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A Scheid

Publications and source records attributed to A Scheid.

At least 19 recordsLinked to original sources
PubMed2001

The sequence complementarity between HIV-1 5' splice site SD4 and U1 snRNA determines the steady-state level of an unstable env pre-mRNA.

HIV-1 env expression from certain subgenomic vectors requires the viral regulatory protein Rev, its target sequence RRE, and a 5' splice site upstream of the env open reading frame. To determine the role of this splice site in the 5'-splice-site-dependent Rev-mediated env gene expression, we have subjected the HIV-1 5' splice site, SD4, to a mutational analysis and have analyzed the effect of those mutations on env expression. The results demonstrate that the overall strength of hydrogen bonding between the 5' splice site, SD4, and the free 5' end of the U1 snRNA correlates with env expression efficiency, as long as env expression is suboptimal, and that a continuous stretch of 14 hydrogen bonds can lead to full env expression, as a result of stabilizing the pre-mRNA. The U1 snRNA-mediated stabilization is independent of functional splicing, as a mismatch in position +1 of the 5' splice site that led to loss of detectable amounts of spliced transcripts did not preclude stabilization and expression of the unspliced env mRNA, provided that Rev enables its nuclear export. The nucleotides capable of participating in U1 snRNA:pre-mRNA interaction include positions -3 to +8 of the 5' splice site and all 11 nt constituting the single-stranded 5' end of U1 snRNA. Moreover, env gene expression is significantly decreased upon the introduction of point mutations in several upstream GAR nucleotide motifs, which are mediating SF2/ASF responsiveness in an in vitro splicing assay. This suggests that the GAR sequences may play a role in stabilizing the pre-mRNA by sequestering U1 snRNP to SD4.

Base Pair Mismatch↗
PubMed2000

Natural variation in the amino acid sequence around the HIV type 1 glycoprotein 160 cleavage site and its effect on cleavability, subunit association, and membrane fusion.

To assess the natural variation of the structure of the cleavage site as well as the N-terminal region of gp41 for the cytopathogenicity of HIV-1, syncytium-inducing (SI) and non-syncytium-inducing (NSI) virus isolates were obtained from HIV-1-infected patients. In addition, the coreceptor usage of the isolates was determined by infection of primary macrophages and PM-1 cells. DNA sequences encoding the C-terminal 41 amino acid residues of gp120 and the 64 amino acid N-terminal residues of gp41 were amplified by the polymerase chain reaction and inserted into the Env expression vector pNLA1. When transfected into HeLa-T4(+) cells, all the recombinant plasmids, including those with inserts from NSI isolates, led to the formation of processed glycoprotein and to syncytium formation. One construct displayed significant lowered fusion capacity and had an amino acid exchange in the first position of the gp41 N terminus (gp41, 512A-->S) leading to a decreased association of the SU and TM subunits. Four constructs derived from two isolates of the same patient showed an unusual gp41 N terminus (gp41, 514G-->P) and a slightly diminished fusion capacity due to a decreased cleavability. This indicates that the major determinants for the SI and NSI phenotypes are not located around the gp160 cleavage site and that the N terminus of gp41 plays a minor role in the processing and fusion capacity of wild-type HIV-1 isolates.

Amino Acid Sequence↗
PubMed2000

Epolones induce erythropoietin expression via hypoxia-inducible factor-1 alpha activation.

Induction of erythropoietin (Epo) expression under hypoxic conditions is mediated by the heterodimeric hypoxia-inducible factor (HIF)-1. Following binding to the 3' hypoxia-response element (HRE) of the Epo gene, HIF-1 markedly enhances Epo transcription. To facilitate the search for HIF-1 (ant)agonists, a hypoxia-reporter cell line (termed HRCHO5) was constructed containing a stably integrated luciferase gene under the control of triplicated heterologous HREs. Among various agents tested, we identified a class of substances called epolones, which induced HRE-dependent reporter gene activity in HRCHO5 cells. Epolones are fungal products known to induce Epo expression in hepatoma cells. We found that epolones (optimal concentration 4-8 micromol/L) potently induce HIF-1 alpha protein accumulation and nuclear translocation as well as HIF-1 DNA binding and reporter gene transactivation. Interestingly, the activity of a compound related to the fungal epolones, ciclopirox olamine (CPX), was blocked after addition of ferrous iron. This suggests that CPX might interfere with the putative heme oxygen sensor, as has been proposed for the iron chelator deferoxamine mesylate (DFX). However, about 10-fold higher concentrations of DFX (50-100 micromol/L) than CPX were required to maximally induce reporter gene activity in HRCHO5 cells. Moreover, structural, functional, and spectrophotometric data imply a chelator:iron stoichiometry of 1:1 for DFX but 3:1 for CPX. Because the iron concentration in the cell culture medium was determined to be 16 micromol/L, DFX but not CPX function can be explained by complete chelation of medium iron. These results suggest that the lipophilic epolones might induce HIF-1 alpha by intracellular iron chelation. (Blood. 2000;96:1558-1565)

Animals↗
PubMed2000

Hypoxia-regulated gene expression in fetal wound regeneration and adult wound repair.

Fetal skin wounds heal scarlessly while adult wounds scar. Fetal wound healing occurs in a physiologically hypoxic environment whereas in adult wound healing, cells have to acutely adapt to hypoxia caused by locally impaired blood supply. We examined the expression of hypoxia-inducible factor 1 (HIF-1), a potent transcriptional regulator of oxygen-dependent genes such as vascular endothelial growth factor (VEGF), and transforming growth factor-beta (TGF-beta), a potentially HIF-1-regulated scarring cytokine, on fetal and adult responses to wounding. Incisional skin wounds were created in four sheep fetuses (twins served as controls) and two ewes at 100 days of gestation (term = 150 days). Fetal and adult wounds as well as non-wounded control tissues were harvested 2 days post-wounding. Intraoperative arterial blood gas analyses and invasive subcutaneous pO2 measurements revealed that the fetuses were indeed hypoxic while the mothers were normoxic. Expression patterns of HIF-1alpha were investigated by Western blot analyses. HIF-1alpha expression in fetal wounds and fetal control skin was similar, whereas HIF-1alpha was only detected in adult wounds but not in adult control skin. Exposure of cultured fetal and adult dermal fibroblasts to hypoxia (1% O2) showed a marked induction of VEGF mRNA. In contrast, exposure of these cell types to hypoxia did not significantly affect TGF-beta1 mRNA expression in comparison to their normoxic controls. The presence of HIF-1alpha in fetal but not in adult normal skin indicates that HIF-1alpha might be involved in fetal skin development. Conversely, the upregulation of HIF-1alpha in adult but not early fetal wound repair might represent a pathway in the pathogenesis of scarring, since several growth factors overexpressed in, and associated, with scarring are hypoxia-inducible. Further studies need to be performed in order to identify hypoxia-regulated HIF-1alpha target genes involved in the pathogenesis of scarring.

Age Factors↗
PubMed2000

Genetically modified mouse models in studies on cutaneous wound healing.

Genetically modified mice have become an invaluable tool in modern biomedical and basic research. This review provides an overview of knockout and transgenic mice studied with regard to their cutaneous wound healing properties. In addition, several gene transfer studies are briefly introduced, which have further highlighted our knowledge on individual gene function in wound healing.

Animals↗
PubMed1999

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Journal Article↗
PubMed1998

Congenital infections with human herpesvirus 6.

Cord blood DNA was tested for the presence of human herpesvirus 6 (HHV-6) DNA by the polymerase chain reaction. Specific DNA could be detected in the specimens of 5 (1.6%) of 305 babies born to ostensibly healthy mothers, indicating that intrauterine infection had occurred. These transmissions would not have been detected by serologic methods, because no specific IgM antibody could be found in the fetal sera. These results indicate that, in addition to infections acquired in early childhood, congenital infections may account for the HHV-6 seropositivity in children.

Antibodies, Viral↗
PubMed1997

Exon 1 leader sequences downstream of U5 are important for efficient human immunodeficiency virus type 1 gene expression.

In previous studies, little attention has been paid to maintaining the native HIV-1 leader sequence in reporter constructs analyzing the human immunodeficiency virus type 1 (HIV-1) promoter activity. To investigate a possible influence of the leader sequence on HIV-1-driven gene expression in the presence as well as in the absence of Tat, an expression vector was designed for transcripts consisting of the native HIV-1 tat 1.4 mRNA leader followed by the open reading frame for the bacterial chloramphenicol acetyltransferase (CAT). Deletion mutants with mutations within the leader sequence downstream of U5 (lsdU5) were constructed, as well as a mutant containing a mutation with a reverse orientation of this region. Quantification of CAT protein in HeLa-T4+ cells transiently transfected with wild-type and mutant leader constructs showed that the exon 1-derived lsdU5 region has an influence on basal as well as Tat-induced protein expression. The dramatic decrease in the level of CAT protein upon deletion of lsdU5 was paralleled by a drop in the steady-state level of CAT mRNA. Deletion of the exon 1-derived lsdU5 region also decreased the expression of mRNAs containing authentic HIV-1 sequences instead of CAT. The effect observed with the reporter constructs was not due to the loss of binding sites for nuclear factors, as could be shown with DBF1 and Sp1 mutant constructs. Nuclear run-on transcription assays showed that the presence or absence of lsdU5 did not influence the rate of transcription. This indicates that the exon 1 lsdU5 element functions at the posttranscriptional level in the processing, nucleocytoplasmic export, or stabilization of HIV-1 transcripts.

Binding Sites↗
PubMed1996

Lipid membrane fusion induced by the human immunodeficiency virus type 1 gp41 N-terminal extremity is determined by its orientation in the lipid bilayer.

The amino-terminal extremity of the human immunodeficiency virus type 1 transmembrane protein (gp41) is thought to play a pivotal role in the fusion of virus membranes with the plasma membrane of the target cell and in syncytium formation. Peptides with sequences taken from the human immunodeficiency virus type 1 gp41 fusogenic (synthetic peptides SPwt and SP-2) and nonfusogenic (SP-3 and SP-4) glycoproteins adopt mainly a beta-sheet conformation in the absence of lipid, as determined by attenuated total reflection Fourier transform infrared spectroscopy, and after interaction with large unilamellar liposomes, the beta-sheet is partly converted into an alpha-helical conformation. Peptides SPwt and SP-2 but not SP-3 or SP-4 were able to promote lipid mixing as assessed by fluorescence energy transfer assay and dye leakage in a vesicle leakage assay. By using polarized attenuated total reflection Fourier transform infrared spectroscopy, SPwt and SP-2 were found to adopt an oblique orientation in the lipid membrane whereas SP-3 and SP-4 were oriented nearly parallel to the plane of the membrane. These findings confirm the correlation between the membrane orientation of the alpha-helix and the lipid mixing ability in vitro. Interestingly, the data provide a direct correlation with the fusogenic activity of the parent glycoproteins in vivo.

Amino Acid Sequence↗
PubMed1996

Rapid tracheal infusion of surfactant versus bolus instillation in rabbits: effects on oxygenation, blood pressure and surfactant distribution.

UNLABELLED: Surfactant bolus instillation may be associated with a drop in blood pressure. Platelet-activating factor (PAF) has been found in surfactant preparations. The aim of this study was to evaluate rapid tracheal infusion of surfactant during 5 min as an alternative to bolus instillation and to examine whether a PAF receptor antagonist is able to prevent the decrease in blood pressure. METHODS: Surfactant deficiency was induced in 16 adult rabbits by lung lavages with saline. Six animals received a bolus of a porcine surfactant preparation (Curosurf (CS); 200 mg/kg), labeled with red microspheres to assess pulmonary distribution. In another 5 rabbits, the same amount of labelled CS was instilled by tracheal infusion within 5 min. A third group of 5 animals received 3 mg/kg body weight of the PAF antagonist WEB 2170 before CS bolus instillation. RESULTS: After CS bolus administration, mean PaO2 increased by 44.7 +/- 8.3 kPa (mean +/- SD) within 2 min and remained at this level. Mean arterial blood pressure dropped transiently by 2.3 +/- 2 kPa within 5 min. Pulmonary distribution of surfactant was even. After infusion, mean PaO2 rose by 22.4 +/- 16.3 kPa within 15 min. Blood pressure dropped by 1.8 +/- 1.1 kPa within 15 min. The distribution was extremely uneven. Blood pressure decreases also occurred after pretreatment with PAF receptor antagonist. CONCLUSION: Rapid tracheal infusion of surfactant results in poorer oxygenation, an inhomogeneous distribution and a similar decrease in blood pressure compared to the bolus instillation method. Blood pressure changes could not be prevented by a PAF receptor-specific antagonist.

Animals↗
PubMed1995

Requirement of N-terminal amino acid residues of gp41 for human immunodeficiency virus type 1-mediated cell fusion.

An expression vector was designed to test the structural requirements of the gp41 N terminus for human immunodeficiency virus type 1-induced membrane fusion. Mutations in the region coding for the N terminus of gp41 were found to disrupt glycoprotein expression because of deleterious effects on the Rev-responsive element (RRE). Insertion of an additional RRE in the 3'-noncoding sequence of env made possible efficient glycoprotein expression, irrespective of the mutations introduced into the RRE in the natural location. This permitted the insertion of the unique restriction site SpeI within the N-terminal sequences of gp41, allowing convenient and efficient mutation of the gp41 N terminus by using double-stranded synthetic oligonucleotides. Mutants with deletions of 1 to 7 amino acids of the N terminus were constructed. Expression and cleavage of all mutants were confirmed by Western immunoblot analysis with anti-gp41 antibodies. The capability of mutants to induce membrane fusion was monitored following transfection of HeLa-T4+ cell lines with wild-type and mutant expression vectors by electroporation and microinjection. The efficiency of cell-fusing activity decreased drastically with deletion of 3 and 4 amino acids and was completely lost with deletion of 5 amino acids. Cotransfection of the parent and mutant expression vectors resulted in reduced cell-fusing activity. The extent of this dominant interference by mutant glycoprotein paralleled the decrease in cell-fusing activity of the mutants alone. This suggests the existence of a specific N-terminal structure required for fusing activity. However, there does not appear to be a stringent requirement for the precise length of the N terminus. This finding is supported by the length variation of this region among natural human immunodeficiency virus type 1 isolates and is in contrast to the apparent stringency in the length of analogous N-terminal structures of influenza A virus and paramyxovirus fusion glycoproteins.

Amino Acid Sequence↗
PubMed1993

Use of DNA end joining activity of a Xenopus laevis egg extract for construction of deletions and expression vectors for HIV-1 Tat and Rev proteins.

We have developed a cloning strategy which combines conventional T4 DNA ligation with the highly efficient nonhomologous DNA end joining (EJ) activity of an extract from Xenopus laevis eggs. The nonhomologous EJ activity allowed the rapid construction of deletion mutants by the intramolecular rejoining of nonhomologous DNA ends generated for the purpose of deleting restriction fragments from the vector. The combined use of T4 DNA ligase for intermolecular ligation and X. laevis egg extracts for intramolecular nonhomologous EJ proved to be a powerful tool, as demonstrated here for the construction of expression vectors for HIV-1 Tat and Rev.

Animals↗
PubMed1990

Detection of HIV RNA by in situ hybridization in peripheral blood mononuclear cells of seronegative children born to HIV-infected mothers.

Of 44 children born to human immunodeficiency virus (type 1) (HIV)-infected mothers, 11 have become seronegative. After the loss of maternal antibodies all children were analysed for several immunological functions and virological parameters in order to determine their HIV status. All children to date are clinically healthy and have normal immune functions. HIV-1 was detected by p24 antigen in one child, by in situ hybridization in nine children while viral cultures were all negative. These data suggest that the rate of vertical transmission of HIV-1 may be underestimated if seronegative children are considered to be not infected. They also suggest that molecular biological techniques are more sensitive than HIV antigen assay or viral cultures.

AIDS Serodiagnosis↗
PubMed1990

[Serologic studies in the diagnosis of pneumonia].

The importance of serological determination procedures for the diagnosis of pathogens was investigated in 207 episodes of pneumonia. The pathogenic organisms were detected in 138 cases; in 40 cases serological analysis helped to establish the diagnosis, while in 11 cases of pneumonia, the diagnosis was possible only with serology. The organisms most commonly found by serological investigations were Cytomegalovirus (n = 10), Aspergillus fumigatus (n = 7), and the influenza B virus (n = 7). Multiple infections, usually triggered by bacterial pathogens, were found in 48% of the cases of pneumonia with serological evidence of pathogens. In 71 episodes of pneumonia, the patients were immunosuppressed; in 11 cases, the causative organism was detected serologically.

Adult↗
PubMed1989

Histomorphological and lectin-histochemical confirmation of the antidegenerative effect of diclofenac in experimental osteoarthrosis.

The integrity of cartilage matrix depends on the homeostasis of synthetic and degradative processes. Any disturbance of the rate of synthesis and catabolism may alter the amount of matrix components (e.g. proteoglycans). Based upon a biochemically induced osteoarthrosis (OA) in the knee joints of rats we investigated the histomorphological alterations under therapy with diclofenac sodium by histological-histochemical grading. Lectin-binding techniques using labelled wheat germ agglutinin (WGA), concanavalin A (Con A), Ulex europeus agglutinin I (UEA I), soybean agglutinin (SBA), and peanut agglutinin (PNA) were applied to analyze the cellular as well as the extracellular glycoconjugates in situ. Lectin-binding patterns quantitatively describe the topographical localization of structural components of the cartilage matrix which carry certain sugar residues. The therapy of the experimental OA with diclofenac sodium (2.0 mg/kg s.c.) led to a marked reduction of cartilage degenerations. Our results indicate antidegenerative properties of this compound. These findings are consistent with the fluorescent analytical data which show a stimulating effect on the anabolic activity of chondrocytes in the osteoarthritic joints under the treatment with diclofenac sodium. Fluorescein isothiocyanate labelled lectins are useful histochemical tools to determine alterations in the integrity of cartilage by their specific binding patterns, because zonal differentiation in physiological function and morphological structure of cartilage tissue as well as cellular, pericellular, and interterritorial local events are characterized.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Protease activation mutants of Sendai virus: sequence analysis of the mRNA of the fusion protein (F) gene and direct identification of the cleavage-activation site.

Trypsin cleaves the fusion protein (F) of wild-type Sendai virus into two disulfide-linked polypeptides, F1 and F2, and thereby activates the membrane fusion activity of the virus. A. Scheid and P.W. Choppin [1976). Virology, 265-277) selected mutant viruses of which the F protein could be activated by different proteases, either elastase, chymotrypsin, or plasmin. Herein, we have further characterized five of these mutants. Sequencing of each mutant mRNA encoding the 60-70 amino acids surrounding the cleavage site revealed one or two amino acid changes near or at the cleavage sites. Virions cleaved in vitro by the appropriate proteases were assayed of their fusion activity by hemolysis, and the cleavage sites were determined by amino acid sequencing. In three cases, the change of protease specificity can be accounted for by changed amino acids right at the cleavage site, whereas several other mutations that potentiate cleavage at new sites by new proteases are somewhat removed from the actual cleavage site. We surmise that such mutations might alter local polypeptide conformation, thereby allowing the proteases access to existing sites. Cleavage at new sites produced fusion proteins with novel F1 NH-termini. We found that a mutant with a charged residue at the third position of this normally hydrophobic NH-terminal sequence retains activity in the hemolysis assay, whereas a mutant with a charged residue at the first position does not.

Amino Acid Sequence↗