Calcium-dependent Enzyme Research Articles

VOLTAGE-GATED CALCIUM CHANNELS: CLASSIFICATION AND PHARMACOLOGICAL PROPERTIES (PART I).

Calcium influx though voltage-gated calcium channels mediate a huge amount of physiological events and cellular responses. Numerous scientific reports indicate that calcium channels are involved in synaptic transmission, neurotransmitter release, regulation of gene expression, cellular membrane voltage oscillations, pacemaker activity, secretion of specific substances from nerve and secretory cells, morphological differentiation, activation of calcium-dependent enzymes, etc. This review represents the modern classification, molecular structure, physiological and pharmacological properties of voltage-gated calcium channels expressed in mammalian cells.

A calcium-dependent acyltransferase that produces N-acyl phosphatidylethanolamines.

More than 30 years ago, a calcium-dependent enzyme activity was described that generates N-acyl phosphatidylethanolamines (NAPEs), which are precursors for N-acyl ethanolamine (NAE) lipid transmitters, including the endocannabinoid anandamide. The identity of this calcium-dependent N-acyltransferase (Ca-NAT) has remained mysterious. Here, we use activity-based protein profiling to identify the poorly characterized serine hydrolase PLA2G4E as a mouse brain Ca-NAT and show that this enzyme generates NAPEs and NAEs in mammalian cells.

Effect of cadmium administration in hyperhomocysteinemic mice due to cystathionine beta synthase deficiency

Homocysteine, a sulfur-containing amino acid formed during the metabolism of methionine, is commonly slightly elevated in the plasma of the general population. Additionally, we previously found that cystathionine beta synthase-deficient mice, a murine model of hyperhomocysteinemia, exhibit altered activities of xenobiotic metabolizing enzymes (XME), which dispose of foreign chemicals, in the liver. Thus, hyperhomocysteinemia may result in susceptibility to xenobiotics like cadmium, a heavy-metal toxicant found in drinking water, atmospheric air, and food. Consequently, we exposed hyperhomocysteinemic mice to cadmium via their drinking water for one month to analyze the combined effects of hyperhomocysteinemia and cadmium exposure in liver. No difference in plasma homocysteine level was found after cadmium administration in control and hyperhomocysteinemic mice, but the glutathione level was significantly lower in exposed hyperhomocysteinemic mice compared to control mice, reflecting oxidative stress. We therefore analyzed the effect of Cd administration on hepatic XMEs known to be dysregulated in hyperhomocysteinemic mice: paraoxonase 1, a phase I XME, and NAD(P)H:quinone oxidoreductase, a phase II XME. Cadmium exposure negatively affected activity of paraoxonase 1, a calcium-dependent enzyme. Thus, we analyzed another calcium-dependent enzyme known to be dysregulated in liver of hyperhomocysteinemic mice, calpain, which was also significantly lower after cadmium administration. A comparison of the calculated affinities of cadmium docking versus calcium redocking suggested that cadmium ions may inhibit enzymatic activities by preventing the binding of calcium ions. Moreover, the increased NAD(P)H:quinone oxidoreductase activity observed after cadmium administration could indicate the presence of protective mechanisms in liver of mice. In conclusion, although cadmium administration had no effect on plasma homocysteine level, its effects on plasma glutathionine level suggest a susceptibility to cadmium in the condition of hyperhomocysteinemia, which could be countered by an increased NAD(P)H:quinone oxidoreductase activity.

Anti-peptidyl-arginine deaminase 3 (PAD3) antibodies as a promising marker to measure joint damage in patients with rheumatoid arthritis.

Recently, antibodies directed against peptidyl arginine deiminase 3 and 4 (anti-PAD3/PAD4 antibodies), calcium-dependent enzymes that catalyze the conversion from arginine to citrulline, have been described. Furthermore, antibodies that cross-react between PAD3 and PAD4 cause increased PAD4 activity and consequently correlate with joint damage. This study analyzes the correlation of anti-PAD3 antibodies with joint damage. To validate the novel chemiluminescent immunoassay (CIA) for the detection of anti-PAD3 antibodies, 20 samples were tested by CIA and by immunoprecipitation (IP). Next, 39 RA patients with available joint erosion score (JES), Total Sharp Score (TSS) and Joint Space Narrowing Score (JSNS) were tested for anti-CCP (using different methods) and anti-PAD3 antibodies by CIA. Excellent correlation was observed between the CIA and IP for the detection of anti-PAD3 antibodies (rho=0.85, p<0.0001). The median JES of our 39 patients was 14.1 with a standard deviation of 11.5. Anti-PAD3 antibody levels (rho=0.39, 95% CI=0.1-0.6; p=0.0149) were correlated with JES. No correlation was found with TSS and JSNS. In this cohort, ACPA measured using different anti-CCP assays did not correlate with the JES. In our cohort, anti-PAD3 antibodies correlate with joint erosion score. Therefore, anti-PAD3 antibodies might represent promising markers to predict joint damage in RA patients.

XoxF Acts as the Predominant Methanol Dehydrogenase in the Type I Methanotroph Methylomicrobium buryatense.

Many methylotrophic taxa harbor two distinct methanol dehydrogenase (MDH) systems for oxidizing methanol to formaldehyde: the well-studied calcium-dependent MxaFI type and the more recently discovered lanthanide-containing XoxF type. MxaFI has traditionally been accepted as the major functional MDH in bacteria that contain both enzymes. However, in this study, we present evidence that, in a type I methanotroph, Methylomicrobium buryatense, XoxF is likely the primary functional MDH in the environment. The addition of lanthanides increases xoxF expression and greatly reduces mxa expression, even under conditions in which calcium concentrations are almost 100-fold higher than lanthanide concentrations. Mutations in genes encoding the MDH enzymes validate our finding that XoxF is the major functional MDH, as XoxF mutants grow more poorly than MxaFI mutants under unfavorable culturing conditions. In addition, mutant and transcriptional analyses demonstrate that the lanthanide-dependent MDH switch operating in methanotrophs is mediated in part by the orphan response regulator MxaB, whose gene transcription is itself lanthanide responsive. Aerobic methanotrophs, bacteria that oxidize methane for carbon and energy, require a methanol dehydrogenase enzyme to convert methanol into formaldehyde. The calcium-dependent enzyme MxaFI has been thought to primarily carry out methanol oxidation in methanotrophs. Recently, it was discovered that XoxF, a lanthanide-containing enzyme present in most methanotrophs, can also oxidize methanol. In a methanotroph with both MxaFI and XoxF, we demonstrate that lanthanides transcriptionally control genes encoding the two methanol dehydrogenases, in part by controlling expression of the response regulator MxaB. Lanthanides are abundant in the Earth's crust, and we demonstrate that micromolar amounts of lanthanides are sufficient to suppress MxaFI expression. Thus, we present evidence that XoxF acts as the predominant methanol dehydrogenase in a methanotroph.

Distribution of Paraoxonase-1 enzyme Activity in some Camels Tissues

Paraoxonase-1 (PON1,aryldialkylphosphatase) is a calcium-dependent esterase enzyme synthesized in the liver and it is mainly associated with a high density lipoprotein (HDL).This study aimed to investigate the PON1 enzyme activities in male camel's serum& tissues and its distribution among camel tissues (liver, heart and kidney) in immature and mature camels. PON1 activity in serum (32 dromedary camels, 17one of them immature ≤ 5 years; 15 one mature > 5years) were 115.89 i?½ 11.37 U/L and 114.43 i?½ 11.87 U/L, respectively. The results showed that PON1 was highly distributed in the liver then kidney and heart tissues in immature camels (2.74 i?½ 0.42, 1.66 i?½ 0.14, 0.017 i?½ 0.005 U/g) as well in mature camels (2.41 i?½ 0.29, 1.41 i?½ 0.12, 0.019 i?½ 0.005 U/g), respectively. Also, Glutathione peroxidase (GPx) and Superoxide dismutase (SOD) were highly distributed in liver then kidney and heart tissues. Moreover, GPx activity in liver was higher with increased camel's age. PON1 is correlated positively with GPx & SOD and HDL-c but negatively correlated with MDA and total cholesterol. Furthermore, was found positive correlation between total cholesterol and LDL-c in serum and hepatic tissues.

Tissue Transglutaminase Regulates Steroid Resistance in Acute Lymphoblastic Leukemia Via Nuclear Factor-Kappa B Related Signaling Pathways

Steroid resistance is lethal to the patients with acute lymphoblastic leukemia (ALL). To improve survival for steroid-resistant ALL patients, it is necessary to develop innovative therapeutic strategies to overcome steroid resistance. Tissue transglutaminase (TG2) is an intracellular calcium-dependent protein cross-linking enzyme reported to be over-expressed in various metastatic or chemotherapy-resistant cancer cells including lymphoma cells. Chronic expression of TG2 constitutively activates nuclear factor kappa B (NF-kappaB) and it is directed linked to NF-kappaB-related signaling networks including hypoxia-inducible factor-1 (HIF-1), Zeb, Snail, or Twist that influence drug resistance or metastasis in inflammatory or cancer cells. In the present study, we used biochemical and molecular methodologies to demonstrate that steroid resistance was associated with TG2-regulated signaling pathways including NF-kappaB and its downstream transcriptional factors in steroid-resistant ALL. We generated steroid-adapted subclones of ALL cell lines that were extremely more resistant to steroid than the parent cells. We found that steroid-resistant ALL cells expressed elevated levels of TG2 and the modification of TG2 activities altered NF-kappaB expression in this cells. TG2-related NF-kappaB activation modulated the expression of HIF-1 and NF-kappaB-targeted genes such as Zeb, Snail, and Twist in steroid-resistant ALL cells. Also, TG2 inhibition improved the cytotoxicity of steroid in steroid-resistant ALL cells. This is the first study to show the link between TG2, NF-kappaB, its downstream signaling networks, and steroid resistance in ALL. TG2 inhibition could be used as an alternative target to overcome steroid resistance in ALL. Furthermore, TG2 and HIF-1 expression could serve as new biomarkers of steroid resistance evolution or cancer recurrence in ALL. DisclosuresNo relevant conflicts of interest to declare.

Orthogonal optimization of prokaryotic expression of a natural snake venom phospholipase A2 inhibitor from Sinonatrix annularis

Phospholipase A2 (PLA2) is a calcium-dependent enzyme that is involved in inflammatory processes such as the liberation of free arachidonic acid from the membrane pool for the biosynthesis of eicosanoids. Snake venom are known containing PLA2s (svPLA2s) which exhibit a wide variety of pharmacological effects including neurotoxicity, cardiotoxicity, myotoxicity and hemorrhage. Therefore, inhibition of svPLA2 would be advantageous to successful envenomation treatment. A gamma type PLI (PLA2 inhibitor) has been extracted from the serum of Sinonatrix annularis, a non-venomous snake indigenous to China. This showed strong inhibition of Deinagkistrrodon acutus PLA2, however, the PLIγ level in the serum and snake resource are not sufficiently sustainable for further research. To overcome these limitations, we constructed a His6-PLIγ pET28 fusion expression vector and transformed Escherichia coli BL21. To improve the expression of PLIγ, an orthogonal experiment [L16(4)5] was performed to optimize induction parameters. The optimized condition was determined to be: induction by 0.4 mM isopropyl-β-d-thiogalactoside (IPTG) for 6 h to the recombinant BL21 after its OD600 was 0.8, with continuous shaking cultivation at 190 rpm and 35 °C. Under these conditions, the amount of expressed protein could reach 57 mg/L. The His6-PLIγ was purified by nickel affinity chromatography and renatured by On-column refolding. The resulting PLIγ showed a good inhibitory effect of enzymatic activities to venom PLA2 isolated from D. acutus. Moreover, the PLIγ had a wide anti-hemorrhage activities to D. acutus, Naja atra and Agkistrodon halys venom.

Ankyrin exposure induced by activated protein kinase C plays a potential role in erythrophagocytosis

BackgroundIn physiological and pathological conditions activated protein kinace C (PKC) has been observed in the erythrocytes. Externalization of ankyrin followed by Arg–Gly–Asp (RGD)/integrin recognition also triggers erythrophagocytosis. In the present study, to test whether activated PKC is associated with ankyrin exposure in erythrophagocytosis. MethodsPhorbol 12-myristate-13-acetate (PMA)-induced PKC activation and ankyrin phosphorylation were tested, and under different treatment conditions the subpopulation of erythrocytes with ankyrin exposure and the levels of intracellular calcium were analyzed by flow cytometry. ResultsResults showed that treatment of erythrocytes with PMA in a calcium-containing buffer led to ankyrin exposure. In the absence of extracellular calcium, no ankyrin exposure was observed. PKC inhibition with calphostin C, a blocker of the PMA binding site, completely prevented the calcium entry, protein phosphorylation and ankyrin exposure. PKC inhibition with chelerythrine chloride, an inhibitor of the active site, diminished the level of ankyrin-exposing cells and ankyrin phosphorylation; however it even led to a higher percentage of cells with increased levels of calcium than with PMA treatment alone. Although PKC was activated and ankyrin phosphorylation occurred, no ankyrin exposure was observed in the absence of extracellular calcium. ConclusionAnalyses of results suggested that PMA induces calcium influx into the erythrocytes, leading to the activation of calcium-dependent enzymes and the phosphorylation of membrane proteins, ultimately inducing ankyrin exposure and erythrophagocytosis. This study may provide insights into the molecular mechanisms of removing aged or diseased erythrocytes.

Tissue Transglutaminase and Its Product Isopeptide Are Increased in Alzheimer's Disease and APPswe/PS1dE9 Double Transgenic Mice Brains.

Alzheimer's disease (AD) is characterized by intracellular and extracellular protein aggregates, including microtubule-associated protein tau and cleavage product of amyloid precursor protein, β-amyloid (Aβ). Tissue transglutaminase (tTG) is a calcium-dependent enzyme that cross-links proteins forming a γ-glutamyl-ε-lysine isopeptide bond. Highly resistant to proteolysis, this bond can induce protein aggregation and deposition. We set out to determine if tTG may play a role in pathogenesis of AD. Previous studies have shown that tTG and isopeptide are increased in advanced AD, but they have not addressed if this is an early or late feature of AD. In the present study, we measured tTG expression levels and enzyme activity in the brains of individuals with no cognitive impairment (NCI), mild cognitive impairment (MCI), and AD, as well as a transgenic mouse model of AD. We found that both enzyme expression and activity were increased in MCI as well as AD compared to NCI. In the transgenic model of AD, tTG expression and enzyme activity increased sharply with age and were relatively specific for the hippocampus. We also assessed overlap of isopeptide immunoreactivity with neurodegeneration-related proteins with Western blots and found neurofilament, tau, and Aβ showed co-localization with isopeptide in both AD and transgenic mice. These results suggest that tTG might be a key factor in pathogenesis of abnormal protein aggregation in AD.

2015 Lucian Award: Jeffery Molkentin.

The 38th Louis and Artur Lucian Award for Research in Circulatory Diseases has been presented to Jeffery Molkentin, PhD, of Cincinnati Children’s Hospital Medical Center. Molkentin is being honored for several paradigm-changing discoveries about the molecular mechanisms of cardiac disease. Jeffery Molkentin, PhD, received his BS in biology from Marquette University and his PhD in physiology from the Medical College of Wisconsin in Milwaukee. During a postdoctoral fellowship with Eric Olson, PhD, at the University of Texas Southwestern Medical Center in Dallas, he and Olson discovered a key role for the calcium-dependent enzyme calcineurin as a driver of pathological hypertrophy—the first of 4 critical findings in distinct areas of research for which he is receiving the Lucian award. In 1997, Molkentin established an independent laboratory at Children’s Hospital Medical Center in Cincinnati. Appointed as an assistant professor of pediatrics at the University of Cincinnati, he became a full professor in 2006. Since 2008, Molkentin has been an investigator of the Howard Hughes Medical Institute, allowing him to pursue discovery-based research in addition to hypothesis-testing studies. In 2012, Molkentin was awarded the Basic Science Research Prize by the American Heart Association for his “transformational studies of signaling mechanisms involved in heart cell death and hypertrophy as well as potential triggers for disease.” Molkentin serves on the editorial board and as a consulting editor at Circulation Research . Fifty years ago, Dame Olga Leibovici established the Lucian award honoring her brothers—both engineers who died prematurely—when she bequeathed $2 million to McGill University.1 The $60 000 CAN prize recognizes the contributions of active investigators or teams whose work has had a significant impact on the understanding of cardiovascular disease (Table). The Lucian Award places no restrictions on the sex or nationality of recipients, who are selected by a committee of scientists that …

Abstract 2271: Peptidylarginine deiminase 2 as a novel therapeutic target for breast cancer

Abstract Peptidylarginine deiminases (PADIs) enzymes are calcium-dependent enzymes that post-translationally convert positively charged protein arginine residues to a neutrally charged citrulline in a process known as citrullination or deimination. This loss of charge can alter the tertiary structure of proteins and affect protein-protein and protein-DNA interactions. We, and others, have found that PADI-mediated histone tail citrullination alters chromatin structure and regulates gene expression. For example, we found that PADI2 interacts with estrogen receptor (ERα) and that PADI2-mediated citrullination of histone H3 arginine 26 residues at ERα binding sites regulates target gene expression. In addition, we also found that PADI2 is often co-expressed with HER2 in both breast cancer cell lines and in tumor tissue. Our molecular studies suggest that PADI2 activates HER2 expression via histone citrullination at the HER2 gene locus and that HER2 signaling appears to induce PADI2 expression, thus potentially forming an oncogenic positive feedback loop with HER2. These findings suggest an important role for PADI2 in breast cancer progression. This prediction is supported by our finding that PADI2 expression is upregulated in cancer cells and that depletion of PADI2 from breast cancer cells results in reduced tumorigenicity. In addition, we found that transgenic overexpression of PADI2 promotes carcinogenesis and that the PADI inhibitor, Cl-Amidine, slows tumor growth in mouse models of breast cancer. Furthermore, we found that Cl-Amidine also maintains basement membrane integrity in xenograft tumors, preventing initiation of metastasis. Together, these findings suggest that PADI2 represents a novel therapeutic target and biomarker for early stage breast cancer. Citation Format: Sachi Horibata, John L. McElwee, David Sadegh, Katherine Rogers, Dalton McLean, Scott A. Coonrod. Peptidylarginine deiminase 2 as a novel therapeutic target for breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2271. doi:10.1158/1538-7445.AM2015-2271

Abstract 238: Citrullination of miRNA regulators by PADs: A potential mechanism for the promotion of colitis and colitis-associated colorectal cancer

Abstract Ulcerative Colitis (UC) is a chronic, relapsing inflammatory bowel disease that affects millions of people worldwide. As a result of DNA damage caused by the chronic inflammatory state of UC, people suffering from UC are at a higher risk of developing colorectal cancer (CRC). Although the cause of UC is still unknown, the protein arginine deiminase (PAD) family of enzymes has been found to be a key component in many human inflammatory diseases and cancer, including UC and CRC. PADs are calcium-dependent enzymes that post-translationally convert positively charged peptidyl-Arginine to neutral peptidyl-Citrulline through a process called ‘citrullination’. In hopes of treating UC and preventing CRC, we developed Chlor-amidine (Cl-amidine), a novel small molecule inhibitor of PADs. Previous results from our group have shown that miR-16, which is often downregulated in cancers, had increased expression levels in mice treated with Cl-amidine. MiR-16 targets (i.e. Cyclin D1 and E1) and colon carcinogenesis were also suppressed in mice treated with Cl-amidine. Our goal is to determine the mechanism(s) by which Cl-amidine targets miR-16 and other tumor suppressor miRNAs. One hypothesis is that Cl-amidine is preventing the citrullination of DNA methyltransferases (DNMTs). Citrullinated DNMTs show hyperactivity, resulting in hypermethylation and subsequent silencing of miRNAs. Evidence suggests that PADs can citrullinate DNMT3A, resulting in ‘methylation-mediated gene silencing’. We predict that this silencing negatively regulates tumor suppressor miRNAs and Cl-amidine treatment will restore these miRNA levels. Initial results suggest citrullination of DNMT3A in our in vitro colon cancer cell line model. Experiments are ongoing to confirm the ability of Cl-amidine to prevent citrullination-induced activation of upstream miRNA regulators. Results from our study will elaborate on the mechanism of action of Cl-amidine and provide further insight into using Cl-amidine as an anti-cancer therapy. Citation Format: Erin E. Witalison, Xiangli Cui, Corey P. Causey, Paul R. Thompson, Lorne J. Hofseth. Citrullination of miRNA regulators by PADs: A potential mechanism for the promotion of colitis and colitis-associated colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 238. doi:10.1158/1538-7445.AM2015-238

Abstract 1348: CD4 T cells targeting citrullinated vimentin reject advanced tumors

Abstract CD4 cells are potent effectors but CD4 responses to tumour associated antigens are attenuated. Cellular stress induces autophagy which leads to modification of proteins recognised by the immune system. In the absence of inflammation, immunity is regulated, whereas in its presence CD4 responses to modified self-antigens are stimulated. T cells commonly target modified self-antigens and have been shown to play a role in the pathophysiology of several autoimmune diseases. In this study the ability of these CD4 cells to target cancer has been explored. One modification is conversion of arginine to citrulline. Peptidylarginine deiminases (PADs), are a family of calcium dependent enzymes found in a variety of tissues, where their role is post translation citrullination of proteins. Citrullination is conversion of the positively charge aldimine group ( = NH) group of arginine to the neutrally charged ketone group ( = O) of citrulline. Immunisation with citrullinated vimentin peptides induced IFNγ and granzyme B secreting CD4 T cells in response to autophagic tumour targets. The induced CD4 responses to citrullinated self-peptide epitopes show minimal reactivity to the unmodified sequence. A single immunization with modified peptide, up to 14 days after tumour implant, resulted in long term survival in 60-90% of animals with no associated toxicity. The anti-tumour responses were dependent upon CD4 but not on CD8 T cells. This study provides the first evidence that tumours can present citrullinated peptides that can be targets for CD4 cells. This presentation is dependent upon autophagy and PAD enzymes. The CD4 T cells release IFNγ and show direct cytotoxicity which results in potent anti-tumour responses in vivo. This approach is being fast tracked into the clinic in patient's whose tumours express vimentin. This could be patients with mesenchymal tumours or in patients whose tumours undergo epithelial to mesenchymal transition as vimentin is one of the first proteins to be expressed in this transition. Citation Format: Lindy G. Durrant, Victoria A. Brentville, Rachael L. Metheringham, Ian Daniels, Peter Symonds, Mohamed Gijon, Wei Xue. CD4 T cells targeting citrullinated vimentin reject advanced tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1348. doi:10.1158/1538-7445.AM2015-1348

Transglutaminase and neurodegenerative diseases

Transglutaminase (TG) is a kind of calcium-dependent enzymes. The TGase family found in rodents and human contains 9 types, including TG1-7, blood coagulation factor XIIIa and erythrocyte membrane protein 4.2, with the former 8 types possessing catalytic activity. TG catalyzes various conversion reactions of glutamine, including transamination, deamination and esterification, and participates in post-transcriptional modification of proteins such as cross-linking peptides glutamine residue and lysyl-residue, stabilizing protein structure and catalyzing formation of protein aggregates. TGase has been found to contribute to a variety of important physiological and pathological processes and play a role in the pathogenesis of multiple diseases. Notably, neurodegenerative diseases such as Huntington's disease, spinocerebellar ataxia, Alzheimer's disease and Parkinson's disease, have a close connection with TGase's role in the human body.

Characterization of an extremely salt-tolerant and thermostable phytase from Bacillus amyloliquefaciens US573

The extracellular phytase produced by the Bacillus amyloliquefaciens US573 strain, isolated from geothermal soil located in Southern Tunisia was purified and characterized. This calcium-dependent and bile-stable enzyme (PHY US573) was optimally active at pH 7.5 and 70°C. It showed a good stability at pH ranging from 4 to 10, and especially, an exceptional thermostability as it recovered 50 and 62% of activity after heating for 10min at 100 and 90°C, respectively. In addition, PHY US573 was found to be extremely salt-tolerant since it preserved 80 and 95% of activity in the presence of 20g/l of NaCl and LiCl, respectively. The gene corresponding to PHY US573 was cloned. It encodes a 383 amino acids polypeptide exhibiting 99% identity with the highly thermostable phytases from Bacillus sp. MD2 and B. amyloliquefaciens DS11 (3 and 5 residues difference, respectively), suggesting the existence of common molecular determinants responsible for their remarkable heat stability. Overall, our findings illustrated that in addition to its high potential for application in feed industry, the salt tolerance of the PHY US573 phytase, may represent an exciting new avenue for improvement of phosphorus-use efficiency of salt-tolerant plants in soils with high salt and phytate content.

Regulation of prohormone convertase 2 protein expression via GPR40/FFA1 in the hypothalamus

Previous studies have shown that the administration of docosahexaenoic acid (DHA) or GW9508, a GPR40/FFA1 (free fatty acid receptor) agonist, facilitates β-endorphin release in the arcuate nucleus of the hypothalamus in mice. However, the mechanisms mediating β-endorphin release induced by GPR40/FFA1 agonists remain unknown. In this study, we focused on the changes in expression of hypothalamic prohormone convertase (PC) 2, which is a calcium-dependent subtilisin-related proteolytic enzyme. The intracerebroventricular injection of DHA or GW9508 significantly increased PC2 protein expression in the hypothalamus. This increase in PC2 expression was inhibited by pretreatment with GW1100, a GPR40/FFA1 antagonist. Furthermore, PC2 protein expression gradually increased over time after complete Freund's adjuvant. These increase in PC2 expression were inhibited by pretreatment with GW1100. However, GW1100 by itself had no effect on PC2 levels. Taken together, our findings suggest that activation of the hypothalamic GPR40/FFA1 signaling pathway may regulate β-endorphin release via PC2, and regulate the endogenous pain control system.

In vitro inhibition effect of some coumarin compounds on purified human serum paraoxonase 1 (PON1)

Human serum paraoxonase 1 (PON1; EC 3.1.8.1) is a high-density lipoprotein associated, calcium-dependent enzyme that hydrolyses aromatic esters, organophosphates and lactones and can protect the low-density lipoprotein against oxidation. In this study, in vitro effect of some hydroxy and dihydroxy ionic coumarin derivatives (1–20) on purified PON1 activity was investigated. Among these compounds, derivatives 11–20 are water soluble. In investigated compounds, compounds 6 and 13 were found the most active (IC50 = 35 and 34 µM) for PON1, respectively. The present study has demonstrated that PON1 activity is very highly sensitive to studied coumarin derivatives.

Production and Characterization of α-Amylase from an Extremely Halophilic Archaeon, Haloferax sp. HA10.

Haloarchaea are found at very high concentrations in salt-conditioned environments, hence produce enzymes which are able to catalyze reactions under harsh conditions, typical of many industrial processes. In the present study, culture conditions for extracellular amylase production from Haloarchaea isolated from a solar saltern were optimized and the purified enzyme was characterized. Haloferax sp. HA10 showed maximum amylase production at 3 M NaCl, 37 °C, pH=7 and 1% starch content. Purified α-amylase was a calcium-dependent enzyme with an estimated molecular mass of about 66 kDa and many industrially useful properties. It was found to be stable in a broad range of pH (from 5 to 9) and NaCl concentrations (from 0.5 to 3.0 M), retaining 48% activity even at 4 M. The optimal temperature for Haloferax sp. HA10 amylase activity was 55 °C (99% activity), and 57% activity was retained at 80 °C, which dropped to 44% with the increase of temperature to 90 or 100 °C. It was able to sustain various surfactants and detergents. To the best of our knowledge the detergent-stable α-amylases from halophilic archaeon have not been reported yet.

The importance of Ca(2+)-dependent mechanisms for the initiation of the heartbeat.

Mechanisms underlying pacemaker activity in the sinus node remain controversial, with some ascribing a dominant role to timing events in the surface membrane (“membrane clock”) and others to uptake and release of calcium from the sarcoplasmic reticulum (SR) (“calcium clock”). Here we discuss recent evidence on mechanisms underlying pacemaker activity with a particular emphasis on the many roles of calcium. There are particular areas of controversy concerning the contribution of calcium spark-like events and the importance of I(f) to spontaneous diastolic depolarisation, though it will be suggested that neither of these is essential for pacemaking. Sodium-calcium exchange (NCX) is most often considered in the context of mediating membrane depolarisation after spark-like events. We present evidence for a broader role of this electrogenic exchanger which need not always depend upon these spark-like events. Short (milliseconds or seconds) and long (minutes) term influences of calcium are discussed including direct regulation of ion channels and NCX, and control of the activity of calcium-dependent enzymes (including CaMKII, AC1, and AC8). The balance between the many contributory factors to pacemaker activity may well alter with experimental and clinical conditions, and potentially redundant mechanisms are desirable to ensure the regular spontaneous heart rate that is essential for life. This review presents evidence that calcium is central to the normal control of pacemaking across a range of temporal scales and seeks to broaden the accepted description of the “calcium clock” to cover these important influences.