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Biomedical subjects

U Lauer

Publications and source records attributed to U Lauer.

At least 19 recordsLinked to original sources
PubMed2000

Physical map of the chromosome of the apple proliferation phytoplasma.

A physical map of the apple proliferation phytoplasma strain AT chromosome was constructed from genomic DNA extracted from diseased tobacco plants. The map was generated with single and double digestions of the chromosome with BssHII, SmaI, MluI, and ApaI restriction endonucleases and resolving the fragments by pulsed-field gel electrophoresis. Partial digestion and Southern blot analysis were used to assist in the arrangement of the 14 contiguous restriction fragments obtained. From the restriction fragments generated by double digestions, the size of the circular chromosome was calculated to be approximately 645 kb. Locations of the two rRNA operons, the operon including the fus and tuf genes, and three other genes were placed on the map. Genome sizes and BssHII restriction profiles of apple proliferation strain AP15 and the pear decline and European stone fruit yellows phytoplasmas were different from that of strain AT.

Chromosomes, Bacterial↗
PubMed1999

Sendai virus infection induces apoptosis through activation of caspase-8 (FLICE) and caspase-3 (CPP32).

Sendai virus (SV) infection and replication lead to a strong cytopathic effect with subsequent death of host cells. We now show that SV infection triggers an apoptotic program in target cells. Incubation of infected cells with the peptide inhibitor z-VAD-fmk abrogated SV-induced apoptosis, indicating that proteases of the caspase family were involved. Moreover, proteolytic activation of two distinct caspases, CPP32/caspase-3 and, as shown for the first time in virus-infected cells, FLICE/caspase-8, could be detected. So far, activation of FLICE/caspase-8 has been described in apoptosis triggered by death receptors, including CD95 and tumor necrosis factor (TNF)-R1. In contrast, we could show that SV-induced apoptosis did not require TNF or CD95 ligand. We further found that apoptosis of infected cells did not influence the maturation and budding of SV progeny. In conclusion, SV-induced cell injury is mediated by CD95- and TNF-R1-independent activation of caspases, leading to the death of host cells without impairment of the viral life cycle.

Animals↗
PubMed1998

Magnetic cell sorting for parietal cell purification using a new monoclonal antibody without influence on cell function.

Conventional isolation protocols for the parietal type of the gastric mucosal cell population are based on physical criteria such as cell size and cell density. Time-consuming centrifugation techniques are required, which decrease the overall yield and exert a negative influence on the functional integrity of purified cells. As an alternative, we now have developed a phenotype-dependent magnetic selection method. A monoclonal antibody (P1.4D5) was raised against rat parietal cells. After immunohistochemical, biochemical and functional characterization, P1.4D5 was used for rapid antibody-mediated magnetic cell sorting (MACS) of enzymatically dispersed gastric mucosal cells, which resulted in a 2.2- to 3-fold enrichment of parietal cells. Characterization of P1.4D5 demonstrated its applicability for parietal cell-specific immunostaining of formalin treated and paraffin-embedded tissue samples and cryosections from both rat and man. In vitro studies of the 14C-aminopyrine accumulation showed no intrinsic effect of P1.4D5 on the acid production of the parietal cell or on the histamine-stimulated acid production. This absence of negative influence on the parietal cell function qualifies P1.4D5 for immunomagnetic cell sorting procedures.

Aminopyrine↗
PubMed1998

Sendai virus-like particles devoid of haemagglutinin-neuraminidase protein infect cells via the human asialoglycoprotein receptor.

Virus-like particles with genetically defined envelope proteins were generated from cDNA in order to examine the requirement of Sendai virus haemagglutinin-neuraminidase (HN) protein for particle formation, and the role of fusion protein (F) in receptor binding and membrane fusion. Characterization of particles devoid of HN protein showed that particle formation was unimpaired by the absence of HN protein, indicating that HN protein is dispensable for virus assembly and budding. Infection studies further demonstrated that virus adsorption and penetration can be mediated solely by the F protein when the human asialoglycoprotein receptor is present at the surface of host cells.

3T3 Cells↗
PubMed1998

Pseudotype formation of Moloney murine leukemia virus with Sendai virus glycoprotein F.

Mixed infection of cells with both Moloney murine leukemia virus (MoMLV) and related or heterologous viruses produces progeny pseudotype virions bearing the MoMLV genome encapsulated by the envelope of the other virus. In this study, pseudotype formation between MoMLV and the prototype parainfluenza virus Sendai virus (SV) was investigated. We report for the first time that SV infection of MoMLV producer cells results in the formation of MoMLV(SV) pseudotypes, which display a largely extended host range compared to that of MoMLV particles. This could be associated with SV hemagglutinin-neuraminidase (SV-HN) glycoprotein incorporation into MoMLV envelopes. In contrast, solitary incorporation of the other SV glycoprotein, SV fusion protein (SV-F), resulted in a distinct and narrow extension of the MoMLV host range to asialoglycoprotein receptor (ASGP-R)-positive cells (e.g., cultured human hepatoma cells). Since stably ASGP-R cDNA-transfected MDCK cells, but not parental ASGP-R-negative MDCK cells, were found to be transduced by MoMLV(SV-F) pseudotypes and transduction of ASGP-R-expressing cells was found to be inhibited by ASGP-R antiserum, a direct proof for the ASGP-R-restricted tropism of MoMLV(SV-F) pseudotypes was provided. Cultivation of ASGP-R-positive HepG2 hepatoma cells on Transwell-COL membranes led to a significant enhancement of MoMLV(SV-F) titers in subsequent flowthrough transduction experiments, thereby suggesting the importance of ASGP-R accessibility at the basolateral domain for MoMLV(SV-F) pseudotype transduction. The availability of such ASGP-R-restricted MoMLV(SV-F)-pseudotyped vectors opens up new perspectives for future liver-restricted therapeutic gene transfer applications.

Animals↗
PubMed1997

Shock wave permeabilization as a new gene transfer method.

Uptake of naked functional DNA into mammalian cells can be achieved by a number of physical methods. However, for most of these techniques possibilities for therapeutic in vivo applications--especially to solid organs--are often limited. In this report, we describe shock wave permeabilization as a new physical gene transfer method, which can be easily applied, provides great flexibility in the size and sequence of the DNA molecules to be delivered, and which should exhibit an advantageous security profile in vivo. Upon exposure to lithotripter-generated shock waves eukaryotic cells display a temporary increase in membrane permeability. This effect was shown to be caused by cavitation resulting in the transient generation of cell pores which allows the direct transfer of naked plasmid DNA. Shockwave transfection of a variety of cell lines was demonstrated. Since shock waves can be well focused within particular body regions, future applications of extracorporally generated shock waves to tissues simultaneously perfused with DNA solutions might open up the possibility of achieving a regionally enhanced in vivo gene transfer.

Animals↗
PubMed1997

Sendai virus efficiently infects cells via the asialoglycoprotein receptor and requires the presence of cleaved F0 precursor proteins for this alternative route of cell entry.

Biochemical evidence suggests that the asialoglycoprotein receptor (ASGP-R) can be used as an alternative receptor for a temperature-sensitive Sendai virus (SV) mutant. We now have investigated this possible alternative route of infection for SV wild-type (SV-wt) strain Fushimi by using a pair of cell lines which differ only with regard to ASGP-R expression. Infection studies after enzymatic destruction of conventional sialic acid-containing SV receptors (SA-R) revealed that only ASGP-R-expressing cells could be infected by SV-wt. This alternative route of cell entry could be completely blocked by incubation of cells with ASGP-R-specific antibodies prior to infection. Furthermore, cleavage of SV-F0 precursor protein into the subunits F1 and F2 was necessary to establish infection via ASGP-R, suggesting a fusion-mediated cell entry after binding of SV-wt to the ASGP-R on host cells. Interestingly, infection via ASGP-R was found to be nearly as efficient as infection via conventional sialic acid-containing SV receptors. A possible physiological role of the ASGP-R-mediated route of SV infection is discussed.

3T3 Cells↗
PubMed1996

Hepatoma-derived integrated HBV DNA causes multi-stage transformation in vitro.

The hepatoma-derived hepatitis B virus (HBV) DNA insert HU-a has recently been shown to contain two viral transactivator genes, X and preS2 /S. We report here that HU-a induces malignant transformation after stable transfection of the fetal mouse hepatocyte line FMH202, as indicated by soft agar growth and nude mouse tumorigenicity. Transfections with HU-a subclones, containing the X gene of the preS2 /S gene alone or sequences without transactivator gene, respectively, suggested that the X gene is essential for transformation. Sequential stages of transformation and tumor progression were analysed by injection of the stably transfected FMH202 lines into nude mice, explanation of the resulting tumors and re-establishment of cell lines from the tumors. Comparison of two HU-a-transformed cell lines by HBV mRNA hybridization, Southern analysis and chromosomal in situ hybridization revealed that integrated HBV DNAs were involved in major chromosomal rearrangements in both cases. Interestingly, recombination of the HBV Dna insert during the nude mouse passage had completely abolished HBV-specific transcription in one case, indicating that expression of integrated HBV genes, while presumably involved in early transformation, is dispensable at later stages of tumor progression. The sequential transformation observed in this experimental system suggests that expression of the X gene by integrated viral DNA and subsequent hepatocyte genome mutations might both contribute to HBV-associated liver carcinogenesis.

Animals↗
PubMed1996

Keratinocyte growth factor is highly overexpressed in inflammatory bowel disease.

Recently we demonstrated an important function of keratinocyte growth factor (KGF) in wound re-epithelialization. As KGF is mitogenic for various epithelial cells, we speculated about a role of KGF in epithelial repair processes of other organs as seen in a variety of inflammatory diseases. Here we demonstrate a strikingly increased expression of KGF in surgical specimens from patients suffering from Crohn's disease and ulcerative colitis. The levels of KGF expression strongly correlated with the degree of inflammation as assessed by histological analysis of adjacent tissue and expression analysis of the pro-inflammatory cytokine interleukin-1 beta. The highest levels of KGF mRNA and protein were found in mesenchymal cells of the lamina propria, particularly in highly inflamed areas. As the KGF receptor is expressed in intestinal epithelial cells, KGF seems to act in a paracrine manner to stimulate proliferation of these cells. These data suggest a crucial role of KGF in epithelial repair after injury caused by inflammatory processes.

Blotting, Western↗
PubMed1994

The hepatitis B virus preS2/St transactivator utilizes AP-1 and other transcription factors for transactivation.

Integrated hepatitis B virus DNA cloned from hepatitis B virus-associated hepatocellular carcinoma frequently contains 3'-truncated middle surface genes (preS2/St), which were recently found to have a transcriptional transactivator function. Because preS2/St, among others, is able to transactivate the promoters of the cellular oncogenes c-myc and c-fos, it has been speculated that integrated preS2/St genes might contribute to hepatitis B virus-associated liver carcinogenesis. In this study, we investigated the mechanism of target gene stimulation by preS2/St. It was found that deletion of a fragment containing the binding site for transcription factor AP-1 (Jun-Fos) substantially decreases inducibility of the human c-myc promoter by preS2/St. A subsequent investigation of AP-1 activation by preS2/St revealed the following: (a) insertion of multimeric AP-1 binding sites confers inducibility to an otherwise unstimulatable test promoter; (b) transactivation of AP-1 sites is dramatically increased when Jun and Fos are overexpressed by cotransfected expression plasmids; and (c) inhibitors of AP-1 activation also impair transactivation by preS2/St. Besides AP-1, preS2/St was also able to utilize the unrelated transcription factors NF-kappa B and AP-2 for transactivation, suggesting that the gene product of preS2/St acts indirectly through one or several general cellular pathways rather than as a bona fide transcription factor. Because AP-1 conveys induction of a large panel of tumor-relevant genes, its preS2/St-dependent activation implies a possible causative role in hepatitis B virus-associated hepatocarcinogenesis.

Animals↗
PubMed1993

Hepatitis B virus transactivator HBx uses a tumour promoter signalling pathway.

The hepatitis B virus (HBV) transactivator protein HBx is enigmatic in that it stimulates a striking variety of promoters which do not share a common cis-regulatory element. As it does not bind to DNA, it has been speculated that HBx acts indirectly through cellular pathways. Under certain conditions HBx can have an oncogenic potential, which may be relevant for HBV-associated liver carcinogenesis, but until now the mechanism for transactivation and cell transformation by HBx was unclear. We report here that HBx uses a complex signal transduction pathway for transactivation. An increase in the endogenous protein kinase C (PKC) activator sn-1,2-diacylglycerol and the subsequent activation of PKC give rise to activation of the transcription factor AP-1 (Jun-Fos). As a result, HBx transactivates through binding sites for AP-1 and other PKC-dependent transcription factors (AP-2, NF-kappa B), thereby explaining the as-yet incomprehensible variety of HBx-inducible genes. As the PKC signal cascade also mediates cell transformation by tumour-promoting agents, the mechanism presented here might account for the oncogenic potential of HBx.

Animals↗
PubMed1993

ER-localization and functional expression of the HBV transactivator MHBst.

In two recently reported cases, integrated hepatitis B virus (HBV) DNAs cloned from hepatocellular carcinoma were found to express a transcriptional transactivator from 3'-terminally truncated HBV surface (preS/S) genes. In this study, we characterized the transactivator at the protein level. Expression of a 3'-truncated preS2/S gene in Spodoptera frugiperda (Sf9) insect cells resulted in a C-terminally truncated middle surface protein of 76 amino acids (MHBst76), which was found to be associated with membranes of the endoplasmic reticulum and retained from Golgi processing and secretion. Accordingly, the microsome fraction of MHBst76-expressing Sf9 cells displayed transactivator activity after electric field-mediated transfer into Chang liver cells. In contrast to full-length MHBs, MHBst76 is unglycosylated, and glycosylation is not required for transactivation as shown by mutation of the glycosylation site at asparagine-4. Since highly purified MHBst76 derived from an E. coli expression system also showed transactivator activity, it is concluded that unglycosylated MHBst76 protein is the authentic transactivating factor. As the transactivator protein derives from inactive MHbs by rearrangements of integrated HBV DNA, it may be important for HBV-associated liver carcinogenesis.

Amino Acids↗
PubMed1992

The hepatitis B virus pre-S/S(t) transactivator is generated by 3' truncations within a defined region of the S gene.

Recently, it was reported that 3' truncation of an integrated surface gene (pre-S2/S) cloned from a hepatitis B virus (HBV)-associated hepatoma gave rise to the generation of a C-terminally truncated middle surface protein (MHBst), which surprisingly exerted a transcriptional transactivator function. To define the molecular requirements for the generation of transactivating MHBs(t) proteins, a 3' deletion analysis of the HBV pre-S2/S gene was performed. In cotransfection experiments with reporter plasmid pSV2CAT, full-length pre-S2/S genes or pre-S2/S genes with minor 3'-terminal deletions did not exhibit transactivator activity. In contrast, deletion of C-terminal hydrophobic region III of the S domain generated the transactivator function. Further stepwise 3' deletions, removing hydrophobic region II and both hydrophilic regions of the S domain, did not interfere with the transactivator activity; it was completely abolished, however, after additional deletion of hydrophobic region I. The results of this study define a range within the S open reading frame (between HBV nucleotides 221 and 573), termed the trans-activity-on region, in which 3' deletions give rise to the generation of transactivating MHBs(t) proteins. Within this region, not only 3' deletions but also the introduction of a stop codon activated the transactivator function, indicating that point mutations of integrated HBV DNA also may give rise to the synthesis of transactivating MHBs(t) proteins in vivo.

Animals↗
PubMed1991

Monoclonal antibodies EBU-141 (CDw75) and EBU-65 allow reliable distinction between mature and pre-B-cell tumors in suspension and on tissue sections.

Two new monoclonal antibodies, EBU-65 and EBU-141, were raised by immunization with plasma cell line U-266. Both antibodies strongly react with B lymphocytes in immunofluorescent staining as well as on paraffin-embedded sections. More than 200 leukemias and lymphomas were tested, and for both antibodies reactivity was found only with "mature" B-cell tumors but not with precursor B-cell leukemias. None of the non-B-lineage hematolymphatic tumors tested was stained by EBU-141 or EBU-65. A subpopulation of T lymphocytes particularly present in nonmalignant pleural effusions was detected by EBU-65 additionally. Although EBU-141 was clustered as CDw75 and EBU-65 as "unique," a close relationship of the staining pattern was found and both antibodies react with a sialyltransferase. In particular, CDw75 antibody EBU-141 was demonstrated to be very useful for immunophenotyping of B-cell neoplasias, while EBU-65 reacted with most multiple myelomas and a subgroup of "activated"-appearing T cells.

Animals↗
PubMed1991

Antibiotics from basidiomycetes. XXXVIII. 2-Methoxy-5-methyl-1,4-benzoquinone, a thromboxane A2 receptor antagonist from Lentinus adhaerens.

In the course of our screening for antithrombotic compounds using platelet rich plasma from bovine slaughter blood, 2-methoxy-5-methyl-1,4-benzoquinone (1) has been isolated from mycelial cultures of Lentinus adhaerens. The compound inhibits the U46619-induced aggregation of human blood platelets with an IC50 of 2.5 micrograms/ml (16.45 microM) and is a new thromboxane A2 receptor antagonist. This is the first report on an inhibitor of platelet aggregation derived from secondary metabolism of basidiomycetes.

Animals↗