Isolation and characterization of a 40-kDa cyclophilin-related protein.

Major and minor isoforms of cyclophilin (CyP-18), a 17.8-kDa protein with peptidyl-prolyl cis-trans isomerase activity, comprise the primary intracellular binding proteins for cyclosporin A. Additional CyP-like proteins with approximate molecular masses of 22 (CyP-22) and 40 kDa (CyP-40) have been recovered from the soluble fraction of calf brain along with CyP-18 by adsorption onto a cyclosporin A affinity column and elution with cyclosporin A. Based on a limited number of peptide sequences from CyP-22, it appears that we have isolated from tissue CyPB, a protein whose sequence was deduced previously from cloned cDNA. The 40-kDa protein was separated from CyP-18 and CyP-22 on a molecular sieving column. Isoelectric focusing of CyP-40 yielded two bands at pI 5.3 and 5.5, in contrast to the basic pI values of CyP-18. Some tryptic peptides from CyP-40 were found to be highly homologous but not identical to bovine CyP-18; others were not significantly homologous to CyP-18 or any other protein in the data base. Unlike the major and minor isoforms of Cyp-18, monospecific polyclonal anti-CyP-18 antibodies did not cross-react with CyP-22 and CyP-40. Likewise, anti-CyP-40 serum minimally cross-reacts with CyP-18 and CyP-22. Cyp-40 possesses peptidyl-prolyl cis-trans isomerase activity which is less sensitive to inhibition by cyclosporin A (IC50 = 300 nM) than is CyP-18 (IC50 = 20 nM).     

Selenocysteine confers the biochemical properties characteristic of the type I iodothyronine deiodinase.

The conversion of thyroxine to 3,5,3'-triiodothyronine (T3) is the first step in thyroid hormone action, and the Type I iodothyronine deiodinase supplies most of this extrathyroidal T3 in the rat. We found that the cDNA coding for this enzyme contains an in-frame UGA encoding the rare amino acid selenocysteine. Using site-directed mutagenesis, we have converted selenocysteine to cysteine and expressed the wild-type and cysteine mutant enzymes in JEG-3 cells by transient transfection. The kinetic properties of the transiently expressed wild-type enzyme are nearly identical to those reported for rat liver Type I deiodinase. Substitution of sulfur for selenium causes a 10-fold increase in the Km of the enzyme for the favored substrate 3,3',5'-triiodothyronine (rT3), a 100-fold decrease in the sensitivity of rT3 deiodination to competitive inhibition by gold and a 300-fold increase in the apparent Ki for uncompetitive inhibition by 6-n-propylthiouracil. These results demonstrate that selenium is responsible for the biochemical properties which characterize Type I iodothyronine monodeiodination.     

Differences in learning by foraging juvenile pumpkinseed and bluegill sunfish in a structured habitat

Synopsis We investigated the ability of two congeneric species of sunfish to learn to forage on a novel prey item in feeding arenas containing structured habitats. Eight bluegill sunfish and eight pumpkinseed sunfish were given the opportunity to forage on whiteworms daily for 10 days. Each day, several behavioural measures were recorded for each fish. Both species of sunfish learned to feed over the 10-day period but the bluegill sunfish learned to feed more quickly than the pumpkinseed sunfish. Pumpkinseeds, however, attained a higher level of foraging efficiency. The differences in learning and foraging efficiency were related to body morphology.     

Use of a monoclonal antibody to measure the surface expression of thrombospondin following platelet activation

The radiolabelled monoclonal antibody, 5G11, directed against native thrombospondin, has been used to assess the surface expression of secreted thrombospondin on human blood platelets. Emphasis has been placed on studying the role of fibrinogen in this process. Unstimulated platelets bound low amounts of 5G11 (about 2000 molecules/platelet). Binding increased 2-fold and 5-7-fold after stimulation of platelets with ADP or thrombin (or ionophore A23187) respectively. Unstimulated platelets from patients deficient in alpha-granule proteins (gray platelet syndrome) bound baseline levels of 5G11. However, binding was not increased after activation. Thrombospondin expression on thrombin-stimulated normal platelets was for a large part divalent-cation-dependent and was not affected by AP-2, a monoclonal antibody to GPIIb-IIIa complexes. However, binding of 5G11 was some 50% lower when platelets were stimulated in the presence of Fab fragments of a polyclonal rabbit antibody to fibrinogen. This suggested either a direct binding of thrombospondin to surface-bound fibrinogen or a steric inhibition due to a close proximity of the two proteins. The fact that binding of 5G11 was at the lower limit of the normal range to the stimulated platelets of an afibrinogenaemic patient specifically lacking detectable fibrinogen favoured the latter explanation. Thus, a major fibrinogen-independent pathway for thrombospondin expression must exist.     

Expression of platelet glycoprotein Ib alpha in HEL cells

We have previously shown that platelet glycoprotein Ib is expressed in a minority of cells of the human leukemic cell line HEL (Tabilio, A., Rosa, J. P., Testa, U., Kieffer, N., Nurden, A. T., Del Canizo, M. C., Breton-Gorius, J., and Vainchenker, W. (1984) EMBO J. 3, 453-459). In this report, we have selected a stable HEL subclone with increased expression of glycoprotein (GP) Ib as assessed by 6 different monoclonal antibodies in order to investigate the biochemical characteristics of this glycoprotein. A single polypeptide chain of apparent Mr = 60,000 was precipitated under reducing and nonreducing conditions by a specific polyclonal anti-platelet glycocalicin antibody and two anti-GPIb alpha monoclonal antibodies (AN51 and AP1), both from surface-labeled and metabolically labeled HEL cells. We were unable to demonstrate the presence of a polypeptide corresponding to the beta subunit of GPIb or GPIX which is closely associated with GPIb. Competitive immunoprecipitation performed in the presence of an excess amount of cold platelet glycocalicin completely displaced the Mr = 60,000 polypeptide. Synthesis of N-linked oligosaccharide chains on this Mr = 60,000 polypeptide was inhibited by the antibiotic tunicamycin, and a shift of the apparent Mr from 60,000 to 48,000 was observed. O-Linked oligosaccharide chains identical to platelet GPIb hexasaccharides were deficient or incomplete since no peanut agglutinin binding to the Mr = 60,000 polypeptide was observed after neuraminidase treatment of HEL cells. Thus, our results provide evidence that the Mr = 60,000 polypeptide expressed on the surface membrane of HEL cells is closely related to platelet GPIb and corresponds to an incompletely or abnormally O-glycosylated GPIb alpha subunit.     

Discrimination of respiratory dysfunction in yeast mutants by confocal microscopy, image, and flow cytometry

Living yeast cells can be selectively stained with the lipophilic cationic cyanine dye DiOC6(3) in a mitochondrial membrane potential-dependent manner. Our study extends the use of flow cytometric analysis and sorting to DiOC6(3)-stained yeast cells. Experimental conditions were developed that prevented the toxic side effect of the probe and gave a quantitative correlation between fluorescence and mitochondrial membrane potential, without any staining of other membranes. The localization of the fluorochrome was checked by confocal microscopy and image cytometry. The mitochondrial membrane alterations were also tested through cardiolipin staining with nonyl acridine orange. Differences in light scattering and in fluorescence were detected in mutants (rho-, rho degrees, mit-, or pet-) and wild-type (rho+mit+) populations of yeast. The dye uptake of respiratory-deficient yeast strains was significantly reduced as compared to that of the wild-type. Application of an uncoupler (mCICCP), which collapsed the mitochondrial membrane potential (alphapsi(m)), led to a drastic reduction of the dye uptake. It was observed that a decrease in deltapsi(m), was usually correlated with a decrease in cardiolipin stainability by nonyl acridine orange (NAO). Quantitative flow cytometry is a fast and reproducible technique for rapid screening of yeast strains that might be suspected of respiratory dysfunction and/or mitochondrial structural changes. We give evidence that it is an adequate method to characterize and isolate respiratory mutants through sorting procedure, with selective enrichment of the population studied in respiring or non-respiring yeast cells. Confocal microscopy and image cytometry corroborate the flow cytometry results.     

Expression of platelet membrane glycoproteins and alpha-granule proteins by a human erythroleukemia cell line (HEL)

We demonstrate that HEL, a human erythroleukemic cell line, has numerous megakaryocytic markers which were markedly enhanced following the addition of the inducers dimethyl sulfoxide or 12-O-tetradecanoylphorbol-13-acetate to the culture medium. Ultrastructural and cytochemical studies showed: (i) the presence of organelles morphologically resembling the platelet alpha-granules; and (ii) a peroxidase activity with the same characteristics as that specifically found in platelets. The platelet alpha-granule proteins (von Willebrand factor, platelet factor-4 and beta-thromboglobulin) were immunologically detected in the HEL cell cytoplasm and their amounts increased after induction. Of particular interest was the presence of platelet membrane proteins. A monoclonal antibody specific for glycoprotein Ib bound to HEL cells. Platelet membrane glycoproteins IIb and IIIa were identified on intact cells using specific antibodies in a binding assay or in cell lysates using either crossed immunoelectrophoresis or an immunoblotting procedure following SDS-polyacrylamide gel electrophoresis. Most HEL cells also expressed the platelet alloantigen PIA1. All of the platelet membrane proteins were present in higher amounts after induction. Glycophorin A, specific for the erythroid lineage, was also detected on HEL cells. Thus, while confirming the presence of erythroid markers, our studies provide evidence that the HEL cell line also expresses platelet antigens. As such, HEL cells represent a unique system with which to study the biosynthesis of platelet-specific proteins and glycoproteins.     

Differential response of cycling and noncycling cells to inducers of DNA synthesis and mitosis

The objective of this study was to determine whether cells in G(0) phase are functionally distinct from those in G(1) with regard to their ability to respond to the inducers of DNA synthesis and to retard the cell cycle traverse of the G(2) component after fusion. Synchronized populations of HeLa cells in G(1) and human diploid fibroblasts in G(1) and G(0) phases were separately fused using UV-inactivated Sendai virus with HeLa cells prelabeled with [(3)H]ThdR and synchronized in S or G(2) phases. The kinetics of initiation of DNA synthesis in the nuclei of G(0) and G(1) cells residing in G(0)/S and G(1)/S dikaryons, respectively, were studied as a function of time after fusion. In the G(0)/G(2) and G(1)/G(2) fusions, the rate of entry into mitosis of the heterophasic binucleate cells was monitored in the presence of Colcemid. The effects of protein synthesis inhibition in the G(1) cells, and the UV irradiation of G(0) cells before fusion, on the rate of entry of the G(2) component into mitosis were also studied. The results of this study indicate that DNA synthesis can be induced in G(0)nuclei after fusion between G(0)- and S-phase cells, but G(0) nuclei are much slower than G(1) nuclei in responding to the inducers of DNA synthesis because the chromatin of G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells differ from G(1) cells with regard to their effects on the cell cycle progression of the G(2) nucleus into mitosis. This difference between G(0) and G(1) cells appears to depend on certain factors, probably nonhistone proteins, present in G(1) cells but absent in G(0) cells. These factors can be induced in G(0) cells by UV irradiation and inhibited in G(1) cells by cycloheximide treatment.     

Exercise training in childhood-onset systemic lupus erythematosus: a controlled randomized trial

Introduction

Exercise training has emerged as a promising therapeutic strategy to counteract physical dysfunction in adult systemic lupus erythematosus. However, no longitudinal studies have evaluated the effects of an exercise training program in childhood-onset systemic lupus erythematosus (C-SLE) patients. The objective was to evaluate the safety and the efficacy of a supervised aerobic training program in improving the cardiorespiratory capacity in C-SLE patients.

Methods

Nineteen physically inactive C-SLE patients were randomly assigned into two groups: trained (TR, n = 10, supervised moderate-intensity aerobic exercise program) and non-trained (NT, n = 9). Gender-, body mass index (BMI)- and age-matched healthy children were recruited as controls (C, n = 10) for baseline (PRE) measurements only. C-SLE patients were assessed at PRE and after 12 weeks of training (POST). Main measurements included exercise tolerance and cardiorespiratory measurements in response to a maximal exercise (that is, peak VO₂, chronotropic reserve (CR), and the heart rate recovery (ΔHRR) (that is, the difference between HR at peak exercise and at both the first (ΔHRR1) and second (ΔHRR2) minutes of recovery after exercise).

Results

The C-SLE NT patients did not present changes in any of the cardiorespiratory parameters at POST (P > 0.05). In contrast, the exercise training program was effective in promoting significant increases in time-to-exhaustion (P = 0.01; ES = 1.07), peak speed (P = 0.01; ES = 1.08), peak VO₂ (P = 0.04; ES = 0.86), CR (P = 0.06; ES = 0.83), and in ΔHRR1 and ΔHRR2 (P = 0.003; ES = 1.29 and P = 0.0008; ES = 1.36, respectively) in the C-SLE TR when compared with the NT group. Moreover, cardiorespiratory parameters were comparable between C-SLE TR patients and C subjects after the exercise training intervention, as evidenced by the ANOVA analysis (P > 0.05, TR vs. C). SLEDAI-2K scores remained stable throughout the study.

Conclusion

A 3-month aerobic exercise training was safe and capable of ameliorating the cardiorespiratory capacity and the autonomic function in C-SLE patients.

Trial registration

NCT01515163.