Biomedical subjects

W E Müller

Publications and source records attributed to W E Müller.

At least 19 recordsLinked to original sources

PubMed1992

Purification and characterization of a pore-forming protein from the marine sponge Tethya lyncurium.

A pore-forming protein was detected and purified for the first time from a marine sponge (Tethya lyncurium). The purified protein has a polypeptide molecular mass of 21 kDa and a pI of 6.4. Tethya pore-forming protein (also called Tethya hemolysin) rapidly lysed erythrocytes from a variety of organisms. After binding to target membranes, the hemolysin resisted elution with EDTA, salt or solutions of low ionic strength and hence resembled an integral membrane protein. Erythrocytes could be protected from hemolysis induced by Tethya hemolysin by addition of 30 mM dextran 4 (4-6 kDa; equivalent hydrodynamic diffusion radius, 1.75-2.3 nm) to the extracellular medium, but not by addition of uncharged molecules of smaller size [sucrose, raffinose and poly(ethylene glycol) 1550; equivalent hydrodynamic diffusion radii, 0.46, 0.57 and 1.2 nm, respectively]. This result indicates that hemolysin is able to form stable transmembrane pores with an effective diameter of about 2-3 nm. Treatment of osmotically protected erythrocytes with Tethya hemolysin caused a rapid efflux of intracellular K+ and ATP, and a rapid influx of extracellularly added Ca2+ and sucrose. In negative-staining electron microscopy, target erythrocyte membranes exposed to purified Tethya hemolysin displayed ultrastructural lesions but without visible pores.

Adenosine Triphosphate↗

PubMed1992

Localization of Epstein-Barr virus-encoded RNAs EBER-1 and EBER-2 in interphase and mitotic Burkitt lymphoma cells.

The subcellular distribution of the small Epstein-Barr virus-encoded RNAs EBER-1 and EBER-2 has been investigated by using a high-resolution in situ hybridization technique. The distribution patterns in Raji cells of fluorescent oligodeoxynucleotides complementary to each RNA were detected by confocal laser scanning microscopy. Both RNAs were found in the cytoplasm as well as in the nuclei of interphase cells. In contrast, use of the same technique indicated an exclusively nuclear location for cellular U2 RNA. In the cytoplasm distribution of the EBERs was similar to that of the double-stranded RNA-dependent protein kinase, to which these RNAs can bind, and was coincident with the rough endoplasmic reticulum. In cells undergoing mitosis the EBERs became localized around the chromosomes, whereas the protein kinase remained uniformly distributed in the cytoplasm. A cytoplasmic location for EBER-1 and EBER-2 in interphase cells is consistent with the evidence for a role for these small RNAs in translational control.

Base Sequence↗

PubMed1992

Association of AUUUA-binding protein with A+U-rich mRNA during nucleo-cytoplasmic transport.

Resealed nuclear envelope (NE) vesicles from rat liver containing entrapped exogenous RNA were used to study the effect of adenosine+uridine binding factor (AUBF), present in cytosolic cell extracts, on ATP-dependent transport of A+U-rich RNA (AU+RNA) and A+U-free RNA (AU-RNA) across the NE. This factor specifically binds to A+U-rich sequences present in the 3' untranslated regions of lymphokine and cytokine mRNAs, containing overlapping AUUUA boxes (granulocyte-macrophage colony stimulating factor, interleukin-3). Addition of AUBF to the extravesicular compartment markedly increased the efflux of the in vitro transcribed, capped and polyadenylated AU+ RNAs. Export of entrapped AU- control RNA, such as beta-globin RNA, was not affected by AUBF, in contrast to chimeric AU+ beta-globin RNA containing the A+U-rich sequence of human interferon-alpha mRNA (6 reiterated AUUUA motifs). Competition experiments revealed that AUBF enhances the affinity of poly(A)-containing AU+ RNAs to the NE poly(A)-binding component (poly(A)-recognizing mRNA carrier p106), and thereby accelerates nuclear export of these RNAs. We could demonstrate that AUBF added to the extravesicular space forms stable complexes with polyadenylated AU+ RNA with relative molecular masses of about 45,000, 62,000 and 70,000 inside the vesicles or during ATP-dependent export. In addition we determined that AUBF may affect mRNA stability by protecting A+U-rich RNA against degradation by trans-acting, nuclear matrix-associated and A+U-specific endoribonuclease V.

Adenosine↗

PubMed1992

Age-related alterations of NMDA-receptor properties in the mouse forebrain: partial restoration by chronic phosphatidylserine treatment.

The effect of aging on the properties of N-methyl-D-aspartate (NMDA) receptors in the forebrain of female NMRI mice was investigated using the antagonist [3H]MK-801 as radioligand. Compared to young (3 months) mice, aged (20 months) mice showed changes of the properties of the NMDA receptor at three different levels: (1) the density was reduced by about 35%; (2) the efficacy of L-glutamate and glycine for stimulating specific [3H]MK-801 binding was enhanced, probably because more NMDA receptor-associated ion channels are closed under baseline conditions in the aged brain; (3) the affinity of L-glutamate and glycine to its binding sites at the NMDA receptor complex was also enhanced. Chronic treatment of aged mice with phosphatidylserine (20 mg/kg, i.p., once daily) for three weeks completely normalized enhanced efficacy and affinity of L-glutamate and glycine and elevated NMDA receptor density by approximately 25%. These findings are consistent with the assumptions that deficits of the NMDA receptor are one of the mechanisms of age-related cognitive impairment and that the beneficial effects of phosphatidylserine treatment on cognitive deficits of aged individuals might be partially due to the effects of this drug on age-related NMDA receptor deficits.

Aging↗

PubMed1992

gp120 of HIV-1 induces apoptosis in rat cortical cell cultures: prevention by memantine.

After incubation of rat cortical cell cultures with the human immunodeficiency virus type 1 (HIV-1) coat protein gp120 for 12 h, cells showed fragmentation of DNA at internucleosomal linkers, the characteristic feature of apoptosis. In a quantitative approach, it was determined that the percentage of DNA fragmentation increased from 7%, in the absence of gp120, to 62% following incubation with 24 ng/ml of gp120. Simultaneously, the percentage of viable cells decreased from 94% to 33%. Memantine (1-amino-3,5-dimethyladamantane), a drug currently used in the therapy of spasticity and Parkinson's disease as well as the NMDA antagonist MK-801 both prevented the effects of gp120 at micromolar concentrations. In human cultured astrocytes, gp120 was ineffective with respect to DNA fragmentation and cell toxicity. From these data, we conclude that the gp120-induced apoptosis may contribute to the neurological complications frequently associated with the immunodeficiency syndrome. The cytoprotective effect of memantine in cortical cell cultures may qualify the drug for the treatment of AIDS-related dementia.

Animals↗

PubMed1992

Accumulation of transcripts coding for prion protein in human astrocytes during infection with human immunodeficiency virus.

The abnormal isoforms of the normal cellular prion protein (PrP), also termed Scrapie-associated fibril protein, are assumed to be one causative factor of spongiform encephalopathies. The mRNA of PrP contains stem-loop structures which are very similar to the human immunodeficiency virus-1 (HIV-1) cis-acting sequence TAR within the LTR; both structures contain the pentanucleotide CUGGG in the loop, and the uridine- and adenine-bulge in the stem. In this study, using purified HIV-encoded trans-activator, Tat, and HIV-1 TAR-RNA or PrP-mRNA containing the stem-loop structure, we demonstrate by use of gel-retardation and filter binding assays that Tat binds to TAR- and PrP-RNA with the dissociation constants of 2.9 or 37.0 nM, respectively, at a molar ratio of 0.7 mol of Tat to 1 mol of RNA fragment. The Tat-RNA (TAR or PrP) complexes bind to protein(s) in the nuclear matrix, isolated from human astrocytes (glial fibrillary acidic protein positive brain cells). Infection of astrocytes with HIV-1 resulted in an increased level of PrP mRNA. The data presented led us to assume that certain sequences in the PrP mRNA might be targets for proteins acting in trans.

Astrocytes↗

PubMed1992

Purification of a glucose-binding protein from rat liver nuclei. Evidence for a role in targeting of nuclear mRNP to nuclear pore complex.

A nuclear carbohydrate-binding protein with a molecular mass of 67 kDa (CBP67), which is specific for glucose residues, was purified to essential homogeneity from rat liver nuclear extracts. This protein could also be isolated from nuclear ribonucleoprotein (RNP) complexes by extraction in the presence of 0.6 M or 2 M NaCl, but it was absent in polysomal RNP complex. The binding of the purified protein, which has an isoelectric point of 7.3, to glucose-containing glycoconjugates depends on the presence of Ca2+ and Mg2+. Using closed nuclear envelope vesicles as a system to study nuclear transport of RNA, it was shown that both entrapped polysomal mRNA and nuclear RNA precursors are readily exported from the vesicles in an ATP-dependent manner. The transport was unidirectional and strongly promoted by the poly(A) segment attached to these RNAs. In contrast, nuclear RNP complexes entrapped into the vesicles together with glucose-conjugated bovine serum albumin or nucleoplasmin, or bird nest glycoprotein, were not exported into the extravesicular space. However, transport of nuclear RNP complexes could be achieved in the presence of glucose or after co-addition of a glucose-recognizing lectin from Pellina semitubulosa. In Western blots, radioiodinated CBP67 binds to an 80-kDa polypeptide both in isolated rat liver nuclear envelopes and pore-complex laminae. From these results we postulate that CBP67 may direct nuclear RNP complexes to the nuclear pore.

Animals↗

PubMed1992

Inhibition of hepatitis B virus production by modified 2',3'-dideoxy-thymidine and 2',3'-dideoxy-5-methylcytidine derivatives. In vitro and in vivo studies.

The effect of analogues of both 2',3'-dideoxy-3'-fluorothymidine (FddThd) [2',3'-dideoxy-3'-fluorouridine (FddUrd), 2',3'-dideoxy-3'-fluoro-5-chlorouridine (FddClUrd), 2',3'-dideoxy-3'- fluoro-5-bromouridine (FddBrUrd) and 2',3'-dideoxy-3'-fluoro-5-bromovinyluridine (FddBVUrd)] and 2',3'-dideoxy-3'-fluorocytidine (FddCyt) [2',3'-dideoxy-3'-fluoro-5-fluorocytidine (FddFCyt), 2',3'-dideoxy-3'-fluoro-5-chlorocytidine (FddClCyt), 2',3'-dideoxy-3'-fluoro-5-methylcytidine (FddMeCyt), 2',3'-dideoxy-3'-fluoro-5-ethylcytidine (FddEtCyt), 2',3'-dideoxy-3'-chloro-5-methylcytidine (ClddMeCyt), 2',3'-dideoxy-3'-amino-5-methylcytidine (AmddMeCyt), 2',3'-dideoxy-3'-azido-5- methylcytidine (AzddMeCyt) and arabinosyl-5-methylcytosine (AraMeCyt)] were tested for their potential antiviral activity in vitro using the human hepatoblastoma cell line, Hep G2 2.2.15, which was transfected with a vector containing hepatitis B virus (HBV). It was found that FddThd, FddMeCyt, FddEtCyt, ClddMeCyt, AmddMeCyt and AraMeCyt display cytostatic activity at concentrations (CD50 values) between 0.54 (FddMeCyt) and 3.93 microM (FddEtCyt), while FddUrd, FddClUrd, FddBrUrd, FddBVUrd, FddCyt, FddFCyt, FddClCyt and AzddMeCyt do not affect cell growth at concentrations of up to 25 microM. Among the thymidine analogues tested, FddThd is the most effective antiviral agent: at a concentration of 0.03 microM a more than 90% reduction of HBV DNA synthesis was measured. On the other hand, the antiviral indexes displayed by FddClUrd, FddBrUrd and FddBVUrd are higher than tht of FddThd; FddUrd was completely inactive. The most powerful antiviral agents in the group of cytidine analogues tested in vitro were FddMeCyt (more than 90% reduction of HBVDNA synthesis at 0.10 microM) and ClddMeCyt (0.10 microM); FddClCyt, FddEtCyt, AmddMeCyt and AraMeCyt were of intermediate activity. None of the negligible antiviral activity was determined for FddUrd, FddCyt, FddFCyt and AzddMeCyt. FddThd and FddMeCyt displayed in vivo an antiviral effect in the duck/duck HBV (DHBV) animal system. Administration of 10 or 20 mg/kg (total daily dose) of FddThd and 5 or 10 mg/kg of FddMeCyt (i.m. daily) to ducks infected with DHBV for 12 days blocked virus production. Termination of treatment with FddThd of infected animals led to reappearance of the virus in the serum though at lower levels. The in vitro and the in vivo data suggest that FddThd and FddMeCyt might be promising antiviral agents for the treatment of infection caused by HBV in humans.

Animals↗

PubMed1992

Cytokine-mediated regulation of monocyte/macrophage cytotoxicity in human immunodeficiency virus-1 infection.

Monocyte/macrophage-mediated tumor cytotoxicity was studied in patients infected with human immunodeficiency virus-1 (HIV-1) at various stages [Center for disease control (CDC) classification] of the disease. using the P-815 tumor cell line as target cells, the results demonstrated reduced monocyte/macrophage cytotoxicity early in HIV-1-related disease (CDCIII, P < 0.01). This cellular dysfunction sustained during the progression of the disease. Evidence could be presented that neither exogenous application of macrophage-stimulating cytokines (e.g. interferons) nor their endogenous induction in vitro restored monocyte/macrophage cytotoxicity. However, enhanced tumor necrosis factor (TNF)-alpha production, which parallels the observed reduced capacity to lyse P-815 tumor cells, might be the major source for monocyte/macrophage-mediated cell lysis. TNF-alpha-induced cytotoxicity can be inhibited by addition of anti-TNF-alpha. Other experimental models using TNF-sensitive tumor target cells may, therefore, mimic monocyte/macrophage-mediated lysis. Suppression of monocyte/macrophage cytotoxicity in later stages of HIV-1 infection (AIDS-related complex, AIDS) could partly be reverted by treatment with the cyclooxygenase blocker, indomethacin. The responsible arachidonic acid product mediating suppression was found to be prostaglandin E2, suggesting that in addition to the direct viral interference cellular dysfunction is at least in part a result of altered cytokine regulation.

Adult↗

PubMed1992

Expression of the HIV-1 Nef protein in the baculovirus system: investigation of anti-Nef antibodies response in human sera and subcellular localization of Nef.

The nef gene of HIV-1 was expressed in insect cells using the eucaryotic baculovirus system. The recombinant Nef protein frequently reacted with seropositive sera of HIV-1 and HIV-2 infected patients. Anti-Nef antibodies in HIV-1 seronegative high risk groups individuals were only occasionally seen. Confocal laser scanning microscopy demonstrated that Nef is present both in the cytoplasm and in the nucleus, indicating that Nef might directly function on gene expression.

Baculoviridae↗

PubMed1992

Expression and cellular localization of the Nef protein from human immunodeficiency virus-1 in stably transfected B-cells.

Nef protein, encoded by the regulatory nef gene of human immunodeficiency virus type 1 (HIV-1), was expressed in the B-cell line Raji. The cells were stably transfected with plasmids containing the nef transcriptional cassette. They expressed Nef with an Mr of 27,000; the yield could be augmented by incubation with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. The intracellular localization of Nef was analyzed applying immunofluorescence microscopy using a confocal laser scanning microscope. The antigen was stained with a monoclonal antibody directed against the N-terminal part of Nef. The experiments revealed that in non-dividing cells Nef is present both in the cytoplasm and the nucleus while in dividing cells the viral protein is present in the cytoplasm and at the nuclear membrane.

B-Lymphocytes↗

PubMed1992

Urinary metabolites of amitriptylinoxide and amitriptyline in single-dose experiments and during continuous therapy.

In a cross-over design, six healthy volunteers received 50 mg amitriptylinoxide (AT-NO) IV and orally and 50 mg amitriptyline (AT) IV. Urine was collected completely for 8 h and occasionally up to 48 h. In addition, five patients each under treatment with AT-NO or AT for tension headache collected 24-h urine samples. The following compounds were analysed by HPLC: AT-NO, E- and Z-10-hydroxy-AT-NO (E- and Z-10-OH-AT-NO), free and conjugated AT, E- and Z-10-OH-AT and their mono- and didemethylated analogues, and 2-OH-nortriptyline (2-OH-NT). Unchanged AT-NO in urine accounted for an average of 34% and 22% of the single IV and oral doses, respectively, and for 28% in continuous therapy, with a further 8-9% being excreted as E- and Z-10-OH-AT-NO. The remaining part was converted to the same metabolites as was AT. In the steady state the measured compounds accounted for 74% and 77% of the daily AT-NO and AT doses, respectively. The renal plasma clearance of AT-NO varied between 75 and 265 ml/min in the six volunteers. Tubular secretion must play an important part in the renal excretion of AT-NO.

Adult↗

PubMed1992

Determination of lectin characteristics by a novel agglutination technique.

A technique generally applicable for the determination of lectin characteristics is described. A sensitive light transmission/scattering method was adapted for the determination of lectin levels and lectin activity. Applying this procedure Geodia cydonium lectin-mediated agglutination was studied in an agglutimeter device using erythrocytes and even T-lymphocytes. In the Geodia lectin/T-lymphocyte system chosen, (i) a lectin concentration as low as 0.57 micrograms/ml could be measured accurately, (ii) the observed cell agglutination velocity constant with a maximal value of 0.75 min-1 was calculated, and (iii) the size of the agglutinates at a given lectin concentration and time period was estimated. The Geodia lectin activity was determined in parallel also in the erythrocyte system. Here, compared to the lectin/T-lymphocyte system the agglutination efficiency of the Geodia lectin-mediated agglutination was more than 10-fold higher and the lowest detectable lectin concentration was 0.06 micrograms/ml. Compared to the hemagglutination assay the lectin/erythrocyte system turns out to be more sensitive and to give much more information on agglutination behavior; this conclusion is supported by additional data using a second lectin isolated from Pellina semitubulosa. The superiority of the agglutination method described here over other known methods must be seen in its accuracy; moreover more lectin characteristics can be determined.

Agglutination↗

PubMed1992

The autoantigen La/SSB: detection on and uptake by mitotic cells.

The nuclear autoantigen La, a transcription/termination factor of RNA polymerase III, was recently shown to translocalize to the cell surface of growth-stimulated cells during transition from G0- to G1-phase. Here we describe the staining of living mitotic cells with the anti-La mab La11G7. Moreover, La protein added to cell culture medium was able to enter into synchronized mitotic cells. Uptake was inhibited by the anti-La mab. La protein taken up into prophase cells assembled into a fibrillar network. Taken up byu ana/telophase cells, La protein was preferentially transported into the newly forming or formed nuclei. This import allowed us to study directly the intranuclear localization of La protein in living cells by the use confocal laser scanning microscopy (cLSM). Adsorbed La protein was found in the nucleoplasm but also assembled into nuclear speckles. Some of these speckles surrounded the nucleolus like a ring.

Anaphase↗

PubMed1992

(2'-5')Oligoadenylate and intracellular immunity against retrovirus infection.

1. The double-stranded RNA-dependent 2',5'-oligoadenylate (2-5A) synthetase/ribonuclease L (RNase L) system plays an essential role in the establishment of the antiviral state of a cell exposed to virus infection. 2. Until recently, the application of 2-5A derivatives to reinforce this system seemed to be limited mainly due to the low specificity of RNase L for viral RNA. 3. Two new strategies have been developed which yield a selective antiviral effect of 2-5As at least against human immunodeficiency virus-1 (HIV-1) infection: (i) an "intracellular immunization" approach using 2-5A synthetase cDNA linked to HIV trans-acting response element (TAR) and (ii) inhibition of retroviral reverse transcriptase activity by 2-5A analogues.

2',5'-Oligoadenylate Synthetase↗

PubMed1992

The sleep structure of patients with anxiety disorders in comparison to that of healthy controls and depressive patients under baseline conditions and after cholinergic stimulation.

This study investigated sleep EEG during placebo and after cholinergic stimulation with RS 86 in 36 healthy subjects, 34 patients with major depression and 20 patients with anxiety disorders. Cholinergic stimulation with RS 86 led to a decrease of slow wave sleep and REM latency. RS 86 had a more profound impact on REM latency in patients with major depression than in healthy controls and patients with anxiety disorders. Six out of 36 healthy controls, three out of 20 patients with anxiety disorders and 24 of 34 patients with depression displayed sleep onset REM periods after cholinergic stimulation. Also effects on REM density and duration of the first REM period were more pronounced in major depression. Even in those patients with anxiety disorders and a secondary major depression no depression-like sleep abnormalities could be provoked. The results underline the usefulness of the cholinergic REM induction test to differentiate patients with major depression from those with other psychiatric disorders. The results can be interpreted as further evidence for the cholinergic-aminergic imbalance model of depression and for the reciprocal interaction model of nonREM-REM regulation.

Adult↗

PubMed1992

A novel galactose- and arabinose-specific lectin from the sponge Pellina semitubulosa: isolation, characterization and immunobiological properties.

A new lectin from the sponge Pellina semitubulosa is derived which was extracted and purified to homogeneity. The purified lectin is probably a hexamer of polypeptide chains (each M(r) 34,000) which are covalently linked via disulfide linkages; the isoelectric point is 6.1. The lectin displays the following specificities: D-galactose (50% inhibition of hemagglutination at 0.2 mM) = L-arabinose (0.2 mM) greater than D-fucose (1.5 mM) greater than D-glucose (3.0 mM). It precipitates human erythrocytes (A1, A2, A1B, B, and O) with a titer between 2(8) and 2(11) and erythrocytes from sheep and rabbits with a titer between 2(5) and 2(10). The Pellina lectin displays a strong mitogenic effect on spleen lymphocytes from mice. Immunochemical analyses revealed that both murine T- and B-lymphocytes display a capping of the lectin receptors on their cell surfaces after lectin treatment. Murine macrophages were found to endocytose the lectin. Pellina lectin at concentrations between 0.3 and 10.0 micrograms/ml potently enhances interleukin 1 (IL-1) release from mouse peritoneal macrophages and interleukin 2 (IL-2) production in mixed murine lymphocyte cultures.

Amino Acids↗

PubMed1992

Production of the cytostatic agent aeroplysinin by the sponge Verongia aerophoba in in vitro culture.

1. The marine sponge Verongia aerophoba contains two bioactive secondary metabolites from tyrosine, (+)-aeroplysinin-1 [3',5'-dibromo-1',2'-dihydroxy-4'- methoxycyclohexa-3',5'-dien-1'-yl-methyl-cyanide; abbreviated AP] and dibromoverongia-quinol [3',5'-dibromo-1'-hydroxy- 4'-oxocyclohexa-2',5'-dien-1'-yl-acetamide; abbreviated DV], which display strong cytostatic activity. 2. The concentrations causing 50% inhibition of cell growth are 0.47 microM (AP) and 1.21 microM (DV), resp. 3. Depending on depth regions from which the sponges were collected, differences in occurrence of metabolites were observed. 4. AP and DV were found to be present in sponges collected at a depth of 5-10 m, whereas only DV could be detected in material from deeper regions (20-30 m). 5. AP is present only in the surface layers (both the outer and oscular region) of the sponge, while in the centre of the sponge only DV is detected. 6. Cubes from sponges, collected at a depth of 30 m, were cultivated in seawater in vitro and were found to have the capacity (i) to synthesize AP, and (ii) to release this bioactive material into the medium under defined conditions. Under optimal conditions (light and aeration) 100 g of sponge synthesize and release 13.02 mg of AP during a 10-day incubation period. 7. In the dark and without aeration this synthesis was prevented. 8. These data show that also under in vitro conditions sponges retain the capability of producing bioactive compounds and can be induced to produce even substances which they did not secrete in their natural environment.

Acetamides↗