Synthesis, maturation, and trafficking of human Na+-dicarboxylate cotransporter NaDC1 requires the chaperone activity of cyclophilin B

Renal excretion of citrate, an inhibitor of calcium stone formation, is controlled mainly by reabsorption via the apical Na(+)-dicarboxylate cotransporter NaDC1 (SLC13A2) in the proximal tubule. Recently, it has been shown that the protein phosphatase calcineurin inhibitors cyclosporin A (CsA) and FK-506 induce hypocitraturia, a risk factor for nephrolithiasis in kidney transplant patients, but apparently through urine acidification. This suggests that these agents up-regulate NaDC1 activity. Using the Xenopus lævis oocyte and HEK293 cell expression systems, we examined first the effect of both anti-calcineurins on NaDC1 activity and expression. While FK-506 had no effect, CsA reduced NaDC1-mediated citrate transport by lowering heterologous carrier expression (as well as endogenous carrier expression in HEK293 cells), indicating that calcineurin is not involved. Given that CsA also binds specifically to cyclophilins, we determined next whether such proteins could account for the observed changes by examining the effect of selected cyclophilin wild types and mutants on NaDC1 activity and cyclophilin-specific siRNA. Interestingly, our data show that the cyclophilin isoform B is likely responsible for down-regulation of carrier expression by CsA and that it does so via its chaperone activity on NaDC1 (by direct interaction) rather than its rotamase activity. We have thus identified for the first time a regulatory partner for NaDC1, and have gained novel mechanistic insight into the effect of CsA on renal citrate transport and kidney stone disease, as well as into the regulation of membrane transporters in general.

A cyclophilin from the polycentric anaerobic rumen fungus Orpinomyces sp. strain PC-2 is highly homologous to vertebrate cyclophilin B.

A cyclophilin (CyP) purified to homogeneity from the polycentric anaerobic rumen fungus Orpinomyces sp. strain PC-2 had a molecular mass of 20.5 kDa and a pI of 8.1. The protein catalyzed the isomerization of the prolyl peptide bond of N-succinyl-Ala-Ala-(cis,trans)-Pro-Phe p-nitroanilide with a kcat/Km value of 9.3 x 10(6) M-1.s-1 at 10 degrees C and pH 7.8. Cyclosporin A strongly inhibited this peptidylprolyl cis-trans isomerase activity with an IC50 of 19.6 nM. The sequence of the first 30 N-terminal amino acids of this CyP had high homology with the N-terminal sequences of other eukaryotic CyPs. By use of a DNA hybridization probe amplified by PCR with degenerate oligonucleotide primers designed based on the amino acid sequences of the N terminus of this CyP and highly conserved internal regions of other CyPs, a full-length cDNA clone was isolated. It possessed an open reading frame encoding a polypeptide of 203 amino acids with a calculated molecular weight of 21,969, containing a putative hydrophobic signal peptide sequence of 22 amino acids preceding the N terminus of the mature enzyme and a C-terminal sequence, Lys-Ala-Glu-Leu, characteristic of an endoplasmic reticulum retention signal. The Orpinomyces PC-2 CyP is a typical type B CyP. The amino acid sequence of the Orpinomyces CyP exhibits striking degrees of identity with the corresponding human (70%), bovine (69%), mouse (68%), chicken (66%), maize (61%), and yeast (54%) proteins. Phylogenetic analysis based on the CyP sequences indicated that the evolutionary origin of the Orpinomyces CyP was closely related with CyPs of animals.

Swiss warmblood horse with symptoms of hereditary equine regional dermal asthenia without mutation in the cyclophylin B gene (PPIB)

Hereditary equine dermal asthenia (HERDA) is an autosomal recessive skin disease that affects predominantly Quarter Horses and related breeds. Typical symptoms are easy bruising and hyperextensible skin on the back. The prognosis is guarded, as affected horses cannot be ridden normally and are often euthanised. In the Quarter Horse, HERDA is associated with a mutation in cyclophilin B (PPIB), an enzyme involved in triple helix formation of collagen. Here we describe the case of a Swiss Warmblood filly with symptoms of HERDA without PPIB-mutation and in which we also could exclude Ehlers-Danlos syndrome Type IV, VI, VIIA, VIIB and VIIC (dermatosparaxis type) as etiological diseases.

Allele frequency of hereditary equine regional dermal asthenia in American Quarter horses in Brazil determined by quantitative real-time PCR with high resolution melting analysis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ocular dimensions, corneal thickness, and corneal curvature in quarter horses with hereditary equine regional dermal asthenia

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A novel fusion gene, TRIMS-Cyclophilin A in the pig-tailed macaque determines its susceptibility to HIV-1 infection

Objective: In Old World monkeys, the tripartite motif Sec (TRIM5 alpha) protein confers resistance to HIV-1 infection following virus entry into host cells. However, the pig-tailed macaque (Macaca nemestrina) is an exception and is susceptible to HIV-1 in

Cyclophilin catalyzes protein folding in yeast mitochondria.

Cyclophilins are a family of ubiquitous proteins that are the intracellular target of the immunosuppressant drug cyclosporin A. Although cyclophilins catalyze peptidylprolyl cis-trans isomerization in vitro, it has remained open whether they also perform this function in vivo. Here we show that Cpr3p, a cyclophilin in the matrix of yeast mitochondria, accelerates the refolding of a fusion protein that was synthesized in a reticulocyte lysate and imported into the matrix of isolated yeast mitochondria. The fusion protein consisted of the matrix-targeting sequence of subunit 9 of F1F0-ATPase fused to mouse dihydrofolate reductase. Refolding of the dihydrofolate reductase moiety in the matrix was monitored by acquisition of resistance to proteinase K. The rate of refolding was reduced by a factor of 2-6 by 2.5 microM cyclosporin A. This reduced rate of folding was also observed with mitochondria lacking Cpr3p. In these mitochondria, protein folding was insensitive to cyclosporin A. The rate of protein import was not affected by cyclosporin A or by deletion of Cpr3p.

The S.T.A.B. Trial - Standardised Testing of Artificial Blood. A comparative study of various products that may be used as artificial blood for high fidelity simulation training in the critical care setting

Aim: In the current climate of medical education, there is an ever-increasing demand for and emphasis on simulation as both a teaching and training tool. The objective of our study was to compare the realism and practicality of a number of artificial blood products that could be used for high-fidelity simulation. Method: A literature and internet search was performed and 15 artificial blood products were identified from a variety of sources. One product was excluded due to its potential toxicity risks. Five observers, blinded to the products, performed two assessments on each product using an evaluation tool with 14 predefined criteria including color, consistency, clotting, and staining potential to manikin skin and clothing. Each criterion was rated using a five-point Likert scale. The products were left for 24 hours, both refrigerated and at room temperature, and then reassessed. Statistical analysis was performed to identify the most suitable products, and both inter- and intra-rater variability were examined. Results: Three products scored consistently well with all five assessors, with one product in particular scoring well in almost every criterion. This highest-rated product had a mean rating of 3.6 of 5.0 (95% posterior Interval 3.4-3.7). Inter-rater variability was minor with average ratings varying from 3.0 to 3.4 between the highest and lowest scorer. Intrarater variability was negligible with good agreement between first and second rating as per weighted kappa scores (K = 0.67). Conclusion: The most realistic and practical form of artificial blood identified was a commercial product called KD151 Flowing Blood Syrup. It was found to be not only realistic in appearance but practical in terms of storage and stain removal.

Can B.I.M be civil?

After many years of development BIM (Building Information Modelling) is starting to achieve significant penetration into the building sector of the construction industry. This paper describes the current status of BIM and the drivers that are motivating the change from 2D CAD to BIM within the building sector. The paper then discusses what the implications of the technology underlying BIM may be for the civil construction sector of the construction industry. A project carried out by the Cooperative Research Centre for Construction Innovation is used as an example of this technology as well as several international examples.

Crystallographic characterization of the first reported crystalline form of the potent hallucinogen (R)-2-amino-1-(8-bromobenzo[1,2-b;5,4-b']difuran-4-yl)propane or `bromodragonfly': the 1:1 anhydrous proton-transfer compound with 3,5-dinitrosalicylic acid

The 1:1 proton-transfer compound of the potent substituted amphetamine hallucinogen (R)-1-(8-bromobenzo[1,2-b; 4,5-b']difuran-4-yl)-2-aminopropane (common trivial name 'bromodragonfly') with 3,5-dinitrosalicylic acid, 1-(8-bromobenzo[1,2-b;4,5-b']difuran-4-yl)-2-mmoniopropane 2-carboxy-4,6-dinitrophenolate, C13H13BrNO2+ C7H3N2O7- forms hydrogen-bonded cation-anion chain substructures comprising undulating head-to-tail anion chains formed through C(8) carboxyl O-H...O(nitro) associations and incorporating the aminium groups of the cations. The intra-chain cation-anion hydrogen-bonding associations feature proximal cyclic R33(8) interactions involving both a N+-H...O(phenolate) and the carboxyl O--H...O(nitro)associations. Also present are aromatic pi-pi ring interactions [minimum ring centroid separation, 3.566(2)A; inter-plane dihedral angle, 5.13(1)deg]. A lateral hydrogen-bonding interaction between the third aminium proton and a carboxyl O acceptor link the chain substructures giving a two-dimensional sheet structure. This determination represents the first of any form of this compound and confirms that it has the (R) absolute configuration. The atypical crystal stability is attributed both to the hydrogen-bonded chain substructures provided by the anions, which accommodate the aminium proton-donor groups of the cations and give cross-linking, and to the presence of cation--anion aromatic ring pi-pi interactions.