Biomedical subjects

G Jung

Publications and source records attributed to G Jung.

At least 379 records · Page 21Linked to original sources

PubMed1991

Myosin IB null mutants of Dictyostelium exhibit abnormalities in motility.

Cellular and intracellular motility are compared between normal Dictyostelium amoebae and amoebae lacking myosin IB (DMIB-). DMIB- cells generate elongated cell shapes, form particulate-free pseudopodia filled with F-actin, and exhibit an anterior bias in pseudopod extension in a fashion similar to normal amoebae. DMIB- cells also exhibit a normal response to the addition of the chemoattractant cAMP, including a depression in cellular and intracellular particle velocity, depolymerization of F-actin in pseudopodia, and a concomitant increase in cortical F-actin. DMIB- cells do, however, form lateral pseudopodia roughly three times as frequently as normal cells, turn more often, and exhibit depressed average instantaneous cell velocity. DMIB- cells also exhibit a decrease in the average instantaneous velocity of intracellular particle movement and an increase in the degree of randomness in particle direction. These findings indicate that if there is functional substitution for myosin IB by other myosin I isoforms, it is at best only partial, with myosin IB being necessary for maintenance of the normal rate and persistence of cellular translocation, suppression of lateral pseudopod formation and subsequent turning, rapid intracellular particle motility, and the normal anterograde bias of intracellular particle movement. Furthermore, it is likely that the behavioral abnormalities observed here for DMIB- cells underlie the delay in the onset of chemotactic aggregation, the increase in the time required to complete streaming, and the abnormalities in morphogenesis exhibited by DMIB- cells.

Actins↗

PubMed1991

Target cell-induced T cell activation with bi- and trispecific antibody fragments.

Previously we proposed a concept for tumor immunotherapy in which two different bispecific antibody conjugates, an anti-target x anti-CD3 and an anti-target x anti-CD28 conjugate, induce the activation of resting human T cells upon binding to the respective tumor target cells. After in vivo application of these reagents, this model of a "target cell-induced T cell activation" envisages the destruction of target cells by in situ activated T cells. Obviously however, for in vivo application, the use of Fc-free antibody fragments is mandatory to prevent binding of the conjugates to Fc receptor-positive cells which would lead to Fc-mediated T cell activation. Here we report a simplification of published procedures for the generation of bispecific Fab-hybrid fragments, univalent for each antigen. We demonstrate that an anti-target x anti-CD3/anti-target x anti-CD28 combination of such hybrids, as well as an identical combination of covalently coupled F(ab')2 fragments, mediate "target cell-induced T cell activation" in an in vitro test system. Thus, these reagents may be capable of inducing an in situ activation of human T cells upon systemic in vivo application according to the concept outlined above. A trispecific conjugate with anti-target, anti-CD3- and anti-CD28 specificity appears to be unsuitable for this purpose because it activates resting T cells in soluble form without requiring immobilization through binding via its anti-target portion.

Antibodies, Monoclonal↗

PubMed1991

Efficiency of peptides and lipopeptides for in vivo priming of virus-specific cytotoxic T cells.

Synthetic peptides and a novel type of lipopeptide vaccine, both containing T cell epitopes recognized by Kd-restricted, influenza virus-specific cytotoxic T cells (CTL) were compared in their efficiency to induce virus-specific CTL in vivo. All attempts to induce virus-specific CTL with synthetic peptide (in the absence of adjuvants) failed. However, a latent immunization was observed, resulting in an increased response to the injected peptide seen only after boosting the recipients with immunogenic virus. In contrast, priming with synthetic lipopeptide vaccine (tripalmitoyl-S-glycerylcysteinyl-seryl-serine [P3CSS] coupled to peptide) was successful under most conditions, and matched the priming efficiency seen with infectious virus. The requirements for in vivo priming of virus-specific CTL using lipopeptide suggest that attachment of the lipopeptide to a hydrophobic entity, such as the cell membrane, is responsible for its efficiency.

Animals↗

PubMed1991

Exact prediction of a natural T cell epitope.

T lymphocytes recognize their antigen as peptides associated with major histocompatibility complex (MHC) molecules. Peptides naturally presented by MHC class I molecules are uniform in length and have a specific motif, both defined by the respective MHC allele (Falk, K. et al. Nature 1991. 351:290). These allele-specific motifs should allow exact prediction of natural T cell epitopes. H-2Kb-restricted epitopes, for example, have a length of eight amino acid residues and conserved anchor residues at positions 5 and 8. According to this information, we predicted the natural Kb-restricted epitope of ovalbumin, thought to be contained in the 19-mer IINFEKLTEWTSSNVMEER, to be SIINFEKL. Here we show that this prediction is correct. Thus, exact prediction of natural T cell epitopes is possible.

Amino Acid Sequence↗

PubMed1991

Intracellular localization of a lipopeptide macrophage activator: immunocytochemical investigations and EELS analysis on ultrathin cryosections of bone marrow-derived macrophages.

Synthetic lipopeptides, structurally derived from the N-terminal part of bacterial lipoprotein, constitute macrophage and B-lymphocyte activators. The molecular mechanism of macrophage activation by lipopeptides still remains unclear. The purpose of our study was to determine the route and kinetics of lipopeptide distribution in bone marrow-derived macrophages. The intracellular localization of the C-terminally biotinylated lipodipeptide Pam3Cys-Ser was investigated on ultrathin cryosections using the biotinstreptavidin-gold system. Our findings indicate that the lipopeptide penetrates the plasma membrane and can already be found within the cytoplasm, the nuclear membrane, and within the nucleus after 2 min of stimulation. The pattern of lipopeptide distribution obtained 2 min after stimulation resembles that obtained after longer incubation times (8 and 20 min). Correlating distribution patterns were observed when using the method of electron energy loss spectroscopy (EELS). These findings are a clear indication for the rapid uptake of lipopeptides into eukaryotic cells, and are of importance for further studies of the immunostimulating properties of the bacterial lipopeptides and vaccines derived therefrom.

Animals↗

PubMed1991

Histopathological studies on the local reactions induced by complete Freund's adjuvant (CFA), bacterial lipopolysaccharide (LPS), and synthetic lipopeptide (P3C) conjugates.

The inflammatory reactions following subcutaneous application of adjuvants revealed characteristic pathological patterns. The injection of complete Freund's adjuvant (CFA) resulted in the formation of large lipid deposits encircled by an inflammatory reaction and concentrically arranged collagen bundles. Bacterial lipopolysaccharide (LPS) caused granulomatous aggregations of mononuclear cells with thrombotic vessel occlusions. Inoculation of the lipopeptide adjuvants induced accumulation of mononuclear cells with only minimal fibrotic changes which were resolved after day 28. Lipopeptide conjugates based on the head group tripalmitoyl-S-glyceryl-cysteinyl-serin (P3CS) can thus be used as effective immunogens and adjuvants without long-term tissue damage.

Animals↗

PubMed1991

Identification of enterobactin and linear dihydroxybenzoylserine compounds by HPLC and ion spray mass spectrometry (LC/MS and MS/MS).

The production of catecholate siderophores was studied in some selected species of Enterobacter (Enterobacteriaceae). The extracted catecholates were separated as iron-free compounds by HPLC on a C18 reversed-phase column using methanol/0.1% phosphoric acid or methanol/0.1% formic acid as a solvent system and identified by ion spray mass spectrometry (LC/MS, MS/MS). Five catecholate compounds were identified which include 2,3-dihydroxybenzoylserine, its linear dimer and trimer, the cyclic enterobactin and an unidentified isomer of enterobactin. In addition, a new large-scale method for the isolation of catecholate siderphores is described which is based on adsorption on XAD-2 and subsequent purification on Sephadex LH20.

Biopolymers↗

PubMed1991

Epitope mapping of the Dermatophagoides pteronyssinus house dust mite major allergen Der p II using overlapping synthetic peptides.

Fourteen synthetic peptides of 15 amino acid residues length, overlapping by five residues and spanning the entire sequence of the major allergen Der p II from the house dust mite Dermatophagoides pteronyssinus were synthesized. These peptides were coupled to CNBr-activated Sepharose-4B and used as solid-phase antigens in epitope mapping studies using human IgE antisera. These antibodies bound predominantly to the peptide comprising residues 65-78, the binding of which was inhibited by native Der p II. In addition these antisera bound, to a lesser extent, to the peptide that comprised residues 1-15, which binding was not inhibited by native Der p II. Thus, we found one sequential epitope for a number of IgE sera.

Allergens↗

PubMed1991

Lipopeptides containing 2-(palmitoylamino)-6,7-bis(palmitoyloxy) heptanoic acid: synthesis, stereospecific stimulation of B-lymphocytes and macrophages, and adjuvanticity in vivo and in vitro.

Lipopeptides, carrying the N-terminal lipoamino acid 2-(palmitoylamino)-6,7-bis(palmitoyloxy) heptanoic acid (Pam3Adh-OH, 1), were obtained by solid-phase synthesis and by synthesis in solution. 2-Amino-6,7-dihydroxyheptanoic acid (Adh) can be regarded as a methylene analogue of S-glycerylcysteine, the N-terminal amino acid of lipoprotein from the outer cell membrane of Escherichia coli (a methylene group substitutes for the sulfur atom). The lipopeptides Pam3Adh-Ser-Ser-Asn-Ala 2a-d, in which the four possible stereoisomers of Pam3Adh-OH (2S,6S)-1 (1a), (2S,6R)-1 (1b), (2R,6S)-1 (1c), and (2R,6R)-1 (1d) are linked to the naturally occurring sequence Ser-Ser-Asn-Ala of the N-terminus of lipoprotein, and also Pam3Adh-Ser-(Lys)4 [2S,6S)-3), with a peptide part rendering the molecule water soluble, were capable of stimulating murine splenocytes polyclonally in vitro, as determined in a proliferation assay and in a hemolytic plaque assay against trinitrophenylated sheep erythrocytes. The diastereomers (2S,6S)-2 and (2R,6S)-2 with S-configurated C-6 were more active than the diastereomers (2S,6R)-2 and (2R,6R)-2 with R-configurated C-6; a change of the configuration at C-2 had less effect on the stimulatory activity. (2S,6S)-2 and (2S,6S)-3 are potent immunoadjuvants. A significantly enhanced primary immune response against trinitrophenylated sheep erythrocytes was obtained in vitro at lipopeptide concentrations of about 5 micrograms/mL and an immunization dose of 10(7) sheep erythrocytes/mL. Balb/c mice, which were immunized with a mixture of ovalbumin and (2S,6S)-2 or (2S,6S)-3, respectively, had a substantially higher antiovalbumin titer 28 days after immunization than mice which had received ovalbumin, (2S,6S)-2 or (2S,6S)-3 alone. Finally, the novel lipopeptides constitute potent macrophage activators: (2S,6S)-3 was able to induce tumor cytotoxicity against the tumor cell line L929 in bone marrow derived macrophages.

Adjuvants, Immunologic↗

PubMed1991

Stable multicopy vectors for high-level secretion of recombinant human serum albumin by Kluyveromyces yeasts.

We have designed stable pKD1 derivatives for efficient secretion of recombinant human serum albumin (rHSA) by industrial strains of Kluyveromyces yeasts. A comparison of this multi-copy expression system with isogenic cassettes integrated at chromosomal loci demonstrated that high level secretion of rHSA is a function of gene dosage in K. lactis. Various signal sequences could be used, and the secretion levels were independent of the presence of the native pro peptide. The mitotic stability of the pKD1-based expression vectors was found to be species and strain dependent and was influenced by promoter strength and culture conditions. Vector stability was drastically enhanced when the HSA gene was expressed from an inducible promoter: 90% of the transformed cells still harbored the vector after 100 generations of non-selective growth in uninduced culture conditions. Secretion levels in the range of several grams per liter of correctly folded and processed rHSA were obtained at the pilot scale, thus making the industrial production of pharmaceutical-grade, Kluyveromyces-derived rHSA economically feasible.

Base Sequence↗

PubMed1991

Synthesis of novel immunologically active tripalmitoyl-S-glycerylcysteinyl lipopeptides as useful intermediates for immunogen preparations.

The synthesis and characterization of lipopeptides consisting of the lipoamino acid N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteine (Pam3Cys-OH) and different peptide segments and/or spacer molecules is described. Pam3Cys-peptides, which are derived from the immunologically active N-terminus of bacterial lipoprotein, were obtained either by solution or solid phase peptide synthesis. In particular, the amphiphilic and water-soluble lipohexapeptides Pam3Cys-Ser-(Lys)4 and Pam3Cys-Ser-(Glu)4 proved to be potent macrophage and B-cell activators and non-toxic, non-pyrogenic immune adjuvants in combination with or covalently linked to antigens and haptens.

Adjuvants, Immunologic↗

PubMed1991

Sulfonation of arginine residues as side reaction in Fmoc-peptide synthesis.

Several arginine-rich peptides containing the C-terminus of neuropeptide Y (NPY) were prepared by solid phase peptide synthesis using Fmoc chemistry and cleaved from the resin with trifluoroacetic acid (TFA). The products were characterized by fast atom bombardment-MS, LC-thermospray-MS, ion spray-MS/MS, and Edman degradation. The side products could be identified as peptides with sulfonated arginine residues resulting from an unexpected cleavage of Mtr or Pmc protecting groups. The degree of sulfonation depended on the choice and composition of the cleavage solution. Several scavenger mixtures were used and a mixture of thioanisole/thiocresol was found to be the most efficient for suppressing sulfonation. Furthermore treatment with the enzyme arylsulfate-sulfohydrolase desulfonated the peptides yielding the correct sequence.

Amino Acid Sequence↗

PubMed1991

Cleavage kinetics and anchor linked intermediates in solid phase peptide amide synthesis.

Kinetics and cleavage conditions of peptide amide synthesis were studied using the anchor molecules 5-(4'-aminomethyl-3',5'-dimethoxyphenoxy)valeric acid (4-ADPV-OH) and 5-(2'-aminomethyl-3'-5'-dimethoxyphenoxy) valeric acid (2-ADPV-OH). Unexpectedly the anchor amide alanyl-4-ADPV-NH2 was isolated and characterized as an intermediate during the cleavage with trifluoroacetic acid (TFA) of alanyl-4-ADPV-alanyl-aminomethyl-polystyrene to yield the alanine amide. As a matter of fact the NH--CH alpha bond of the alanyl spacer has to be cleaved to form this intermediate. Using TFA-dichloromethane (1:9) alanyl-4-ADPV-NH2 was obtained as a cleavage product in 50% yield within 60 min, whereas the isomeric alanyl-2-ADPV-NH2 was formed more slowly under these mild conditions. At high TFA concentration no difference between the 2- and 4-ADPV anchor was observed in the rate of formation of the free alanine amide. The presence of tryptophan amide in the cleavage mixture resulted in an anchor alkylated tryptophan amide, which remains stable in acidic solution but disappears rapidly in the presence of the resin. A low TFA/high TFA cleavage procedure is recommended for peptide amid synthesis applying the ADPV anchor.

Alkylation↗

PubMed1991

Synthesis of N alpha-Fmoc protected derivatives of S-(2,3-dihydroxypropyl)-cysteine and their application in peptide synthesis.

Acylated derivatives of S-(2,3-dihydroxypropyl)-cysteine (S-glycerylcysteine) form the N-terminus of structural and functional proteins of bacterial origin. Synthetic lipopeptides containing tripalmitoyl-S-glycerylcysteine are derived from bacterial lipoprotein and constitute potent immunoadjuvants activating both B-lymphocytes and macrophages. There is increasingly interest in conjugates consisting of tripalmitoyl-S-glycerylcysteine linked to appropriate viral and bacterial antigens, because of their capability of inducing antigen specific antibodies and T-helper and T-killer cell specific immune responses. A new convenient synthetic pathway for the preparation of these tripalmitoyl-S-glycerylcysteinyl peptides is described. The use of N alpha-Fmoc-protected S-(2,3-dihydroxypropyl)-cysteine and its O,O'-bis acylated derivatives for the synthesis of triacyl-S-glycerylcysteinyl, O,O'-bis-acyl-S-glycerylcysteinyl and S-glycerylcysteinyl peptides of high diastereomeric purity by solid phase peptide synthesis or synthesis in solution is demonstrated.

Amino Acid Sequence↗

PubMed1991

Molecular cloning of protozoan myosin I heavy chain genes.

Myosins I are ubiquitous, nonfilamentous, actin-based mechanoenzymes originally discovered in protozoa. The extensive in vitro biochemical studies of purified protozoan myosins I are now being complemented with in vivo studies using cloned myosin I heavy chain genes and gene targeting techniques. Here we review briefly the systems and methods being used in these efforts to dissect protozoan myosin I structure and function using molecular genetic approaches.

Acanthamoeba↗

PubMed1991

The antibody response in BALB/c mice to the Plasmodium falciparum circumsporozoite repetitive epitope covalently coupled to synthetic lipopeptide adjuvant.

The repetitive epitope of Plasmodium falciparum circumsporozoite protein (Asn-Ala-Asn-Pro)3 [(NANP)3] was coupled to tripalmitoyl-S-glyceryl-cysteine (P3C) and tripalmitoyl-S-glyceryl-cysteinyl-serine (P3CS). The lipopeptide P3CS is a potent B-cell and macrophage activator. The resulting immunogenic lipopeptides were used for immunization of the low responder mouse strain BALB/c. These low molecular weight conjugates induced specific anti-(NANP)3 IgG and IgM levels without any carrier proteins or admixed adjuvants after a single administration.

Adjuvants, Immunologic↗

PubMed1991

Patterns of actin filaments during cell shaping in developing mesophyll of wheat (Triticum aestivum L.).

Young leaves of wheat exhibit a smooth developmental gradient with meristematic cells at the base and highly differentiated cells at the tip. During differentiation, mesophyll cells attain a lobed outline resembling tube-shaped balloons with almost regularly spaced isthmi. Microfilament patterns in developing wheat mesophyll cells were investigated using fluorescent-labeled phalloidin. Various patterns were found, including delicate arrays of transversely oriented microfilaments in the cortex of the cytoplasm. A close correlation between changes in the patterns of cortical microfilaments, microtubules, cell wall microfibrils, and cell shape was observed. The fine arrays of transversely oriented microfilaments coaligned with bands of microtubules occurring during cell elongation. These bands were found beneath sites of intense wall deposition. It has recently been proposed that the resulting hoops of wall reinforcement prevent cell expansion in the corresponding regions and thus give rise to the peculiar cell shape. When cell expansion ceased, and the typical lobed cell shape was attained, a dense network of microfilaments was retained in the cytoplasm, which was in contrast to what has been described for the microtubular arrays.

Actin Cytoskeleton↗

PubMed1991

Ambiguous patterns in cellular differentiation in a case of "M3 variant" leukemia.

We report a case of acute non lymphoblastic leukemia in which clinical and cytological patterns corresponded closely to the M3 variant as defined in the FAB classification, although we did not find the characteristic t (15; 17) chromosomal translocation. However, cytochemistry, DNA content studies and immunophenotyping showed unusual patterns suggesting a monocytic differentiation of most of the blast cells, while a smaller population of blasts showed in contrast typical markers of granulocytic lineage.

Antigens, Neoplasm↗