Biomedical subjects

C Kunz

Publications and source records attributed to C Kunz.

At least 91 records · Page 5Linked to original sources

PubMed1995

Roe deer as sentinels for endemicity of tick-borne encephalitis virus.

The suitability of serological surveys of roe deer (Capreolus capreolus) in determining the spread of tick-borne encephalitis virus (TBEV) was tested in a south German area with a low risk of TBEV infection to humans. Sera obtained from 192 hunted roe were screened by an haemagglutination-inhibition test (HAI) and in an ELISA developed in our laboratory. Those found positive were tested in a neutralization test (NT). Fifty (26.0%) sera reacted positive by ELISA and 43 (86.0%) of these were confirmed by HAI or NT. Forty-seven (24.5%) samples were positive by HAI, 44 (93.6%) of which were also positive in NT or ELISA. Only insignificant increase of the antibody prevalence with age (P = 0.17 for HAI antibodies) suggests that most infections occur at an early age in scattered natural foci. The antibody prevalence in females was lower than in males (OR = 0.63; P = 0.02 for HAI antibodies). In determining the distribution of seropositive roe we increased the sample size to 235 sera. No antibodies were detected in 56 (23.8%) sera collected in the eastern third of the county. The areas of high antibody prevalence in roe match those in which humans have been infected. We conclude that serosurveys of roe deer are useful in marking out areas in which humans face the risk of infection, provided that an adequate number of sera, preferably from males, is available.

Age Distribution↗

PubMed1995

Oligomeric rearrangement of tick-borne encephalitis virus envelope proteins induced by an acidic pH.

The flavivirus envelope protein E undergoes irreversible conformational changes at a mildly acidic pH which are believed to be necessary for membrane fusion in endosomes. In this study we used a combination of chemical cross-linking and sedimentation analysis to show that the envelope proteins of the flavivirus tick-borne encephalitis virus also change their oligomeric structure when exposed to a mildly acidic environment. Under neutral or slightly alkaline conditions, protein E on the surface of native virions exists as a homodimer which can be isolated by solubilization with the nonionic detergent Triton X-100. Solubilization with the same detergent after pretreatment at an acidic pH, however, yielded homotrimers rather than homodimers, suggesting that exposure to an acidic pH had induced a simultaneous weakening of dimeric contacts and a strengthening of trimeric ones. The pH threshold for the dimer-to-trimer transition was found to be 6.5. Because the pH dependence of this transition parallels that of previously observed changes in the conformation and hydrophobicity of protein E and that of virus-induced membrane fusion, it appears likely that the mechanism of fusion with endosomal membranes involves a specific rearrangement of the proteins in the viral envelope. Immature virions in which protein E is associated with the uncleaved precursor (prM) of the membrane protein M did not undergo a low-pH-induced rearrangement. This is consistent with a protective role of protein prM for protein E during intracellular transport of immature virions through acidic compartments of the trans-Golgi network.

Animals↗

PubMed1995

Synthesis and secretion of recombinant tick-borne encephalitis virus protein E in soluble and particulate form.

A quantitative study was performed to investigate the requirements for secretion of recombinant soluble and particulate forms of the envelope glycoprotein E of tick-borne encephalitis (TBE) virus. Full-length E and a carboxy terminally truncated anchor-free form were expressed in COS cells in the presence and absence of prM, the precursor of the viral membrane protein M. Formation of a heteromeric complex with prM was found to be necessary for efficient secretion of both forms of E, whereas only low levels of anchor-free E were secreted in the absence of prM. The prM-mediated transport function could also be provided by coexpression of prM and E from separate constructs, but a prM-to-E ratio of greater than 1:1 did not further enhance secretion. Full-length E formed stable intracellular heterodimers with prM and was secreted as a subviral particle, whereas anchor-free E was not associated with particles and formed a less stable complex with prM, suggesting that prM interacts with both the ectodomain and anchor region of E.

Animals↗

PubMed1994

Loss of a histidine residue at the active site of S-locus ribonuclease is associated with self-compatibility in Lycopersicon peruvianum.

Gametophytic self-incompatibility in the Solanaceae is controlled by a single, multiallelic locus, the S locus. We have recently described an allele of the S locus of Lycopersicon peruvianum that caused this normally self-incompatible plant to become self-compatible. We have now characterized two glycoproteins present in the styles of self-compatible and self-incompatible accessions of L. peruvianum: one is a ribonuclease that cosegregates with a functional self-incompatibility allele (S6 allele); the other cosegregates with the self-compatible allele (Sc allele) but has no ribonuclease activity. The derived amino acid sequences of the cDNAs encoding the S6 and Sc glycoproteins resemble sequences of other ribonucleases encoded by the S locus. The derived sequence for the Sc glycoprotein differs from the others by lacking one of the histidine residues found in all other S-locus ribonucleases. These findings demonstrate the essential role of ribonuclease activity in self-incompatibility and lend further weight to evidence that this histidine residue is involved in the catalytic site of the enzyme.

Alleles↗

PubMed1994

Prevalence of hepatitis-C virus RNA in serum and throat washings of children with chronic hepatitis.

Serum samples from 46 children with chronic and probably transfusion acquired hepatitis were tested for the presence of hepatitis C virus (HCV) RNA by a "nested" polymerase chain reaction (PCR) assay, to judge a possible risk of HCV transmission from these patients. In 73% of the samples, viral RNA was detected, indicating a high virus prevalence in this patient group. High titers of HCV-RNA were observed in some sera as shown by the detection of virus in some samples even at dilutions of 10(-3). Comparison of simultaneously obtained PCR results and ALT values revealed no significant correlation between virus presence in serum and higher ALT levels. It was, however, shown that unusually high ALT values may reflect a high titer of viral RNA in serum. To investigate the prevalence of viral RNA in saliva, which could be a vehicle of virus transmission, 35 throat washing samples from the HCV-infected children were screened by PCR. Using three different sample preparation procedures, 20% of the throat washings were found to be positive for HCV-RNA. This indicates a prevalence of virus in this fluid lower than that reported previously.

Alanine Transaminase↗

PubMed1994

Clinical and immunological investigation of a new combined hepatitis A and hepatitis B vaccine.

As with hepatitis B vaccines, the recently developed hepatitis A vaccine is suitable not only for individual protection, but also for public health control measures. For introduction into routine immunisation programmes, however, hepatitis A vaccine should preferably be combined with other already established vaccines. In particular, a combination of hepatitis A and hepatitis B vaccines would be appropriate. We investigated a new combined hepatitis A/hepatitis B vaccine comparing its tolerability and immunogenicity with that obtained after separate or mixed simultaneous administration of the two components. Three groups of healthy volunteers, each of approximately 50 persons, were included. All were negative for hepatitis A and hepatitis B markers and had normal liver enzyme values. They received hepatitis A (720 ELISA units) and hepatitis B (20 micrograms) vaccines in the deltoid muscle, combined, mixed or separately, according to a 0, 1, 6-month schedule. Blood samples for determination of antibodies to hepatitis A virus (anti-HAV) and hepatitis B virus (anti-HBs) and of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were drawn at months 0, 1, 2, 6, and 7. Local and systemic reactions were monitored by means of questionnaires. The results of our study demonstrate that the combined hepatitis A and B vaccine is well tolerated and highly immunogenic. The seropositivity and seroprotection rates were 100% for both antigens in all groups. Surprisingly, anti-HAV and anti-HBs antibody titres after the combined and mixed vaccines were significantly higher compared with the respective monovalent vaccines injected separately.

Adult↗

PubMed1994

Structural changes and functional control of the tick-borne encephalitis virus glycoprotein E by the heterodimeric association with protein prM.

We have used tick-borne encephalitis virus to study the involvement of acidic compartments during the entry and release phases of flavivirus infection and to elucidate the role of protein prM in immature virions. Elevation of the pH in acidic intracellular compartments by either bafilyomycin A1, a specific inhibitor of the vacuolar type H(+)-ATPase or by NH4Cl had a strong inhibitory effect during virus penetration and also prevented the cleavage of prM when added in the late phase of the viral life cycle. In the latter case the release of virus particles was not impaired. These immature (prM-containing) virions exhibited a 20- to 50-fold lower specific infectivity and HA activity than mature virions and in contrast to these did not undergo low pH-triggered aggregation. The presence of prM also affected the binding of monoclonal antibodies to protein E, especially at sites which have been shown to undergo acid pH-induced conformational changes in mature virions. Crosslinking, solubilization, and sedimentation analyses revealed the existence of prM-E heterooligomeric complexes, suggesting that the function of prM is to protect protein E from undergoing the irreversible conformational changes in acidic compartments of the secretory pathway that are necessary for triggering fusion activity in the endosome during virus entry.

Ammonium Chloride↗

PubMed1994

The interactions of the flavivirus envelope proteins: implications for virus entry and release.

Viral membrane proteins play an important role in the assembly and disassembly of enveloped viruses. Oligomerization and proteolytic cleavage events are involved in controlling the functions of these proteins during virus entry and release. Using tick-borne encephalitis virus as a model we have studied the role of the flavivirus envelope proteins E and prM/M in these processes. Experiments with acidotropic agents provide evidence that the virus is taken up by receptor-mediated endocytosis and that the acidic pH in endosomes plays an important role for virus entry. The envelope glycoprotein E undergoes irreversible conformational changes at acidic pH, as indicated by the loss of several monoclonal antibody-defined epitopes, which coincide with the viral fusion activity in vitro. Sedimentation analysis reveals that these conformational changes lead to aggregation of virus particles, apparently by the exposure of hydrophobic sequence elements. None of these features are exhibited by immature virions containing E and prM rather than E and M. Detergent solubilization, sedimentation, and crosslinking experiments provide evidence that prM forms a complex with protein E which prevents the conformational changes necessary for fusion activity. The functional role of prM before its endoproteolytic cleavage by a cellular protease thus seems to be the protection of protein E from acid-inactivation during its passage through acidic trans Golgi vesicles in the course of virus release.

Acids↗

PubMed1994

Expression of cloned envelope protein genes from the flavivirus tick-borne encephalitis virus in mammalian cells and random mutagenesis by PCR.

The structural membrane proteins prM and E of the flavivirus tick-borne encephalitis (TBE) virus were expressed in mammalian cells for the purpose of probing the structure and molecular interactions of these proteins. Advantage was taken of the natural error frequency of the Taq polymerase used in the PCR amplification to generate a randomly mutated population of genes that were then cloned directly into plasmid expression vectors under the control of an SV40 promoter. Analysis of the mutation frequency by direct sequencing of 22 separate clones showed that the PCR produced mutations at a rate yielding an average of one to two amino acid changes per clone in the 496 amino acid long protein E. This is an ideal rate for assessing the importance of individual amino acid residues within protein domains, thus demonstrating the potential value of the PCR as a random mutagenesis method. Clones encoding wild-type prM and E proteins, and a truncated form of E, were also constructed by recombining portions of selected PCR clones. Transfection of COS-1 cells with these constructs resulted in expression of the prM and E proteins, which was demonstrated by indirect immunofluorescence using monoclonal antibodies (Mabs). The intracellular level of TBE virus antigen, measured in lysates of transfected cells by ELISA, reached approximately 25% of that found in virus-infected COS cells. Furthermore, it was shown by immunofluorescence using a panel of 19 anti-E Mabs that the antigenic structure of the expressed E proteins was nearly identical to that of E protein in infected cells, thus confirming the suitability of this model system as a tool for studying flavivirus protein structure.

Animals↗

PubMed1994

Isolation and characterization of a 21 kDa whey protein in rhesus monkey (Macaca mulatta) milk.

A soluble protein in Rhesus monkey milk was isolated to apparent homogeneity by FPLC gel filtration, anion-exchange and reverse-phase chromatography. It is a major milk protein and is present at 2.5-3.0 mg/ml milk throughout lactation. It is only found in the whey fraction of milk; acid precipitation of casein does not result in any significant change in its concentration. A molecular weight (MW) of about 21.6 kDa was estimated from gel filtration and SDS gel electrophoresis and also calculated from its amino acid composition. The amino acid composition of this protein is similar to that of bovine beta-lactoglobulin (beta-Lg), but it is larger in size, possibly representing a family of primate beta-Lgs.

Amino Acids↗

PubMed1994

Increased renal net acid excretion in prematures below 1,600 g body weight compared with prematures and small-for-date newborns above 2,100 g on alimentation with a commercial preterm formula.

In 76 low birth weight infants with an actual body weight ranging from 1,210 to 2,540 g and fed a commercial preterm formula, urine samples were collected and blood acid base status was measured on day 38 (+/- 17, mean +/- SD) of life. Infants with an actual body weight below 1,600 g demonstrated a higher daily weight gain (22 +/- 3 vs. 14 +/- 5 g/kg/day), lower blood pCO2 (35.4 +/- 5.0 vs. 38.9 +/- 3.8 mm Hg), lower urine pH (5.8 +/- 0.5 vs. 6.5 +/- 0.3), higher renal net acid (1.86 +/- 0.38 vs. 1.28 +/- 0.55 mmol/kg/day) and higher phosphorus excretion (0.67 vs. 0.52 mmol/kg/day) than infants with an actual body weight above 2,100 g. Urinary ionogram data of these 2 groups of infants show that the increased renal net acid excretion of the smaller prematures is the result of a lower urinary excretion of sodium, potassium and chloride, due to a higher daily weight gain, probably a higher retention of these minerals, and a higher urinary phosphorus excretion probably due to an age-specific lower intestinal calcium absorption, and therefore a lower rate of calcium and phosphorus retention. Considering the low renal capacity for hydrogen ion excretion, very low birth weight infants still run a considerable risk for disturbances of acid base metabolism due to the high mean level of net acid excretion in nutrition with preterm formulas and an additional age-specific augmentation of renal acid load.

Acid-Base Equilibrium↗

PubMed1994

Detection of varicella zoster virus (VZV) DNA in fetal tissue by polymerase chain reaction.

Primary Varicella Zoster Virus (VZV) infection during pregnancy can be associated with severe fetal malformation. The virological diagnosis of congenital VZV syndrome has not yet been successful, however, as VZV could not be isolated from fetal tissue. We have now applied a polymerase chain reaction (PCR) assay to allow the detection of VZV DNA in fetuses whose mothers had acquired chickenpox in the first trimester of pregnancy. In 2 of 3 fetuses investigated viral DNA was identified by PCR independently in 2 laboratories, thus confirming a direct fetal VZV infection.

Base Sequence↗

PubMed1994

Why are some pamphlets hard for patients to read?

In this study with lung cancer patients, we describe not only text characteristics that hinder comprehension, including unfamiliar words and poor organization, but also reader characteristics that hinder comprehension, such as educational level, and what the patient wants to know. Based on our and other's research we recommend writing what the reader wants to know, relating new learning to what the learner already knows, and using organizational cues for clarity and emphasis.

Aged↗

PubMed1994

High-performance liquid chromatography for routine analysis of hepatitis C virus cDNA/PCR products.

Ion-pair reversed-phase high-performance liquid chromatography on alkylated nonporous polystyrene-divinylbenzene particles with a mean diameter of 2.1 microns was used to analyze PCR products according to their chain length within a few minutes. The simple and reliable procedure allows the simultaneous separation and isolation of DNA fragments differing in chain length by 1%-5% up to a size of 500 base pairs with recovery rates exceeding 97%. A greater than 70-fold increase in sensitivity could be achieved through the use of a fluorescein-labeled primer, which allowed the determination of a 127-bp hepatitis C virus cDNA/PCR product with a lower mass detection limit of 2 fmol. Calibration curves showed excellent linearity over a range of at least 4 magnitudes. Finally, the stationary phase allowed the routine analysis of hundreds of PCR products with high reproducibility of both retention times and peak areas.

Base Sequence↗

PubMed1993

Ets transcription factor binding site is required for positive and TNF alpha-induced negative promoter regulation.

Thrombomodulin (TM) is expressed on vascular endothelial cells and plays an important role in the anticoagulant pathway by maintaining the thrombo-resistance of the blood vessel wall. We show that in primary human endothelial cells TM gene expression is repressed at the transcriptional level by Tumour necrosis factor (TNF alpha) through a protein kinase C independent pathway. The TM promoter is highly active in endothelial cells and is inhibited by TNF alpha. The -76/-56 region mediates both specific high basal activity and TNF alpha-repression. It binds a nuclear factor specific to endothelial cells, that appears to belong to the Ets-family by various criteria. The -76/-56 region contains three direct repeats of the ets-core sequence GGAA that are important for specific high basal activity, TNF alpha repression and trans-activation by expression of Ets-1 and 2. Although human Ets-1 (h-Ets-1) and chicken c-Ets-1 and 2 stimulate the TM promoter through the -76/-56 element, their activity is not suppressed by TNF alpha. c-Ets-1 competes and overrides TNF alpha repression in a concentration dependent manner. We propose that either a different member of the Ets domain protein family, or an Ets-associated co-factor, is the target of the TNF alpha signalling cascade in endothelial cells.

Animals↗

PubMed1993

A rat histone H2B pseudogene is closely associated with the histone H1d gene.

A 9 kb EcoRI restriction fragment was isolated from a recombinant phage out of a rat genomic library. This DNA fragment contains a rat H1d histone gene, its flanking sequences and a H2B histone pseudogene closely associated with the H1d gene. A comparison of the H2B pseudogene with human H2B genes flanking regions reveals sequence homologies to a human H2B histone gene (Albig, W. et al. (1991) Genomics 10, 940-948).

Amino Acid Sequence↗

PubMed1993

Complete genomic sequence of Powassan virus: evaluation of genetic elements in tick-borne versus mosquito-borne flaviviruses.

The complete nucleotide sequence of the positive-stranded RNA genome of the tick-borne flavivirus Powassan (10,839 nucleotides) was elucidated and the amino acid sequence of all viral proteins was derived. Based on this sequence as well as serological data, Powassan virus represents the most divergent member of the tick-borne serocomplex within the genus flaviviruses, family Flaviviridae. The primary nucleotide sequence and potential RNA secondary structures of the Powassan virus genome as well as the protein sequences and the reactivities of the virion with a panel of monoclonal antibodies were compared to other tick-borne and mosquito-borne flaviviruses. These analyses corroborated significant differences between tick-borne and mosquito-borne flaviviruses, but also emphasized structural elements that are conserved among both vector groups. The comparisons among tick-borne flaviviruses revealed conserved sequence elements that might represent important determinants of the tick-borne flavivirus phenotype.

Amino Acid Sequence↗

PubMed1993

Evaluation of 3 nonradioactive DNA detection systems for identification of herpes simplex DNA amplified from cerebrospinal fluid.

The efficiencies of 4 different hybridization assays, using probes labeled with 32P, digoxigenin (DIG) and biotin, were compared for the detection of herpes simplex virus (HSV) DNA amplified from cerebrospinal fluid during herpes simplex encephalitis (HSE). The biotinylated probe was shown to provide a clearly lower sensitivity than the radioactive hybridization system, regardless of whether the biotin-labeled probe was detected by a colorimetric or by a photobiotin assay. In contrast, the DIG system, which includes a luminescent detection step was shown to be equivalent to the 32P system, at least at lower template concentrations. Although generally higher background signals were observed with the DIG assay the analysis of 73 CSF samples yielded identical results with the 32P and the DIG test systems in all cases, identifying concordantly an HSE in 6 patients. Since the time necessary for the performance of both assays is similar the DIG system seems to be a powerful alternative to the radioactive detection of amplified HSV DNA.

Azides↗