A quantitative real-time polymerase chain reaction assay for the seagrass pathogen Labyrinthula zosterae.

The protist Labyrinthula zosterae (Phylum Bigyra, sensu Tsui et al. 2009) has been identified as a causative agent of wasting disease in eelgrass (Zostera marina), of which the most intense outbreak led to the destruction of 90% of eelgrass beds in eastern North America and western Europe in the 1930s. Outbreaks still occur today, albeit at a smaller scale. Traditionally, L. zosterae has been quantified by measuring the necrotic area of Z. marina leaf tissue. This indirect method can however only lead to a very rough estimate of pathogen load. Here, we present a quantitative real-time polymerase chain reaction (qPCR) approach to directly detect and quantify L. zosterae in eelgrass tissue. Based on the internal transcribed spacer (ITS) sequences of rRNA genes, species-specific primers were designed. Using our qPCR, we were able to quantify accurately and specifically L. zosterae load both from culture and eelgrass leaves using material from Europe and North America. Our detection limit was less than one L. zosterae cell. Our results demonstrate the potential of this qPCR assay to provide rapid, accurate and sensitive molecular identification and quantification of L. zosterae. In view of declining seagrass populations worldwide, this method will provide a valuable tool for seagrass ecologists and conservation projects.

Molecular investigation of the functional relevance of missense variants of ICAM-1.

In genome-wide studies, the intercellular adhesion molecule-1 (ICAM-1) locus has been associated with cardiovascular and inflammatory bowel diseases. To determine the functional relevance of five missense ICAM-1 variants (G241R; I316V; P352L; K469E; R478W), we generated wild-type and variant proteins [M2(241R); M3(469E); M4(352L); M5(478W); M6(316V); M7(352L/469E)] and transiently transfected CV1 cells. Reverse transcription PCR, western blot, and ELISA did not reveal any differences in mRNA and protein expression levels for any construct. Conversely, in pulse-chase experiments, compared with wild-type (90-120 min), M3 and M5 possessed a prolonged half-life of approximately 150 min, whereas M2, M4, and M7 displayed a decreased half-life of approximately 60-75 min, implying differences in protein degradation. Our results do not indicate a major impact of missense variants on ICAM-1 biological function, even if G241R and K469E were functional in pulse-chase experiments. Whether these differences in protein stability exert measurable functional consequences needs to be elucidated further.

Neutrophil elastase gene variation and coronary heart disease.

AIMS: Identification and functional characterization of variants in the neutrophil elastase (ELA2) gene in cardiovascular disease. METHODS: From participants of the ECTIM (Etude Cas-Témoins sur l'infarctus du Myocarde) Study with myocardial infarction (MI) 2082 chromosomes were genetically scanned; 990 patients with MI and 904 controls were genotyped for the common polymorphisms G-761A and S173S (C4890A). Expression vectors for Ela2 variants were transiently transfected, followed by Northern and Western blot analyses. Promoter variants were analyzed by transfection/reporter gene assays. RESULTS: We identified 11 genetic variants, two in the 5'-flanking (G-761A, -852/del53 bp), six in exons (R49H, N81N, G93V, S173S, D222Y, P228L) and three in introns (C+29/in3T, C+149/in3T, C+137/in4T). In Belfast, 4890A allele carriers had a risk for MI with an odds ratio (OR) of 1.44 (95% CI 1.12-1.86; P=0.005), the OR for MI associated with the -761G/-4890A haplotype with reference to -761G/-4890C amounting to 2.38 (95% CI 1.23-4.57; P=0.01). Transcript or protein expression of both allelic constructs (4890A and 4890C) did not, however, differ. Conversely, transcriptional activity was significantly elevated (<35%) by -852/del53 bp in THP-1 monocytes compared with the nondeleted promoter (P=0.001); the deletion was observed in one patient with premature MI at the age of 28 years, whose mother had had an MI at the age of 48 years. CONCLUSIONS: The association of C4890A with MI in Belfast exclusively, and the presumed absence of its functionality, provides little support for a substantial implication of common ELA2 gene variants in overall MI risk. Whether -852/53del plays a role in cardiovascular pathophysiology or not should be evaluated further.

SAH gene variants are associated with obesity-related hypertension in Caucasians: the PEGASE Study.

OBJECTIVE: The SAH gene locus has recently been proposed to be involved in obesity-related hypertension in Japanese individuals. METHODS: To replicate independently the initial findings in another ethnic group, we scanned the entire SAH gene in 190 Caucasian chromosomes. A total of 651 patients with essential hypertension and 776 controls (PEGASE Study) were genotyped for all identified variants using allele-specific oligonucleotides, and single nucleotide polymorphism as well as haplotype analyses were carried out. We also performed transient transfection experiments, northern and western blots, immunoprecipitation, and acyl-coenzyme A synthetase activity assays. RESULTS: We identified five polymorphisms in the promoter region (C-1808T, G-1606A, -962ins/del, G-451A, T-67C), two in introns 5 and 7 (T+9/In5C, A+20/In7T), and one missense variant (K359N). Carriage of the -1606A allele was significantly associated with hypertension [odds ratio (OR) 1.28, P = 0.049] as was 359N (OR 1.35, P = 0.048) compared with non-carriers. Conversely, for -962del, the OR for hypertension was 0.80 (P = 0.042). The SAH alleles -1606A and 359N, but not -962ins/del, displayed a raising effect on body mass index (BMI; P = 0.004 and P = 0.030, respectively) in hypertensive as well as in control individuals. After adjustment for BMI in hypertensive individuals, only the OR associated with -962ins/del remained significant (OR 0.77, P = 0.028). Functional analyses in BHK did not reveal differences for SAH 359N or 359K-containing constructs, formally excluding K359N as the functional variant. CONCLUSION: We confirm recent evidence that the SAH locus is associated with obesity-related hypertension, in which pathophysiological context SAH variants affecting blood pressure remain, however, to be shown.