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Biomedical subjects

B Becher

Publications and source records attributed to B Becher.

At least 37 records · Page 2Linked to original sources
PubMed1996

Soluble tumor necrosis factor receptor inhibits interleukin 12 production by stimulated human adult microglial cells in vitro.

IL-12 is a cytokine detected in active lesions in multiple sclerosis (MS) and promotes the acquisition of a Th1 cytokine profile by CD4+ T cells. Autoreactive T cells recovered from the central nervous system of animals with experimental autoimmune encephalomyelitis (EAE), a disease model for MS, display this phenotype. We demonstrate that human central nervous system-derived microglia, but not astroglia, can produce IL-12 in vitro. Under basal culture conditions, human adult microglia do not express detectable levels of IL-12, although these cells show some degree of activation as assessed by expression of the immunoregulatory surface molecules HLA-DR and B7 as well as low levels of TNF-alpha mRNA. Following activation with LPS, IL-12 p40 mRNA and p70 protein can be readily detected. IL-12 production is preceded by TNF-alpha production and is inhibited by recombinant soluble human TNF receptor (II)-IgG1 fusion protein (shu-TNF-R). These data indicate regulation of IL-12 by an autocrine-dependent feedback loop, providing an additional mechanism whereby shu-TNF-R, now used in clinical trials in MS, may be exerting its effect.

Adult↗
PubMed1996

Regulation of CD14 expression on human adult central nervous system-derived microglia.

Microglial cells function as regulators of immune reactivity within the CNS and may contribute to tissue injury under inflammatory conditions. Such functions are correlated with their state of activation. In this study, we report the de novo expression of CD14 by adult human CNS-derived microglia which acquire a bipolar activated morphologic phenotype in dissociated tissue culture. Surface CD14 expression can be down-regulated by interaction with its ligand lipopolysaccharide (LPS), and by the T-helper (Th1) cytokine interferon-gamma (IFN-gamma) or the Th2 cytokine interleukin-4 (IL-4). Semiquantitative polymerase chain reaction (PCR) analysis of CD14 mRNA expression under each condition suggests a different mechanism accounting for the reduced surface expression. LPS down-regulates CD14 mRNA, consistent with a feed-back signal preventing over-stimulation. IFN-gamma augments CD14 transcription, suggesting cleavage of surface CD14 consequent to general cell activation. IL-4 decreases mRNA production likely reflecting a generalized suppressive effect. The effect of LPS, IFN-gamma and IL-4 on CD14 expression differes from their effect on expression of the immune-accessory molecules B7-1 and HLA-DR, and on production of tumor necrosis factor-alpha (TNF-alpha), whose secretory pathway is similar to that of CD14. These results indicate the selective effects of molecules, likely to be present in the infected or inflamed CNS, on regulating CD14 expression and that there can be differential regulation of immune response relevant molecules expressed by activated microglia.

Adult↗
PubMed1996

Expression of the activation marker urokinase plasminogen-activator receptor in cultured human central nervous system microglia.

The ability of microglia to migrate through central nervous system (CNS) tissue requires proteolytic degradation of components of the extracellular matrix. Urokinase plasminogen activator (uPA), when bound to its cell surface receptor (uPAR), is an active cell surface protease. uPAR expression has been associated with cell activation. Cultured human microglia express surface uPAR. uPAR expression was found to be associated predominately with spindle- or bipolarshaped microglia. The addition of lipopolysaccharide (LPS) to microglial cultures enhanced the proportion of uPAR expression and shifted cell morphology to the elongated spindle or bipolar shape. When microglia were examined immediately ex vivo, uPAR surface expression could not be detected. Similarly, uPAR transcripts detected by reverse transcription-polymerase chain reaction techniques were found in cultured, but not ex vivo, microglia. Microglia isolated from a patient with multiple sclerosis (MS) displayed a large amount of uPAR+ cells. These cells were predominantly spindle or bipolar in nature. These findings suggest that uPAR surface expression is associated with microglial activation. Surface expression of uPAR and associated cell surface protease activity may provide a mechanism for microglial migration and may be important in the pathophysiology of MS.

Adult↗
PubMed1996

Sodium ion translocation by N5-methyltetrahydromethanopterin: coenzyme M methyltransferase from Methanosarcina mazei Gö1 reconstituted in ether lipid liposomes.

The N5-methyltetrahydromethanopterin (H,MPT):coenzyme M methyltransferase is a membrane associated, corrinoid-containing protein that uses the methylation of coenzyme M (HS-CoM) by methyl-tetrahydromethanopterin to drive an energy-conserving sodium ion pump. The enzyme was purified from acetate-grown Methanosarcina mazei Gö1 by a two-step solubilization with n-octyl-beta-glucoside, chromatography on hydroxyapatite, and by gel filtration on Superdex 200 or Sepharose CL-6B. The highly purified protein was apparently composed of six different subunits of 34, 28, 20, 13, 12, and 9 kDa. The N-terminal amino acid sequences of these polypeptides were determined. The native enzyme exhibited an apparent molecular mass of about 380 kDa. During purification, the enzyme was stabilized with 10 microM hydroxocobalamin. The highest specific activity reached during purification was 10.4 U/mg. The purified enzyme was reconstituted in monolayer liposomes prepared from ether lipids of M. mazei Gö1. In experiments with radioactive sodium ions, it was shown that the methyltransferase catalyzes the vectorial translocation of sodium ions across the membrane. Methyltransferase activity was stimulated by sodium ions. 1.7 mol Na-/mol methyl groups transferred were translocated. Methyltetrahydrofolate and methyl-cobalamin could substitute for methyl-H,MPT.

Amino Acid Sequence↗
PubMed1996

Comparison of phenotypic and functional properties of immediately ex vivo and cultured human adult microglia.

Microglial cells are resident cells of the CNS and are implicated as regulators and effectors of immune responses which occur within this compartment. The precise role of parenchymal microglia remains speculative because distinctions between these cells, perivascular "microglia," and blood-derived monocytes/macrophages are not well defined. The current study describes the phenotype and function of microglia immediately upon isolation from the non-inflamed adult human CNS and the phenotypic changes which occur in these cells when maintained in tissue culture. We find that the characteristic phenotype of immediately ex vivo parenchymal microglia (CD11c+/CD45low/CD14) corresponds to that found in situ in the "normal" human brain. The phenotype differs from that of perivascular "microglia" in situ and PBDM (both CD45hi/CD14++). The immediately ex vivo microglia express B7-2 and HLA class II molecules and can support alloantigen-induced proliferation by CD4+ T cells freshly isolated from peripheral blood. Following in vitro culture, the cells are characterized by a bipolar morphology, continued lower levels of CD45 expression compared to PBDM, and slight upregulation of B7-1 and HLA-DR antigen expression. CD14 becomes expressed at high levels on the cells, suggesting that CD14 can serve as an apparent marker of microglia activation which is not based on changes in morphology or APC capacity. Further, treatment of the cells with IFN-gamma and LPS causes further upregulation of HLA-DR and clear expression of B7-1 molecules on the surface. The capacity to characterize phenotypic and functional properties of microglia before and after activation provides an opportunity to determine means to manipulate the immune regulatory and effector properties of this cell type.

Adult↗
PubMed1996

IL-10 production by adult human derived microglial cells.

Microglia, a population of central nervous system (CNS) macrophages, have been demonstrated to support immune accessory and effector functions in the CNS. Numerous studies support the role of microglia in CNS development and pathology, where activation of microglia is consistently noted. The current study investigated microglial immune functions under basal and activation conditions and assessed the ability of interleukin-10 (IL-10), added exogenously or produced by microglia, to down-regulate microglial functions. This report demonstrates that microglia from the adult human brain produce IL-10 following interferon-gamma/lipopolysaccharide activation. Functionally, recombinant human IL-10 down-regulated basal HLA-DR expression by microglia and inhibited, in a dose-dependent response, the ability of microglia to stimulate CD4+ T-cells in antigen presentation assays. These data, together with recent observations of the inhibition of experimental allergic encephalomyelitis (EAE) following IL-10 administration and reduced CNS infection by Listeria monocytogenes after anti-IL-10 treatment, suggest that IL-10 production by microglia may have important immune-regulatory functions in CNS disease and disease models.

Adult↗
PubMed1995

Electron paramagnetic resonance spectroscopic and electrochemical characterization of the partially purified N5-methyltetrahydromethanopterin:coenzyme M methyltransferase from Methanosarcina mazei Gö1.

The N5-methyltetrahydromethanopterin:coenzyme M methyltransferase is a membrane-bound cobalamin-containing protein of Methanosarcina mazei Gö1 that couples the methylation of coenzyme M by methyltetra-hydrosarcinopterin to the translocation of Na+ across the cell membrane (B. Becher, V. Müller, and G. Gottschalk, J. Bacteriol. 174:7656-7660, 1992). We have partially purified this enzyme and shown that, in addition to the cobamide, at least one iron-sulfur cluster is essential for the transmethylation reaction. The membrane fraction or the partly purified protein contains a "base-on" cobamide with a standard reduction potential (Eo') for the Co2+/1+ couple of -426 mV. The iron-sulfur cluster appears to be a [4Fe-4S]2+/1+ type with an Eo' value of -215 mV. We have determined the methyltransferase activity at various controlled redox potentials and demonstrated that the enzyme activity is activated by a one-electron reduction with half-maximum activity occurring at -235 mV in the presence of ATP and -450 mV in its absence. No activation was observed when ATP was replaced by other nucleoside triphosphates or nonhydrolyzable ATP analogs.

Adenosine Triphosphate↗
PubMed1994

Delta mu Na+ drives the synthesis of ATP via an delta mu Na(+)-translocating F1F0-ATP synthase in membrane vesicles of the archaeon Methanosarcina mazei Gö1.

Methanosarcina mazei Gö1 couples the methyl transfer from methyl-tetrahydromethanopterin to 2-mercaptoethanesulfonate (coenzyme M) with the generation of an electrochemical sodium ion gradient (delta mu Na+) and the reduction of the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreoninephosphate with the generation of an electrochemical proton gradient (delta muH+). Experiments with washed inverted vesicles were performed to investigate whether both ion gradients are used directly for the synthesis of ATP. delta mu Na+ and delta mu H+ were both able to drive the synthesis of ATP in the vesicular system. ATP synthesis driven by heterodisulfide reduction (delta mu H+) or an artificial delta pH was inhibited by the protonophore SF6847 but not by the sodium ionophore ETH157, whereas ETH157 but not SF6847 inhibited ATP synthesis driven by a chemical sodium ion gradient (delta pNa) as well as the methyl transfer reaction (delta mu Na+). Inhibition of the Na+/H+ antiporter led to a stimulation of ATP synthesis driven by the methyl transfer reaction (delta mu Na+), as well as by delta pNa. These experiments indicate that delta mu Na+ and delta mu H+ drive the synthesis of ATP via an Na(+)- and an H(+)-translocating ATP synthase, respectively. Inhibitor studies were performed to elucidate the nature of the ATP synthase(s) involved. delta pH-driven ATP synthesis was specifically inhibited by bafilomycin A1, whereas delta pNa-driven ATP synthesis was exclusively inhibited by 7-chloro-4-nitro-2-oxa-1,3-diazole, azide, and venturicidin. These results are evidence for the presence of an F(1)F(0)-ATP synthase in addition to the A(1)A(0)-ATP synthase in membranes of M. Mazei Gö1 and suggest that the F(1)F(0)-type enzyme is an Na+-translocating ATP synthase, whereas the A(1)A(0)-ATP synthase uses H+ as the coupling ion.

Acetamides↗
PubMed1993

In vitro activation of the 20S proteasome.

The effect of chemical compounds like sodium dodecyl sulfate (SDS), fatty acid esters of glycerol, carnitine and coenzyme A, phospholipids, histones, polylysines as well as homobifunctional chemical cross-linkers on the various proteolytic activities of mammalian proteasomes have been tested. Most of the reagents enhance these activities, and some, e.g. fatty acid CoA esters, histones and the chemical cross-linkers, exert dual effects, i.e. activation and inhibition at the same time, depending on the activity measured. With optimally activating concentrations of SDS, no structural changes in proteasomes can be detected by electron microscopy. Formation of micelles at supra-optimal detergent concentrations may be a reason for irreversible denaturation of the proteasome.

Amino Acid Sequence↗
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

N5-methyl-tetrahydromethanopterin:coenzyme M methyltransferase of Methanosarcina strain Gö1 is an Na(+)-translocating membrane protein.

To determine the cellular localization of components of the methyltransferase system, we separated cell extracts of Methanosarcina strain Gö1 into cytoplasmic and inverted-vesicle fractions. Measurements demonstrated that 83% of the methylene-tetrahydromethanopterin reductase activity resided in the cytoplasm whereas 88% of the methyl-tetrahydromethanopterin:coenzyme M methyltransferase (methyltransferase) was associated with the vesicles. The activity of the methyltransferase was stimulated 4.6-fold by ATP and 10-fold by ATP plus a reducing agent [e.g., Ti(III)]. In addition, methyltransferase activity depended on the presence of Na+ (apparent Km = 0.7 mM) and Na+ was pumped into the lumen of the vesicles in the course of methyl transfer from methyl-tetrahydromethanopterin not only to coenzyme M but also to hydroxycobalamin. Both methyl transfer reactions were inhibited by 1-iodopropane and reconstituted by illumination. A model for the methyl transfer reactions is presented.

Carrier Proteins↗
PubMed1990

Quaternary N-glucuronides of 10-hydroxylated amitriptyline metabolites in human urine.

1. Conjugated metabolites were isolated from the urine of patients receiving amitriptyline treatment using a combination of solid-phase extraction, h.p.l.c. and t.l.c. 2. By n.m.r. and mass spectrometry, N-glucuronides of E- and Z-10-hydroxyamitriptyline and of trans-10,11-dihydroxyamitriptyline were identified in addition to the previously described O-glucuronides of E- and Z-10-hydroxyamitriptyline and -nortriptyline and amitriptyline-N-glucuronide. 3. The quaternary ammonium glucuronides proved to be resistant to acid hydrolysis, but could be cleaved enzymatically. 4. In urine samples from three patients, 35-60% of conjugated 10-hydroxyamitriptyline was found in the form of N-glucuronides. 5. A volunteer given an i.v. infusion of amitriptyline-N-glucuronide excreted E- and Z-10-hydroxyamitriptyline-N-glucuronide; following ingestion of E-10-hydroxyamitriptyline its N-glucuronide could be measured in urine.

Adult↗
PubMed1980

The photoconversion of lumirhodopsin at 77 degrees K. Estimation of the quantum efficiency.

Evidence is presented that lumirhodopsin (containing all-trans retinal) is not directly photoconverted to bathorhodopsin (all-trans) at 77 degrees K as previously suggested (Yoshizawa and Wald. 1963. Nature (Lond.) 197:1279-1286). Rather, lumirhodopsin is converted to a new species, L' (11-cis and/or 9-cis retinal) which, on warming to room temperature, is indistinguishable from rhodopsin or isorhodopsin. The quantum efficiency for the conversion of lumirhodopsin to L' is estimated to be 0.5 +/- 0.1. This value is significantly higher than that of other all-trans to cis conversions for bovine rhodopsin intermediates, indicating that the opsin conformation has a significant effect on a pigment's quantum efficiency.

Animals↗
PubMed1979

Resonance Raman study of the dark-adapted form of the purple membrane protein.

The resonance Raman spectrum of the dark-adapted form of the purple membrane protein (bacteriorhodopsin) has been obtained and is compared to the light-adapted pigment and model chromophore spectra. As in the light-adapted form, the chromophore-protein linkage is found to be a protonated Schiff base. Electron delocalization appears to play the dominant role in color regulation. The dark-adapted spectrum indicates a conformation closer to 13-cis than the light-adapted spectrum.

Bacteriorhodopsins↗
PubMed1978

The fluorescence from the chromophore of the purple membrane protein.

The fluorescence from the purple membrane protein (PM) of Halobacterium halobium and its relation to the primary photochemical events have been studied. The emission spectrum at 77 degrees K has structure, with peaks at 680, 710-715, and 730-735 nm. The excitation spectrum shows a single peak centered at 580 nm. This and a comparison of the fluorescence intensity at 77 degrees K under a variety of conditions with the amounts of the bathoproduct (or K, the only photoproduct seen at this temperature) formed suggest that the source of the fluorescence is the purple membrane itself, not the photoproduct. From the difference in several of their properties, we suggest that the fluorescing state of the pigment is different from the excited state which leads to photoconversion.

Bacteriorhodopsins↗