Sodium/hydrogen exchanger NHA2 in osteoclasts: subcellular localization and role in vitro and in vivo

NHA2 was recently identified as a novel sodium/hydrogen exchanger which is strongly upregulated during RANKL-induced osteoclast differentiation. Previous in vitro studies suggested that NHA2 is a mitochondrial transporter required for osteoclast differentiation and bone resorption. Due to the lack of suitable antibodies, NHA2 was studied only on RNA level thus far. To define the protein's role in osteoclasts in vitro and in vivo, we generated NHA2-deficient mice and raised several specific NHA2 antibodies. By confocal microscopy and subcellular fractionation studies, NHA2 was found to co-localize with the late endosomal and lysosomal marker LAMP1 and the V-ATPase a3 subunit, but not with mitochondrial markers. Immunofluorescence studies and surface biotinylation experiments further revealed that NHA2 was highly enriched in the plasma membrane of osteoclasts, localizing to the basolateral membrane of polarized osteoclasts. Despite strong upregulation of NHA2 during RANKL-induced osteoclast differentiation, however, structural parameters of bone, quantified by high-resolution microcomputed tomography, were not different in NHA2-deficient mice compared to wild-type littermates. In addition, in vitro RANKL stimulation of bone marrow cells isolated from wild-type and NHA2-deficient mice yielded no differences in osteoclast development and activity. Taken together, we show that NHA2 is a RANKL-induced plasmalemmal sodium/hydrogen exchanger in osteoclasts. However, our data from NHA2-deficient mice suggest that NHA2 is dispensable for osteoclast differentiation and bone resorption both in vitro and in vivo.

Regulation and structure of YahD, a copper-inducible / serine hydrolase of Lactococcus lactis IL1403

Lactococcus lactis IL1403 is a lactic acid bacterium that is used widely for food fermentation. Copper homeostasis in this organism chiefly involves copper secretion by the CopA copper ATPase. This enzyme is under the control of the CopR transcriptional regulator. CopR not only controls its own expression and that of CopA, but also that of an additional three operons and two monocistronic genes. One of the genes under the control of CopR, yahD, encodes an α/β-hydrolase. YahD expression was induced by copper and cadmium, but not by other metals or oxidative or nitrosative stress. The three-dimensional structure of YahD was determined by X-ray crystallography to a resolution of 1.88 Å. The protein was found to adopt an α/β-hydrolase fold with the characteristic Ser-His-Asp catalytic triad. Functional testing of YahD for a wide range of substrates for esterases, lipases, epoxide hydrolases, phospholipases, amidases and proteases was, however, unsuccessful. A copper-inducible serine hydrolase has not been described previously and YahD appears to be a new functional member of this enzyme family.

Pseudomyotonia, a muscle function disorder associated with an inherited ATP2A1 (SERCA1) defect in a Dutch Improved Red and White cross-breed calf

A Dutch Improved Red and White cross-breed heifer calf was evaluated for a muscular disorder resulting in exercise induced muscle stiffness. Clinical findings included generalized exercise-induced muscle spasms with normal response to muscle percussion. Electromyography showed no myotonic discharges, thus ruling out myotonia. Whereas histological examination of muscle tissue was unremarkable, Ca(2+)-ATPase activity of sarcoplasmatic reticulum membranes (SERCA1) was markedly decreased compared to control animals. Mutation analysis revealed the presence of a missense mutation in the ATP2A1 gene encoding the SERCA1 protein (p.Arg559Cys). The present case presents similarities to human Brody's disease, but also to pseudomyotonia and congenital muscular dystonia previously described in different cattle breeds.

Ca(2+) release from the sarcoplasmic reticulum activated by the low affinity Ca(2+) chelator TPEN in ventricular myocytes

The Ca(2+) content of the sarcoplasmic reticulum (SR) of cardiac myocytes is thought to play a role in the regulation and termination of SR Ca(2+) release through the ryanodine receptors (RyRs). Experimentally altering the amount of Ca(2+) within the SR with the membrane-permeant low affinity Ca(2+) chelator TPEN could improve our understanding of the mechanism(s) by which SR Ca(2+) content and SR Ca(2+) depletion can influence Ca(2+) release sensitivity and termination. We applied laser-scanning confocal microscopy to examine SR Ca(2+) release in freshly isolated ventricular myocytes loaded with fluo-3, while simultaneously recording membrane currents using the whole-cell patch-clamp technique. Following application of TPEN, local spontaneous Ca(2+) releases increased in frequency and developed into cell-wide Ca(2+) waves. SR Ca(2+) load after TPEN application was found to be reduced to about 60% of control. Isolated cardiac RyRs reconstituted into lipid bilayers exhibited a two-fold increase of their open probability. At the low concentration used (20-40muM), TPEN did not significantly inhibit the SR-Ca(2+)-ATPase in SR vesicles. These results indicate that TPEN, traditionally used as a low affinity Ca(2+) chelator in intracellular Ca(2+) stores, may also act directly on the RyRs inducing an increase in their open probability. This in turn results in an increased Ca(2+) leak from the SR leading to its Ca(2+) depletion. Lowering of SR Ca(2+) content may be a mechanism underlying the recently reported cardioprotective and antiarrhythmic features of TPEN.

Consequences of depleted SERCA2-gated calcium stores in the skin

Sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2) pumps belong to the family of Ca2+-ATPases responsible for the maintenance of calcium in the endoplasmic reticulum. In epidermal keratinocytes, SERCA2-controlled calcium stores are involved in cell cycle exit and onset of terminal differentiation. Hence, their dysfunction was thought to provoke impaired keratinocyte cohesion and hampered terminal differentiation. Here, we assessed cultured keratinocytes and skin biopsies from a canine family with an inherited skin blistering disorder. Cells from lesional and phenotypically normal areas of one of these dogs revealed affected calcium homeostasis due to depleted SERCA2-gated stores. In phenotypically normal patient cells, this defect compromised upregulation of p21(WAF1) and delayed the exit from the cell cycle. Despite this abnormality it failed to impede the terminal differentiation process in the long term but instead coincided with enhanced apoptosis and appearance of chronic wounds, suggestive of secondary mutations. Collectively, these findings provide the first survey on phenotypic consequences of depleted SERCA-gated stores for epidermal homeostasis that explain how depleted SERCA2 calcium stores provoke focal lesions rather than generalized dermatoses, a phenotype highly reminiscent of the human genodermatosis Darier disease.

Cadaver dogs--a study on detection of contaminated carpet squares

INTRODUCTION: Cadaver dogs are known as valuable forensic tools in crime scene investigations. Scientific research attempting to verify their value is largely lacking, specifically for scents associated with the early postmortem interval. The aim of our investigation was the comparative evaluation of the reliability, accuracy, and specificity of three cadaver dogs belonging to the Hamburg State Police in the detection of scents during the early postmortem interval. MATERIAL AND METHODS: Carpet squares were used as an odor transporting media after they had been contaminated with the scent of two recently deceased bodies (PMI<3h). The contamination occurred for 2 min as well as 10 min without any direct contact between the carpet and the corpse. Comparative searches by the dogs were performed over a time period of 65 days (10 min contamination) and 35 days (2 min contamination). RESULTS: The results of this study indicate that the well-trained cadaver dog is an outstanding tool for crime scene investigation displaying excellent sensitivity (75-100), specificity (91-100), and having a positive predictive value (90-100), negative predictive value (90-100) as well as accuracy (92-100).

Point mutations in the stem region and the fourth AAA domain of cytoplasmic dynein heavy chain partially suppress the phenotype of NUDF/LIS1 loss in Aspergillus nidulans

Cytoplasmic dynein performs multiple cellular tasks but its regulation remains unclear. The dynein heavy chain has a N-terminal stem that binds to other subunits and a C-terminal motor unit that contains six AAA (ATPase associated with cellular activities) domains and a microtubule-binding site located between AAA4 and AAA5. In Aspergillus nidulans, NUDF (a LIS1 homolog) functions in the dynein pathway, and two nudF6 partial suppressors were mapped to the nudA dynein heavy chain locus. Here we identified these two mutations. The nudAL1098F mutation resides in the stem region, and nudAR3086C is in the end of AAA4. These mutations partially suppress the phenotype of nudF deletion but do not suppress the phenotype exhibited by mutants of dynein intermediate chain and Arp1. Surprisingly, the stronger DeltanudF suppressor, nudAR3086C, causes an obvious decrease in the basal level of dynein's ATPase activity and an increase in dynein's distribution along microtubules. Thus, suppression of the DeltanudF phenotype may result from mechanisms other than simply the enhancement of dynein's ATPase activity. The fact that a mutation in the end of AAA4 negatively regulates dynein's ATPase activity but partially compensates for NUDF loss indicates the importance of the AAA4 domain in dynein regulation in vivo.

Characterization of the CopR regulon of Lactococcus lactis IL1403

To identify components of the copper homeostatic mechanism of Lactococcus lactis, we employed two-dimensional gel electrophoresis to detect changes in the proteome in response to copper. Three proteins upregulated by copper were identified: glyoxylase I (YaiA), a nitroreductase (YtjD), and lactate oxidase (LctO). The promoter regions of these genes feature cop boxes of consensus TACAnnTGTA, which are the binding site of CopY-type copper-responsive repressors. A genome-wide search for cop boxes revealed 28 such sequence motifs. They were tested by electrophoretic mobility shift assays for the interaction with purified CopR, the CopY-type repressor of L. lactis. Seven of the cop boxes interacted with CopR in a copper-sensitive manner. They were present in the promoter region of five genes, lctO, ytjD, copB, ydiD, and yahC; and two polycistronic operons, yahCD-yaiAB and copRZA. Induction of these genes by copper was confirmed by real-time quantitative PCR. The copRZA operon encodes the CopR repressor of the regulon; a copper chaperone, CopZ; and a putative copper ATPase, CopA. When expressed in Escherichia coli, the copRZA operon conferred copper resistance, suggesting that it functions in copper export from the cytoplasm. Other member genes of the CopR regulon may similarly be involved in copper metabolism.

Gender modulates the expression of calcium-regulating proteins in pediatric atrial myocardium

A differential expression of sarcoplasmic reticulum calcium-ATPase (SERCA2a) and phospholamban (PLB) characterizes the remodeling process in heart failure and atrial arrhythmias in adult patients. Gender is known to modulate the course and prognosis of different forms of heart disease. We hypothesized that gender plays a role in molecular changes of myocardial calcium regulating components already in childhood. Moreover, we studied the influence of volume overloaded (VO) on SERCA2a and PLB in pediatric patients. Quantitative reverse transcription-polymerase chain reaction was used to measure mRNA expression of SERCA2a and PLB in atrial myocardium from 30 pediatric patients (12 girls, 18 boys). Eighteen patients had VO right atria, and 12 patients had not-overloaded atria (NO). Protein expression was studied by Western blot. In the entire population, SERCA2a and PLB expression was not different between girls and boys. If hemodynamic overload was taken into account, SERCA2a mRNA expression was significantly reduced in the VO group compared with the NO group (P = 0.021). The VO versus NO difference was restricted to boys, which corresponds to a highly significant interaction of gender versus VO status (P = 0.002). The PLB to SERCA2a protein ratio was significantly lower in girls (P = 0.028). The decrease in SERCA2a mRNA expression in VO atrial myocardium and the PLB to SERCA2a ratio of protein expression was modulated by gender in this pediatric population. To our knowledge, this study is the first to show the impact of gender on the differential expression of calcium-regulating components in pediatric cardiac patients.

Age-dependent suppression of SERCA2a mRNA in pediatric atrial myocardium

Differential expression of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) and phospholamban (PLB) has been shown in heart failure and atrial arrhythmias. We investigated the influence of volume overload and age on their expression in pediatric atrial myocardium. Right atrial specimens from 18 children with volume overloaded right atrium (VO) and 12 patients without overload were studied. Each group was further divided into patients less than and older than 12 months of age. Only in the younger patients SERCA2a was significantly reduced in the VO group. In younger patients PLB mRNA level tended to be lower in VO. The PLB:SERCA protein ratio was significantly reduced in the VO group. Age itself did not influence the SERCA2a and PLB expression, if the hemodynamic overload was not taken into account. This study is the first to show a combined influence of volume overload and age on atrial SERCA2a expression.

Alpha1-syntrophin mutations identified in sudden infant death syndrome cause an increase in late cardiac sodium current.

BACKGROUND Sudden infant death syndrome (SIDS) is a leading cause of death during the first 6 months after birth. About 5% to 10% of SIDS may stem from cardiac channelopathies such as long-QT syndrome. We recently implicated mutations in alpha1-syntrophin (SNTA1) as a novel cause of long-QT syndrome, whereby mutant SNTA1 released inhibition of associated neuronal nitric oxide synthase by the plasma membrane Ca-ATPase PMCA4b, causing increased peak and late sodium current (I(Na)) via S-nitrosylation of the cardiac sodium channel. This study determined the prevalence and functional properties of SIDS-associated SNTA1 mutations. METHODS AND RESULTS Using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing of SNTA1's open reading frame, 6 rare (absent in 800 reference alleles) missense mutations (G54R, P56S, T262P, S287R, T372M, and G460S) were identified in 8 (approximately 3%) of 292 SIDS cases. These mutations were engineered using polymerase chain reaction-based overlap extension and were coexpressed heterologously with SCN5A, neuronal nitric oxide synthase, and PMCA4b in HEK293 cells. I(Na) was recorded using the whole-cell method. A significant 1.4- to 1.5-fold increase in peak I(Na) and 2.3- to 2.7-fold increase in late I(Na) compared with controls was evident for S287R-, T372M-, and G460S-SNTA1 and was reversed by a neuronal nitric oxide synthase inhibitor. These 3 mutations also caused a significant depolarizing shift in channel inactivation, thereby increasing the overlap of the activation and inactivation curves to increase window current. CONCLUSIONS Abnormal biophysical phenotypes implicate mutations in SNTA1 as a novel pathogenic mechanism for the subset of channelopathic SIDS. Functional studies are essential to distinguish pathogenic perturbations in channel interacting proteins such as alpha1-syntrophin from similarly rare but innocuous ones.

Syntrophin mutation associated with long QT syndrome through activation of the nNOS-SCN5A macromolecular complex.

Mutations in 11 genes that encode ion channels or their associated proteins cause inherited long QT syndrome (LQTS) and account for approximately 75-80% of cases (LQT1-11). Direct sequencing of SNTA1, the gene encoding alpha1-syntrophin, was performed in a cohort of LQTS patients that were negative for mutations in the 11 known LQTS-susceptibility genes. A missense mutation (A390V-SNTA1) was found in a patient with recurrent syncope and markedly prolonged QT interval (QTc, 530 ms). SNTA1 links neuronal nitric oxide synthase (nNOS) to the nNOS inhibitor plasma membrane Ca-ATPase subtype 4b (PMCA4b); SNTA1 also is known to associate with the cardiac sodium channel SCN5A. By using a GST-fusion protein of the C terminus of SCN5A, we showed that WT-SNTA1 interacted with SCN5A, nNOS, and PMCA4b. In contrast, A390V-SNTA1 selectively disrupted association of PMCA4b with this complex and increased direct nitrosylation of SCN5A. A390V-SNTA1 expressed with SCN5A, nNOS, and PMCA4b in heterologous cells increased peak and late sodium current compared with WT-SNTA1, and the increase was partially inhibited by NOS blockers. Expression of A390V-SNTA1 in cardiac myocytes also increased late sodium current. We conclude that the A390V mutation disrupted binding with PMCA4b, released inhibition of nNOS, caused S-nitrosylation of SCN5A, and was associated with increased late sodium current, which is the characteristic biophysical dysfunction for sodium-channel-mediated LQTS (LQT3). These results establish an SNTA1-based nNOS complex attached to SCN5A as a key regulator of sodium current and suggest that SNTA1 be considered a rare LQTS-susceptibility gene.

Design, synthesis and pharmacological characterization of analogs of 2-aminoethyl diphenylborinate (2-APB), a known store-operated calcium channel blocker, for inhibition of TRPV6-mediated calcium transport

2-Aminoethyl diphenylborinate (2-APB) is a known modulator of the IP3 receptor, the calcium ATPase SERCA, the calcium release-activated calcium channel Orai and TRP channels. More recently, it was shown that 2-APB is an efficient inhibitor of the epithelial calcium channel TRPV6 which is overexpressed in prostate cancer. We have conducted a structure-activity relationship study of 2-APB congeners to understand their inhibitory mode of action on TRPV6. Whereas modifying the aminoethyl moiety did not significantly change TRPV6 inhibition, substitution of the phenyl rings of 2-APB did. Our data show that the diaryl borinate moiety is required for biological activity and that the substitution pattern of the aryl rings can influence TRPV6 versus SOCE inhibition. We have also discovered that 2-APB is hydrolyzed and transesterified within minutes in solution.

Transient Neonatal Zinc Deficiency Caused by a Heterozygous G87R Mutation in the Zinc Transporter ZnT-2 (SLC30A2) Gene in the Mother Highlighting the Importance of Zn (2+) for Normal Growth and Development

Suboptimal dietary zinc (Zn(2+)) intake is increasingly appreciated as an important public health issue. Zn(2+) is an essential mineral, and infants are particularly vulnerable to Zn(2+) deficiency, as they require large amounts of Zn(2+) for their normal growth and development. Although term infants are born with an important hepatic Zn(2+) storage, adequate Zn(2+) nutrition of infants mostly depends on breast milk or formula feeding, which contains an adequate amount of Zn(2+) to meet the infants' requirements. An exclusively breast-fed 6 months old infant suffering from Zn(2+) deficiency caused by an autosomal dominant negative G87R mutation in the Slc30a2 gene (encoding for the zinc transporter 2 (ZnT-2)) in the mother is reported. More than 20 zinc transporters characterized up to date, classified into two families (Slc30a/ZnT and Slc39a/Zip), reflect the complexity and importance of maintaining cellular Zn(2+) homeostasis and dynamics. The role of ZnTs is to reduce intracellular Zn(2+) by transporting it from the cytoplasm into various intracellular organelles and by moving Zn(2+) into extracellular space. Zips increase intracellular Zn(2+) by transporting it in the opposite direction. Thus the coordinated action of both is essential for the maintenance of Zn(2+) homeostasis in the cytoplasm, and accumulating evidence suggests that this is also true for the secretory pathway of growth hormone.

Reconstructing the subsurface ocean decadal variability using surface nudging in a perfect model framework

Initialising the ocean internal variability for decadal predictability studies is a new area of research and a variety of ad hoc methods are currently proposed. In this study, we explore how nudging with sea surface temperature (SST) and salinity (SSS) can reconstruct the threedimensional variability of the ocean in a perfect model framework. This approach builds on the hypothesis that oceanic processes themselves will transport the surface information into the ocean interior as seen in ocean-only simulations. Five nudged simulations are designed to reconstruct a 150 years ‘‘target’’ simulation, defined as a portion of a long control simulation. The nudged simulations differ by the variables restored to, SST or SST + SSS, and by the area where the nudging is applied. The strength of the heat flux feedback is diagnosed from observations and the restoring coefficients for SSS use the same time-scale. We observed that this choice prevents spurious convection at high latitudes and near sea-ice border when nudging both SST and SSS. In the tropics, nudging the SST is enough to reconstruct the tropical atmosphere circulation and the associated dynamical and thermodynamical impacts on the underlying ocean. In the tropical Pacific Ocean, the profiles for temperature show a significant correlation from the surface down to 2,000 m, due to dynamical adjustment of the isopycnals. At mid-tohigh latitudes, SSS nudging is required to reconstruct both the temperature and the salinity below the seasonal thermocline. This is particularly true in the North Atlantic where adding SSS nudging enables to reconstruct the deep convection regions of the target. By initiating a previously documented 20-year cycle of the model, the SST + SSS nudging is also able to reproduce most of the AMOC variations, a key source of decadal predictability. Reconstruction at depth does not significantly improve with amount of time spent nudging and the efficiency of the surface nudging rather depends on the period/events considered. The joint SST + SSS nudging applied verywhere is the most efficient approach. It ensures that the right water masses are formed at the right surface density, the subsequent circulation, subduction and deep convection further transporting them at depth. The results of this study underline the potential key role of SSS for decadal predictability and further make the case for sustained largescale observations of this field.