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Biomedical subjects

B Hildebrand

Publications and source records attributed to B Hildebrand.

At least 19 recordsLinked to original sources
PubMed1999

90-day subchronic toxicity study in rats and mice fed N-methylpyrrolidone (NMP) including neurotoxicity evaluation in rats.

In mice, there were no effects on body weight or food consumption. As observed in rats, mice fed 2,500 or 7,500 ppm exhibited a change in urine coloration which was not associated with morphological changes in cholesterol, triglycerides, calcium, and alkaline phosphatase occurred at 28 days but not 90 days. These changes are thus assessed as being of minor toxicological relevance. Liver weights were elevated in males fed 2,500 or 7,500 ppm and centrilobular hypertrophy was seen in both sexes fed 7,500 ppm. These changes may be regarded as an adaptation process but are clearly related to NMP exposure. Other toxicological endpoints examined were unaffected by NMP. The NOAEL was 3,000 ppm for both sexes of rats based on body weight effects and changes in 3 neurobehavioral parameters (males only) at higher feeding levels. In mice, the NOAEL was 1,000 ppm based on liver responses to higher concentrations.

Animals↗
PubMed1999

[Expert assessment of medical treatment errors--a new responsibility for the medical service of health insurance].

During the past few years there has been an enormous increase of inquiries for medical certificates concerning suspected mistakes in medical treatment (medical malpractice). The profound changes of the social circumstances and the social law (e.g. section 66 SGB V) are responsible for this situation. The legal involvement of the "Gesetzliche Krankenversicherung" (GKV) and the "Medizinischer Dienst der Krankenversicherung" (MDK) into social legislation concerning the expert opinion are mentioned. The general development of the judgement of medical malpractice and its legal background, which bases on the contract between doctor and patient, is shown. As a practical example serves the description of the procedure of judging medical malpractice in the MDK. First statistical results, which have been developed since summer 1997 in Hamburg are presented. Some interesting tendencies are obvious: orthopedics and surgery are involved with more than 50%. "Characteristic mistakes in therapy" are the most important question for the expert opinion. Creating a medical certificate concerning medical malpractice takes about 6-12 weeks. This tendency is decreasing because of fast learning procedures, caused by the increasing demand of expert opinions. The medical judgement of medical malpractice will become one of the main tasks of the medical service in health insurance. The "MDK's" have to adapt to this change in tasks in medical, legal and organisation-technical respect, if they want to offer competent and up to date judgement in future times.

Expert Testimony↗
PubMed1998

Subchronic inhalation toxicity study of 2-ethylhexanol vapour in rats.

A 90-day subchronic inhalation toxicity study was performed on Wistar rats in accordance to OECD testing guidelines to evaluate the toxicological profile of 2-ethylhexanol, potential target organs, and a no-observable-adverse-effect-level (NOAEL). 10 males and 10 females per group were exposed to 2-ethylhexanol vapours at concentrations of 15, 40 and 120 ppm (the latter corresponding to the vapour saturation at 20 degrees C) 6 hours/day for 90 days. The respective controls inhaled clean air under the same conditions. No substance-related adverse effects were observed for body weight, body weight gain, mortality, organ weights, clinical biochemistry and haematological parameters including clotting time. Cyanide-insensitive palmitoyl-CoA oxidation, a marker for peroxisome proliferation, was found elevated in a subchronic study in Fischer 344 rats after gavage application of 500 mg/kg but not under the conditions of this 90-day subchronic inhalation study. There were no findings related to the treatment with 2-ethylhexanol either at necropsy or at histological examination. The highest concentration tested under these conditions (120 ppm) was found to be the NOAEL for male and female rats.

Administration, Inhalation↗
PubMed1997

Expression of the proenkephalin gene in cultured astroglial cells: analysis of cell cycle dependence.

Glial progenitors strongly express the proenkephalin (PEnk) gene during their proliferation in the subventricular zone of the neocortex. Also in primary culture, astroglial cells from rat neocortex produce PEnk mRNA. Since the basal expression sharply declines after the cultured cells reach confluence, it seems to be related to cell proliferation. In contrast, activation of protein kinases A and C strongly enhances the levels of PEnk mRNA in confluent cultures. Therefore, it was investigated in cultured neocortical astroglial cells, whether the basal and stimulated expression of the PEnk gene occurred during different phases of the cell cycle. Activation of protein kinases A and C with 8Br.cAMP and tetradecanoylphorbolacetate, respectively, enhanced the PEnk gene expression only during the G1 phase. Unstimulated astroglial cells contained PEnk mRNA during the G1 as well as the S phase of the cell cycle. Since confluent astroglial cells are arrested in the G1 phase, they should have a basal expression of the gene which is comparable to that in preconfluent cells. Thus, the reduced basal expression after confluence indicates that other mechanisms may play a role, such as humoral factors or contact inhibition.

Animals↗
PubMed1997

Neurons are generated in confluent astroglial cultures of rat neonatal neocortex.

Cells of the telencephalon are generated in specific proliferative zones from which neuronal and glial precursors migrate to their destinations. Recent evidence indicates that some precursors do not turn into differentiated cells but keep their ability to proliferate. Here, we report that neurons can originate in primary cultures of astroglial cells prepared from neocortex of newborn rats. The first neuronal cells appeared shortly before confluence, when a glial monolayer was being formed. After confluence, these still undifferentiated cells increased in number. Later, they became immunohistochemically positive for the neuron-specific marker microtubule-associated protein 2a,b. They also contained neurofilament-L protein as well as the specific messenger RNA coding for neurofilament-H. The observation that they took up bromo-deoxyuridine indicated that they synthesized DNA, i.e. they proliferated. When Dulbecco's modified essential medium was substituted with fetal calf serum, the appearance of neurons depended on the seeding density of the dispersed cells. This was no longer the case, when the cultures were maintained in Dulbecco's modified essential medium/F12 medium to which transferrin, insulin and selenium chloride had been added. It is concluded that neuronal precursors can survive in primary astroglial cultures. After confluence of the astroglial cells the precursors proliferate if appropriate conditions are present. Our observation provides a new model for the investigation of cultured neurons and neuronal-glial interactions.

Animals↗
PubMed1996

Regulation of the expression of the proenkephalin gene in cultured meningeal fibroblasts: opposite effects of alpha 1- and beta 2-adrenoceptors.

Meningeal fibroblasts express the proenkephalin gene during embryonal development but terminate the expression shortly before birth. When brought into primary culture at postnatal day 1, the fibroblasts again express the gene. Activation of protein kinase A reduces this expression and thus may contribute to its prenatal termination. Since the noradrenergic innervation of the meninges begins around the time of birth, it was investigated in the present study, how adrenergic agonists affected the levels of proenkephalin mRNA in cultured fibroblasts. The beta 2-adrenoceptor agonists salbutamol and procaterol increased the levels of endogenous cAMP and diminished the concentration of proenkephalin mRNA indicating that the cultured fibroblasts possessed this beta-subtype. In contrast, noradrenaline increased the level of proenkephalin mRNA in a concentration-dependent manner. This effect was independent of endogenous cAMP and was mediated by alpha 1-adrenoceptors. The data indicate that the noradrenergic innervation of the meninges at the time of birth is not responsible for the termination of the proenkephalin gene expression.

Adrenergic alpha-1 Receptor Agonists↗
PubMed1996

Noradrenaline increases the expression of the proenkephalin gene in cultured astroglial cells by acting on beta 1- and alpha 1-adrenoceptors.

In primary culture, type I astroglial cells from neocortex of newborn rats express the proenkephalin gene. The glial cells are not homogeneous but differ in their morphology; i.e., polygonal and process-bearing cells are found. Transcription of the proenkephalin gene is increased via protein kinase A upon stimulation with cyclic AMP (cAMP) analogues. In the present study, how noradrenaline affected the expression of the proenkephalin gene in both cell types was investigated. Noradrenaline enhanced the levels of proenkephalin mRNA in a concentration-dependent manner. Experiments with subtype-selective antagonists suggested that beta 1-adrenoceptors were involved. In situ hybridization showed that proenkephalin mRNA was induced only in polygonal cells. Nor-adrenaline also increased the levels of cAMP. However, concentrations of noradrenaline that produced a maximal increase in cAMP caused only submaximal elevations of proenkephalin mRNA. This discrepancy was explained by the finding that noradrenaline increased the expression of the proenkephalin gene also via alpha 1-adrenoceptors. It is concluded that beta 1- and alpha 1-adrenoceptors can act in a synergistic manner on the expression of the proenkephalin gene in astroglial cells.

Animals↗
PubMed1995

Rat meningeal fibroblasts in primary culture express the proenkephalin gene.

When brought into primary culture, rat meningeal fibroblasts contained proenkephalin-mRNA detected with Northern blot hybridization. In contrast, splenic fibroblasts did not express the gene under the same culture conditions. In situ hybridization showed that the meningeal fibroblasts did not uniformly express the gene: groups of positive cells were surrounded by cells with low or no proenkephalin-mRNA. Some fibroblasts which contained the mRNA species took up bromo-deoxyuridine indicating that the expression of the gene also occurred in proliferating cells, but was not restricted to this group. In chromaffin and astroglial cells, activation of protein kinase A or C with 8 Br.cAMP or O-tetradecanoyl 13-phorbolacetate, respectively, increases the expression of the gene. In meningeal fibroblasts, however, both agents reduced the levels of proenkephalin-mRNA. In the case of 8Br.cAMP, this effect was blocked by the protein synthesis inhibitor cycloheximide indicating that a newly-synthesized protein was involved. Cultured meningeal fibroblasts appear to be useful for studies on the cell specificity of the expression of this peptide gene as well on its regulation.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

Evidence for the involvement of 5-lipoxygenase products in the regulation of the expression of the proenkephalin gene in cultured astroglial cells.

Cultured astroglial cells secrete eicosanoids which are produced by the cyclooxygenase and lipoxygenases. These cells also transcribe the proenkephalin gene. In the present study, it was investigated whether agents which inhibit the metabolism of arachidonic acid affect the basal and stimulated expression of the gene. Tetradecanoyl phorbol acetate (TPA; 1-1000 nmol/l) increases the concentration of proenkephalin mRNA in these cells by activating protein kinase C. The enhancement in proenkephalin mRNA caused by TPA (10 nmol/l) was not affected by the cyclooxygenase inhibitor indomethacin (5 mumol/l). However, nordihydroguaiaretic acid, which blocks cyclooxygenase and lipoxygenases, potentiated the effect of TPA on proenkephalin mRNA, when used at concentrations of 0.5-50 mumol/l. Two selective inhibitors of 5-lipoxygenase, i.e. MK886 (5 mumol/l) and BAY X1005 (1 mumol/l), also enhanced the effect of TPA (10 nmol/l) without affecting the basal expression of the gene. When added to the incubation medium, leukotriene E4 (10-1000 nmol/l) diminished in a dose-dependent manner the basal and TPA-induced expression of the proenkephalin gene. It is concluded that in astroglial cells derived from cortex of new-born rats products of 5-lipoxygenase can diminish the action of protein kinase C on the proenkephalin gene.

Animals↗
PubMed1994

Inherited resistance to activated protein C is corrected by anticoagulant cofactor activity found to be a property of factor V.

Recently, our laboratory described a defect in anticoagulant response to activated protein C (APC). This response, APC resistance, was shown to be inherited and associated with familial thrombophilia. As other possible mechanisms were excluded, APC resistance was hypothesized to be due to deficiency of a previously unrecognized cofactor of APC. The aim of the present study was to isolate and characterize this factor. Plasma from an individual with pronounced inherited APC resistance was used as test plasma in a biological assay which monitored APC cofactor activity during its isolation from normal plasma. A purification procedure was devised that yielded a protein which was shown to be identical to coagulation factor V. It proved impossible to separate the APC cofactor activity from factor V, even by affinity chromatography using a monoclonal antibody against factor V. The affinity-purified factor V corrected the poor anticoagulant response to APC of APC-resistant plasma in a dose-dependent manner. Because the APC-resistant plasma contained normal levels of factor V procoagulant activity, the results indicated APC resistance to be due to a selective defect in the anticoagulant function of factor V. The present results show factor V not only to express procoagulant properties after its activation by thrombin but also to play an important part in the anticoagulant system as cofactor to APC.

Blood Coagulation↗
PubMed1990

Novel type of very high affinity calcium-binding sites in beta-hydroxyasparagine-containing epidermal growth factor-like domains in vitamin K-dependent protein S.

Vitamin K-dependent protein S is shown to contain four very high affinity Ca2(+)-binding sites. The number of sites and their affinities were determined from Ca2+ titration in the presence of the chromophoric chelator Quin 2. In 0.15 M NaCl, pH 7.5, the four macroscopic binding constants are K1 greater than or equal to 1 x 10(8) M-1, K2 = 3 +/- 2 x 10(7) M-1, K3 = 4 +/- 2 x 10(6) M-1, and K4 = 9 +/- 4 x 10(5) M-1. At low ionic strength, the corresponding values are K1 greater than or equal to 2 x 10(9) M-1, K2 = 9 +/- 4 x 10(8) M-1, K3 = 2 +/- 1 x 10(8) M-1, and K4 = 9 +/- 4 x 10(7) M-1. To localize the Ca2(+)-binding sites, protein S was subjected to proteolysis using lysyl endopeptidase. This yielded a 20-21-kDa fragment which comprised the third and fourth epidermal growth factor (EGF)-like domains and remained high affinity Ca2(+)-binding site(s). The susceptibility of the EGF-like domains to proteolysis increased when Ca2+ was removed from protein S indicating that the Ca2+ binding is important for the stability and/or conformation of the EGF domains. Three of the four EGF-like domains in protein S contain beta-hydroxyasparagine. In each of these domains there is a cluster of three or four negatively charged amino acid residues which are likely to contribute to the extraordinary high Ca2+ affinity. From sequence homology it is suggested that this novel type of high affinity Ca2(+)-binding site is present in several other proteins, e.g. in the EGF-like domains in the low sensity lipoproteins receptor, thrombomodulin, the Notch protein of Drosophila melanogaster, and transforming growth factor beta 1-binding protein.

Amino Acid Sequence↗
PubMed1990

Characterization of functionally important domains in human vitamin K-dependent protein S using monoclonal antibodies.

Vitamin K-dependent protein S is an anticoagulant plasma protein functioning as a cofactor to activated protein C in the degradation of coagulation factors Va and VIIIa. To determine which regions in protein S are important for its cofactor activity, we have raised and characterized a large panel of monoclonal antibodies against human protein S. Several of the antibodies were directed against Ca2(+)-dependent epitopes, and they were found to be located either in the domain containing gamma-carboxyglutamic acid (Gla), the thrombin-sensitive region, or in the first epidermal growth factor (EGF)-like domain. The first two types of epitopes were exposed at approximately 1 mM Ca2+, whereas the epitope(s) in the EGF-like domains required less than 1 microM Ca2+, suggesting the presence of one or more high affinity Ca2(+)-binding site(s). The antibodies, as well as their Fab' fragments, against all three types of Ca2(+)-dependent epitopes efficiently inhibited the activated protein C cofactor function of protein S, but through different mechanisms. The antibodies against the Gla domain exerted their effects through inhibition of protein S binding to negatively charged phospholipid. Fab'-fragments of antibodies against the thrombin-sensitive region and the first EGF-like domain were the most potent inhibitors of the activated protein C cofactor function but did not inhibit phospholipid binding of protein S. In conclusion, we have identified the domains in protein S that are important for the activated protein C cofactor activity. The Gla domain is instrumental in the binding of protein S to phospholipid, whereas the thrombin-sensitive region and the first EGF-like domain may be directly involved in protein-protein interactions on the phospholipid surface.

1-Carboxyglutamic Acid↗
PubMed1986

Isolation and characterization of human blood-coagulation factor X cDNA.

Using synthetic oligodeoxynucleotides as probes, we have isolated factor X cDNA from human liver cDNA library. We sequenced the 1430-bp cDNA which spans the coding region of the mature factor X and contains the polyadenylation signal and poly(A) tail. The amino acid (aa) sequence is in agreement with the published aa sequence. The nucleotide (nt) sequence of cDNA confirmed that factor X is synthesized and secreted as a single-chain precursor, and then converted into dimeric form by proteolytic cleavage of an internal tripeptide. From the nt sequence, it was also predicted that like other secretory proteins, human factor X is synthesized with a leader sequence (prepro-protein). The 5'-coding region of factor X cDNA is 60 and 40% homologous to the corresponding regions of factor IX and prothrombin genes, respectively. This supports the hypothesis of gene evolution by gene duplication followed by divergence.

Amino Acid Sequence↗
PubMed1983

Degradation of human complement component C4b in the presence of the C4b-binding protein-protein S complex.

Vitamin K-dependent protein S and the higher-molecular-weight form of C4b-binding protein (C4bp-high) interact, forming a 1:1 complex with a KD of approx. 1 X 10(-7) M [Dahlbäck (1983) Biochem. J. 209, 847-856]. In the present study the effect of protein S on the degradation of C4b by Factor I (C3b inactivator) and C4bp was investigated both in fluid phase and on cell surfaces, with the use of highly purified components. Fluid-phase degradation of C4b was monitored on sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis, and the effect on surface-bound C4b was estimated by haemolytic assay. No effect of protein S could be demonstrated in any of the systems used. Thus, although bound to C4bp, protein S is neither involved in, nor does it affect, the interaction between C4bp and C4b. This indicates that the binding sites on the C4bp molecule for protein S and for C4b are independent and different.

Binding Sites↗