PubMed Health⌕ Search

Biomedical subjects

P Overath

Publications and source records attributed to P Overath.

At least 73 records · Page 4Linked to original sources
PubMed1990

Suppressive macrophages occurring in murine Trypanosoma brucei infection inhibit T-cell responses in vivo and in vitro.

Intraperitoneal injection of Trypanosoma brucei AnTat 1.1 into mice of the C3H.He, BALB/c or C57BL/6 strains resulted in impaired immune responses from day 3 onwards, as measured by the reduction in DNA synthesis in spleen cell populations stimulated with concanavalin A (Con-A) in vitro. Adherent cells from the peritoneum (PC) or from the spleen of infected mice, consisting predominantly of macrophages, caused a 60-80% reduction of the Con-A response in spleen cells from syngeneic recipients 3-4 days after transfer in vivo. Adherent PC from irradiated or athymic mice were equally suppressive. Spleen cells from infected mice reduced the proliferative response of spleen cells from uninfected mice upon co-cultivation in vitro. This dominant suppressive effect was abolished after the selective removal of macrophages from the spleen cell population by treatment with L-leucine methylester. Moreover, the macrophage-depleted spleen cells from infected mice responded normally to Con-A provided they were supplemented with splenic adherent cells from naive mice as a source of accessory cells. Both the cell transfer and co-cultivation experiments suggest that infection with African trypanosomes changes the properties of macrophages to a state which allows them actively to suppress immune responses.

Animals↗
PubMed1989

Sequence and expression of the glycosyl-phosphatidylinositol-specific phospholipase C of Trypanosoma brucei.

Trypanosoma brucei contains a membrane-bound phospholipase C which converts the variant surface glycoprotein (VSG), anchored in the membrane by a C-terminal glycosyl-phosphatidylinositol moiety, into a soluble form and diacylglycerol. The amino acid sequence (358 residues) of this enzyme, derived from the nucleotide sequence of the cDNA and the gene, reveals a polypeptide which lacks an obvious N-terminal signal sequence and stretches of exclusively hydrophobic residues. These properties suggest that the phospholipase is synthesized in the cytoplasm and subsequently associates with or translocates across intracellular membranes. There are much higher levels of glycosyl-phosphatidylinositol specific phospholipase C mRNA in bloodstream form than in procyclic form trypanosomes. The phospholipase gene is probably present in one or two copies per haploid genome, probably not associated with VSG expression sites.

Amino Acid Sequence↗
PubMed1989

Fast measurement of galactoside transport by lactose permease.

Lactose permease of Escherichia coli was reconstituted into vesicles of dimyristoylphosphatidylcholine, and the rate of galactoside counterflow was measured in the millisecond time range. The turnover number and the half-saturation constant for transport agree with the values known for cells. This result demonstrates that lactose permease is the sole protein necessary for galactoside transport. Furthermore, lactose permease seems not to require a high level of negatively charged lipids or a certain degree of unsaturation of the lipid hydrocarbon chains. However, the lipids must be in the fluid state, because the transport rate drastically decreases below the lipid ordered fluid phase transition.

Biological Transport, Active↗
PubMed1989

Release of the variant surface glycoprotein during differentiation of bloodstream to procyclic forms of Trypanosoma brucei.

Investigations on the turnover of the membrane-form variant surface glycoprotein (mfVSG) of Trypanosoma brucei during cultivation in vitro of the monomorphic variant clones MIT at 1.2 and MIT at 1.4 showed that bloodstream forms slowly released the surface coat into the medium (time required to decline to half the initial amount, t50% = 32 +/- 3 h). VSG appeared in the medium in its soluble form (sVSG) which lacked the dimyristoylglycerol membrane anchor as judged by electrophoretic mobility and exposure of the cross-reacting determinant. The total VSG in the culture was very stable with a t50% = 189 +/- 24 h, compared to the other cellular proteins with a t50% approximately 28 h. Coat release during differentiation of bloodstream forms to procyclic cells could be distinguished from this turnover both by its more rapid kinetics (t50% = 13 +/- 1 h) and by the appearance in the medium of a predominant proteolytic fragment in addition to sVSG. Coat release during the transition to procyclic forms was not inhibited by the lysosomotropic agents ammonium chloride or chloroquine, by the proton ionophore monensin, or by the protease inhibitor tosyl-L-lysine chloromethyl ketone. The experiments demonstrate that coat release during differentiation is a specific cellular event distinct from simple turnover. The possibility is discussed that VSG release under both conditions occurs by endocytosis of mfVSG, degradation by a phospholipase C or a protease or both in a non-acidic intracellular compartment and recycling to the surface by exocytosis.

Ammonium Chloride↗
PubMed1989

Intracellular localization of the glycosyl-phosphatidylinositol-specific phospholipase C of Trypanosoma brucei.

Glycosyl-phosphatidylinositol-specific phospholipase C (GPI-PLC) is a membrane-bound enzyme of bloodstream forms of Trypanosoma brucei, which cleaves the GPI-membrane anchor of the variant surface glycoprotein forming diacylglycerol and 1,2-cyclic phosphate on the inositol ring. The cellular localization of the enzyme was studied by fractionation of sub-cellular organelles and immunofluorescence microscopy and was found to be primarily cytoplasmic. This was confirmed by immuno-electron microscopy using cryo-sections, which showed that the labelling was predominantly on the cytoplasmic side of intracellular membranes but was absent from the plasma membrane including the region lining the flagellar pocket. The significance of these results for the possible function of the phospholipase is discussed.

Animals↗
PubMed1988

Rapid lateral diffusion of the variant surface glycoprotein in the coat of Trypanosoma brucei.

The membrane form of the variant surface glycoprotein (mfVSG) is anchored in the plasma membrane of Trypanosoma brucei by a dimyristoylphosphatidylinositol residue connected via a glycan to the COOH-terminal amino acid. The glycoprotein molecules are tightly packed, forming a coat that is impenetrable to lytic serum components. Lateral diffusion of mfVSG was measured by the fluorescence recovery after photobleaching technique. mfVSG labeled on the cell surface with rhodamine-conjugated anti-VSG Fab fragments showed a diffusion coefficient of 1 X 10(-10) cm2/s at 37 degrees C and of 0.7 X 10(-10) cm2/s at 27 degrees C. About 80% of the molecules were mobile. Affinity-purified mfVSG molecules implanted into the plasma membrane of baby hamster kidney cells exhibited a similar mobility to that found in the trypanosome coat [D = (0.4-0.7) X 10(-10) cm2/s at 4 degrees C]. Phospholipid mobility in the plasma membrane of trypanosomes was characterized by a diffusion coefficient of 2.2 X 10(-9) cm2/s at 37 degrees C. It is concluded that mfVSG mobility in the surface coat of the parasite is rapid and comparable to that of other membrane-bound glycoproteins but slower than that of phospholipids.

Animals↗
PubMed1987

Repression and reactivation of the variant surface glycoprotein gene in Trypanosoma brucei.

Rapid repression of variant surface glycoprotein (VSG) synthesis is an early event during the in vitro transformation of Trypanosoma brucei from coated bloodstream forms to uncoated procyclic cells. Repression occurs at the transcriptional level and is triggered by the combined action of two signals: a reduction in temperature from 37 to 27 degrees C and the addition of the citric acid cycle intermediates citrate and cis-aconitate. It is shown that synthesis of VSG mRNA can be reactivated up to 8 h after triggering differentiation by releasing either one or both of the signals. After 30 h repression is irreversible. The results suggest that transformation of bloodstream forms to procyclic cells proceeds through a reversible phase to an irreversible committed state. A reversible repression of VSG mRNA synthesis is also observed upon inhibition of protein synthesis in bloodstream forms at 37 degrees C.

Aconitic Acid↗
PubMed1987

Secondary structure of the variant surface glycoproteins of trypanosomes.

The secondary structure of seven variant surface glycoproteins (VSGs) of trypanosomes has been determined by Raman spectroscopy. They are all predominantly alpha-helical, the alpha-helix content varying between 50 and 60%. The beta-strand content varies between 20 and 25%, and the content of beta-turn and nonregular structures is about 25%. For three VSGs the N-terminal domain obtained by proteolytic cleavage was found to have essentially the same secondary structure as the complete VSGs. For three VSGs a secondary structure prediction has been performed applying the rules of Chou and Fasman. In all cases, two long alpha-helices extending over about 50 residues or 80 A are predicted in agreement with the X-ray diffraction data of Freymann et al. [(1984) Nature 311, 167-169] and Metcalf et al. [(1987) Nature 325, 84-86]. The region between the two alpha-helical segments exhibits a high potential of beta-turns, suggesting that this segment may be exposed on the cell surface and carry major antigenic determinants.

Amino Acid Sequence↗
PubMed1987

Lactose permease of Escherichia coli: properties of mutants defective in substrate translocation.

Mutants of lactose permease of Escherichia coli with amino acid changes (Gly-24----Glu; Gly-24----Arg; Pro-28---Ser; Gly-24, Pro-28----Glu-Ser and Gly-24, Pro-28----Arg-Ser) within a putative membrane-spanning alpha-helix (Phe-Gly-Leu-Phe-Phe-Phe-Phe-Tyr-Phe-Phe-Ile-Met-Gly- Ala-Tyr-Phe-Pro-Phe-Phe-Pro-Ile) are incorporated into the cytoplasmic membrane. The mutant proteins retain the ability to bind galactosides, and the affinity for several substrates is actually increased. However, the rate of active transport is decreased to 0.01% of the wild-type rate in the mutants carrying Arg-24 or Arg-24, Ser-28. Kinetic analysis demonstrates that the two mutants require 10 min to cause occupied binding sites for galactoside and H+ to change their exposure from the periplasm to the cytoplasm as compared to 50 ms in the wild type. The effect is less pronounced when these sites are unoccupied.

Amino Acid Sequence↗
PubMed1987

RNA turnover in Trypanosoma brucei.

Regulation of variant surface glycoprotein (VSG) mRNA turnover in Trypanosoma brucei was studied in bloodstream forms, in procyclic cells, and during in vitro transformation of bloodstream forms to procyclic cells by approach-to-equilibrium labeling and pulse-chase experiments. Upon initiation of transformation at 27 degrees C in the presence of citrate-cis-aconitate, the half-life of VSG mRNA was reduced from 4.5 h in bloodstream forms to 1.2 h in transforming cells. Concomitantly, an approximately 25-fold decrease in the rate of transcription was observed, resulting in a 100-fold reduction in the steady-state level of de novo-synthesized VSG mRNA. This low level of expression was maintained for at least 7 h, finally decreasing to an undetectable level after 24 h. Transcription of the VSG gene in established procyclic cells was undetectable. For comparison, the turnover of polyadenylated and nonpolyadenylated RNA, beta-tubulin mRNA, and mini-exon-derived RNA (medRNA) was studied. For medRNA, no significant changes in the rate of transcription or stability were observed during differentiation. In contrast, while the rate of transcription of beta-tubulin mRNA in in vitro-cultured bloodstream forms, transforming cells, and established procyclic cells was similar, the half life was four to five times longer in procyclic cells (t1/2, 7 h) than in cultured bloodstream forms (t1/2, 1.4 h) or transforming cells (t1/2, 1.7 h). Inhibition of protein synthesis in bloodstream forms at 37 degrees Celsius caused a dramatic 20-fold decrease in the rate of VSG mRNA synthesis and a 6-fold decrease in half-life to 45 min, while beta-tubulin mRNA was stabilized 2- to 3-fold and mRNA stability remained unaffected. It is postulated that triggering transformation or inhibiting protein synthesis induces changes in the abundance of the same regulatory molecules which effect the shutoff of VSG gene transcription in addition to shortening the half-life of VSG mRNA.

Animals↗
PubMed1986

The effect of citrate/cis-aconitate on oxidative metabolism during transformation of Trypanosoma brucei.

Monomorphic bloodstream forms of Trypanosoma brucei, grown in the mammal, are deficient in aconitase and 2-oxoglutarate dehydrogenase and they do not respire in the presence of the substrates citrate, cis-aconitate, succinate, proline or 2-oxoglutarate. When grown in vitro low levels of aconitase, succinate oxidase and proline oxidase are detected. Addition of citrate/cis-aconitate at 37 degrees C to bloodstream forms leads to the formation of aconitase and proline oxidase. Most cells undergo an 'abortive' transformation to non-dividing procyclic-like cells while some cells adapt to the presence of the citric acid cycle intermediates and continue to multiply as bloodstream forms. At 27 degrees C and in the presence of citrate/cis-aconitate bloodstream forms transform synchronously to dividing procyclic cells. Within 72 h the rate of respiration with proline, succinate and 2-oxoglutarate becomes similar to that in established procyclic cells while the rate of glucose oxidation decreases. The possible role of citric acid cycle intermediates in determining whether a trypanosome will retain the properties of a bloodstream trypomastigote or differentiate to a procyclic trypomastigote is discussed.

Aconitic Acid↗
PubMed1986

Purification and characterization of the membrane-form variant surface glycoprotein hydrolase of Trypanosoma brucei.

The conversion of the membrane-form variant surface glycoprotein (mfVSG) of the unicellular parasitic flagellate Trypanosoma brucei to soluble variant surface glycoprotein and sn-1,2-dimyristoyl glycerol is catalyzed by an endogeneous, membrane bound phospholipase C-like hydrolase. Using a monoclonal antibody against the enzyme the hydrolase was purified 3,000-fold with a yield of 32%. The enzyme has a molecular weight of 39,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The rate with which mfVSG hydrolase cleaves phosphatidylinositol is 170 times lower than the cleavage rate for mfVSG, whereas phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylcholine cannot serve as substrates. Reconstitution experiments into phospholipid vesicles show that the enzyme can hydrolyze mfVSG when present in the same phospholipid bilayer but not when present in separate bilayers.

Animals↗
PubMed1986

Anti-peptide antibodies and proteases as structural probes for the lactose/H+ transporter of Escherichia coli: a loop around amino acid residue 130 faces the cytoplasmic side of the membrane.

From the amino acid sequence of the Escherichia coli lactose/H+ transporter, 7 hydrophilic segments were selected, 8-13 amino acids in length, and chemically synthesized, and anti-peptide antibodies were raised in rabbits. Apart from the antiserum to the synthetic COOH terminus (P408-417), which reacted strongly with the lactose/H+ transporter and has previously been used to localize the COOH terminus on the cytoplasmic face of the membrane, only those antibodies directed against the peptide corresponding to amino acid residues 125-135 (P125-135) exhibited a marked reaction with the transporter, while antibodies to the five other peptides reacted very weakly or not at all, suggesting that most of the hydrophilic segments are conformationally restricted or buried in the interior of the protein. Thermolysin treatment destroys the epitope on the transporter which is recognized by anti-P125-135 antibodies. Comparison of the kinetics and the extent of proteolysis of the transporter in right-side-out or inside-out cytoplasmic membrane vesicles or in reconstituted proteoliposomes suggests that the hydrophilic sequence from amino acid 125 to amino acid 135 is accessible to thermolysin only from one side, corresponding to the cytoplasmic face of the membrane. Furthermore, the experiments demonstrate that the transporter is inserted bimodally in a nonpreferential fashion into the proteoliposomes, confirming earlier results using antibodies to the synthetic COOH terminus of the transporter in conjunction with carboxypeptidase A treatment.

Amino Acid Sequence↗
PubMed1986

Biosynthesis of Trypanosoma brucei variant surface glycoproteins. N-glycosylation and addition of a phosphatidylinositol membrane anchor.

The variant surface glycoproteins (VSGs) of Trypanosoma brucei are synthesized with a hydrophobic COOH-terminal peptide that is cleaved and replaced by a glycophospholipid, which anchors VSG to the surface membrane. The kinetics of VSG processing were studied by metabolic labeling with [35S]methionine and [3H]myristic acid. The COOH-terminal oligosaccharide-containing structure remaining after phospholipase removal of dimyristyl glycerol from membrane-form VSG could be detected serologically within 1 min of polypeptide synthesis in two T. brucei variants studied. Addition of the oligosaccharide-containing structure was resistant to tunicamycin. VSGs synthesized in the presence of tunicamycin displayed lower apparent molecular weights, consistent with the complete inhibition of N-glycosylation at one (variant 117), two (variant 221), or at least three (variant 118) internal asparagine sites. In most experiments, N-glycosylation appeared to occur during or immediately after polypeptide synthesis but in a few cases N-glycosylation was delayed or incomplete. In all cases, addition of the COOH-terminal oligosaccharide-containing structure occurred normally. In dual-labeling studies, cycloheximide caused rapid inhibition of both [35S]methionine and [3H]myristic acid incorporation, suggesting that myristic acid addition also occurs immediately after polypeptide synthesis. Our data suggest that the complex ethanolamine-glycosyl-dimyristylphosphatidylinositol structure of membrane-form VSG is added en bloc within 1 min of completion of the polypeptide.

Animals↗
PubMed1986

Trypanosoma brucei: cis-aconitate and temperature reduction as triggers of synchronous transformation of bloodstream to procyclic trypomastigotes in vitro.

Synchronous transformation of the monomorphic Trypanosoma brucei 427 variant clone MITat 1.4 (117) from bloodstream to procyclic trypomastigotes was studied in modified minimum essential medium plus 15% inactivated horse serum. Repression of variant surface glycoprotein synthesis, subsequent morphological transformation, and growth of procyclic cells was triggered by the simultaneous action of two signals: a reduction in temperature from 37 to 27 C and the addition of cis-aconitate. Repression of variant surface glycoprotein synthesis initiated by these two signals is reversible during the first hours, but becomes irreversible after about 1 day. Thereafter, cells are committed to differentiation at 27 C.

Aconitic Acid↗
PubMed1985

Synthesis of a hydrolase for the membrane-form variant surface glycoprotein is repressed during transformation of Trypanosoma brucei.

A membrane-bound phospholipase C-like hydrolase present in lysates of bloodstream forms of Trypanosoma brucei rapidly converts the membrane form of the variant surface protein to the soluble form and 1,2-dimyristoylglycerol [(1985) M.A.J. Ferguson et al. J. Biol. Chem., 260, 4963-4968]. The hydrolase is inhibited by p-chloromercuribenzenesulfonate. The synthesis of the enzyme is rapidly repressed upon differentiation of bloodstream forms to procyclic cells and the enzyme activity declines to an undetectable level during subsequent growth of procyclic forms.

4-Chloromercuribenzenesulfonate↗