Modulation of Sarco/Endoplasmic Reticulum Ca2+-ATPase 2 (SERCA2) function by acetylation following the treatment with histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA)

Background: Acetylation and deacetylation at specific lysine (K) residues is mediated by histone acetylases (HATs) and deacetylases (HDACs), respectively. HATs and HDACs act on both histone and non-histone proteins, regulating various processes, including cardiac impulse propagation. Aim of the present work was to establish whether the function of the Ca2+ ATPase SERCA2, one of the major players in Ca2+ reuptake during excitation-contraction coupling in cardiac myocytes (CMs), could be modulated by direct K acetylation. Materials and methods: HL-1 atrial mouse cells (donated by Prof. Claycomb), zebrafish and Streptozotocin-induced diabetic rat CMs were treated with the pan-inhibitor of class I and II HDACs suberanilohydroxamic acid (SAHA) for 1.5 hour. Evaluation of SERCA2 acetylation was analyzed by co-immunoprecipitation. SERCA2 activity was measured on microsomes by pyruvate/NADH coupled reaction assay. SERCA2 mutants were obtained after cloning wild-type and mutated sequences into the pCDNA3 vector and transfected into HEK cells. Ca2+ transients in CMs (loading with Fluo3-AM, field stimulation, 0.5 Hz) and in transfected HEK cells (loading with FLUO-4, caffeine pulse) were recorded. Results: Co-Immunoprecipitation experiments performed on HL-1 cells demonstrated a significant increase in the acetylation of SERCA2 after SAHA-treatment (2.5 µM, n=3). This was associated with an increase in SERCA2 activity in microsomes obtained from HL-1 cells, after SAHA exposure (n=5). Accordingly, SAHA-treatment significantly shortened the Ca2+ reuptake time of adult zebrafish CMs. Further, SAHA 2.5 nM restored to control values the recovery time of Ca2+ transients decay in diabetic rat CMs. HDAC inhibition also improved contraction parameters, such as fraction of shortening, and increased pump activity in microsomes isolated from diabetic CMs (n=4). Notably, the K464, identified by bioinformatic tools as the most probable acetylation site on human SERCA2a, was mutated into Glutamine (Q) or Arginine (R) mimicking acetylation and deacetylation respectively. Measurements of Ca2+ transients in HEK cells revealed that the substitution of K464 with R significantly delayed the transient recovery time, thus indicating that deacetylation has a negative impact on SERCA2 function. Conclusions: Our results indicate that SERCA2 function can be improved by pro-acetylation interventions and that this mechanism of regulation is conserved among species. Therefore, the present work provides the basis to open the search for novel pharmacological tools able to specifically improve SERCA2 activity in diseases where its expression and/or function is impaired, such as diabetic cardiomyopathy.

The β domain is required for Vps4p oligomerization into a functionally active ATPase

Endocytic and biosynthetic trafficking pathways to the lysosome/vacuole converge at the prevacuolar endosomal compartment. During transport through this compartment, integral membrane proteins that are destined for delivery to the lysosome/vacuole lumen undergo multivesicular body (MVB) sorting into internal vesicles formed by invagination of the endosomal limiting membrane. Vps4 is an AAA family ATPase which plays a key role in MVB sorting and facilitates transport through endosomes. It possesses an N-terminal microtubule interacting and trafficking domain required for recruitment to endosomes and an AAA domain with an ATPase catalytic site. The recently solved 3D structure revealed a P domain, which protrudes from the AAA domain, and a final C-terminal alpha-helix. However, the in vivo roles of these domains are not known. In this study, we have identified motifs in these domains that are highly conserved between yeast and human Vps4. We have mutated these motifs and studied the effect on yeast Vps4p function in vivo and in vitro. We show that the P domain of the budding yeast Vps4p is not required for recruitment to endosomes, but is essential for all Vps4p endocytic functions in vivo. We also show that the P domain is required for Vps4p homotypic interaction and for full ATPase activity. In addition, it is required for interaction with Vta1p, which works in concert with Vps4p in vivo. Our studies suggest that assembly of a Vps4p oligomeric complex with full ATPase activity that interacts with Vta1p is essential for normal endosome function.

Modulation of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase activity and oxidative modification during the development of adjuvant arthritis

Adjuvant arthritis (AA) was induced by intradermal administration of Mycobacterium butyricum to the tail of Lewis rats. In sarcoplasmic reticulum (SR) of skeletal muscles, we investigated the development of AA. SR Ca(2+)-ATPase (SERCA) activity decreased on day 21, suggesting possible conformational changes in the transmembrane part of the enzyme, especially at the site of the calcium binding transmembrane part. These events were associated with an increased level of protein carbonyls, a decrease in cysteine SH groups, and alterations in SR membrane fluidity. There was no alteration in the nucleotide binding site at any time point of AA, as detected by a FITC fluorescence marker. Some changes observed on day 21 appeared to be reversible, as indicated by SERCA activity, cysteine SH groups, SR membrane fluidity, protein carbonyl content and fluorescence of an NCD-4 marker specific for the calcium binding site. The reversibility may represent adaptive mechanisms of AA, induced by higher relative expression of SERCA, oxidation of cysteine, nitration of tyrosine and presence of acidic phospholipids such as phosphatidic acid. Nitric oxide may regulate cytoplasmic Ca(2+) level through conformational alterations of SERCA, and decreasing levels of calsequestrin in SR may also play regulatory role in SERCA activity and expression.

A heavy metal P-type ATPase OsHMA4 prevents copper accumulation in rice grain

Acknowledgements We thank B. Lahner, E. Yakubova and S. Rikiishi for ICP-MS analysis, N. Komiyama, Iowa State University Plant Transformation Facility and Prashant Hosmani for generation of transgenic rice, K. Wang for providing pTF101.1 vector and N. Verbruggen for providing pYES2 and pYEC2/CT-GFP vectors. We also thank Rice T-DNA Insertion Sequence Database center for providing the T-DNA insertion line and X. Wang, T. Zheng and Z. Li for accessing 3 K rice genome sequence, and Graeme Paton for helpful discussions on Cu bioavailability in water-logged soils. This research was supported by a Grant-in-Aid for Specially promoted Research (JSPS KAKENHI Grant Number 16H06296 to J.F.M), and the US National Science Foundation, Plant Genome Research Program (Grant #IOS 0701119 to D.E.S., M.L.G. and S.R.M.P.).

Towards a single crystal Raman spectrum of kaolinite at 77 K

The Raman spectra at 77 K of the hydroxyl stretching of kaolinite were obtained along the three axes perpendicular to the crystal faces. Raman bands were observed at 3616, 3658 and 3677 cm−1 together with a distinct band observed at 3691 cm−1 and a broad profile between 3695 and 3715 cm−1. The band at 3616 cm−1 is assigned to the inner hydroxyl. The bands at 3658 and 3677 cm−1 are attributed to the out-of-phase vibrations of the inner surface hydroxyls. The Raman spectra of the in-phase vibrations of the inner-surface hydroxyl-stretching region are described in terms of transverse and longitudinal optic splitting. The band at 3691 cm−1 is assigned to the transverse optic and the broad profile to the longitudinal optic mode. This splitting remained even at liquid nitrogen temperature. The transverse optic vibration may be curve resolved into two or three bands, which are attributed to different types of hydroxyl groups in the kaolinite.

K-tree : large scale document clustering

We introduce K-tree in an information retrieval context. It is an efficient approximation of the k-means clustering algorithm. Unlike k-means it forms a hierarchy of clusters. It has been extended to address issues with sparse representations. We compare performance and quality to CLUTO using document collections. The K-tree has a low time complexity that is suitable for large document collections. This tree structure allows for efficient disk based implementations where space requirements exceed that of main memory.

The knowledge advantage (K-Adv): unleashing creativity and innovation

The K-Adv has been developed around the concept that it comprises an ICT enabling infrastructure that encompasses ICT hardware and software infrastructure facilities together with an enabling ICT support system; a leadership infrastructure support system that provides the vision for its implementation and the realisation capacity for the vision to be realised; and the necessary people infrastructure that includes the people capabilities and capacities supported by organisational processes that facilitates this resource to be mobilised.

Document clustering with K-tree

This paper describes the approach taken to the XML Mining track at INEX 2008 by a group at the Queensland University of Technology. We introduce the K-tree clustering algorithm in an Information Retrieval context by adapting it for document clustering. Many large scale problems exist in document clustering. K-tree scales well with large inputs due to its low complexity. It offers promising results both in terms of efficiency and quality. Document classification was completed using Support Vector Machines.

The role of timber plantations in U.K. investment portfolios

Although timber plantations and forests are classified as forms of agricultural production, the ownership of this land classification is not limited to rural producers. Timber plantations and forests are now regarded as a long-term investment with both institutional and absentee owners. While the NCREIF property indices have been the benchmarks for the measurement of the performance of the commercial property market in the UK, for many years the IPD timberland index has recently emerged as the U.K. forest and timberland performance indicator. The IPD Forest index incorporates 126 properties over five regions in the U.K. This paper will utilise the IPD Forestry Index to examine the performance of U.K. timber plantations and forests over the period 1981-2004. In particular, issues to be critically assessed include plantation and forest performance analysis, comparative investment analysis, and the role of plantations and forests in investment portfolios, the risk reduction and portfolio benefits of plantations and forests in mixed-asset portfolios and the strategic investment significance of U.K. timberlands.

Random Indexing K-tree

Random Indexing K-tree is the combination of two algorithms suited for large scale document clustering.