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Biomedical subjects

R Graf

Publications and source records attributed to R Graf.

At least 127 records · Page 7Linked to original sources
PubMed1997

Histochemical evaluation of placental dipeptidyl peptidase IV (CD26) in pre-eclampsia: enzyme activity in villous trophoblast indicates an enhanced likelihood of gestational hypertensive disorders.

PROBLEM: To determine whether differences in placental dipeptidyl peptidase IV (DPP IV, CD26) activities occurred in hypertensive complicated pregnancies as compared with uncomplicated pregnancies. METHOD: DPP IV activity was detected with H-Gly-Pro-4M2NA as the substrate in placental cryostat sections from 65 patients with gestational hypertension and 67 patients with uncomplicated pregnancies. The graduated intensities of the reaction product in the villous trophoblast were scored semiquantitatively by light microscopy and were related to the relative frequencies of hypertensive disorders (proportional odds model). After detection of enzyme activity, the same tissue samples were homogenized and used for kinetic fluorometric measurements. RESULTS: Enhanced villous trophoblastic DPP IV activity was significantly associated with an increased frequency of proteinuric hypertension in pregnant women (cumulative odds ratio theta1 = 1.6; P < 0.01). CONCLUSION: This study demonstrates for the first time that increased villous trophoblastic DPP IV activity indicates an increased likelihood of the presence and of the severity of hypertensive disorders in pregnancy.

Adult↗
PubMed1997

Early detection of irreversibly damaged ischemic tissue by flumazenil positron emission tomography in cats.

BACKGROUND AND PURPOSE: Ligands for cerebral benzodiazepine receptors were used in the past to indicate the intactness of cortical neurons in subacute to chronic states after stroke and thus to differentiate among brain regions with complete or incomplete infarction and with functional deactivation. For planning acute interventional therapy, however, a marker of irreversible damage in early ischemia is needed. We studied the applicability of [11C]flumazenil (FMZ) for differentiation between tissue with and without potential of recovery in the first hours after focal experimental ischemia. METHODS: In 11 cats, cerebral blood flow, cerebral metabolic rate for oxygen, oxygen extraction fraction, and FMZ binding were studied repeatedly by positron emission tomography before, during, and up to 12 hours after transient middle cerebral artery occlusion (MCAO) (30 minutes in 2, 60 minutes in 7, and 120 minutes in 2 cats, respectively). Development of the defects in energy metabolism were compared with the defects in FMZ binding (2 to 3 hours and 8 to 9 hours after MCAO), with the pattern of disturbed glucose metabolism (determined 12 hours after MCAO), and with the size of the infarcts (determined approximately 15 hours after MCAO). RESULTS: Irrespective of the level of reperfusion, defects in FMZ binding (2 to 3 hours after MCAO) were closely related to areas with severely depressed oxygen consumption and predicted the size of the final infarcts, whereas preserved FMZ binding indicated intact cortex. Depression of glucose metabolism was in all animals larger than the defects in FMZ binding and the infarcts, indicating functional deactivation of brain areas beyond the permanent morphological damage. In addition, FMZ distribution within 2 minutes after injection was significantly correlated to flow and yielded reliable perfusion images. CONCLUSIONS: The reduction of FMZ binding early after focal ischemia reflects irreversible neuronal damage that otherwise only can be detected by multitracer studies. Our experimental data and first clinical applications suggest that FMZ has potential as an indicator of developing infarction. Since FMZ distribution additionally images perfusion, this tracer might be useful for the selection of patients who would benefit from acute therapeutic intervention.

Animals↗
PubMed1997

Villous cytotrophoblast proliferating potential in complete and partial hydatidiform mole: diagnostic value of silver-stained nucleolar organizer region (AgNOR)-associated proteins.

Aside from their typical morphologic features, complete (CHM) and partial hydatidiform mole (PHM) are characterized by variable trophoblastic proliferation and/or atypia. CHM and PHM usually present little diagnostic difficulty. However, some may be extremely difficult to distinguish by morphologic features alone. Therefore, we investigated the diagnostic value of silver-stained nucleolar organizer region (AgNOR)-associated proteins in cytotrophblasts as compared to cytogenetic features of nine CHM, nine PHM and six non-molar spontaneous embryonic abortions (controls), as well as of two suspected CHM and two histologically suspected PHM. Tissue sections were submitted to autoclave pretreatment and to silver colloid solution. The proliferating potential of cytotrophoblasts was determined by the analysis of mean number and mean area of AgNORs per nucleus using a PC-based image analysis system. Mean values of AgNOR parameters were significantly different from each other (p < 0.001). Each of the four cases of tentative diagnosis could be assigned to the corresponding group of examined trophoblastic lesions. The evaluation of AgNORs in cytotrophoblasts contributes to a reliable discrimination of CHM and PHM; this fairly simple and economical method could serve as an useful addition to conventional methods of diagnosis in gestational trophoblastic disease.

Cell Division↗
PubMed1997

[Redefinition of the proximal perichondrium and perichondrial gap in hip ultrasound imaging].

As a result of the improvement in the resolution of diagnostic sonography equipment it is possible to depict sonographic echoes of structures in the area of the preformed cartilage acetabular roof, especially in the region of the proximal perichondrium, perichondrial gap and the joint capsule that up to now have not been anatomically identified. Therefore, the question arises as to which anatomical structures these newly observed sonographic echoes can be classed with. Hip-joint NMR images of corpses were made and compared to anatomical specimen, anatomical sections and sonograms of high-resolution ultrasound equipment. Through this comparative study it was possible to demonstrate that the echo of the proximal perichondrium is anatomically consistent with the proximal perichondrium itself, parts of the joint capsule and the insertion of the musculus rectus femoris tendon. Identification of the ligamentum ischiofemorale and its separation from the labrum acetabulare, the joint capsule and the musculus rectus femoris tendon leads to better interpretation of the perichondrial gap in the sonogram. Common terms, such as "proximal perichondrium" and "perichondrial gap" obtain a new anatomical assessment. These terms, taken as a basis for knowledge of the anatomical structures, can be used as usual. There are no consequences for hip sonography today if the definition of the distinction between type III and type IV, and of the standard section is used further as the basis of sonography even if these terms have received a new anatomical classification.

Acetabulum↗
PubMed1996

Early nitric oxide increase in depolarized tissue of cat focal cerebral ischaemia.

Tissue nitric oxide (NO) concentration was investigated in relation to ion-homeostasis disturbance in the cat model of focal cerebral ischaemia. An NO electrode, a Ca2+ microelectrode and a laser Doppler probe were applied to the cerebral cortex in the core and periphery of the middle cerebral artery. NO concentration increased by 25.1 +/- 6.3 nM at 5 min in severely ischaemic regions exhibiting anoxic depolarization (n = 5, p < 0.0005). This occurred with no reduction in extracellular Ca2+ concentration and before a massive Ca2+ influx into cells started several minutes later. The NO increase was abolished by NG-nitro-L-arginine treatment (n = 6, p < 0.05) and was absent in regions with no depolarization (n = 5, p < 0.0005). We conclude that the early increase in NO associated with depolarization is achieved by activation of constitutive NO synthase, possibly triggered by intracellular Ca2+ release.

Acute Disease↗
PubMed1996

Random circular permutation of genes and expressed polypeptide chains: application of the method to the catalytic chains of aspartate transcarbamoylase.

Recent studies on proteins whose N and C termini are in close proximity have demonstrated that folding of polypeptide chains and assembly of oligomers can be accomplished with circularly permuted chains. As yet no methodical study has been conducted to determine how extensively new termini can be introduced and where such termini cannot be tolerated. We have devised a procedure to generate random circular permutations of the catalytic chains of Escherichia coli aspartate transcarbamoylase (ATCase; EC 2.1.3.2) and to select clones that produce active or stable holoenzyme containing permuted chains. A tandem gene construct was made, based on the desired linkage between amino acid residues in the C- and N-terminal regions of the polypeptide chain, and this DNA was treated with a suitable restriction enzyme to yield a fragment containing the rearranged coding sequence for the chain. Circularization achieved with DNA ligase, followed by linearization at random with DNase I, and incorporation of the linearized, repaired, blunt-ended, rearranged genes into a suitable plasmid permitted the expression of randomly permuted polypeptide chains. The plasmid with appropriate stop codons also contained pyrI, the gene encoding the regulatory chain of ATCase. Colonies expressing detectable amounts of ATCase-like molecules containing permuted catalytic chains were identified by an immunoblot technique or by their ability to grow in the absence of pyrimidines in the growth medium. Sequencing of positive clones revealed a variety of novel circular permutations. Some had N and C termini within helices of the wild-type enzyme as well as deletions and insertions. Permutations were concentrated in the C-terminal domain and only few were detected in the N-terminal domain. The technique, which is adaptable generally to proteins whose N and C termini are near each other, can be of value in relating in vivo folding of nascent, growing polypeptide chains to in vitro renaturation of complete chains and determining the role of protein sequence in folding kinetics.

Aspartate Carbamoyltransferase↗
PubMed1996

Elastic fibres are an essential component of human placental stem villous stroma and an integrated part of the perivascular contractile sheath.

The stroma of human placental stem villi is believed to consist only of reticular and collagen fibres. In the present study we were able to show for the first time by light (orcein staining) and electron microscopy large amounts of elastic fibres in the stem villous stroma. Electron microscopically, homogeneous elastin was found alone or in association with microfibrils. In addition, microfibrils were observed forming long bands. These three structures, generally known to form elastic connective tissue, were seen in close connection with placental extravascular smooth muscle cells, which belong to the perivascular contractile sheath (PVCS) of stem villi. Elastin was associated with these smooth muscle cells and connected to collagen fibres via microfibrils. Collagen fibres were additionally interconnected by spike-like structures. Extravascular smooth muscle cells revealed numerous adhesion plaques which occupied conspicuously long cytoplasmic faces of the plasma membrane. In cryostat sections, immunoreactivity of talin, an attachment protein of adhesion plaques linking intracellular alpha-actin filaments with extracellular fibronectin, was detected in extravascular and vascular (media) smooth muscle cells. The arrangement of placental extravascular smooth muscle cells, elastic and collagen fibres suggests a functional myofibroelastic unit within the PVCS, which surrounds the large foetal blood vessels possibly contributing to elasticity and supporting tensile and/or contracting forces within the stem villi.

Actins↗
PubMed1996

Nitric oxide synthase I immunoreactivity and NOS-associated NADPHd histochemistry in the visceral epithelial cells of the intraplacental mouse yolk sac.

In the course of our studies on the local blood flow modulation in the NMRI-mouse placenta we have focussed on regulatory pathways involving recently appreciated gaseous messenger molecules nitric oxide (NO) and carbon monoxide (CO), which are generated by NO synthase (NOS) and heme oxygenase (HO)-2, respectively. The distribution of NOS was investigated by immunohistochemistry using an antiserum to the neuronal isoform (NOS-I) and by NADPH diaphorase (NADPHd) histochemistry, supplemented with procedures (permanganate and formaldehyde method) serving to enhance the specificity of the enzyme histochemical method for NOS visualization. HO-2 was demonstrated immunohistochemically. In addition, cyclic guanosine monophosphate (cGMP)-forming soluble guanylate cyclase (sGC) and dehydrogenases generating the NOS co-substrate NADPH were analysed either by immunohistochemistry or enzyme histochemistry. NOS-I immunostaining was observed in the intraplacental visceral yolk sac epithelial cells but not in the placenta and extraplacental visceral epithelial yolk sac cells. Co-localization of NOS-I immunolabeling and NOS-associated NADPHd was exclusively found in the intraplacental visceral epithelial cells, while NADPHd activity not associated to NOS was present in other placental and extraplacental cells additionally analysed for control reasons. HO-2 and sGC immunoreactivity could not be detected in the placenta including the intraplacental visceral epithelial cells but were expressed in several extraplacental cells. Dehydrogenases producing the NOS co-substrate NADPH were present in the intraplacental visceral epithelium as well as in other placental and extraplacental cells. Since the intraplacental visceral epithelial yolk sac layer closely accompanies large fetal blood vessels entering the placental labyrinth from the chorionic plate it may be assumed that NO, generated by the NADPH-consuming NOS-I in the intraplacental yolk sac epithelium, acts to regulate the blood flow by relaxing smooth muscle cells in the wall of these fetal vessels. The lack of immunoreactivity to the NO-effector molecule sGC may be due to methodological reasons. The absence of the HO-2/CO system suggests its insignificant role as a potential gas signaling pathway in the vascular smooth muscle system of the intraplacental visceral yolk sac of mice.

Animals↗
PubMed1996

[Experiences with a 3-D ultrasound system in infant hip joints].

AIM: To test the possibilities of 3-dimensional sonography in diagnosing congenital hip dysplasia. METHOD: 128 infant hip joints were examined by 4 investigators with different levels of experience. The Kretz Combison 530 machine additionally equipped with an automatic 5 MgHz transducer and volume box system was employed. RESULTS: The average time to determine the standard plane was 21 seconds. The single standard deviation for alpha values in the 3-dimensional system was 3.0 degrees, compared to 1.37 degrees in the conventional system. Observer experience was not a crucial factor. CONCLUSION: The described 3-dimensional system makes it quicker and simpler to determine the standard plane when evaluating infant hips. The higher standard deviation is possibly caused by the obliquity of the ultrasonic beam. Ossification defects on the acetabular roof are better visualized with this approach than with conventional ultrasound or radiological methods. Even though 3-dimensional ultrasound represents a new approach to infant hips, it seems worthy of rapid development.

Acetabulum↗
PubMed1996

Prolonged transient ischemia results in impaired CBF recovery and secondary glutamate accumulation in cats.

Effects of prolonged focal ischemia [middle cerebral artery occlusion (MCAO)] of 1, 2, and 4 h followed by 15-h reperfusion on CBF, extracellular amino acids, purine catabolites, evoked potentials, and infarction were studied in core (A:auditory cortex) and border zone (SF: somatosensory cortex) areas of halothane-anesthetized cats. Following MCAO, CBF reduction was severe in A (<15 ml 100 g-1 min-1) and mild to moderate in SF. Prominent elevation of glutamate and abolition of evoked potentials in A contrasted with milder and more variable disturbances in SF. After reperfusion, recovery of CBF, glutamate, and evoked potentials was fast and persistent in the 1- and 2-h groups. In the 4-h group, immediate recovery of CBF, glutamate, and evoked potentials was incomplete, and secondary deterioration of all parameters was obtained at the end of the experiments. Infarction in the 4-h group was significantly larger than in the 1- and 2-h groups. Persistent recovery of extracellular glutamate concentration and electrical function and salvage of neuronal tissue from infarction therefore seem to depend on successful restoration of CBF, which in turn depends on the magnitude and the duration of CBF reduction and of exposure to potentially harmful substances such as glutamate during the ischemic attack.

Amino Acids↗
PubMed1996

Cloning and expression analysis of Aedes aegypti opsin: adaptation of an in situ hybridization protocol for mosquitoes.

Opsin is a G protein coupled photoreceptor that activates a signal transduction cascade in the ommatidia. Its primary and secondary structure is conserved both in insects and vertebrates as exemplified by the Drosophila opsins. Through serendiplious cloning of a PCR fragment, we have identified an opsin cDNA. The latter was used to clone full length cDNAs from a mosquito head library. The main purpose of cloning was to have a positive control probe to establish an in situ hybridization protocol for less abundant probes. Opsin-mRNA is localized specifically to the visual receptor cells in the ommatida. No other cells in the brain or the remainder of the body are positive. This is confirmed by Northern blot analysis. The sequence of the receptor, of which we have found two different transcripts, confirms its typical topology, including the seven transmembrane spanning regions and the intracellular carboxy terminus that has potential phosphorylation sites. Our in situ hybridization protocol combines several procedures: the most important points are: (a) the immediate processing of sections after cutting, and (b) the sections are never allowed to dry out once the procedure was started. Our protocol has a much higher sensitivity, using approximately 50 x lower concentrations of probe compared to published protocols. In addition to the detection of opsin-mRNA, it has been successfully applied to the detection of the low abundant insulin receptor homologue. Furthermore, Aedes aegypti probes were visualizing a similar tissue specificity when applied to the malaria mosquito Anopheles albimanus.

Aedes↗
PubMed1995

Halothane, but not alpha-chloralose, blocks potassium-evoked cortical spreading depression in cats.

The effects of two anesthetics, halothane and alpha-chloralose, on induction of spreading depression and on extracellular glutamate elevation after intracortical potassium administration were investigated in artificially ventilated (30% oxygen/70% nitrous oxide) cats. High potassium concentrations were achieved using either direct KCl injections (7 microliters, 150 mM via a micropipette) or microdialysis by supplementing 100, 300 or 500 mM KCl, respectively, for 10 min to the perfusion solution (Ringer's). Changes of the cortical DC potential were recorded adjacent (1-2 mm: electrode DC1) and distant (6-7 mm: electrode DC2) to the injection site. Either under halothane (0.75% in the respiratory gas mixture) or under alpha-chloralose (60 mg/kg i.v.) anesthesia, prolonged negative shifts of the DC potential reflecting the elevated potassium levels after KCl injection were measured near the injection site (electrode DC1). In contrast, spreading depressions (transient short DC deflections) were almost exclusively observed under alpha-chloralose. Spreading depressions recorded with electrode DC1 were superimposed on the prolonged negative DC shifts and they propagated frequently to the more distant site (DC2). Upon KCl administration, dose dependent elevations of extracellular glutamate were measured. These elevations were not significantly altered by the type of anesthesia. Our results suggest that in cats, spreading depression induction is affected by anesthesia, i.e., spreading depression induction is inhibited by halothane as compared to alpha-chloralose. Furthermore, factors other than glutamate or high potassium seem to contribute to spreading depression induction.

Animals↗
PubMed1995

Protein insertion into the endoplasmic reticulum of permeabilized cells.

We have established efficient translocation of newly synthesized proteins into the endoplasmic reticulum of permeabilized Mel Juso cells. By site-specific photo-crosslinking we show that translocating polypeptide chains contact the same components of permeabilized cells ER as in dog pancreas rough microsomes. This cellular assay system has the potential to overcome the limitations of isolated microsomes in investigating the molecular environment of a newly synthesized protein after they have left the ER translocation site.

Animals↗
PubMed1995

Photolabeling identifies a putative fusion domain in the envelope glycoprotein of rabies and vesicular stomatitis viruses.

Vesicular stomatitis and rabies viruses enter cells through receptor-mediated endocytosis, followed by fusion of the viral with the endosomal membrane. The latter step is catalyzed by the viral envelope glycoprotein, which, in the low pH environment of the endosome, undergoes a conformational transition to a fusion-competent state. To investigate whether fusion competence involves the low pH exposure of a hydrophobic fusion region(s), we have applied hydrophobic photolabeling using the recently developed phospholipid analogue 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromethyl-3H- diazirin-3-yl)benzyl]oxy]carbonyl] nonanoyl]-sn-glycero-3-phosphocholine ([125I]TID-PC/16) (Weber, T., and Brunner, J. (1995) J. Am. Chem. Soc. 117, 3084-3095). Rosettes of rabies virus glycoprotein, whole rabies virus, or vesicular stomatitis virus were incubated with large unilamellar vesicles containing [125I]TID-PC/16. Following reagent activation, the labeled glycoprotein was isolated and analyzed. In all cases, labeling of the glycoprotein strongly increased as the pH was lowered from 7.0 to 6.0, suggesting the exposure at acidic pH of a domain capable of interacting with membranes. To identify the labeled region(s), CNBr fragments were generated and analyzed by SDS-polyacrylamide followed by autoradiography. In rabies glycoprotein, the labeled segment was found to be contained within fragment RCr5 (residues 103-179). Glycoprotein from vesicular stomatitis virus was labeled within fragment VCr1 (residues 59-221). These results demonstrate that rhabdovirus glycoprotein contains a domain that at low pH is capable of interacting with a target membrane in a hydrophobic manner. This domain may play a role similar to that of the fusion peptide found in many other viral fusion proteins.

Amino Acid Sequence↗
PubMed1995

Localisation of the high affinity facilitative glucose transporter protein GLUT 1 in the placenta of human, marmoset monkey (Callithrix jacchus) and rat at different developmental stages.

In the present study, the facilitative D-glucose transporter protein GLUT 1 was localised by immunohistochemistry in the placenta of human, marmoset (Callithrix jacchus) and rat at different developmental stages. A polyclonal antiserum against a 13-amino-acid peptide of the GLUT 1 carboxy terminus was used. It identified a protein of around 50 kDa molecular weight in immunoblotting of the placental tissues. GLUT 1 was located in the syncytiotrophoblast, in cytotrophoblast cells and in fetal endothelium. Similar staining patterns, except in human extravillous cytotrophoblast cells, were observed at all differentiation stages, despite differences in the internal placental architecture of the species. In the marmoset placenta, GLUT 1 was undetectable in endothelial cells of maternal vessels. In rat placentae, trophoblastic giant cells, epithelial cells of both visceral and parietal yolk sac, yolk sac vessels and the stratum spongiosum were stained. Reichert's membrane did not immunoreact. Preadsorption of the antiserum with a 13-amino-acid peptide resulted in the loss of immunoreactivity. The results suggest that GLUT 1 is a prominent isoform of glucose transporters in mammalian placentae. It is generally abundant in placental cell populations bordering on the maternal and fetal circulations and may therefore facilitate an effective glucose supply to the fetus and placenta.

Animals↗
PubMed1995

The perivascular contractile sheath of human placental stem villi: its isolation and characterization.

Former studies have shown that fetal blood vessels of stem villi in the human placenta are enclosed by sheaths of presumed contractile cells. Efforts to prove the contractility of these sheaths by isometric and biochemical investigations still suffer from the difficulty of differentiating between extravascular and vascular (media) contractile cells. The present study describes a method for the selective dissection of perivascular tissue sheaths in stem villi of approximately 2-4 mm thickness, which contained abundant alpha-actin immunoreactive cells and were free of adherent vascular smooth muscle cells. These extravascular contractile cells are part of the 'fibrous paravascular sheath', which, in placental pathology, is used as an index of maturity. To emphasize the high number of contractile cells and their location within this sheath, we propose the common term perivascular contractile sheath (PVCS). The isolation method offers the possibility of selectively investigating the contractile forces of the PVCS and of obtaining more insight into its functional role.

Actins↗
PubMed1995

Decidua and placenta in mice after treatment with a synthetic glucocorticoid.

To investigate a possible long-term effect of glucocorticoids on decidua and placenta of mice, a single dose of 24 mg kg-1 body weight triamcinolone acetonide in crystalline suspension was given subcutaneously to NMRI mice on gestational day (GD) 2. Deciduae and placentae, as well as corticosterone and triamcinolone concentrations in maternal plasma of GDs 10 and 17 were examined. NADPH-cytochrome P450 reductase involved in drug biotransformation was detected immunocytochemically and showed co-localization with NADPH diaphorase histochemistry in the decidua and placenta. Both reactions were higher in endothelial cells of decidual sinusoids on GD 10, but were lower on GD 17 in the trophoblast, spongiotrophoblast and extraplacental visceral yolk-sac epithelial cells of treated mice than in untreated animals. Histochemistry of 11 beta-hydroxysteroid dehydrogenase, an enzyme that metabolizes biologically active adrenocortical steroids and their synthetic congeners in the placenta, showed higher activity on GD 17 in enlarged labyrinthic trophoblast I cells of treated mice than in untreated animals. As corticosterone concentrations were still decreased on GD 17, when triamcinolone concentrations were no longer detectable, a long-term suppression of adrenal gland function seems obvious.

11-beta-Hydroxysteroid Dehydrogenases↗