[Course of disease after surgery for hypoplastic left heart with mitral valve atresia and aortic isthmus stenosis--report by the mother].
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Adaptation, Psychological↗
Biomedical subjects
S Fischer
Publications and source records attributed to S Fischer.
Comparison of the recombinant Escherichia coli-produced protease domain of tissue-type plasminogen activator with alteplase, reteplase and streptokinase in a canine model of coronary artery thrombolysis.
Recent in vitro studies have shown that although recombinant Escherichia coli-produced protease domain of tissue-type plasminogen activator (t-PA) has no appreciable fibrin binding and less plasminforming activity compared to the wild-type, it is nevertheless an effective fibrinolytic agent in a dynamic in vitro plasma clot lysis system. The purpose of the present study was to evaluate the pharmacological profile of the protease in a canine model of coronary artery thrombosis. The effects of a single i.v. bolus injection of 1 mg/kg protease were compared with those of alteplase, reteplase and streptokinase at clinically relevant doses and dosing regimens in eight dogs per group. The protease rapidly restored coronary blood flow at 12 +/- 1 min in all treated dogs with a significantly higher maximal coronary blood flow than in the reference groups, but was associated with short cycles of reocclusion in 4/8 animals. Overall, the coronary blood flow quality of the protease was not significantly different from that of the reference thrombolytics. Although fibrinogen was nearly completely degraded during protease treatment, the bleeding time was not significantly more prolonged than in reference groups. In conclusion, the protease domain is a rapidly acting, effective, bolus-injectable thrombolytic agent associated with a systemic lytic state and does not appear to cause significantly more bleeding than the reference thrombolytic agents.
Animals↗
[Quality of diabetes management in the free Saxony state--scientific evaluation of the Saxony model for managing diabetes. EVA group].
After the societal change in the "New Federal States" of Germany Saxon diabetologists developed an integrated concept for care of diabetic patients: the Saxon Care Model (SCM). It aims at quality assurance in diabetes care and is based on services by cooperating general practitioners and specialists. This model was evaluated. First results were obtained describing the quality of care for 510 patients at baseline. This sample was collected at two specialized diabetologists (level 2), two specialized out-patient units at universities (level 2) and two general practitioners (level 1). The design of the study is a descriptive evaluation. Process and outcome quality at specialized and primary care units working according to the SCM is relatively high. HbA1c measurements were taken in order to quantify outcome quality. The results for primary as well as specialized care units ranged from acceptable to very good. They unfortunately cannot be generalized. The patients expressed that their quality of life was limited only scarcely. The presented preliminary results indicate a high effectiveness of the SCM with regard to relevant process-related outcome-variables. The large variance between single practices of both levels demonstrates that deficits in early diagnosis of diabetes-related complications still exist. On the other hand this points towards resources of quality improvement. The influence of quality assuring measures, e.g. quality circles, will be assessed in a three-year follow up.
Delivery of Health Care↗
[Rational therapy of Type II diabetes].
Noninsulin-dependent diabetes mellitus is a genetically determined form of diabetes, due to impaired insulin secretion by the B-cells as well as to insulin resistance of the peripheral tissues. According to the glucose toxicity theory hyperglycemia and hyperinsulinemia exist in a vicious circle. Therefore, it is a major therapeutical aim to put the B-cell to rest and improve insulin sensitivity by a strict control of fasting blood glucose and of postprandial hyperglycemia. Furthermore, associated abnormalities within the metabolic syndrome, such as hypertension, dyslipoproteinemia and hemostatic disorders should be corrected to avoid vessel complications. Therefore, it should be started with basic measures as body weight reduction, carbohydrate-rich and fat-poor diet and exercise. If these measures fail to achieve acceptable glycemic control, antihyperglycemic drugs (acarbose, metformin) are indicated, eventually in a combination with small doses of short-acting sulfonylureas. Further impairment of insulin secretion is the indication for sulfonylurea and/or insulin application. HbA1c of 7 to 7.5% should be the goal of antidiabetic therapy, also for patients in advanced age. The main criterion for the choice of antidiabetics is the present insulin secretion capacity. Simple indicators in this respect are changes of body weight, plasma triglycerides and C-Peptide after i.v. glucagon stimulation. Application of insulin in combination with other antidiabetics or in the form of intensified insulin therapy should not be too much postponed.
Adult↗
The proline-rich region of Vav binds to Grb2 and Grb3-3.
Vav has structural features found in signaling proteins and is expressed only in hematopoietic cells. The recent development of mice Vav -/- has confirmed a major role of Vav in early blood cell development. We previously showed that Vav constitutively interacts with glutathione-S-transferase-Grb2. Coimmunoprecipitation experiments supported the idea of a complex formed by Vav-Grb2 in vivo. This complex is of potential interest in signaling of hematopoietic cells. In this work we localize the domains of Vav and Grb2 involved in this interaction. By the use of an in vivo genetic approach (the double hybrid system) and through in vitro experiments (glutathione-S-transferase fusion proteins) we furnish evidence that the interaction between Vav and Grb2 involves the C-SH3 domain of Grb2 and the proline-rich region located in the N-SH3 of Vav. Furthermore this was confirmed by the use of both Vav and Sos derived proline-rich peptides which blocked the binding. In addition we show that Vav also interacts with Grb3-3, a naturally occurring Grb2 isoform wich lacks functional SH2 domain.
Adaptor Proteins, Signal Transducing↗
Enhanced sensitivity for peptide mapping with electrospray liquid chromatography-mass spectrometry in the presence of signal suppression due to trifluoroacetic acid-containing mobile phases.
A method is described for improving the sensitivity of peptide mapping with electrospray liquid chromatography--mass spectrometry using trifluoroacetic acid (TFA) containing HPLC mobile phases. The signal suppressing effects of TFA are shown to be due to the combined effect of ion-pairing and surface tension modifications. The post-column addition of a propionic acid-2-propanol (75:25, v/v) in a 1:2 proportion with the HPLC mobile phase counteracts the deleterious effects of TFA resulting in 10-100 x improvement of the signal-to-noise ratio. The system described introduces total HPLC flow (plus additive) directly into the electrospray source without splitting. Using 2.1 mm I.D. HPLC columns, minimum detectable quantities are below 40 pmol total protein. As examples, separations of proteolytic enzyme digests of several proteins are shown using standard HPLC conditions, comparing results with and without the addition of propionic acid. The application of the technique is shown in more depth in the identification of oxidative modification sites in glutamine synthetase. In this application, the enhanced sensitivity allowed location of a modified residue by comparison endoproteinase Lys C digest of native and oxidized forms of the protein without extensive sample preparation or concentration. A third application demonstrates the identification of glycosylation sites in an endoproteinase Arg C digest of single-chain plasminogen activator through the use of in-source collisionally induced dissociation.
Chromatography, High Pressure Liquid↗
Structure of the human aggrecan gene: exon-intron organization and association with the protein domains.
The complete exon-intron organization of the human aggrecan gene has been defined, and the exon organization has been compared with the individual domains of the protein core. A yeast artificial chromosome containing the aggrecan gene was selected from the Centre d'Etude du Polymorphisme Humaine yeast artificial chromosome library. A cosmid sulibrary was created from this, and direct sequencing of individual cosmids was used to provide the exon-intron organization. The human aggrecan gene was found to be composed of 19 exons ranging in size from 77 to 4224 bp. Exon 1 is non-coding, whereas exons 2-19 code for a protein core of 2454 amino acids with a calculated mass of 254379 Da. Intron 1 of the gene is at least 13 kb. Overall, the sizes of the 18 introns range from 0.5 to greater than 13 kb. Each intron begins with a GT and ends with an AG, thus obeying the GT/AG rule of splice-junction sequences. The entire coding region is contained in 39.4 kb of the gene. The organization of exons is strongly related to the specific domains of the protein core. The A loop of G1 and the interglobular domain are encoded by exons 3 and 7 respectively. The B and B' loops of G1 are encoded by exons 4-6, and those of G2 are encoded by exons 8-10. These sets of exons, coding for the B and B' loops, are identical in size and organization. This is supported by the intron classes associated with these exons. Exon 11 codes for the 5' half of the keratan sulphate-rich region, and exon 12 codes for the 3' half of the keratan sulphate-rich region as well as the entire chondroitin sulphate-rich region. G3 is encoded by exons 13-18, including the alternatively spliced epidermal growth factor-like and complement regulatory protein-like domains. The correspondence between the exon organization and the protein domains argues strongly for modular assembly of the aggrecan gene.
Aggrecans↗
The src-family protein-tyrosine kinase p59hck is located on the secretory granules in human neutrophils and translocates towards the phagosome during cell activation.
The src-family protein-tyrosine kinase p59hck is mainly expressed in neutrophils; however, its functional role in these cells is unknown. Several other src-family members are localized on secretory vesicles and have been proposed to regulate intracellular traffic. We have established here the subcellular localization of p59hck in human neutrophils. Immunoblotting of subcellular fractions showed that approx. 60% of the p59hck per cell is localized on the secretory granules; the other 40% is distributed equally between non-granular membranes and the cytosol. Immunofluorescence of neutrophils and HL60 cells suggests that the p59hck-positive granules are azurophil granules. Granular p59hck is highly susceptible to degradation by an azurophil-granule proteinase. Different forms of p59hck occur in the three subcellular compartments: a 61 kDa form is mainly found in the granules, a 59 kDa form is predominant in the non-granular membranes, whereas cytosolic p59hck migrates as a doublet at 63 kDa. During the process of phagocytosis-linked degranulation, induced by serum-opsonized zymosan in neutrophils or HL60 cells, granular p59hck translocates towards the phagosome. The subcellular localization of p59hck suggests that the enzyme could be involved in the regulation of the degranulation process.
Biological Transport↗
Heterogeneity of the T-cell receptor delta gene indicating subclone formation in acute precursor B-cell leukemias.
Precursor B-cell acute lymphoblastic leukemias (B-ALLs) have been shown to be oligoclonal at the Ig heavy-chain (IgH) gene level in up to 40% of cases by Southern blot hybridization. In contrast, oligoclonality as deduced from diversity of T-cell receptor (TcR)-delta gene rearrangements of the immature types (ie, V delta 2-D delta 3, D delta 2-D delta 3) has not been reported, so far. We detected oligoclonality characterized by the coexistence of different junctional regions of identical V delta 2-D delta 3 rearrangements in four childhood precursor B-ALLs. No variation was found in the IgH gene status. Therefore, we define these populations as subclones. Two leukemias displayed the variants in an unequal proportion. In the other two leukemias, for which similar quantities of the coexisting rearrangements were detected, single cell-nuclei polymerase chain reaction (PCR) showed two separate leukemic populations. Subclone formation could not be demonstrated by Southern blot hybridization, but was detectable after PCR amplification of the V delta 2-D delta 3 rearrangement and separation by polyacrylamide gel electrophoresis. The variants arose independently from each other, as deduced from their individual sequences. Using subclone-specific oligonucleotides for hybridization to amplified DNA obtained at diagnosis and during follow-up from bone marrow samples, we demonstrate, (1) specificity of all subclone-deduced probes, (2) that one residual leukemic cell can be detected in 10(4) to 10(5) normal mononuclear cells in a semiquantitative assay, and (3) that none of the subclones persisted after induction therapy. We propose that in a leukemic cell population, TcR-delta gene diversity arises after rearrangements of the IgH genes resulting in apparent clonality at the IgH gene level. However, cells are oligoclonal, if the TcR-delta gene rearrangements are considered. As various subclones may respond differently to chemotherapy, they may hamper the detection of minimal residual disease. Therefore, we use all subclone-specific oligonucleotides for hybridization to amplified DNA from follow-up samples.
Base Sequence↗
Active site labeling with dansyl-glutamyl-glycyl-arginyl chloromethyl ketone demonstrates the full activity of the refolded and purified tissue-type plasminogen activator variant BM 06.022.
BM 06.022 is a tissue-type plasminogen activator deletion variant that is comprised of the kringle 2 and the protease domain of the native molecule. BM 06.022 is expressed as inactive inclusion bodies in E. coli and transferred into the active enzyme by an in vitro folding process. Active site labeling with dansyl-glutamyl-glycyl-arginyl chloromethyl ketone provides evidence that the purified BM 06.022 is fully active and that misfolded species are completely removed by affinity chromatography on ETI-Sepharose. The comparison of the kinetics of the inhibition of BM 06.022 with that of CHO-t-PA indicates that the active centers of both enzymes are rather similar. The further evaluation of the site of interaction of BM 06.022 and DnsEGRck by mass spectroscopy and amino acid sequence analysis revealed that the inhibitor is bound selectively to His322, which is part of the catalytic triad of this serine protease.
Amino Acid Chloromethyl Ketones↗
Peptidyl-Asp metalloendopeptidase.
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Amino Acid Sequence↗
Expression of vascular permeability factor/vascular endothelial growth factor in pig cerebral microvascular endothelial cells and its upregulation by adenosine.
Porcine brain-derived microvascular endothelial cells (BMEC) express the mRNA of the polypeptide mitogen vascular permeability factor/vascular endothelial growth factor (VPF/VEGF). The VEGF mRNA expression in BMEC could be upregulated 2.5 fold after 6 h of treatment with 5 microM adenosine and adenosine agonists. Adenosine A1 and A2 receptor antagonists completely abolished the upregulation of the VEGF mRNA caused by adenosine. Agents like forskolin and cAMP phosphodiesterase inhibitors which are known to increase the cAMP level decreased the VEGF mRNA expression slightly whereas agents like phorbolester which activate the proteinkinase C (PKC) pathway enhanced the VEGF mRNA expression 3.2 fold. The specific inhibitor of the PKC bisindolymaleimide (BIM) abolished the upregulation of the VEGF mRNA by adenosine completely. The BMEC conditioned medium stimulated the proliferation of BMEC itself and Western blot analysis of the BMEC conditioned medium using a polyclonal antibody to human VEGF showed one band at 18 kDa which was slightly upregulated after treatment with adenosine. Results suggest that the effect of adenosine on the VEGF mRNA expression is mediated via the A1 receptor and that an activation of the PKC may be involved in the observed effects of adenosine on the VEGF mRNA expression. VEGF produced by BMEC and which is inducible by adenosine may function via the autocrine pathway and may be involved in repair reactions of brain blood vessels and/or the maintenance of these cells.
Adenosine↗
Bone mass at lumbar spine and tibia in young males--impact of physical fitness, exercise, and anthropometric parameters: a prospective study in a cohort of military recruits.
Bone mineral density (BMD) and bone mineral content (BMC) were measured using DXA at lumbar spine and tibial diaphyses at the beginning and at the end of a 15-week training period in 151 military recruits of the Swiss army belonging to 5 different troop categories (infantry grenadiers, tank drivers, tank gunners, signalmen, and privates) who each were exposed to physical training of various intensity. At baseline, height, body mass index, and degree of physical fitness independently correlated with vertebral and tibial BMD. Over the 15 weeks of physical training BMD at tibial diaphyses increased by 2.2 +/- 0.3% at the left leg (p = 0.0001) and by 1.1% at the right leg (p = 0.002) with differences between troop categories. At lumbar spine, BMD decreased significantly in tank drivers (-1.2 +/- 0.4%, p = 0.001) and particularly in infantry grenadiers (-2.1 +/- 0.4%) who had the most strenuous weight-bearing training, but not in other troop categories. This decrease was twice as large at the center of the vertebra than for the whole vertebra. These BMD changes were associated with increments in serum levels of osteocalcin and alkaline phosphatase activity. From the initial cohort, 48 subjects volunteered for a third investigation carried out 2 years after the end of the military training period. At this time, lumbar BMD and BMC had risen back to baseline, whereas at tibial diaphyses bone width and BMC but not BMD increased by 5.8 +/- 1.1% and 6.2 +/- 0.9%, respectively, vs. baseline (p = 0.0001 for both).(ABSTRACT TRUNCATED AT 250 WORDS)
Absorptiometry, Photon↗
In vitro effects of fentanyl, methohexital, and thiopental on brain endothelial permeability.
BACKGROUND: The use of anesthetics can lead to changes of the permeability of the blood-brain barrier (BBB). To eliminate those factors, such as varying hemodynamic effects that are associated with anesthesia, an in vitro model of the BBB consisting of brain microvascular endothelial cells (BMEC) was used to study the direct effects of the opiate, fentanyl, and the barbiturates methohexital and thiopental, which are widely used in the clinical setting, on the permeability of confluent monolayers. METHODS: BMEC isolated from porcine brains were grown to confluence on collagen-coated polycarbonate membranes, which were placed into 24 well dishes, thus forming a two-compartment chamber. The permeability of the BMEC monolayer to ions--determined by measurements of the transendothelial resistance (TER)--the passage of sucrose, Evans Blue albumin (EBA), and alpha-aminoisobutyric acid (AIB) across the BMEC monolayer were assessed in the presence and absence of fentanyl (25-100 ng/ml), methohexital (10-50 micrograms/ml), and thiopental (25-100 micrograms/ml). RESULTS: The permeability of cultured BMEC to the tracers used increased significantly after exposure of the monolayer to arabinose and after removal of calcium ions. Fentanyl, methohexital, and thiopental did not change the permeability of the cell monolayer to ions, sucrose, albumin, and AIB. Only thiopental at the concentration of 100 micrograms/ml increased the flux of AIB. CONCLUSIONS: At the concentrations tested, there is little evidence of changes in the permeability of the in vitro BBB caused by fentanyl, methohexital, and thiopental regarding the para- and transcellular route of ions, sucrose, and albumin. Only thiopental at a concentration of 100 micrograms/ml increased the passage of AIB across the BMEC monolayer.
Albumins↗
Susceptibility and resistance alleles of human leukocyte antigen (HLA) DQA1 and HLA DQB1 are shared in endocrine autoimmune disease.
Because particular human leukocyte antigen (HLA) DQ alleles are the major predisposing factors for type 1 diabetes mellitus (IDDM), we investigated whether they are shared by other endocrine autoimmune diseases. We, therefore, analyzed the HLA DQ genotypes of 171 patients with IDDM, 271 with Graves' disease (GD), 65 with Hashimoto's thyroiditis, 51 with postpartum thyroiditis, 53 with Addison's disease (AD), and 271 healthy controls. HLA DQA1 and DQB1 alleles were defined by polymerase chain reaction and sequence-specific oligonucleotide hybridization as well as by single strand conformational polymorphism analysis. HLA DQA1*0501 was significantly more frequent in IDDM (60%), GD (65%), and AD (70%) than in controls (43%); DQA1*0301 was significantly more frequent only in IDDM (67% vs. 30% controls). The heterozygous state DQA1*0301/*0501 was found in 9% of controls and 35% of IDDM (relative risk, 5.6). An arginine at position 52 on either DQA1 allele was significantly more frequent in patients with IDDM (94%), GD (80%), and AD (89%) compared with controls (66%). HLA DQB1*0201 and DQB1*0302 were more frequent in IDDM patients (*0201, 62% vs. 36% in controls, *0302, 59% vs. 19% controls), whereas DQB1*0602 was less frequent in IDDM (4%) and GD (18% vs. 31% of controls). In conclusion, endocrine autoimmunity has a common immunogenetic background; susceptibility is conferred by DQA1*0501 as well as an arginine at position 52 of DQA1 alleles, and protection against IDDM and GD is conferred by DQB1*0602.
Addison Disease↗
Naturally occurring anti-idiotypic antibodies to anti-phosphotyrosine in systemic lupus erythematosus interact with SRC-homology 2 domains.
We have recently identified in SLE sera naturally occurring anti-idiotypic antibodies against anti-phosphotyrosine antibodies. Analysis of immunochemical properties of these anti-idiotypic antibodies suggest that they are of beta/gamma type mimicking the antigen. The interaction between these anti-idiotypes and SH2 domains of various fusion proteins was analysed by immunoprecipitation and immunoblotting. Our data demonstrate that these anti-idiotypic antibodies specifically bind SH2 domains, with the highest affinity for SH2 domain of lck protein tyrosine kinase. The significance of this interaction is discussed.
Antibodies, Anti-Idiotypic↗
[Domestic animals as patients in a small animal practice. 2. Handling, sex determination, blood collection, drug application].
The importance of small pets as domestic animals and patients in veterinary practice increased in the last years. Differences in physiology and behaviour require exact knowledge of appropriate handling and sex determination, techniques of blood collection as well as drug application methods. Therefore, a survey of rabbit, guinea-pig, chinchilla, hamster, gerbil, mouse, and rat as patients in veterinary practice is given.
Animals↗
Endothelin induces tyrosine phosphorylation and GRB2 association of Shc in astrocytes.
While the mitogen-activated protein kinase (MAPK) pathway coupled to receptor tyrosine kinases has been largely clarified, little is known about MAPK activation mediated by heterotrimeric G protein-coupled receptors. In a previous study, it has been shown that endothelin-1 (ET-1) signaling through heterotrimeric G protein-coupled receptors stimulates MAPK activity in primary cultures of astrocytes (Cazaubon, S., Parker, P. J., Strosberg, A.D., and Couraud, P.O. (1993) Biochem. J. 293, 381-386). To clarify the molecular mechanism responsible for this response, involvement of the adapter proteins, Shc and Grb2, has now been investigated. It is shown here that in these cells, ET-1 stimulates tyrosine phosphorylation of Shc, resulting in its stable association with Grb2 but not with Grb3-3, a Grb2 isoform with partially deleted SH2 domain. These results demonstrate that tyrosine-phosphorylated Shc specifically interacts with the SH2 domain of Grb2. This response was rapid and transient, showing a maximum at 10 min and declining at 60 min. Interestingly, direct activation of G proteins by fluoroaluminate mimics the ET-1 effect. In addition, a shift to a higher apparent molecular mass of Raf-1 kinase, likely reflecting its hyperphosphorylation, was also detected in ET-1-treated cells. These data strongly suggest that ET-1-induced MAPK activation is a G protein-coupled pathway that involves Shc, Grb2, and probably Raf-1. In conclusion, the Shc-Grb2 complex may be involved in the activation of the MAPK pathway, not only by several receptor tyrosine kinases but also by heterotrimeric G protein-coupled receptors, such as ET-1 receptors.
Adaptor Proteins, Signal Transducing↗