Biomedical subjects

Hans Wolf

Publications and source records attributed to Hans Wolf.

At least 37 records · Page 2Linked to original sources

PubMed2004

The reading frame BPLF1 of Epstein-Barr virus: a homologue of herpes simplex virus protein VP16.

The open reading frame BPLF1 of Epstein-Barr virus (EBV) shows homology to the Herpes simplex virus 1 (HSV1) protein VP16. This protein is a structural tegument component playing a pivotal role for HSV replication as trans-activator of viral immediate-early genes. An EBV gene with a comparable function has not been described so far. However, computer analysis indicated that BPLF1 may be a tegument protein homologous to VP16. This is the first report on the characterisation of the BPLF1 gene, its transcription, and expression of its gene product in vitro and in vivo. Using RT-PCR and Northern blot assays we demonstrated that the BPLF1 gene belongs to the class of late lytic cycle genes of EBV. Besides a full length transcript of 9.5 kb also a polyadenylated transcript of approximately 3 kb is synthesised. However, no consensus splice sites could be identified. Northern blot experiments using partially overlapping probes and sequencing of a BPLF1-specific cDNA revealed 1,550 nucleotides of the BPLF1 transcript, collinear in sequence with the viral genome from position 64547 to 66097. A recombinant Western blot assay detected BPLF1-specific antibodies in seropositive individuals, in particular in cases with elevated viral replication like infectious mononucleosis, chronic active infection, and nasopharyngeal carcinoma. This demonstrated expression of the BPLF1 protein in vivo. Thus, experimental data and computer analysis strongly support the hypothesis of BPLF1 being a tegument protein of the EBV homologous to VP16 of HSV1 and ORF22 of Varicella zoster virus.

Animals↗

PubMed2004

Plasma volume and blood pressure regulation in hypertensive pregnancy.

BACKGROUND: Pre-eclampsia is a multisystem disorder, peculiar to and frequent in human pregnancy. It remains a leading cause of maternal and neonatal morbidity and mortality. Hemodynamic disturbances are the most prominent features of the syndrome. PURPOSE: To provide an overview of plasma volume regulation and blood pressure control mechanisms outside pregnancy, and of the changes in normal pregnancies and in pregnancies complicated by hypertensive disorders. Furthermore, to discuss the rationale of several hemodynamic interventions. RESULTS: In normal pregnancy, large cardiovascular changes take place. A generalized fall in vascular tone by systemic vasorelaxation causes increased blood volume, heart rate and cardiac output. In the preclinical phase, differences have been observed between normal and hypertensive pregnancies in the function of the autonomic nervous system, cardiac output and plasma volume, the volume remaining at the non-pregnant level. In the clinical phase of pre-eclampsia the typical case picture is one of a vasoconstrictive state with low plasma volume and cardiac output, high blood pressure and systemic vascular resistance in combination with signs of organ damage [proteinuria, hemolysis elevated liver enzymes low platelets (HELLP) syndrome]. Hemodynamic management is necessary in severe disease to prevent maternal complications. Management primarily focuses on pharmacological treatment of blood pressure. Clinicians make educated choices from a limited array of available drugs: beta-receptor antagonists, nifedipine, dihydralazine, methyldopa or ketanserine. Other drugs have restricted use in pregnancy. Management of low circulating volume with plasma expanders remains a subject of controversy.

Blood Pressure↗

PubMed2004

Real-time fluorescence PCR assays for detection and characterization of heat-labile I and heat-stable I enterotoxin genes from enterotoxigenic Escherichia coli.

To facilitate the diagnosis of enterotoxigenic Escherichia coli (ETEC) infections in humans, we developed and evaluated real-time fluorescence PCR assays for the Roche LightCycler (LC) against the enterotoxin genes commonly present in strains associated with human illness. Separate LC-PCR assays with identical cycling conditions were designed for the type I heat-labile enterotoxin (LT I) and the type I heat-stable enterotoxin (ST I) genes, using the LC hybridization probe format. A duplex assay for ST I with two sets of amplification primers and three hybridization probes was required to detect the major nucleotide sequence variants of ST I, ST Ia and ST Ib. LC-PCR findings from the testing of 161 E. coli isolates of human origin (138 ETEC and 23 non-ETEC) were compared with those obtained by block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the LT I and ST I genes. The LC-PCR and block cycler PCR assays were also compared for their abilities to detect LT I and ST I genes in spiked stool specimens with different methods of sample preparation. Findings from these experiments revealed that the limits of detection for the LC-PCR assays were the same or substantially lower than those observed for the block cycler PCR assay. Melting curve analysis of the amplified LT I and ST I genes revealed sequence variation within each gene, which for the ST I genes correlated with the presence of ST Ia and ST Ib. The rapidity, sensitivity, and specificity of the LC-PCR assays make them attractive alternatives to block cycler PCR assays for the detection and characterization of ETEC.

Bacterial Toxins↗

PubMed2004

Affinity measurements of biological molecules by a quartz crystal microbalance (QCM) biosensor.

We present a immunosensing system based on a quartz crystal microbalance (QCM) that allows detection and evaluation of a broad range of molecules relevant to medicine and biology. Soluble analyte is applied by injection into a flow-through chamber where receptor molecules are immobilized, thus enabling real-time measurement of the specific intermolecular interactions. This system can be used for detection purposes, e.g., screening for antibodies, and for determination of affinity constants including affinity evaluation of newly selected or designed aptamers, as kd and ka rates can be derived from the sensograms.

Acoustics↗

PubMed2003

Regulation of transcription by the heterogeneous nuclear ribonucleoprotein E1B-AP5 is mediated by complex formation with the novel bromodomain-containing protein BRD7.

E1B-AP5 was initially identified as a target of the early adenovirus E1B-55 kDa protein during the course of lytic infection. E1B-AP5 belongs to the heterogeneous nuclear ribonucleoprotein family and was demonstrated to be involved in mRNA processing and transport [Gabler, Schutt, Groitl, Wolf, Shenk and Dobner (1998) J. Virol. 72, 7960-7971]. In the present paper, we demonstrate that E1B-AP5 differentially regulates basic and ligand-dependent transcription. We found that E1B-AP5 represses basic transcription driven by several virus and cellular promoters, and mapped the repression activity to the N-terminal part of the protein. In contrast with basic repression, E1B-AP5 activated the glucocorticoid-dependent promoter in the absence of dexamethasone, but did not contribute to the dexamethasone-induced activation. Mutant analysis indicated the presence of an additional cellular factor that modulates E1B-AP5 transcriptional activity. Using yeast two-hybrid screening, we identified a novel chromatin-associated bromodomain-containing protein, BRD7, as an E1B-AP5 interaction partner. We confirmed E1B-AP5-BRD7 complex formation in vivo and in vitro. We found that, although BRD7 binds to histones H2A, H2B, H3 and H4 through its bromodomain, this domain was not necessary for the interaction with E1B-AP5. Indeed, the triple complex formation of E1B-AP5, BRD7 and histones was demonstrated. Disruption of the E1B-AP5-BRD7 complex increased E1B-AP5 repression activity for basic transcription and converted it from being an activator of the hormone-dependent promoter into being a strong repressor. We conclude that complex formation between BRD7 and E1B-AP5 links chromatin events with mRNA processing at the level of transcriptional regulation.

Animals↗

PubMed2003

High-resolution methylation analysis and in vivo protein-DNA binding at the promoter of the viral oncogene LMP2A in B cell lines carrying latent Epstein-Barr virus genomes.

Latency protein LMP2A of Epstein-Barr virus (EBV) has been implicated in EBV related tumorigenesis. To understand the host cell dependent expression of the LMP2A gene, it is necessary to analyse the regulatory mechanisms of the LMP2A promoter (LMP2Ap). By transient transfection and in vitro binding analyses two CBF1 sites have previously been shown to be involved in the regulation of LMP2Ap. However, the promoter structure has not been examined at the nucleotide level in vivo. Therefore we undertook a comprehensive analysis of in vivo protein binding and of CpG-methylation patterns at LMP2Ap in a panel of B cell lines carrying latent EBV genomes. The presence of characteristic footprints on two CBF1 and further binding-sites, together with overall hypomethylation of CpG dinucleotides correlated well with promoter activity. In contrast, the absence of several genomic footprints, as well as the presence of patches of highly methylated CpG dinucleotides were characteristic of silent LMP2Aps.

B-Lymphocytes↗

PubMed2003

The BZLF1 promoter of Epstein-Barr virus is controlled by E box-/HI-motif-binding factors during virus latency.

The BZLF1 open reading frame of Epstein-Barr virus (EBV) encodes an important transactivator of replication. During latency, transcription of this gene is switched off. HI motifs have been shown to cause negative regulation of the promoter. Using yeast one-hybrid assays, we isolated the E box-binding protein, E2-2, interacting with these motifs. Electrophoretic mobility shift assays demonstrated that E2-2 binds to HI alpha, HI beta and HI gamma, which contain E box consensus binding sites. Deletion of the HI-associated E boxes and overexpression of E2-2 in transfection assays revealed that these elements act as repressors in lymphoid cells. In contrast, in epithelial cells they contribute to the increased responsiveness of the promoter to transactivation by the BZLF1 protein. The data presented are in accord with an alternative and exclusive binding of different cell type- and differentiation-specific factors, such as E2-2, to the HI-associated E boxes in lymphoid and epithelial cells. This implies a role in cell type-specific virus replication.

Cell Line↗

PubMed2003

Characterization of Mycobacterium tuberculosis complex DNAs from Egyptian mummies by spoligotyping.

Bone and soft tissue samples from 85 ancient Egyptian mummies were analyzed for the presence of ancient Mycobacterium tuberculosis complex DNA (aDNA) and further characterized by spoligotyping. The specimens were obtained from individuals from different tomb complexes in Thebes West, Upper Egypt, which were used for upper social class burials between the Middle Kingdom (since ca. 2050 BC) and the Late Period (until ca. 500 BC). A total of 25 samples provided a specific positive signal for the amplification of a 123-bp fragment of the repetitive element IS6110, indicating the presence of M. tuberculosis DNA. Further PCR-based tests for the identification of subspecies failed due to lack of specific amplification products in the historic tissue samples. Of these 25 positive specimens, 12 could be successfully characterized by spoligotyping. The spoligotyping signatures were compared to those in an international database. They all show either an M. tuberculosis or an M. africanum pattern, but none revealed an M. bovis-specific pattern. The results from a Middle Kingdom tomb (used exclusively between ca. 2050 and 1650 BC) suggest that these samples bear an M. africanum-type specific spoligotyping signature. The samples from later periods provided patterns typical for M. tuberculosis. This study clearly demonstrates that spoligotyping can be applied to historic tissue samples. In addition, our results do not support the theory that M. tuberculosis originated from the M. bovis type but, rather, suggest that human M. tuberculosis may have originated from a precursor complex probably related to M. africanum.

DNA, Bacterial↗

PubMed2003

The in vivo binding site for oncoprotein c-Myc in the promoter for Epstein-Barr virus (EBV) encoding RNA (EBER) 1 suggests a specific role for EBV in lymphomagenesis.

BACKGROUND: Epstein-Barr virus (EBV) was isolated in the 1960s from the African childhood tumor, Burkitt's Lymphoma (BL), characterized by the translocation of the c-myc gene into one of the immunoglobulin loci. Due to the extreme discrepancy between the widespread dissemination of EBV infection and the overall rarity of EBV-related tumors, it remains an open question whether EBV is really a human tumor virus, and if so, what specific contribution EBV may have to tumorigenesis. MATERIAL/METHODS: Protein binding at the EBER locus of EBV was analyzed by genomic footprinting electrophoretic mobility shift, reporter gene assay, and chromatin immunoprecipitation in a panel of six B-cell lines. RESULTS: Several novel in vivo protein binding sites were found in the EBER locus. Among those, a prominent binding site, 130 base pairs upstream of the EBER1 gene, contains two E-boxes providing a consensus sequence for binding of the transcription factor and oncoprotein c-Myc to the EBV genome. CONCLUSIONS: Based on the discovery of a binding site for c-Myc in the EBV genome, a new molecular model for the specific role of EBV as a causal factor in the origin of endemic Burkitt's Lymphoma is proposed. Translocated and deregulated c-myc directly activates and maintains the antiapoptotic functions of the EBER locus in a single EBV-infected B cell undergoing the germinal center (GC) reaction. With the balance shifted towards cell survival, the oncogenic potential of the pro-apoptotic c-Myc protein is unmasked in the translocated GC cell. This single translocated and surviving cell is the founder cell of an endemic BL. The new model reinstitutes EBV as a real human tumor virus.

Binding Sites↗

PubMed2003

HIV-1 gag expression is quantitatively dependent on the ratio of native and optimized codons.

There is a significant variation of codon usage bias among different species and even among genes within the same organisms. Codon optimization, this is, gene redesigning with the use of codons preferred for the specific expression system, results in improved expression of heterologous genes in bacteria, plants, yeast, mammalian cells, and transgenic animals. The mechanisms preventing expression of genes with rare or low-usage codons at adequate levels are not completely elucidated. Human immunodeficiency virus (HIV) represents an interesting model for studying how differences in codon usage affect gene expression in heterologous systems. Construction of synthetic genes with optimized codons demonstrated that the codon-usage effects might be a major impediment to the efficient expression of HIV gag/pol and env gene products in mammalian cells. According to another hypothesis, the poor expression of HIV structural proteins even without HIV context is attributed to the so-called cis-acting inhibitory elements (INS), which are located within the protein-coding region. They consist of AU-rich sequences and may be inactivated through the introduction of multiple mutations over the large regions of gag gene. In our work, we evaluated expression of hybrid HIV-1 gag mRNAs where wild-type (A-rich) gag sequences were combined with artificial sequences. In such "humanized" gag fragments with adapted codon usage, AT-content was significantly reduced in favor of G and C nucleotides without any changes in protein sequence. We show that wild-type gag sequences negatively influence expression of gag-reporter, and the addition of fragments with optimized codons to gag mRNA partially rescues its expression. The results demonstrate that the expression of HIV-1 gag is determined by the ratio of optimized and rare codons within mRNA. Our data also indicates that some wtgag fragments counteract the influence of the other wtgag sequences, which cause the inhibition of gag expression. The presented data do not contradict the concept of INS; yet, it makes the definition of INS more complex. This supports the idea of a broader role of the selected codon usage in influencing the expression of HIV proteins in mammalian cells.

Codon↗

PubMed2003

Codon usage-mediated inhibition of HIV-1 gag expression in mammalian cells occurs independently of translation.

Codon usage is considered one of the critical factors that limit the expression rate of heterologous genes. Impaired translation efficiency, specifically insufficient amount of corresponding tRNAs and changed startcodon context, are believed to account for the low translation initiation and elongation rates during the protein biosynthesis in unicellular organisms. Translational efficiency is probably not the primary factor influencing codon usage diversity in mammalian cells. However, the other possible mechanisms preventing expression of genes with low-usage such as mRNA stability, processing and nucleocytoplasmic transport, are not adequately explored. In our work, we addressed the question of whether codon usage differences affect exclusively translational efficiency of mammalian gene products. We demonstrated that the CMV-induced expression of gag-reporter in human H1299 cell line was influenced by the nucleotide composition of the mRNA, and the limitation of gag expression appeared to be inversely related to the level of codon optimization. However, cytoplasmic expression of the gag-reporter driven by vaccinia virus/T7 RNA polymerase hybrid system rescued its expression independently of HIV-1 gag mRNA nucleotide content. We concluded that impaired HIV-1 gag expression may be caused by translation-independent mechanisms, which probably play a major role in codon usage-mediated defects in heterologous gene expression in mammalian cells.

Codon↗

PubMed2003

Technical evaluation of cDNA microarrays.

The aims were to evaluate the common reference design approach in microarray experiments and to evaluate the technical performance and the normalisation of cDNA microarrays with a limited number of spots. Total RNA from 3 normal and 3 tumour sample biopsies were used for synthesis of amino-allyl labelled cRNA. Equal amounts of cRNA from all samples were mixed as reference. After conjugation of cRNA with fluorophores (Cy3/Cy5), each individual tumour cRNA was hybridised to a cDNA microarray together with reference cRNA, scanned and analysed. We show that our procedures for producing cDNA microarrays are reproducible. The concordance between duplicated spots and replicate hybridisation was found to be high. We have demonstrated that our cDNA microarrays are of a high technical quality. The majority of the cDNA microarrays had low local spot background levels. Despite the high background levels for some local spots, variation could be minimized by locally weighted scatter plot smooth normalisation (LOWESS), which we showed was also suitable for normalisation of cDNA microarrays with a limited number of probes.

DNA, Complementary↗

PubMed2003

Co-influence of transgene expression in mammalian cells. Mutual influence of transgenes on their expression in mammalian cells.

It becomes increasingly clear that therapeutic gene delivery should provide not only for the sustained high level of gene expression but also, in most cases, for the regulated expression of transgenes as much as it occurs under natural conditions. Over the past few years a variety of different systems have been developed in order to regulate the amounts of transcribed RNA upon administration of exogenous agents, or in autoregulated manner. While efforts were focused on optimizing gene expression at the transcriptional level, other levels are still overlooked. In the meantime, regulation of gene expression is not restricted to transcription, but is also executed at the post-transcriptional level, i.e. mRNA stability, processing, transport, translation, protein stability, and modification. Codon usage is considered to be one of the critical factors that limit the expression rate of heterologous genes in different organisms at the posttranscriptional level. HIV-1 structural genes gag, pol, and env represent one of the most extensively utilized models for studying codon usage-mediated effects on transgene expression. In the current work we demonstrate that the codon content affects not only CMV-driven HIV-1 gag expression but also the expression of luciferase reporter gene transcribed independently from the SV40 promoter. The expression levels of both transgenes co-transfected into the human H1299 were inversely co-dependent. The observed phenomenon may be described as sequence-independent post-transcriptional gene silencing, which reflects the existing limitation of transgene expression in mammalian cells at the post-transcriptional level. Optimization of the codon usage may provide for the additional level of regulation of transgene expression in gene transfer experiments in order to maintain the concentration of the protein at the therapeutic levels.

Cell Line, Tumor↗

PubMed2002

Identifying distinct classes of bladder carcinoma using microarrays.

Bladder cancer is a common malignant disease characterized by frequent recurrences. The stage of disease at diagnosis and the presence of surrounding carcinoma in situ are important in determining the disease course of an affected individual. Despite considerable effort, no accepted immunohistological or molecular markers have been identified to define clinically relevant subsets of bladder cancer. Here we report the identification of clinically relevant subclasses of bladder carcinoma using expression microarray analysis of 40 well characterized bladder tumors. Hierarchical cluster analysis identified three major stages, Ta, T1 and T2-4, with the Ta tumors further classified into subgroups. We built a 32-gene molecular classifier using a cross-validation approach that was able to classify benign and muscle-invasive tumors with close correlation to pathological staging in an independent test set of 68 tumors. The classifier provided new predictive information on disease progression in Ta tumors compared with conventional staging (P < 0.005). To delineate non-recurring Ta tumors from frequently recurring Ta tumors, we analyzed expression patterns in 31 tumors by applying a supervised learning classification methodology, which classified 75% of the samples correctly (P < 0.006). Furthermore, gene expression profiles characterizing each stage and subtype identified their biological properties, producing new potential targets for therapy.

Disease Progression↗

PubMed2002

An aptamer-based quartz crystal protein biosensor.

We developed a quartz crystal biosensor designed to detect concentrations and ligand affinity parameters of free unlabeled proteins in real time. Using a model system with human IgE as the analyte and single-stranded DNA aptamers or an anti-IgE antibody as immobilized ligands, we could demonstrate that aptamers were equivalent to antibodies in terms of specificity and sensitivity. Both receptor types selectively detected 0.5 nmol/L of IgE. In addition, the aptamer receptors tolerated repeated affine layer regeneration after ligand binding and recycling of the biosensor with little loss of sensitivity. Because of the small size and nonprotein nature of the aptamers, they were immobilized in a dense, well-oriented manner, thus extending the linear detection range to 10-fold higher concentrations of IgE. In addition to demonstrating for the first time that an aptamer-based biosensor can specifically and quantitatively detect an analyte in various complex protein mixes, the aptamer-ligand proved to be relatively heat resistant and stable over several weeks. Since aptamers consist of nucleic acids, well-established chemistry can be applied to produce optimized affine layers on biosensors that may be developed to specifically detect proteins in solution for analysis of proteomes.

Antibodies, Anti-Idiotypic↗

PubMed2002

Molecular analysis of ancient microbial infections.

The detection of ancient microbial DNA offers a new approach for the study of infectious diseases, their occurrence, frequency and host-pathogen interaction in historic times and populations. Moreover, data obtained from skeletal and mummified tissue may represent an important completion of contemporary phylogenetic analyses of pathogens. In the last few years, a variety of bacterial, protozoal and viral infections have been detected in ancient tissue samples by amplification and characterization of specific DNA fragments. This holds particularly true for the identification of the Mycobacterium tuberculosis complex, which seems to be more robust than other microbes due to its waxy, hydrophobic and lipid-rich cell wall. These observations provided useful information about the occurrence, but also the frequency of tuberculosis in former populations. Moreover, these studies suggest new evolutionary models and indicate the route of transmission between human and animals. Until now, other pathogens, such as Mycobacterium leprae, Yersinia pestis, Plasmodium falciparum and others, have occasionally been identified - mostly in single case studies or small sample sizes - as well, although much less information is available on these pathogens in ancient settings. The main reason therefore seems to be the degradation and modification of ancient DNA by progressive oxidative damage. Furthermore, the constant risk of contamination by recent DNA forces to take time and cost effective measures and renders the analysis of ancient microbes difficult. Nevertheless, the study of microbial ancient DNA significantly contributes to the understanding of transmission and spread of infectious diseases, and potentially to the evolution and phylogenetic pathways of pathogens.

Animals↗

PubMed2002

Muscle specific versus ubiquitous expression of Gag based HIV-1 DNA vaccines: a comparative analysis.

Most studies on DNA-based immunization have used viral promoters to drive antigen expression. In this study, we analyzed the properties of the commonly used CMV promoter, the tissue specific murine muscle creatine kinase (MCK) promoter as well as a hybrid MCK/CMV promoter regarding promoter activity and tissue specificity in vitro. Furthermore, the efficiency of inducing HIV-1 Gag specific immune responses in vivo following intramuscular immunization of naked DNA containing a codon optimized synthetic gene was compared. Although antibody titers and cellular immune responses using the MCK construct were slightly reduced as compared to conventional CMV based vector modules, the utilization of nonviral promoters may add significantly to the safety of future DNA vaccines.

AIDS Vaccines↗

PubMed2002

Efficiency of a myogenic DNA vaccine is strictly dependent upon cellular localization of HIV-1 Pr55(gag).

Most studies on DNA-based immunization have used viral promoters to drive antigen expression. Recently, the use of tissue-specific DNA vaccines has been favored regarding safety issues. In this study, we determined the impact of antigen localization and tissue-specific expression on the induction of humoral as well as cellular immune responses in a BALB/c mouse model. Thereby, we show that using the muscle-specific muscle creatine kinase (MCK) promoter/enhancer the efficiency of immune stimulation is strictly dependent on the ability of HIV-1 Pr55(gag) to be released from cells. By contrast, localization of Pr55(gag) and derivatives thereof plays only a minor role when antigen is constitutively expressed using the ubiquitous viral CMV promoter.

AIDS Vaccines↗