The two common polymorphic forms of human NRH-quinone oxidoreductase 2 (NQO2) have different biochemical properties

There are two common forms of NRH-quinone oxidoreductase 2 (NQO2) in the human population resulting from SNP rs1143684. One has phenylalanine at position 47 (NQO2-F47) and the other leucine (NQO2-L47). Using recombinant proteins, we show that these variants have similar steady state kinetic parameters, although NQO2-L47 has a slightly lower specificity constant. NQO2-L47 is less stable towards proteolytic digestion and thermal denaturation than NQO2-F47. Both forms are inhibited by resveratrol, but NQO2-F47 shows negative cooperativity with this inhibitor. Thus these data demonstrate, for the first time, clear biochemical differences between the variants which help explain previous biomedical and epidemiological findings. © 2014 Federation of European Biochemical Societies.

Molecular pedigree reconstruction and estimation of evolutionary parameters in a wild Atlantic salmon river system with incomplete sampling: a power analysis

Background: Pedigree reconstruction using genetic analysis provides a useful means to estimate fundamental population biology parameters relating to population demography, trait heritability and individual fitness when combined with other sources of data. However, there remain limitations to pedigree reconstruction in wild populations, particularly in systems where parent-offspring relationships cannot be directly observed, there is incomplete sampling of individuals, or molecular parentage inference relies on low quality DNA from archived material. While much can still be inferred from incomplete or sparse pedigrees, it is crucial to evaluate the quality and power of available genetic information a priori to testing specific biological hypotheses. Here, we used microsatellite markers to reconstruct a multi-generation pedigree of wild Atlantic salmon (Salmo salar L.) using archived scale samples collected with a total trapping system within a river over a 10 year period. Using a simulation-based approach, we determined the optimal microsatellite marker number for accurate parentage assignment, and evaluated the power of the resulting partial pedigree to investigate important evolutionary and quantitative genetic characteristics of salmon in the system.

Results: We show that at least 20 microsatellites (ave. 12 alleles/locus) are required to maximise parentage assignment and to improve the power to estimate reproductive success and heritability in this study system. We also show that 1.5 fold differences can be detected between groups simulated to have differing reproductive success, and that it is possible to detect moderate heritability values for continuous traits (h(2) similar to 0.40) with more than 80% power when using 28 moderately to highly polymorphic markers.

Conclusion: The methodologies and work flow described provide a robust approach for evaluating archived samples for pedigree-based research, even where only a proportion of the total population is sampled. The results demonstrate the feasibility of pedigree-based studies to address challenging ecological and evolutionary questions in free-living populations, where genealogies can be traced only using molecular tools, and that significant increases in pedigree assignment power can be achieved by using higher numbers of markers.

Donor leukemia following allogeneic bone marrow transplantation

Although allogeneic bone marrow transplantation has been shown to be a highly effective treatment for acute and chronic leukemia, leukemic relapse remains a significant problem. Leukemic relapse occurs in recipient cells in the majority of cases, but the paucity of donor cell leukemias may reflect the sensitivity of the investigative technique. We have developed a highly sensitive technique to identify the origin of all hematopoietic cells in the post transplant state which is based on PCR amplification of microsatellites, polymorphic tandem repetitive elements. We have identified donor leukemia (AML M5) following a sex matched BMT for severe aplastic anemia, verified a previously reported case of donor leukemia following BMT for chronic granulocytic leukemia and recently identified an acquired cytogenetic abnormality(del 11q23) in donor cells four years following an apparently successful BMT for AML. In all cases the donors have remained healthy. Postulated mechanisms include transfer to the transplanted marrow of a dormant oncogene residing in the DNA of either a virus, the chromosomes of degenerating irradiation damaged host leukemic cells or in the marrow stroma which is radioresistant and host in origin following BMT. Using sensitive techniques donor leukemia has been shown to be a more common event than was previously thought and an understanding of its pathogenesis may allow us to elucidate leukemogenic mechanisms in man.

Localization of polymorphic N-acetyltransferase (NAT2) in tissues of inbred mice

Like humans, mice exhibit polymorphism in the N-acetylation of aromatic amines, many of which are toxic and/or carcinogenic. Mice have three N-acetyltransferase (Nat) genes, Nat1, Nat2 and Nat3, and Nat2 is known to be polymorphic. There is a dramatic difference in the acetylation of NAT2 substrates by blood from fast (C57BL/6J) compared with slow acetylator (A/J) mice. However, the acetylation of these substrates by liver cytosols from the two strains is very similar. In order to determine whether the expression of the NAT2 protein corresponded with the activities measured, a polyclonal antipeptide antisera was raised against the C-terminal decapeptide of NAT2 and characterized using recombinant murine NAT2 antigen. Enzyme-linked immunosorbent assays (ELISAs) demonstrated that the anti-NAT2 antiserum bound in a concentration-dependent fashion to recombinant NAT2. Immunochemical analysis of mouse liver cytosols from C57BL/6J or A/J livers indicated that the level of NAT2 protein expressed in the two strains was similar. Immunohistochemical staining of C57BL/6J liver with anti-NAT2 antiserum showed that NAT2 was expressed in hepatocytes throughout the liver although the intensity of staining in the perivenous (centrilobular) region was higher than that in the periportal region. NAT2 was also detected in epithelial cells in the lung, kidney, bladder, small intestine and skin as well as in erythrocytes and lymphocytes in the spleen and hair follicles and sebaceous glands in the skin. Characterization of the distribution of NAT2 will be of value in elucidating the role of polymorphic N-acetylation in protecting the organism from environmental insults as well as in endogenous metabolism.

Genetic conectivity patterns in Holothuria Mammata considering different spatial scales

Dissertação de Mestrado, Biologia Marinha, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

Postpacing abnormal repolarization in catecholaminergic polymorphic ventricular tachycardia associated with a mutation in the cardiac ryanodine receptor gene.

BACKGROUND Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmogenic disease for which electrophysiological studies (EPS) have shown to be of limited value.OBJECTIVE This study presents a CPVT family in which marked postpacing repolarization abnormalities during EPS were the only consistent phenotypic manifestation of ryanodine receptor (RyR2) mutation carriers.METHODS The study was prompted by the observation of transient marked QT prolongation preceding initiation of ventricular fibrillation during atrial fibrillation in a boy with a family history of sudden cardiac death (SCD). Family members underwent exercise and pharmacologic electrocardiographic testing with epinephrine, adenosine, and flecainide. Noninvasive clinical test results were normal in 10 patients evaluated, except for both epinephrine- and exercise-induced ventricular arrhythmias in 1. EPS included bursts of ventricular pacing and programmed ventricular extrastimulation reproducing short-long sequences. Genetic screening involved direct sequencing of genes involved in long QT syndrome as well as RyR2.RESULTS Six patients demonstrated a marked increase in QT interval only in the first beat after cessation of ventricular pacing and/or extrastimulation. All 6 patients were found to have a heterozygous missense mutation (M4109R) in RyR2. Two of them, presenting with aborted SCD, also had a second missense mutation (I406T- RyR2). Four family members without RyR2 mutations did not display prominent postpacing QT changes.CONCLUSION M4109R- RyR2 is associated with a high incidence of SCD. The contribution of I406T to the clinical phenotype is unclear. In contrast to exercise testing, marked postpacing repolarization changes in a single beat accurately predicted carriers of M4109R- RyR2 in this family.

The Implications of Forager Behaviour for Social Organisation in a Socially Polymorphic Carpenter Bee (Xylocopa virginica)

In social Hymenoptera, the division of labour is a major step in the evolution of sociality. Bees, which express many different kinds of sociality, can be classified according to how individuals share or do not share foraging and reproductive activities (Michener, 1974). The large carpenter bee, Xylocopa virginica, lives in populations with both solitary and social nests. In social nests, reproduction is controlled by the dominant female, who does all of her own foraging and egg-laying, while the subordinates guard the nest only. This study examined foraging behaviour as a way to classify the social hierarchy. Individual females were marked, measured and intensely observed for the foraging season. It was found that a large number of subordinates forage and likely obtain more reproductive fitness than previously thought. The dominance hierarchy is very likely a social queue, in which bees take turns foraging and egg-laying.

Microsatellites from Fosterella christophii (Bromeliaceae) by de novo transcriptome sequencing on the Pacific Biosciences RS platform

• Premise of the study: Microsatellite markers were developed in Fosterella christophii (Bromeliaceae) to investigate the genetic diversity and population structure within the F. micrantha group, comprising F. christophii, F. micrantha, and F. villosula. • Methods and Results: Full-length cDNAs were isolated from F. christophii and sequenced on a Pacific Biosciences RS platform. A total of 1590 high-quality consensus isoforms were assembled into 971 unigenes containing 421 perfect microsatellites. Thirty primer sets were designed, of which 13 revealed a high level of polymorphism in three populations of F. christophii, with four to nine alleles per locus. Each of these 13 loci cross-amplified in the closely related species F. micrantha and F. villosula, with one to six and one to 11 alleles per locus, respectively. • Conclusions: The new markers are promising tools to study the population genetics of F. christophii and to discover species boundaries within the F. micrantha group.