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Biomedical subjects

R Graf

Publications and source records attributed to R Graf.

At least 145 records · Page 8Linked to original sources

Antibodies to cytokeratins bind to epitopes in human uterine smooth muscle cells in normal and pathological pregnancies.

Cytokeratin antibodies have been widely used for the identification of trophoblast cells in the placental bed, following their invasion from the developing conceptus. Their identification centres upon the expression of cytokeratin in epithelial cells, from which trophoblast cells are derived. Our recent observations indicate that this strict relationship may be more complex than was thought. Cryostat and paraffin sections of human decidua and myometrium, taken from the placental bed and the uterotomy cut, were examined immunocytochemically for cytokeratins using ten antibody clones selected to identify different cytokeratin proteins and antigenic epitopes. Biopsy specimens were obtained from normal and pathological pregnancies (pre-eclampsia, fetal retardation, amnioninfection, hysterorrhexis, placenta praevia) at the time of caesarean section (26-41 weeks of pregnancy). Antibodies against nine clones, CAM 5.2, MNF 116, AE1/AE3, CK5, KS-B17.2, CY-90, M20, E3, and 34 beta E12 identified, as expected, syncytial giant cells and mononuclear trophoblasts within the placental bed and glandular epithelial cells throughout the uterus. In addition, they stained numerous fusiform cells that were classified by established criteria to represent smooth muscle cells, both within blood vessels and myometrium. No staining differences were observed between normal and pathological disorders. These results indicate that cytokeratin antibodies CAM 5.2, MNF 116 and AE1/AE3, and other antibodies targeting proteins 8 and 18, cross-react with epitopes expressed in cells other than giant trophoblastic cells and mononuclear trophoblasts in the uterus and, thus, caution has to be used when such antibodies are used for the diagnostic characterization of tissues related to the placental bed.

Actins↗

Modulation of the allosteric equilibrium of yeast chorismate mutase by variation of a single amino acid residue.

Chorismate mutase (EC 5.4.99.5) from the yeast Saccharomyces cerevisiae is an allosteric enzyme which can be locked in its active R (relaxed) state by a single threonine-to-isoleucine exchange at position 226. Seven new replacements of residue 226 reveal that this position is able to direct the enzyme's allosteric equilibrium, without interfering with the catalytic constant or the affinity for the activator.

Allosteric Regulation↗

High-resolution PET in cats: application of a clinical camera to experimental studies.

UNLABELLED: A commercial high-resolution scanner designed for clinical PET studies was tested for its applicability to investigate cerebral metabolism and blood flow in cats. METHODS: Cerebral blood flow, CMRO2, CBV and CMRglc were determined repeatedly using 15O steady-state oxygen methods and 18F-fluorodeoxyglucose (FDG). Metabolic and blood flow images of 14 contiguous 3-mm PET slices were compared to histological sections in four control animals. In another six cats, hemodynamic and metabolic changes were followed by serial multi-tracer PET for 24 hr after permanent occlusion of the left middle cerebral artery (MCA). Pattern and extent of changes of the physiological variables were related to the final infarct verified in matched histological sections. RESULTS: At spatial resolutions (FWHM) of 3.6 mm in transaxial planes and 4.0 mm axially, details of the gross anatomy of the cat brain were distinguished best in the FDG images. Cerebral blood flow, CMRO2 and CMRglc values measured in the cortex, white matter and basal ganglia were in the range of common autoradiographic results. Immediately after MCA occlusion, there was widespread decrease in blood flow, but metabolism was preserved at values, which suggest viable tissue. With time, the areas of increased oxygen extraction fraction (OEF) moved from the center to the periphery of the MCA territory. CONCLUSION: High-resolution PET can be used for repeat, quantitative imaging of blood flow and metabolism in small animals such as the cat. After MCA occlusion, the changes in blood flow and metabolism can be followed over time and can be related to the final morphological lesion.

Animals↗

[Early detection of opiate-induced respiratory depression in the postoperative phase].

We examined in 30 patients the efficacy of regular assessments of respiratory rate (every 15 minutes) and blood gas analysis (at 30, 60, 120, 180 minutes) and continuous monitoring via pulsoximeter and capnometer in recognizing early ventilatory problems. For postoperative analgesia the patients received randomly and double-blind patient-controlled intravenous or epidural analgesia with sufentanil. Within 15 minutes after the initial intravenous bolus injection of 15 micrograms sufentanil respiratory depression occurred in 4 patients. This was alerted by the Oscar-CO2-Monitor and -Pulsoximeter. Oxygen saturation time patterns of pulsoximetry and blood gas analysis correlated significantly (p < 0.001), although the mean values of the methods differed (NS). In contrast, carbon-dioxide pressure time patterns of capnometry and blood gas analysis correlated less significantly (p < 0.01) although the mean values of the methods correlated significantly (p < 0.01). Concomittant monitoring via pulsoximeter and capnometer is therefore superior to regulary assessments of respiratory rate and blood gas analysis and potentially useful for the clinical routine.

Adult↗

The crystal structure of allosteric chorismate mutase at 2.2-A resolution.

The crystal structure of an allosteric chorismate mutase, the Thr-226-->Ile mutant, from yeast Saccharomyces cerevisiae has been determined to 2.2-A resolution by using the multiple isomorphous replacement method. Solvent-flattening and electron-density modification were applied for phase improvement. The current crystallographic R factor is 0.196. The final model includes 504 of the 512 residues and 97 water molecules. In addition, two tryptophan molecules were identified in the interface between monomers. The overall structure is completely different from the reported structure of chorismate mutase from Bacillus subtilis. This structure showed 71% helices with essentially no beta-sheet structures.

Allosteric Regulation↗

CHIRBASE, a molecular database for the separation of enantiomers by chromatography.

A selected review on the separation of the stereoisomers of menthol by gas chromatography serves to demonstrate a new approach to tackle the increasing number of publications on enantiomer separation. The graphical molecular database CHIRBASE covers information (structural, bibliographic and chromatographic data) on liquid chromatography, supercritical fluid chromatography and gas chromatography. CHIRBASE is based on standard database software, thus meeting the requirements of "state of the art" information management in the chemical and pharmaceutical industries and academia. The scrutiny required to create and maintain a factual database revealed that the information content of the contemporary literature with respect to chromatographic data requires improvement.

Chromatography↗

The extravascular contractile system in the human placenta. Morphological and immunocytochemical investigations.

In the human placenta, besides the fetal blood vessel system a second extravascular contractile system exists. It is localized in the chorionic plate and runs in a longitudinal direction and adjacent to fetal blood vessels into the stem villi, where it forms perivascular contractile sheaths. Characteristically, cells of the extravascular contractile system are extremely long and spindle-shaped and give rise to fine cell processes, by which they obviously contact each other or insert into the basement membrane of the trophoblast. They show immunoreactivity with desmin, vimentin, alpha-actin, myosin, nitric oxide synthase type I (brain form) and dipeptidyl peptidase IV. The ultrastructure suggests that cells of the extravascular contractile system are related to smooth muscle cells, including subpopulations with myofibroblastic features. In stem villi a few cells are nitric oxide synthase type I immunoreactive. These cells are thought to be specialized smooth-muscle-like cells of the extravascular contractile system or cells of the extravascular contractile system related to paraneurons that generate nitric oxide, which, in turn, may modulate the tone of perivascular contractile sheaths. The high dipeptidyl peptidase IV activity suggests that modulation of the extravascular contractile system may also occur by substance P.

Actins↗

Effects of the synthetic glucocorticoid triamcinolone acetonide on vasoactive hydrolases of the human placenta in vitro.

Therapy with glucocorticoids during pregnancy is still debated. Previously reported effects of glucocorticoid application in rats resemble certain symptoms of preeclampsia. Therefore, we studied in vitro the effects of the synthetic glucocorticoid triamcinolone acetonide soluble (0.1-10 mM) on placental alpha-glutamyl amino-peptidase, microsomal alanyl aminopeptidase, dipeptidyl peptidase IV, acetylcholinesterase and butyrylcholinesterase in purified trophoblast monolayers and villous explants from first trimester (n = 5) and term placentae (n = 9) using bio- and histochemical methods. In term placentae quantitative histochemistry (microdensitometry) of trophoblast monolayers revealed an increase of alpha-glutamyl aminopeptidase and microsomal alanyl aminopeptidase activity up to 149% and 126% respectively, after treatment with supraphysiological doses. In trophoblast monolayers from first trimester alpha-glutamyl aminopeptidase activity was not affected, whereas microsomal alanyl aminopeptidase activity increased by 25%. Dipeptidyl peptidase IV staining was reduced to 26%. Biochemical measurements of alpha-glutamyl aminopeptidase and microsomal alanyl aminopeptidase activity in homogenates of cultured villi revealed effects similar to those found by microdensitometry in trophoblast monolayers. In contrast, dipeptidyl peptidase IV activity increased in explants of term placentae by 47%. Acetyl- and butyrylcholinesterase activities were reduced in term placental villi by 38% and 40%, respectively. The data indicate that glucocorticoids may affect the activity of hydrolases which are thought to be involved in local placental blood pressure modulation.

Acetylcholinesterase↗

Dynamic penumbra demonstrated by sequential multitracer PET after middle cerebral artery occlusion in cats.

Experimental models of focal cerebral ischemia have provided important data on early circulatory and biochemical changes, but typically their correspondence with metabolic and hemodynamic findings in stroke patients has been poor. To fill the gap between experimental studies at early time points and rather late clinical studies, we repeatedly measured CBF, CMRO2, oxygen extraction fraction (OEF), cerebral blood volume (CBV), and CMRglc in six cats before and up to 24 h after permanent middle cerebral artery (MCA) occlusion (MCAO), using the 15O steady state and [18F]fluorodeoxy-glucose methods and a high-resolution positron emission tomography (PET) scanner. Likewise, three sham-operated control cats were studied during the same period. Final infarct size was determined on serial histologic sections. In the areas of final glucose metabolic depression that were slightly larger than the histologic infarcts, mean CBF dropped to approximately 40% of control values immediately on arterial occlusion. If further decreased to < 20% during the course of the experiment. This progressive ischemia was most conspicuous in border zones. CMRO2 fell to a lesser degree (55%), eventually reaching approximately 25% of its control level. At early stages, OEF increased mainly in the center of ischemia. With time, areas of increased OEF moved from the center to the periphery of the MCA territory. Concurrently, progressive secondary decreases in OEF in conjunction with further reductions of CBF and CMRO2 indicated the development of central necrosis. The findings are highly suggestive of a dynamic penumbra. In five cats with complete MCA infarcts, CBF decreased and OEF increased in the contralateral hemisphere after 24 h, suggesting whole-brain damage. This effect may be explained by the widespread brain edema found histologically in addition to the nonspecific CBF reductions and OEF elevations observed also in the sham-operated controls after 1 day in the experimental condition. In one cat, cortical OEF increased only transiently. Normal CMRO2 and CMRglc were eventually restored, and the final infarct was small. This study demonstrates that acute regional pathophysiologic changes can be repeatedly assessed by multivariate PET in cats. Viable tissue can be detected up to several hours after MCA occlusion, and the transition of misery-perfused regions into necrosis or preserved tissue can be followed over time. The present results support the concept of a dynamic penumbra, in which for up to 24 h tissue damage spreads progressively from the center to the periphery of ischemia. Sequential high-resolution PET provides insight into the dynamics of regional pathophysiology and may thus further the development of rational therapeutic strategies.

Animals↗

The ischemic penumbra.

The term ischemic penumbra, originally applied to brain tissue perfused at values between the functional and morphologic thresholds, has recently been extended to characterize ischemically affected but still viable tissue with uncertain chances for infarction or recovery. Results have accumulated supporting the concept of the ischemic penumbra as a dynamic process of impaired perfusion and metabolism eventually propagating with time from the center of ischemia to the neighboring tissue. As mediators and modulators of this process, waves of depolarization, extracellular increases in excitatory amino acids, activation of Ca++ channels, induction of immediate early genes and expression of heat-shock proteins, among others, have been discussed. The contribution of the various electrophysiologic and biochemical/molecular events to the complex cascade, eventually leading to neuronal damage, is still controversial. The demonstration of viable (penumbra) tissue by positron emission tomography up to several hours after ischemic stroke renders the rationale for therapeutic interventions. A short therapeutic window of a few hours is relevant for re-establishment of perfusion; the time-dependent propagation of the ischemic penumbra suggests an extended period for effective intervention with biochemical/molecular processes.

Amino Acids↗

[The extravascular contractile system of the human placenta: a new aspect for function of the organ?].

In the chorionic plate and stem villi of the human placenta besides the fetal blood vessel system a second extravascular contractile system (EVCS) exists. The cells of this system contain contractile and intermediate filaments and are dipeptidyl peptidase-IV- and NO-synthase-type-I-(NOS)-immunoreactive. Therefore it can be assumed that these cells cleave the vasodilator Substance P (by DPP IV) and produce NO (by NOS) and may contribute to a modulation of the EVCS.

Amino Acid Oxidoreductases↗

[Sonography of the infant hip. Sources of error, progress and current clinical relevance].

Effective prevention of late dysplastic dislocation of the hip (DDH) has become a reality since reliable diagnosis has been possible at an early stage by means of the original sonographic method according to Graf. A correct sonographic diagnosis must be instantly followed by adequate, biomechanically rational, and consistent therapeutic management and long-term follow-up. The results of a strictly comparative follow-up study emphasize the enormous progress in the quality of therapeutic results gained when routine sonographic newborn hip "screening" is performed directly in the maternity wards: successful anatomical healing with conservative treatment is regularly obtained without femoral head necrosis, without surgery and without late DDH, and costs less when sonographic assessment is done within the first days of life. For this reason, the Austrian government has included sonographic neonatal hip screening in its general health prevention program-and also reimbursed the cost of this preventive procedure-since 1992.

Austria↗

YMC1, a yeast gene encoding a new putative mitochondrial carrier protein.

We have cloned and sequenced a Saccharomyces cerevisiae gene coding for a protein with significant similarities to the mitochondrial carrier family. The gene we termed YMC1 (yeast mitochondrial carrier) is located on chromosome XVI, closely downstream of ARO7 encoding chorismate mutase.

Amino Acid Sequence↗

Enzyme histochemical evidence for the presence of potential blood pressure regulating proteases in cultured villous explants from human first trimester placentae.

The proteases dipeptidyl peptidase IV, angiotensinase A and microsomal alanyl aminopeptidase are present in the human term placenta where they may be involved in the local modulation of placental blood pressure. In order to establish an in vitro model system to study the significance of these proteases in disorders related to pregnancy-induced hypertension, the activity of the proteases was localized histochemically in cultured explants of villi from human first trimester placentae. These studies revealed a similar distribution pattern of the activity of the proteases of cryostat sections of first trimester placental villi and in cultured tissue of the same placentae. Dipeptidyl peptidase IV and angiotensinase A activity were present in cytotrophoblast cells and dipeptidyl peptidase IV activity was found in the syncytiotrophoblast, respectively. Additionally, the activity of the proteases was visualized in various populations of stromal cells. Comparing our results with former studies, the protease activity pattern in first trimester placentae was found to be the same as in term placentae. Despite morphological changes of the tissue after 14 d in culture the localization of the proteases remained unchanged up to 52 d of culture. The results suggest that placental explants may serve as a suitable in vitro model for experimental studies on the role of proteases in pregnancy-induced hypertension.

Aminopeptidases↗

Elevation of neuroactive substances in the cortex of cats during prolonged focal ischemia.

Sustained accumulation of excitatory amino acids and other neuroactive substances may contribute to the delayed progression of infarction in focal ischemia. Following occlusion of the left middle cerebral artery (MCAO), extracellular amino acid and purine catabolite concentrations as well as local CBF were repeatedly monitored for up to 15 h in auditory (A) and somatosensory (SF) cortices of seven halothane-anesthetized cats using microdialysis/HPLC and hydrogen clearance. MCAO resulted in persistent reduction of local CBF, which was more severe in A (n = 6) than in SF (n = 6). Accordingly, higher transmitter amino acid and purine catabolite concentrations were found in A than in SF during ischemia. Aspartate, glutamate, and gamma-aminobutyrate (GABA) as well as hypoxanthine and inosine reached maximum levels 1-2 h after onset of ischemia (15-, 7-, 31-, 8-, and 14-fold increases, respectively). Maximum levels remained almost constant, with the exception of inosine, which decreased subsequently. Glycine seemed to increase with prolonged ischemia and reached maximum levels (10-fold) 15 h after occlusion. Adenosine peaked 30 min after occlusion (54-fold) and decreased thereafter to control levels within 1-2 h. One hour after occlusion, CBF thresholds for amino acid elevation were lower (glutamate and GABA approximately 20 ml 100 g-1 min-1 and glycine approximately 10 ml 100 g-1 min-1) than 6 and 15 h after occlusion (thresholds for all amino acids at approximately 30 ml 100 g-1 min-1). These results indicate that in prolonged ischemia, excitotoxicity is an important factor, particularly in border zones of ischemic foci.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

Ischemia-induced accumulation of extracellular amino acids in cerebral cortex, white matter, and cerebrospinal fluid.

In a global model of brain ischemia, accumulation of amino acids was studied in the extracellular space of the auditory cortex and the internal capsule using microdialysis, and in CSF of halothane anesthetized cats. In both brain regions, blood flow determined by hydrogen clearance decreased below 10 ml/100 g/min after extracranial multiple-vessel occlusion, and extracellular potassium activity (Ke) measured in the dialysate increased significantly. A delayed rise in Ke was observed in CSF. In contrast, ischemic amino acid accumulation differed markedly between the two brain regions investigated. In cortex, transmitter amino acids glutamate, aspartate, and gamma-aminobutyric acid (GABA) rose almost immediately after onset of ischemia, and increased 30-, 25-, and 250-fold, respectively, after 2 h of ischemia. The nontransmitter amino acids taurine, alanine, and serine increased 10-, seven-, and fourfold, respectively, whereas glutamine and essential amino acids (valine, phenylalanine, isoleucine, and leucine) increased only 1.5-fold. In the internal capsule, increases in amino acids, if any, were delayed and much smaller than in cortex. The largest alteration was a fivefold elevation of GABA. In CSF, changes in amino acids were small and comparable to those in the internal capsule. Our results demonstrate that ischemia-induced extracellular amino acid accumulation is a well localized phenomenon restricted to gray matter structures that possess release and reuptake systems for these substances. We assume that amino acids diffuse slowly into adjacent while matter structures, and into CSF.

Amino Acids↗