Search PubMed⌕ Search

Biomedical subjects

C Scholz

Publications and source records attributed to C Scholz.

At least 37 records · Page 2Linked to original sources
PubMed2000

Failure of potassium siphoning by Müller cells: a new hypothesis of perfluorocarbon liquid-induced retinopathy.

PURPOSE: To determine the effect of perfluorocarbon liquid (PFCL)-induced abolition of potassium siphoning by the vitreal end feet of Miller cells. METHODS: Porcine eyecups were filled with stained balanced salt solution and PFCLs (perfluorodecalin, perfluorooctane, perfluoroperhydrophenanthrene or the semifluorocarbon perfluorohexylhexane). With optical coherence tomography, the distance between PFCL and retina was determined, and the size of the aqueous space covering the retinal surface was estimated. The data were used to calculate the retinal potassium siphoning into small aqueous volumes. RESULTS: The distance between PFCL and retinal surface was found to be less than 5 to 10 microm with any PFCL tested. The resultant volume of the aqueous space was too small to act as a sufficient sink for K+ ion siphoning. CONCLUSIONS: A certain threshold volume of vitreal fluid seems to be necessary for efficient buffering of intraretinal increases of K+ and perhaps other (e.g., H+) ions through the Müller cells. When the aqueous fluid is replaced by a PFCL (or by silicone oil) for longer periods, the outer retina becomes subject to long-lasting K+ accumulation, and consequent neurodegeneration and reactive gliosis occurs. The authors propose to search for new vitreous-substituting fluids with the capability to dissolve ions.

Animals↗
PubMed1999

Interaction of the capsid protein p24 (HIV-1) with sequence-derived peptides: influence on p24 dimerization.

Protein-protein interactions of the p24 (HIV-1) capsid protein play an essential role in the production of infectious virus particles. To map the putative p24 dimerization site, a set of overlapping peptides spanning the p24 sequence was prepared using spot synthesis on a cellulose membrane and probed with recombinant p24 (rp24). Three sequence regions interacting with rp24 were identified. Peptides from each region were synthesized, but only one peptide was effectively able to inhibit rp24 dimerization in solution. Amino acids that were exposed in the corresponding p24 region were mutated in rp24, resulting in a significant decrease of rp24 dimerization. Thus, participation of this region in virus capsid assembly can be assumed.

Animals↗
PubMed1999

R73A and H144Q mutants of the yeast mitochondrial cyclophilin Cpr3 exhibit a low prolyl isomerase activity in both peptide and protein-folding assays.

Previously we reported that the R73A and H144Q variants of the yeast cyclophilin Cpr3 were virtually inactive in a protease-coupled peptide assay, but retained activity as catalysts of a proline-limited protein folding reaction [Scholz, C. et al. (1997) FEBS Lett. 414, 69-73]. A reinvestigation revealed that in fact these two mutations strongly decrease the prolyl isomerase activity of Cpr3 in both the peptide and the protein-folding assay. The high folding activities found previously originated from a contamination of the recombinant Cpr3 proteins with the Escherichia coli protein SlyD, a prolyl isomerase that co-purifies with His-tagged proteins. SlyD is inactive in the peptide assay, but highly active in the protein-folding assay.

Arginine↗
PubMed1999

Low density lipoprotein receptor as a candidate receptor for hepatitis C virus.

Hepatitis C virus (HCV) binds to different human cell lines in vitro. However, the efficiency of adsorption is very low due mainly to a relatively small fraction of the virus being able to bind to these cells. Free low density lipoprotein (LDL > 200 microg/ml) is able to block the attachment of HCV to human fibroblasts in vitro completely. COS-7 cells being primarily not able to bind HCV were transfected with a vector containing the entire coding sequence of the human LDL-receptor (LDLR). HCV was now bound to these cells. We propose that HCV and LDL are competitive for the cellular LDLR and that LDL in sera of patients may regulate the binding of HCV to this target.

Adsorption↗
PubMed1999

Characterization of neutralizing anti-pre-S1 and anti-pre-S2 (HBV) monoclonal antibodies and their fragments.

Single-chain Fv fragments (scFv) were generated from two murine monoclonal antibodies directed to the neutralizing epitopes of the pre-S1 and pre-S2 region of hepatitis B virus, respectively, using different assembly cloning strategies. The scFv fragments were solubly expressed in E. coli. Dissociation constants were in the nanomolar range for all forms (whole IgG antibodies, Fab fragment and scFv fragments). The epitopes of both antibodies were mapped using solid phase peptide synthesis on continuous cellulose membranes and turned out to be linear determinants. The minimal epitope for the anti-pre-S2 antibody 1F6 was identified to be DPRVRGLYF (amino acid 133-141 of the pre-S region). For the anti-pre-S1 antibody MA 18/7 the minimal epitope proved to be the hexamer LDPAFR (amino acid 30-35 of the pre-S region). Complete substitutional analyses as well as truncation experiments revealed key residues for these antibody-antigen interactions. On the basis of those results we used computer-assisted modeling techniques to suggest models for both antibody-peptide interactions providing insight into the structural basis of these molecular recognitions.

Amino Acid Sequence↗
PubMed1999

CD95/Fas-triggered apoptosis of activated T lymphocytes is prevented by dendritic cells through a CD58-dependent mechanism.

T-cell apoptosis is a mechanism regulating T-cell homeostasis. Activation renders T cells susceptible to activation-induced cell death, a process mediated through CD95 ligand/CD95 (Apo-1/Fas) ligation. The aim of this study was to test whether antigen-presenting cells can inhibit CD95/Fas-triggered activation-induced cell death. Dendritic cells (DC), which are highly effective antigen-presenting cells, were generated in vitro from human peripheral blood monocytes by culture in granulocyte-macrophage colony-stimulating factor and interleukin 4. Subsequently, DC were cocultured with activated T cells and the effect of DC on CD95/Fas-mediated apoptosis was determined. Coculture with increasing amounts of DC prevented CD95/Fas-triggered apoptosis in a dose-dependent fashion by inhibiting activation of caspase 8 and caspase 3. This protective effect of the DC on T-cell death could be blocked by 50% by adding an anti-CD58 antibody, whereas further addition of anti-CD80 (B7.1) and anti-CD86 (B7.2) led to an even more pronounced effect. Our findings suggest that DC can protect T cells from activation-induced cell death, with CD58 ligation playing a key role.

Antibodies, Monoclonal↗
PubMed1998

Cyclophilin and trigger factor from Bacillus subtilis catalyze in vitro protein folding and are necessary for viability under starvation conditions.

Cyclophilin (the product of the ppiB gene) and the trigger factor (the product of the tig gene) are the only cytosolic peptidyl-prolyl cis-trans isomerases that are known in Bacillus subtilis. Both enzymes catalyze the in vitro refolding of ribonuclease T1, a reaction that is limited in rate by a prolyl cis/trans isomerization. The efficiency of cyclophilin as a folding catalyst is only modest with a kcat/KM value of 3.8 x 10(4) M-1 s-1, but the trigger factor shows an almost 40-fold higher specific activity with a kcat/KM value of 1.4 x 10(6) M-1 s-1. This high catalytic activity originates from the tight binding to the protein substrate as reflected in both the low KM value of 0.5 microM and in the strong inhibition of the trigger factor by unfolded proteins. By use of a protein-folding assay, the concentrations of cyclophilin and the trigger factor in the cytosol of B. subtilis could be determined as 26 and 35 microM, respectively. Together they account for the entire folding activity that is detectable in crude extracts of wild-type B. subtilis cells. The genes encoding cyclophilin and the trigger factor in the B. subtilis chromosome were disrupted individually and simultaneously. Even in combination, these disruptions had no effect on cell viability in rich medium or under several stress conditions, such as heat, osmotic, or oxidative stress. However, in poor medium and, in particular, in the absence of amino acids, the growth of the double mutant strain was strongly decelerated, indicating that the prolyl isomerases become essential for growth under starvation conditions. It is not yet known whether this function relates to the catalysis of the proline-limited folding of essential proteins.

Amino Acids↗
PubMed1998

The glycoinositolphospholipids from Leishmania panamensis contain unusual glycan and lipid moieties.

The cell surface of Leishmania parasites is coated by glycosylphosphatidylinositol (GPI)-anchored macromolecules (glycoproteins and a lipophosphoglycan) and a polymorphic family of free GPI glycolipids or glycoinositolphospholipids (GIPLs). Here we show that GIPLs with unusual glycan and lipid moieties are likely to be major cell surface components of L. panamensis (subgenus Viannia) promastigotes. These glycolipids were purified by high performance thin layer chromatography and their structures determined by gas-liquid chromatography-mass spectrometry, fast-atom bombardment mass spectrometry, methylation analysis and chemical and enzymatic sequencing of the glycan headgroups. The major GIPLs contained two glycan core sequences, Manalpha1-3Manalpha1-4GlcN-phosphatidylinositol (type-2 series) or Manalpha1-3[Manalpha1-2Manalpha1-6]Manalpha1- 4GlcN-phosphatidylinosit ol (hybrid series), which were elaborated with Galalpha1-2Galbeta1- or Galalpha1-2/3Galalpha1-2Galbeta1- extensions that were attached to the 3-position of the alpha1-3 linked mannose. The phosphatidylinositol moiety contained exclusively diacylglycerol with palmitoyl, stearoyl and heptadecanoyl chains. Non-galactosylated GIPL species with the same core structures were also found. The galactose extensions and the presence of diacylglycerol in the lipid moieties are novel features for the GIPLs of Leishmania spp. The implications of these structures for the biosynthesis of leishmanial GIPLs and their putative function in the mammalian host are discussed.

Animals↗
PubMed1998

Recognition of protein substrates by the prolyl isomerase trigger factor is independent of proline residues.

The trigger factor is associated with bacterial ribosomes and catalyzes proline-limited protein folding reactions. Its folding activity is very high and conserved in evolution, as shown for the homologous enzymes from Escherichia coli and Mycoplasma genitalium. The folding protein substrate (a variant of ribonuclease T1) binds with high affinity to the trigger factors, and permanently unfolded proteins are strong, competitive inhibitors. We used this inhibition to characterize the substrate binding sites of the trigger factors. Unfolded alpha-lactalbumin binds very tightly and inhibits the trigger factor from M. genitalium with a KI value of 50 nM. The binding of inhibitory proteins is independent of proline residues, as shown for unfolded tendamistat, which binds to the trigger factor with equal affinity in the presence and in the absence of its three proline residues. The good inhibition by a non-folding variant of ribonuclease T1 that lacks Pro39 showed that this proline, at which the catalysis of folding occurs, is dispensable for substrate binding. The trigger factors cannot catalyze prolyl isomerization when proteins are partially folded already. They preferentially recognize unstructured protein chains, which bind with high affinity to a site distinct from the catalytic prolyl isomerase center in the FKBP domain.

Catalysis↗
PubMed1998

Expansion of autoreactive T cells in multiple sclerosis is independent of exogenous B7 costimulation.

Multiple sclerosis (MS) is an inflammatory disease of the myelinated central nervous system that is postulated to be induced by myelin-reactive CD4 T cells. T cell activation requires an antigen-specific signal through the TCR and a costimulatory signal, which can be mediated by B7-1 or B7-2 engagement of CD28. To directly examine the activation state of myelin-reactive T cells in MS, the costimulation requirements necessary to activate myelin basic protein (MBP) or tetanus toxoid (TT)-reactive CD4 T cells were compared between normal controls and MS patients. Peripheral blood T cells were stimulated with Chinese hamster ovary (CHO) cells transfected either with DRB1*1501/DRA0101 chains (t-DR2) alone, or in combination with, B7-1 or B7-2. In the absence of costimulation, T cells from normal subjects stimulated with the recall antigen TT p830-843 were induced to expand and proliferate, but stimulation with MBP p85-99 did not have this effect. In marked contrast, T cells from patients with MS stimulated with MBP p85-99 in the absence of B7-1 or B7-2 signals expanded and proliferated. Thus, MBP-reactive CD4 T cells in patients with MS are costimulation independent and have been previously activated in vivo. These experiments provide further direct evidence for a role of activated MBP-specific CD4 T cells in the pathogenesis of MS.

Abatacept↗
PubMed1998

Dendritic cells prevent CD95 mediated T lymphocyte death through costimulatory signals.

T cell apoptosis is a mechanism regulating T cell homeostasis. Prolonged stimulation renders T cells susceptible to activation induced cell death (AICD), a process mediated through CD95 (Apo-1/Fas). While under some circumstances AICD can be prevented, little is known about molecules involved. Here, we wanted to assess whether dendritic cells (DC) have the capacity to prevent CD95-dependent AICD. T cells activated with PHA/PMA or anti-CD3 monoclonal antibody (mAb) were cocultured with increasing amounts of DC. While spontaneous T cell apoptosis amounted to 25%, the presence of an agonistic anti-CD95 antibody increased cell death to 64%. Addition of scalar amounts of DC prevented T cell apoptosis in a dose dependent fashion, where coculture of 10(5) DC/ml with 10(6) T cells/ml reduced apoptosis almost to baseline level (33%). Further addition of an anti-CD58 antibody partially abolished this protective effect. This was even more pronounced if anti-CD80 and anti-CD86 antibodies were added. Our findings suggest that dendritic cells are able to rescue T cells from AICD, with CD58 ligation playing a key role.

Antigens, CD↗
PubMed1998

A phage library-derived single-chain Fv fragment in complex with turkey egg-white lysozyme: characterization, crystallization and preliminary X-ray analysis.

Using phage-display, an anti-turkey egg-white lysozyme single-chain Fv fragment was selected from a naive light chain variable region repertoire in combination with a heavy chain variable region 'mini library' of anti-hen egg-white lysozyme single-domain binders (Ward et al., 1989). Whereas the selected VH domain alone binds somewhat better hen egg-white lysozyme than turkey egg-white lysozyme, but both with comparatively low affinity, the specificity of VH is converted by addition of the VL domain. Thus, the single-chain Fv fragment is more specific for turkey egg-white lysozyme, with markedly increased affinities towards both lysozymes. The complex of single-chain Fv with turkey lysozyme has been crystallized and characterized by preliminary X-ray analysis.

Amino Acid Sequence↗
PubMed1998

Vascular anastomosis using a biodegradable device with a heat-shrinking sleeve: a preliminary report.

PURPOSE: The objective of this study is to present a simple mechanical anastomosis system capable of uniting vessels of differing diameters and to evaluate its applicability. MATERIALS AND METHODS: A new mechanical system for anastomosis of small vessels was used in 30 rabbits to join 100 sectioned carotid arteries and jugular veins. The system is resorbable and consists of a cuff and a shrinkable sleeve. To carry out the anastomosis, one vessel end is pulled through the cuff and everted over it. The second vessel end is then slipped over the everted vessel end and the cuff. A heat-shrinking sleeve is positioned over the site and shrunk to produce permanent fixation. RESULTS: The overall patency rate of the anastomoses was 96.6% in the arteries and 93.3% in veins. Microscopically, the intima-to-intima contact sites were bridged by an endothelial layer in the first postoperative weeks. CONCLUSION: Advantages of the system include a short insertion time, simple instrumentation, a high patency rate, and resorbability. However, the method requires additional vessel length for eversion, and it cannot be used for end-to-side anastomoses.

Absorbable Implants↗
PubMed1998

Prolyl isomerases do not catalyze isomerization of non-prolyl peptide bonds.

Prolyl isomerases accelerate the cis <--> trans isomerization of prolyl peptide bonds during protein folding and probably also in folded proteins. We asked whether this catalytic function is in fact restricted to prolyl bonds or whether the isomerizations of 'normal' non-prolyl peptide bonds are catalyzed as well. By using the P39A variant of ribonuclease T1 as a substrate we find that the trans --> cis isomerization of the Tyr38-Ala39 bond in the refolding of this protein is not catalyzed by prolyl isomerases of the cyclophilin, FKBP and parvulin families. These enzymes are neither able to catalyze amide bond isomerizations in the proline-free model peptide Ala-Ala-Tyr-Ala-Ala.

Amides↗
PubMed1998

[Metastasis of a sigmoid carcinoma to the breast].

Breast metastases are usually a rare event. We present the case of a 42-year-old patient with a primary carcinoma of the sigmoid metastatic to the breast and metastasis in the liver as well. Although seldom, it is important that metastatic cancer to the breast be differentiated from primary breast carcinoma as both the treatment and the prognosis differ significantly.

Adenocarcinoma↗
PubMed1998

Modified pentamer formation assay for measurement of tacrolimus and its active metabolites: comparison with liquid chromatography-tandem mass spectrometry and microparticle enzyme-linked immunoassay (MEIA-II).

A modified pentamer formation assay (PFA) for quantification of tacrolimus and active metabolites after extraction from whole blood is described. The lower limit of detection was 2 microg/L. Intraassay precision (CV) was 5.7-13.7%, and the interassay CV was 6. 1-14.9%. Tacrolimus trough concentrations in 104 whole blood specimens from liver and kidney transplant recipients were compared with results from HPLC-tandem mass spectrometry (LC/MS/MS) and microparticle enzyme immunoassay (MEIA-II). Data were analyzed by difference plots and are presented as median (95% confidence intervals) of the method differences. MEIA-II results were on average 2.00 microg/L (range, -0.08 to 5.17 microg/L) higher than LC/MS/MS, whereas PFA results were only 1.07 microg/L (range, -2.62 to 5.33 microg/L) higher. Of 104 specimens tested, 25 displayed differences >/=3 microg/L between MEIA-II and PFA: median difference, 4.65 microg/L (range, 3.01-8.79 microg/L). The corresponding median difference between PFA and LC/MS/MS was -0.91 microg/L (range, -4.11 to 0.85 microg/L), and the difference between MEIA-II and LC/MS/MS was 3.67 microg/L (range, 1.88-6.34 microg/L), suggesting the presence of inactive metabolites that caused a positive bias in the immunoassay. In contrast, similar median differences were observed for the remaining 79 specimens: MEIA-II minus LC/MS/MS, 1.78 microg/L (range, -0.45 to 4.11 microg/L); PFA minus LC/MS/MS, 1.90 microg/L (range, -1.70 to 5.50 microg/L). Active tacrolimus metabolites may have contributed to the higher apparent tacrolimus concentrations in these specimens.

Chromatography, High Pressure Liquid↗
PubMed1997

Crystallographic analysis of anti-p24 (HIV-1) monoclonal antibody cross-reactivity and polyspecificity.

The X-ray crystal structures of an anti-p24 (HIV-1) monoclonal antibody Fab fragment alone and in complexes with the epitope peptide GATPQDLNTnL (n = norleucine), an epitope-homologous peptide GATPEDLNQKLAGN, as well as two unrelated peptides GLYEWGGARITNTD and efslkGpllqwrsG (D-peptide), are presented to a maximum resolution of 2.6 A. The latter three peptides were identified from screening synthetic combinatorial peptide libraries. Although all peptides bind to the same antigen combining site, the nonhomologous peptides adopt different binding conformations and also form their critical contacts with different antibody residues. Only small readjustments are observed within the framework of the Fab fragment upon binding.

Amino Acid Sequence↗