Taxonomy and host-specificity of Gyrodactylus aideni n. sp. and G. pleuronecti (Monogenea: Gyrodactylidae) from Pseudopleuronectes americanus (Walbaum) in Passamaquoddy Bay, New Brunswick, Canada.

Wild and cultured winter flounder Pseudopleuronectes americanus (Walbaum) from Passamaquoddy Bay were surveyed for species of Gyrodactylus Nordmann, 1832. Two species were found: G. pleuronecti Cone, 1981 and G. aideni n. sp, both members of Malmberg's 'groenlandicus group'. Although the hard parts in the haptor are very similar in the two species, hamuli of G. aideni are consistently shorter than those of G. pleuronecti. The two species differed by 35 base pairs in the ITS 1, 5.8 and ITS 2 region. A BLAST search identified a variety of species of Gyrodactylus from marine fishes in the Atlantic Ocean as closest matches, indicating the 'groenlandicus group' is part of a major marine lineage within Gyrodactylus (sensu lato) that has successfully radiated among coastal percid, pleuronectid, cottid and anarhichadid fishes. Exposure experiments suggested that winter flounder is the primary host of both species of parasites and that three other pleuronectid species in the bay may potentially serve only as occasional transport hosts.

Quantitative myocardial cytokine expression and activation of the apoptotic pathway in patients who require left ventricular assist devices.

BACKGROUND: Molecular mechanisms underlying the deterioration of patients undergoing LV assist device (LVAD) implantation remain poorly understood. We studied the cytokines tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta and IL-6 and the terminal stage of the apoptotic pathway in patients with decompensating heart failure who required LVAD support and compared them with patients with less severe heart failure undergoing elective heart transplantation. METHODS AND RESULTS: Myocardial and serum samples from 23 patients undergoing LVAD implantation were compared with those from 36 patients undergoing elective heart transplantation. Myocardial TNF-alpha mRNA (1.71-fold; P<0.05) and protein (3.43+/-0.19 versus 2.95+/-0.10 pg/mg protein; P<0.05) were elevated in the LVAD patients. Immunocytochemistry demonstrated TNF expression in the myocytes. Serum TNF-alpha was also elevated (12.5+/-1.9 versus 4.0+/-0.4 pg/mL; P<0.0001) in the LVAD patients. IL-6 mRNA (2.57-fold higher; P<0.005) and protein (27.83+/-9.35 versus 4.26+/-1.24 pg/mg protein; P<0.001) were higher in the LVAD candidates, as was serum IL-6 (79.3+/-23.6 versus 7.1+/-1.6 pg/mL; P<0.0001). Interleukin-1beta mRNA expression was 9.78-fold higher in the LVAD patients (P<0.001). iNOS mRNA expression was similar to that in advanced heart failure patients and was not further elevated in the LVAD patients. Levels of procaspase-9 (8.02+/-0.91 versus 6.16+/-0.43 oligodeoxynucleotide [OD] units; P<0.01), cleaved caspase-9 (10.02+/-1.0 versus 7.34+/-0.40 OD units; P<0.05), intact and spliced DFF-45 (4.58+/-0.75 versus 2.84+/-0.23 OD units; P<0.05) were raised in LVAD patients, but caspase-3 and human nuclease CPAN were not. CONCLUSIONS: Elevated TNF-alpha, IL-1beta, and IL-6 and alterations in the apoptotic pathway were found in the myocardium and elevated TNF-alpha and IL-6 in serum of deteriorating patients who required LVAD support. These occurrences may have therapeutic implications and influence the timing of LVAD insertion.

Expression of RGS3, RGS4 and Gi alpha 2 in acutely failing donor hearts and end-stage heart failure.

BACKGROUND: Regulators of G-protein Signalling (RGS) proteins have been shown to limit in vitro signalling of G proteins. In common with end-stage heart failure, we have recently shown that upregulation of the inhibitory G-protein, Gialpha, occurs in acutely failing donor hearts unused for transplantation due to severe myocardial dysfunction. In light of recent data on RGS proteins, we have evaluated mRNA and protein expression of RGS3, RGS4 and Gialpha2 in the myocardium from normal, end-stage failing and acutely failing unused donor hearts. METHODS AND RESULTS: Myocardial samples were obtained from end-stage failing hearts explanted prior to transplantation (n=19), unused donor hearts with ejection fractions < 30% (n=14) and used donor hearts with good function (ejection fraction > 60%) (n=4-7). mRNA levels were quantified using quantitative reverse transcriptase polymerase chain reaction. Levels of RGS3 and RGS4 mRNA were found to be significantly upregulated in unused donor and end-stage failing myocardium (P < 0.05 and 0.01, and P < 0.05 and 0.02, respectively) compared to non-failing hearts. Protein abundance of RGS3 and RGS4 was found to be higher in myocardium from end-stage failing hearts, and relative RGS4 expression higher in unused donor hearts. CONCLUSIONS: We show here that RGS3 and RGS4 mRNA and protein expression is upregulated in human heart failure. These observations suggest that RGS4 may be induced in the heart to regulate cell signalling pathways in response to hypertrophy, and support the existence of a negative feedback loop for the long-term regulation of hypertrophy.

Elevated tumor necrosis factor-alpha and interleukin-6 in myocardium and serum of malfunctioning donor hearts.

BACKGROUND: Myocardial dysfunction is a common and important problem in donor hearts. The mechanisms responsible remain unclear. We have studied the cytokines tumor necrosis factor (TNF)-alpha and interleukin-6 (IL-6) in the myocardium and serum from donors with myocardial dysfunction (unused donors) and compared them with donors with good ventricular function (used donors) and patients with advanced heart failure (HF). METHODS AND RESULTS: Clinical details and ventricular function were assessed in 46 donors (31 used, 15 unused). Real-time reverse transcription-polymerase chain reaction, Western blotting, and immunocytochemistry were performed on myocardium and immunoassays on serum. TNF-alpha mRNA was 1.6-fold higher in unused than in used donors (P:<0.005) and 1.74-fold higher than in 36 patients with HF. IL-6 mRNA was 2.4-fold higher in unused than in used donors (P:<0.0001) and 4.67-fold higher than in HF (P:<0.0001). Western blotting showed higher TNF-alpha in unused (218. 3+/-6.4, n=4 versus 187.3+/-5.4, n=3 OD units) than used donors (P:<0.05). TNF-alpha expression was localized to cardiac myocytes. Serum TNF-alpha was higher in unused (8.72+/-1.3 pg/mL, n=13) than in used (6.12+/-0.8 pg/mL, n=25, P:<0.05) donors and HF (4.0+/-0.4 pg/mL, n=17, P:<0.005). Serum TNF-alpha receptors did not differ between unused (4.3+/-0.8 and 8.6+/-1.6 ng/mL, n=10) and used (3. 5+/-0.4 and 6.5+/-1.1 ng/mL, n=24) donors. There was a trend for higher serum IL-6 in unused (16.5+/-2.9 pg/mL, n=9) compared with used (13.9+/-1.6 pg/mL, n=26, P:=NS) donors. CONCLUSIONS: This study documented an increase in the expression of TNF-alpha and IL-6 in the myocardium of all donor hearts that was more marked in the dysfunctional (unused) donor hearts. This was accompanied by similar changes in the serum. This might have important therapeutic implications.

Structural characterization of the human fast skeletal muscle troponin I gene (TNNI2).

Three troponin I genes have been identified in vertebrates that encode the isoforms expressed in adult cardiac muscle (TNNI3), slow skeletal muscle (TNNI1) and fast skeletal muscle (TNNI2), respectively. While the organization and regulation of human cardiac and slow skeletal muscle genes have been investigated in detail, the fast skeletal troponin I gene has to date only been examined in birds. Here, we describe the structure and complete sequence of the human fast skeletal muscle troponin I gene (TNNI2) and identify putative regulatory elements within both the 5' flanking region and the first intron. In particular, a region containing MEF-2, E-box, CCAC and CAGG elements was identified in intron 1 that closely resembles the fast internal regulatory element (FIRE) of the quail intronic enhancer. We have previously shown that the fast skeletal muscle troponin I gene is located at 11p15.5 and noted potential close linkage with the fast skeletal muscle troponin T gene (TNNT3). Here, we have isolated two independent human PAC genomic clones that contain either TNNI2 or TNNT3 and demonstrate by interphase FISH mapping that they are less than 100 kb apart in the genome. The results demonstrate that the human TNNI2 gene is closely related to its avian counterparts with conserved elements within both the putative promoter and first intron. Our data further confirm close physical linkage of TNNI2 and TNNI3 on 11p15.5.

Close physical linkage of human troponin genes: organization, sequence, and expression of the locus encoding cardiac troponin I and slow skeletal troponin T.

Based on chromosomal mapping data, we recently revealed an unexpected linkage of troponin genes in the human genome: the six genes encoding striated muscle troponin I and troponin T isoforms are located at three chromosomal sites, each of which carries a troponin I-troponin T gene pair. Here we have investigated the organization of these genes at the DNA level in isolated P1 and PAC genomic clones and demonstrate close physical linkage in two cases through the isolation of individual clones containing a complete troponin I-troponin T gene pair. As an initial step toward fully characterizing this pattern of linkage, we have determined the organization and complete sequence of the locus encoding cardiac troponin I and slow skeletal troponin T and thereby also provide the first determination of the structure and sequence of a slow skeletal troponin T gene. Our data show that the genes are organized head to tail and are separated by only 2.6 kb of intervening sequence. In contrast to other troponin genes, and despite their close proximity, the cardiac troponin I and slow skeletal troponin T genes show independent tissue-specific expression. Such close physical linkage has implications for the evolution of the troponin gene families, for their regulation, and for the analysis of mutations implicated in cardiomyopathy.