The N-terminus of Drosophila SU(VAR)3-9 mediates dimerization and regulates its methyltransferase activity

In most eukaryotes, the histone methyltransferase SU(VAR)3-9 and its orthologues play a major role in the function of centromeric heterochromatin. Although the methyltransferase domain is required for the formation of a fully functional centromere, mutations within other regions of the gene such as the N-terminus also have a strong impact on its in vivo function. To analyze the contribution of the N-terminus on the methyltransferase activity, we have expressed the full-length Drosophila SU(VAR)3-9 (dSU(VAR)3-9) together with various N-terminal deletions in Escherichia coli and analyzed the structural and enzymatic properties of the purified recombinant enzymes. Full-length dSU(VAR)3-9 specifically methylates lysine 9 within histone H3 on peptides, on intact histones, and, to a lesser extent, on nucleosomes. A detailed analysis of the reaction products shows that dSU(VAR)3-9 adds two methyl groups to an unmethylated H3 tail peptide in a nonprocessive manner. The full-length enzyme elutes with an apparent molecular weight of 160 kDa from a gel filtration column, which indicates the formation of a dimer. This property is dependent on an intact N-terminus. In contrast to the full-length enzymes, proteins lacking the N-terminus fail to dimerize, and show a 10-fold lower specific activity and a linear dependence of methyltransferase activity on enzyme concentration. A N-terminal peptide containing amino acids 1-152 of dSU(VAR)3-9 is sufficient to mediate this interaction in vitro. The dimerization of dSU(VAR)3-9 and the subsequent increase of its methyltransferase activity provide a starting point to understand the molecular details of the formation of heterochromatic structures in vivo.     

Identification of a specific inhibitor of the histone methyltransferase SU(VAR)3-9

Histone methylation plays a key role in establishing and maintaining stable gene expression patterns during cellular differentiation and embryonic development. Here, we report the characterization of the fungal metabolite chaetocin as the first inhibitor of a lysine-specific histone methyltransferase. Chaetocin is specific for the methyltransferase SU(VAR)3-9 both in vitro and in vivo and may therefore be used to study heterochromatin-mediated gene repression.     

Antibodies to Aeromonas salmonicida Lipopolysaccharide and cell wall antigens in rabbit and salmon immune sera

Atlantic salmon (Salmo salar L.) immunised with A-layer positive or A-layer negative strains ofAeromonas salmonicida did not produce antibodies reactive with proteinase K-digested LPS in the low molecular weight area corresponding to the core-region of LPS. The salmon produced antibody titres as high as those produced by rabbit when assayed against whole bacteria or LPS in ELISA. The salmon antibodies against the A-layer positive strain of A. salmonicida lysed rabbit erythrocytes sensitised with LPS from the A-layer positive strain of A. salmonicida. This was in contrast to the non-haemolytic activity of the salmon antibodies against the A-layer negative strain, indicating differences in epitopes between the two strains.     

Alarmone (p)ppGpp regulates the transition from pathogenicity to mutualism in Photorhabdus luminescens

The enteric gamma‐proteobacterium Photorhabdus luminescens kills a wide range of insects, whilst also maintaining a mutualistic relationship with soil nematodes from the family Heterorhabditis. Pathogenicity is associated with bacterial exponential growth, whilst mutualism is associated with post‐exponential (stationary) phase. During post‐exponential growth, P. luminescens also elaborates an extensive secondary metabolism, including production of bioluminescence, antibiotics and pigment. However, the regulatory network that controls the expression of this secondary metabolism is not well understood. The stringent response is a well‐described global regulatory system in bacteria and mediated by the alarmone (p)ppGpp. In this study, we disrupted the genes relA and spoT, encoding the two predicted (p)ppGpp synthases of P. luminescens TTO1, and we showed that (p)ppGpp is required for secondary metabolism. Moreover, we found the (p)ppGpp is not required for pathogenicity of P. luminescens, but is required for bacterial survival within the insect cadaver. Finally, we showed that (p)ppGpp is required for P. luminescens to support normal nematode growth and development. Therefore, the regulatory network that controls the transition from pathogenicity to mutualism in P. luminescens requires (p)ppGpp. This is the first report outlining a role for (p)ppGpp in controlling the outcome of an interaction between a bacteria and its host.     

Familial Risk of Chronic Musculoskeletal Pain and the Importance of Physical Activity and Body Mass Index: Prospective Data from the HUNT Study,Norway

The main objectives of the current study was i) to prospectively examine if chronic musculoskeletal pain in parents is associated with risk of chronic musculoskeletal pain in their adult offspring, and ii) to assess if these parent-offspring associations are modified by offspring body mass index and leisure time physical activity. We used data on 4,742 adult offspring linked with their parents who participated in the population-based HUNT Study in Norway in 1995–97 and in 2006–08. Family relations were established through the national Family Registry. A Poisson regression model was used to estimate relative risk (RR) with 95% confidence interval (CI). In total, 1,674 offspring (35.3%) developed chronic musculoskeletal pain during the follow-up period of approximately 11 years. Both maternal (RR: 1.26, 95% CI: 1.03, 1.55) and paternal chronic musculoskeletal pain (RR: 1.29, 95% CI: 1.06, 1.57) was associated with increased risk of offspring chronic musculoskeletal pain. Compared to offspring of parents without chronic musculoskeletal pain, the adverse effect of parental pain was somewhat stronger among offspring who reported a low (RR: 1.82, 95% CI: 1.32, 2.52) versus high (RR: 1.32, 95% CI: 0.95, 1.84) level of leisure time physical activity. Offspring of parents with chronic musculoskeletal pain and who were classified as obese had more than twofold increased risk (RR: 2.33, 95% CI: 1.68, 3.24) of chronic musculoskeletal pain compared to normal weight offspring of parents without pain. In conclusion, parental chronic musculoskeletal pain is positively associated with risk of chronic musculoskeletal pain in their adult offspring. Maintenance of normal body weight may reduce the risk of chronic musculoskeletal pain in offspring of pain-afflicted parents.     

Coexpression and nuclear colocalization of metastasis-promoting protein S100A4 and p53 without mutual regulation in colorectal carcinoma

Nuclear localization of the metastasis-associated protein S100A4 has been shown to correlate with advanced disease stage in primary colorectal carcinomas (CRC), but nuclear function and its relevance for the metastatic capacity of tumor cells is still unclear. Among several nuclear interacting protein partners suggested for S100A4, the tumor suppressor protein p53 has attracted particular interest, and previous studies suggest direct and indirect modes of interaction between the two proteins. The present study was undertaken to assess coexpression and potential interaction in CRC. TP53 mutational status and S100A4 expression were investigated in a selected series of primary CRC specimens (n = 40) and cell lines (n = 17) using DNA sequencing, western blot, and double immunostaining. Additionally, S100A4 and p53 were experimentally up- and down-regulated in vitro to assess reciprocal effects. For the first time, S100A4 and p53 coexpression was demonstrated in individual CRC cells, with nuclear colocalization as a particularly interesting feature. In contrast to previous studies, no correlation was observed between TP53 mutational status and S100A4 expression, and no evidence was obtained to support reciprocal regulation between the two molecules in the HCT116 isogenic cell line model. In conclusion, S100A4 and p53 were shown to be colocalized in individual nuclei of CRC cells, and it might be speculated whether the proteins interact in this subcellular compartment.     

A microdialysis study in rat brain of dihydrokainate,a glutamate uptake inhibitor

Microdialysis in neostriatum of anaesthetized rats was performed to study effects on amino acid efflux of the glutamate uptake-inhibitor dihydrokainate (DHK). Both basal and K⁺-evoked (100 mM) efflux of glutamate increased in the presence of DHK. The increase in the basal glutamate efflux occurred at lower DHK concentrations than during K⁺-depolarization (when the extracellular glutamate concentration was several-fold higher), confirming that DHK is a competitive inhibitor. The increase in basal efflux caused by DHK did not exhibit Ca²⁺-dependency, whereas ∼50% of the increase in glutamate efflux during K⁺-depolarization was Ca²⁺-dependent. The Ca²⁺-dependent efflux is related to transmitter release, whereas the Ca²⁺-independent efflux is probably due to metabolic events and/or transport of DHK into cells in exchange for glutamate. Taurine efflux in response to DHK increased both during basal conditions and K⁺-depolarization, probably secondary to the increase in glutamate concentration, whereas aspartate, GABA, glutamine and alanine effluxes did not change.     

Host Restrictions of Avian Influenza Viruses: In Silico Analysis of H13 and H16 Specific Signatures in the Internal Proteins

Gulls are the primary hosts of H13 and H16 avian influenza viruses (AIVs). The molecular basis for this host restriction is only partially understood. In this study, amino acid sequences from Eurasian gull H13 and H16 AIVs and Eurasian AIVs (non H13 and H16) were compared to determine if specific signatures are present only in the internal proteins of H13 and H16 AIVs, using a bioinformatics approach. Amino acids identified in an initial analysis performed on 15 selected sequences were checked against a comprehensive set of AIV sequences retrieved from Genbank to verify them as H13 and H16 specific signatures. Analysis of protein similarities and prediction of subcellular localization signals were performed to search for possible functions associated with the confirmed signatures. H13 and H16 AIV specific signatures were found in all the internal proteins examined, but most were found in the non-structural protein 1 (NS1) and in the nucleoprotein. A putative functional signature was predicted to be present in the nuclear export protein. Moreover, it was predicted that the NS1 of H13 and H16 AIVs lack one of the nuclear localization signals present in NS1 of other AIV subtypes. These findings suggest that the signatures found in the internal proteins of H13 and H16 viruses are possibly related to host restriction.     

Aerobic Interval Training Partly Reverse Contractile Dysfunction and Impaired Ca2+ Handling in Atrial Myocytes from Rats with Post Infarction Heart Failure

Background

There is limited knowledge about atrial myocyte Ca²⁺ handling in the failing hearts. The aim of this study was to examine atrial myocyte contractile function and Ca²⁺ handling in rats with post-infarction heart failure (HF) and to examine whether aerobic interval training could reverse a potential dysfunction.

Methods and results

Post-infarction HF was induced in Sprague Dawley rats by ligation of the left descending coronary artery. Atrial myocyte shortening was depressed (p<0.01) and time to relaxation was prolonged (p<0.01) in sedentary HF-rats compared to healthy controls. This was associated with decreased Ca²⁺ amplitude, decreased SR Ca²⁺ content, and slower Ca²⁺ transient decay. Atrial myocytes from HF-rats had reduced sarcoplasmic reticulum Ca²⁺ ATPase activity, increased Na⁺/Ca²⁺-exchanger activity and increased diastolic Ca²⁺ leak through ryanodine receptors. High intensity aerobic interval training in HF-rats restored atrial myocyte contractile function and reversed changes in atrial Ca²⁺ handling in HF.

Conclusion

Post infarction HF in rats causes profound impairment in atrial myocyte contractile function and Ca²⁺ handling. The observed dysfunction in atrial myocytes was partly reversed after aerobic interval training.