A cladistic analysis of phenotypic associations with haplotypes inferred from restriction endonuclease mapping. I. Basic theory and an analysis of alcohol dehydrogenase activity in Drosophila

Because some genes have been cloned that have a known biochemical or physiological function, genetic variation can be measured in a population at loci that may directly influence a phenotype of interest. With this measured genotype approach, specific alleles or haplotypes in the probed DNA region can be assigned phenotypic effects. In this paper we address several problems encountered in implementing the measured genotype approach with restriction site data. A number of analytical problems arise in part as a consequence of the linkage disequilibrium that is commonly encountered when dealing with small DNA regions: 1) different restriction site polymorphisms are not statistically independent, 2) the sites being measured are not likely to be the direct cause of the associated phenotypic effects, 3) haplotype classes may be phenotypically heterogeneous, and 4) the sites that are most strongly associated with phenotypic effects are not necessarily the most closely linked to the actual genetic cause of the effects. When recombination and gene conversion are rare, the primary cause of linkage disequilibrium is history (mutational origin, genetic drift, hitchhiking, etc.). We deal with historical association directly by producing a cladogram that partially reconstructs the evolutionary history of the present-day haplotype variability. The cladogram defines a nested analysis of variance that simultaneously detects phenotypic effects, localizes the effects within the cladogram, and identifies haplotypes that are potentially heterogeneous in their phenotypic associations. The power of this approach is illustrated by an analysis of the associations between alcohol dehydrogenase (ADH) activity and restriction site variability in a 13-kb fragment surrounding the ADH locus in Drosophila melanogaster.     

Apolipoprotein E phenotyping with a single gel method: application to the study of informative matings

Two-dimensional gel electrophoresis or isoelectric focusing before and after treatment with cysteamine are currently used to determine the six apolipoprotein E isomorphic phenotypes from isolated very low density lipoproteins. A technique is described that makes this possible by performing isoelectric focusing on a single polyacrylamide cylindrical gel under standardized conditions. The technique is simple and accurate enough to obtain 99.5% concordance when the gels are interpreted independently by four different skilled and unskilled observers in the absence of any knowledge of the origin of the samples. There was complete agreement between our technique and the bidimensional method carried out independently in another laboratory on 74 aliquots of plasma very low density lipoproteins. Its application to 16 informative matings involving 101 subjects confirmed the recent demonstration that the apolipoprotein E phenotype inheritance is autosomal and compatible with three common alleles acting at a single genetic locus. Analyses of the contribution of apoE polymorphism to lipid and lipoprotein variability demonstrated a recessive allelic effect of epsilon 2 on plasma very low density lipoprotein cholesterol and a dominant epsilon 4 effect on low density lipoprotein cholesterol. As much as 30% of the variability in low density lipoprotein cholesterol was attributable to this polymorphic gene locus. A simplified scheme is proposed for the symbolic representation of the six phenotypes for clinical and genetic applications.     

Evidence for nonrandom multiplicity of gene products in 22 plant genera

Correlations in the number (multiplicity) of molecular forms among 10 enzymes have been estimated from a sample of plants representing 22 genera. Significant correlations in multiplicity among some of the 10 enzyme systems suggest a nonrandom organization of variation in number of molecular forms. An analysis of the correlations among enzymes of a glycolytic and Krebs cycle subset supports the occurrence of patterns of influence on multiplicity which correspond to the known metabolic relatedness of the enzymes. We interpret these data as evidence for organization of enzyme multiplicity. This property may be of adaptive value to the species.This research was supported by U.S.A.E.C. Contract AT(11-1)-1552, U.S.P.H.S. Grant AM 09381 and Rackham Faculty Award NSF-GU-3470-Project 131, and U.S.P.H.S. Career Development Award 1-K3-AM7959 (GJB).     

Side effects of chronic lithium therapy in Hong Kong Chinese: An ethnopsychiatric perspective

A biocultural study of the side effects of chronic lithium treatment among 70 Hong Kong Chinese psychiatric patients, using a self-report 33-item checklist and semi-structured interviews, revealed an imperfect correspondence between biomedically prescribed and culturally endorsed psychotropic side effects. Although polidipsia and polyuria (47%) were the biomedically most real side effects, they were not usually regarded as bothersome or translated into metaphors to express undesirable side effects.Complaints such as tiredness (38%), drowsiness (36%) and poor memory (31%) were also common but their frequency was significantly lower than that of normal control subjects. The item loss of creativity had no conceptual equivalent in Chinese and was usually misinterpreted. As no patient was aware that lithium was a metal, the side effect metallic taste was variously labelled. Contrary to Western findings, complaints of missing of highs, loss of assertiveness and fear of weight gain were rarely encountered. Active elicitation was required for indigenous complaints, with 38% of patients considering lithium to cause mild hotness. This was readily neutralized by drinking more water which had a cooling effect. Expectedly, concurrent neuroleptics and antidepressants amplified most lithium side effects.This study affirms Western data on the biomedically universalizable effects of chronic lithium treatment, but also supports the thesis that culturally constituted cognitive styles affect patients' recognition, labelling, experiencing and reporting the total drug effect. Further, it demonstrates that the lived experience and clinical negotiation of lithium associated side effects reproduce, authenticate, and at times critique, core cultural and moral premises of Western and Chinese societies.     

Genotypes with the apolipoprotein ε4 allele are predictors of coronary heart disease mortality in a longitudinal study of elderly Finnish men

Earlier we reported that allelic variation in the gene coding for apolipoprotein (apoE) is a significant predictor of variation in the risk of coronary heart disease (CHD) death in a longitudinal study of elderly Finnish men. Here we address the question: which of the apoE genotypes confers the risk information in these men, and whether such information persists after other CHD risk factors are considered? We followed two cohorts of elderly Finnish men aged 65 to 84 years, one in Eastern (n = 281) and the other in the Southwestern (n = 344) Finland for 5 years during which 26 (9.3%) of the men from the Eastern cohort and 40 (11.6%) of the men in the Southwestern cohort died from CHD. Baseline high density lipoprotein (HDL) cholesterol and (HDL cholesterol)² in the Eastern cohort and age, and total and HDL cholesterol and smoking status in the Southwestern cohort were significant predictors of CHD death (P < 0.05). The apoE genotypes were significant predictors in the Southwestern cohort atP = 0.02 and in the Eastern cohort atP = 0.18. In multivariable models, information about apoE genotypes improved the prediction atP = 0.10 level of statistical significance in both cohorts. When genotypes were considered separately, the ε2/4 combined with the ε4/4 in the Eastern cohort (odds ratio = 7.69, 95% CI = 1.67-35.52) and the ε3/4 in the Southwestern cohort (odds ratio = 2.44, 95% CI = 1.16–5.10) had sigificanctly greater odds of CHD death compared to the common ε3/3 genotype. We conclude that apoE genotypes confer risk information about CHD death in two cohorts of elderly Finnish men in a longitudinal study, and this information persists after adjustment for other CHD risk factors. Because different genotypes were predictors in these two cohorts, we further conclude that the utility of a particular genotype as a predictor of CHD death in other populations may depend on the distribution of risk factor profiles at baseline, geographically defined environmental exposures, the CHD mortality history, and the evolutionary history of background genotypes in the population considered.     

A Cladistic Analysis of Phenotypic Associations with Haplotypes Inferred from Restriction Endonuclease Mapping and DNA Sequence Data. III. Cladogram Estimation

We previously developed a cladistic approach to identify subsets of haplotypes defined by restriction endonuclease mapping or DNA sequencing that are associated with significant phenotypic deviations. Our approach was limited to segments of DNA in which little recombination occurs. In such cases, a cladogram can be constructed from the restriction site or sequence data that represents the evolutionary steps that interrelate the observed haplotypes. The cladogram is used to define a nested statistical design to identify mutational steps associated with significant phenotypic deviations. The central assumption behind this strategy is that any undetected mutation causing a phenotypic effect is embedded within the same evolutionary history that is represented by the cladogram. The power of this approach depends upon the confidence one has in the particular cladogram used to draw inferences. In this paper, we present a strategy for estimating the set of cladograms that are consistent with a particular sample of either restriction site or nucleotide sequence data and that includes the possibility of recombination. We first evaluate the limits of parsimony in constructing cladograms. Once these limits have been determined, we construct the set of parsimonious and nonparsimonious cladograms that is consistent with these limits. Our estimation procedure also identifies haplotypes that are candidates for being products of recombination. If recombination is extensive, our algorithm subdivides the DNA region into two or more subsections, each having little or no internal recombination. We apply this estimation procedure to three data sets to illustrate varying degrees of cladogram ambiguity and recombination.     

The influence of glucocorticoid on albumin synthesis and its messenger RNA in rat in vivo and in hepatocyte suspension culture

Corticosteroids are known to stimulate the synthesis of a number of liver-specific proteins. The reports regarding the effect of glucocorticoid on albumin synthesis in vivo and in vitro are controversial. In an attempt to determine the mechanism by which glucocorticoid exerts its influence on hepatic albumin synthesis and to find an explanation for the conflicting data, we have studied the effect of dexamethasone disodium phosphate on albumin synthesis and albumin messenger RNA as determined by the molecular hybridization technique in hepatocytes in rat in vivo and in suspension culture. In hepatocyte suspension culture, addition of 0.48 μM dexamethasone in medium at zero time led to a significant increase (20%) in incorporation of labeled precursor into albumin as compared to control experiments; this was accompanied by a maintainance of the initial level of full-length albumin mRNA for a 9 h period. In hepatocytes cultured without dexamethasone in the medium there was a progressive loss of albumin mRNA content. Despite this finding, dexamethasone was not able to increase the albumin mRNA content in hepatocyte to a level higher than the initial value. Moreover, administration of this hormone either intraperitoneally or intravenously into rats did not lead to enhanced cell-free albumin synthesis or to an increased level of albumin mRNA. These findings suggest that glucocorticoid does not play an essential role in the regulation of albumin synthesis in vivo. In vitro, however, glucocorticoid leads to a preservation of the initial level of albumin mRNA and thus plays a role in the control of spontaneous dedifferentiation of liver cells in culture.     

Identification of 3′,4′,5′-trimethoxychalcone analogues as potent inhibitors of Helicobacter pylori-induced inflammation in human gastric epithelial cells

Efforts to identify potent small molecule inhibitors of Helicobacter pylori led to the evaluation of 23 3′,4′,5′-trimethoxychalcone analogues. Some of the compounds displayed potent antibacterial activity against H. pylori. Three most active and selective compounds 1, 7, and 13 also showed the bactericide activity against the reference as well as multidrug-resistant strains of H. pylori. Additionally, the aforementioned three compounds potentially inhibited the H. pylori adhesion and invasion to human gastric epithelial (AGS) cells. Furthermore, these selective compounds inhibited the H. pylori-induced gastric inflammation by reduced inflammatory mediator’s nuclear factor kappa B activation, and the secretion of interleukin-8.     

Caspase activation, sialidase release and changes in sialylation pattern of recombinant human erythropoietin produced by CHO cells in batch and fed-batch cultures

The activation of caspases represents a crucial turning point during a batch or a fed-batch culture of mammalian cells. It not only affects the quantity but also the quality of the recombinant glycoprotein produced. In this study, the activation of various caspases, the release of intracellular sialidase and the changes in sialylation pattern of a recombinant product, erythropoietin (EPO), in the culture medium were analyzed in both batch and fed-batch cultures. In both setups, all caspase activities peaked at the culture time point at which decline of cell viability was most pronounced. In addition, the release of intracellular lactate dehydrogenase (LDH) was also tracked during these cultures. The increase in LDH activity in the medium coincided with the increase of intracellular caspase activities, the release of sialidase and the observed decline in cell viability, suggesting that the LDH activity in the medium can be used as an indirect indicator of apoptotic cell death in bioreactors. Isoelectric focusing (IEF) coupled with double blotting was employed to analyze the changes in sialylation pattern of the recombinant EPO. This assay resulted in a prompt resolution of secreted EPO isoforms in a time course format. IEF profile of batch culture showed relatively consistent product sialylation compared to fed-batch culture, which showed gradual band shifts towards the isoforms with fewer sialic acid as the culture progressed. These data provided a guideline for the optimal time point to terminate the culture and collect products in batch and fed-batch cultures.Kok Hwee Chuan and Sing Fee Lim contributed equally to this work.