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Biomedical subjects

G Weber

Publications and source records attributed to G Weber.

At least 325 records · Page 18Linked to original sources
PubMed1991

Determination of thiazole-4-carboxamide adenine dinucleotide (TAD) levels in mononuclear cells of leukemic patients treated with tiazofurin.

Tiazofurin is an oncolytic agent which has shown therapeutic activity in end-stage acute nonlymphocytic leukemia (ANLL) and blast crisis of chronic granulocytic leukemia (CGL-BC). Tiazofurin is anabolized to the active metabolite, thiazole-4-carboxamide adenine dinucleotide (TAD), which inhibits IMP dehydrogenase activity, leading to reduction of guanylate pools and cessation of cancer cell proliferation. The concentration of TAD in neoplastic cells of patients treated with tiazofurin should be a good indicator of sensitivity to the drug and also might herald the emergence of drug-resistant cells. Therefore, the precise quantitation of TAD in cancer cells during tiazofurin treatment is essential. In this paper we report a highly sensitive method for the determination of TAD in biological samples. With this technique, in addition to TAD, thirteen other biologically relevant adenine, guanine, cytosine and uridine nucleotides can be separated and quantitated accurately. TAD standard was separated on a Waters Partisil 10-SAX column in a RCM-10 module using an ammonium phosphate buffer system. TAD eluted at 21 min with a limit of detection of 15 pmol and linearity up to 3 nmol. The coefficient of variation was 0.6 +/- 0.1% for retention time and 2 +/- 0.3% for TAD concentration. Recovery of TAD was 96% with reproducibility of 98%. To examine the applicability of this method to a clinical setting, blood samples were obtained from a patient with CGL-BC and leukocytes were separated on a Ficoll-Hypaq gradient, extracted with trichloroacetic acid, and an aliquot was analyzed on HPLC. The TAD peak was identified by comparing the retention time and spectral analysis of the standard. After the patient was treated with a 2200 mg/m2 (12.7 mM) dose of tiazofurin, the TAD concentrations in the mononuclear cells at 2, 6, and 24 hr were 23.1, 13.6, and 0.8 microM. TAD levels at 2, 6, and 24 hr after a tiazofurin dose of 3300 mg/m2 (21.1 mM) were 42.8, 26.1, and 1.4 microM respectively.

Adenine Nucleotides↗
PubMed1991

Expression of human IMP dehydrogenase types I and II in Escherichia coli and distribution in human normal lymphocytes and leukemic cell lines.

Two distinct cDNAs encoding proteins with 84% sequence identity have been isolated for human IMP dehydrogenase (EC 1.1.1.205) (Natsumeda, Y., Ohno, S., Kawasaki, H., Konno, Y., Weber, G., and Suzuki, K. (1990) J. Biol. Chem. 265, 5292-5295), an important target in antileukemic chemotherapy. We constructed expression plasmids containing these cDNAs in full length with pUC plasmids and produced lacZ'-IMP dehydrogenase fusion proteins in Escherichia coli. Both synthesized proteins exhibited IMP dehydrogenase activity and were partially separated from endogenous E. coli IMP dehydrogenase. By injecting the fusion proteins into mice we generated a polyclonal antibody specific to type I IMP dehydrogenase and an antibody which reacted with both types. Immunoblot analysis revealed that the total amounts of types I and II enzymes increased in human leukemic cell lines K562 and HL-60 in agreement with the increase in IMP dehydrogenase activity to 7.8- and 9.4-fold, respectively, above that of normal lymphocytes. The extent of expression of type I IMP dehydrogenase was similar in these cells, however, indicating that the increase in IMP dehydrogenase amount in leukemic cells was due to specific up-regulation of type II enzyme. Northern blot analysis also showed specific and predominant expression of type II in the leukemic cells. Thus, de novo GTP biosynthesis may be controlled differently in normal and neoplastic cells by different IMP dehydrogenase molecular species.

Cell Line↗
PubMed1991

Regulation of de novo and salvage pathways in chemotherapy.

An overview was presented of our approach of inhibition of de novo and salvage pathways in pyrimidine and purine metabolism. 1. Combination of acivicin, an inhibitor of de novo biosynthesis, and dipyridamole, a transport inhibitor, provided synergistic cytotoxicity in hepatoma and colon carcinoma cells. 2. AZT, a competitive inhibitor of the salvage enzyme, thymidine kinase, and 5-FU or MTX provided synergistic cytotoxicity in hepatoma 3924A. In human colon carcinoma HT-29 cells AZT and methotrexate yielded synergistic cytotoxicity and thymidine and hypoxanthine together provided protection from the action of these drugs. 3. These observations are significant because in rat hepatoma 3924A and in human cell lines HT-29, HL-60 and K562 thymidine kinase activity was 16- to 67-fold higher than that of dTMP synthase. Therefore, inhibition of dTMP synthase activity alone may provide poor responses because the salvage pathways can circumvent this block. 4. In leukemic patients treated with tiazofurin, an inhibitor of IMP dehydrogenase, the rate-limiting enzyme of GTP biosynthesis, and with allopurinol, which inhibits GPRT activity through raising plasma hypoxanthine levels, synergistic therapeutic results were obtained. The responses in sensitive patients entailed a decrease in IMP dehydrogenase activity and GTP concentration in leukemic cells and down-regulation of the ras and myc oncogenes. The down-regulation of the ras oncogene by tiazofurin through the decrease of GTP concentration has now been shown in K562, HL-60 and hepatoma cells and in patients with chronic granulocytic leukemia in blast crisis. Tiazofurin may be useful in studies on selective depression of the expression of the ras oncogene. 5. In 27 consecutive patients 50% responded positively to tiazofurin treatment. From this group, 10 out of 12 patients (83%) with chronic granulocytic leukemia in blast crisis responded to tiazofurin treatment.

Antimetabolites, Antineoplastic↗
PubMed1991

Molecular characterization and functional analysis in Aspergillus nidulans of the 5'-region of the Penicillium chrysogenum isopenicillin N synthetase gene.

The isopenicillin N synthetase gene (pcbC) was isolated from a genomic library of Penicillium chrysogenum BC39813, a penicillin production strain. The nucleotide sequence, including 555 bp upstream of the translation start site was determined. Various deletions within the pcbC 5'-region were constructed and linked to the Escherichia coli lacZ gene. An Aspergillus nidulans argB strain was transformed with DNA of these constructions. The region essential for promoter function could be localized between positions -307 and -89 by analyzing beta-galactosidase expression of transformants containing a single copy of the corresponding plasmid integrated at the homologous argB locus. A region responsible for regulatory effects concerning nitrogen metabolism was identified by determining beta-galactosidase activities in cell-lysates of transformants cultivated under varying growth conditions. Two major transcription start sites at positions -131 and -132, as well as a further upstream located site at position -397 +/- 1 could be located by primer extension studies employing RNA isolated from P. chrysogenum BC39813.

Amino Acid Sequence↗
PubMed1991

[Placental maturity at term and functional placental performance: CTG changes in relation to histologic detection of placental maturation reserves].

Cardiotocography (CTG) is to be considered today's most sensitive monitoring tool for the functional surveillance of placental performance during parturition. 351 cardiotocographically monitored singleton pregnancies were used as patient material for fine tissue examination of the state of maturity of the villi of the relevant placentas. In histological assessment, placental diagnoses are allocated to defined CTG changes; in particular, however, the identification of reserves, capable of maturing (immature intermediate villi in the centres of the placental subdivisions or placentones), is subject to separate scrutiny. The following results emerge clearly: terminal placentas without maturing potentials, prematurely matured placentas and placentas with deficiency of terminal villi, as well as the absence of immature intermediate villi in the centers of the placentones, are connected with a suspected prepathological or pathological CTG assessment. The histological groups have the lowest incidence of the normal oscillatory type and normal oscillatory frequency and have the highest proportion of abnormal CTG assessments according to the Hammacher score. The absence of the identification of potential maturing reserves (immature intermediate villi) is associated with the highest incidence of Caesarean sections (29.8%). These results show, that the physiological maturing potentials (immature intermediate villi) which can be identified up to term are a histological indication towards regular placental performance, even in labour.

Cardiotocography↗
PubMed1991

A followup study of manic-depressive and schizoaffective psychoses after systemic family therapy.

The authors report the results of a followup study of manic-depressive and schizoaffective psychoses after systemic family therapy. They describe the development of inpatient relapse rates, familial interactional patterns, and prescriptions of medication in a sample of 30 cases treated with family therapy and followed up after a mean interval of 3 years. Finally, they assess the effectiveness of systemic family therapy with manic-depressive and schizoaffective psychoses.

Adolescent↗
PubMed1991

AZT: a biochemical response modifier of methotrexate and 5-fluorouracil cytotoxicity in human ovarian and pancreatic carcinoma cells.

In ovarian and pancreatic carcinoma cell lines, the activity of the salvage enzyme, thymidine kinase (EC 2.7.1.21), was 2- to 13-fold higher than that of the key enzyme of thymidylate de novo biosynthesis, thymidylate synthase (dTMP synthase, EC 2.1.1.45). AZT (3'-azido-3'-deoxythymidine, zidovudine) competitively inhibited thymidine kinase activity in extracts of human ovarian and pancreatic carcinoma cells, with Dixon plots yielding Ki = 1.1 microM in both cell lines. AZT (20 microM) yielded synergistic cytotoxicity with methotrexate (0.4 microM) in human pancreatic carcinoma cells in clonogenic assay and also with methotrexate (0.02 microM) in human ovarian carcinoma cells, as measured by cell counts. Thymidine (10 microM) and hypoxanthine (100 microM) reversed these inhibitions. AZT (20 or 40 microM) also provided synergistic cytotoxicity with 5-fluorouracil (0.5 and 1.0 microM) in human pancreatic carcinoma cells in clonogenic assay. These studies suggest a new role for AZT, which, as an inhibitor of thymidine salvage, should be useful as a biochemical response modifier to provide a synergistic clinical anticancer impact on de novo biosynthesis of thymidylates in conjunction with methotrexate or 5-fluorouracil.

Animals↗
PubMed1991

Tiazofurin down-regulates expression of c-Ki-ras oncogene in a leukemic patient.

The increased activity in cancer cells of inosine 5'-monophosphate dehydrogenase (IMP DH, EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis, was suggested as a sensitive target for chemotherapy. Tiazofurin (NSC 286193), through its conversion to the active metabolite, thiazole-4-carboxamide adenine dinucleotide (TAD), is a strong inhibitor of IMP DH. In our clinical trial, tiazofurin caused return to the chronic phase in patients with chronic granulocytic leukemia in blast crisis (Tricot, G.; Jayaram, H.N.; Weber, G.; Hoffman, R. Tiazofurin: Biological effects and clinical uses. Int. J. Cell Cloning 8:161-170; 1990). In K562 human leukemic cells, tiazofurin down-regulated the expression of c-Ki-ras and c-myc oncogenes, which was followed by induced differentiation. We now report down-regulation by tiazofurin of the c-Ki-ras oncogene in a patient with chronic granulocytic leukemia in blast crisis. A single tiazofurin infusion (2,200 mg/m2) on days one and two decreased IMP dehydrogenase activity (the apparent t1/2 was 30 min), GTP concentration (the apparent t1/2 was 6 hr), and expression of ras (the apparent t1/2 was 8 hr) and c-myc (the apparent t1/2 was 38.5 hr) oncogenes in the leukemic cells. No further tiazofurin was given, because on days three and four the chemotherapeutic impact became evident in a tumor-lysis syndrome and the blast cells were cleared from the periphery by day five. The decrease in IMP DH activity, GTP concentration, and expression of c-Ki-ras oncogene were early markers of the successful chemotherapeutic impact of tiazofurin in a patient with chronic granulocytic leukemia in blast crisis.

Adenine Nucleotides↗
PubMed1991

Neuropsychiatric side effects after the use of mefloquine.

This study describes neuropsychiatric side effects in patients after treatment with mefloquine. Reactions consisted mainly of seizures, acute psychoses, anxiety neurosis, and major disturbances of sleep-wake rhythm. Side effects occurred after both therapeutic and prophylactic intake and were graded from moderate to severe. In a risk analysis of neuropsychiatric side effects in Germany, it is estimated that one of 8,000 mefloquine users suffers from such reactions. The incidence calculation revealed that one of 215 therapeutic users had reactions, compared with one of 13,000 in the prophylaxis group, making the risk of neuropsychiatric reactions after mefloquine treatment 60 times higher than after prophylaxis. Therefore, certain limitations for malaria prophylaxis and treatment with mefloquine are recommended.

Adult↗
PubMed1991

Thromboresistance of the arterial wall: the role of heparin and glycosaminoglycans.

Thromboresistance of the arterial wall is the result of many factors which dynamically interact with each other. Glycosaminoglycans (GAG), among which heparin, synthesized by endothelial cells, smooth muscle cells, and fibroblasts, play an important role in determining vessel wall non-thrombogenicity. We are describing morphological-morphometrical characterization (increase in endothelial cell density and nucleus/cytoplasm ratio) of bovine aortic endothelial cells grown in the presence of heparin and other GAGs.

Animals↗
PubMed1991

[Foreign body aspiration in children. The advantages of emergency endoscopy and foreign body removal].

In the last 15 years, 124 children with a history and clinical signs of aspiration have been managed by emergency rigid endoscopy under general anesthesia. In 115 (93%) of them a foreign body could be identified, which was successfully removed by forceps extraction in 114. Only 1 patient required a thoracotomy for removal of a distally located aspirated needle. The remaining 9 patients (7%) had a typical history and clinical signs of an aspiration, but no aspirated foreign body was found on endoscopy. Most of the children (102, or 82%) were admitted within 12 h after aspiration, while 22 (18%) had a history of foreign body aspiration between 2 days and 5 weeks before. Complications occurred in only 3% (3 children) of the 102 who underwent endoscopy within 12 h as against 50% (11 patients) of the 22 children in whom this was delayed. In all cases of foreign body aspiration and endoscopical removal within 12 h, the patients were discharged after a plain chest X-ray the following day. In children with chronic endotracheal foreign bodies, in 80% a second endoscopy after 48 h was indicated, and the mean stay in hospital was extended to 7 days. Emergency rigid tracheobronchoscopy and forceps removal of aspirated foreign bodies under general anesthesia and with meticulous perioperative monitoring is a safe and effective procedure with no mortality. Even in suspected aspiration or chronic bronchopulmonary infections, liberal use of endoscopy is recommended.

Anesthesia, General↗
PubMed1990

Abelson murine leukemia virus transforms preneoplastic Emu-myc transgene-carrying cells of the B-lymphocyte lineage into plasmablastic tumors.

E mu-myc transgenic mice were back-crossed to BALB/c mice up to back-cross generation 3. The offspring that included transgene-carrying and -negative mice in approximately equal proportions were randomly divided into 2 groups. Thirty-four mice (group I) were treated with pristane, followed by A-MuLV, and 40 (group II) were injected with A-MuLV alone. Altogether, 16 lymphoid tumors developed in group I and 17 in group II. Nine of the tumors in group I and 4 in group II appeared as ascitic tumors. The ascites contained lymphoblasts and 10 to 45% plasmacytoid cells. These tumors were designated as plasmablastic lymphomas (PLs). All tumors except one were transgene-positive and did not carry translocations. An exceptional tumor in group I carried a variant 6;15 translocation but not the transgene. It obviously corresponds to the regular Abelson + pristane-induced plasmacytoma. Among 11 tested PLs, 10 had a single retroviral insertion site, while one tumor showed 3. Among 18 untreated transgenic descendants (group III), chosen randomly during serial back-crosses, 15 (83%) developed lymphomas, with no sign of plasmacytoid differentiation. The incidence was comparable in all 3 groups, assuming 50% of the mice in groups I and II to be transgenic. The time distribution of tumor development was also similar. Spleen cells from transgene-carrying mice with no clinical sign of lymphoma were infected in vitro with A-MuLV and transplanted i.p. into BALB/c recipients. PLs developed in 26 of 31 pristane-treated recipients, but in only one of 18 untreated recipients. One of 6 PLs tested was monoclonal, whereas the remaining 5 were oligoclonal. They all expressed v-abl. These results show that some of the preneoplastic B-cells that expressed constitutively active myc transgene turned into plasmablasts after infection with A-MuLV. Full development of their neoplastic potential was facilitated by the presence of pristane-granuloma.

Animals↗
PubMed1990

Vagally induced release of gastrin, somatostatin and bombesin-like immunoreactivity from perfused rat stomach. Effect of stimulation frequency and cholinergic mechanisms.

The isolated stomach of rats was vascularly perfused to measure the secretion of gastrin, somatostatin (SLI) and bombesin-like immunoreactivity (BLI). The gastric lumen was perfused with saline pH 7 or pH 2, and electrical vagal stimulation was performed with 1 ms, 10 V and 2, 5 or 10 Hz, respectively. Atropine was added in concentrations of 10(-9) or 10(-7) M to evaluate the role of cholinergic mechanisms. In control experiments, vagal stimulation during luminal pH 2 elicited a significant increase of BLI secretion only at 10 Hz but not at 2 and 5 Hz. Somatostatin release was inhibited independent of the stimulation frequency employed. Gastrin secretion at 2 Hz was twice the secretion rates observed at 5 and 10 Hz, respectively. At luminal pH 7 BLI rose significantly at 5 and 10 Hz. SLI secretion was decreased by all frequencies. Gastrin secretion at 2 and 5 Hz was twice as high as during stimulation with 10 Hz. Atropine at doses of 10(-9), 10(-8), 10(-7) and 10(-6) M had no effect on basal secretion of BLI, SLI and gastrin. At luminal pH 2, atropine increased dose-dependently the BLI response at 2 and 5 but not at 10 Hz. The decrease of SLI during 2 and 5 Hz but not 10 Hz was abolished by atropine 10(-9) M. SLI was reversed to stimulation during atropine 10(-7) M at all frequencies. The rise of gastrin at 2 Hz was reduced by 50%. At luminal pH 7, atropine had comparable effects with a few differences: the BLI response at 10 Hz was augmented and the gastrin response to 2 and 5 Hz was reduced. In conclusion the present data demonstrate a frequency and pH-dependent stimulation of BLI and gastrin release. The stimulation of BLI is predominantly due to atropine-insensitive mechanisms while muscarinic cholinergic mechanisms exert an inhibitory effect on BLI release during lower stimulation frequencies (2 and 5 Hz) independent of the intragastric pH and also during higher frequencies at neutral pH. Both, atropine sensitive and insensitive mechanisms are activated frequency dependent. The atropine-sensitive cholinergic mechanisms but not the noncholinergic mechanisms involved in regulation of G-cell function are pH and frequency dependent. Somatostatin is regulated largely independent of stimulation frequency and pH by at least two pathways involving cholinergic mechanisms of different sensitivity to atropine. These data suggest a highly differentiated regulation of BLI, gastrin and SLI secretion and the interaction between these systems awaits further elucidation.

Animals↗
PubMed1990

Subunit interactions in hemoglobin probed by fluorescence and high-pressure techniques.

The dissociation of the subunits of human adult oxyhemoglobin has been investigated by using steady-state fluorescence anisotropy, multifrequency phase fluorometry, and high hydrostatic pressure. Human hemoglobin obtained by using two purification procedures (bulk preparation by centrifugation or further fractionation using anion-exchange chromatography) was labeled with an extrinsic fluorescent probe, 5-(dimethylamino)naphthalene-1-sulfonyl chloride (DNS-Cl). The long fluorescence lifetime of this probe allows for the observation of the macromolecular tumbling, and thus provides a method for observing changes in the size of the complex upon subunit dissociation under differing solution conditions of proton and organic phosphate concentration. At pH 7, the dansylated preparations of bulk and fractionated hemoglobin showed a concentration-dependent decrease in the anisotropy which though not identical can only arise from the tetramer to dimer dissociation. We observed primarily the dimer at pH 9 and a small destabilization of the tetramer in the presence of saturating inositol hexaphosphate (IHP). High-pressure experiments allowed for the observation of the dissociation of the hemoglobin dimer into monomers. From these measurements, we estimate the dimer dissociation constant to be between 0.1 and 1 nM. We compare the present results on the subunit affinities in hemoglobin obtained from steady-state and time-resolved fluorescence data with those obtained previously by using gel filtration, sedimentation, and kinetic techniques. These comparisons are indicative of a certain degree of conformational heterogeneity in the hemoglobin preparations.

Chromatography, Gel↗
PubMed1990

High concentration of neutral endopeptidase (enkephalinase E.C. 3.4.24.11) in a malignant tumor: rat hepatoma 3924A.

The activity of the membrane-bound neutral endopeptidase 24.11 was low in the normal liver (21 +/- 3 pmol/h/mg protein, mean +/- SE) but it increased 56-fold in rapidly-growing rat hepatoma 3924A. The identity of the enzyme in the tumor was established by immunoprecipitation and by using a specific inhibitor of neutral endopeptidase. The endopeptidase concentration in the differentiating and regenerating liver was lower than in normal tissue, 39 and 8% of the corresponding control. The activity of a plasma membrane marker enzyme carboxypeptidase M in the normal liver was 1.0 +/- 0.2 nmol/h/mg protein, it increased about 2-fold in the rapidly-growing hepatoma and in the differentiating liver, but was unchanged in regenerating liver. The function of the strikingly increased neutral endopeptidase activity in the rapidly growing hepatoma may relate to activation of autocrine or exocellular growth factors or to inactivation of cell proliferation-inhibitory factors. Such a biochemical change should confer selective advantages to the cancer cells.

Animals↗