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Biomedical subjects

C Kunz

Publications and source records attributed to C Kunz.

At least 73 records · Page 4Linked to original sources
PubMed1996

Characterization and complete genome sequences of high- and low- virulence variants of tick-borne encephalitis virus.

The entire genomic sequences of two strains (Hypr and 263) of the flavivirus tick-borne encephalitis (TBE) virus differing in virulence from the prototypic strain Neudoerfl were determined. Strain Hypr is a human isolate of TBE virus with a high laboratory passage history which exhibits a significantly higher neuro-invasiveness in mice compared to the prototype strain. Strain 263 is a low-passage tick-isolate with a temperature-sensitive and attenuated phenotype. Except for the heterogeneous 3' non-coding regions strains Hypr and 263 share, respectively, 97.2% and 97.6% nucleotide sequence identity with strain Neudoerfl, and differ by a total of 42 and 36 amino acids from the prototypic strain. Of these, only 12 amino acids for each of the two strains represent non-conservative differences unique to an individual strain and some of these are located at positions highly conserved among flaviviruses. Based on these observations, the potential biological significance of particular sequence differences is discussed in the context of the current knowledge about molecular determinants of flavivirus virulence.

Animals↗
PubMed1996

Urinary excretion of lactose and oligosaccharides in preterm infants fed human milk or infant formula.

At present, not much is known about the absorption and metabolism of human milk (HM) oligosaccharides in term and preterm infants. We investigated the renal excretion of lactose and complex oligosaccharides in preterm infants fed HM (n = 9, mean actual body weight 2290 g) or a cow's milk-based infant formula (n = 9, mean actual body weight 2470 g). We found that the renal excretion of lactose in HM-fed infants was slightly lower than in formula-fed infants (14.0 +/- 7.4 versus 20.4 +/- 8.7 mg kg-1 day-1, mean +/- SD). The excretion of neutral sugars deriving from oligosaccharides was similar in HM-fed and formula-fed infants (3.8 +/- 2.1 versus 2.9 +/- 0.9 mg kg-1 day-1); the difference between means was not statistically significant. The separation and characterization of oligosaccharides by high-pH anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) and subsequent analysis by fast atom bombardment-mass spectrometry (FAB-MS) revealed a more complex pattern in HM-fed infants compared to the formula-fed group. Lactose-derived oligosaccharides characteristic for HM (e.g. lacto-N-tetraose, and lacto-N-fucopentaoses I and II) were excreted in HM-fed but not in formula-fed infants. These results indicate that nutrition has a significant impact on the oligosaccharide composition in urine of preterm infants.

Chromatography, High Pressure Liquid↗
PubMed1996

Presence of human herpesvirus 6 variants A and B in saliva and peripheral blood mononuclear cells of healthy adults.

Saliva and peripheral blood mononuclear cells (PBMCs) from 44 healthy young adults were tested for human herpesvirus 6 variants A and B (HHV-6A and -6B) DNA by a sensitive nested PCR. HHV-6B infection was ascertained in 98% of the subjects, and 95% were found to excrete variant B in their saliva. HHV-6A was found in the PBMCs of 16%, but was not detected in saliva samples.

Adult↗
PubMed1996

Structural requirements for low-pH-induced rearrangements in the envelope glycoprotein of tick-borne encephalitis virus.

The exposure of the flavivirus tick-borne encephalitis (TBE) virus to an acidic pH is necessary for virus-induced membrane fusion and leads to a quantitative and irreversible conversion of the envelope protein E dimers to trimers. To study the structural requirements for this oligomeric rearrangement, the effect of low-pH treatment on the oligomeric state of different isolated forms of protein E was investigated. Full-length E dimers obtained by solubilization of virus with the detergent Triton X-100 formed trimers at low pH, whereas truncated E dimers lacking the stem-anchor region underwent a reversible dissociation into monomers without forming trimers. These data suggest that the low-pH-induced rearrangement in virions is a two-step process involving a reversible dissociation of the E dimers followed by an irreversible formation of trimers, a process which requires the stem-anchor portion of the protein. This region contains potential amphipathic alpha-helical and conserved structural elements whose interactions may contribute to the rearrangements which initiate the fusion process.

Amino Acid Sequence↗
PubMed1996

Recombinant subviral particles from tick-borne encephalitis virus are fusogenic and provide a model system for studying flavivirus envelope glycoprotein functions.

Recombinant subviral particles (RSPs) obtained by coexpression of the envelope (E) and premembrane (prM) proteins of tick-borne encephalitis virus in COS cells (S. L. Allison, K. Stadler, C. W. Mandl, C. Kunz, and F. X. Heinz, J. Virol. 69:5816-5820, 1995) were extensively characterized and shown to be ordered structures containing envelope glycoproteins with structural and functional properties very similar to those in the virion envelope. The particles were spherical, with a diameter of about 30 nm and a buoyant density of 1.14 g/cm3 in sucrose gradients. They contained mature E proteins with endoglycosidase H-resistant glycans as well as fully cleaved mature M proteins. Cleavage of prM, which requires an acidic pH in exocytic compartments, could be inhibited by treatment of transfected cells with ammonium chloride, implying a common maturation pathway for RSPs and virions. RSPs incorporated [14C]choline but not [3H]uridine, demonstrating that they contain lipid but probably lack nucleic acid. The envelope proteins of RSPs exhibited a native antigenic and oligomeric structure compared with virions, and incubation at an acidic pH (pH <6.5) induced identical conformational changes and structural rearrangements, including an irreversible quantitative conversion of dimers to trimers. The RSPs were also shown to be functionally active, inducing membrane fusion in a low-pH-dependent manner and demonstrating the same specific hemagglutination activity as whole virions. Tick-borne encephalitis virus RSPs thus represent an excellent model system for investigating the structural basis of viral envelope glycoprotein functions.

Animals↗
PubMed1996

Potential ligands for cell adhesion molecules in human milk.

In this study, glycoproteins and oligosaccharides with sialyl Lewis a, sialyl Lewis x, Lewis x, and Lewis y epitopes were isolated by ultracentrifugation and fast-protein liquid chromatography from human milk of mothers with term or preterm infants. The identification of these epitopes on whey proteins was achieved by monoclonal antibodies and lectins after Western blotting. Lactose-derived oligosaccharides were characterized by high-performance thin-layer chromatography and high-pH anion-exchange chromatography with pulsed amperometric detection. These carbohydrate epitopes which are potential ligands for selections are not of cellular origin but appear in the soluble fraction of milk. Here, they are present as lactose-derived oligosaccharides (molecular weight < 10 kD) or bound to proteins with a high molecular weight (> 100 kD). Lewis antigens might represent another category of protective nonimmunological substances in human milk with the potential to influence inflammatory processes in human milk fed infants.

Antibodies, Monoclonal↗
PubMed1995

Variations of soluble intercellular cell adhesion molecule 1 (sICAM-1) in serum of adult volunteers.

Soluble cell adhesion molecules such as sICAM-1 in serum and other biological fluids are suggested as being useful diagnostic parameters for a variety of diseases. Since increased concentrations during diseases are frequently less pronounced compared to other parameters, we tested whether it would be necessary to align the time of blood collection during the course of a clinical trial. In the 9 volunteers of our trial we found a statistically significant effect at the point in time of blood collection and corresponding serum concentrations of sICAM-1 (p < 0.01). The deviation of the concentrations at a certain time from the daily mean in each individual was seen to be as high as 15%. Our data suggest that daytime variations of serum sICAM-1 concentrations should be taken into consideration when longitudinal observations are planned.

Adult↗
PubMed1995

The flavivirus 3'-noncoding region: extensive size heterogeneity independent of evolutionary relationships among strains of tick-borne encephalitis virus.

The sequences of the 3'-noncoding regions (NCR) of 12 strains of tick-borne encephalitis (TBE) virus were analyzed and found to vary in length from 350 to approximately 750 nucleotides. The size heterogeneity is restricted to a variable region following the stop codon, whereas the most 3'-terminal 350 nucleotides form a highly conserved core element containing several potentially important sequence motifs and secondary structure elements. A homoadenosine tract previously thought to form the 3'-terminus of some TBE virus strains was now shown to be an internal part of the variable region of certain strains. The strains included in this study were isolated from both humans and ticks over a time period of more than 40 years at various locations throughout the entire endemic area of TBE virus, but there was no correlation between these parameters and the observed lengths of the 3'-NCRs. Identity data calculated from common 3'-NCR sequences and also from short sections of the open reading frame indicated that coding and noncoding sequences were linked during evolution, but the lengths of the 3'-NCRs were independent of these relationships. These observations together with detailed analyses and alignments of the sequences suggest that the variable region was originally acquired through duplication and recombination events, but--much more recently during evolution--various portions of this region were lost again, resulting in the now observed heterogeneous 3'-NCRs.

Animals↗
PubMed1995

Differential regulation of plasminogen activator and inhibitor gene transcription by the tumor suppressor p53.

The ability of p53 to activate or repress transcription suggests that its biological function as tumor suppressor is in part accomplished by regulating a number of genes including such required for inhibition of cell growth. We here give evidence that p53 also may regulate genes responsible for the proteolytic degradation of the extracellular matrix, which is considered a crucial feature for local invasion and metastasis of neoplastic cells. An important and highly regulated cascade of such proteolytic events involves the plasminogen activator system. We show that wild-type p53 represses transcription from the enhancer and promoter of the human urokinase-type (u-PA) and the tissue-type plasminogen activator (t-PA) gene through a non-DNA binding mechanism. Oncogenic mutants lost the repressing activity. In contrast, wild-type but not mutant p53 specifically binds to and activates the promoter of the plasminogen activator inhibitor type-1 (PAI-1) gene. Interestingly, one of the p53 mutants (273his) inhibited PAI-1 promoter activity. Our results suggest that altered function of oncogenic forms of p53 may lead to altered expression of the plasminogen activators and their inhibitor(s) and thus to altered activation of the plasminogen/plasmin system during tumor progression.

Base Sequence↗
PubMed1995

The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution.

The crystallographically determined structure of a soluble fragment from the major envelope protein of a flavivirus reveals an unusual architecture. The flat, elongated dimer extends in a direction that would be parallel to the viral membrane. Residues that influence binding of monoclonal antibodies lie on the outward-facing surface of the protein. The clustering of mutations that affect virulence in various flaviviruses indicates a possible receptor binding site and, together with other mutational and biochemical data, suggests a picture for the fusion-activating, conformational change triggered by low pH.

Amino Acid Sequence↗
PubMed1995

A comparison of nucleotide sequences of measles virus L genes derived from wild-type viruses and SSPE brain tissues.

The nucleotide sequences of the large protein (L) gene derived from two wild-type measles viruses (MV) and two SSPE brain-derived viruses have been determined. All sequences have single large open reading frames encoding 2183 amino acid residues. The deduced L proteins are well conserved and the proposed functional domains which have been identified for rhabdo- and paramyxoviruses are completely conserved in all strains. The degree of variability of L proteins is the lowest of all structural proteins of MV, reflecting its role in virus reproduction and persistence. Biased hypermutation was not observed in the L genes derived from SSPE brain tissue. None of the nucleotide changes can be associated with the attenuated phenotype of the Edmonston vaccine viruses.

Amino Acid Sequence↗
PubMed1995

Tick-borne encephalitis virus envelope protein E-specific monoclonal antibodies for the study of low pH-induced conformational changes and immature virions.

A set of ten monoconal antibodies (mabs) specific for the tick-borne encephalitis (TBE) virus envelope protein E were prepared and characterized with respect to their functional activities, the location of their binding sites on protein E and the involvement of their epitopes in acid pH-induced conformational changes and interactions with the precursor to the membrane protein (prM) in immature virions. The majority of these mabs mapped to the previously defined antigenic domain A. All of the mabs recognize parts of the E protein which undergo low pH-induced structural rearrangements believed to be necessary for the fusion activity of the virus, and six of the mabs define epitopes which are affected by the prM-E interaction in immature virions. They are therefore of potential value as specific reagents for studying the structure and function of protein E, as well as the function of the prM-E association. Five of the mabs exhibited neutralizing activity, and can therefore be used for the selection of escape mutants.

Animals↗
PubMed1995

Effects of a high protein intake on renal acid excretion in bodybuilders.

Bodybuilders often prefer a high protein diet to achieve maximum skeletal muscle hypertrophy. In this study the effect of a high protein diet on renal acid load and renal handling of proton excretion was studied comparing dietary intake and urinary ionograms in 37 male bodybuilders and 20 young male adults. Energy intake (+ 7%), protein intake (128 vs 88 g/d/1.73 m2), and renal net acid excretion (95 vs 64 mmol/d/1.73 m2) were higher in the bodybuilders than in the controls, however, urine-pH was only slightly lower (5.83 vs 6.12). In the bodybuilders renal ammonium excretion was higher at any given value of urine pH than in the controls. In a regression analysis protein intake proved to be an independent factor modulating the ratio between urine-pH and renal ammonium excretion. The concomitant increase of renal net acid excretion and maximum renal acid excretion capacity in periods of high protein intake appears to be a highly effective response of the kidney to a specific food intake leaving a large renal surplus capacity for an additional renal acid load.

Adolescent↗
PubMed1995

Recombinant and virion-derived soluble and particulate immunogens for vaccination against tick-borne encephalitis.

Using different forms of the envelope glycoprotein E from tick-borne encephalitis virus we investigated the influence of physical and antigenic structure on the efficacy of vaccination. Different protein E-containing preparations were either derived from purified virions or were produced as recombinant proteins in COS cells. These included soluble dimeric forms (virion-derived protein E dimers with and without membrane anchor; recombinant protein E dimers without membrane anchor), micellar aggregates of protein E (rosettes), and recombinant subviral particles (RSPs). The structural differences between these immunogens were verified by sedimentation analysis, immunoblotting and epitope mapping with a panel of monoclonal antibodies. Specific immunogenicities were determined in mice in comparison to formalin-inactivated whole virus. Rosettes and RSPs were excellent immunogens and exhibited similar efficacies as inactivated virus in terms of antibody induction and protection against challenge, whereas all of the soluble forms were much less immunogenic. These data emphasize the importance of the immunogen's antigenic and physical structure for an effective stimulation of the immune system and indicate that RSPs represent an excellent candidate for a recombinant vaccine against tick-borne encephalitis.

Animals↗
PubMed1995

Laboratory acquired tick-borne meningoencephalitis: characterisation of virus strains.

BACKGROUND: The handling of tick-borne encephalitis (TBE) virus is potentially hazardous, as indicated by a number of laboratory-acquired infections in the prevaccination era. OBJECTIVES: (1) To reemphasize the hazard of handling TBE virus without being vaccinated by describing the case of a laboratory-acquired full-blown TBE in a microbiologist who isolated the virus from a blood sample. (2) To molecularly characterize the causative virus strain isolated in Slovenia in comparison with the European prototype strain Neudoerfl. STUDY DESIGN: The virological diagnosis of the laboratory infection was established by serology and virus isolation. The virus was characterized by restriction fragment analysis of PCR products of amplified genomic sequences and a panel of monoclonal antibodies reacting with the major envelope protein. RESULTS: The laboratory infection, most probably acquired by aerosol, resulted in a biphasic course of the disease with a severe meningoencephalitis in the second phase. Both by restriction fragment and monoclonal antibody analysis the Slovenian virus strains involved were indistinguishable from the European prototype strain. CONCLUSIONS: This report confirms the potential hazard of handling TBE virus in the laboratory without being vaccinated. The similarity of the virus isolates from Slovenia with the European prototype strain confirms the previously observed homogeneity of TBE virus strains from different European countries.

Journal Article↗
PubMed1995

Identification of tick-borne encephalitis virus ribonucleic acid in tick suspensions and in clinical specimens by a reverse transcription-nested polymerase chain reaction assay.

BACKGROUND: Tick-borne encephalitis virus (TBEV) is a major human pathogenic flavivirus. Sensitive assays for the detection of viral RNA may be valuable both for the identification of virus in ticks as well as for diagnostic purposes. OBJECTIVES: (1) The development of a sensitive polymerase chain reaction (PCR) test system for the detection of TBEV-RNA and its application to the identification of infected ticks; and (2) evaluation of the PCR assay for diagnostic purposes, i.e., detection of TBE virus RNA in blood and in cerebrospinal fluid (CSF) of TBE patients. STUDY DESIGN: (1) Establishment of a TBEV-specific reverse transcription (RT)-nested PCR assay and evaluation of its sensitivity; (2) comparison of the PCR assay with that of virus isolation from tick suspensions; and (3) investigation of 105; serum and CSF samples from patients with serologically confirmed TBE by RT-nested PCR. RESULTS: An RT-nested PCR assay was established with a detection limit of 100-1000 copies of TBEV RNA. All tick suspensions from which the virus could be isolated by inoculation of suckling mice also screened positive in the PCR assay. Of the 105 clinical samples investigated, only one serum and one CSF sample were positive by PCR assay, and these were both obtained very early in the course of the disease. CONCLUSIONS: The PCR assay described is valuable for the detection of TBEV in tick suspensions and can substitute for the usual virus isolation procedure in which suckling mice are inoculated. Its application for diagnostic purposes, however, does not seem to provide a significant improvement over serological diagnosis. Only in very rare cases, when a sample is drawn extremely early in the course of disease, may TBEV RNA be detected in serum or CSF before the appearance of specific IgM antibodies and thus allow an earlier diagnosis.

Journal Article↗