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Biomedical subjects

J Pohl

Publications and source records attributed to J Pohl.

At least 37 records · Page 2Linked to original sources
PubMed1999

[Megaloblastic anemia in a psychogenic eating disorder].

HISTORY AND ADMISSION FINDINGS: A 68-year-old woman had progressively lost strength over the past 5 years and become bed-ridden. She reported gradually reducing her diet during the last few years, for the last two years restricting herself to oatmeal, biscuits and water. Acute exacerbation of dyspnoea, dizziness, tachycardia and cardiac arrhythmias for the last 3 month necessitated her emergency admission when she was found to have oedema of the limbs and definite jaundice, as well as predominantly distal sensory deficits in the legs, a slightly ataxic gait and poor reflexes in the limbs. INVESTIGATIONS: She was found to have pancytopenia with marked megaloblastic anaemia (haemoglobin 4.8 mg/dl, mean red cell volume 141.8 fl, leukocytopenia 41/nl), marked haemolysis (lactate dehydrogenase 1629 U/l, haptohaemoglobin < 0.1 g/l, total bilirubin concentration 4.5 mg/dl). Pernicious anaemia was excluded: no antibodies against parietal cells and intrinsic factor, and no atrophic gastritis in a gastric biopsy. DIAGNOSIS, TREATMENT AND COURSE: Megaloblastic anaemia due to dietary deficiencies with early funicular myelosis was suspected. After infusion of four erythrocyte concentrates and vitamin B12 administration the symptoms and blood picture improved within days. CONCLUSION: Although dietary causes of megaloblastic anaemia are rare in Central Europe, they must be considered in the differential diagnosis, especially in the elderly and those with psychogenic disorders.

Aged↗
PubMed1999

Chemical stability and fate of the cytostatic drug ifosfamide and its N-dechloroethylated metabolites in acidic aqueous solutions.

31P NMR spectroscopy was used to study the products of the decomposition of the antitumor drug ifosfamide (IF, 1d) and its N-dechloroethylated metabolites, namely, 2,3-didechloroethylIF (1a) and 2- (1b) and 3-dechloroethylIF (1c), in buffered solutions at acidic pH. The first stage of acid hydrolysis of these four oxazaphosphorines is a P-N bond cleavage of the six-membered ring leading to the phosphoramidic acid monoesters (2a-d) of type R'HN(CH(2))(3)OP(O)(OH)NHR, with R and/or R' = H or (CH(2))(2)Cl. The electron-withdrawing chloroethyl group at the endocyclic and/or exocyclic nitrogens counteracts the endocyclic P-N bond hydrolysis. This effect is even more marked when the N-chloroethyl group is in the exocyclic position since the order of stability is 1d > 1c > 1b > 1a. In the second stage of hydrolysis, the remaining P-N bond is cleaved together with an intramolecular attack at the phosphorus atom by the non-P-linked nitrogen of the compounds 2a-d. This leads to the formation of a 2-hydroxyoxazaphosphorine ring with R = H (3a coming from compounds 2a,c) or (CH(2))(2)Cl (3b coming from compounds 2b,d) and to the release of ammonia or chloroethylamine. The third step is the P-N ring opening of the oxazaphosphorines 3a,b leading to the phosphoric acid monoesters, H(2)N(CH(2))(3)OP(O)(OH)(2) (4a) and Cl(CH(2))(2)HN(CH(2))(3)OP(O)(OH)(2) (4b-1), respectively. For the latter compound, the chloroethyl group is partially (at pH 5.5) or totally (at pH 7.0) cyclized into aziridine (4b-2), which is then progressively hydrolyzed into an N-hydroxyethyl group (4b-3). Compounds 3a,b are transient intermediates, which in strongly acidic medium are not observed with (31)P NMR. In this case, cleavage of the P-N bond of the type 2 phosphoramidic acid monoesters leads directly to the type 4 phosphoric acid monoesters. The phosphate anion, derived from P-O bond cleavage of these latter compounds, is only observed at low levels after a long period of hydrolysis. Compounds 1a-c and some of their hydrolytic degradation products (4b-1, 4b-2, diphosphoric diester [Cl(CH(2))(2)NH(CH(2))(3)OP(O)(OH)](2)O (5), and chloroethylamine) did not exhibit, as expected, any antitumor efficacy in vivo against P388 leukemia. (31)P NMR determination of the N-dechloroethylated metabolites of IF or its structural isomer, cyclophosphamide (CP), and their degradation compounds could provide an indirect and accurate estimation of chloroacetaldehyde amounts formed from CP or IF.

Amides↗
PubMed1999

Neuroprotective effects of growth/differentiation factor 5 depend on the site of administration.

Growth/differentiation factor 5 (GDF5) is a neurotrophin which protects the rat nigrostriatal dopaminergic pathway from 6-hydroxydopamine-induced damage. Here we used amphetamine-induced rotational testing, high-performance liquid chromatography and immunocytochemistry to investigate the minimum effective dose of GDF5. We also compared the effectiveness of injecting GDF5 into either the substantia nigra pars compacta (SNpc), the lateral ventricle (LV) or the striatum (or combinations of these sites).

3,4-Dihydroxyphenylacetic Acid↗
PubMed1999

Modulation of expression of endothelial nitric oxide synthase by nordihydroguaiaretic acid, a phenolic antioxidant in cultured endothelial cells.

Retrospective epidemiological studies have suggested that antioxidant therapy may decrease cardiovascular morbidity and mortality rates, although the mechanisms for this effect remain unclear. In the present study, we demonstrate that selective antioxidants can enhance expression of endothelial nitric oxide synthase (eNOS). We found that the antioxidants nordihydroguaiaretic acid (NDGA), catechol, glutaryl probucol, and N-acetylcysteine increased eNOS expression in cultured bovine aortic endothelial cells (BAECs). NDGA seemed to be the most potent of the phenolic antioxidants, producing a 3-fold increase in eNOS mRNA. This effect of NDGA was enhanced by nonphenolic antioxidants such as N-acetylcysteine and ascorbic acid. Nuclear run-on studies indicated that NDGA increased eNOS transcription. A similar increase in eNOS protein content was observed with Western blot analysis after treating BAECs or human aortic endothelial cells with NDGA. Exposure of BAECs to NDGA enhanced NO production, as measured by electron paramagnetic resonance spin trapping and eNOS activity, as measured by [14C]arginine-to-[14C]citrulline assay. Methylation of the phenolic hydroxyl groups completely inhibited the NDGA effect on eNOS mRNA levels. This effect of NDGA was not due to inhibition of lipoxygenase because cis-5,8,11,14-eicosatetraynoic acid did not alter eNOS expression. We conclude that antioxidants may not only increase the bioactivity of nitric oxide but also enhance expression of the eNOS enzyme. Such an effect may prove useful in conditions such as hypertension and atherosclerosis, in which nitric oxide production and/or biological activity is impaired.

Acetylcysteine↗
PubMed1999

Bone morphogenetic proteins and growth and differentiation factors in the human cornea.

PURPOSE: To investigate transcription of members of the transforming growth factor (TGF)-beta superfamily and corresponding receptors in human corneal epithelium and stroma. METHODS: Transcription of bone morphogenetic proteins (BMP)-2, BMP-3, BMP-4, BMP-5, and BMP-7; growth- differentiation factor (GDF)-5), and BMP receptors (BMPR) types I (BMPR-IA, BMPR-IB) and II (BMPR-II) was investigated by reverse transcription-polymerase chain reaction (RT-PCR) in ex vivo and cultured cells. For verification, PCR fragments were cloned and sequenced. DNA dot blot analysis was performed to estimate the level of transcription. RNA dot blots were performed to determine expression of GDF-5. Expression of BMP receptor proteins was investigated by immunohistochemistry. Single-cell clonal growth proliferation assays were performed using recombinant human GDF-5 and TGF-beta1. RESULTS: Transcription of BMP-2, BMP-3, BMP-4, BMP-5, and BMP-7 and receptors of BMPR-IA, BMPR-IB and BMPR-II was detected in ex vivo and cultured epithelium and stroma. The level of transcription was higher in cultured stroma for all factors, but the level for the receptors was higher in cultured epithelium. In contrast GDF-5 was transcribed only in stromal cells, suggesting that this cytokine may be an important mediator between keratocytes and epithelial cells. Furthermore, GDF-5 inhibited proliferation of corneal epithelial and stromal cells. CONCLUSIONS: Given the importance of the TGF-beta family during embryonic development, the results suggest that its members may be components of the corneal cytokine network and may participate in the regulation of cellular proliferation and differentiation.

Animals↗
PubMed1998

14-3-3zeta binds a phosphorylated Raf peptide and an unphosphorylated peptide via its conserved amphipathic groove.

14-3-3 proteins bind a variety of molecules involved in signal transduction, cell cycle regulation and apoptosis. 14-3-3 binds ligands such as Raf-1 kinase and Bad by recognizing the phosphorylated consensus motif, RSXpSXP, but must bind unphosphorylated ligands, such as glycoprotein Ib and Pseudomonas aeruginosa exoenzyme S, via a different motif. Here we report the crystal structures of the zeta isoform of 14-3-3 in complex with two peptide ligands: a Raf-derived phosphopeptide (pS-Raf-259, LSQRQRSTpSTPNVHMV) and an unphosphorylated peptide derived from phage display (R18, PHCVPRDLSWLDLEANMCLP) that inhibits binding of exoenzyme S and Raf-1. The two peptides bind within a conserved amphipathic groove on the surface of 14-3-3 at overlapping but distinct sites. The phosphoserine of pS-Raf-259 engages a cluster of basic residues (Lys49, Arg56, Arg60, and Arg127), whereas R18 binds via the amphipathic sequence, WLDLE, with its two acidic groups coordinating the same basic cluster. 14-3-3 is dimeric, and its two peptide-binding grooves are arranged in an antiparallel fashion, 30 A apart. The ability of each groove to bind different peptide motifs suggests how 14-3-3 can act in signal transduction by inducing either homodimer or heterodimer formation in its target proteins.

14-3-3 Proteins↗
PubMed1998

The effects of tamoxifen on endometrial insulin-like growth factor-1 expression.

OBJECTIVE: To determine whether modulation of insulin-like growth factor-1 and insulin-like growth factor-binding protein-1 expression underlies the uterotropic effects associated with tamoxifen therapy in postmenopausal breast cancer patients. METHODS: Using immunohistochemical techniques, we analyzed 37 endometrial specimens from biopsies (n = 18) or hysterectomies (n = 19) for Ki-67, insulin-like growth factor-1, and insulin-like growth factor-binding protein-1 expression. Specifically, five secretory- and three proliferative-phase endometrial specimens were used as controls; 20 specimens (including two endometrial adenocarcinomas) were analyzed from postmenopausal breast cancer patients treated with tamoxifen (20 mg/day) for at least 6 months; and nine endometrial adenocarcinoma specimens from patients not treated with tamoxifen were studied. Intensity of immunostaining was quantified using digitized imaging techniques. RESULTS: Insulin-like growth factor-1 and insulin-like growth factor-1-binding protein-1 were found to be expressed in normal and neoplastic endometrium of all patients, regardless of tamoxifen treatment. However, insulin-like growth factor-1 expression varied cyclically in histologically normal endometrium, was reduced in undifferentiated endometrial tumors, and was upregulated in tamoxifen-treated specimens. Insulin-like growth factor-binding protein-1 immunostaining did not vary during the menstrual cycle, but it was reduced significantly in benign tamoxifen-exposed tissue and endometrial adenocarcinomas, regardless of degree of differentiation or tamoxifen exposure. No correlation was found between the expression of insulin-like growth factor-1 and insulin-like growth factor-binding protein-1 and the proliferative indices of the tissues examined. CONCLUSION: The expression of insulin-like growth factor-1 and insulin-like growth factor-binding protein-1 in the uterus supports an autocrine and/or paracrine role for these proteins in endometrial physiology. Although further studies are needed, our investigation suggests that altered expression of insulin-like growth factor-1 and insulin-like growth factor-binding protein-1 may contribute to the uterotropic effects of tamoxifen.

Adenocarcinoma↗
PubMed1998

Expression of human activin C protein in insect larvae infected with a recombinant baculovirus.

In order to generate dimeric recombinant transforming growth factor-beta (TGF-beta) proteins, expensive eucaryotic cell systems, such as CHO cells, are usually used. An alternative represents the expression of such proteins in insects using a baculovirus expression system. In this study, recombinant human activin C protein was expressed in Noctuidae larvae. On SDS-PAGE, the expressed protein has a size of about 15 kD under reducing conditions and of about 20 kD under non-reducing conditions. This suggests that activin C is expressed as a dimer and disulfide bridges can be formed. Compared with expression in eucaryotic cell culture systems, expression in insect larvae presents a rapid and low cost method, without the need for expensive tissue culture scale-ups or special equipment.

3T3 Cells↗
PubMed1998

Growth/differentiation factor 5 and glial cell line-derived neurotrophic factor enhance survival and function of dopaminergic grafts in a rat model of Parkinson's disease.

Growth/differentiation factor 5 is a member of the transforming growth factor beta superfamily, which has neurotrophic and neuroprotective effects on dopaminergic neurons both in vitro and in vivo. Here we investigate the effects of growth/differentiation factor 5 on foetal mesencephalic grafts transplanted into a rat model of Parkinson's disease, and compare them with those of glial cell line-derived neurotrophic factor. Mesencephalic tissue was suspended in solutions containing either growth/differentiation factor 5 or glial cell line-derived neurotrophic factor prior to transplantation into the left striatum of rats with 6-hydroxydopamine lesions of the left medial forebrain bundle. Both proteins enhanced graft-induced compensation of amphetamine-stimulated rotations. Positron emission tomography studies showed that both neurotrophins increased graft-induced recovery of striatal binding of [11C]RTI-121, a marker for dopaminergic nerve terminals. Post mortem analysis at 8 weeks after transplantation showed that both neurotrophins significantly increased the survival of grafted dopaminergic neurons. This study shows that growth/differentiation factor 5 is at least as effective as glial cell line-derived neurotrophic factor in enhancing the survival and functional activity of mesencephalic grafts, and thus is an important candidate for use in the treatment of Parkinson's disease.

Animals↗
PubMed1998

Time course and cellular localization of interleukin-10 mRNA and protein expression in autoimmune inflammation of the rat central nervous system.

Experimental autoimmune encephalomyelitis of the Lewis rat is a T-cell-mediated autoimmune disease of the central nervous system characterized by a self-limiting monophasic course. In this study, we analyzed the expression of the anti-inflammatory cytokine interleukin (IL)-10 at the mRNA and protein level in experimental autoimmune encephalomyelitis actively induced with the encephalitogenic 68-86 peptide of guinea pig myelin basic protein. Semiquantitative reverse transcriptase-polymerase chain reaction revealed that IL-10 mRNA expression peaked during the acute phase of the disease at days 11 and 13. IL-10 mRNA was synchronously induced with mRNA for the proinflammatory cytokine interferon-gamma. Immunocytochemistry with a monoclonal antibody against rat IL-10 showed that the peak of IL-10 mRNA was accompanied by an abundant expression of IL-10 protein during the acute stage of the disease. Both in situ hybridization and double labeling immunocytochemistry in combination with confocal microscopy identified T cells, macrophages/microglia, and astrocytes as major cellular sources of IL-10 in vivo. The early peak of IL-10 production was unexpected in light of its well-documented anti-inflammatory properties. Additional studies are required to determine whether endogenous IL-10 contributes to rapid clinical remission typical for Lewis rat experimental autoimmune encephalomyelitis or if it plays other, yet undefined, roles in central nervous system autoimmunity.

Animals↗
PubMed1997

Cell death during autoimmune demyelination: effector but not target cells are eliminated by apoptosis.

The Fas and FasL apoptotic pathway was investigated by protein immunohistochemistry, flow cytometry, and reverse transcriptase-PCR analysis to assess whether it is involved in the elimination of target and/or effector cells from the central nervous system (CNS) during adoptively transferred chronic relapsing experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. In addition to Fas and FasL, we studied Bax, an intracellular protein of the apoptotic cascade, the Bax antagonist and anti-apoptotic molecule Bcl-2, and DNA fragmentation, the final step in the apoptotic pathway. Infiltrating CD4+ T cells and parenchymal microglia expressed Fas, FasL, and Bax, and about half of these cells showed DNA fragmentation, a combination indicative of ongoing apoptosis. Using flow cytometry and reverse transcriptase-PCR, a positive correlation was seen between disease activity and up-regulation of the Fas system; in fact, Fas and FasL were expressed at low levels at the onset of EAE and increased at the height of disease to involve about one-third of all infiltrating lymphocytes. In the normal CNS, Fas immunoreactivity was constitutively present at low levels on oligodendrocytes and was up-regulated in the CNS during the course of EAE. However, oligodendrocytes showed no Bax reactivity or DNA fragmentation and expressed high levels of Bcl-2, as did the majority of infiltrating CD3+ cells, a pattern inconsistent with apoptosis. Thus, while molecules of the apoptotic cascade are well represented in the CNS during EAE, their expression correlates with elimination of infiltrating cells and microglia, not the myelinating cell, the oligodendrocyte.

Animals↗
PubMed1997

Characterization of growth/differentiation factor 5 (GDF-5) as a neurotrophic factor for cultured neurons from chicken dorsal root ganglia.

Growth/differentiation factor-5 (GDF-5), a morphogenetic protein, has previously been shown to act as a neurotrophic factor for midbrain dopaminergic neurons. To further elucidate the neurotrophic potential of GDF-5, serum free cultures of dorsal root ganglionic (DRG) neurons from developing chick embryos were treated with GDF-5 with or without the simultaneous addition of other trophic factors. Our results show that GDF-5 has a minor promoting effect on its own, but it can enhance the survival promoting effect of neurotrophin-3 (NT-3) and nerve growth factor (NGF) on cultured DRG neurons. Our finding fits well into the concept that neurotrophic factors may act synergistically in ensuring survival of different neuronal populations. The capacity of GDF-5 to reduce the requirement of a subpopulation of sensory neurons for NT-3 may have implications for the treatment of peripheral neuropathies.

Animals↗
PubMed1997

Growth/differentiation factor 5 protects nigrostriatal dopaminergic neurones in a rat model of Parkinson's disease.

Growth/differentiation factor 5 (GDF5), a novel member of the transforming growth factor beta superfamily, promotes the survival of dopaminergic neurones in vitro. We present here the first evidence for a neuroprotective action of GDF5 in vivo. We investigated the effects of intracerebral administration of GDF5 on a rat model of Parkinson's disease. GDF5 was administered just above the substantia nigra and into the lateral ventricle immediately before ipsilateral injection of 6-hydroxydopamine into the medial forebrain bundle. GDF5 prevented the development of amphetamine-induced rotations and preserved the integrity of striatal dopaminergic nerve terminals, as measured by positron emission tomography. Post-mortem studies showed that GDF5 spared dopamine levels in the striatum and tyrosine hydroxylase positive neurones in the midbrain. This study suggests that GDF5 has potential for the treatment of Parkinson's disease.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1997

Subunit structure and function of porcine factor Xa-activated factor VIII.

Factor Xa and thrombin (factor IIa) activate factor VIII (fVIII) by different proteolytic pathways. Thrombin cleaves fVIII at Arg372 between the A1 and A2 domains, at Arg740 between the A2 and B domains, and at Arg1689 between the B and A3 domains to form an A1/A2/A3-C1-C2 heterotrimer. We now report a stable porcine fVIIIaXa preparation obtained by Mono S HPLC at pH 6. NH2-terminal sequence analysis of purified subunits of fVIIIaXa revealed that factor Xa cleaves fVIII at Arg219 within the A1 domain and at Arg490 within the A2 domain, as well as at Arg372, Arg740, and Arg1689. Analytical ultracentrifugation of the fVIIIaXa preparation yielded results consistent with a single, 148 kDa species, similar to previous results with fVIIIaIIa [Lollar, P., & Parker, C. G. (1989) Biochemistry 28, 666-674]. Thus, the major species in the fVIIIaXa preparation contains five subunits, including fragments of the A1 and A2 domains that remain noncovalently bound. Fluorescence anisotropy measurements indicated there was no difference in the affinity of fVIIIaXa and fVIIIaIIa for a fluorescent dye-labeled, active-site-blocked derivative of porcine factor IXa. Additionally, the fVIIIaXa preparation bound dye-labeled factor IXa with 1:1 stoichiometry, indicating that all fVIIIaXa molecules in the preparation can bind factor IXa. However, fVIIIaXa had 4-fold less procoagulant activity than fVIIIaIIa. Kinetic analysis of fVIIIa cofactor activity using purified factor IXa and factor X suggested this difference is due to greater activity of fVIIIaIIa relative to fVIIIaXa within the intrinsic fXase complex, rather than a difference in their stabilities.

Amino Acid Sequence↗
PubMed1997

Induction of DNA crosslinks and DNA strand lesions by cyclophosphamide after activation by cytochrome P450 2B1.

Cyclophosphamide requires metabolic activation by cytochrome P450 to exert its genotoxic effects. Therefore in vitro studies on its mechanism of action have been limited to the use of self-activating derivatives of cyclophosphamide or to hepatocytes as an activating system. In this study we used a cell line of Chinese hamster lung fibroblasts (V79 cells), genetically engineered to express active cytochrome P450 2B1 as the sole observable cytochrome P450 (SD1 cells). An increase in DNA strand lesions (SL: DNA single-strand breaks and alkali labile sites) was observed between 0.5 and 1.5 mM cyclophosphamide (24 h incubation) which could be classified as alkali labile sites using a modified alkaline elution assay. Compared to cyclophosphamide, its active metabolite 4-hydroperoxycyclophosphamide (4-OOH-CY) was about 250-fold more effective in induction of SL. Equimolar concentrations of phosphoramide mustard (50 microM), the ultimate DNA binding metabolite of cyclophosphamide, caused only about 50% of SL compared to 4-OOH-CY. A minimum of 12 h of incubation of SD1 cells was needed for cyclophosphamide (1 mM) until SL were detectable, compared to only 2 h for 4-OOH-CY and 1.5 h for phosphoramide mustard (50 microM). DNA crosslinks were observable after shorter incubation periods than single-strand breaks (6 h for cyclophosphamide and 1 h for 4-OOH-CY and phosphoramide mustard) and were no longer detectable at incubation periods of more than 20 h. Treatment of SD1 cells with ionizing radiation only, cyclophosphamide only, and radiation plus cyclophosphamide showed that SL induced by cyclophosphamide were not repaired during incubation with fresh culture medium (24 h). However, an efficient repair of SL caused by ionizing radiation was observed and was not inhibited by cyclophosphamide. These observations give strong evidence that different types of SL were induced by cyclophosphamide and radiation. SD1 cells were able to repair the special kind of SL induced by radiation but not the SL caused by cyclophosphamide.

Animals↗
PubMed1997

The development of alkylphosphocholines as signal transduction inhibitors: experimental and clinical challenges.

Alkylphosphocholines are a new class of anticancer agents. Their mode of action is considered to be related to the inhibition of phospholipase C and protein kinase C. These enzymes play a major role in intracellular signalling pathways. Their inhibition by alkylphosphocholines leads in the dimethylbenzanthracene-induced mammary carcinoma of the rat to a response pattern similar to that of the antiestrogen zindoxifene. This suggests that the inhibition of transcription factor formation might be the common pathway for alkylphosphocholines and antihormones. Based on the experimental dose-response pattern, new clinical strategies for dose finding and response evaluation will have to be developed for inhibitors of signal transduction, such as alkylphosphocholines.

Animals↗