Mechanism of urinary calcium regulation by urinary magnesium and pH.

Urinary magnesium and pH are known to modulate urinary calcium excretion, but the mechanisms underlying these relationships are unknown. In this study, the data from 17 clinical trials in which urinary magnesium and pH were pharmacologically manipulated were analyzed, and it was found that the change in urinary calcium excretion is directly proportional to the change in magnesium excretion and inversely proportional to the change in urine pH; a regression equation was generated to relate these variables (R(2) = 0.58). For further exploration of these relationships, intravenous calcium chloride, magnesium chloride, or vehicle was administered to rats. Magnesium infusion significantly increased urinary calcium excretion (normalized to urinary creatinine), but calcium infusion did not affect magnesium excretion. Parathyroidectomy did not prevent this magnesium-induced hypercalciuria. The effect of magnesium loading on calciuria was still observed after treatment with furosemide, which disrupts calcium and magnesium absorption in the thick ascending limb, suggesting that the effect may be mediated by the distal nephron. The calcium channel TRPV5, normally present in the distal tubule, was expressed in Xenopus oocytes. Calcium uptake by TRPV5 was directly inhibited by magnesium and low pH. In summary, these data are compatible with the hypothesis that urinary magnesium directly inhibits renal calcium absorption, which can be negated by high luminal pH, and that this regulation likely takes place in the distal tubule.

Surface modification of biomaterials for conjugation with human fetal osteoblasts.

Surface functionalization of hydroxyapatite (HA) and beta-tricalcium phosphate (TCP) bioceramics with chemical ligands containing a pyrrogallol moiety was developed to improve the adhesion of bone cell precursors to the biomaterials. Fast and biocompatible copper-free click reaction with azido-modified human fetal osteoblasts resulted in improved cell binding to both HA and TCP bioceramics, opening the way for using this methodology in the preparation of cell-engineered bone implants.

PDZ domain-binding motif regulates cardiomyocyte compartment-specific NaV1.5 channel expression and function.

BACKGROUND: Sodium channel NaV1.5 underlies cardiac excitability and conduction. The last 3 residues of NaV1.5 (Ser-Ile-Val) constitute a PDZ domain-binding motif that interacts with PDZ proteins such as syntrophins and SAP97 at different locations within the cardiomyocyte, thus defining distinct pools of NaV1.5 multiprotein complexes. Here, we explored the in vivo and clinical impact of this motif through characterization of mutant mice and genetic screening of patients. METHODS AND RESULTS: To investigate in vivo the regulatory role of this motif, we generated knock-in mice lacking the SIV domain (ΔSIV). ΔSIV mice displayed reduced NaV1.5 expression and sodium current (INa), specifically at the lateral myocyte membrane, whereas NaV1.5 expression and INa at the intercalated disks were unaffected. Optical mapping of ΔSIV hearts revealed that ventricular conduction velocity was preferentially decreased in the transversal direction to myocardial fiber orientation, leading to increased anisotropy of ventricular conduction. Internalization of wild-type and ΔSIV channels was unchanged in HEK293 cells. However, the proteasome inhibitor MG132 rescued ΔSIV INa, suggesting that the SIV motif is important for regulation of NaV1.5 degradation. A missense mutation within the SIV motif (p.V2016M) was identified in a patient with Brugada syndrome. The mutation decreased NaV1.5 cell surface expression and INa when expressed in HEK293 cells. CONCLUSIONS: Our results demonstrate the in vivo significance of the PDZ domain-binding motif in the correct expression of NaV1.5 at the lateral cardiomyocyte membrane and underline the functional role of lateral NaV1.5 in ventricular conduction. Furthermore, we reveal a clinical relevance of the SIV motif in cardiac disease.

Mineralocorticoid receptor is essential for corticosteroid-induced up-regulation of L-type calcium currents in cultured neonatal cardiomyocytes.

Despite the fact that mineralocorticoid receptor (MR) antagonist drugs such as spironolactone and eplerenone reduce the mortality in heart failure patients, there is, thus far, no unambiguous demonstration of a functional role of MR in cardiac cells. The aim of this work was to investigate the activation pathway(s) mediating corticosteroid-induced up-regulation of cardiac calcium current (ICa). In this study, using neonatal cardiomyocytes from MR or glucocorticoid receptor (GR) knockout (KO) mice, we show that MR is essential for corticosteroid-induced up-regulation of ICa. This study provides the first direct and unequivocal evidence for MR function in the heart.

Effects of pioglitazone on renal calcium excretion.

Context: Glitazones increase fracture risk in long-term users and in postmenopausal women. Studies have demonstrated deleterious effects of glitazones on bone metabolism. Glitazones also have direct renal tubular effects increasing sodium reabsorption. We hypothesized that glitazones may also regulate renal calcium excretion. Design: In this double-blind, randomized, placebo-controlled, four-way, crossover study, we examined the effects of pioglitazone (45 mg/d for 6 wk) or placebo on renal calcium and phosphate excretion and PTH levels during different sodium intakes in 16 individuals (eight with type 2 diabetes and eight with essential hypertension). Results: Pioglitazone had no effect on corrected plasma calcium and phosphate levels but decreased significantly the alkaline phosphatase and PTH levels. Pioglitazone induced on average a 45% increase in urinary calcium excretion. The fractional excretion of calcium rose to the same extent, suggesting a glomerular filtration rate-independent effect. Sodium intake did not influence the calciuric effect of pioglitazone. Changes in diurnal and nocturnal calciuria were similar. There was no effect of pioglitazone on phosphate excretion. Conclusion: Pioglitazone decreases PTH levels and increases urinary calcium excretion, independently from changes in glomerular filtration rate and from the sodium load, suggesting an inhibitory effect of pioglitazone on the tubular reabsorption of calcium. These effects may contribute to the increased fracture risk with glitazone treatment.

Activation of human meiosis-specific recombinase Dmc1 by Ca2+.

Rad51 and its meiotic homolog Dmc1 are key proteins of homologous recombination in eukaryotes. These proteins form nucleoprotein complexes on single-stranded DNA that promote a search for homology and that perform DNA strand exchange, the two essential steps of genetic recombination. Previously, we demonstrated that Ca2+ greatly stimulates the DNA strand exchange activity of human (h) Rad51 protein (Bugreev, D. V., and Mazin, A. V. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 9988-9993). Here, we show that the DNA strand exchange activity of hDmc1 protein is also stimulated by Ca2+. However, the mechanism of stimulation of hDmc1 protein appears to be different from that of hRad51 protein. In the case of hRad51 protein, Ca2+ acts primarily by inhibiting its ATPase activity, thereby preventing self-conversion into an inactive ADP-bound complex. In contrast, we demonstrate that hDmc1 protein does not self-convert into a stable ADP-bound complex. The results indicate that activation of hDmc1 is mediated through conformational changes induced by free Ca2+ ion binding to a protein site that is distinct from the Mg2+.ATP-binding center. These conformational changes are manifested by formation of more stable filamentous hDmc1.single-stranded DNA complexes. Our results demonstrate a universal role of Ca2+ in stimulation of mammalian DNA strand exchange proteins and reveal diversity in the mechanisms of this stimulation.

Effects of epitope modification on T cell receptor-ligand binding and antigen recognition by seven H-2Kd-restricted cytotoxic T lymphocyte clones specific for a photoreactive peptide derivative.

We tested for antigen recognition and T cell receptor (TCR)-ligand binding 12 peptide derivative variants on seven H-2Kd-restricted cytotoxic T lymphocytes (CTL) clones specific for a bifunctional photoreactive derivative of the Plasmodium berghei circumsporozoite peptide 252-260 (SYIPSAEKI). The derivative contained iodo-4-azidosalicylic acid in place of PbCS S-252 and 4-azidobenzoic acid on PbCS K-259. Selective photoactivation of the N-terminal photoreactive group allowed crosslinking to Kd molecules and photoactivation of the orthogonal group to TCR. TCR photoaffinity labeling with covalent Kd-peptide derivative complexes allowed direct assessment of TCR-ligand binding on living CTL. In most cases (over 80%) cytotoxicity (chromium release) and TCR-ligand binding differed by less than fivefold. The exceptions included (a) partial TCR agonists (8 cases), for which antigen recognition was five-tenfold less efficient than TCR-ligand binding, (b) TCR antagonists (2 cases), which were not recognized and capable of inhibiting recognition of the wild-type conjugate, (c) heteroclitic agonists (2 cases), for which antigen recognition was more efficient than TCR-ligand binding, and (d) one partial TCR agonist, which activated only Fas (C1)95), but not perforin/granzyme-mediated cytotoxicity. There was no correlation between these divergences and the avidity of TCR-ligand binding, indicating that other factors than binding avidity determine the nature of the CTL response. An unexpected and novel finding was that CD8-dependent clones clearly incline more to TCR antagonism than CD8-independent ones. As there was no correlation between CD8 dependence and the avidity of TCR-ligand binding, the possibility is suggested that CD8 plays a critical role in aberrant CTL function.

Revisiting spatial distribution and biochemical composition of calcium-containing crystals in human osteoarthritic articular cartilage.

INTRODUCTION: Calcium-containing (CaC) crystals, including basic calcium phosphate (BCP) and calcium pyrophosphate dihydrate (CPP), are associated with destructive forms of osteoarthritis (OA). We assessed their distribution and biochemical and morphologic features in human knee OA cartilage. METHODS: We prospectively included 20 patients who underwent total knee replacement (TKR) for primary OA. CaC crystal characterization and identification involved Fourier-transform infra-red spectrometry and scanning electron microscopy of 8 to 10 cartilage zones of each knee, including medial and lateral femoral condyles and tibial plateaux and the intercondyle zone. Differential expression of genes involved in the mineralization process between cartilage with and without calcification was assessed in samples from 8 different patients by RT-PCR. Immunohistochemistry and histology studies were performed in 6 different patients. RESULTS: Mean (SEM) age and body mass index of patients at the time of TKR was 74.6 (1.7) years and 28.1 (1.6) kg/m², respectively. Preoperative X-rays showed joint calcifications (chondrocalcinosis) in 4 cases only. The medial femoro-tibial compartment was the most severely affected in all cases, and mean (SEM) Kellgren-Lawrence score was 3.8 (0.1). All 20 OA cartilages showed CaC crystals. The mineral content represented 7.7% (8.1%) of the cartilage weight. All patients showed BCP crystals, which were associated with CPP crystals for 8 joints. CaC crystals were present in all knee joint compartments and in a mean of 4.6 (1.7) of the 8 studied areas. Crystal content was similar between superficial and deep layers and between medial and femoral compartments. BCP samples showed spherical structures, typical of biological apatite, and CPP samples showed rod-shaped or cubic structures. The expression of several genes involved in mineralization, including human homolog of progressive ankylosis, plasma-cell-membrane glycoprotein 1 and tissue-nonspecific alkaline phosphatase, was upregulated in OA chondrocytes isolated from CaC crystal-containing cartilages. CONCLUSIONS: CaC crystal deposition is a widespread phenomenon in human OA articular cartilage involving the entire knee cartilage including macroscopically normal and less weight-bearing zones. Cartilage calcification is associated with altered expression of genes involved in the mineralisation process.

Amphibian albumins as members of the albumin, alpha-fetoprotein, vitamin D-binding protein multigene family.

The Xenopus laevis 68-kd and 74-kd albumin amino acid sequences are examined with respect to their relationship to the other known members of the albumin/alpha-fetoprotein/vitamin D-binding protein gene family. Each of the three members of this family presents a unique pattern of conserved regions indicating a differential selective pressure related to specific functional characteristics. Furthermore, an evolutionary tree of these genes was deduced from the divergence times calculated from direct nucleotide sequence comparisons of individual gene pairs. These calculations indicate that the vitamin D-binding protein/albumin separation occurred 560-600 million years (Myr) ago and the albumin/alpha-fetoprotein divergence 280 Myr ago. This observation leads to the hypothesis according to which the albumin/alpha-fetoprotein gene duplication occurred shortly after the amphibian/reptile separation. Consequently, and unlike mammals, amphibians and fishes should lack an alpha-fetoprotein in their serum at larval stages, which is consistent with a recent analysis of serum proteins in Xenopus laevis larvae. This hypothesis now will have to be tested further in additional lower vertebrates.

Effets des supplémentations en calcium et vitamine D sur la maladie cardiovasculaire. [Effects of calcium and vitamin D supplementations on cardiovascular disease: review article].

Studies exploring the effect of calcium supplementation on cardiovascular risk suggest that systolic blood pressure decreases with supplementation. A lower calcium intake has been associated with an increased risk of stroke. By contrast, calcium supplementation may increase the risk of myocardial infarction. The effect of vitamin D supplementation on blood pressure is still unclear and no effect of vitamin D supplementation on coronary heart disease or stroke has been clearly demonstrated. There is a lack of randomized clinical trials primarily addressing the effect of these parameters on CVD. Currently, the use of calcium and vitamin D supplementations for the prevention of cardiovascular disease is not justified.

Association of calcemia and serum vitamin D with 24H-urinary calcium excretion in a swiss population-based study

Background: Elevated urinary calcium excretion is associated with reduced bone mineral density. Population-based data on urinary calcium excretion are scarce. We explored the association of serum calcium and circulating levels of vitamin D (including 25(OH)D2 and 25(OH)D3) with urinary calcium excretion in men and women in a population-based study. Methods: We used data from the "Swiss Survey on Salt" conducted between 2010 and 2012 and including people aged 15 years and over. Twenty-four hour urine collection, blood analysis, clinical examination and anthropometric measures were collected in 11 centres from the 3 linguistic regions of Switzerland. Vitamin D was measured centrally using liquid chromatography - tandem mass spectrometry. Hypercalciuria was defined as urinary calcium excretion >0.1 mmol/kg/24h. Multivariable linear regression was used to explore factors associated with 24-hour urinary calcium excretion (mmol/24h) squared root transformed, taken as the dependant variable. Vitamin D was divided into monthspecific tertiles with the first tertile having the lowest value and the third tertile having the highest value. Results: The 669 men and 624 women had mean (SD) age of 49.2 (18.1) and 47 (17.9) years and a prevalence of hypercalciuria of 8.9% and 8.0%, respectively. In adjusted models, the association of urinary calcium excretion with protein-corrected serum calcium was (β coefficient } standard error, according to urinary calcium squared root transformed) 1.125 } 0.184 mmol/L per square-root (mmol/24h) (P<0.001) in women and 0.374 } 0.224 (P=0.096) in men. Men in the third month-specific vitamin D tertile had higher urinary calcium excretion than men in the first tertile (0.170 } 0.05 nmol/L per mmol/24h, P=0.001) and the corresponding association was 0.048 } 0.043, P= 0.272 in women. Conclusion: About one in eleven person has hypercalciuria in the Swiss population. The positive association of serum calcium with urinary calcium excretion was steeper in women than in men, independently of menopausal status. Circulating vitamin D was associated positively with urinary calcium excretion only in men. The reasons underlying the observed sex differences in the hormonal control of urinary calcium excretion need to be explored in further studies.

Binding free energy differences in a TCR-peptide-MHC complex induced by a peptide mutation: a simulation analysis.

Recognition by the T-cell receptor (TCR) of immunogenic peptides presented by class I major histocompatibility complexes (MHCs) is the determining event in the specific cellular immune response against virus-infected cells or tumor cells. It is of great interest, therefore, to elucidate the molecular principles upon which the selectivity of a TCR is based. These principles can in turn be used to design therapeutic approaches, such as peptide-based immunotherapies of cancer. In this study, free energy simulation methods are used to analyze the binding free energy difference of a particular TCR (A6) for a wild-type peptide (Tax) and a mutant peptide (Tax P6A), both presented in HLA A2. The computed free energy difference is 2.9 kcal/mol, in good agreement with the experimental value. This makes possible the use of the simulation results for obtaining an understanding of the origin of the free energy difference which was not available from the experimental results. A free energy component analysis makes possible the decomposition of the free energy difference between the binding of the wild-type and mutant peptide into its components. Of particular interest is the fact that better solvation of the mutant peptide when bound to the MHC molecule is an important contribution to the greater affinity of the TCR for the latter. The results make possible identification of the residues of the TCR which are important for the selectivity. This provides an understanding of the molecular principles that govern the recognition. The possibility of using free energy simulations in designing peptide derivatives for cancer immunotherapy is briefly discussed.

Heterologously expressed Staphylococcus aureus fibronectin-binding proteins are sufficient for invasion of host cells.

Staphylococcus aureus invasion of mammalian cells, including epithelial, endothelial, and fibroblastic cells, critically depends on fibronectin bridging between S. aureus fibronectin-binding proteins (FnBPs) and the host fibronectin receptor integrin alpha(5)beta(1) (B. Sinha et al., Cell. Microbiol. 1:101-117, 1999). However, it is unknown whether this mechanism is sufficient for S. aureus invasion. To address this question, various S. aureus adhesins (FnBPA, FnBPB, and clumping factor [ClfA]) were expressed in Staphylococcus carnosus and Lactococcus lactis subsp. cremoris. Both noninvasive gram-positive microorganisms are genetically distinct from S. aureus, lack any known S. aureus surface protein, and do not bind fibronectin. Transformants of S. carnosus and L. lactis harboring plasmids coding for various S. aureus surface proteins (FnBPA, FnBPB, and ClfA) functionally expressed adhesins (as determined by bacterial clumping in plasma, specific latex agglutination, Western ligand blotting, and binding to immobilized and soluble fibronectin). FnBPA or FnBPB but not of ClfA conferred invasiveness to S. carnosus and L. lactis. Invasion of 293 cells by transformants was comparable to that of strongly invasive S. aureus strain Cowan 1. Binding of soluble and immobilized fibronectin paralleled invasiveness, demonstrating that the amount of accessible surface FnBPs is rate limiting. Thus, S. aureus FnBPs confer invasiveness to noninvasive, apathogenic gram-positive cocci. Furthermore, FnBP-coated polystyrene beads were internalized by 293 cells, demonstrating that FnBPs are sufficient for invasion of host cells without the need for (S. aureus-specific) coreceptors.

Odorant binding proteins of the red imported fire ant, Solenopsis invicta: an example of the problems facing the analysis of widely divergent proteins.

We describe the odorant binding proteins (OBPs) of the red imported fire ant, Solenopsis invicta, obtained from analyses of an EST library and separate 454 sequencing runs of two normalized cDNA libraries. We identified a total of 18 putative functional OBPs in this ant. A third of the fire ant OBPs are orthologs to honey bee OBPs. Another third of the OBPs belong to a lineage-specific expansion, which is a common feature of insect OBP evolution. Like other OBPs, the different fire ant OBPs share little sequence similarity (∼ 20%), rendering evolutionary analyses difficult. We discuss the resulting problems with sequence alignment, phylogenetic analysis, and tests of selection. As previously suggested, our results underscore the importance for careful exploration of the sensitivity to the effects of alignment methods for data comprising widely divergent sequences.

Nuclear translocation and DNA recognition signals colocalized within the bZIP domain of cyclic adenosine 3',5'-monophosphate response element-binding protein CREB.

CREB is a cAMP-responsive nuclear DNA-binding protein that binds to cAMP response elements and stimulates gene transcription upon activation of the cAMP signalling pathway. The protein consists of an amino-terminal transcriptional transactivation domain and a carboxyl-terminal DNA-binding domain (bZIP domain) comprised of a basic region and a leucine zipper involved in DNA recognition and dimerization, respectively. Recently, we discovered a testis-specific transcript of CREB that contains an alternatively spliced exon encoding multiple stop codons. CREB encoded by this transcript is a truncated protein lacking the bZIP domain. We postulated that the antigen detected by CREB antiserum in the cytoplasm of germinal cells is the truncated CREB that must also lack its nuclear translocation signal (NTS). To test this hypothesis we prepared multiple expression plasmids encoding carboxyl-terminal deletions of CREB and transiently expressed them in COS-1 cells. By Western immunoblot analysis as well as immunocytochemistry of transfected cells, we show that CREB proteins truncated to amino acid 286 or shorter are sequestered in the cytoplasm, whereas a CREB of 295 amino acids is translocated into the nucleus. Chimeric CREBs containing a heterologous NTS fused to the first 248 or 261 amino acids of CREB are able to drive the translocation of the protein into the nucleus. Thus, the nine amino acids in the basic region involved in DNA recognition between positions 287 and 295 (RRKKKEYVK) of CREB contain the NTS. Further, mutation of the lysine at position 290 in CREB to an asparagine diminishes nuclear translocation of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)