Biomedical subjects

G Jung

Publications and source records attributed to G Jung.

At least 181 records · Page 10Linked to original sources

PubMed1997

Identification of high potency microbial and self ligands for a human autoreactive class II-restricted T cell clone.

CD4+ class II-restricted T cells specific for self antigens are thought to be involved in the pathogenesis of most human autoimmune diseases and molecular mimicry between foreign and self ligands has been implicated as a possible mechanism for their activation. In this report we introduce combinatorial peptide libraries as a powerful tool to identify cross-reactive ligands for these T cells. The antigen recognition of a CD4+ T cell clone (TCC) specific for myelin basic protein peptide (MBP) (86-96) was dissected by the response to a set of 220 11-mer peptide sublibraries. Based on the results obtained with the libraries for each position of the antigen, artificial peptides were found that induced proliferative responses at much lower concentrations than MBP(86-96). In addition stimulatory ligands derived from protein sequences of self and microbial proteins were identified, some of them even more potent agonists than MBP(86-96). These results indicate that: (a) for at least some autoreactive CD4+ T cells antigen recognition is highly degenerate; (b) the autoantigen used to establish the TCC represents only a suboptimal ligand for the TCC; (c) a completely random and unbiased approach such as combinatorial peptide libraries can decrypt the spectrum of stimulatory ligands for a T cell receptor (TCR).

Amino Acid Sequence↗

PubMed1997

RNase H activity of human hepatitis B virus polymerase expressed in Escherichia coli.

Human Hepatitis B Virus (HBV) replication is accomplished by its own polymerase. The HBV RNase H domain of HBV polymerase has been expressed in Escherichia coli and purified by affinity column chromatography. The MBP-RNase H fusion protein (43 kDa MBP plus 17 kDa HBV RNase H domain) was proved to be RNase H by in vitro activity assay, inhibitor studies, and mutagenesis. The HBV RNase H domain represented the optimal RNase H activity in the presence of either 8 mM MgCl2 or 16 mM MnCl2. In Tris-Cl buffer, the optimum pH for MBP-RNase H fusion protein is between 7.7 and 8.2. The MBP-RNase H fusion protein required 40 mM monovalent cation for its enzyme activity, whereas it showed lower activity at a salt concentration of more than 100 mM. Ribonucleoside Vanadyl complex (RAV) and 2'-deoxyadenosine 5'-monophosphate (dAMP) inhibited the RNase H activity. Moreover, the mutation of highly conserved amino acids in the HBV RNase H domain diminished the RNase H activity. These results clearly suggest that the RNase H activity is separable from viral HBV polymerase enzymatic activities.

Blotting, Western↗

PubMed1997

Effect of leader peptide mutations on biosynthesis of the lantibiotic Pep5.

Lantibiotics are lanthionine-containing antibiotic peptides which are synthesized from ribosomal prepeptides by post-translational modification. In order to elucidate the function of a conserved motif in the N-terminal leader sequence of lantibiotic prepeptides, three amino acids were exchanged in the leader peptide sequence of the lantibiotic Pep5. Exchanging Phe-19 for Ser and Glu-16 for Lys in the FDLEI-motif, reduced Pep5 production to 35 and 38% of the control whereas, after exchanging Asp-6 for Ser and Glu-16 for Lys in the FDLEI-motif, reduced Pep5 production to 35 and 38% of the control whereas, after exchanging ASp-6 for Lys, the production was decreased only to 82%. Proteolytic fragments of Pep5 or incorrectly modified Pep5 molecules, indicative of incorrect modifications, were not found in the culture supernatant. Thus, in contrast to the biosynthesis of the lantibiotic nisin, the FDLEI-motif is not essential for biosynthesis of Pep5 and has no influence on correct ring formation or processing, but seems to be important for optimal biosynthesis rates.

Anti-Bacterial Agents↗

PubMed1997

C/EBP alpha is a major activator for the transcription of rat Cu/Zn superoxide dismutase gene in liver cell.

The rat Cu/Zn superoxide dismutase (SOD1) is expressed in all tissues. Sequence analysis of the SOD1 promoter region showed that none of the cis-elements of hepatocyte-specific nuclear factors (HNF) were observed. The cis-element of C/EBP alpha in the proximal region of the SOD1 promoter and the high level of C/EBP alpha in the liver tissue led us to focus on the transcriptional regulation of the SOD1 gene by C/EBP alpha. Cotransfection assays with the plasmid expressing transcription factor C/EBP alpha showed that C/EBP alpha transactivated SOD1 gene by 27 fold. The marked transactivation and direct binding of C/EBP alpha to the SOD1 promoter were confirmed by deletion analyses and mobility shift assays. These results suggested that C/EBP alpha plays a major role in the tissue distribution of SOD1.

Animals↗

PubMed1997

Early antibody response against hypervariable region 1 is associated with acute self-limiting infections of hepatitis C virus.

Antibodies directed to hypervariable region 1 (HVR1) of hepatitis C virus (HCV) have recently been shown to neutralize the corresponding HCV isolate in vitro. We analyzed the appearance of antibodies directed to HVR1 during the course of infection in a large group of patients who have been infected by the same isolate of a HCV contaminated anti-D immunoglobulin (HCV-AD78). An enzyme-linked immunosorbent assay (ELISA) was established using a synthetic peptide to detect antibodies against the main HVR1 variant of HCV-AD78. 207 sera obtained at different time points post infection (p.i.) of 51 patients having either acute self-limiting (n = 28) or chronic infection (n = 23) were studied. Antibodies directed to HVR1 were found at least at one time point during the infection course in 15 of 28 patients (53%) having acute self-limiting infections and in 17 of 23 patients (74%) with chronic disease. The time of appearance of anti-HVR1 was significantly different between these two patient groups (P < .025) although appearance and titers of other HCV-specific antibodies were found to be similar at early time points p.i. In acute self-limiting infections 9 of 21 sera (43%) of respective patients with sera available within the first 6 months p.i. were anti-HVR1 positive. The highest prevalence of anti-HVR1 in this group of patients was within month 6 to 12 p.i. (64%). None of the sera available after 24 months p.i. had such antibodies. In contrast, only 2 of 15 sera (13%) of chronically infected patients with respective time points of sera were anti-HVR1 positive within the first 6 months p.i. and only 5 of 18 sera (28%) were positive within month 7 to 12 p.i. Seven patients with chronic HCV infections showed at least two consecutive anti-HVR1 negative early time points up to month 18 p.i. Prevalence of anti-HVR1 after 24 months p.i. was high (84%) in this group of patients and most of the patients maintained high levels of anti-HVR1 for up to 17 years p.i. Our findings suggest clearance of virus by respective neutralizing antibodies resulting in a self-limiting infection and may have implications for prognosis of the disease and also for any future vaccine development.

Adult↗

PubMed1997

High affinity presentation of an autoantigenic peptide in type I diabetes by an HLA class II protein encoded in a haplotype protecting from disease.

Polymorphism of the genes coding for the human histocompatibility leukocyte antigen class II DR and DQ molecules makes the single largest genetic contribution to the risk of developing insulin-dependent diabetes mellitus (IDDM) and can be associated with highly elevated as well as decreased disease frequency. The mechanism of IDDM risk modification by HLA polymorphism is likely to involve differential presentation of autoantigenic peptides by HLA class II proteins. We have generated T cell lines (TCL) with specificity for the IDDM autoantigen 65 kDa glutamic acid decarboxylase (GAD65) from lymphocytes of two patients carrying HLA class II alleles associated with distinct risk of IDDM (DRB1*0101/0401 and 1302/1501). For both patients, TCL generated at various time points all recognized single epitopes mapped as GAD 88-99 and 248-257, respectively. These epitopes are presented by the DRB1*0101 and DRB5*0101, HLA class II molecules associated with a moderately elevated risk of IDDM, or carried in a strongly protective haplotype, respectively. In an HLA/peptide binding assay, epitope GAD 248-257 was shown to possess high affinity for DRB5*0101. This epitope overlaps with a central GAD peptide binding to the high risk allele DQB1*0302 and containing a Coxsackie P2C-identical mimicry sequence, raising the possibility of competition of DRB5*0101 and DQB1*0302 for binding of a central GAD65 fragment.

Adult↗

PubMed1997

Two types of modified cardiac Na+ channels after cytosolic interventions at the alpha-subunit capable of removing Na+ inactivation.

Failure of inactivation is the typical response of voltage-gated Na+ channels to the cytosolic presence of proteolytic enzymes, protein reagents such as N-bromoacetamide (NBA) or iodate, and antibodies directed against the linker between domains III and IV of the alpha-subunit. The present patch clamp experiments with cardiac Na+ channels aimed to test the hypothesis that these interventions may provoke the occurrence of non-inactivating Na+ channels with distinct kinetic properties. A site-directed polyclonal antibody (anti-SLP2, target sequence 1481-1496 of the cardiac Na+ channel alpha-subunit) eliminated fast Na+ inactivation to induce burst activity which was accompanied by the occurrence of two open states. A deactivation process terminated channel activity during membrane depolarization proceeding with time constants of close to 40 ms (at -40 mV). NBA-modified and iodatemodified Na+ channels were kinetically indistinguishable from the anti-SLP2-modified type since they likewise deactivate and, thus, attain an only moderate Po of close to 20%. This is fundamentally different from the behaviour of enzymatically-modified Na+ channels: after cytosolic proteolysis with alpha-chymotrypsin, trypsin or pronase, mean Po during membrane depolarization amounted to approximately 40% because deactivation operated extremely slowly and less efficiently (time constants 100-200 ms at -40 mV, as a minimum) or was virtually non-operating. Invitro cleavage of the synthetic linker sequence 1481-1496 confirmed that this part of the alpha-subunit provides a substrate for these peptidases or reactants for NBA but cannot be chemically modified by iodate. This iodate resistance indicates that iodate-modified Na+ channels are based on a structural alteration of still another region which is also involved in Na+ inactivation, besides the linker between domains III and IV of the alpha-subunit. Endogenous peptidases such as calpain did not affect Na+ inactivation. This stresses the stochastic nature of a kinetic peculiarity of cardiac Na+ channels, mode-switching to a non-inactivating mode.

Acetamides↗

PubMed1997

Late results after resection of benign hepatic tumors: clinical and radiological findings.

The purpose of our work was to provide data on the recurrence of resected benign hepatic lesions and to evaluate the value of follow-up examinations in this group of patients. From August to October 1993, 75 patients who had been admitted for liver surgery for benign tumors between 1975 and 1993 were controlled by physical examinations, serological tests, US, and, in the case of equivocal US findings, by CT. The histological diagnoses of the operative specimen included hydatidosis in 43 patients, focal nodular hyperplasia (FNH) in 12 patients, liver cell adenoma in 8 patients, cavernous hemangioma in 8 patients, and congenital cyst in 4 patients. Hepatic scars were observed in 36 of the 75 patients. Four cases of intrahepatic recurrence and 1 case of intraperitoneal spread were observed in the 42 patients with recent hydatosis. Long-term postoperative controls (specific serological tests, US) are necessary in the management of patients with hydatid disease. Follow-up examinations are not indicated in asymptomatic patients who have been operated on for FNH, hemangioma, or congenital cysts.

Adult↗

PubMed1997

Drug specific antibodies: T-cell epitope-lipopeptide conjugates are potent adjuvants for small antigens in vivo and in vitro.

To generate conventional or monoclonal antibodies for the serological detection of drugs, antibiotics, toxins and other low molecular mass substances, a suitable and effective adjuvant is needed. Lipopeptides derived from a major component of the bacterial cell wall constitute potent nontoxic and nonpyrogenic immunoadjuvants when mixed with conventional antigens. Here we demonstrate that the synthetic lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)-cysteinyl- serine (P3CS) coupled to a Th-cell epitope (P3CS-Th) can efficiently enhance the specific immune response against low molecular weight compounds in different species. In the presence of the synthetic lipopeptide P3CS-Th, the peptides which are per se non-immunogenic stimulated a specific humoral immune response in mice after intraperitoneal application. Mixtures containing adjuvants without the Th sequence showed no significant antibody induction. A marked enhancement of the humoral immune response was obtained with the low molecular mass antigens Iturin AL, Herbicolin A and Microcystin (MLR) coupled to poly-l-lysin (MLR-PLL), in rabbits and in chickens. Lipopeptide-Th cell epitope conjugates also constituted adjuvants for the in vitro immunization of either human mononuclear cells or mouse B-cells with MLR-PLL; after fusion of the immunized cultures with the heteromyeloma cell lines CB-F7 or the mouse myeloma cell line SP 2/0, respectively, we observed a significantly increased yield of antibody secreting hybridomas.

Adjuvants, Immunologic↗

PubMed1997

Electropolymerization of a phenol-modified peptide for use in receptor-ligand interactions studied by surface plasmon resonance.

The combination of surface plasmon resonance (SPR) with an electrochemical method for surface modification is presented. The SLP1 sequence of the sodium channel protein of rat cardiac muscle cells was N-terminally modified with an electropolymerizable group and immobilized on a gold-coated glass slide by oxidative polymerization. The resulting peptide-functionalized substrate was incubated with a polyclonal-specific anti-SLP1 serum. Growth of the peptide layer and the immunological reaction between ligand and receptor were detected on-line by SPR. The applicability of this approach for the rapid and selective analysis of receptor-ligand interactions is demonstrated.

Amino Acid Sequence↗

PubMed1997

Amplification of 11q13 DNA markers in head and neck squamous cell carcinomas: correlation with clinical outcome.

This study was performed on 282 patients with primary head and neck squamous cell carcinomas to evaluate the prognostic importance of 11q13 amplification. Amplification of the 11q13 DNA markers, HST-1/FGF-4 and BCL-1, evaluated by Southern and slot blot hybridisation, was detected in 52% of tumours. 11q13 amplification was associated with tumour site since this alteration occurred in 76% of tumours arising in the hypopharynx, versus 40% in the other sites (P = 0.0007). 11q13 amplification was also significantly related to the presence of involved neck lymph nodes (P = 0.013). The relationship between 11q13 amplification and risk of progression was studied in two subgroups of head and neck cancer patients with regard to treatment modalities. The presence of 11q13 amplification in the tumour was not significantly associated with a shorter event-free survival (P = 0.82) and crude survival (P = 0.61) of the 201 patients treated by surgery and postoperative radiotherapy. Similarly, absence of a relationship was observed for the group of 79 patients treated by surgery alone. These results confirm that 11q13 amplification is a prominent event in head and neck squamous cell carcinoma, indicating that it may be a common genetic event in the development of these neoplasms, but is not a reliable prognostic marker.

Adult↗

PubMed1997

Prospective controlled study of hydroxyzine and cetirizine in pregnancy.

BACKGROUND: Hydroxyzine has been used for many years for the treatment of allergic symptoms. Cetirizine, an active metabolite of hydroxyzine, has become very popular for the treatment of allergy symptoms because of its efficacy without the sedating effects of the parent compound. Little is known about the safety of hydroxyzine use during pregnancy, and there are no published reports on the effects of cetirizine on pregnancy outcome. OBJECTIVE: To determine whether hydroxyzine and cetirizine are associated with any increased risk of malformations in humans. METHODS: All pregnant women counseled by the Motherisk Program in Toronto on the use of hydroxyzine or cetirizine during their pregnancies were enrolled in a prospective, controlled, observational study. The control group consisted of pregnant women matched for age, smoking, and alcohol consumption who were counseled for non-teratogenic drug. RESULTS: One hundred twenty women were followed after exposure to either hydroxyzine or cetirizine during pregnancy. Of these, 53 were exposed to hydroxyzine during organogenesis and 39 to cetirizine. There were no significant differences found between the hydroxyzine or cetirizine groups and the control groups in the pregnancy outcome: rate of livebirths, spontaneous or therapeutic abortion, or stillbirth. There was also no difference in the rates of major or minor anomalies, mean birth weight, mode of delivery, gestational age, or presence of neonatal distress. CONCLUSIONS: The use of hydroxyzine and cetirizine does not appear to be associated with increased teratogenic risk.

Abortion, Spontaneous↗

PubMed1997

Composition and purity of combinatorial aryl ether collections analyzed by electrospray mass spectrometry.

Electrospray mass spectrometry (ESI-MS), tandem mass spectrometry and on-line RP-HPLC-ESI-MS were used to evaluate the composition and purity of three different aryl ether mixtures consisting of 10 and 45 aryl ethers synthesized on solid support by Williamson etherification. The libraries feature two potential pharmacophores connected with three different spacers and serve as models for a detailed component analysis. Individual members of the library and by-products were identified rapidly and conveniently by product ion scans. Compound collections obtained by two different synthetic methods, the split/combine approach and the premix method, showed different mass distributions in the ESI-MS spectra. Some components were not detected in direct ESI-MS measurements, but were found by MS/MS experiments. Precursor ion and constant neutral loss scans allowed the identification of components with common structural features.

Chromatography, High Pressure Liquid↗

PubMed1997

ATP-dependent ferric hydroxamate transport system in Escherichia coli: periplasmic FhuD interacts with a periplasmic and with a transmembrane/cytoplasmic region of the integral membrane protein FhuB, as revealed by competitive peptide mapping.

The Escherichia coli iron transport system via ferrichrome belongs to the group of ATP-dependent transporters that are widely distributed in prokaryotes and eukaryotes. Transport across the cytoplasmic membrane is mediated by three proteins: FhuD in the periplasm, FhuB in the cytoplasmic membrane and FhuC (ATPase) associated with the inside of the cytoplasmic membrane. Interaction of FhuD with FhuB was studied in vitro with biotinylated synthetic 10 residue and 20-24 residue peptides of FhuB by determining the activity of beta-galactosidase linked to the peptides via streptavidin. Peptides identical in sequence to only one of the four periplasmic loops (loop 2), predicted by a transmembrane model of FhuB, and peptides representing a transmembrane segment and part of the adjacent cytoplasmic loop 7 of FhuB bound to FhuD. Decapeptides were transferred into the periplasm of cells through a FhuA deletion derivative that forms permanently open channels three times as large as the porins in the outer membrane. FhuB peptides that bound to FhuD inhibited ferrichrome transport, while peptides that did not bind to FhuD did not affect transport. These data led us to propose that the periplasmic FhuD interacts with a transmembrane region and the cytoplasmic segment 7 of FhuB. The transmembrane region may be part of a pore through which a portion of FhuD inserts into the cytoplasmic membrane during transport. The cytoplasmic segment 7 of FhuB contains the conserved amino acid sequence EAA...G (in FhuB DTA ...G) found in ABC transporters, which is predicted to interact with the cytoplasmic FhuC ATPase. Triggering of ATP hydrolysis by substrate-loaded FhuD may occur by physical interaction between FhuD and FhuC, which bind close to each other on loop 7. Although FhuB consists of two homologous halves, FhuB(N) and FhuB(C), the sites identified for FhuD-mediated ferrichrome transport are asymmetrically arranged.

Adenosine Triphosphate↗

PubMed1997

The transcriptional repression of the human Cu/Zn superoxide dismutase(sod1) gene by the anticancer drug, mitomycin C(MMC).

The Cu/Zn superoxide dismutase(sod1) is one of the key enzymes that protects cells against oxidative stress. In order to investigate the effects of mitomycin C(MMC) on the induction of apoptotic cell death and on the sod1 transcription level, the CATs activity of HepG2 cells transfected with sod1 promoter-CAT(chloramphenicol acetyl transferase) fusion reporter was measured after MMC treatment. The CAT assay showed that exposure of HepG2 cells to MMC decreased the transcription level of the sod1 gene. The accumulation of p53 tumor suppressor protein by MMC treatment of HepG2 cells was noted. In order to investigate the p53-negative response element in its promoter region, a p53 cotransfection experiment with serially deleted sod1 promoter/CAT reporter constructs was performed. The results show a significant reduction of CAT activity in all deletion reporter constructs. The results show that MMC treatment inhibited sod1 gene transcription through p53-mediated transcriptional repression.

Antibiotics, Antineoplastic↗

PubMed1997

Plasmid DNA expressing a secreted or a nonsecreted form of hepatitis C virus nucleocapsid: comparative studies of antibody and T-helper responses following genetic immunization.

In a murine model, we have compared humoral and T-helper (Th) responses induced following genetic immunization with two hepatitis C virus (HCV) plasmid-derived immunogens: a plasmid expressing the full-length nucleocapsid (CAP) as a nonsecreted antigen (pCMVC2) and a plasmid expressing the amino-terminal part of CAP as a secreted antigen (pS2S.C2N). In BALB/c mice, intramuscular injection of either plasmid induced IgG2a antibodies associated with a Th1-like profile characterized by the in vitro splenic production of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma). The pS2S.C2N plasmid induced antibody titers three- to five-fold higher than those obtained with the pCMVC2 plasmid (maximal titers 1:1,500 versus 1:500). In control experiments, immunization using purified CAP antigen induced a predominant, but not exclusive, Th2-like profile as determined by the splenic production of IL-4 and IL-10. Six putative Th determinants were identified using a panoply of overlapping synthetic peptides in in vitro stimulation assays: amino acids 20-44, 39-63, 79-113, 89-113, 118-142, and 138-152. For all CAP immunogens, MHC haplotype of immunized mice was found to influence seroconversion rates but not the type of cytokines produced in vitro. H-2d mice were faster responders and displayed recall T-cell activation by a larger number of peptides than H-2b mice, whereas H-2s mice were overall very poor responders. Splenic stimulation by at least one determinant, amino acids 79-103, appeared to be highly specific of the H-2b background and of DNA immunization only. These data indicate that DNA immunogens expressing different forms of HCV-CAP are not associated with different Th profiles but rather different seroconversion rates and antibody titers and that collaboration of distinct T-help epitopes can be restricted by the MHC background.

Animals↗

PubMed1997

Evidence of acute demyelination around a developmental venous anomaly: magnetic resonance imaging findings.

The authors report on the occurrence of a focal demyelination showing a mass effect around a developmental venous anomaly in the cerebellum. Because the latter presented as a single lesion, the differentiation from intraparenchymal neoplasms or infarction was difficult. Follow-up magnetic resonance imaging and histology from a biopsy specimen give useful information to find the appropriate diagnosis.

Acute Disease↗

PubMed1997

Antibodies to a new linear site at the topographical or functional interface between the haemagglutinin and fusion proteins protect against measles encephalitis.

The haemagglutinin protein (H) of measles virus (MV) binds to susceptible cells and collaborates with the fusion protein (F) to mediate fusion of the virus with the cell membrane. Binding and fusion activity of the virus can be monitored by haemagglutination and haemolysis, respectively, of monkey erythrocytes. Most monoclonal antibodies (MAbs) with haemolysis inhibiting activity (HLI+) are either MV-F specific and do not inhibit haemagglutination (HI-), or they bind to MV-H and are HI+ by interfering with virus binding. We describe here a small panel of H-specific MAbs (BH47, BH59, BH103, BH129) which bind to a new linear neutralizing epitope, H244-250 (SELSQLS; NE domain), and which prevent virus-cell fusion (HLI+) but not virus binding (HI-). These antibodies also protect against MV encephalitis in an animal model. They do not compete with an HLI+/HI+ antibody (BH216) which binds to the haemagglutinin noose epitope (HNE). The antibodies described here and the HNE-specific antibodies are functionally distinct and define two topographically non-overlapping interfaces, supposedly with a bias towards the host cell MV-receptor and the fusion protein respectively. The proximity of the CD46 downregulating amino acid Arg-243 may suggest a functional link between the domain described here and the CD46 binding domain. This new protective linear site is also of potential interest for the design of a subunit-based vaccine.

Animals↗