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G Weber

Publications and source records attributed to G Weber.

At least 253 records · Page 14Linked to original sources
PubMed1994

Increased expression of neprilysin (neutral endopeptidase 24.11) in rat and human hepatocellular carcinomas.

BACKGROUND: Neprilysin (EC 3.4.24.11) (NEP), a membrane metallopeptidase, is identical with common acute lymphoblastic leukemia antigen or cluster differentiation antigen 10. This antigen is present in blast cells in acute lymphoblastic leukemias and is implicated in differentiation of B lymphocytes. NEP cleaves a variety of peptides including bradykinin, substance P, bombesin, enkephalins, and atrial natriuretic peptide. We investigated its expression in several variants of rat hepatomas and a human hepatocellular carcinoma cell line. Normal rat and human livers were used as controls. EXPERIMENTAL DESIGN: The expression of NEP (common acute lymphoblastic leukemia antigen) was determined with: (a) enzyme assays; (b) high performance liquid chromatography analysis of bradykinin metabolism; (c) immunoprecipitation; and (d) mRNA characterization. RESULTS: NEP activity increased by 2 to 3 orders of magnitude in all rat hepatomas and in the human SK-HEP1 cell line, compared with normal tissues. Antiserum against rat NEP precipitated 93% of endopeptidase activity in rat hepatomas, whereas monoclonal antibody to common acute lymphoblastic leukemia antigen immunoprecipitated 99% of that in human hepatocarcinoma cells. Solubilized rat hepatoma membranes cleaved bradykinin to a hepta- and dipeptide; the reaction was inhibited by an NEP inhibitor. Activity of three other membrane peptidases did not increase in rat hepatomas. Northern hybridization revealed the presence of NEP mRNA in rat hepatoma, but not in normal liver. Reverse transcriptase-polymerase chain reaction showed that hepatomas have higher amounts of NEP mRNA than normal liver of the same strain. CONCLUSIONS: Rat hepatomas and a human hepatocarcinoma cell line express high amounts of NEP, in contrast to normal rat and human livers, which have very little. The increase in NEP activity could be due to increased transcription by tumor cells and may signal malignant transformation of liver cells.

Animals↗
PubMed1993

Sequential impact of tiazofurin and ribavirin on the enzymic program of the bone marrow.

Tiazofurin and ribavirin are clinically used inhibitors of IMP dehydrogenase (DH), binding to the NAD and IMP sites, respectively, of the target enzyme. In patients with chronic granulocytic leukemia in blast crisis, daily tiazofurin infusions decreased the high IMP DH activity in blast cells and resulted in 77% response (G. Weber. In: R. A. Harkness et al., Purine and Pyrimidine Metabolism in Man, Vol. VII, Part B, pp. 287-292, 1991). However, patients relapsed in a few weeks with emergence of high IMP DH activity (G. Tricot et al., Int. J. Cell Cloning, 8: 161-170, 1990). The present study showed that the tiazofurin-induced depression of IMP DH activity in rat bone marrow can be maintained by ribavirin injection. Tiazofurin (150 mg/kg, i.p., once a day for 2 days) decreased IMP DH activity to 10% and ribavirin (250 mg/kg, i.p., once a day for the subsequent 3 days) maintained the enzymic activity at 20 to 30% of control values. In control rats where no ribavirin was given, IMP DH activity of the tiazofurin-treated rats rapidly returned to the range of untreated animals. The decrease of IMP DH activity (t1/2 = 2.6 h) sharply preceded that of the bone marrow cellularity (t1/2 = 17.4 h). In addition to the target enzyme, IMP DH, tiazofurin also decreased activities of the guanylate metabolic enzymes, guanine phosphoribosyltransferase and GMP reductase, and the pyrimidine salvage enzymes, deoxycytidine and thymidine kinases with t1/2 of 2.6, 4.7, 6.0, 3.4, and 6.5 h, respectively. In cycloheximide-treated rats, where much of protein biosynthesis was blocked, the t1/2(8) of these five enzymes in bone marrow were shorter, 1.6, 4.3, 3.0, 0.6, and 0.8 h, respectively. Thus, the impact of tiazofurin in the bone marrow entails a decrease in the activity of the target enzyme, IMP DH, and also of other enzymes in purine and pyrimidine biosynthesis as a result of the enzyme half-lives shortened by this drug. These novel observations should assist in achieving better protection and recovery of bone marrow during and after chemotherapy.

Animals↗
PubMed1993

Plurality of protein conformations of ribulose-1,5-bisphosphate carboxylase/oxygenase monomers probed by high pressure electrophoresis.

We used hydrostatic pressure in the range of 1 to 2 kbar, coupled with polyacrylamide gel electrophoresis, to investigate the properties of monomers of dimeric ribulose bisphosphate carboxylase/oxygenase. At temperatures below -5 degrees C or pressures above 1.5 kbar, only a diffuse band with low electrophoretic mobility was observed, which is assigned to a denatured monomer. In gels run at 1.0 kbar and temperatures above 0 degree C, both the wild type and a mutant in which a positively charged Lys at the dimer interface is replaced by a negatively charged glutamic acid displayed several discrete bands with retardation coefficients larger than that of the dimer. Cross-linking due to oxidation of cysteines was not the reason for the multiplicity of bands, which in addition were independent on the length of the electrophoretic run in the range of 1-3 h. Binding of 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid to the pressure dissociated monomers stabilized the unfolded conformations. We propose that the dissociated monomers adopt various expanded conformations, which, under the experimental conditions, are stabilized to the extent necessary to be considered as distinct chemical species. Gel filtration high performance liquid chromatography analysis of bands eluted from nonstained gels run at 1 kbar (15 degrees C) and 1.5 kbar (-5 degrees C), respectively, was performed. In both cases the dimeric structure was fully recovered, along with the spectroscopic properties and catalytic activity characteristic of the native dimer, indicating that the unresolved unfolded conformers that appear at -5 degrees C, as well as the set of discrete conformers obtained at 15 degrees C, are able to reconstitute a single active conformation on reassociation.

Electrophoresis, Polyacrylamide Gel↗
PubMed1993

Tiazofurin-induced changes in inositol lipid cycle in nuclei of Friend erythroleukemia cells.

When murine erythroleukemia cells are treated up to 96 hrs with the antitumor drug tiazofurin the induction of erythroid differentiation is accompanied by changes in both synthesis and breakdown of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5 bisphosphate in the nucleus. These changes, which parallel the effect of dimethylsulfoxide, a well known inducer of erythroid differentiation in vitro, are due to the inhibition of the nuclear phosphoinositidase C activity. Therefore the present data indicate that nuclear inositol lipid cycle as a target of tiazofurin in addition to its inhibition of IMP dehydrogenase (EC 1.1.1.205) activity.

Animals↗
PubMed1993

Nutritional and hormonal regulation of liver deoxycytidine kinase activity.

Administration of CdR (50 mg/100 g every 12 h) elevated hepatic CdR kinase (EC 2.7.1.74) activity in normal or adrenalectomized rats over the control values 2- to 2.7-fold. Actinomycin (5 micrograms/100 g) or cycloheximide (50 micrograms/100 g) blocked the increase in enzyme activity. In starved rats, liver CdR kinase decreased to 64% compared to fed controls; refeeding restored enzyme activity to normal range. Steroid treatment (triamcinolone 1 mg/100 g/day) elevated liver CdR kinase activity to 138%, 169% and 193% of controls in 1, 2 and 3 days, respectively. Actinomycin or cycloheximide prevented this rise. These data suggest that the substrate and steroid induction of hepatic CdR kinase activity may be due to increased mRNA production and enhanced protein biosynthesis.

Adrenalectomy↗
PubMed1993

Differential modulation of the effects of lipopolysaccharide on macrophages by a major outer membrane protein of Proteus mirabilis.

We previously showed that a major protein isolated from purified cell walls of Proteus mirabilis (39-kDa protein) is a strong modulator of the specific immune responses to LPS from this bacterium in mice. When mixed with LPS before immunization, this protein enhances T cell-dependent, IgG antibody-producing cell responses specific for LPS. Furthermore, complexes of the 39-kDa protein with LPS drastically inhibit the production of oxygen radicals by murine macrophages activated with LPS, as measured in a chemiluminescence assay. In the present report, we have further investigated possible modulating effects of the protein at the level of LPS-macrophage interaction. When mixed with LPS, the 39-kDa protein inhibited IL-1 production by murine macrophages derived from bone marrow in a dose-dependent manner, as determined in an IL-2-dependent IL-1 assay. On the other hand, the protein had little effect on LPS-mediated suppression of MHC class II expression on the surface of macrophages induced with IFN-gamma. Some abrogation of suppression was observed, but the amounts of protein needed for this effect were quite large, in comparison with the amounts rendering inhibition of IL-1 production. In contrast, the 39-kDa protein enhanced the LPS-induced cytotoxicity of macrophages against L929 target cells, primarily as the result of production of TNF. These results show that the 39-kDa protein is a potent modulator of the interaction of LPS with macrophages, exerting its effects in a differential manner with respect to various parameters of LPS-induced activation of macrophages.

Animals↗
PubMed1993

Physical heterogeneity of muscle glycogen phosphorylase revealed by hydrostatic pressure dissociation.

Four independent methods that employ fluorescence spectroscopy show that the tetramer of glycogen phosphorylase A (GPA) from rabbit muscle is reversibly dissociated into monomers by hydrostatic pressures under 2.5 kbar, if aggregation of the monomers is prevented by the addition of 8% glycerol. The free energy of association at 20 degrees C (-32 kcal mol-1) depends upon a large entropy increase (T delta S = +65 kcal mol-1) that counteracts an unfavorable enthalpy of association of +33 kcal mol-1. The association volumes calculated from the pressure dependence of the dissociation are nearly 4-fold smaller than those calculated from the shift in dissociation pressure with concentration. The dimer obtained by dilution of GPA at atmospheric pressure differs from the hypothesized dimer intermediate in the pressure dissociation by the much larger monomer affinity of the former. Like other tetramers, GPA shows hysteresis of the pressure profile upon decompression and conformational drift of the dissociated monomers. By use of the energy transfer method it is demonstrated that the relaxation time for half-dissociation (5 min) is over an order of magnitude shorter than that for subunit exchange (118 min). In all three tetramers studied, lactate dehydrogenase, glyceraldehyde phosphate dehydrogenase, and glycogen phosphorylase, the deterministic character of the dissociation equilibrium under pressure and the anomalous concentration dependence of the pressure dissociation demonstrate that these tetramers are heterogeneous populations with regard to their free energy and/or volumes of association.

Animals↗
PubMed1993

Reversible pressure dissociation of R17 bacteriophage. The physical individuality of virus particles.

In the absence of urea, pressures up to 2.5 kbar promote only 10% dissociation of the whole particles of R17 bacteriophage. In the presence of concentrations of urea between 1.0 and 5.0 M, pressure promotes complete, reversible dissociation of the virus particles. At the lower urea concentrations reversible dissociation of R17 virus particles shows no dependence on protein concentration indicating a high degree of heterogeneity of the particles, but higher urea concentrations, 2.5 to 5.0 M, result in progressive restoration of the protein concentration dependence of the pressure dissociation. At still higher urea concentrations, 5.0 to 8.0 M, irreversible dissociation of virus takes place at atmospheric pressure. In contrast, the dissociation of the isolated dimers of the capsid protein was dependent on protein concentration to the extent predicted for a stochastic equilibrium, and dimers were much less stable than the whole virus both to dissociation by pressure or urea. In contradistinction, the reversible whole-virus dissociation observed at urea concentrations below 2.5 M appears to be a typical deterministic equilibrium, without appreciable dynamic exchange between whole particle and subunits during the lengthy experiments. The experiments demonstrate that the "thermodynamic individuality" of the virus particles arises in conformational differences in the assembled viruses, and that there is a direct relation between the stability of the particles and their heterogeneity.

Capsid↗
PubMed1993

Characterization of the sporulation-related gamma-D-glutamyl-(L)meso-diaminopimelic-acid-hydrolysing peptidase I of Bacillus sphaericus NCTC 9602 as a member of the metallo(zinc) carboxypeptidase A family. Modular design of the protein.

The sporulation-related gamma-D-glutamyl-(L)meso-diaminopimelic-acid-hydrolysing peptidase I of Bacillus sphaericus NCTC 9602 has been analysed by proton-induced X-ray emission. It contains 1 equivalent Zn2+ per mol of protein. As derived from gene cloning and sequencing, the B. sphaericus Zn peptidase I is a two-module protein. A 100-amino-acid-residue N-terminal domain consisting of two tandem segments of similar sequences, is fused to a 296-amino-acid-residue C-terminal catalytic domain. The catalytic domain belongs to the Zn carboxypeptidase A family, the closest match being observed with the Streptomyces griseus carboxypeptidase [Narahashi (1990) J. Biochem. 107, 879-886] and with the family prototype, bovine carboxypeptidase A. The catalytic domain of the B. sphaericus peptidase I possesses, distributed along the amino-acid sequence, peptide segments, a triad His162-Glu165-His307 and a dyad Tyr347-Glu366 that are equivalent to secondary structures, the zinc-binding triad His69-Glu72-His196 and the catalytic dyad Tyr248-Glu270 of bovine carboxypeptidase A respectively. The N-terminal repeats of the B. sphaericus peptidase I have similarity with the C-terminal repeats of the Enterococcus hirae muramidase 2, the Streptococcus (now Enterococcus) faecalis autolysin and the Bacillus phi PZA and phi 29 lysozymes, to which a role in the recognition of a particular moiety of the bacterial cell envelope has been tentatively assigned. Detergents enhance considerably the specific activity of the B. sphaericus peptidase I.

Amino Acid Sequence↗
PubMed1993

Reversible dissociation and conformational stability of dimeric ribulose bisphosphate carboxylase.

Dimer-monomer dissociation of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum was investigated using hydrostatic pressure in the range 1-2 kbar to promote dissociation. Intrinsic fluorescence emission and polarization, along with the polarization of the fluorescence of single-labeled AEDANS conjugates, were used to follow the dissociation. Full reversibility after dissociation was observed to depend on the presence of small ligands: glycerol, Mg2+, and NaHCO3, the last two being required to activate the enzyme. The free energy of association at 15 degrees C, -12.9 kcal mol-1, was made up of a positive change in enthalpy on association of 6.0 kcal mol-1 and an entropic contribution (T delta S) of 18.9 kcal mol-1; thus the monomer association is entropy driven. No dissociation of the quaternary complex formed by the dimer, 2-carboxy-D-arabinitol 1,5-diphosphate (CADP), Mg2+, and NaHCO3 was observed at pressures up to 2.0 kbar; the magnitude of stabilization by the inhibitor binding was estimated as 2.3 kcal mol-1. Pressurization in the presence of bis-ANS results in a time-dependent increase in fluorophore emission, indicating changes in monomer conformation with exposure of hydrophobic surfaces upon dissociation. Reactivity against the fluorescent probe 1,5-I-AEDANS was also used as a conformational probe: HPLC of a trypsin digest of rubisco labeled at atmospheric pressure revealed a single fluorescent peptide, whereas more extensive labeling was observed when the reaction was carried out at 2.0 kbar, indicative of exposure of internal cysteines.(ABSTRACT TRUNCATED AT 250 WORDS)

Bicarbonates↗
PubMed1993

A fast and sensitive method for detection of phospholipid-binding proteins on nitrocellulose membranes.

We describe a sensitive new method for specific detection and quantitation of phospholipid-binding proteins using two purified lipocortins as model proteins. The method consists of blotting proteins to nitrocellulose membranes followed by incubation with radiolabeled phospholipids and autoradiographic detection of bound phospholipids. It allows specific detection of phospholipid binding proteins to a threshold of about 100 ng/spot and their quantitation up to several micrograms as well as rapid analysis of specific phospholipid binding properties of individual proteins. If performed as a dot-blotting technique, the method permits the simultaneous analysis of a large number of samples and the establishment of Ca2+ and phospholipid binding curves. If blotting is preceded by electrophoresis, the method also allows specific detection and quantitative estimation of phospholipid binding proteins in crude biological preparations and may thus be useful for monitoring and analysis of these proteins in various biological samples.

Animals↗
PubMed1993

Assignment of the human FAU gene to a subregion of chromosome 11q13.

The FAU gene is the cellular homologue of the fox sequence in the Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV). FAU (for FBR-MuSV associated ubiquitously expressed gene) encodes the ribosomal protein S30 fused to a ubiquitin-like protein. A cosmid clone containing the human FAU gene was used for fluorescence in situ hybridization to metaphase chromosomes. The obtained localization to 11q13 was confirmed by hybridization against a panel of somatic cell hybrids containing different parts of chromosome 11 on a hamster background. FAU was then further mapped, both on a panel of radiation-reduced somatic cell hybrids designed to carry different parts of the 11q13 region and by pulsed-field gel electrophoresis. This fine mapping assigned FAU close to the skeletal muscle glycogen phosphorylase gene (PYGM), in a region that contains several oncogenes as well as the putative tumor suppressor genes MEN1 and ST3.

Animals↗
PubMed1993

Regulation of deoxycytidine kinase activity and inhibition by DFDC.

(1) Deoxycytidine kinase activity increased in a transformation- and progression-linked fashion in rat hepatomas of different proliferation rates. The activity also increased and was growth rate-linked in a series of tissue culture cell lines of human and animal tumors. (2) Deoxycytidine kinase activity was stringently linked with expression of the neoplastic proliferative program as it sharply increased in log phase in tissue culture cells of hepatoma 3924A and several human carcinoma strains. (3) Deoxycytidine kinase is subject to nutritional and hormonal regulation. On starvation the activity in liver decreased and on refeeding it returned to normal. Steroid hormone increased liver enzymic activity. Deoxycytidine kinase is substrate-inducible, since deoxycytidine injections in rat led to a 2- to 3-fold increase in hepatic enzyme activity. (4) Actinomycin or cycloheximide treatment blocked the increase in liver deoxycytidine kinase activity induced by steroid or deoxycytidine treatment. Therefore, it is assumed that the rise in deoxycytidine kinase activity requires new RNA and protein synthesis. (5) Cycloheximide treatment of rats carrying hepatomas yielded a t1/2 = 3.4 hr in the tumor for deoxycytidine kinase activity which was the shortest among the examined enzymes of purine and pyrimidine biosynthesis. (6) Actinomycin treatment of rats carrying hepatomas yielded a t1/2 of 5.8 hr for deoxycytidine kinase activity in the tumor which was one of the shortest in the examined enzymes of purine and pyrimidine biosynthesis. (7) Difluorodeoxycytidine (DFDC) is a competitive inhibitor (Ki = 7-28 microM) of deoxycytidine kinase from rat hepatoma and from human pancreatic carcinoma and ovarian carcinoma cells in culture.

Animals↗
PubMed1993

Cloning and characterization of a surface antigen of Eimeria tenella merozoites and expression using a recombinant vaccinia virus.

A rabbit serum raised against Eimeria tenella merozoites was used to screen a lambda gt11 cDNA library made from merozoite mRNA of E. tenella. The insert of the phage clone lambda Mz 5-7 revealed an open reading frame consisting of 945 nucleotides, encoding a 33-kDa protein. This size is consistent with the size of a protein translated in vitro from merozoite mRNA and immunoprecipitated with monospecific anti-Mzp 5-7 antibodies. A smaller protein of 24 kDa, located on the surface of the parasite, also reacted with the monospecific antiserum and is the potential processed form of the Mzp 5-7. Furthermore, a recombinant vaccinia virus expressing the Mzp 5-7 antigen was constructed and used to immunize chickens.

Amino Acid Sequence↗
PubMed1993

[Prenatal diagnosis of fetal teratomas].

Teratomas are the most frequent tumours in a newborn child. Prenatal ultrasound allows an early diagnosis with high significance. Six cases are described by prenatal sonographic signs, clinical management and pathologic-anatomic findings. Since teratomas are mostly benign and surgery is often successful, sufficient cooperation between obstetricians, surgeons and neonatologists is important.

Adult↗
PubMed1993

Blood cell activation: new perspectives from ultrastructural morphometry.

The new computerized approaches for image analysis available for a few years permit, in an excellent and detailed way, monitoring cellular structural modifications during activation processes. Here, we presented morphometric data obtained on blood cells (platelets, monocytes, and PMN cells) after in vitro activation and under in vivo conditions related to atherosclerosis studied by means of electron microscopy. Area and perimeter form factors, nucleus to cytoplasma area ratio, and the surface density of platelet plasma membrane openings of SCCS presented significant differences compared with the respective significant differences compared with the respective controls. Ultrastructural markers of blood cell activation were revealed by the computerized morphometry applied on ultrastructural images.

Animals↗