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Biomedical subjects

M Fuhrmann

Publications and source records attributed to M Fuhrmann.

At least 19 recordsLinked to original sources
PubMed2002

Sulfur polymer solidification/stabilization of elemental mercury waste.

Elemental mercury, contaminated with radionuclides, presents a waste disposal problem throughout the Department of Energy complex. In this paper we describe a new process to immobilize elemental mercury wastes, including those contaminated with radionuclides, in a form that is non-dispersible, will meet EPA leaching criteria, and has low mercury vapor pressure. In this stabilization and solidification process, elemental mercury is combined with an excess of powdered sulfur polymer cement (SPC) and sulfide additives in a mixing vessel and heated to approximately 40 degrees C for several hours, until all of the mercury is converted into mercuric sulfide (HgS). Additional SPC is then added and the temperature of the mixture raised to 135 degrees C, resulting in a molten liquid which is poured into a mold where it cools and solidifies. The final treated waste was characterized by powder X-ray diffraction and found to be a mixture of the hexagonal and orthorhombic forms of mercuric sulfide. The Toxicity Characteristic Leaching Procedure was used to assess mercury releases, which for the optimized process averaged 25.8 microg/l, with some samples being well below the new EPA Universal Treatment Standard of 25 microg/l. Longer term leach tests were also conducted, indicating that the leaching process was dominated by diffusion. Values for the effective diffusion coefficient averaged 7.6x10(-18) cm2/s. Concentrations of mercury vapor from treated waste in equilibrium static headspace tests averaged 0.6 mg/m3.

Diffusion↗
PubMed2002

Spectroscopic characterization of flavin mononucleotide bound to the LOV1 domain of Phot1 from Chlamydomonas reinhardtii.

The absorption and emission behavior of flavin mononucleotide (FMN) in the light-, oxygen- and voltage-sensitive (LOV) domain LOV1 of the photoreceptor Phot1 from the green alga Chlamydomonas reinhardtii was studied. The results from the wild-type (LOV1-WT) were compared with those from a mutant in which cysteine 57 was replaced by serine (LOV1-C57S), and with free FMN in aqueous solution. A fluorescence quantum yield of phi(F) = 0.30 and a fluorescence lifetime of tau(F) = 4.6 ns were determined for FMN in the mutant LOV1-C57S, whereas these quantities are reduced to about phi(F) = 0.17 and tau(F) = 2.9 ns for LOV1-WT, indicating an enhanced intersystem crossing in LOV1-WT because of the adjacent sulfur of C57. A single-exponential fluorescence decay was observed in picosecond laser time-resolved fluorescence measurements for both LOV1-WT and LOV1-C57S as expected for excited singlet state relaxation by intersystem crossing and internal conversion. An excitation intensity dependent fluorescence signal saturation was observed in steady-state fluorescence measurements for LOV1-WT, which is thought to be because of the formation of a long-lived intermediate flavin-C(4a)-cysteinyl adduct in the triplet state (few microseconds triplet lifetime, adduct lifetime around 150 s). No photobleaching was observed for LOV1-C57S, because no thiol group is present in the vicinity of FMN for an adduct formation.

Animals↗
PubMed2001

Human and rat alveolar macrophages express multiple EDG receptors.

Endothelial differentiation gene (EDG) receptors are a new family of eight G protein-coupled receptors for the lysophospholipids lysophosphatitic acid and sphingosine-1-phosphate. In the present experiments, the expression of EDG receptors in rat and human alveolar macrophages was studied by reverse transcription-polymerase chain reaction (RT-PCR). In alveolar macrophages of both species, mRNA for multiple EDG receptors could be detected, but the pattern of expression was different in both species. In human alveolar macrophages, mRNA for EDG1, EDG2, EDG4, EDG7 receptors and, to a lesser extent, for the EDG7 receptor was detected, whereas in rat macrophages, mRNA for EDG2, EDG5 receptors and, to a lesser extent, for the EDG6 receptor was found. In functional experiments, it was observed that lysophosphatitic acid and sphingosine-1-phosphate can stimulate O(2)(-) generation in rat and human alveolar macrophages suggesting that lysophosphatitic acid and sphingosine-1-phosphate possibly acting via EDG receptors may play a role in controlling the activation of macrophages.

Animals↗
PubMed2001

A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii.

Although Chlamydomonas reinhardtii serves as the most popular algal model system, no efficient enzymatic selection marker for the nuclear transformation of wild-type cells is available. We sequenced an aminoglycoside 3'-phosphotransferase gene (aph) from Streptomyces rimosus. Though the derived protein sequence is homologous to members of APH type V, it constitutes a new type, named APHVIII. Since the aphVIII gene has a codon bias similar to that of the nuclear genome of green algae, the aphVIII coding sequence was fused to the 5'- and 3'-untranslated regions of the C. reinhardtii rbcS2 gene. C. reinhardtii transformants were capable of inactivating the antibiotics paromomycin, kanamycin, and neomycin, to which wild-type cells are sensitive. After addition of the 5'-region of hsp70A as a second promoter and insertion of the rbcS2 intron I, the transformation rate increased to two transformants per 1 x 10(5) cells, which is close to the efficiency of transforming auxotrophic strains with the homologous marker arg7. Transformation with the promoter-less aphVIII led to random gene fusion at high frequency. In an aphVIII-based reporter gene assay we have found a so far unknown promoter activity of the 3'-untranslated region of rbcS2, that may promote antisense RNA synthesis from the rbcS2 gene in vivo. We conclude that the aphVIII gene is a useful marker for nuclear transformation and promoter tagging of C. reinhardtii wild-type and probably other green algae.

5' Untranslated Regions↗
PubMed2001

Glucocorticoids inhibit lipopolysaccharide-induced up-regulation of arginase in rat alveolar macrophages.

1. As arginase by limiting nitric oxide (NO) synthesis may play a role in airway hyperresponsiveness and glucocorticoids are known to induce the expression of arginase I in hepatic cells, glucocorticoid effects on arginase in alveolar macrophages (AM Phi) were studied. 2. Rat AM Phi were cultured in absence or presence of test substances. Thereafter, nitrite accumulation, arginase activity, and the expression pattern of inducible NO synthase, arginase I and II mRNA (RT - PCR) and proteins (immunoblotting) were determined. 3. Lipopolyssacharides (LPS, 20 h) caused an about 2 fold increase in arginase activity, whereas interferon-gamma (IFN-gamma), like LPS a strong inducer of NO synthesis, had no effect. 4. Dexamethasone decreased arginase activity by about 25% and prevented the LPS-induced increase. Mifepristone (RU-486) as partial glucocorticoid receptor agonist inhibited LPS-induced increase by 45% and antagonized the inhibitory effect of dexamethasone. 5. Two different inhibitors of the NF-kappa B-pathway also prevented LPS-induced increase in arginase activity. 6. Rat AM Phi expressed mRNA and protein of arginase I and II, but arginase I expression was stronger. Arginase I mRNA and protein was not affected by IFN-gamma, but increased by LPS and this effect was prevented by dexamethasone. Both, LPS and IFN-gamma enhanced the levels of arginase II mRNA and protein, effects also inhibited by dexamethasone. As IFN-gamma did not affect total arginase activity, arginase II may represent only a minor fraction of total arginase activity. 7. In rat AM Phi glucocorticoids inhibit LPS-induced up-regulation of arginase activity, an effect which may contribute to the beneficial effects of glucocorticoids in the treatment of inflammatory airway diseases.

Animals↗
PubMed2001

The abundant retinal protein of the Chlamydomonas eye is not the photoreceptor for phototaxis and photophobic responses.

The chlamyopsin gene (cop) encodes the most abundant eyespot protein in the unicellular green alga Chlamydomonas reinhardtii. This opsin-related protein (COP) binds retinal and was thought to be the photoreceptor controlling photomovement responses via a set of photoreceptor currents. Unfortunately, opsin-deficient mutants are not available and targeted disruption of non-selectable nuclear genes is not yet possible in any green alga. Here we show that intron-containing gene fragments directly linked to their intron-less antisense counterpart provide efficient post-transcriptional gene silencing (PTGS) in C. reinhardtii, thus allowing an efficient reduction of a specific gene product in a green alga. In opsin-deprived transformants, flash-induced photoreceptor currents (PC) are left unchanged. Moreover, photophobic responses as studied by motion analysis and phototaxis tested in a light-scattering assay were indistinguishable from the responses of untransformed wild-type cells. We conclude that phototaxis and photophobic responses in C. reinhardtii are triggered by an as yet unidentified rhodopsin species.

Algal Proteins↗
PubMed2000

MioC is an FMN-binding protein that is essential for Escherichia coli biotin synthase activity in vitro.

Biotin synthase is required for the conversion of dethiobiotin to biotin and requires a number of accessory proteins and small molecule cofactors for activity in vitro. We have previously identified two of these proteins as flavodoxin and ferredoxin (flavodoxin) NADP(+) reductase. We now report the identification of MioC as a third essential protein, together with its cloning, purification, and characterization. Purified MioC has a UV-visible spectrum characteristic of a flavoprotein and contains flavin mononucleotide. The presence of flavin mononucleotide and the primary sequence similarity to flavodoxin suggest that MioC may function as an electron transport protein. The role of MioC in the biotin synthase reaction is discussed, and the structure and function of MioC is compared with that of flavodoxin.

Apoproteins↗
PubMed2000

Nuclear factor-kappaB mediates simultaneous induction of inducible nitric-oxide synthase and Up-regulation of the cationic amino acid transporter CAT-2B in rat alveolar macrophages.

The connection between the regulation of L-arginine transport and nitric oxide (NO) synthesis was studied in rat alveolar macrophages. Lipopolysaccharides (LPSs) and interferon-gamma stimulated in the same concentration- and time-dependent manner NO synthesis (measured by nitrite accumulation) and L-[(3)H]arginine uptake. This correlated with an increased mRNA expression for iNOS and the cationic amino acid transporter CAT-2B (analyzed by reverse transcription-polymerase chain reaction), with the same kinetics observed for the up-regulation of both mRNAs. Because nuclear factor-kappaB (NF-kappaB) is essential for induction of iNOS its role for the regulation of CAT-2B expression and L-arginine transport was investigated. The NF-kappaB inhibitors pyrrolidine dithiocarbamate and N(alpha)-p-tosyl-L-lysine chloromethyl ketone abrogated LPS- and interferon-gamma-induced increase of nitrite accumulation and L-[(3)H]arginine uptake as well as up-regulation of iNOS and CAT-2B mRNA. LPS-induced increase in iNOS and CAT-2B mRNA was also suppressed by specific NF-kappaB decoy oligodesoxynucleotides, confirming the essential role of NF-kappaB for iNOS and CAT-2B expression. Dexamethasone did not affect the initial (5 h) LPS-induced increase of iNOS and CAT-2B mRNA, but down-regulated both mRNAs after prolonged (20 h) exposure and this was accompanied by partial inhibition of LPS-stimulated nitrite accumulation and L-[(3)H]arginine uptake. These findings demonstrate parallel regulation of the expression of iNOS and CAT-2B, and of NO synthesis and L-arginine uptake in rat alveolar macrophages. NF-kappaB is an essential transcription factor not only for the induction of iNOS, but also for the up-regulation of CAT-2B. The simultaneous up-regulation of CAT-2B with iNOS is considered as a mechanism to ensure a high substrate supply for iNOS.

Amino Acid Transport Systems, Basic↗
PubMed1999

Purification, characterization, DNA sequence and cloning of a pimeloyl-CoA synthetase from Pseudomonas mendocina 35.

A pimeloyl-CoA synthetase from Pseudomonas mendocina 35 was purified and characterized, the DNA sequence determined, and the gene cloned into Escherichia coli to yield an active enzyme. The purified enzyme had a pH optimum of approximately 8.0, Km values of 0.49 mM for pimelic acid, 0.18 mM for CoA and 0.72 mM for ATP, a subunit Mr of approximately 80000 as determined by SDS/PAGE, and was found to be a tetramer by gel-filtration chromatography. The specific activity of the purified enzyme was 77.3 units/mg of protein. The enzyme was not absolutely specific for pimelic acid. The relative activity for adipic acid (C6) was 72% and for azaleic acid (C9) was 18% of that for pimelic acid (C7). The N-terminal amino acid was blocked to amino acid sequencing, but controlled proteolysis resulted in three peptide fragments for which amino acid sequences were obtained. An oligonucleotide gene probe corresponding to one of the amino acid sequences was synthesized and used to isolate the gene (pauA, pimelic acid-utilizing A) coding for pimeloyl-CoA synthetase. The pauA gene, which codes for a protein with a theoretical Mr of 74643, was then sequenced. The deduced amino acid sequence of the enzyme showed similarity to hypothetical proteins from Archaeoglobus fulgidus, Methanococcus jannaschii, Pyrococcus horikoshii, E. coli and Streptomyces coelicolor, and some limited similarity to microbial succinyl-CoA synthetases. The similarity with the protein from A. fulgidus was especially strong, thus indicating a function for this unidentified protein. The pauA gene was cloned into E. coli, where it was expressed and resulted in an active enzyme.

Acyl Coenzyme A↗
PubMed1999

Biotin production under limiting growth conditions by Agrobacterium/Rhizobium HK4 transformed with a modified Escherichia coli bio operon.

The E. coli biotin (bio) operon was modified to improve biotin production by host cells: (a) the divergently transcribed wild-type bio operon was re-organized into one transcriptional unit; (b) the wild-type bio promoter was replaced with a strong artificial (tac) promoter; (c) a potential stem loop structure between bioD and bioA was removed; and (d) the wild-type bioB ribosomal binding site (RBS) was replaced with an artificial RBS that resulted in improved bioB expression. The effects of the modifications on the bio operon were studied in E. coli by measuring biotin and dethiobiotin production, and bio gene expression with mini-cells and two-dimensional polyacrylamide gel electrophoresis. The modified E. coli bio operon was introduced into a broad host-range plasmid and used to transform Agrobacterium/Rhizobium HK4, which then produced 110 mg L-1 of biotin in a 2-L fermenter, growing on a defined medium with diaminononanoic acid as the starting material. Biotin production was not growth-phase dependent in this strain, and the rate of production remained high under limiting (maintenance) and zero growth conditions.

Journal Article↗
PubMed1999

A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii.

The use of Chlamydomonas reinhardtii as a model system has been hindered by difficulties encountered in expressing foreign genes. We have synthesised a gene encoding green fluorescent protein (GFP) adapted to the codon usage of C. reinhardtii (cgfp). After verifying the gene was functional in Escherichia coli, the cgfp was fused in frame to the phleomycin resistance gene ble from Streptoalloteichus hindustanus and expressed in C. reinhardtii under control of the rbcS2 promoter and intron sequences. The GFP-fluorescence was seen only in the nucleus demonstrating the nuclear accumulation of the Ble-GFP fusion protein. The cgfp was also fused to the chlamyopsin gene, cop, and expressed in C. reinhardtii under control of the cop promoter. The eyespot became fluorescent indicating that the opsin-GFP fusion protein was correctly directed into the eyespot along with the endogenous unmodified opsin. We conclude that cgfp provides a useful tool to visualize protein synthesis and localisation in vivo in C. reinhardtii and possibly in related green algal species.

Algal Proteins↗
PubMed1999

Identification and function of cyclic nucleotide phosphodiesterase isoenzymes in airway epithelial cells.

Epithelial cells actively participate in inflammatory airway disease by liberating mediators such as arachidonate metabolites and cytokines. Inhibition of phosphodiesterases (PDEs) may be a useful anti-inflammatory approach. The PDE isoenzyme pattern and the effects of PDE inhibition on mediator generation were analyzed in primary cultures of human and porcine airway epithelial cells (AEC) and in the bronchial epithelial cell line BEAS-2B. PDE4 and PDE5 were detected in lysates of all cell types studied. In primary cultures of human AEC, the PDE4 variants PDE4A5, PDE4C1, PDE4D2, and PDE4D3 were identified by polymerase chain reaction analysis. Evidence of the recently described PDE7 was obtained by rolipram- insensitive cyclic adenosine monophosphate (cAMP) degradation, and its presence was verified by the demonstration of PDE7 messenger RNA. Primary cultures of human airway epithelium also expressed PDE1. Enhanced epithelial cAMP levels, induced by forskolin and PDE4 inhibition, increased formation of prostaglandin E2 (PGE2), but not of interleukin (IL)-8 or 15-hydroxyeicosatetraenoic acid (15-HETE) in airway epithelial cells. Increased cyclic guanosine monophosphate levels in these cells provoked by sodium nitroprusside and the PDE5 inhibitor zaprinast reduced the PGE2 synthesis, whereas 15-HETE and IL-8 formation were unchanged. The data suggest that PDE isoenzymes are important in airway inflammation and that PDE inhibitors exert anti-inflammatory effects by acting on AEC.

Animals↗
PubMed1997

Sorption/desorption of radioactive contaminants by sediment from the Kara Sea.

To understand the long term impact of the disposal of radioactive waste on the Kara Sea, partition coefficients (Kd) for several important radionuclides, the mineralogy of the sediment, and the relationship of Kd to liquid-to-solid ratio were quantified. Sediment was obtained from four locations in the Kara Sea area. Slow sorption kinetics were observed for 85Sr, 232U, 125I and 99Tc, whilst sorption was rapid (less than 50 h to steady-state) for 137Cs, 210Pb, and 241Am. Partition coefficients (Kd) were determined using batch type experiments and sorption isotherms which were developed for 85Sr, 99Tc, 125I, U and 137Cs. Partition coefficients for 137Cs were approx. 350 ml/g for sediment from the Trough and 180 ml/g for Stepovogo Fjord. This difference may be caused by the lower fraction of expandable clay in sediment from the fjord. Uptake of 85Sr, 99Tc, 125I, and U were all similar for both locations, with Kd values averaging 4, 3, 17 and 60 ml/g, respectively. The Kd for 137 Cs varied non-linearly from 40 to 3800 ml/g as the liquid-to-solid ratio varied from 3.4 to 6500, but only when the sorption capacity was high compared to the mass of 137Cs in the closed system of the experiment. Under identical conditions, sediment with lower Kd values showed no effect. Oxidation of sediment effectively desorbed 99Tc from the solid phase, whilst it caused increased uptake of 85Sr and U. In sequential rinses with fresh seawater, desorption was limited to 60% of 137Cs and 85Sr, and 35% of uranium.

Absorption↗
PubMed1996

Hyperpermeability of pulmonary endothelial monolayer: protective role of phosphodiesterase isoenzymes 3 and 4.

The regulation of endothelial permeability is poorly understood. An increase in endothelial permeability in the pulmonary microvasculature, however, is critical in noncardiogenic pulmonary edema and other diffuse inflammatory reactions. In the present study thrombin and Escherichia coli hemolysin (HlyA), a membrane-perturbing bacterial exotoxin, were used to alter hydraulic permeability of porcine pulmonary artery and human endothelial cell monolayers. We also investigated the pharmacological approach of adenylyl cyclase activation/phosphodiesterase (PDE) inhibition to block endothelial hyperpermeability. Thrombin (1-5 units/ml) and HlyA (0.5-3 hemolytic units/ml) dose and time dependently (> 15 min) increased endothelial permeability. Forskolin, cholera toxin, and prostaglandin E1, which all stimulate adenylyl cyclase activity, abrogated this effect. One mM dibutyryl cAMP, a cell membrane-permeable cAMP analogue, was similarly active. Endothelial hyperpermeability was also reduced dose dependently by inhibitors of different PDE isoenzymes (motapizone, rolipram, and zardaverine, which block PDE3 and/or PDE4). The effectiveness of PDE inhibitors was increased in the presence of adenylyl cyclase activators. Analysis of cyclic nucleotide hydrolyzing PDE activity in lysates of human umbilical vein endothelial cells showed high activities of PDE isoenzymes 2, 3, and 4. Consistent with the functional data PDE3 and PDE4 were the major cAMP hydrolysis enzymes in intact endothelial cells. We conclude that the hyperpermeability of pulmonary endothelial monolayers, evoked by thrombin or HlyA, can be blocked by the simultaneous activation of adenylyl cyclase and inhibition of PDEs, especially of PDE3 and PDE4. The demonstration of PDE isoenzymes 2-4 in human endothelial cells will help optimize this therapeutic approach.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1996

Role of nitric oxide and phosphodiesterase isoenzyme II for reduction of endothelial hyperpermeability.

Regulation of endothelial permeability is poorly understood. Previous studies have shown that endothelial cells contain phosphodiesterase (PDE) isoenzymes II-IV and that simultaneous adenylate cyclase activation and/or PDE inhibition blocked endothelial hyperpermeability (J.Clin.Invest. 91: 1421-1428, 1993). We now focused on a possible role for guanosine 3',5'-cyclic monophosphate (cGMP)-dependent mechanisms and studied H2O2-exposed porcine pulmonary artery endothelial cell monolayers. Pretreatment of cells with different nitric oxide (NO) donors or atrial natriuretic peptide (ANP) increased endothelial cGMP-content severalfold and blocked H2O2-related effects on permeability; opposite results were obtained with a NO synthase inhibitor. Determination of cGMP degradation in nitroprusside-exposed endothelial cells identified PDE II as the major cGMP metabolizing pathway, whereas PDE III and IV contributed little or nothing. Inhibition of PDE II reduced H2O2-related endothelial hyperpermeability, an effect that could be enhanced synergistically by simultaneous guanylate cyclase activation. In summary, these studies indicate that cGMP-dependent mechanisms (NO donors, ANP, and dibutyryl-cGMP) blocked H2O2-related increases in endothelial permeability. The major cGMP degrading pathway in endothelial cells was PDE II, thereby substituting the missing PDE V in these cells. Simultaneous guanylate cyclase activation and/or PDE II inhibition may be a valuable approach to treat endothelial hyperpermeability.

Animals↗
PubMed1995

Biotin synthase from Escherichia coli, an investigation of the low molecular weight and protein components required for activity in vitro.

We have developed a radiochemical method for the measurement of biotin synthase activity in vitro. A cell-free extract from an Escherichia coli strain containing a cloned bioB (biotin synthase) gene was incubated with [14C]dethiobiotin, which was converted to [14C] biotin. The assay was used to identify the low molecular weight compounds and two of the proteins that, in addition to the bioB gene product, are required for biotin synthase activity in vitro. The low molecular weight compounds are cysteine; S-adenosylmethionine; thiamine pyrophosphate; Fe2+; a pyridine nucleotide (the most effective being NADPH); and one of the amino acids asparagine, aspartate, glutamine, or serine. The proteins ae flavodoxin and ferredoxin (flavodoxin)-NADP+ reductase (EC 1.18.1.2). A third thiamine pyrophosphate-dependent protein is also required for activity. When the cell-free extract was incubated with nonlabeled dethiobiotin and either [35S]cysteine or [35S]cystine, 35S was incorporated into biotin, and we present further evidence that cysteine, and not S-adenosylmethionine or methionine, is the sulfur donor for the biotin synthase reaction.

Amino Acid Sequence↗
PubMed1993

Pore-forming bacterial toxins potently induce release of nitric oxide in porcine endothelial cells.

Nitric oxide (NO) is believed to play an important role in sepsis-related hypotension. We examined the effects of two pore-forming bacterial exotoxins, Escherichia coli hemolysin and Staphylococcus aureus alpha-toxin, on NO formation in cultured porcine pulmonary artery endothelial cells. NO was quantified using a difference-spectrophotometric method based on the rapid and stoichiometric reaction of NO with oxyhemoglobin. Endothelial cyclic guanosine monophosphate levels were also monitored. Both exotoxins increased NO synthesis in endothelial cells in a time- and dose-dependent manner to an extent exceeding that observed with the ionophore A23187 or thrombin. The capacity of exotoxins to induce NO formation may be relevant in patients with severe local or systemic bacterial infections.

Animals↗