The cyclophilin multigene family of peptidyl-prolyl isomerases. Characterization of three separate human isoforms.

Cyclophilin (CyP), a major cytosolic protein possessing peptidyl-prolyl cis-trans isomerase activity, has been implicated as the specific receptor of the immunosuppressive drug cyclosporin A (CsA). To identify other potential CsA receptors related to CyP, two human cDNA libraries were screened under low stringency conditions using human CyP cDNA (encoding hCyP1) as a probe. Two cDNAs were identified which encode distinct proteins related to human hCyP1. These two novel proteins, designated hCyP2 and hCyP3, share 65 and 76% amino acid sequence homology with hCyP1, respectively. Both hCyP2 and hCyP3 contain NH2-terminal hydrophobic extensions of 32 and 42 amino acids, respectively. Protein-specific antibodies revealed the predominant association of hCyP2 and hCyP3 with membranes and subcellular organelles, which suggests that the amino-terminal leader sequences of the two CyP isoforms may act as signal peptides. In contrast to the results with hCyP1, Southern blot analysis indicated that both hCyP2 and hCyP3 gene sequences are represented infrequently in the human genome. Northern and Western blot analysis showed that the distribution of mRNA and proteins of the three hCyPs in differing tissues and cell types was similar. Each hCyP protein was expressed in Escherichia coli, purified, and shown to be an active peptidyl-prolyl isomerase. Substrate specificity was examined with 11 synthetic peptides (Suc-Xaa-Yaa-Pro-Phe-4-nitroanilide), and inhibition of the peptidyl-prolyl isomerase activities associated with hCyP1, hCyP2, and hCyP3 was studied with CsA, MeAla6-CsA and MeBm2t1-CsA. From both equilibrium considerations and the results of kinetic characterizations it is proposed that of these three CyP proteins, hCyP1 is the most likely intracellular target for CsA.     

Antiviral activity of acyclic nucleoside phosphonates PMEA, (S)-HPMPC, PMEDAP and ribavirin against Cauliflower mosaic virus in Brassica pekinensis

Antiviral effects of acyclic nucleoside phosphonates PMEA, (S)-HPMPC, PMEDAP, and ribavirin on double-stranded DNA Cauliflower mosaic virus (CaMV) were evaluated in Brassica pekinensis plants grown in vitro on liquid medium. A double-antibody sandwich ELISA was used for relative quantification of viral protein and PCR for detection of CaMV nucleic acid in plants. Ribavirin and PMEA had no significant antiviral effect. (S)-HPMPC at concentration 50?mg?l^?1 and PMEDAP at concentrations 50 and 12.5?mg?l^?1 significantly (P?<?0.05) reduced CaMV concentration in plants within 42?C63?days to levels detectable neither by ELISA nor by PCR. A phytotoxicity experiment resulted in progressive yellowing of leaves and dwarfing in plants cultured 42?days on media with concentrations 12.5, 25 and 50?mg?l^?1 of (S)-HPMPC and PMEDAP. Reduction in fresh and dry weights of plants was significant (P?<?0.05) already at 12.5?mg?l^?1 with both compounds.     

An antigenic HIV-1 peptide sequence engineered into the surface structure of transferrin does not elicit an antibody response.

One novel approach for the biological delivery of peptide drugs is to incorporate the sequence of the peptide into the structure of a natural transport protein such as human serum transferrin (HST). However, a potential drawback is that the HST may increase the immunoreactivity of the peptide, in the same way that carrier proteins can be used to generate highly immunogenic peptide hapten conjugates. In this study we have generated a recombinant HST carrier protein that contains a peptide substrate of HIV-1 protease (VSQNYPIVL). The protein retained native HST function, and the peptide was surface exposed since it was immunoreactive in native dot blots, and was cleaved by HIV-1 protease. Immunisation of rabbits with the recombinant protein elicited only a very poor anti-peptide immune response. In contrast, strong anti-peptide immune responses were raised against both the peptide alone, and a chemical conjugate of the peptide with HST. These data demonstrate that it is possible to attenuate the immune response normally directed against an immunogenic peptide sequence by engineering into a surface exposed loop of HST. These findings may have an important impact on the future design of peptide delivery systems.     

Effects of high sucrose diets and 4-aminopyrazolopyrimidine on serum lipids and lipoproteins in the rat

The effect of feeding a semipurified diet high in sucrose on serum lipid and lipoprotein concentrations was studied. In rats fed this diet the serum triglyceride concentration doubled, and liver triglyceride concentration increased by 30%. A fivefold increase in VLDL protein concentration and a small but significant increase of HDL protein concentration was also observed. In these rats there was increased incorporation of labeled amino acids into the proteins of plasma VLDL and HDL. Fatty livers developed in the animals receiving 4-aminopyrazolopyrimidine, and levels of serum triglyceride and cholesterol fell markedly. The concentration of all lipoprotein classes decreased, with VLDL showing the most marked effect. Incorporation of labeled amino acids into lipoproteins and other plasma proteins was depressed.     

Characterization of the specific binding of rat apolipoprotein E-deficient HDL to rat hepatic plasma membranes

We have used a preparation of rat liver plasma membranes to study the binding of rat apolipoprotein E-deficient HDL to rat liver. The membranes were found to bind HDL by a saturable process that was competed for by excess unlabeled HDL. The binding was temperature-dependent and was 85% receptor-mediated when incubated at 4, 22 and 37 degrees C. The affinity of the binding site for the HDL was consistent at all temperatures, while the maximum binding capacity increased at higher temperatures. The specific binding of HDL to the membranes did not require calcium and was independent of the concentration of NaCl in the media. The effect of varying the pH of the media on HDL binding was small, being 30% higher at pH 6.5 than at pH 9.0. Both rat HDL and human HDL3 were found to compete for the binding of rat HDL to the membranes, whereas rat VLDL remnants and human LDL did not compete. At 4 degrees C, complexes of dimyristoylphosphatidylcholine (DMPC) and apolipoproteins A-I, A-IV and the C apolipoproteins, but not apolipoprotein E, competed for HDL binding to the membranes. At 22 and 37 degrees C, all DMPC-apolipoprotein complexes competed to a similar extent, DMPC vesicles that contained no protein did not compete for the binding of HDL. These results suggest that the rat liver possesses a specific receptor for apolipoprotein E-deficient HDL that recognizes apolipoproteins A-I, A-IV and the C apolipoproteins as ligands.     

Genotoxic and cell-transforming properties of (trans,trans)-muconaldehyde

(trans,trans)-Muconaldehyde, a putative metabolite of benzene, should be expected to have mutagenic properties by virtue of its twin alpha,beta-unsaturated carbonylic function. It displayed definitely mutagenic properties in S. typhimurium TA100 without metabolic activation and with a 5-fold concentration of tester organisms in the preincubation assay and induced SOS response in E. coli. It induced micronucleus formation and morphological transformation in a dose-dependent manner in Syrian hamster embryofibroblasts. No DNA single-strand breaks or interstrand cross-links could be detected using the alkaline elution technique; however, strand-break generation by subsequent gamma-irradiation was found to be increased.     

Functional analysis of a single chain chimeric alpha/beta-granulocyte-macrophage colony-stimulating factor receptor. Importance of a glutamate residue in the transmembrane region.

To analyze the function of each subunit of the receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF), GMR, we previously generated a single-chain chimeric receptor by fusion of the extracellular and transmembrane domain from the alpha-subunit (alpha-GMR) to the intracellular part of the beta-subunit (beta-GMR) introducing an additional glutamate residue at the fusion site (alpha/beta-GMR). We demonstrated the capacity of alpha/beta-GMR to bind GM-CSF with low affinity and to induce GM-CSF-dependent activation of tyrosine kinase activity and proliferation in transfected Ba/F3 cells. To further compare the functions of wild type and chimeric receptors, we now report that this alpha/beta-GMR is sufficient to mediate morphological changes, expression of alpha(4)- and beta(1)-integrin receptor subunits, and serine-phosphorylation of Akt kinase. To analyze the function of the glutamate residue at the fusion region of alpha/beta-GMR various point mutants changing this amino acid and its position were expressed in Ba/F3 cells. None of these mutants was capable of supporting GM-CSF-dependent proliferation; however, when beta-GMR was coexpressed, GM-CSF mediated short and long term proliferation. Interestingly, some mutants but not alpha/beta-GMR can induce proliferation in the presence of an anti-alpha-GMR antibody. These data demonstrate the significance of a glutamate residue in the transmembrane region of alpha/beta-GMR for ligand-induced receptor activation.     

Long-term effects of dietary isoflavones on uterine gene expression profiles

Isoflavones (ISOs) are bioactive food ingredients of the traditional East Asian diet and currently discussed as alternatives to classical hormone replacement therapies and for reducing the prevalence of hormone-dependent cancers. Although there are many studies on ISOs, not much is known about their long-term effects.Therefore, we performed an animal experiment analyzing the effects of three different diets: a phytoestrogen-free diet, a diet supplemented with genistein (700 μg/g diet) and an ISO-high diet (232 μg daidzein and 240 μg genistein/g) at two distinct time points, juvenile (21 days) and adult (97 days). Exposure started prior to mating of the parents and throughout the life of the offspring.We observed a stronger increase of uterine wet weights in juvenile offspring with genistein exposure (1018 ± 350 mg/kg BW) than with ISO-high diet (497 ± 133 mg/kg BW). Whereas the expression of proliferation related genes (PCNA; Ki67; IGF-1; IGF-1R), analyzed by real-time-qPCR and Western blot, were significantly down-regulated in juvenile animals exposed to genistein. Additionally, genistein exposure led to estrogenic responses, observed upon increase of complement C3 and decrease of estrogen receptors gene expressions, while the exposure to ISO-high diet did not show these effects.In conclusion, both the time point on which phytoestrogen exposure starts together with the composition of the ingested phytoestrogen containing diet are of great importance for the biological response of the offspring.     

Über eigenartige organische Membraneinschlüsse in der Epidermis vonPortulaca Gilliesii Hook

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