthetase (ACS) isoforms have been implicated in the channeling of fatty acids either toward lipid synthesis or toward oxidation. Although ACS1 had been localized to three different subcellular regions in rat liver, endoplasmic reticulum, mitochondria, and peroxisomes, the study had used an antibody raised against the full-length ACS1 protein which cross-reacts with other isoforms, probably because all ACS family members contain highly conserved amino acid sequences. Therefore, we examined the subcellular location of ACS1, ACS4, and ACS5 in rat liver to determine which isoform was present in peroxisomes, whether the ACSs were intrinsic membrane proteins, and which ACS isoforms were up-regulated by PPAR alpha ligands. Non-cross-reacting ACS1, ACS4, and ACS5 peptide antibodies showed that ACS4 was the only ACS isoform present in peroxisomes isolated from livers of gemfibrozil-treated rats. ACS4 was also present in fractions identified as mitochondria-associated membrane (MAM). ACS1 was present in endoplasmic reticulum fractions and ACS5 was present in mitochondrial fractions. Incubation with troglitazone, a specific inhibitor of ACS4, decreased ACS activity in the MAM fractions 30-45% and in the peroxisomal fractions about 30%. Because the signal for ACS4 protein in peroxisomes was so strong compared to the MAM fraction, we examined ACS4 mRNA abundance in livers of rats treated with the PPAR alpha agonist GW9578. Treatment with GW9578 increased ACS4 mRNA abundance 40% and ACS1 mRNA 25%. Although we had originally proposed that ACS4 is linked to triacylglycerol synthesis, it now appears that ACS4 may also be important in activating fatty acids destined for peroxisomal oxidation. We also determined that, unlike ACS1 and 5, ACS4 is not an intrinsic membrane protein. This suggests that ACS4 is probably targeted and linked to MAM and peroxisomes by interactions with other proteins.     

Salt stress induces up-regulation of an efficient chloroplast antioxidant system in the salt-tolerant wild tomato species Lycopersicon pennellii but not in the cultivated species

The response of the chloroplastic antioxidant system of the cultivated tomato Lycopersicon esculentum (Lem) and its wild salt-tolerant related species L. pennellii (Lpa) to NaCl stress was studied. An increase in H₂O₂ level and membrane lipid peroxidation was observed in chloroplasts of salt-stressed Lem. In contrast, a decrease in these indicators of oxidative stress characterized chloroplasts of salt-stressed Lpa plants. This differential response of Lem and Lpa to salinity, correlates with the activities of the antioxidative enzymes in their chloroplasts. Increased activities of total superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), glutathione-S-transferase (GST), phospholipid hydroperoxide glutathione peroxidase (PHGPX) and several isoforms of non-specific peroxidases (POD) were found in chloroplasts of salt-treated Lpa plants. In these chloroplasts, in contrast, activity of lipoxygenase (LOX) decreased while in those of salt-stressed Lem it increased. Although total SOD activity slightly increased in chloroplasts of salt-treated Lem plants, differentiation between SOD types revealed that only stromal Cu/ZnSOD activity increased. In contrast, in chloroplasts of salt-treated Lpa plants FeSOD activity increased while Cu/ZnSOD activity remained unchanged. These data indicate that salt-dependent oxidative stress and damage, suffered by Lem chloroplasts, was effectively alleviated in Lpa chloroplasts by the selective up-regulation of a set of antioxidative enzymes. Further support for the above idea was supplied by leaf discs experiments in which pre-exposure of Lpa plants to salt-treatment conferred cross-tolerance to paraquat-induced oxidative stress while increased oxidative damage by paraquat-treatment was found in salt-stressed Lem plants.     

Isolation and partial characterization of the structural gene for human acid alpha glucosidase.

Genetic deficiency of acid alpha glucosidase (GAA) results in glycogen storage disease type II. To study the disease at the molecular level, we have previously isolated and sequenced the cDNA (3.6 kb) for human GAA. We have now isolated the structural gene, mapped and determined the position and size of the exons containing the entire cDNA, and determined the sequence of the intron-exon junctions. The structural gene is approximately 28 kb and contains 20 exons. The first exon has only 5' untranslated sequence and is separated by an approximately 2.7-kb intron from the second exon that contains the initiation ATG. The second as well as the last exon are quite large (578 and 607 bp) with the remainder of the exons ranging from 85-187 bp. Additionally, two new restriction fragment length (RFLPs) for Xba I and Stu I are described at the GAA locus, one of which is most 5' of the eight RFLPs we have previously described.