Preferential amplification of rearranged sequences near amplified adenylate deaminase genes.

In a previous study of three independent families of mutants selected for overproduction of adenylate deaminase (AMPD), we were not able to isolate a cDNA probe for the gene and so could not demonstrate its amplification directly. In addition to overproduction of AMPD, four proteins of unknown function, designated W, X, Y1, and Y2, accumulated, and by using the corresponding cDNA probes, we demonstrated amplification of all four genes. In independent mutant clones, sometimes all and sometimes only a subset of these genes were amplified. Assuming that all five genes are linked, the pattern of their coamplification suggested a genetic map in which AMPD lies between W and Y1. We show here that a two-step chromosome walk joins the W and Y1 genes, that the AMPD gene is the only expressed sequence between them, and that its amplification is indeed responsible for overproduction of the AMPD protein. In the course of this work, we cloned and studied two novel joints which mark rearrangements on either side of the AMPD gene. Each joint was generated independently in a single first-step mutant at single or low copy number. Remarkably, each joint was amplified preferentially in every second- and third-step mutant derived from the first-step line in which it was originally present, suggesting that the two independent rearrangements each generated amplification-prone structures.     

Preferential PCR amplification of parasitic protistan small subunit rDNA from metazoan tissues

A "universal non-metazoan" polymerase chain reaction (UNonMet-PCR) that selectively amplifies a segment of nonmetazoan Small Subunit (SSU) rDNA gene was validated. The primers used were: 18S-EUK581-F (5'-GTGCCAGCAGCCGCG-3') and 18S-EUK1134-R (5'-TTTAAGTTTCAGCCTTGCG-3') with specificity provided by the 19-base reverse primer. Its target site is highly conserved across the Archaea, Bacteria, and eukaryotes (including fungi), but not most Metazoa (except Porifera, Ctenophora, and Myxozoa) which have mismatches at bases 14 and 19 resulting in poor or failed amplification. During validation, UNonMet-PCR amplified SSU rDNA gene fragments from all assayed protists (n = 16 from 7 higher taxa, including two species of marine phytoplankton) and Fungi (n = 3) but amplified very poorly or not at all most assayed Metazoa (n = 13 from 8 higher taxa). When a nonmetazoan parasite was present in a metazoan host, the parasite DNA was preferentially amplified. For example, DNA from the parasite Trypanosoma danilewskyi was preferentially amplified in mixtures containing up to 1,000 x more goldfish Carassius auratus (host) DNA. Also, the weak amplification of uninfected host (Chionoecetes tanneri) SSU rDNA did not occur in the presence of a natural infection with a parasite (Hematodinium sp.). Only Hematodinium sp. SSU rDNA was amplified in samples from infected C. tanneri. This UNonMet-PCR is a powerful tool for amplifying SSU rDNA from non-metazoan pathogens or symbionts that have not been isolated from metazoan hosts.     

Preferential amplification and molecular characterization of junction sequences of a pathogenetic deletion in human mitochondrial DNA

Deletions of mitochondrial DNA have been detected in skeletal muscle of some patients with mitochondrial encephalomyopathies, but their junctions have been defined only approximately. We developed a procedure, using widely spaced primers for the polymerase chain reaction, that amplifies preferentially the sequences bracketing the deletion. This procedure permits detection of minor proportions, not detectable by Southern analysis, of deleted mitochondrial DNA species in a heteroplasmic mixture. Different proportions of intact mitochondrial DNA and species deleted from nucleotide 8708 to 13,722 were found in skeletal muscle, blood, and urinary epithelial cells from a patient with chronic progressive external ophthalmoplegia. These data indicate that the mutation occurred at or before early embryonic development and provide the first definition at the nucleotide level of a human disease caused by a deletion of mitochondrial DNA.     

Epidemiological studies of HCMV infection in children by restriction analysis of long-PCR products

Sequences of MIE, pol, gB gene longer fragments (2.0-2.6 kb) were amplified in two stage PCR (nested PCR) from 46 PEG-urine extracts. Restriction of particular amplicons with selected endonucleases identified 19 distinct HCMV genotypes: 8 for MIE, 6 for gB and 5 for pol. MIE profiling revealed highest differentiation power. Our data showed the correlation among MIE, pol, and gB genotypes and clinical disease, indicating that genotypes may contribute the course of HCMV infection. For newborns with symptomatic infection, the highest level of variability among three genes analysed was obtained in comparison to samples of newborns with asymptomatic infection. Long PCR technique coupled with restriction analysis may be successfully applied in HCMV genotypes differentiation in samples taken directly from HCMV infected individuals, thus may be useful in prognosis of clinical infection.     

Preferential and absolute interactions of solvent components with proteins in mixed solvent systems

An equation is derived relating total and preferential interactions of solvent components with macromolecules in a three-component system. Application of this equation to literature data shows that binding of non-polar solvents to proteins parallels the unfolding of the latter. This observation is discussed in terms of local inter-residue and residue-solvent interactions.     

Preferential mating in symmetric multilocus systems: stability conditions of the central equilibrium

Several multilocus models that incorporate both preferential mating and viability selection are studied. Specifically, a class of symmetric heterozygosity models are considered that assign individuals to phenotypic classes according to which loci are in heterozygous state regardless of the actual allelic content. Otherwise, an arbitrary number of loci, number of alleles per locus, and arbitrary recombination scheme, viability parameters and preferential mating pattern based on phenotypes are allowed. The conditions for the stability of a central polymorphism are indicated and interpreted. The effects of viability and preference selection may be summarized in a single quantity for each phenotypic class, a generalized fitness. Preferential assortative mating alone can produce stability for a central polymorphism as in the case of viability selection when sexual attractiveness or general fitness increases with higher levels of heterozygosity. The situation is more complex with sexual selection.     

Preferential target is mitochondria in alpha-mangostin-induced apoptosis in human leukemia HL60 cells

Our previous study has shown that alpha-mangostin, a xanthone from the pericarps of mangosteen, induces caspase-3-dependent apoptosis in HL60 cells. In the current study, we investigated the mechanism of apoptosis induced by alpha-mangostin in HL60 cells. Alpha-mangostin-treated HL60 cells demonstrated caspase-9 and -3 activation but not -8, which leads us to assume that alpha-mangostin may mediate the mitochondrial pathway in the apoptosis. Parameters of mitochondrial dysfunction including swelling, loss of membrane potential (deltapsim), decrease in intracellular ATP, ROS accumulation, and cytochrome c/AIF release, were observed within 1 or 2 h after the treatment. On the other hand, alpha-mangostin-treatment did not affect expression of bcl-2 family proteins and activation of MAP kinases. These findings indicate that alpha-mangostin preferentially targets mitochondria in the early phase, resulting in indication of apoptosis in HL60 cells. Furthermore, we examined the structure-activity relationship between xanthone derivatives including alpha-mangostin and the potency of deltapsim-loss in HL60 cells. Interestingly, replacement of hydroxyl group by methoxy group remarkably decreased its potency. It was also shown that the cytotoxicity substantially correlated with deltapsim decrease. These results indicate that alpha-mangostin and its analogs would be candidates for preventive and therapeutic application for cancer treatment.     

Highly-sensitive electrochemical immunosensing method based on dual amplification systems

In order to rapidly and simultaneously quantify and screen trace levels of multiple biomarkers in a single sample, rapid 1,1'-oxalyldiimidazole chemiluminescence (ODI CL) was applied as a biosensor of immunoassays using various enzymes such as alkaline phosphatase (ALP) and horseradish peroxidise (HRP). (1) Fluorescein was formed from the reaction of fluorescein diphosphate (FDP) and immuno-complex conjugated with ALP. (2) Resorufin was formed from the reaction between Amplex Red and H(2)O(2) in the presence of immuno-complex conjugated with HRP. When ODI CL reagents (H(2)O(2) in isopropyl alcohol, ODI in ethyl acetate) were injected in a test tube or strip-well containing fluorescein and resorufin formed from above two reactions a bright CL emission spectrum having two peaks (518 nm for fluorescein and 602 nm for resorufin) was observed. The two peaks can be independently quantified with an appropriate statistical tool capable of deconvoluting multiple emission peaks. In conclusion, we expect that ODI chemiluminescent enzyme immunoassays (CLEIAs) using a couple of enzymes conjugated with antigen or antibody and substrates can rapidly and simultaneously quantify and screen multiple biomarkers in a single sample.     

Preferential mating in symmetric multilocus systems: limits for multiallelism and for many Loci

Models in which general forms of preferential mating have been superimposed on the framework of the symmetric heterozygosity selection regime have been examined previously with respect to the existence and local stability of a central polymorphic equilibrium. The results are now extended to produce the limiting form of the stability conditions in two cases: First, where the number of alleles per locus is assumed to be very large; second, where the number of loci affecting the character is very large. It is argued that some type of frequency dependence in the mating pattern must be included, and a particular case is examined in detail. It is shown that multiallelism is ambiguous in its effect on stability, while an increasing number of loci, at least under zero linkage, leads to a simple stability condition which is analogous to the one-locus heterosis principle. Assortative mating appears to be more likely to produce a stable central polymorphism under high levels of allelism than is sexual selection, but is relatively very much weaker than sexual or viability selection if the number of loci involved is large.     

Preferential ligand binding to multi-state acceptor systems: the unexplored paradox of acceptor self-association that is ligand-mediated but detrimental to ligand binding

Consideration is given to the interactions of ligand with self-associating acceptor systems for which preferential ligand binding is an ambiguous term, in that the acceptor species with greater affinity for ligand possesses relatively fewer binding sites. A paradoxical situation wherein ligand-mediated self-association is seemingly detrimental to ligand binding is shown to be the predicted outcome for a transient range of ligand concentrations. This outcome reflects the existence of a critical point in the dependence of the extent of acceptor self-association upon ligand concentration that coincides with a cross-over point of ligand-binding curves for different, fixed total concentrations of acceptor. By classical differentiation methods the conditions for the existence of these critical points are established not only for two-state acceptor systems but also for three-state acceptor systems in which the ligand-binding form of monomer also undergoes reversible isomerization to an inactive state. Similar procedures are used to comment upon the forms of binding curves for the three-state acceptor systems, the Scatchard representations of which may exhibit as many as three critical points (two maxima and a minimum). This delineation of quantitative expressions for critical points and other distinctive features associated with the conflicting interplay of ligand-binding and self-association behaviour should provide a more definitive means of characterizing systems with one acceptor state the preferred binding form on affinity grounds but with the other the preferred state from the viewpoint of binding-site numbers.     

Preferential retrotransposition in aging yeast mother cells is correlated with increased genome instability

Retrotransposon expression or mobility is increased with age in multiple species and could promote genome instability or altered gene expression during aging. However, it is unclear whether activation of retrotransposons during aging is an indirect result of global changes in chromatin and gene regulation or a result of retrotransposon-specific mechanisms. Retromobility of a marked chromosomal Ty1 retrotransposon in Saccharomyces cerevisiae was elevated in mother cells relative to their daughter cells, as determined by magnetic cell sorting of mothers and daughters. Retromobility frequencies in aging mother cells were significantly higher than those predicted by cell age and the rate of mobility in young populations, beginning when mother cells were only several generations old. New Ty1 insertions in aging mothers were more strongly correlated with gross chromosome rearrangements than in young cells and were more often at non-preferred target sites. Mother cells were more likely to have high concentrations and bright foci of Ty1 Gag–GFP than their daughter cells. Levels of extrachromosomal Ty1 cDNA were also significantly higher in aged mother cell populations than their daughter cell populations. These observations are consistent with a retrotransposon-specific mechanism that causes retrotransposition to occur preferentially in yeast mother cells as they begin to age, as opposed to activation by phenotypic changes associated with very old age. These findings will likely be relevant for understanding retrotransposons and aging in many organisms, based on similarities in regulation and consequences of retrotransposition in diverse species.     

Distinct mechanisms for two amplification systems of insulin release.

The mechanisms whereby activation of the cyclic AMP-dependent protein kinase A or the Ca2+-phospholipid-dependent protein kinase C amplifies insulin release were studied with mouse islets. Forskolin and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) were used to stimulate adenylate cyclase and protein kinase C respectively. The sulphonylurea tolbutamide was used to initiate insulin release in the presence of 3 mM-glucose. Tolbutamide alone inhibited 86Rb+ efflux, depolarized beta-cell membrane, triggered electrical activity, accelerated 45Ca2+ influx and efflux and stimulated insulin release. Forskolin alone only slightly inhibited 86Rb+ efflux, but markedly increased the effects of tolbutamide on electrical activity, 45Ca2+ influx and efflux, and insulin release. In the absence of Ca2+, only the inhibition of 86Rb+ efflux persisted. TPA (100 nM) alone slightly accelerated 45Ca2+ efflux and insulin release without affecting 45Ca2+ influx or beta-cell membrane potential. It increased the effects of tolbutamide on 45Ca2+ efflux and insulin release without changing 86Rb+ efflux, 45Ca2+ influx or electrical activity. Omission of extracellular Ca2+ suppressed all effects due to the combination of TPA and tolbutamide, but not those of TPA alone. Though ineffective alone, 10 nM-TPA amplified the releasing action of tolbutamide without affecting its ionic and electrical effects. In conclusion, the two amplification systems of insulin release involve at least partially distinct mechanisms. The cyclic AMP but not the protein kinase C system initiating signal (Ca2+ influx) triggered by the primary secretagogue.