Biomedical subjects

S Pfeiffer

Publications and source records attributed to S Pfeiffer.

At least 55 records · Page 3Linked to original sources

PubMed2000

Barrier characteristics of different human skin types investigated with X-ray diffraction, lipid analysis, and electron microscopy imaging.

The stratum corneum requires ceramides, cholesterol, and fatty acids to provide the cutaneous permeability barrier. The lipids are organized in intercellular membranes exhibiting short- and long-periodicity lamellar phases. In recent years, the phase behavior of barrier lipid mixtures has been studied in vitro. The relationship of human stratum corneum lipid composition to membrane organization in vivo, however, has not been clearly established. Furthermore, the special function of the different ceramide species in the stratum corneum is largely unknown. We examined lipid organization and composition of stratum corneum sheets from different subtypes of healthy human skin (normal, dry, and aged skin). Lipid organization was investigated using X-ray diffraction and demonstrated that the 4.4 nm peak attributed to the long periodicity phase was frequently missing for skin with a low Cer(EOS)/Cer(total) ratio, indicating an important part for Cer(EOS), which contains omega-hydroxy fatty acid (O) ester-linked to linoleic acid (E) and amide-linked to sphingosine (S). A deficiency in the 4. 4 nm peak was predominantly observed in young dry skin. In one case of aged skin, however, and less often in young normal skin this peak was also missing. Furthermore, the ceramide composition of samples without the 4.4 nm peak showed a deficiency of Cer(EOH), which contains 6-hydroxy-4-sphingenine (H), and an increase in Cer(NS) and Cer(AS), which contain nonhydroxy (N) or alpha-hydroxy fatty acids (A). In addition, a 3.4 nm peak attributed to crystalline cholesterol occurred in most cases of aged and dry skin, but was not observed in young normal skin. Our results do not indicate a definite pattern of correlation between lipid organization and types of human skin. They demonstrate, however, that Cer(EOS) and Cer(EOH) are key elements for the molecular organization of the long periodicity lamellar phase in the human stratum corneum.

Adult↗

PubMed2000

High-pressure freezing provides new information on human epidermis: simultaneous protein antigen and lamellar lipid structure preservation. Study on human epidermis by cryoimmobilization.

Current transmission electron microscopy techniques do not permit simultaneous visualization of skin ultrastructure and stratum corneum extracellular lipids. We developed a new procedure, which entails application of high-pressure freezing followed by freeze-substitution with acetone containing uranyl acetate, followed by low temperature embedding in HM20. Electrospray ionization mass spectrometry showed that the amount of lipids lost during preparation was minimal. The ultrastructure of cryoprocessed skin was compared with that of conventionally prepared skin samples. Cryoprocessing, but not conventional processing, enabled visualization of lipid stacks within epidermal lamellar bodies, as well as the extracellular lipid domains of the stratum corneum and the ultrastructure within keratinocytes. Anti-filaggrin immunocytochemistry also showed, e.g., excellent preservation of filaggrin on cryoprocessed samples. Additionally, the cytosol of keratinocytes appeared to be organized in "microdomain"-like areas. Finally, the stratum corneum appeared more compact with smaller intercellular spaces and hence tighter cell-cell interactions, after cryoprocessing, than after conventional tissue preparation for transmission electron microscopy. We conclude here that only cryoprocessing preserves skin in a close to native state.

Adult↗

PubMed1999

Inducible nitric oxide synthase expression in cultures enriched for mature oligodendrocytes is due to microglia

Hewett JA, Hewett SJ, Winkler S, Pfeiffer SE. 1999. Inducible nitric oxide synthase expression in cultures enriched for mature oligodendrocytes is due to microglia. J Neurosci Res 56:189-198. In the article referenced above, the LPS concentration employed in all studies was 1 &mgr;g/ml, not 1 mg/ml as published. This correction appears: in the Materials and Methods section on page 190, column 1, line 47; in the Results section on page 191, column 2, legend to Figure 1, line 3; on page 192, column 2, legend to Figure 2, line 3; on page 194, column 2, legend to Figure 5, line 4; on page 195, column 1, legend to Figure 6, line 3; and in the Discussion section on page 196, column 1, line 9. The publisher regrets this error.

Journal Article↗

PubMed1999

Impact of Cl- and Na+ ions on simulated structure and dynamics of betaARK1 PH domain.

A nonzero net charge of proteins at pH 7 is usually compensated by the addition of charge-balancing counter ions during molecular dynamics simulation, which reduces electrostatic interactions. For highly charged proteins, like the betaARK1 PH domain used here, it seems reasonable to also add explicit salt ions. To assess the impact of explicit salt ions, two molecular dynamics simulations of solvated betaARK1 PH domain have been carried out with different numbers of Cl- and Na+ ions, based on the Cornell et al. force field and the Ewald summation, which was used in the treatment of long-range electrostatic interactions. Initial positions of ions were obtained from the AMBER CION program. Increasing the number of ions alters the average structure in loop regions, as well as the fluctuation amplitudes of dihedral angles. We found unnaturally strong interactions between side chains in the absence of salt ions. The presence of salt ions reduces these electrostatic interactions. The time needed for the equilibration of the ionic environment around the protein, after initial placement of ions close to oppositely charged side chains, is in the nanosecond time range, which can be shortened by using a higher ionic strength. Our results also suggest selecting those methods that do not place the ions initially close to the protein surface.

Blood Proteins↗

PubMed1999

Signalling at a distance: transport of Wingless in the embryonic epidermis of Drosophila.

Secreted signalling molecules affect the behavior of cells at a distance. Here we discuss how the Wnt family member Wingless reaches distant cells within the embryonic epidermis of Drosophila. We consider three possible mechanisms: free diffusion, restricted diffusion and active transport. We argue that free diffusion is unlikely to occur. However, a variant of restricted diffusion may account for Wingless transport. It may be that Wingless is carried from one side of a cell to the other by a drifting transmembrane protein such as a specific receptor or a glycosaminoglycan. Transfer from cell-to-cell would involve release from the donor cell and recapture in an adjacent cell. Alternatively, Wingless might be transported by a mechanism akin to transcytosis. This would involve the packaging of Wingless in specialized vesicles at one end of a cell, active transport across the cell, and vesicle fusion and Wingless release on the other side. We describe the evidence in favor and against these two alternatives.

Animals↗

PubMed1998

Expression and renaturation of the N-terminal extracellular domain of torpedo nicotinic acetylcholine receptor alpha-subunit.

The N-terminal extracellular region (amino acids 1-209) of the alpha-subunit of the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata electric tissue was expressed as inclusion bodies in Escherichia coli using the pET 3a vector. Employing a novel protocol of unfolding and refolding, in the absence of detergent, a water-soluble globular protein of 25 kDa was obtained displaying approximately 15% alpha-helical and 45% beta-structure. The fragment bound alpha-[3H]bungarotoxin in 1:1 stoichiometry with a KD value of 0.5 nM as determined from kinetic measurements (4 nM from equilibrium binding). The kinetics of association of toxin and fragment were of second order, with a similar rate constant (8.2 x 10(5) M-1 s-1) as observed previously for the membrane-bound heteropentameric nAChR. Binding of small ligands was demonstrated by competition with alpha-[3H]bungarotoxin yielding the following KI values: acetylcholine, 69 microM; nicotine, 0.42 microM; anatoxin-a, 3 miroM; tubocurarine, 400 microM; and methyllycaconitine, 0.12 microM. The results demonstrate that the N-terminal extracellular region of the nAChR alpha-subunit forms a self-assembling domain that functionally expresses major elements of the ligand binding sites of the receptor.

Amino Acid Sequence↗

PubMed1998

Lack of tyrosine nitration by peroxynitrite generated at physiological pH.

Nitration of tyrosine residues of proteins has been suggested as a marker of peroxynitrite-mediated tissue injury in inflammatory conditions. The nitration reaction has been extensively studied in vitro by bolus addition of authentic peroxynitrite, an experimental approach hardly reflecting in vivo situations in which the occurrence of peroxynitrite is thought to result from continuous generation of .NO and O-2 at physiological pH. In the present study, we measured the nitration of free tyrosine by .NO and O-2 generated at well defined rates from the donor compound (Z)-1-[N-[3-aminopropyl]-N-[4-(3-aminopropylammonio)butyl]-amino]- dia zen-1-ium-1,2-diolate] (spermine NONOate) and the xanthine oxidase reaction, respectively. The results were compared with the established nitration reaction triggered by authentic peroxynitrite. Bolus addition of peroxynitrite (1 mM) to tyrosine (1 mM) at pH 7.4 yielded 36.77 +/- 1.67 microM 3-nitrotyrosine, corresponding to a recovery of about 4%. However, peroxynitrite formed from .NO and O-2, which were generated at equal rates ( approximately 5 microM x min-1) from 1 mM spermine NONOate, 28 milliunits/ml xanthine oxidase, and 1 mM hypoxanthine was much less efficient (0.67 +/- 0.01 microM; approximately 0.07% of total product flow). At O-2 fluxes exceeding the .NO release rates, 3-nitrotyrosine formation was below the detection limit of the high performance liquid chromatography method (<0.06 microM). Nitration was most efficient (approximately 0.3%) with the .NO donor alone, i.e. without concomitant generation of O-2. Nitration by .NO had a pH optimum of 8.2, increased progressively with increasing tyrosine concentrations (0.1-2 mM), and was not enhanced by NaHCO3 (up to 20 mM), indicating that it was mediated by .NO2 rather than peroxynitrite. Our results argue against peroxynitrite produced from .NO and O-2 as a mediator of tyrosine nitration in vivo.

Carbon Dioxide↗

PubMed1998

Electrochemical determination of S-nitrosothiols with a Clark-type nitric oxide electrode.

Low-molecular-mass thiols and nitric oxide (NO) form S-nitrosothiols (thionitrites) in the presence of oxygen. Thionitrites play an integral role in a variety of NO-dependent physiological processes. This study describes a sensitive analytical method for the quantitative determination of thionitrites. The method is based on the Cu+-catalyzed homolytic cleavage of thionitrites and electrochemical detection of the released NO with a Clark-type electrode. Cu+ was generated by addition of Cu(NO3)2 to samples containing 1 mM GSH or 4 mM L-cysteine as reducing agents. The effect of Cu(NO3)2 on the release of NO from GSNO was concentration-dependent. In the presence of 1 mM GSH, the EC50 for Cu(NO3)2 was 1.34 +/- 0.08 mM. Using cysteine instead of GSH, NO release was quantitative at much lower concentrations of Cu(NO3)2 (EC50 = 8.5 +/- 2.8 microM. NO release was not significantly affected by pH (7.0-9.0) and was inhibited by the Cu+-selective chelator neocuproine, whereas the Cu2+ chelator cuprizone was approximately 16-fold less potent. Calibration of the method with GSNO, S-nitroso-N-acetyl-penicillamine, or S-nitrosated bovine serum albumin yielded linear plots of initial rates of NO release versus thionitrite concentration from 50 nM to 5 microM. This method may be useful for the quantitative determination of thionitrites in biological samples.

Animals↗

PubMed1998

Reaction of peroxynitrite with HEPES or MOPS results in the formation of nitric oxide donors.

We investigated the effects of organic buffers on the NO-like biological activities of ONOO-. In HEPES buffer (50 mM), ONOO- (1 mM) induced a 20-fold increase in endothelial cGMP accumulation and the effect was comparable to that elicited by a maximally active concentration of the NO donor DEA/NO. ONOO- produced a 12-fold increase of cGMP in MOPS buffer (50 mM), but was virtually inactive in phosphate buffer (50 mM). Electrochemical detection of NO showed that the biological effects of ONOO- in HEPES or MOPS were due to accumulation of compounds that released NO in the presence of copper ions. CuCl2-induced formation of NO was completely blocked by the Cu(I) chelator neocuproine but unaffected by the Cu(II) chelator cuprizone, pointing to a Cu(I)-catalyzed decomposition pathway. Formation of NO from ONOO- was not detectable in phosphate buffer, in agreement with the lack of effect of ONOO- on cGMP accumulation in this buffer. These data demonstrate that certain buffer components present in cell culture media may yield artificial results in experiments with authentic ONOO-.

Animals↗

PubMed1998

A new pathway of nitric oxide/cyclic GMP signaling involving S-nitrosoglutathione.

Nitric oxide (NO), a physiologically important activator of soluble guanylyl cyclase (sGC), is synthesized from L-arginine and O2 in a reaction catalyzed by NO synthases (NOS). Previous studies with purified NOS failed to detect formation of free NO, presumably due to a fast inactivation of NO by simultaneously produced superoxide (O-2). To characterize the products involved in NOS-induced sGC activation, we measured the formation of cyclic 3',5'-guanosine monophosphate (cGMP) by purified sGC incubated in the absence and presence of GSH (1 mM) with drugs releasing different NO-related species or with purified neuronal NOS. Basal sGC activity was 0.04 +/- 0.01 and 0.19 +/- 0.06 micromol of cGMP x mg-1 x min-1 without and with 1 mM GSH, respectively. The NO donor DEA/NO activated sGC in a GSH-independent manner. Peroxynitrite had no effect in the absence of GSH but significantly stimulated the enzyme in the presence of the thiol (3.45 +/- 0.60 micromol of cGMP x mg-1 x min-1). The NO/O-2 donor SIN-1 caused only a slight accumulation of cGMP in the absence of GSH but was almost as effective as DEA/NO in the presence of the thiol. The profile of sGC activation by Ca2+/calmodulin-activated NOS resembled that of SIN-1; at a maximally active concentration of 200 ng/0.1 ml, NOS increased sGC activity to 1.22 +/- 0.12 and 8.51 +/- 0.88 micromol of cGMP x mg-1 x min-1 in the absence and presence of GSH, respectively. The product of NOS and GSH was identified as the thionitrite GSNO, which activated sGC through Cu+-catalyzed release of free NO. In contrast to S-nitrosation by peroxynitrite, the novel NO/O-2-triggered pathway was very efficient (25-45% GSNO) and insensitive to CO2. Cu+-specific chelators inhibited bradykinin-induced cGMP release from rat isolated hearts but did not interfere with the direct activation of cardiac sGC, suggesting that thionitrites may occur as intermediates of NO/cGMP signaling in mammalian tissues.

Animals↗

PubMed1998

Bound water in apo and holo bovine heart fatty-acid-binding protein determined by heteronuclear NMR spectroscopy.

Two- and three-dimensional heteronuclear NMR experiments have been performed to identify internally bound water molecules in the solution structure of bovine heart fatty-acid-binding protein (heart FABP). NOE and rotating-frame NOE (ROE) cross peaks between protein protons and protons of bound water molecules were observed in two-dimensional H2O-ROE/NOE-1H,15N-heteronuclear single quantum coherence spectra recorded from a uniformly 13C/15N-enriched sample of bovine heart FABP. Contacts between water protons and 23 NH protons of the protein backbone were identified. The protein structure consists of 10 antiparallel beta-strands (betaA-betaJ), forming two nearly orthogonal beta-sheets, and a short helix-turn-helix motif connecting beta-strands A and B. The spatial folding resembles a beta-barrel. Most of the water molecules are localized in the gap between beta-strands D and E, and near the two alpha-helices. In the delipidated heart FABP additional contacts between water molecules and NH protons could be observed using a three-dimensional rotating frame Overhauser 1H,15N heteronuclear single quantum coherence experiment obtained with a 15N-labeled sample of apo-heart FABP.

Animals↗

PubMed1998

Variability in osteon size in recent human populations.

The possibility of smaller osteons in the cortical bone of Late Pleistocene human populations begs the question of how these histological features vary within individual skeletons among and between populations. The distributional characteristics of total osteon area (On.Ar) and Haversian canal area (H.Ar) are explored using data from three samples of historically known individuals: ribs and femora from eighteenth-century Huguenots in England (Spitalfields, n = 20), ribs and femora from nineteenth-century British settlers in Canada (St. Thomas, n = 21), and ribs from twentieth-century South African cadavers (University of Cape Town; following curatorial classifications, n = 10 white, 10 black, 10 colored). Neither histological variable is normally distributed. About 96% of the random variation is within the individual bone sample. There are no significant differences between sexes for either variable in any sample, and age has no effect in most instances. Femoral osteons are significantly larger than rib osteons within individuals and across samples. Haversian canal area is more variable than On.Ar, especially in the twentieth-century sample, where within-sample coefficients of variation are frequently >100%. Using modern centiles developed here, some Late Pleistocene long bone samples have On.Ar values below the range of modern variation. Because of ribs' smaller cross-sectional areas and less broadly ranging values for On.Ar, ribs would provide a preferable site for future comparative studies.

Adolescent↗

PubMed1998

Hydration water molecules of nucleotide-free RNase T1 studied by NMR spectroscopy in solution.

The hydration of uncomplexed RNase T1 was investigated by NMR spectroscopy at pH 5.5 and 313 K. Two-dimensional heteronuclear NOE and ROE difference experiments were employed to determine the spatial proximity and the residence times of water molecules at distinct sites of the protein. Backbone carbonyl oxygens involved in intermolecular hydrogen bonds to water molecules were identified based on 1J(NC) coupling constants. These coupling constants were determined from 2D-H(CA)CO and 15N-HSQC experiments with selective decoupling of the 13C alpha nuclei during the t1 evolution time. Our results support the existence of a chain of water molecules with increased residence times in the interior of the protein which is observed in several crystal structures with different inhibitor molecules and serves as a space filler between the alpha-helix and the central beta-sheet. The analysis of 1J(NC) coupling constants demonstrates that some of the water molecules seen in crystal structures are not involved in hydrogen bonds to backbone carbonyls as suggested by crystal structures. This is especially true for a water molecule, which is probably hydrogen bonded by the protonated carboxylate group of D76 and the hydroxyl group of T93 in solution, and for a water molecule, which was reported to connect four different amino acid residues in the core of the protein by intermolecular hydrogen bonds.

Amino Acid Sequence↗

PubMed1998

Arginine availability controls the N-methyl-D-aspartate-induced nitric oxide synthesis: involvement of a glial-neuronal arginine transfer.

The neuronal nitric oxide (NO) synthase generates NO from arginine. NO mediates its physiological effects mainly by stimulating the synthesis of cyclic GMP. We have investigated the role of the arginine availability on the NMDA-induced cyclic GMP accumulation in immature rat brain slices. The effect of NMDA was blocked by the inhibitor of the NO synthase, N(G)-nitro-L-arginine, and by the antagonist of ionotropic non-NMDA receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). This inhibition was not due to a direct interaction of CNQX with the NMDA receptor, and it was overcome by the presence of exogenously applied arginine. CNQX also blocked the NMDA-evoked release of [3H]arginine from cerebellar slices. Moreover, the arginine uptake inhibitor L-lysine reduced the cyclic GMP response to NMDA significantly. Therefore, the extracellular arginine availability, which is dependent on the activation of ionotropic non-NMDA receptors, determines the rate of the NO biosynthesis by the neuronal NO synthase. Together with the reported release of arginine from glial cells upon activation of glial ionotropic non-NMDA receptors and the predominant glial localization of arginine, these data provide the first evidence of an essential role of the arginine transfer from glial cells to neurons for the biosynthesis of NO.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1998

Molecular actions of a Mn(III)Porphyrin superoxide dismutase mimetic and peroxynitrite scavenger: reaction with nitric oxide and direct inhibition of NO synthase and soluble guanylyl cyclase.

Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), described as a superoxide dismutase mimetic and peroxynitrite scavenger, has been used previously to investigate the cytotoxic potential of superoxide and peroxynitrite in several pathological models. Here we report on the interference of MnTMPyP with NO/cGMP signaling using cultured endothelial cells as well as purified soluble guanylyl cyclase (sGC) either activated by the NO donor 2,2-diethyl-1-nitroso-oxyhydrazine sodium salt (DEA/NO) or reconstituted with nitric oxide synthase (NOS). MnTMPyP inhibited endothelial cGMP accumulation induced by A23187 (0.3 microM) with an IC50 of 75.0 +/- 10.4 microM but had no significant effect on the potency of the Ca2+ ionophore. Purified NOS was inhibited by MnTMPyP (IC50 = 5.5 +/- 0.8 microM) because of an interference of the Mn-porphyrin with the reductase domain of the enzyme. The most pronounced actions of MnTMPyP were direct inhibition of sGC and scavenging of NO. Purified sGC stimulated with either Ca2+/calmodulin-activated NOS (in the presence of GSH) or DEA/NO (in the absence of GSH) was inhibited with IC50 values of 0.8 +/- 0.09 microM and 0.6 +/- 0.2 microM, respectively. In the presence of GSH, MnTMPyP was reduced to the Mn(II) complex, resulting in efficient scavenging of NO under these conditions. Our data demonstrate that MnTMPyP (i) interferes with the reductase domain of NOS, (ii) scavenges NO in the presence of GSH, and (iii) is a potent direct inhibitor of sGC. These results cast doubt on the usefulness of MnTMPyP and related Mn-porphyrin complexes as probes to study the involvement of peroxynitrite/superoxide in biological systems.

Animals↗

PubMed1998

Activation of soluble guanylyl cyclase by the nitrovasodilator 3-morpholinosydnonimine involves formation of S-nitrosoglutathione.

Soluble guanylyl cyclase (sGC) is the major physiological target of sydnonimine-based vasodilators such as molsidomine. Decomposition of sydnonimines results in the stoichiometric formation of nitric oxide (NO) and superoxide (O2-), which rapidly react to form peroxynitrite. Inasmuch as sGC is activated by NO but not by peroxynitrite, we investigated the mechanisms underlying sGC activation by 3-morpholinosydnonimine (SIN-1). Stimulation of purified bovine lung sGC by SIN-1 was found to be strongly dependent on glutathione (GSH). By contrast, GSH did not affect sGC activation by NO released from 2,2-diethyl-1-nitroso-oxyhydrazine, indicating that NO/O2- released from SIN-1 converted GSH to an activator of sGC. High performance liquid chromatography identified this product as the thionitrite S-nitrosoglutathione. Further, the reaction product decomposed to release NO upon addition of Cu(NO3)2 in the presence of GSH. Activation of sGC was antagonized by the Cu(I)-specific chelator neocuproine, whereas the Cu(II)-selective drug cuprizone was less potent. Carbon dioxide (delivered as NaHCO3) antagonized S-nitrosation by peroxynitrite but not by SIN-1. Thus, NO/O2- released from SIN-1 mediates a CO2-insensitive conversion of GSH to S-nitrosoglutathione, a thionitrite that activates sGC via trace metal-catalyzed release of NO. These results may provide novel insights into the molecular mechanism underlying the nitrovasodilator action of SIN-1.

Animals↗

PubMed1998

The natural decomposition of adipocere.

Adipocere is a waxy substance which sometimes forms from the adipose tissue of dead bodies, especially when they are under water. A disinterment in southern Ontario lead to the recovery of extensive adipocere from an interment which occurred in AD 1869. Subsequent laboratory research was designed to explore the conditions under which adipocere will disappear, the goal being to identify strategies for estimating a range of time since death in cases where adipocere is present. Varieties of aerobic or facultatively anaerobic microorganisms from the surface of the adipocere were separated and identified. In culture, the gram positive bacteria were able to degrade the adipocere. We propose that the persistence of adipocere is related to the exclusion of gram positive bacteria from the burial environment. The role of bacteria in adipocere formation and degradation must be understood before we can use the presence of adipocere to extrapolate information about the post-death interval.

Adipose Tissue↗

PubMed1998

[A comparative study of minimal invasive harvesting of vena saphena magna segments].

From July 1997 to February 1998 we performed either minimally invasive or endoscopic saphenous vein harvesting in 55 patients and compared the results with those of 46 patients after conventional saphenous vein harvesting. Minimally invasive and endoscopical vein harvesting can be safely performed after a longer learning curve. Patients after minimally invasive and endoscopical saphenous vein harvesting showed fewer wound healing problems and better cosmetic results than after conventional vein harvesting.

Aged↗