bluuebunny/crossref_metadata_2025_split huggingface dataset:
Generation of an Electrochemical Proton Gradient by Lactate Efflux in Membrane Vesicles of Escherichia coli
Authors: TEN BRINK Bart , KONINGS Wil N. 
Published: July 2010

The ‘energy recycling model’ (Michels et al. (1979) FEMS Microbiol. Lett. 5, 357–364] postulates the generation of an electrochemical gradient across the bacterial cytoplasmic membrane by carrier‐mediated efflux of metabolic endproducts in symport with protons.Experimental evidence for this model is presented. In membrane vesicles from Escherichia coli ML 308–225 l‐lactate translocation (both uptake and efflux) is carrier‐mediated. The H+/t‐lactate stoichiometry varies, depending on the external pH, between 1 and 2. This change in stoichiometry is most likely the result of a protonation of the lactate carrier protein. This process has a pK of 6.75. l‐Lactate efflux from membrane vesicles, loaded with 50 mM potassium l‐lactate, results at an external pH of 6.6 in an 11‐fold accumulation of proline inside the vesicles. This accumulation is completely inhibited by the uncoupler carbonyl cyanide p‐trifluoromethoxyphenylhydrazone. The uptake of proline is not the result of a potassium or an osmotic gradient.At an external pH of 6.6 efflux of l‐lactate from the vesicles leads to the generation of an electrical potential across the membrane of ‐ 55 mV, as is demonstrated from the accumulation of the lipophilic cation tetraphenylphosphonium.
Non‐specific Biosynthesis of Gammacerane Derivatives by a Cell‐Free System from the Protozoon Tetrahymena pyriformis
Authors: BOUVIER Pierrette , BERGER Yves , ROHMER Michel , OURISSON Guy 
Published: July 2010

A cell‐free system from the protozoon Tetrahymena pyriformis was incubated with either [12‐3H]squalene or (RS)‐2,3‐epoxy‐2,3‐dihydro‐[12,13‐3H]squalene. Squalene was cyclized into tetrahymanol whereas racemic squalene epoxide was transformed into gammacerane‐3α,21α‐diol and gammacerane‐3β,21α‐diol. After cyclization of (RS)‐2,3‐epoxy‐2,3‐dihydro‐[3‐3H]squalene, both epimeric gammaceranediols were labelled with a tritium atom located at C‐3, showing that no isomerization via a 3‐oxo compound occurred.
The proton NMR spectra of the cyclization products of synthetic (2E, 22E)‐(1,1,1,24,24,24‐2H6) squalene and (RS)‐(22E)‐2,3‐epoxy‐2,3‐dihydro‐(1,1,1,24,24,24‐2H6) squalene show that squalene and the (3S)enantiomer of its epoxide are cyclized in an all pre‐chair conformation, whereas tae (3R) enantiomer of squalene epoxide is cyclized in a pre‐boat conformation as concerns the cycle A.
The squalene cyclase of T. pyriformis presents the sane lack of substrate specificity as the cyclase of Acetobacter pasteurianum: in addition to squalene, its normal substrate, it also cyclizes both enantiomers of its epoxide. This conformational versatility is characteristic of squalene cyclases but no longer exists in the squalene epoxide cyclases from eukaryotes.
Evidence for Two Different Reactive Sulfhydryl Groups in the ATP‐Binding Sites of (Na++ K+)‐ATPase
Authors: PATZELT‐WENCZLER Rosemarie , SCHONER Wilhelm 
Published: July 2010

The disulfide of thioinosine triphosphate, (SnoPPP)2 is a substrate of (Na++ K+)‐ATPase from pig kidney.
Binding of (SnoPPP)2 and of the β,γ‐imido or β,γ‐methylene analogues, (SnoPP[NH]P)2 or (SnoPP[CH2]P)2, to (Na++ K+)‐ATPase in the presence of 10 mM EDTA results in an inactivation of the enzyme. The inactivation is temperature‐dependent and has an activation energy of 95.5 kJ/mol with (SnoPPP)2 as substrate.
The inactivation proceeded with all disulfide analogues of ATP with two different velocities. Identical inactivation rate constants k2 were found for all substances for the rapidly inactivating part (k′2= 2.9–4.7 × 10−3 s−1) and the slowly inactivating part (k″2= 2.0−2.3 × 10−4 s−1). From the analysis of the inactivation kinetics identical dissociation constants were found for both sites. The dissociation constants of the complexes between enzyme and ATP analogue inactivating the enzyme slowly were: (SnoPPP)2= 18.6 μM, (SnoPP[NH]P)2= 38.8 μM and (SnoPP[CH2]P)2= 63.3 μM.
ATP protected both sites against the inactivation by the disulfide analogues of ATP. From the protective action of ATP at 37° C against the inactivation, a dissociation constant of the enzyme‐ATP complex of 2.95 μM was calculated for the rapidly inactivating site and of 77 μM for the slowly reacting site.
The total capacity of the sulfhydryl groups reacting with the ATP analogue (SnoPPP)2 is four‐times higher than the capacity of the phosphointermediate and the with (SnoPPP)2 rapidly reacting site (high, affinity ATP‐binding site).
It is concluded that (SnoPPP)2 as well as (SnoPP[NH]P)2 and (StoPP[CH2]P)2 are ATP affinity labels of the high‐affinity and low‐affinity ATP‐binding sites of (Na++ K+)‐ATPase. Since dithiothreitol reactivates (Na++ K+)‐ATPase after inactivation by (SnoPPP)2 [Patzelt‐Wenezler and Schoner (1975) Biochim. Biophys, Acta, 403, 538–513] it is assumed that (Na++ K+)‐ATPase contains two different reactive types of sulfhydryl groups in the ATP‐binding sites. Tte sulfhydryl group, may interact with the 6‐amino group of the purine moiety of ATP.
Role of Sialic Acid in the Mobility of Membrane Proteins Containing O‐Linked Oligosaccharides on Polyacrylamide Gel Electrophoresis in Sodium Dodecyl Sulfate
Authors: GAHMBERG Carl G. , ANDERSSON Leif C. 
Published: July 2010

The major sialoglycoprotein of the human red‐cell membrane, glycophorin A, contains 15 O‐glycosidically linked oligosaccharides and one N‐glycosidic oligosaccharide. The protein shows a decreased mobility on polyacrylamide gel electrophoresis in sodium dodecyl sulfate after neuraminidase treatment of thc nondenatured protein.The molecular mechanism behind this phenomenon has been elucidated. Neuraminidase treatment of glycophorin A in intact cells or after solubilization in buffers containing Triton X‐100 resulted in conversion of the predominant tetrasaccharide N‐acetylneuraminosylα2 ‐3galactosylβl‐ 3(N‐acetylneuraminosylα2 ‐ 6)‐N‐acetylgalactosamine to the trisaccharide galactosylβl ‐ 3(N‐acetylneuraminosylα2 ‐ 6)‐N‐acetylgalactosamine and the disaccharide galactosylβl ‐ 3‐N‐acetylgalactosamine. After denaturation with sodium dodecyl sulfate, Vibrio cholerae neuraminidase also liberated the N‐acetylgalactosamine‐bound sialic acids. Such treatment resulted in increased electrophoretic mobility.The results show that distal sialic acids linked to galactose are readily available to neuraminidase, and that their negative charge gives an increased electrophoretic mobility in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. In contrast, most of thc N‐acetylgalactosamine‐linked sialic acids of glycophorin A are not liberated by neuraminidase without denaturation of the substrate. Like sialic acids of complex‐type oligosaccharides the decreased electrophoretic mobility caused by them is exclusively due to their mass while no significant contribution by the charge was seen.
Routes of Flavodoxin and Ferredoxin Reduction in Escherichia coli
Authors: BLASCHKOWSKI Hans P. , KNAPPE Joachim , LUDWIG‐FESTL Monika , NEUER Gabriele 
Published: July 2010

Flavodoxin and ferredoxin become reduced in Escherichia coli cells by oxidoreductase reactions which use pyruvate and NADPH as electron donor substrates. The two enzymes, which are minor proteins of this organism, were measured through the reduced flavodoxin‐dependent activation of pyruvate formate‐lyase. The NADPH‐dependent enzyme, obtained homogeneously through Procion‐red affinity chromatography, was identified as the flavoprotein ‘component R’ described previously by Fujii and Huennekens [J. Biol. Chem. 249, 6745–6753 (1974)]. The pyruvate‐dependent enzyme was identified as CoA‐acetylating pyruvate; flavodoxin (ferredoxin) oxidoreductase. Its catalytic properties in the forward. reverse, and the 14CO2‐pyruvate exchange reaction are reported.The dihydro form of flavodoxin was characterized as the particular species involved in the activation of pyruvate formate‐lyase. The activation process still occurs with 70% of maximal efficiency when the ratio [NADPH]/([NADP] + [NADPH]) is fixed at the intracellular ‘anabolic reduction charge’ value of 0.45, in conjunction with the NADPH‐dependent enzyme. The [2Fe‐2S] ferredoxin, though being readily used as electron acceptor of both oxidoreductases and having a redox potential similar to flavodoxin, proved incompetent in mediating the activation of pyruvate formate‐lyase.
The Pressure Dependence of the Spin Equilibrium in Camphor‐Bound Ferric Cytochrome P‐450
Authors: HUI BON HOA Gaston , MARDEN Michael C. 
Published: July 2010

The spin equilibrium of camphor‐bound ferric cytochrome P‐450 has been measured between 1–1000 bar (105‐108 Pa). Increasing pressure shifts the absorption spectrum from the high‐spin form at 302 nm to the low‐spin form at 417 nm. The molar volume change for the spin states ΔV= ‐RTôln KeôP and the equilibrium coefficient Ke= [high spin]/[low spin] depend on the solvent conditions.At pH 5.6 the equilibrium coefficient at 1 bar, K1= 0.5 and ΔV = 31 cm3/mol. A sample with 10 mM KCl at pH 7 has K1= 7.0 and ΔV = 52 cm3/mol. Solvent changes producing a larger K1 also result in a larger ΔV which ranged over 16–74 cm3/mol. The correlation can be approximated as ΔV = 36 + 18 log K1. which implies that there is a pressure, 3000 bar, for camphor‐bound ferric cytochrome P‐450 at 4°C, at which the changes in ΔV are compensated by the other thermodynamic parameters leaving Ke independent of the solvent conditions. Although the protein is not stable above 1000 bar for most sample conditions, the extrapolated log Ke versus pressure curves for all sample conditions intersect near 3000 bar. Camphor‐bound cytochrome P‐450 appears to be a rather flexible protein, having a low denaturing pressure, a large volume change, and a high sensitivity to the protein environment.
Transport of N‐Acetylglutamate in Rat‐Liver Mitochondria
Authors: MEIJER Alfred J. , VAN WOERKOM George M. , WANDERS Ron J. A. , LOF Cor 
Published: July 2010

The permeability properties of the rat‐liver mitochondrial membrane for N‐acetylglutamate, the activator of carbamoyl‐phosphate synthetase (ammonia), were studied.

Transport of N‐acetylglutamate into the mitochondria was only observed in partially or fully de‐energized mitochondria and when the extramitochondrial concentration was unphysiologically high (in the mM range). However, even under these conditions the intramitochondrial concentration of N‐acetylglutamate was much lower than that outside.
Mitochondria1 N‐acetylglutamate efflux only occurs when the mitochondria are in an energized state. At 25 °C, at an intramitochondrial N‐acetylglutamate concentration of 0.7–1.0 nmol/mg protein, efflux proceeds at a rate of about 0.05 nmol × min−1× mg protein‐′. This is 10‐fold lower than the maximal rate of N‐acetyl‐ glutamate synthesis in the mitochondria.
Homologous exchange between intramitochondrial N‐[14C]acetylglutamate and extrainitochondrial un‐ labelled N‐acetylglutamate could not be demonstrated.
It is concluded that transport of N‐acetylglutamate in vivo is effectively unidirectional, out of the mitochondria. This behaviour is in accordance with the physiological requirement for efflux of N‐acetylglutamate from the mitochondria in order to be degraded in the cytosol.
Purification and Characterization of Amyloid‐Related Transthyretin Associated with Familial Amyloidotic Cardiomyopathy
Authors: Hermansen Leonila F. , Bergman Tomas , Jörnvall Hans , Husby Gunnar , Ranløv Ida , Sletten Knut 
Published: July 2010

Analysis of amyloid fibril material associated with familial amyloidotic cardiomyopathy revealed that it contains a mixture of transthyretin‐related polypeptides. The major protein band in SDS/polyacrylamide gel corresponding to a molecular mass of 14.5 kDa, consists of transthyretin fragments starting at positions 46, 49 and 59, the latter not previously identified, and one blocked fragment derived from the N‐terminal part of transthyretin. In reverse‐phase HPLC, the major fragment recovered was that starting at Thr49, indicating a trypsin‐like cleavage (Lys at position 48). Two minor bands, corresponding to 17 kDa and 35 kDa, contained proteins with blocked N‐termini, and migrated as monomeric and dimeric transthyretin, respectively. A 13‐kDa protein band was found to contain transthyretin with a ragged N‐terminus, mainly starting at positions 2 and 5. Three more bands, corresponding to 10, 25 and 29 kDa, consist of transthyretin molecules with blocked N‐termini and most likely of aggregates of truncated molecules.A point mutation of amyloid transthyretin was identified at position 111 (Met instead of Leu in normal serum transthyretin) which confirms the mutation found for Danish siblings with familial amyloidotic cardiomyopathy. However, the presence of a non‐variant amyloid transthyretin was also observed, indicating that the Danish kindred is heterozygous with respect to this point mutation. Isoelectric focusing of the amyloid fibril material resolved multiple protein bands ranging over pH4.5–6.5, confirming heterogeneities.Methanol extraction of the cardiac amyloid fibril material prior to the purification steps reveals a methanol‐soluble substance amounting to about 10% (by mass dry material) of the amyloid fibril material. A yellow substance in this fraction shows absorbance maxima (270, 280 and 430 nm) similar to those observed for transthyretin in normal serum. Gas chromatography/mass spectrometry of the methanol extract revealed the presence of saturated fatty acids (C14:0, C16:0 and C18:0 in the corresponding ratio 2:8:5) and polyunsaturated fatty acids (C16:1, C18:1, C18:2 and C20:4 in the corresponding ratio of 1:2:1:1) as further constituents of the amyloid fibril material.
A Cytochrome‐b5‐Containing Fusion Protein Similar to Plant Acyl Lipid Desaturases
Authors: Sperling Petra , Schmidt Hermann , Heinz Ernst 
Published: July 2010

The similarity between oleate and linoleate desaturase sequences from several plants was used lo construct degenerate oligonucleotide primers for PCR experiments with cDNA transcribed from mRNA of ripening sunflower embryos. A DNA fragment was amplified and sequenced. Specific primers derived from this partial sequence were used for rapid amplification of the 3′‐ and 5′‐ends of this cDNA. With appropriate primers derived from these sequences, a full‐length clone of 1377 bp was amplified by PCR which, after sequencing, showed an open reading frame of 458 amino acids corresponding to a putative protein of about 52 kDa.Comparison with other desaturases showed the conserved three histidine boxes and the characteristic hydropathy profile of membrane‐bound desaturases, but the amino acid identity was restricted to 18 % and the N‐terminal region was about 100 amino acids longer. This N‐terminal extension showed high similarity with cytochrome b5, and, accordingly, the whole sequence can be considered as coding for a fusion protein between cytochrome b5 and a desaturase‐like enzyme. Furthermore, we detected a similar cytochrome b5 fold in the previously sequenced Δ9 acyl‐CoA desaturase from yeast, but in this enzyme it was located at the C‐terminus. An alignment of these fusion proteins with other heme‐binding proteins revealed desaturases to be novel members of the cytochrome b5, superfamily. A truncated DNA representing 366 bp of the 5′‐end was amplified from the cDNA clone and expressed in Escherichia coli. The truncated cDNA coded for a soluble protein of about 12 kDa as shown by SDS/PAGE and N‐terminal sequencing. The enriched recombinant protein exhibited redox absorbance spectra characteristic of plant microsomal cytochrome b5
Early onset pneumonia in patients with cholinesterase inhibitor poisoning
Authors: WANG Chen‐Yu , WU Chieh‐Liang , TSAN Yu‐Tse , HSU Jeng‐Yuan , HUNG Dong‐Zong , WANG Chih‐Hung 
Published: July 2010

ABSTRACTBackground and objective:  Organophosphates and carbamates are potent cholinesterase inhibitors that are widely used as insecticides in agriculture. Pneumonia is a frequent complication of cholinesterase inhibitor poisoning (CIP) and a risk factor for death. The aim of this retrospective study was to assess the risk factors for pneumonia in patients with CIP.Methods:  The medical records of 155 patients, who were treated for CIP in a 1300‐bed medical centre in central Taiwan, from January 2002 to December 2004, were retrospectively analysed. Pneumonia was diagnosed by a new or persistent infiltrate on CXR, as well as clinical symptoms. Demographic data, comorbidities, acute respiratory failure and in‐hospital mortality were also recorded.Results:  Of the 155 patients, 31 (20%) died and 92 (59.4%) developed acute respiratory failure. Thirty‐four patients (21.9%) were diagnosed with early onset pneumonia during hospitalization. Acute respiratory failure (OR 12.10, 95% CI: 2.55–57.45), underlying cardiovascular disease (OR 3.02, 95% CI: 1.02–8.91), undergoing gastric lavage at peripheral hospitals (OR 6.23, 95% CI: 1.52–25.98) and development of respiratory failure at the study centre after gastric lavage (OR 3.43, 95% CI: 1.17–10.0) were predictive factors for early onset pneumonia. Cardiopulmonary resuscitation (OR 23.58, 95% CI: 6.03–92.29), early onset pneumonia (OR 7.45, 95% CI: 2.02–27.5) and lower Glasgow coma score (OR 1.26, 95% CI: 1.08–1.48) were predictive factors for mortality.Conclusions:  Pneumonia was a significant risk factor for death in patients with CIP. In addition to aggressive management of patients with CIP who develop respiratory failure, careful respiratory evaluation before and after gastric lavage would help to decrease the incidence of early onset pneumonia in patients with CIP.
Exponential Growth and Future of Artificial Organs
Authors: Kolff W. J. 
Published: July 2010

ABSTRACTOne hundred thousand (100,000) people are living—thanks to Artificial Kidneys. We need more emphasis on Home Dialysis. The immediate future will bring us:WAK (Wearable Artificial Kidney)FAK (Filtrating Artificial Kidney)PAK (Peritoneal Artificial Kidney)HAK (Hemoperfusion Artificial Kidney)Intra‐aortic balloon pumps and transapical left ventricular bypass may offer patients now dying from heart failure, a possibility to either recover or be maintained until an artificial heart is available. Our aim is to create an Artificial Heart to totally replace the irreparably sick human heart. We have obtained survival times up to more than six months after total replacement of the natural heart in calves. Dr. Steve Jacobsen's Artificial Arm is activated by electromyographic signals that are derived from the shoulder muscles. The Artificial Eye makes use of direct stimulation of the visual cortex of the brain by arrays of electrodes situated against the visual cortex. For the Artificial Ear, we stimulate the fibers of the eighth nerve by threading platinum wires up into the cochlea. Deaf volunteers can hear rhythm, loudness and some pitch.
Uremic Neuropathy: Evidence of Middle Molecule Toxicity
Authors: Milutinovic Jovan , Babb Albert L. , Eschbach Joseph W. , Follette William C. , Graefe Ulrich , Strand Michael J. , Scribner Belding H. 
Published: July 2010

ABSTRACTTen dialysis patients were followed in a prospective study to determine the neurotoxicity of metabolites in the middle molecule (MM) molecular weight (mol wt) range of 500–2000 daltons/molecule. In the absence of readily available direct serum measurements of MM concentrations, a theoretically calculated dialysis index, D1(MM), which included the combined effects of dialysis and residual glomerular filtration rate (GFR) on MM removal was used to estimate changes in their predialysis concentrations.The ten patients were dialyzed on protocols which yielded a D1(MM) less than 1.0. Evidence of uremic neuropathy developed in six of these ten patients, and five of these six also developed a progression in their anemia. Two additional patients with no signs of neuropathy developed a progression in their anemia. One patient developed pericarditis with tamponade. A total of eight patients developed complications. One additional patient developed increased weakness, tiredness and general malaise without change in objective findings. When the dialysis therapy to reduce MM concentrations by increasing the D1(MM) above 1.0 was instituted, the complications were reversed.Our data support the findings of others, namely, that there are toxic substances in the MM mol wt range of 500–2000 daltons/molecule. However, a synergism between elevated concentrations of small molecules and MM cannot be ruled out.
Sorbent Membranes: Device Designs, Evaluations and Potential Applications
Authors: Malchesky Paul S. , Piatkiewicz Wojciech , Varnes Warren G. , Ondercin Lawrence , Nosé Yukihiko 
Published: July 2010

ABSTRACTFor the biomedical application of sorbents in extracorporeal circulation, the system must be free of particle release and must be biocompatible. In addition, because of the wide range of biochemical abnormalities generally seen in the disease state, the use of multiple sorbents should be possible. Based on these criteria, the Enka sorbent Cuprophan membranes have been selected for evaluation. Sorbent membranes are of two general types: sorption and sorption‐dialysis. Coil, parallel plate and capillary designs have been constructed and tested employing sorption‐dialysis charcoal membranes of film and hollow fiber configurations. The results have indicated potential advantages. By design, the sorbent membranes prevent direct interaction of the sorbent with blood cells and prevent particle release, making chronic applications safe. Devices employing sorbent fibers are particularly suited to select applications where higher sorbent contents are required or ultrafiltration is not desired.In an effort to optimize the application of sorbents and the sorbent membranes with regard to the quantity of sorbent employed and the length of treatment, a general scheme for evaluation of sorbent systems has been established involving three phases: 1) equilibrium isotherm studies to determine the selectivity and capacity of the sorbent system for the solute, 2) kinetic studies to determine mass transfer properties of the sorbent system in the absence of fluid dynamic resistance and 3) device evaluation to determine overall mass transfer properties. Studies show that, in device designs, it is possible to separate the effects of sorption and fluid dynamics and to design around the limiting parameters.
Influence of Middle Molecules on the Anemia of Uremic Patients
Authors: Leber Heinrich‐Wolfgang , Spiegelhalter Rita , Ulm Anne , Goubeaud Gerhard , Rawer Peter 
Published: July 2010

ABSTRACTTo evaluate their toxicity at the cellular level, middle molecules from uremic serum were incubated with erythrocytes from healthy subjects and the activity of the enzyme Delta‐aminolevulinic acid dehydrase (D‐ALA‐D) and peroxidative hemolysis were investigated. Uremic middle molecules caused a significant decrease of the D‐ALA‐D activity of normal erythrocytes which was not due to differences in the concentrations of Pb, Cd or Zn. The decreased enzyme activity could be restored by adding reduced glutathione (GSH; 5 mmol/L) together with the middle molecules to the assay system. Uremic middle molecules caused a significant increase of peroxidative hemolysis in normal erythrocytes.Uremic middle molecules contribute to the anemia of uremic patients by impeding hemoglobin synthesis and by increasing peroxidative hemolysis, possibly by affecting SH‐groups. H2O2‐producing compounds should be avoided in uremic patients.