Biomedical subjects
M Schwab
Publications and source records attributed to M Schwab.
IgG transport across trophoblast-derived BeWo cells: a model system to study IgG transport in the placenta.
In primates, prenatal transfer of IgG from mother to offspring occurs predominantly across the placenta. Although a number of Fcgamma-receptors and IgG binding proteins have been detected in human placental tissue, an involvement of any of these receptors in IgG transport across the syncytiotrophoblast remains to be demonstrated. Therefore, we investigated the mechanism of IgG transcytosis in trophoblast-derived BeWo cells. BeWo cells were not only found to express the MHC class I-related IgG Fc receptor, human FcRn, but also specifically bound fluorescein isothiocyanate (FITC)-labeled human IgG (FITC-hIgG) at the apical surface at mildly acidic pH. The cells preferentially transcytosed FITC-hIgG from the apical to the basolateral side when compared to the fluid-phase marker FITC-dextran and to FITC-hIgG transcytosis in the opposite direction. However, endocytosis of FITC-hIgG at the apical plasma membrane at physiological pH required the continuous presence of FITC-hIgG at concentrations similar to those present in the maternal circulation. These results suggest a mechanism by which IgG is internalized by BeWo cells via fluid-phase endocytosis. Tight binding of IgG to hFcRn may then occur in acidic endosomes, followed by selective sorting into the transcytotic pathway. Thus, the main function of this receptor is to prevent entry of IgG into the degradative pathway in lysosomes.
A versatile bait vector allowing rapid isolation of prey vectors in Gal4-dependent yeast two-hybrid screens.
Two-hybrid screens have been widely used in recent years for identifying and isolating new interaction partners to known proteins. Current Gal4-dependent two-hybrid screens employ mostly bait and library vectors, which both have the ampicillin gene as a selection marker in bacteria. The isolation of the library plasmids takes several days, because library and bait plasmid cannot be separated easily. We have replaced the ampicillin gene by a kanamycin gene in a Gal4 DNA binding domain bait vector. This vector reduces four- to fivefold the time period required for the isolation of library plasmid DNA. In addition we have changed the multicloning site in the modified vector for easy cloning of cDNA inserts. This vector is advantageous not only in standard two-hybrid screens, but also in mass screens that require multiple screening rounds in order to characterize networks of protein-protein interactions.
Oncogene amplification in solid tumors.
The term gene amplification refers to the selective increase of the gene copy number and is better designated as DNA amplification. It should not be confused with elevated gene expression, although amplification generally does result in enhanced levels of the products encoded by the amplified gene. Amplification is one of the mechanisms by which cells can meet the demand for synthesis of specific gene products in amounts exceeding the transcriptional capacity of a single copy gene. Cytogenetic studies of human and animal tumor cells have provided evidence for mysterious chromosomal abnormalities including double minutes (DMs), C-bandless chromosomes (CMs) or homogeneously staining chromosomal regions (HSRs) diagnostic for amplified DNA. MYCN has been the first oncogene found amplified in direct preparations of solid tumors, the group of oncogenes undergoing amplification has considerably expanded since. Today amplification of oncogenes is recognized as a major player in the development of many solid tumors in humans and at the same time is a reflection of the genetic instability of solid tumor cells.
[Spontaneous dissection of the coronary arteries: a rare cardiologic diagnosis].
A 55-year-old woman who was admitted to hospital with acute chest pain as a case of emergency suffered from an acute anteroseptal myocardial infarction. Four weeks later coronary angiography revealed a long dissection of the left anterior descending artery (LAD) as well as a significant stenosis of the left main and the proximal circumflex. Cardiovascular surgery was done subsequently. In addition to myocardial revascularization using coronary artery bypass grafts a readaptation of the dissecting artery walls and a proximal ligation of the LAD before anastomosis were performed. Clinical data, pathogenesis, and indications for medical and surgical treatment of spontaneous artery dissection are presented.
Atrial fibrillation after coronary artery bypass surgery: association with changes in G protein levels in mononuclear leukocytes.
Atrial fibrillation (AFib) is a frequent complication of coronary artery bypass grafting (CABG). Its cause, however, is unknown. As the adrenergic system is involved in some types of AFib, we hypothesized that a change in guanine nucleotide-binding protein (G protein) expression plays a role in the development of post-CABG AFib. In 28 patients undergoing CABG surgery, the G(s alpha)/G(i alpha) ratio (stimulatory/inhibitory G protein alpha) at the protein (Western blotting) and mRNA (reverse transcription polymerase chain reaction) levels was measured before and after surgery. As a suitable test system allowing multiple analysis mononuclear leukocytes (MNL) were chosen. The perioperative change of the G(s alpha)/G(i alpha) ratio of protein and mRNA was significantly different in patients who subsequently developed AFib (eight patients) and in patients who did not (20 patients; P<0.01 and <0.001, respectively). On average, the protein G(s alpha)/G(i alpha) ratio decreased from 1.79+/-1.13 (mean+/-SD) to 1.32+/-0.69 in patients without AFib (P=0.1, n.s.) whereas a significant increase from 0.86+/-0.44 to 1.62+/-0.65 (P<0.01) was observed in patients subsequently developing AFib. The mRNA G(s alpha)/G(i alpha) ratio decreased significantly from 0.53+/-0.24 to 0.36+/-0.11 in patients without AFib (P<0.01) whereas a significant increase from 0.31+/-0.14 to 0.47+/-0.13 was observed in those who subsequently developed AFib (P<0.05). These results indicate that an increase of the G(s alpha)/G(i alpha) ratio in MNL is associated with AFib after CABG surgery and possibly may be used as a prognostic indicator of this complication.
Human neuroblastoma: from basic science to clinical debut of cellular oncogenes.
Neuroblastoma is a childhood embryonic tumor of migrating neuroectodermal cells derived from the neural crest and destined for the adrenal medulla and the sympathetic nervous system. It very often has a rapidly progressive clinical course, and although many advances have been made in understanding the development of this tumor, improving the survival rates particularly in patients with metastatic tumor has been a frustrating experience. The mechanisms leading to neuroblastoma are largely unclear, but nonrandom chromosomal changes discovered early suggested the involvement of genetic alterations. Most prominent among these is the amplification of the oncogene MYCN, which identifies a group of patients who have a particularly dire prognosis. Amplified MYCN is used today as a prognostic marker on which therapy design is based to a large extent. An unusual aspect of neuroblastoma is the high rate at which tumors regress spontaneously, even in infants with extensive liver involvement and numerous subcutaneous nodules. Identifying the molecular and cellular basis of spontaneous regression could result in improved therapeutic approaches. Neuroblastoma is a model tumor with many fascinating aspects but has remained a challenge to the pediatric oncologist.
From fish to FISH: the comparative pathology of neuroblastomas in humans, mice and fish.
Neuroblastoma is a malignant cancer of the sympathetic nervous system and is one of the most frequent solid cancers in young children. Only a few of the many advances in our understanding of basic genetic and cellular mechanisms leading to neuroblastoma development have translated to clinical practice, and the prognosis for children with neuroblastoma, particularly at advanced stages, has remained poor. Major directions for neuroblastoma management and control include the application of prognostic parameters, particularly amplified MYCN, which can be readily visualized by chromosomal fluorescence in situ hybridization (FISH), for individual therapy design, as well as the initiation of a presymptomatic screening program for early tumor detection to reduce the fraction of advanced-stage tumors. In addition, new and innovative therapeutic approaches are being sought. The understanding of molecular and cellular pathways resulting in spontaneous regression in up to 10% of neuroblastoma patients, possibly by apoptosis, could provide the basis for new biologically based therapeutic interventions. Unlike most other pediatric cancers, neuroblastoma can be studied in two experimental animal systems. One is the fish system Xiphophorus, where neuroblastomas can be induced in specific strains by exposure to mutagens/carcinogens; the second is mice that carry MYCN as a transgene. These animal systems demonstrate that neuroblastomas are evolutionarily conserved tumors. Their study could well result in a better understanding of neuroblastoma development. At the same time they represent systems in which experimental therapies can be preclinically tested.
Cloning and sequence of partial cDNAs encoding the human type V and VI adenylyl cyclases and subsequent RNA-quantification in various tissues.
Adenylyl cyclases (Acs) and VI are the predominant form of Acs in mammalian heart where they are part of the beta-adrenergic pathway. Up to now, the sequences for both enzymes from human tissues have not yet been reported. We investigated the mRNA expression AC V and VI in human colon, heart, liver, lung and MNL. Partial cDNAs of human types V and VI Acs were amplified by PCR using oligonucleotides derived from the cytoplasmatic domain sequences of the corresponding enzymes from dog heart. Primers derived from the human sequence were used to detect the mRNAs corresponding to both Acs. For quantification of mRNAs we constructed internal standards for competitive quantitative reverse transcriptase PCR (RT-PCR). Both types of transcripts could be found in all investigated tissues except MNL where only type VI could be detected. Further we demonstrated a more than 60 times higher amount of AC V-mRNA in human heart compared to AC VI-mRNA.
Partial aortectomy in hypothermic circulatory arrest: method of choice in patients with aortic arch atheroma.
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[Quality assurance: the AO documentation at a peripheral hospital].
Follow-up of fracture treatment one year after surgery based on AO Documentation in a small, rural hospital in a region with many tourists is demanding. How many patients can be contacted and assessed at the one year follow-up? From 1.1.95 to 31.12.96 302 patients with 314 fractures were operated on and the results subsequently documented. 255 patients (84%) were assessed personally (221) or by an AO member or family doctor (34). 15 patients died before the appointment, 32 patients could not be contacted or their data was incomplete. To achieve this rate of follow-up, some internal organization in the hospital was necessary, as will be described in this paper. AO Documentation is an important tool for personal quality assurance, especially in small hospitals. Small hospitals might be interesting for the AO since their follow-up rate can be high due to a simple organization and a personal patient-doctor relationship.
Drug-induced hyponatraemia in elderly patients.
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Down-regulation of endothelial cell growth inhibitors by enhanced MYCN oncogene expression in human neuroblastoma cells.
Recent evidence indicates that the genetic alterations of the multistage process of malignant transformation appear to activate tumor neovascularization by altering the balance between stimulators and inhibitors of angiogenesis. In the present study, we have attempted to define the effect of enhanced MYCN oncogene expression on the profile of endothelial cell growth modulators in neuroblastoma cells. We report here that conditioned medium of human neuroblastoma cells with normal MYCN expression contains three inhibitors of endothelial cell proliferation, which appear to be novel proteins as judged by their physicochemical, immunological and biological properties. All three inhibitors are diminished or become undetectable upon experimental increase of MYCN expression. Our results suggest that enhanced MYCN expression in human neuroblastoma cells alters the angiogenic balance by down-regulating endothelial cell growth inhibitors but leaving the expression of the stimulators unaffected. These data shed light on the molecular mechanisms linking the genetic changes of malignant transformation with initiation of tumor angiogenesis. Moreover, our observations might explain the poor prognosis of human neuroblastomas following MYCN oncogene amplification through initiation of angiogenesis and subsequent tumor growth and spread.
Betamethasone-mediated vascular dysfunction and changes in hematological profile in the ovine fetus.
Glucocorticoid administration to fetal sheep induces a sustained systemic blood pressure rise and an associated increase in femoral vascular resistance. We utilized a small vessel myograph to compare isometric vascular responses of small femoral arterial branches from fetal sheep infused intravenously with either betamethasone or vehicle in vivo from 128 days gestation. Changes in hematological parameters were also determined. Betamethasone was infused for 48 h to produce fetal plasma betamethasone concentrations similar to those observed in human fetuses after maternal treatment with betamethasone to accelerate fetal lung maturation. When compared with vessels removed from vehicle-infused fetuses, vessels obtained from betamethasone-treated fetuses exhibited 1) enhanced sensitivity to depolarizing potassium solutions; 2) no differences in response to the thromboxane mimetic U-46619 or norepinephrine; and 3) differential responses to vasodilators, enhanced sensitivity to ACh, but decreased response to bradykinin and forskolin. In addition, erythrocyte and leukocyte counts were increased in betamethasone-infused fetuses. These observations indicate that multiple mechanisms operate to increase fetal vascular resistance during antenatal betamethasone exposure.
Fine mapping of distal 1p loci reveals TP73 at D1S468.
In the present study we establish a FISH fine-map of 1p36.3 loci. This region is frequently altered in different types of human tumors suggesting the existence of cancer-related genes. Identification of cosmids carrying both D1S468 and TP73 sequences leads to the assignment of TP73 to the most frequently deleted locus in colon and breast cancer and integrates this gene in human genetic maps. Localization of other distal loci was determined as follows: distal-CDC2L1-D1Z2-D1S94-TP73/D1S468-D1 S1615-proximal. D1S1615, earlier reported as a telomeric sequence, is considerably more proximal than previously thought.
Retention of polysomy at 9p23-24 during karyotypic evolution in human breast cancer cell line COLO 824.
Somatic genetic alterations of 9p have been seen in a wide range of human cancers, including breast cancer. Loss of heterozygosity analysis of primary breast cancer tumors has revealed a high frequency of deletion of DNA from 9p21-22 encompassing the MTSI (P16/CDKN2A) gene. We report the approximately tenfold increase in copy number of DNA from 9p23-24, which is far distal to P16/CDKN2A in female breast cancer cell line COLO 824, as revealed by fluorescence in situ hybridization, comparative genomic hybridization, and microsatellite analysis. Amplification of DNA has been reported previously to encompass multiple sites of the genome of the breast cancer cell, but increase in DNA copy number has not been seen in distal 9p.
Control of cyclin ubiquitination by CDK-regulated binding of Hct1 to the anaphase promoting complex.
Proteolysis of mitotic cyclins depends on a multisubunit ubiquitin-protein ligase, the anaphase promoting complex (APC). Proteolysis commences during anaphase, persisting throughout G1 until it is terminated by cyclin-dependent kinases (CDKs) as cells enter S phase. Proteolysis of mitotic cyclins in yeast was shown to require association of the APC with the substrate-specific activator Hct1 (also called Cdh1). Phosphorylation of Hct1 by CDKs blocked the Hct1-APC interaction. The mutual inhibition between APC and CDKs explains how cells suppress mitotic CDK activity during G1 and then establish a period with elevated kinase activity from S phase until anaphase.
Cloning of the human aflatoxin B1-aldehyde reductase gene at 1p35-1p36.1 in a region frequently altered in human tumor cells.
Alterations of the distal portion of the short arm of chromosome 1 (1p) are among the earliest abnormalities of human colorectal tumors. Loss of heterozygosity analysis has previously revealed a smallest region of overlapping deletion (SRO) B, at 1p35-36.1, deleted in 48% of sporadic tumors. From this region we have now cloned a gene encoding a protein of 330 amino acids that is 78% identical with the Rattus norvegicus aflatoxin B1 aldehyde reductase (Afar) and, therefore, likely represents its human homologue. In rat liver, Afar is strongly inducible by the antioxidants ethoxyquin and butylated hydroxyanisole, which protect the rat against aflatoxin B1-induced liver tumorigenesis by detoxifying its genotoxic and cytotoxic dialdehyde. Human AFAR is expressed in a broad range of tissues and, therefore, is likely involved in endogenous detoxication pathways. Impaired detoxication of genotoxic aldehydes and ketones, which are involved in tumorigenesis of the colon and breast, may be a crucial factor both for tumor initiation and progression. We here provide a detailed contig of 1.5-2 Mbp/2.7 cM encompassing part of SRO B, including known genes and previously unmapped expressed sequence tags. PLA2G2A (secretory type II phospholipase A2), described previously as a candidate, is localized outside SRO B.