Comparison of statistics for candidate-gene association studies using cases and parents.

Studies of association between candidate genes and disease can be designed to use cases with disease, and in place of nonrelated controls, their parents. The advantage of this design is the elimination of spurious differences due to ethnic differences between cases and nonrelated controls. However, several statistical methods of analysis have been proposed in the literature, and the choice of analysis is not always clear. We review some of the statistical methods currently developed and present two new statistical methods aimed at specific genetic hypotheses of dominance and recessivity of the candidate gene. These new methods can be more powerful than other current methods, as demonstrated by simulations. The basis of these new statistical methods is a likelihood approach. The advantage of the likelihood framework is that regression models can be developed to assess genotype-environment interactions, as well as the relative contribution that alleles at the candidate-gene locus make to the relative risk (RR) of disease. This latter development allows testing of (1) whether interactions between alleles exist, on the scale of log RR, and (2) whether alleles originating from the mother or father of a case impart different risks, i.e., genomic imprinting.     

Polypyrimidine Tract Binding Protein Prevents Activity of an Intronic Regulatory Element That Promotes Usage of a Composite 3��-Terminal Exon

Alternative splicing of 3′-terminal exons plays a critical role in gene expression by producing mRNA with distinct 3′-untranslated regions that regulate their fate and their expression. The Xenopus α-tropomyosin pre-mRNA possesses a composite internal/3′-terminal exon (exon 9A9′) that is differentially processed depending on the embryonic tissue. Exon 9A9′ is repressed in non-muscle tissue by the polypyrimidine tract binding protein, whereas it is selected as a 3′-terminal or internal exon in myotomal cells and adult striated muscles, respectively. We report here the identification of an intronic regulatory element, designated the upstream terminal exon enhancer (UTE), that is required for the specific usage of exon 9A9′ as a 3′-terminal exon in the myotome. We demonstrate that polypyrimidine tract binding protein prevents the activity of UTE in non-muscle cells, whereas a subclass of serine/arginine rich (SR) proteins promotes the selection of exon 9A9′ in a UTE-dependent way. Morpholino-targeted blocking of UTE in the embryo strongly reduced the inclusion of exon 9A9′ as a 3′-terminal exon in the endogenous mRNA, demonstrating the function of UTE under physiological circumstances. This strategy allowed us to reveal a splicing pathway that generates a mRNA with no in frame stop codon and whose steady-state level is translation-dependent. This result suggests that a non-stop decay mechanism participates in the strict control of the 3′-end processing of the α-tropomyosin pre-mRNA.     

The preparation and kinetics of lactate dehydrogenase attached to water-insoluble particles and sheets

1. The preparation of lactate dehydrogenase covalently attached to anion-exchange cellulose particles and sheets by use of a dichloro-sym-triazinyl dyestuff, Procion brilliant orange MGS, is described. 2. The stability and kinetic properties of these preparations were investigated. 3. An equation is derived to describe the change in concentration of a substrate when passed through a uniform bed of a substrate-inhibited enzyme. A number of theoretical curves are shown to illustrate the system. 4. A titrimetric assay for lactate dehydrogenase is described, and shown to be stoicheiometric over the range pH5.0-9.2. 5. The results are discussed in relation to previous work, and the effects of charged groups on the support, and of the diffusion film surrounding any particle in suspension, are treated qualitatively.     

Suppressive effects of soluble histocompatibility antigens on the in vitro generation of cytotoxic T cells to D-end alloantigens

Murine histocompatibility antigens were solubilized from the spleens and lungs of C57BL/6 (H-2b) animals with hypertonic salt (3 M KC1). Aggregate-free soluble antigens were incubated with nonadherent lymph node cells from BALB/c (H-2d) mice for 18 hr prior to their use as responder cells in the mixed-lymphocyte reaction (MLR). It was found that the generation of cytotoxic cells was suppressed while the proliferative response was not affected. The observed suppression was not due to a shift in the kinetics of the generation of cytotoxicity as determined throughout a 10-day culture period. The suppression was specific in that the response in MLR to unrelated H-2f stimulator cells and the subsequent generation of cytotoxic cells were unchanged. Using various H-2 recombinant strains as target cells in the assay of cell-mediated lympholysis, suppression of cytotoxicity was observed when the D end, but not the K end, was shared with the C57BL/6 strain from which the antigens were derived.     

The effect of water-miscible solvents on the Δ1-dehydrogenase activity of free and PAAH-entrapped Arthrobacter simplex

Summary The effect of water-miscible cosolvents on biotransformations of poorly water-soluble substrates by immobilized cells was investigated, using ¹-dehydrogenation of hydrocortisone by Arthrobacter simplex as a model. Criteria for solvent selection on the basis of retention of enzymic activity were postulated and tested. Diols were considered to be the most suitable group of solvents. Substrate solubility increased tenfold in 30% (v/v) ethylene glycol, but reaction rates were significantly slower in such solutions. This was mainly caused by a decrease of oxygen solubility in the presence of the cosolvent and conformational changes imposed on the intracellular enzyme by cosolvent molecules penetrating the cell. The inhibition could be eliminated by the addition of an artificial electron acceptor, phenazine methosulphate (PMS). Reaction rates faster than those for substrate suspensions (no cosolvent added) could thus be achieved. Immobilization of Arthrobacter simplex in cross-linked polyacrylamide hydrazide gave high retentions of activity. PMS exhibited toxic effects on the entrapped cells, leading to reduced activity after extended use.     

Competition between Dunaliella species at high salinity

Information on the distribution of more than 225 species of planktonic, periphytic, and benthic rotifers from diverse waters in south and central Sweden was analyzed for pH preference. No particularly strong correlation was found between pH and any other environmental factor, with regards to rotifer distribution. However, species indicating oligotrophy generally have their pH optima at or below 7.0, while those indicating eutrophy occur at or above this level. Rotifers found in acidic waters are often non-planktonic or semiplanktonic.     

Transbilayer movement of bile acids in model membranes

The ability of bile acids to traverse membranes has important implications for their reabsorption from the gut, recirculation to and uptake into the liver, and resecretion into bile. The rate constant for transbilayer movement, or "flip-flop", of three common, unconjugated bile acids was determined by 13C nuclear magnetic resonance spectroscopy. At high pH, the sodium salts of the bile acids did not appreciably traverse the bilayer; however, upon protonation a rapid equilibration between the inner and outer monolayers occurred. The rate of flip-flop of each bile acid at 37 degrees C was found to be dependent on both number and location of hydroxyl groups but not on concentration in the bilayer over the range studied (2-4 wt%) nor on the presence of a different bile acid in the same bilayer.     

Enzymic interesterification of fats: Laboratory and pilot-scale studies with immobilized lipase from Rhizopus arrhizus

An immobilized lipase suitable for fat interesterification has been prepared by precipitation with acetone of a commercial lipase from Rhizopus arrhizus onto diatomaceous earth. As observed previously with a less active enzyme from Aspergillus sp., the interesterification activity was enhanced by addition of purified lipase or by high loadings of commercial enzyme. The interesterification activities reached maximum values in both cases. For immobilized preparations with purified enzyme, interesterification activity was also enhanced by the presence of a precoat of glutaraldehyde cross-linked commercial lipase. A 2.9-L column of immobilized lipase was used to interesterify batches of shea oleine (67 kg) and shea oil (40 kg). Little activity was lost processing shea oleine, but slow poisoning of the bed occurred when shea oil was fed to the column.