Profile: George Weber. Man of many worlds. Interview by Teresa Amoroso.
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Canada↗
Biomedical subjects
G Weber
Publications and source records attributed to G Weber.
Signal transduction and biochemical targeting of ovarian carcinoma.
UNLABELLED: The purpose was to identify novel targets for the chemotherapy of ovarian carcinoma. METHODS: Assays were worked out to measure the activities of P1 kinase, PIP kinase and PLC in ovarian carcinoma samples and in OVCAR-5 cells and to compare the activities to those in normal ovaries. A method was also designed for measuring the concentration of the end product of signal transduction, IP3. RESULTS AND DISCUSSION: Signal transduction activity was markedly increased in ovarian cancer cells as shown by the increased steady-state activities of the three enzymes and the elevated concentrations of IP3. Inhibitors blocked activities of PI kinase (quercetin), PIP kinase (genistein), and lowered GTP concentration required for PLC (tiazofurin). Combinations of tiazofurin with quercetin, tiazofurin with genistein, and quercetin with genistein yielded a synergistic kill of ovarian cancer cells. Tiazofurin, quercetin and genistein are in various stages of clinical trials. CONCLUSION: The increased signal transduction activity provides novel, sensitive targets to chemotherapy in ovarian cancer cells.
Cell Survival↗
Physical localization of single-copy sequences on pachytene chromosomes in maize (Zea mays L.) by chromosome in situ suppression hybridization.
A new approach for locating single-copy DNA sequences on pachytene chromosomes of maize (Zea mays L.) was developed. A cosmid clone with homologous sequences to a molecular marker (umc105a) linked to a quantitative trait locus (QTL) for resistance against sugarcane borer (SCB) was physically mapped by fluorescence in situ hybridization (FISH) to the short arm of chromosome 9. The marker umc105a was genetically placed in the centromeric region. To suppress signals generated by maize repetitive DNA, competitive in situ suppression (CISS) hybridization was necessary to obtain specific signals from umc105a. A centromere specific DNA probe (CentC) was used in a double-labeling technique as a reference marker. Fluorescence signals generated by umc105a cosmid and CentC were specific and highly reproducible. Thus the single-copy DNA sequence of umc105a was physically localized on the short arm of chromosome 9 near the telomere. This is the first report of physical localization of single-copy DNA sequence by CISS hybridization to a maize pachytene chromosome.
Chromosome Mapping↗
Study of the mechanism of direct laser desorption/ionisation for some small organic molecules (M < 400 Daltons).
Aspects of direct laser desorption/ionisation have been studied for three molecules, aminotriazole (positive ion), dinoterb and ioxynil (negative ion). The samples are deposited on metallic substrates, and a nitrogen laser is used for desorption/ionisation; ion yields are measured with a time-of-flight mass spectrometer. Previous work had shown that ion yields can strongly vary from one substrate to another, and that this variation does not reflect the (calculated) metal surface temperatures. New results obtained in this work indicate that the desorption/ionisation mechanism is linked to the physical state of the substrate surface. Copyright 1999 John Wiley & Sons, Ltd.
Journal Article↗
Characterization of the MEN1 ortholog in zebrafish.
Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant syndrome predisposing to multiple tumors. The responsible gene, MEN1, has been identified and inactivating mutations reported. It encodes a protein named menin, which lacks homology to any known proteins. Comparative genomics is used to ascertain important functional domains via the identification of evolutionary conserved regions. Here we report the sequencing and characterization of the MEN1 gene in zebrafish (Danio rerio) at the cDNA level. Zebrafish menin is a 617 amino acid protein and, when compared with human and rodent proteins, shows 75% and 76% similarity, respectively. The most conserved region is amino acid residues 41-322 which shows a human/zebrafish similarity of 83%. Amino acids affected by inactivating missense mutations in MEN1 patients in this region are completely conserved between human and zebrafish. Such high correlation between conservation throughout evolution and mutation position strongly emphasizes the importance of this region.
Amino Acid Sequence↗
A novel cGMP-regulated K+ channel in immortalized human kidney epitheliall cells (IHKE-1).
1. K+ channels from the apical membrane of immortalized human kidney epithelial (IHKE-1) cells were investigated in the cell-attached membrane configuration as well as in excised membranes using the patch clamp technique. 2. In cell-attached membrane patches the open probability (Po) of the K+ channel was 0.42 +/- 0.06 (mean +/- s.e.m. , n = 22) and its conductance was 94 +/- 5 pS with 145 mM K+ in the pipette (n = 25). In excised membrane patches the Po of the channel was 0.55 +/- 0.03 (n = 86) and its conductance was 65 +/- 2 pS (n = 68) with 145 mM K+ on one side of the membrane and 3.6 mM K+ on the other. The I-V curve of the K+ channel was not rectifying. 3. The channel was inhibited by several blockers of K+ channels such as 1 mM Ba2+ (cell-attached membrane: 78 +/- 8 %, n = 9; excised: 80 +/- 4 %, n = 26), 10 mM TEA+ (excised inside-out: 48 +/- 5 %, n = 34; excised outside-out: 100 +/- 0 %, n = 26), 0.1 mM verapamil (excised: 73 +/- 9 %, n = 12), and 10 nM charybdotoxin (excised outside-out: 67 +/- 9 %, n = 9). 4. The K+ channel was activated by depolarization and rising cytosolic Ca2+. Half-maximal activity occurred at a cytosolic Ca2+ concentration of 200 nM. In the cell-attached membrane configuration the K+ channel was inhibited in a concentration-dependent manner by atrial natriuretic peptide (ANP). Powas blocked equally well by 10 nM ANP (52 +/- 7 %, n = 10), brain natriuretic peptide (BNP; 37 +/- 11 %, n = 6) and C-type natriuretic peptide (CNP; 44 +/- 13 %, n = 8). 8-Bromoguanosine 3',5' cyclic monophosphate (8-Br-cGMP, 0.1 mM) also inhibited Poof this K+ channel, by 70 +/- 10 % (n = 5). 5. In excised membrane patches cGMP inhibited Po of this K+ channel in a concentration-dependent manner. The first significant effects were measured at a concentration of 1 microM (22 +/- 7 %, n = 6), and greatest effects were obtained at 0.1 mM (34 +/- 5 %, n = 15). cAMP (0.1 mM, n = 5) as well as GTP (0.1 mM, n = 5) had no significant effects on Po of this K+ channel. ATP (0.1 mM) had a weak inhibitory effect (17 +/- 5 %, n = 14). Addition of Mg-ATP to cGMP did not increase the inhibitory effect (30 +/- 4 %, n = 14). KT5823 (1 microM), a specific inhibitor of cGMP-dependent protein kinases, did not significantly alter the cGMP-induced reduction in Po of the K+ channel in three excised membrane patches. 6. The results present the first electrophysiological characterization of a mammalian K+ channel that is directly regulated by cGMP.
Atrial Natriuretic Factor↗
Suppression of the neoplastic phenotype by transfection of phospholipase C beta 3 to neuroendocrine tumor cells.
The expression of phospholipase C beta 3 (PLCB3) is low or absent in several neuroendocrine neoplasias. To investigate the role of PLCB3 in the neuroendocrine tumorigenesis, we transfected a PLCB3 construct to three neuroendocrine tumor cell lines with a low PLCB3 expression. The growth rate and tumorigenicity were assessed in vitro by [3H]thymidine incorporation and cell counting, in vivo, by xenografting to nude mice. In vitro, PLCB3 expressing clones showed a significant growth inhibition. The tumor weight was reduced for one of the two xenografted PLCB3-transfected cell lines and in both, a reduced number of proliferating (Ki-67 positive) cells was observed. This study implies an essential role for PLCB3 in the neuroendocrine tumorigenesis.
Animals↗
Separation of plant membranes by electromigration techniques.
The review focuses on the multiple separating regimes that offers the free flow electrophoresis technique: free flow zone electrophoresis, isoelectric focusing, isotachophoresis, free flow step electrophoresis. Also, the feasibility to apply either interval or continuous flow electrophoresis is evaluated. The free flow zone electrophoresis regime is generally selected for the separation of cells, organelles and membranes while the other regimes find their largest fields of applications in the purification of proteins and peptides. The latter regimes present the highest resolution efficiency. Therefore, a large part of this review is devoted to the applicabilities of these different regimes to the purification of organelles and membrane vesicles at the preparative scale. Recent developments, both in instrumentation and procedures, are described. The major achievements in plant membrane fractionation obtained with free flow electrophoresis are outlined. The related procedures are both analytical and preparative: they separate tonoplast and plasma membrane simultaneously from the same homogenate, they discriminate for one type of membrane vesicles of opposite orientation, and process large quantities of membrane material by reason of the continuous flow mode. Recent advances using electromigration techniques that permit confirmation of the dynamic state of membranes, characterisation of complex membrane-dependent functions and discovery of new membrane-localised activities are presented.
Cell Membrane↗
Prenatal sonographic chest and lung measurements for predicting severe pulmonary hypoplasia.
UNLABELLED: Pulmonary hypoplasia was diagnosed sonographically in 32 fetuses from 20 to 33 weeks of gestation. In addition to standard biometry, transverse thoracic diameter (TTD), sagittal thoracic diameter (TSD), thoracic circumference (TC) and lung diameter (LD) were measured in all cases and compared with known nomograms. The fetuses were divided into five groups according to the main sonographic findings: group 1-skeletal dysplasia; group 2-renal agenesis; group 3-diaphragmatic hernia; group 4-hydrothorax; and group 5-others. Severe pulmonary hypoplasia (PH) was diagnosed prenatally in all cases on the basis of LD measurements. In 17 (53.1 per cent) out of 32 cases TTD was below the 5th percentile while lower TSD measurements were recorded in 15 (46.8 per cent) fetuses. A thorax circumference below the 5th percentile for the respective gestational age was found in 15 cases (46.8 per cent) and a decreased LD/TC ratio in 25 cases (78.1 per cent). In 13 out of 32 fetuses pulmonary hypoplasia was diagnosed before, and in 19 cases after 24 weeks of gestation. Pulmonary hypoplasia was confirmed by autopsy in all cases. CONCLUSION: pulmonary hypoplasia can be sonographically detected before 24 weeks of gestation. In cases of skeletal dysplasia and renal agenesis pulmonary hypoplasia can be diagnosed by chest and lung measurements, whereas in diaphragmatic hernia and hydrothorax diagnosis of pulmonary hypoplasia is possible only by lung measurement.
Bone Diseases, Developmental↗
Infective endocarditis due to Fusobacterium nucleatum in an intravenous drug abuser.
Infective endocarditis due to anaerobic non-spore-forming gram-negative bacilli in intravenous drug abusers is exceedingly rare, with only two cases being previously reported in the literature. A case of endocarditis due to Fusobacterium nucleatum in an intravenous drug abuser is reported, accompanied by a review of the literature.
Adult↗
Synergistic down-regulation of signal transduction and cytotoxicity by tiazofurin and quercetin in human ovarian carcinoma cells.
Ovarian carcinoma is one of the most common causes of cancer death in women. Tiazofurin, a C-nucleoside, arrests the cell cycle at S phase and reduces the activities of PI (phosphatidylinositol) utilizing enzymes in signal transduction by depleting cellular GTP concentration. Quercetin (QN), a flavonoid, attacks the cell cycle at the G1 and S phase boundary and mainly inhibits PI kinase (1-phosphatidylinositol 4-kinase, EC 2.7.1.67) activity in the signal transduction pathway. Because tiazofurin and QN attack different biochemical targets and arrest different phases of the cell cycle, we tested the hypothesis that the two drugs might be synergistic against human carcinoma cells. In human ovarian carcinoma OVCAR-5 cells in growth inhibition assay, the IC50s (drug concentration that inhibits 50% of cell proliferation) for tiazofurin and QN were (mean +/- SE) 13 +/- 1.2 and 66 +/- 3.0 microM; in clonogenic assays they were 6 +/- 0.5 and 15 +/- 1.2 microM, respectively. When tiazofurin was added to cells followed 12 h later by QN, synergism was observed in both growth inhibition and clonogenic assays. The combination also yielded synergistic reduction of IP3 (inositol 1,4,5-trisphosphate) concentration in the cells which may explain, at least in part, the synergistic action of tiazofurin and QN in OVCAR-5 cells. The protocols yielding synergism may have implications in the clinical treatment of human ovarian carcinoma.
Antineoplastic Agents↗
17Beta-hydroxy-11alpha-(3'-sulfanylpropyl)oxy-estra-1,3,5(10)- trien-3-yl sulfamate--a novel hapten structure: toward the development of a specific enzyme immunoassay (EIA) for estra-1,3,5(10)-triene-3-yl sulfamates.
The title compound 17 has been synthesized for the use as hapten in the development of a competitive enzyme immunoassay for estrogen sulfamates. The synthesis started from estradiol diacetate 2. Oxyfunctionalization at C-11 to give 11alpha-hydroxy steroid 8 was accomplished by hydroboration/alkaline hydrogen peroxide oxidation of the 9(11)-dehydro derivative 7, which was obtained from compound 2 via 9-hydroxylation with dimethyldioxirane. After transformation of compound 8 into the allyl ether 9, the side chain was thio-functionalized at the omega-position affording the thioate 11 in two steps. Selective silylether deprotection at position 3 followed by sulfamoylation gave the sulfamate 19, which in turn was demasked at position 17 and treated with sodium borohydride/aluminum chloride to liberate the side chain thiol. Alternatively, title compound 17 was synthesized via the disulfides 13-16. For the preparation of the immunogen the title compound 17 was coupled to bovine gamma globulin in a two-step procedure using an amine and thiol specific bifunctional crosslinker. The immunization of rabbits resulted in the formation of antibodies which clearly discriminated the sulfamoylated estrogens from the non-esterified estrogens. The use of a biotinylated hapten derivative as a tracer in combination with a streptavidin-peroxidase-tetramethylbenzidine based detection system allowed the measurement of estradiol 3-sulfamate (1) in the range of about 1 to 1000 pg/well.
Animals↗
Amplification of signal transduction capacity and down-regulation by drugs.
Recent work in this Laboratory showed increased activity of PI 4-kinase, PIP kinase and PLC in various cancer cells, indicating a stepped-up capacity for signal transduction. This elevated potential was paralleled with increased concentration of the end product of signal transduction, IP3. Current investigations showed that in normal cells the activities of the specific phosphatases (which degrade PIP2 and PIP and oppose those of the synthetic enzymes) were 4 to 5 orders of magnitude higher than those of the synthetic kinases. In hepatoma cells the specific phosphatase activities markedly decreased. Thus, in cancer cells the marked elevations in activities of the synthetic enzymes were opposed by a reduction in the activities of the degradative specific phosphatases. This enzymic imbalance is responsible, in part at least, for the elevated capacity of signal transduction and IP3 concentration. Since the enzymic activities measured were proportionate with time elapsed and amount of enzyme added, the alterations in activities should reflect changes in enzyme amounts. These alterations indicate a reprogramming of gene expression which should confer selective advantages to the cancer cells, marking out the elevated synthetic enzyme activities as potentially sensitive targets for drug treatment. We showed earlier that tiazofurin, which curtailed the biosynthesis of enzymes with short half-lives such as PI and PIP kinases, down-regulated signal transduction and brought down IP3 concentration. Quercetin and genistein chiefly inhibited PI-4 kinase and PIP kinase, respectively, and as a result reduced IP3 concentration in cancer cells. Current studies reveal that tiazofurin with quercetin, tiazofurin with genistein, and quercetin with genistein were synergistic in killing human cancer cells and in reducing signal transduction activity. In estrogen receptor-negative MDA-MB-435 human breast carcinoma cells which have elevated signal transduction activity, tamoxifen caused IC50S for growth inhibition and cytotoxicity of 12 and 0.7 microM, respectively. When tiazofurin was added to breast carcinoma cells, followed 12 hr later by tamoxifen, synergism was observed in growth inhibition, in clonogenic assays and in the reduction of IP3 concentration. The synergistic action of tiazofurin and tamoxifen and the other synergistic drug interactions outlined above may have implications in the clinical treatment of neoplasias.
Animals↗
Massive pulmonary haemorrhage caused by leptospirosis successfully treated with nitric oxide inhalation and haemofiltration.
A patient with leptospirosis who developed oliguric renal failure, massive pulmonary haemorrhage and respiratory failure is described. The patient's clinical condition and arterial oxygenation failed to improve despite vigorous supportive measures. Nitric oxide inhalation and haemofiltration resulted in a marked clinical improvement and subsequent full recovery. We suggest that the addition of haemofiltration and nitric oxide inhalation therapy should be considered in patients with pulmonary haemorrhage and renal failure caused by leptospirosis, in whom conventional therapy fails.
Administration, Inhalation↗
Abdominal surgery reduces the ability of rat spleen cells to synthesize and secrete active tumour necrosis factor-alpha (TNF-alpha) by a multilevel regulation.
We have previously shown that abdominal surgery (explorative laparotomy) reduces the ability of lipopolysaccharide (LPS)-triggered spleen macrophages to secrete TNF-alpha. In this study we characterize possible mechanisms which could be responsible for the reduction in splenic production of TNF-alpha. Post-operative and control (unoperated) rat splenocytes or enriched splenic macrophages were cultured with LPS. Steady-state levels of TNF-alpha mRNA were determined by Northern and slot blot analyses, and validated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The amount of TNF-alpha protein was measured by Western blot analysis, and its biological activity was determined by the fibroblast L-929 cytotoxicity assay. Surgery induced a 12-fold inhibition in TNF-alpha activity (P < 0.02), caused up to two-fold reduction in the accumulation of TNF-alpha mRNA (P < 0.01), and suppressed TNF-alpha protein maturation into its 17-kD form in cellular extracts. Post-surgical spleen supernatants revealed mainly a band of a lower molecular weight (14 kD). Our data suggest a multilevel regulation of post-operative inhibition of TNF-alpha response to LPS, at the accumulation of mRNA, translational and secretory levels. We also suggest that the reduced bioactivity could be partially caused by a proteolytic cleavage of TNF-alpha. Since TNF-alpha is an important participant in immune responses, its reduced production and activity may be a central mechanism of post-operative immunosuppression.
Animals↗
Therapeutic apheresis in myasthenia gravis patients: a six year follow-up.
Six years ago 4 patients suffering from myasthenia gravis (MG) types C and E according to Compston with failed drug therapy were initially treated 3 times (1 patient, a total of 11 times) by protein A immunoadsorption (Immunosorba, Excorim AB, Lund, Sweden). No further immunoadsorption treatments have been carried out. In addition, 3 patients were given a thymectomy. The present status of the patients was checked. We could see a beneficial effect in all MG patients. The patients are fit for work; each has an improved Besinger index. The patients were used as their own controls. A higher anti-AChRAb level 6 years after protein A immunoadsorption than at the beginning was seen in all patients, combined with less serious MG. In addition, their immunomodulation could be induced as seen in lymphocyte and inflammatory protein changes during the first 36 days after beginning immunoadsorption treatment. A larger population has to be investigated to verify these results.
Adult↗
A new sonomorphologic scoring-system (Mainz score) for the assessment of ovarian tumors using transvaginal ultrasonography. Part II: A comparison between the scoring-system and the assessment by an experienced sonographer in postmenopausal women.
OBJECTIVE: The early and accurate detection of ovarian carcinomas continues to pose a problem. To what degree can the evaluation of a maximum number of sonographic tumor markers on the basis of a newly developed 10-item score improve the preoperative assessment of adnexal tumors in postmenopausal women? MATERIALS AND METHODS: In a prospective study 195 postmenopausal women with an adnexal tumor were examined by transvaginal sonography. A score allowing the evaluation of 10 sonographic markers was used to predict the adnexal tumor status: 1. Total tumor structure, 2. tumor border, 3. wall thickness, 4. inner echoes in cystic component, 5. septa, 6. shape of echocomplex or of the completely solid tumor, 7. echogenicity of the echocomplex or of the completely solid tumor, 8. acoustic phenomena behind tumor, 9. ascites, 10. detection of liver metastases/peritoneal carcinosis. The different markers were rated on a scale from 0 to 2 points depending upon the degree of expression observed for the individual characteristics. The total score obtained after addition of the point number recorded for each marker served as the basis for the assessment of the sonographic tumor status. The adnexal tumor was then assessed by a second sonographer to confirm the validity of the score. After having been informed of all clinical parameters, this sonographer evaluated the preoperative tumor status assessment based on his previous experience. The preoperative tumor status assessment according to the score as well as that by the experienced sonographer were compared postoperatively with the results of the histological diagnosis. RESULTS: All assessment criteria of the score with the exception of septal thickness showed p-values of < 0.05, thus demonstrating a statistically significant correlation between score point numbers and histological findings. A suitable scoring threshold for the sonographic differentiation between malignant and benign adnexal tumors was determined at > or = 10 points at a sensitivity of 96.8% and a specificity of 91.2%, a positive predictive value of 91.2% and a negative predictive value of 96.9%. Ninety of 93 malignant and 93 of 102 benign tumors were accurately identified. Compared with the tumor status assessment by the experienced sonographer, the score offers a clear advantage in the evaluation of ambiguous cases. In 24 of 38 neoplasms whose tumor status could not be determined definitely by the experienced sonographer, the accurate tumor status could be established on the basis of the score. Using the score a problem continues to exist, however, for a small number of false-positive assessments of benign tumors, which occurred in particular for dermoid cysts and fibromas. CONCLUSION: The 10-item score provides even an inexperienced ultrasound sonographer with a valuable tool allowing the assessment of the tumor status of postmenopausal adnexal tumors with a high degree of diagnostic accuracy. For the experienced sonographer the score represents, in particular in the case of indistinct ultrasound findings, a refined and improved method for the prediction of tumor status.
Adult↗
Bone density and bone metabolism are normal after long-term gluten-free diet in young celiac patients.
OBJECTIVES: Osteoporosis and alterations of bone metabolism are frequent complications of celiac disease. We evaluated the impact of long-term gluten-free diet (GFD) initiated during childhood and adolescence on bone mineralization and bone metabolism. METHODS: We studied 30 celiac patients on GFD for > or = 5 yr. The mean age at diagnosis was 11.4+/-5.0 yr, and the mean duration of GFD was 10.7+/-4.3 yr. Results were compared with those obtained in 240 healthy controls. Bone mineral density (BMD) was measured in the lumbar spine and in the whole skeleton by dual-energy x-ray absorptiometry. Serum levels of bone-specific alkaline phosphatase (BALP) and N-terminal propeptide of type I procollagen (PINP) were measured as bone formation indices, and urine levels of N-telopeptide of type I collagen (NTx) as bone resorption index. RESULTS: BMD measurements of celiac patients (lumbar spine: 1.131+/-0.121 g/cm2; total body: 1.145+/-0.184 g/cm2) did not differ from those of control subjects (lumbar spine: 1.131+/-0.184 g/cm2; total body: 1.159+/-0.118 g/cm2). The levels of BALP, PINP, and NTx of celiac patients did not differ from those of controls. Patients who started GFD before puberty had BMD and bone metabolism measurements comparable to those of patients who started GFD during puberty. CONCLUSIONS: Our data show that long-term dietary treatment ensures normal mineralization and bone turnover.
Absorptiometry, Photon↗