An optimized method for the generation of AFLP markers in a stingless bee (Melipona quadrifasciata) reveals a high degree of intracolonial genetic polymorphism

This study describes an optimized protocol for the generation of Amplified Fragment Length Polymorphism (AFLP) markers in a stingless bee. Essential modifications to standard protocols are a restriction enzyme digestion (EcoRI and Tru1I) in a two-step procedure, combined with a touchdown program in the selective PCR amplification step and product labelling by incorporation of alpha[P-33]dATP. In an analysis of 75 workers collected from three colonies of Melipona quadrifasciata we obtained 719 markers. Analysis of genetic variability revealed that on average 32% of the markers were polymorphic within a colony. Compared to the overall percentage of polymorphism (44% of the markers detected in our bee samples), the observed rates of within-colony polymorphism are remarkably high, considering that the workers of each colony were all of spring of a singly mated queen.

cpDNA-RFLP in Ceramium (Rhodophyta): Intraspecific polymorphism and species-level phylogeny

Restriction fragment length polymorphism (RFLP) analysis of chloroplast (cp) DNA is a powerful tool for the study of microevolutionary processes in land plants, yet has not previously been applied to seaweed populations. We used cpDNA-RFLP, detected on Southern blots using labeled total plastid DNA, to search for intraspecific and intrapopulational cpDNA RFLP polymorphism in two species of the common red algal genus Ceramium in Ireland and Britain. In C. botryocarpum one polymorphism was detected in one individual among 18 from two populations. Twenty-six individuals of C. virgatum from five populations at three locations exhibited a total of four haplotypes. One was frequent (80.8% of individuals); the others were rare (7.7, 7.7 and 4.2%) and were private to particular populations. Polymorphism was observed in two populations. The corrected mean was 2.26 +/- 0.36 haplotypes per population, which was within the typical range determined for higher plants using similar techniques. The spatial distribution of haplotypes was heterogeneous, with highly significant population differentiation (P = 0.00018; Fisher's exact test). Intraspecific polymorphism in C. virgatum had no impact on species-level phylogenetic reconstruction. This is the first unequivocal report of both intraspecific and intrapopulational cpDNA-RFLP polymorphism in algae.

Crystal and liquid crystalline polymorphism in 1-alkyl-3-methylimidazolium tetrachloropalladate(II) salts

1-Alkyl-3-methylimidazolium tetrachloropalladate(ii) salts ([C-n-mim](2)[PdCl4], n = 10, 12, 14, 16, 18) containing a single, linear alkyl-chain substituent on the cation have been synthesised and their behaviour characterised by differential scanning calorimetry, polarising optical microscopy and small-angle X-ray scattering. The salts display thermotropic polymorphism, exhibiting both crystal-crystal transitions and, for n = 14-18, the formation of a thermotropic smectic liquid crystalline phase.

Association of functional haem oxygenase-1 gene promoter polymorphism with polycystic kidney disease and IgA nephropathy

Background: Haem oxygenase-1 (HO-1) is a cytoprotective molecule that is reported to have a protective role in a variety of experimental models of renal injury. A functional dinucleotide repeat (GT)n polymorphism, within the HO-1 promoter, regulates HO-1 gene expression; a short number of repeats (S-allele <25) increases transcription. We report the first assessment of the role of this HO-1 gene promoter polymorphism in chronic kidney disease due to autosomal dominant polycystic kidney disease (ADPKD) and IgA nephropathy (IgAN).

Methods: The DNA from 160 patients (99% Caucasian) on renal replacement therapy (RRT) was genotyped. The primary renal disease was ADPKD in 100 patients and biopsy-proven IgAN in 60 patients.

Results: Overall, the mean age at commencement of RRT was not significantly different between patients with and without an S-allele (44.1 years versus 45.0 years, P = 0.64). In patients with ADPKD, the age at commencement of RRT was comparable regardless of the HO-1 genotype (47.7 years versus 46.7 years, P = 0.59). The same was true in patients with IgAN (38.3 years versus 42.2 years, P = 0.28).

Conclusion: This suggests that the functional HO-1 promoter polymorphism does not influence renal survival in CKD due to ADPKD or IgAN.

Homocysteine, Methylenetetrahydrofolate Reductase C677T Polymorphism, and Risk of Retnal Vein Occlusion: A Meta-analysis

Objective: To assess the role of plasma total homocysteine (tHcy) concentrations and homozygosity for the thermolabile variant of the methylenetetrahydrofolate reductase (MTHFR) C677T gene as risk factors for retinal vascular occlusive disease.

Design: Retinal vein occlusion (RVO) is an important cause of vision loss. Early meta-analyses showed that tHcy was associated with an increased risk of RVO, but a significant number of new studies have been published. Participants and/or Controls: RVO patients and controls.

Methods: Data sources included MEDLINE, Web of Science, and PubMed searches and searching reference lists of relevant articles and reviews. Reviewers searched the databases, selected the studies, and then extracted data. Results were pooled quantitatively using meta-analytic methods.

Main Outcome Measures: tHcy concentrations and MTHFR genotype.

Results: There were 25 case-control studies for tHcy (1533 cases and 1708 controls) and 18 case-control studies for MTHFR (1082 cases and 4706 controls). The mean tHcy was on average 2.8 mol/L (95% confidence
interval [CI], 1.8 –3.7) greater in the RVO cases compared with controls, but there was evidence of between-study heterogeneity (P0.001, I2 93%). There was funnel plot asymmetry suggesting publication bias. There was no evidence of association between homozygosity for the MTHFR C677T genotype and RVO (odds ratio [OR] 1.20; 95% CI, 0.84–1.71), but again marked heterogeneity (P 0.004, I2 53%) was observed.

Conclusions: There was some evidence that elevated tHcy was associated with RVO, but not homozygosity for the MTHFR C677T genotype. Both analyses should be interpreted cautiously because of marked heterogeneity between the study estimates and possible effect of publication bias on the tHcy findings.

Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

The human prion protein residue 129 polymorphism lies within a cluster of epitopes for T cell recognition.

T cell immune responses to central nervous system-derived and other self-antigens are commonly described in both healthy and autoimmune individuals. However, in the case of the human prion protein (PrP), it has been argued that immunologic tolerance is uncommonly robust. Although development of an effective vaccine for prion disease requires breaking of tolerance to PrP, the extent of immune tolerance to PrP and the identity of immunodominant regions of the protein have not previously been determined in humans. We analyzed PrP T cell epitopes both by using a predictive algorithm and by measuring functional immune responses from healthy donors. Interestingly, clusters of epitopes were focused around the area of the polymorphic residue 129, previously identified as an indicator of susceptibility to prion disease, and in the C-terminal region. Moreover, responses were seen to PrP peptide 121-134 containing methionine at position 129, whereas PrP 121-134 [129V] was not immunogenic. The residue 129 polymorphism was also associated with distinct patterns of cytokine response: PrP 128-141 [129M] inducing IL-4 and IL-6 production, which was not seen in response to PrP 128-141 [129V]. Our data suggest that the immunogenic regions of human PrP lie between residue 107 and the C-terminus and that, like with many other central nervous system antigens, healthy individuals carry responses to PrP within the T cell repertoire and yet do not experience deleterious autoimmune reactions.

Crystal polymorphism in 1-butyl-3-methylimidazolium halides: supporting ionic liquid formation by inhibition of crystallization

Crystallization of 1-butyl-3-methylimidazolium chloride from mixed ionic liquid or ionic liquid-aromatic solution, and from the melt yields different crystalline polymorphs, the first direct evidence for inhibition of crystallization in ionic liquids by polymorphism.

Evidence That Interspecies Polymorphism in the Human and Rat Cholecystokinin Receptor-2 Affects Structure of the Binding Site for the Endogenous Agonist Cholecystokinin

The cholecystokinin (CCK) receptor-2 exerts very important central and peripheral functions by binding the neuropeptides cholecystokinin or gastrin. Because this receptor is a potential therapeutic target, great interest has been devoted to the identification of efficient antagonists. However, interspecies genetic polymorphism that does not alter cholecystokinin-induced signaling was shown to markedly affect activity of synthetic ligands. In this context, precise structural study of the agonist binding site on the human cholecystokinin receptor-2 is a prerequisite to elucidating the molecular basis for its activation and to optimizing properties of synthetic ligands. In this study, using site-directed mutagenesis and molecular modeling, we delineated the binding site for CCK on the human cholecystokinin receptor-2 by mutating amino acids corresponding to that of the rat homolog. By doing so, we demonstrated that, although resembling that of rat homolog, the human cholecystokinin receptor-2 binding site also displays important distinct structural features that were demonstrated by susceptibility to several point mutations (F120A, Y189A, H207A). Furthermore, docking of CCK in the human and rat cholecystokinin receptor-2, followed by dynamic simulations, allowed us to propose a plausible structural explanation of the experimentally observed difference between rat and human cholecystokinin-2 receptors.