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.     

Egg weight, batch size and fecundity of the spotted stalk borer, Chilo partellus in relation to weight of females and time of oviposition

The egg laying pattern of the spotted stalk borer on sorghum and maize, Chilo partellus (Swinhoe) (Lepidoptera: Pyralidae) was studied during the first three nights of oviposition under laboratory conditions. More than 50% of both eggs and egg batches are laid during the first night of oviposition. Batch size decreases with time, whereas mean egg weight remains constant.Larger females lay both larger eggs and egg batches than smaller ones. They are also more fecund but it is only the second night of oviposition that contributes significantly to this difference.The reproductive effort decreases much with time. Allocation of reproductive reserves to early eggs seems more important than having eggs evenly distributed over time. Larger females use proportionally more resources to the production of late eggs. Large eggs are likely to contain more yolk which could be of importance for the ballooning behaviour of newly hatched larvae.

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Résumé La ponte de C. partellus Swinhoe a été étudiée au laboratoire pendant les trois premières nuits de la ponte. Plus de 50% des ooplaques et des oefs ont été pondus pendant la première nuit. La taille des ooplaques diminue avec le temps, tandis que le poids moyen des oeufs reste constant.Les grosses femelles pondent des ooplaques et des oeufs plus grands que les petites. Elles sont aussi plus fécondes, mais c'est seulement à la seconde nuit de ponte qu'est due cette différence.Le reproduction diminue beaucoup avec le temps. L'affectation aux premiers oeufs des réserves utilizées pour la reprodution semble plus importante que l'émission d'oeufs régulièrement répartis dans le temps. Les grosses femelles utilisent proportionnellement plus de ressources à la production d'oeufstardifs. Les gros oeufs contiennent vraisemblablement plus de vitellus que pourrait être nécessaire pour le comportement aérostatique des chenilles néonates.

     

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.     

Statistic evaluation of the influence of species variation,culture conditions,surface wettability and fluid shear on attachment and biofilm development of marine bacteria

A budding coccoid bacterium, (CH1), a Vibrio sp. and a Pseudomonas sp. were investigated for factors governing their attachment to glass surfaces in static batch culture and laminar flow continuous culture systems. An analysis of variance showed that the three species exhibited very different responses. For CH1 attachment was dependent on cell density, incubation time and nutrient concentration. The Vibrio sp. was affected by nutrient concentration while the attachment of the Pseudomonas sp. was independent of cell density, incubation time and nutrient concentration. A comparison of attachment to hydrophilic and hydrophobic surfaces showed that attachment of the Vibrio sp. and CH1 to hydrophilic surfaces was 3 and 10 times greater respectively than to hydrophobic surfaces while Pseudomonas attached in equal numbers to both surfaces. The continuous culture system with defined flow hydrodynamics and growth conditions at steady state revealed a random sampling effect 3 times smaller than the batch culture system did. When the biofilm development of Pseudomonas sp. was followed during 46 h at different fluid shear under laminar and turbulent flow conditions, the former biofilm reached 3.3·10⁸ cells·cm^-2 and the latter 8.2·10⁷ cells·cm^-2.Non-common abbreviation NSS Nine salt solution     

Clinical and immunological evaluation of 20 patients with advanced colorectal cancer treated with high dose recombinant leukocyte interferon-αA (rIFNαA)

Summary A total of 20 patients with advanced colorectal cancer received recombinant leukocyte interferon-A (rIFNA) either chronically (group I: twice a week up to 20×10⁶ IU/m² i.m.) or cyclically (group II: 1–4 periods of 8 consecutive days up to 20×10⁶ IU/m² i.m. daily at 20-days intervals) over a period of 12 weeks. There was 1 partial response, 1 mixed response and 1 patient with stable disease, whilst 17 patients had progressive disease. Median survival was 15.5 months. Survival was significantly shorter when the extent of hepatic disease was >25% (P=0.05), extrahepatic disease was extensive (P<0.005), alkaline phosphatase level was >2× normal (P<0.02), or performance status was <100% (P<0.001). Toxicity consisting mainly of fever, fatigue, anorexia and weight loss was serious in group I and minimal in group II. Administration of rIFNA led to a short lived augmentation of natural killer (NK) cell activity. In the cyclically treated group this was a recurrent phenomenon whereas a marked lasting depression of NK cell activity was seen in chronically treated patients. Interferon- production capacity was significantly stimulated during rIFNA therapy. The differences in toxicity and immunostimulatory effects between the two schedules may be of importance in the design of further studies.This trial was supported in part by Hoffmann-La Roche, Basle     

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.     

Drug resistance in rat colon cancer cell lines is associated with minor changes in susceptibility to cytotoxic cells

The development of resistance to anticancer drugs urges the search for different treatment modalities. Several investigators have reported the concomitant development of drug resistance and resistance to natural killer (NK), lymphokine-activated killer (LAK) or monocyte/macrophage cell lysis, while others described unchanged or even increased susceptibility. We investigated this subject in the rat colon carcinoma cell line, CC531-PAR, which is intrinsically multidrug-resistant (MDR), and in three sublines derived from this parental cell line: a cell line with an increased MDR phenotype (CC531-COL), a revertant line from CC531-COL (CC531-REV), which demonstrates enhanced sensitivity to anticancer drugs of the MDR phenotype, and an independently developed cisplatin-resistant line (CC531-CIS). In a 4-h⁵¹Cr-release assay we found no difference in susceptibility to NK cell lysis. No significant differences in lysability by adherent LAK (aLAK) cells were observed in a 4-h assay. In a prolonged 20-h⁵¹Cr-release assay an enhanced sensitivity to aLAK-cell-mediated lysis was observed in the revertant, P-glycoprotein-negative cell line and in the cisplatin-resistant cell line (CC531-CIS). None of the cell lines was completely resistant to lysis by aLAK cells. Therefore, a role for immunotherapy in the treatment of drug-resistant tumors remains a realistic option.