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M F Schmidt

Publications and source records attributed to M F Schmidt.

At least 37 records · Page 2Linked to original sources
PubMed1998

Fusion activity of transmembrane and cytoplasmic domain chimeras of the influenza virus glycoprotein hemagglutinin.

The role of the sequence of transmembrane and cytoplasmic/intraviral domains of influenza virus hemagglutinin (HA, subtype H7) for HA-mediated membrane fusion was explored. To analyze the influence of the two domains on the fusogenic properties of HA, we designed HA-chimeras in which the cytoplasmic tail and/or transmembrane domain of HA was replaced with the corresponding domains of the fusogenic glycoprotein F of Sendai virus. These chimeras, as well as constructs of HA in which the cytoplasmic tail was replaced by peptides of human neurofibromin type 1 (NF1) or c-Raf-1, NF78 (residues 1441 to 1518), and Raf81 (residues 51 to 131), respectively, were expressed in CV-1 cells by using the vaccinia virus-T7 polymerase transient-expression system. Wild-type and chimeric HA were cleaved properly into two subunits and expressed as trimers. Membrane fusion between CV-1 cells and bound human erythrocytes (RBCs) mediated by parental or chimeric HA proteins was studied by a lipid-mixing assay with the lipid-like fluorophore octadecyl rhodamine B chloride (R18). No profound differences in either extent or kinetics could be observed. After the pH was lowered, the above proteins also induced a flow of the aqueous fluorophore calcein from preloaded RBCs into the cytoplasm of the protein-expressing CV-1 cells, indicating that membrane fusion involves both leaflets of the lipid bilayers and leads to formation of an aqueous fusion pore. We conclude that neither HA-specific sequences in the transmembrane and cytoplasmic domains nor their length is crucial for HA-induced membrane fusion activity.

Amino Acid Sequence↗
PubMed1997

Targeted delivery of human neurofibromin and c-Raf-1 mutants to the cytoplasmic membrane by use of the influenza virus hemagglutinin.

Mutants of human neurofibromin and c-Raf-1 genes were fused to the 3' end of the hemagglutinin (HA) gene of influenza A virus by oligonucleotide-directed polymerase chain reaction (PCR). The two resulting chimeric genes, HA (1-534)/NF1 (1441-1518) and HA (1-534)/Raf-1 (51-132) which we designated HN and HR, respectively, were cloned in a vaccinia virus expression vector (pTMI) under the control of a T7 RNA polymerase promoter. The clones were expressed in a monkey cell line (CV-1) and the resulting chimeric proteins analysed. We found that expression levels of the chimeric proteins were similar to that of wild-type HA protein. Comparative endoglycosidase treatment revealed that the expressed chimeric proteins HN and HR were processed as wild-type HA, and FACS-analysis showed that both chimeric expression products localised in the cell membrane as the wild-type control. HN and HR expressing cells showed similar fusogenic activity as CV-1 cells transfected with wild-type HA indicating the correct topology of the fusion inducing portion (HA) of these chimera in the membrane. These findings show that the influenza virus hemagglutinin (HA) is a suitable vehicle to target foreign proteins with therapeutical potential into the cell membrane. In this respect HN and HR could potentially be used to block the abnormal signals generated by particular proteins in the cell membrane that lead to cell transformation.

Animals↗
PubMed1996

Differential fatty acid selection during biosynthetic S-acylation of a transmembrane protein (HEF) and other proteins in insect cells (Sf9) and in mammalian cells (CV1).

The transmembrane glycoprotein HEF and its acylation deficient mutant M1 were expressed in Sf9 insect cells infected with recombinant baculovirus and in CV1 mammalian cells using the vaccinia T7 system. In insect cells (Sf9), both wild type HEF and HEF(M1) are synthesized in their precursor form HEF0, which appears as a double band in SDS gels. Digestion with glycopeptidase F and endoglycosidase H reveals that the larger 84-kDa form is modified by the attachment of unprocessed carbohydrates of the high mannose type whereas the smaller 76-kDa form is non-glycosylated. As revealed by in vitro labeling experiments with palmitic acid another modification of HEF is the attachment of a long chain fatty acid to cysteine residue Cys-652 which is located at the internal border of the cytoplasmic membrane. After labeling with [3H]palmitic acid in both systems only HEF(WT) is acylated, whereas HEF(M1) is not. High performance liquid chromatography analysis of the fatty acids bound to HEF(WT) expressed in Sf9 insect cells reveals nearly 80% of palmitic acid. In contrast to this finding, the acylation pattern of HEF expressed in CV1 cells shows nearly the same amounts of stearic and palmitic acid (40%). Since the interconversion of the input [3H]palmitic acid to stearic acid is even lower in CV1 cells than in insect cells, it follows that only HEF expressed in mammalian, but not in insect cells selects for stearic acid during its biosynthetic acylation. We extended our study to acylation of endogenous proteins in Sf9 cells. In finding only palmitate linked to protein we present evidence that, in contrast to mammalian cells, insect cells (Sf9) cannot transfer stearic acid to polypeptide. This finding favors the hypothesis of enzymatic acylation over non-enzymatic mechanisms of acyl transfer to protein.

Acylation↗
PubMed1996

Cytoplasmic tail length influences fatty acid selection for acylation of viral glycoproteins.

We report remarkable differences in the fatty acid content of thioester-type acylated glycoproteins of enveloped viruses from mammalian cells. The E2 glycoprotein of Semliki Forest virus contains mainly palmitic acid like most other palmitoylated proteins analysed so far. However, the other glycoprotein (E1) of the same virus, as well as the HEF (haemagglutinin esterase fusion) glycoprotein of influenza C virus, are unique in this respect because they are acylated primarily with stearic acid. Comparative radiolabelling of uninfected cells with different fatty acids suggests that stearate may also be the prevailing fatty acid in some cellular acylproteins. To look for further differences between palmitoylated and stearoylated glycoproteins we characterized stearoylation in more detail. We identified the acylation site of HEF as a cysteine residue located at the boundary between the transmembrane region and the cytoplasmic tail. The attachment of stearate to HEF and E1 occurs post-translationally in a pre-Golgi compartment. Thus, stearoylated and palmitoylated proteins cannot be discriminated on the basis of the fatty acid linkage site or the intracellular compartment, where acylation occurs. However, stearoylated acylproteins contain a very short, positively charged cytoplasmic tail, whereas in palmitoylated proteins this molecular region is longer. Replacing the short cytoplasmic tail of stearoylated HEF with the long influenza A virus haemagglutinin (HA) tail in an HEF-HA chimera, and subsequent vaccinia T7 expression in CV-1 cells, yielded proteins with largely palmitic acid bound. The reverse chimera, HA-HEF with a short cytoplasmic tail was not fatty acylated at all during expression, indicating that conformational or topological constraints control fatty acid transfer.

Acylation↗
PubMed1996

Transient elevations in intracellular calcium are sufficient to induce sustained responsiveness to the neurotrophic factor bFGF.

The present study investigates how a neuron's past history of neural activity may alter its responsiveness to subsequent signals. We demonstrate that a depolarizing pulse of extracellular potassium can prime neurons to become responsive to basic fibroblast growth factor (bFGF), even when the pulse is brief and occurs prior to addition of bFGF. Specifically, we subjected cultured embryonic chick ciliary ganglion neurons (E7) to a short pulse of elevated extracellular potassium followed by addition of bFGF and tested the effect of such treatment on neuronal survival. Neurons treated in this manner produced high levels of survival, whereas neurons exposed to either the pulse alone or the continuous presence of bFGF alone failed to promote any significant levels of survival. This priming effect of depolarization on bFGF-induced survival was blocked by calcium channel antagonists. To test the time dependency of this effect, we increased the time interval between termination of the calcium pulse and addition of bFGF. Our results demonstrate that a brief elevation in intracellular calcium has long lasting effects, up to 8 h after cessation of the depolarizing pulse, on neuronal responsiveness to bFGF. These findings suggest how a developing neuron's history of activity can alter its subsequent ability to respond to neurotrophic factors and has significant implications on the mechanisms by which activity may influence neuronal survival.

Analysis of Variance↗
PubMed1996

Intracellular compartmentalization of the glycoprotein B of herpesvirus Simian agent 8 expressed with a baculovirus vector in insect cells.

The intracellular localization of the glycoprotein B of herpesvirus simian agent 8 expressed with a baculovirus system in insect cells was studied. Cell fractionation and immunoprecipitation revealed that gB is present in microsomal as well as in nuclear membranes. Both fractions contain oligomers, probably dimers, of gB with endoglycosidase-H sensitive, mannose-rich carbohydrates. Nuclear transport of gB was further analysed by immuno electron microscopy of recombinant baculovirus-infected cells. The glycoprotein is present both in the outer and the inner nuclear membrane as well as in cytoplasmic structures and at the cell surface. This study precludes the possibility that glycosylation and/or oligomerisation of SA8 gB are responsible for nuclear targeting.

Animals↗
PubMed1996

Deacylation of influenza virus hemagglutinin does not affect the kinetics of low pH induced membrane fusion.

The relevance of palmitoylation of cysteine residues of influenza virus hemagglutinin (HA) for the HA-mediated membrane fusion triggered at low pH is investigated. Either wild-type HA (subtype H7) or mutant HA devoid of fatty acids were expressed in insect cells. The kinetics as well as the extent of fusion of HA-expressing cells with human erythrocyte ghosts were measured by a membrane mixing assay. Fusion was measured continuously at different pH by fluorescence dequenching of the lipid-like fluorophore R18 initially incorporated into the erythrocyte membrane. No significant difference between fusion of wild-type and mutant HA expressing cells with ghosts could be detected showing that deacylation does affect neither the extent nor the kinetics of fusion.

Animals↗
PubMed1995

Palmitoylation of endogenous and viral acceptor proteins by fatty acyltransferase (PAT) present in erythrocyte ghosts and in placental membranes.

Human erythrocyte ghosts were shown to have palmitoylating activity which acylates both endogenous ghost polypeptides and exogenous proteins derived from Semliki Forest virus (SFV). Cell-free fatty acid transfer from [3H]palmitoyl-CoA to endogenous protein was greatly enhanced in ghosts when pre-existing fatty acids linked to the endogenous acyl proteins were removed by hydroxylamine treatment prior to the transfer reaction. In contrast to erythrocyte acyl proteins acceptor proteins present in human placental membranes were palmitoylated in vitro to a similar extent with or without prior deacylation by hydroxylamine treatment. This indicates the presence of large pools of non-acylated proteins in placenta and small pools in erythrocytes. In testing for the protein substrate specificity of the palmitoyl transferase (PAT) present in ghosts we found that the SFV acceptor proteins, which are totally unrelated to erythrocytes, competed with the palmitoylation of endogenous ghost protein acceptors. This palmitoylating enzyme is inhibited by Cibacron Blue, SDS, and heat treatment, but stimulated in the presence of low concentrations of mild detergent (TX-100). Since PAT operating at the surface membrane of red blood cells has properties very similar to those of PAT present in human placental microsomes [1], we suggest that only one type of PAT may transfer fatty acids to various acylproteins that occur at multiple locations in different tissues [2].

Acylation↗
PubMed1995

Assessment of fusogenic properties of influenza virus hemagglutinin deacylated by site-directed mutagenesis and hydroxylamine treatment.

Influenza virus hemagglutinin (HA) subtype H7 expressed from a baculovirus vector in insect cells requires cysteine residues for palmitoylation. Mutant HA devoid of fatty acids shows hemagglutinating and hemolytic activities almost identical to those of the acylated wild-type HA (wt). Using a membrane mixing assay (R18), neither the kinetics nor the pH dependence of fusion induced by wt or mutant HA was significantly different from virus-induced fusion. HA-induced fusion of insect cells with human erythrocyte ghosts could also be demonstrated by a cytoplasmic content mixing assay. Both species of recombinant HA induced the flow of lucifer yellow from preloaded ghosts into the cytoplasm of HA-bearing cells. This indicates that membrane fusion mediated by wild-type and fatty-acid-free HA includes both leaflets of the lipid bilayers. Hydroxylamine treatment of wt HA (H7) and fatty-acid-free mutant HA present in lysates of insect cells led to the complete inhibition of hemolytic activity. Deacylation of spike proteins by NH2OH treatment of virus particles resulted in a block of hemolytic activity in influenza virus subtypes H7 and H10 as well as of that in the togaviruses Semliki Forest and Sindbis virus. However, the same treatment did not affect subtypes H2 and H3 or two vesicular stomatitis virus serotypes. With such a differential effect whether or not fatty acids are present in the spike proteins of the different virus particles, hydroxylamine must have other effects than just deacylation, and therefore seems unsuitable for the study of the biological functions of acylproteins.

Acylation↗
PubMed1995

Depolarization and laminin independently enable bFGF to promote neuronal survival through different second messenger pathways.

This study presents evidence that cellular responsiveness to the neurotrophic factor basic fibroblast growth factor (bFGF) can be achieved by at least two classes of agents using different initial second messenger pathways. Embryonic chick ciliary ganglion neurons plated on polyornithine normally undergo rapid cell death in culture and could not be rescued by addition of bFGF. Similarly, neither exposure to laminin alone nor low levels of depolarization alone was sufficient to promote neuronal survival. In contrast, if ciliary ganglion neurons were exposed to bFGF in the presence of either laminin or low levels of depolarization, nearly all neurons were rescued from cell death. The observed synergistic effect of bFGF and low levels of depolarization required influx of calcium since addition of the L-type calcium channel antagonists PN200-110 completely prevented neuronal survival. The synergistic effect observed between laminin and bFGF was independent of changes in intracellular calcium since PN200-110 failed to block the synergistic effect and because neither addition of bFGF to neurons plated on laminin nor direct addition of laminin caused any significant changes in intracellular calcium. The ability of laminin to enable bFGF to promote neuronal survival could completely be blocked by the addition of antibodies to beta 1-integrin, suggesting a calcium-independent integrin-mediated response. Taken together, these results suggest that both laminin and depolarization can enable bFGF responsiveness by acting through different second messenger pathways.

Animals↗
PubMed1995

Laminin and fibronectin guideposts signal sustained but opposite effects to passing growth cones.

Guidepost cells are known to alter the behavior of growth cones in vivo, yet the nature of communication and the type of signals employed are largely undefined. The present study demonstrates that model guideposts, composed of a single molecular species, are sufficient to change the navigation and the behavior of advancing growth cones well beyond the time of contact. Laminin on model guideposts caused a sustained increase in growth cone velocity, whereas fibronectin led to a sustained decrease. A spatially discrete array of multiple laminin-model guideposts maintained increased growth rates on fibronectin, as expected for homogeneous laminin, and also provided unambiguous directional guidance information. Laminin-evoked growth cone responses required activation of protein kinase C-dependent intracellular signalling mechanisms.

Animals↗
PubMed1995

Rapid changes in thyroid function tests upon treatment of tuberculosis.

SETTING: Inpatient service and tuberculosis (TB) clinic of a public hospital. OBJECTIVES: (1) To test the hypothesis that an hepatic effect of antituberculosis drugs increases serum thyroxine-binding globulin (TBG); (2) to resolve conflicting reports on thyroid function in TB. DESIGN: Measurement of serum thyroid hormones, thyroid hormone binding (T3RU) and binding proteins (TBG, transthyretin [TTR] and albumin) in 38 patients with active TB and in 29 healthy tuberculin-positive controls, before and about 10 days into therapy. RESULTS: With therapy of TB (with isoniazid [INH], rifampin [RIF], ethambutol and/or pyrazinamide), TBG increased above control values and T3RU decreased (P < 0.001). These changes were weakly correlated with liver enzyme activities but did not predict clinical hepatitis, which developed in only 1 patient. T3 was initially subnormal in 61% of 38 TB patients, while T4, thyrotropin (TSH) and TBG were normal. T3, TTR and albumin, all negative acute phase reactants, increased towards normal by day 10 (P < 0.001). Thyroid function remained unaltered in 14 control patients taking INH, whereas T3RU decreased (binding increased) and T3 increased in 15 taking INH and RIF (P < 0.001). CONCLUSIONS: TB patients manifest the expected low T3 of non-thyroid illness, but, unlike most sick patients, usually have normal or increased serum binding of thyroid hormones. Chemotherapy further increases binding by increasing TBG, an effect probably due to RIF.

Antitubercular Agents↗
PubMed1995

Isolated pulmonary mucormycosis in an apparently normal host: a case report.

Mucormycosis is a rare fungal disease commonly affecting individuals with diabetes mellitus, hematological malignancy, and immune deficiency. Isolated pulmonary mucormycosis is extremely rare. This article reports a case of isolated pulmonary mucormycosis that presented as a solitary cavity infiltrate in a patient with no underlying risk factors.

Aged↗
PubMed1994

The alpha-subunits of G-proteins G12 and G13 are palmitoylated, but not amidically myristoylated.

The alpha-subunits of the G-proteins G12 and G13 were expressed with a baculovirus system in insect cells and analysed for acylation. Both proteins incorporated tritiated palmitic and to a lesser extent also tritiated myristic acid. Radiolabel from both fatty acids was sensitive to treatment with neutral hydroxylamine. This result supports a thioester-type fatty acid bond and argues against amidical N-myristoylation. Fatty acid analysis after labeling with [3H]palmitic acid showed that palmitate represents the predominant fatty acid linked to G alpha 12 and G alpha 13. Separation of cells into cytosolic and membranous fractions revealed that palmitoylated alpha-subunits of G12 were exclusively membrane-bound, whereas [35S]methionine-labeled proteins were detected in soluble and particulate fractions. Inhibition of protein synthesis with cycloheximide did not block palmitoylation of the alpha-subunits, which indicates that palmitoylation occurs independently of protein synthesis.

Acylation↗
PubMed1994

Post-translational folding of the influenza C virus glycoprotein HEF: defective processing in cells expressing the cloned gene.

The post-translational processing of the influenza C virus glycoprotein HEF was analysed. In cells infected with influenza C virus, HEF protein is synthesized as a glycosylated 80K polypeptide. A post-translational conformational rearrangement involving the formation of intramolecular disulphide bonds results in a decrease in its electrophoretic mobility. Therefore, SDS-PAGE under non-reducing conditions suggests an Mr of about 100K, whereas under reducing conditions an 80K protein is observed which is in accordance with the sequence data. The 100K form was detected 10 min after synthesis of HEF, and transport to the cell surface took about 60 min. This result indicates that the conformational change presumably occurs in the endoplasmic reticulum. A difference in post-translational processing was observed when the HEF gene was expressed in the absence of other influenza C virus genes. In cells infected with recombinant simian virus 40, the 80K precursor was synthesized, but this protein was neither converted to the 100K form nor transported to the cell surface. Deletion of the short cytoplasmic tail of HEF (Arg-Thr-Lys) or replacement of the two basic amino acids by hydrophobic (Ile) or acidic residues (Glu) resulted in HEF protein which was partially converted to the 100K form. Influenza C virus glycoprotein obtained after transient expression of the HEF gene using the vaccinia virus system was completely converted to the 100K form. However, in neither expression system was HEF transported to the cell surface. The possibility is discussed that the interaction of HEF with another viral protein is required for the post-translational folding and transport of this glycoprotein. The M protein of influenza C virus is suggested as a candidate for the chaperone which might interact with the cytoplasmic tail of HEF.

Amino Acid Sequence↗
PubMed1994

Expression of the Sendai virus fusion protein in insect cells and characterization of its post-translational modifications.

The fusion (F) protein of Sendai virus was expressed with a baculovirus system in insect cells. The F protein is synthesized in its uncleaved form F0, which appears in SDS-containing gels as two bands with M(r) values of 66K and 64K. Digestion of the F protein with endoglycosidase H revealed that the 66K species contains high mannose-type carbohydrates, whereas partially processed oligosaccharides are attached to the 64K species. Pulse-chase experiments showed that the F protein is initially synthesized as its 66K form. After 1 h of chase this precursor glycoprotein is partially converted to the 64K species and exposed at the cell surface. Other modifications of the F protein in insect cells are the formation of intramolecular disulphide linkages and oligomerization. However, processing and plasma membrane transport of the F protein in insect cells are incomplete and retarded compared to the F protein synthesized in Sendai virus-infected mammalian cells.

Animals↗