Novel conserved hydrolase domain in the CLCA family of alleged calcium-activated chloride channels

Advanced protein structure prediction methods combined with structure modeling show that the mammalian proteins, described until now as calcium-activated chloride channels (CLCAs), appear in fact to be membrane anchored metal-dependent hydrolases, possibly proteases. A metallohydrolase structural domain was predicted, unexpectedly, in the CLCA sequences. The well-conserved active site in the modeled structure of this hydrolase domain allows the prediction of catalytic action similar to that of metalloproteases. A number of protein structure prediction methods suggest the overall fold of the N-terminal hydrolase domain to be most similar to that of zinc metalloproteases (zincins), notably matrixins. This is confirmed by analysis of the three-dimensional structure model of the predicted CLCA1 hydrolase domain built using the known structure of the MMP-11 catalytic domain. Fragments of CLCA1 corresponding to the modeled hydrolase domain were expressed in Escherichia coli, and the resulting proteins were readily refolded into monomeric soluble protein, indicating formation of stable independent domains. The homology model was used to predict putative substrate sequences. Homologs of mammalian CLCA genes were detected in the genomes of a vast array of multicellular animals: lower vertebrates, tunicates, insects, crustaceans, echinoderms, and flatworms. The hydrolase prediction is discussed in the context of published experimentally determined effects of CLCA proteins on chloride conductance. Altered proteolytic processing of full-length CLCA1 containing a mutation abolishing the predicted hydrolase activity is shown as initial experimental evidence for a role of the hydrolase domain in processing of mature full-length CLCA1. The hydrolase prediction together with the presented experimental data add to doubts about the function of CLCAs as chloride channels and strengthen the hypothesis of channel-activating and/or channel-accessory roles.     

The gene expression profiles of canine mammary cancer cells grown with carcinoma-associated fibroblasts (CAFs) as a co-culture in vitro

Background

It is supposed that fibroblasts present in tumour microenvironment increase cancer invasiveness and its ability to metastasize but the mechanisms have not been clearly defined yet. Thus, the current study was designed to assess changes in gene expression in five various cancer cell lines grown as a co-culture with the carcinoma-associated fibroblasts (CAFs) in vitro.

Results

A carcinoma-associated fibroblast cell line was isolated from a canine mammary cancer. Then, a co-culture of cancer cells with the CAFs was established and maintained for 72 hrs. Having sorted the cells, a global gene expression in cancer cells using DNA microarrays was examined. The analysis revealed an up-regulation of 100 genes and a down-regulation of 106 genes in the cancer cells grown as a co-culture with the CAFs in comparison to control conditions. The PANTHER binomial statistics tool was applied to determine statistically over-manifested pathways (p < 0.05). Bulk of the up-regulated genes are involved in the adhesion, the angiogenesis, the epithelial-mesenchymal transition (EMT) and generally take part in the developmental processes. These results were further confirmed using real-time qPCR. Moreover, a wound-healing assay and growth characteristics on Matrigel matrix showed that CAFs increase cancer cell migration and matrix invasion.

Conclusion

The results of the current study showed that the co-culturing of cancer cells and the CAFs caused significant changes to the cancer gene expression. The presence of the CAFs in a microenvironment of cancer cells promotes adhesion, angiogenesis and EMT.     

Computationally Driven, Quantitative Experiments Discover Genes Required for Mitochondrial Biogenesis

Mitochondria are central to many cellular processes including respiration, ion homeostasis, and apoptosis. Using computational predictions combined with traditional quantitative experiments, we have identified 100 proteins whose deficiency alters mitochondrial biogenesis and inheritance in Saccharomyces cerevisiae. In addition, we used computational predictions to perform targeted double-mutant analysis detecting another nine genes with synthetic defects in mitochondrial biogenesis. This represents an increase of about 25% over previously known participants. Nearly half of these newly characterized proteins are conserved in mammals, including several orthologs known to be involved in human disease. Mutations in many of these genes demonstrate statistically significant mitochondrial transmission phenotypes more subtle than could be detected by traditional genetic screens or high-throughput techniques, and 47 have not been previously localized to mitochondria. We further characterized a subset of these genes using growth profiling and dual immunofluorescence, which identified genes specifically required for aerobic respiration and an uncharacterized cytoplasmic protein required for normal mitochondrial motility. Our results demonstrate that by leveraging computational analysis to direct quantitative experimental assays, we have characterized mutants with subtle mitochondrial defects whose phenotypes were undetected by high-throughput methods.     

Thyroid hormones and the cardiomyocytes

The authors present the current knowledge on the intracellular mechanisms of thyroid hormone action in the cardiomyocytes. Many of the clinical manifestations of thyroid diseases are due to the ability of thyroid hormone to alter cardiovascular hemodynamics. Triiodothyronine affects the hemodynamic state mainly by its influence on the expression of cardiomyocyte genes. These genes encode both structural and regulatory proteins in the heart (myosin heavy chains, sarcoplasmic reticulum calcium-activated ATP-ase, phospholamban). The impaired myocardium contractile activity in hypothyreosis reminds findings in heart failure and may warrant further exploration of therapeutic approaches using thyroid hormone to improve cardiac function in heart failure.     

Plantlets from encapsulated meristems of Gentiana pneumonanthe L.

The preparation of synthetic seeds of marsh gentian from apical and axillary meristems, using 0.1 M CaCl₂ and 3 % solutions of sodium alginate (aquatic solution, aquatic with IAA, and with MS medium) and the results of regrowth of these seeds are discussed. The best regeneration occurred in the case of apical meristems encapsulated in 3 % sodium alginate prepared on the basis of MS medium.     

Access To Essential Maternal Health Interventions and Human Rights Violations among Vulnerable Communities in Eastern Burma

Background

Health indicators are poor and human rights violations are widespread in eastern Burma. Reproductive and maternal health indicators have not been measured in this setting but are necessary as part of an evaluation of a multi-ethnic pilot project exploring strategies to increase access to essential maternal health interventions. The goal of this study is to estimate coverage of maternal health services prior to this project and associations between exposure to human rights violations and access to such services.

Methods and Findings

Selected communities in the Shan, Mon, Karen, and Karenni regions of eastern Burma that were accessible to community-based organizations operating from Thailand were surveyed to estimate coverage of reproductive, maternal, and family planning services, and to assess exposure to household-level human rights violations within the pilot-project target population. Two-stage cluster sampling surveys among ever-married women of reproductive age (15–45 y) documented access to essential antenatal care interventions, skilled attendance at birth, postnatal care, and family planning services. Mid-upper arm circumference, hemoglobin by color scale, and Plasmodium falciparum parasitemia by rapid diagnostic dipstick were measured. Exposure to human rights violations in the prior 12 mo was recorded. Between September 2006 and January 2007, 2,914 surveys were conducted. Eighty-eight percent of women reported a home delivery for their last pregnancy (within previous 5 y). Skilled attendance at birth (5.1%), any (39.3%) or ≥ 4 (16.7%) antenatal visits, use of an insecticide-treated bed net (21.6%), and receipt of iron supplements (11.8%) were low. At the time of the survey, more than 60% of women had hemoglobin level estimates ≤ 11.0 g/dl and 7.2% were Pf positive. Unmet need for contraceptives exceeded 60%. Violations of rights were widely reported: 32.1% of Karenni households reported forced labor and 10% of Karen households had been forced to move. Among Karen households, odds of anemia were 1.51 (95% confidence interval [CI] 0.95–2.40) times higher among women reporting forced displacement, and 7.47 (95% CI 2.21–25.3) higher among those exposed to food security violations. The odds of receiving no antenatal care services were 5.94 (95% CI 2.23–15.8) times higher among those forcibly displaced.

Conclusions

Coverage of basic maternal health interventions is woefully inadequate in these selected populations and substantially lower than even the national estimates for Burma, among the lowest in the region. Considerable political, financial, and human resources are necessary to improve access to maternal health care in these communities.     

Electrofusion of protoplasts from Solanum tuberosum, S. nigrum and S. bulbocastanum

Leaf protoplasts of two wild species, Solanum nigrum var. gigantea (S. ngr gig) and S. bulbocastanum Dun. (S. blb), were electrofused with leaf protoplasts of two diploid potato clones, H-8105 and ZEL-1136, respectively, in order to confer the late blight-resistance from the wild species to the cultivated potato. The S. ngr gig mesophyll (+) H-8105 mesophyll combination resulted in regenerants of mostly normal ngr phenotype. Two regenerants from this combination were proved to be true hybrids by RAPD analysis but they rooted poorely in vitro and did not survive the transfer to soil. The S. ngr gig (+) H-8105 fusion combination was also performed with H-8105 cell suspension derived protoplasts enabling an easy identification of interspecific fusants on basis of their intermediate morphology. From the S. ngr gig mesophyll (+) H-8105 cultured cell combination, many abnormal shoots were regenerated. The two lines which survived had normal ngr phenotype but the presence of tuberosum (tbr) genome in those regenerants was not confirmed by RAPD analysis. No plants with tbr phenotype were obtained from both of S. ngr gig (+) H-8105 combinations. On the contrary, when S. blb mesophyll protoplasts were electrofused with ZEL-1136 mesophyll protoplasts, all regenerated plants had tbr phenotype, indicating much lower morphogenetic potential of S. bulbocastanum in comparison with that of S. nigrum var. gigantea. However, the hybridity of those regenerants has not been confirmed by RAPD analysis with two different primers. The efficiency of the applied fusion procedure and analysis of the regenerants is discussed.     

The role of benzoate secreted by Desulfotomaculum acetoxidans DSM 771 in sulfate uptake

This work was designed to find the cause of the delay in hydrogen sulfide dissimilation in Desulfotomaculum acetoxidans DSM 771, which is dependent on the sulfate uptake. This bacterium grown without addition of any aromatic compound was shown by spectrum analysis with the methylene method to contain hydroxy-benzoate derivatives. The presence of these compounds was confirmed by HPLC in fractions obtained from cell walls after 15 days of culture. The test with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt seemed to indicate the presence of peroxidase, which probably oxidized benzoate to its hydroxy derivatives. The test with 5-sulfo-salicylic acid proved the ability of the investigated strain to utilize arylsulfates and to reduce sulfate group to hydrogen sulfide. On the basis of the above data, we propose the following sequence of reactions: 1, benzoate secretion; 2, benzoate hydroxylation; 3, sulfonation of hydroxy-benzoate derivatives.