SUMOylation does not affect cardiac troponin I stability but alters indirectly the development of force in response to Ca2.

Post-translational modification of the myofilament protein troponin I by phosphorylation is known to trigger functional changes that support enhanced contraction and relaxation of the heart. We report for the first time that human troponin I can also be modified by SUMOylation at lysine 177. Functionally, TnI SUMOylation is not a factor in the development of passive and maximal force generation in response to calcium, however this modification seems to act indirectly by preventing SUMOylation of other myofilament proteins to alter calcium sensitivity and cooperativity of myofilaments. Utilising a novel, custom SUMO site-specific antibody that recognises only the SUMOylated form of troponin I, we verify that this modification occurs in human heart and that it is upregulated during disease.

A rapid liquid chromatography-tandem mass spectrometry method for the analysis of urinary 5-hydroxyindoleacetic acid (5-HIAA) that incorporates a 13C-labelled internal standard.

BACKGROUND: Urinary 5-hydroxyindoleacetic acid (5-HIAA) is a first-line investigation for gastrointestinal neuroendocrine tumours that secrete serotonin. It also has clinical utility for monitoring disease progression and therapeutic response. AIM: To develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for urinary 5-hydroxyindoleacetic acid that incorporates a supported liquid extraction and 13C-labelled internal standard. METHODS: Samples were diluted in ammonium acetate containing a 13C-labelled internal standard (5-hydroxyindole-3a,4,5,6,7,7a-13C6-3-acetic acid). Supported liquid extraction was performed followed by chromatographic separation using the 2.1 × 30 mm CORTECS® UPLC® T3 column. Mass spectrometry detection (Waters Xevo TQ-XS) was performed in electrospray positive mode using the transitions 192.3 > 146.4 m/z (quantifier) and 192.3 > 118.4 m/z (qualifier) for 5-hydroxyindoleacetic acid and 198.2 > 152.4 m/z for 13C-5-HIAA. RESULTS: A well-defined 5-hydroxyindoleacetic acid peak was observed at 0.8 min with a run time of 2.4 min. The assay was linear (r2 > 0.99) to 382 µmol/L, with a lower limit of quantification of 5.3 µmol/L (CV <15%). Analysis of 29 external quality assurance samples showed good agreement between our method and the UKNEQAS method mean (4.7% positive bias). The intra- and inter-assay precision was within acceptable limits, and the assay was stable up to 96 h postextraction with minimal carryover. CONCLUSION: We have developed a robust LC-MS/MS method with semi-automated extraction that offers an improved run time and performance over the existing, labour-intensive, HPLC method. The method was quick, precise, showed good agreement with UKNEQAS external quality assurance material and is in routine service for clinical samples.

Failure of Parastrongyloides trichosuri daf-7 to complement a Caenorhabditis elegans daf-7 (e1372) mutant: implications for the evolution of parasitism.

DAF-7 is the ligand of the TGF-ß pathway that, in conjunction with the insulin-like and guanylyl cyclase pathways, controls entry into dauer development in Caenorhabditis elegans. Proposed orthologues of Ce-daf-7 have been identified in several species of parasitic nematodes and demonstrate an expression pattern that is consistent between parasitic nematode genera but different to that of Ce-daf-7. This variation in expression pattern is consistent with the current paradigm in evolutionary developmental biology: that regulatory rather than functional change is the primary source of phenotypic diversity. In this work we investigated the proposed orthology of a daf-7 like sequence obtained from Parastrongyloides trichosuri, Pt-daf-7, to Ce-daf-7 via transformation rescue of a C. elegans daf-7 mutant with Pt-daf-7 coding regions. We also investigated further the difference in expression pattern of Pt-daf-7 both by fusing a Pt-daf-7 promoter to a Ce-daf-7 coding region and to a gfp reporter gene. We found that Pt-daf-7 was unable to complement a C. elegans daf-7 mutant, even when reduced to the smallest functional TGF-ß unit possible, the ligand domain, and that this failure appears to be the result of gene silencing. Furthermore, we show that although the Pt-daf-7 promoter is active later in development than the Ce-daf-7 promoter and most likely active in the correct neurons, a Ce-daf-7 coding region under control of a Pt-daf-7 promoter failed to rescue. Together, these results suggest that, if the free-living nematode developmental pathways, such as the DAF-7 TGF- ß pathway, have been co-opted during the evolution of parasitism, this co-option has been both at the protein level and in the control of their transcription.

Heritable transgenesis of Parastrongyloides trichosuri: a nematode parasite of mammals.

Germline transformation of a parasitic nematode of mammals has proven to be an elusive goal. We report here the heritable germline transformation of Parastrongyloides trichosuri, a nematode parasite whose natural hosts are Australian possums of the genus Trichosurus. This parasite can undergo multiple free-living life cycles and these replicative cycles can be maintained indefinitely in the laboratory. Transformation was achieved by microinjection of DNA into the ovary syncytium of either free-living or parasitic adult females. By selecting for the transgenic progeny of successive free-living life cycles, it was possible to establish and maintain transgenic lines. All three transgenic lines tested were shown capable of establishing patent infections in possums and to transmit the functional transgene to their progeny. The transgene, driven by the Pt hsp-1 promoter, was constitutively expressed in intestinal cells at all stages of both parasitic and free-living life cycles, although gene silencing appears to occur in some transgenic progeny. This is the first report of heritable transgenesis in a parasitic nematode of a mammal and we discuss a variety of previously inaccessible experimental avenues that will now be possible with this powerful model system.