bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparison of Clostridium botulinum toxins type D and C1 in molecular property, antigenicity and binding ability to rat‐brain synaptosomes
Authors: MURAYAMA Satoru , SYUTO Bunei , OGUMA Keiji , IIDA Hiroo , KUBO Shuichiro 
Published: March 2005

Botulinum type D neurotoxin was purified 950‐fold from the culture supernatant with an overall yield of 32%. The purified toxin had a specific toxicity of 5.8 × 107 mouse minimal lethal dose per mg of protein and a relative molecular mass of 140000. The purified toxin had a di‐chain structure consisting of heavy and light chains with relative molecular masses of 85000 and 55000, respectively, linked by one disulfide bond. These subunits had different amino acid compositions and antigenicities. A similarity in molecular constructions and amino acid compositions was observed between type D and type C1 toxins as well as between their subunits.Among the seven kinds of monoclonal antibodies against type D toxin, six reacted with the heavy chain of type D toxin, while one of the six also reacted with the heavy chain of type C1 toxin and neutralized the toxicities of the two toxins. The other one of monoclonal antibodies reacted with the light chains of both toxins. This evidence indicates that both toxins have common antigenic sites on their heavy and light chains and that the antigenic site on the heavy chain may contribute to the neutralization of both toxins by antibody.The binding of type D toxin to rat brain synaptosomes was examined by use of 125I‐labelled type D toxin. The binding was competitively inhibited not only by unlabelled type D and C1 toxins, but also by the heavy chains of both toxins, however, it was not inhibited by the light chain of type D toxin. These results suggest that the toxin receptors on synaptosomal membrane are common for type D and C1 toxins, and that the heavy chain contributes to the binding of toxin to synaptosomes and the structure of the binding sites on the heavy chains of both toxins is quite similar.
Binding of adriamycin‐Fe3+ complex to membrane phospholipids
Authors: DEMANT Erland J. F. 
Published: March 2005

Binding of adriamycin‐Fe3+ complex to phospholipids has been examined by phase partitioning in a hexane‐water system. Formation of a stable ternary adriamycin‐Fe3+‐lipid complex with the negatively charged phospholipids cardiolipin and phosphatidylglycerol (both isolated from Escherichia coli) and synthetic dioleoyl glycerophosphoglycerol is demonstrated.Binding of adriamycin‐Fe3+ complex to phospholipid bilayers was assessed by incubation of the complex with aqueous phospholipid dispersions prepared from synthetic dimyristoyl glycerophosphocholine, E. coli phosphatidylethanolamine or E. coli cardiolipin and from mixtures of these lipids. Binding of the complex to the phospholipids took place with an affinity for cardiolipin > phosphatidylethanolamine > phosphatidylcholine. The ternary adriamycin‐Fe3+‐ADP complex formed in the presence of 1 mM ADP (ADP in 25‐fold molar excess to adramycin) showed low affinity for binding to phospholipid.
Antibodies to synthetic peptides as probes of acetylcholine receptor structure
Authors: BARKAS Thomas , JUILLERAT Marcel , KISTLER Joerg , SCHWENDIMANN Bernard , MOODY John 
Published: March 2005

Peptides containing the C‐terminus of the alpha‐chain of the nicotinic acetylcholine receptor (nAChR), as deduced from cDNA data, were synthesised and shown to bind to antibodies to denatured nAChR. Conversely, peptide‐specific antibodies bound both to native and to denatured nAChR. Binding was exclusively to the alpha‐chain.Trypsinization experiments and the use of the unique C‐terminal hexapeptide of the alpha‐chain demonstrated that the proposed C‐terminus does exist on the mature alpha‐chain, and that post‐translational cleavage can be discounted as an explanation of the discrepancy of the molecular masses of the alpha‐chain deduced from SDS gel electrophoresis (40 kDa) and from the DNA sequencing (50 kDa).Cleavage of the alpha‐chain in the membrane occurs at two closely linked sites, resulting in the formation of a large fragment (approximately 35 kDa) and the remainder of the chain (approximately 9–10 kDa).No signs of experimental myasthenia gravis were observed in rabbits immunised with C‐terminal peptide coupled to carrier protein.
5‐Enolpyruvylshikimate‐3‐phosphate synthase of Klebsiella pneumoniae
Authors: STEINRÜCKEN Hans Christian , AMRHEIN Nikolaus 
Published: March 2005

The shikimate pathway enzyme 5‐enolpyruvylshikimate‐3‐phosphate (EPSP) synthase (3‐phosphoshikimate 1‐carboxyvinyltransferase, EC 2.5.1.19) has been purified to apparent homogeneity from Aerobacter aerogenes, strain 62‐1 (=Klebsiella pneumoniae ATCC 25306). A 3300‐fold purification of the enzyme was achieved by ammonium sulfate fractionation, heat precipitation, chromatography on DEAE‐cellulose, Sephadex G‐75, and cellulose phosphate, and chromatofocusing as the final step. The recovery was 49%.An apparent relative molecular mass of 32400 was determined by calibrated gel filtration, while a single peptide chain of Mr= 42900 was found by sodium dodecyl sulfate/acrylamide gel electrophoresis. The isoelectric point was determined to be at pH 4.6. Two distinct pH optima (pH 5.4 and 6.8) were observed for the enzyme‐catalyzed formation of EPSP from phosphoenolpyruvate (PEP) and shikimate 3‐phosphate(S3P). For the reverse reaction, the pH optima were 5.6 and 7.6. No evidence for a metal cofactor was found. While the temperature optimum was at 60°C, the activation energies were calculated to be 54.2 kJ/mol for the forward, and 64.1 kJ/mol for the reverse reaction. At low PEP and S3P concentrations, anions acted as activators of EPSP synthase at low concentrations, and as inhibitors at high concentrations.Non‐linear Lineweaver‐Burk plots were interpreted to result from the activation of EPSP synthase by its anionic substrates. The following dissociation constants were determined for the respective enzyme‐substrate complexes: forward reaction: 43 μM (PEP) and 22 μM (S3P); reverse reaction: 1.3 μM (EPSP) and 2.6 μM (Pi). The kinetic patterns indicate a random sequential mechanism for the forward reaction.
Photo‐induced dimerization of anthracene phospholipids for the study of the lateral distribution of lipids in membranes
Authors: de BONY Jacqueline , TOCANNE Jean‐François 
Published: March 2005

It is shown that photo‐induced cross‐linking reaction between anthracene‐labelled phospholipids can be used for studying, at a molecular level, their lateral distribution in bilayer structures. A simple and versatile method is proposed. It is based on the property of anthracene to form covalently bound dimers upon irradiation in the near ultraviolet (360 nm) and on the possibility of separating the lipid photo‐dimers from the lipid monomers by thin‐layer chromatography. Identification of the photo‐dimers is easily achieved since, upon illumination at a shorter wavelength (250–280 nm), they partially dissociate to the native monomer molecules.The feasibility of the method was tested by checking the effects of cations (sodium, calcium) on the homogeneity of 1/1 mixtures of anthracene‐phosphatidylcholine, i.e. 1‐acyl‐2‐[9‐(2‐anthryl)‐nonanoyl]‐sn‐glycero‐3‐phosphocholine (Anthr‐PC) with anthracene‐phosphatidic acid (Anthr‐PA) and with anthracene‐phosphatidylglycerol (Anthr‐PG) in the form of liposomes. These lipids were anthracene‐labelled by acylation of their glycerol backbone at the sn‐2 position with the synthetic 9‐(2‐anthryl)‐nonanoic acid. Data presented indicate a good miscibility of these lipids in the presence of sodium. For each lipid mixture, the lipid heterodimers were clearly identified and, quantitatively, they dominated the lipid homodimers, as expected for a regular distribution of the lipids in the 1/1 mixture. Addition of calcium ions to the lipid suspensions did not alter the miscibility properties of Anthr‐PC and Anthr‐PG. In contrast, calcium triggered a clear‐cut phase separation in the Anthr‐PC/Anthr‐PA mixture as, in this case, only traces of the heterodimer form of the lipids remained observable on the chromatogram. The three anthracene‐phospholipids, pure or mixed together, exhibit a clear‐cut gel‐to‐liquid phase transition which was detectable by fluorescence intensity measurements. The analysis of, the corresponding phase‐transition temperatures confirms, at a ‘macroscopic’ level, the effects of sodium and calcium on the mixing properties of the anthracene phospholipids which were revealed at a ‘microscopic’ level by the dimerization procedure.
A study of the interactions between residues in the C‐terminal half of calmodulin by one and two‐dimensional NMR methods and computer modelling
Authors: AULABAUGH Ann , NIEMCZURA Walter P. , BLUNDELL Thomas L. , GIBBONS William A. 
Published: March 2005

Assignments of the six sets of aromatic ring protons and four high‐field‐shifted methyl group protons of the C‐terminal fragment of calmodulin, residues 78–148, was achieved by a combination of one and two‐dimensional NMR spectroscopic methods. A full spectral analysis of the aromatic region in terms of chemical shifts and scalar coupling constants was achieved and confirmed by spectral simulation.A three‐dimensional structural model of the C‐terminal fragment was constructed by interactive computer graphics techniques and combined with nuclear Overhauser enhancements to propose sequence assignments for all aromatic and high‐field‐shifted methyl groups. This computer‐generated three‐dimensional model was generally supported by the fact that it qualitatively accounted for many of the ring‐current‐shifted proton resonances and the intraresidue and interresidue nuclear Overhauser enhancements.
Apolipoproteins as the basis for heterogeneity in high‐density lipoprotein2 and high‐density lipoprotein3
Authors: MARCEL Yves L. , WEECH Philip K. , NGUYEN Thanh‐Dung , MILNE Ross W. , McCONATHY Waller J. 
Published: March 2005

A method is described for the isoelectric focusing (IEF) of lipoproteins on thin films of agarose. Within a pH gradient of 4.60–5.30 both high‐density lipoproteins 2 and 3 (HDL2 and HDL3) are resolved into more than 10 fractions which could be stained either for protein or for lipids. The isoelectric focusing patterns for HDL3 and HDL3 are similar although HDL2 appears richer in the more alkaline bands.Narrow film strips from the IEF separation of HDL2 and HDL3 were interfaced with various agarose plates containing antisera against apolipoproteins apoAI, apoAII and apoCIII either alone or in combination, to provide two‐dimensional IEF immunoelectrophoresis patterns. This technique demonstrated that apoAI and apoAII were present throughout the IEF gel for both subclasses of HDL. It also provided evidence for the existence of lipoproteins containing both apoAI and apoAII and other lipoproteins present in the alkaline region of the gel which contained apoAI but no apoAII. ApoCIII was found mostly in acidic lipoproteins and was not distributed identically in HDL2 and HDL3. The lipoproteins separated by IEF on agarose were also analysed by two‐dimensional IEF‐SDS electrophoresis and the individual apo lipoproteins were identified by reaction with antibodies to apolipoproteins AI, AII, CI, CII, CIII, D, and E. This technique confirmed that in IEF of HDL, apoAI extended throughout the spectrum of lipoproteins whereas apoE was only present in alkaline lipoproteins and apoD was only present in acidic lipoproteins.IEF on agarose of either HDL2 or HDL3 allowed us to collect eight different fractions, which have the same pi in either lipoprotein class. The apolipoprotein composition of each isolated band was analysed by electroimmunoassays for apolipoproteins AI, AII, CI, CII, CIII, D, and E and the results expressed as the ratio of the measured apolipoprotein to measured apoAI. In both HDL2 and HDL3, acidic lipoprotein fractions were enriched in apoAII, apoCIII and apoCI. ApoCII and apoCIII were not similarly distributed in HDL2 and HDL3 subfractions whereas the apoCI distribution was similar in both classes. Noteworthy in all experiments was the difference in the distributions of apoCI, apoCII, and apoCIII in HDL2 and HDL3, which indicated that the existence of a lipoprotein containing simultaneously CI, CII and CIII can only account for a small fraction of these apolipoproteins.Therefore these experiments substantiate the theory of the protein basis of HDL heterogeneity and suggest that the majority of apolipoproteins are present in complexes which upon IEF result in lipoprotein fractions of identical pi for both HDL2 or HDL3. In addition, subtle differences in the distribution of apolipoproteins in HDL2 and HDL3 indicate the occurrence of some different lipoprotein species in these two subclasses of HDL.
Regulation of hepatic glutamate metabolism
Authors: HÄUSSINGER Dieter , GEROK Wolfgang 
Published: March 2005

1. In isolated perfused rat liver, addition of the oxoanalogues of leucine, isoleucine, methionine and phenylalanine is followed by a rapid and reversible stimulation of glutamate release. This is not observed with the corresponding amino acids or 2‐oxoisovaIerate, 2‐oxoglutarate or oxaloacetate.2. The increased glutamate release by the liver is accompanied by a decrease in the tissue contents of 2‐oxoglularate and glutamate by about 25% and 50%, respectively.3. During the metabolism of glutamine, i.e. conditions with elevated tissue glutamate concentrations, 2‐oxoacid‐induced glutamate release is stimulated. In the presence of glutamine (5 mM), 2‐oxoisocaproate, 2‐oxo‐4‐melhylvalerate and 2‐oxo‐4‐methylthiobulyrate were found to be most effective and glutamate release by the liver increased linearly from about 80 nmol g−1 min−1 to 600 nmol g−1 min−1 at increasing 2‐oxoacid concentrations up to 1mM, When glutamate [issue levels were decreased by phenylephrine, stimulation of glutamate release by 2‐oxoisocaproate was markedly diminished.4. 2‐Oxoacid‐stimulated glutamate release is independent of oxoacid metabolism, indicating that the effect is probably not explained by a 2‐oxoacid/glutamate exchange across glutamate export predominantly occurs in a sodium‐independent way.5. At low concentrations of 2‐oxoisocaproate (below 0.2 mM), the increased glutamate release was accompanied by a slight inhibition of 14CO2 production from added [14C] glutamate, indicating a simultaneous glutamate uptake and release also under these conditions.6. Stimulation of glutamate release by 2‐oxoisocaproate is followed by a decreased rate of urea and glutamine synthesis from portal ammonia, as a consequence of, an increased glutamate release.
Characterization and function of pig intestinal sucrase‐isomaltase and its separate subunits
Authors: RODRIGUEZ Ignacio R. , TARAVEL Francois R. , WHELAN William J. 
Published: March 2005

1. Papain‐solubilized pig intestinal sucrase‐isomaltase was purified to homogeneity in a four‐step process with a yield of 50%.2. The substrate specificities of the two enzyme activities were studied together and separately after inactivation or inhibition of one of the activities.3. Michaelis constants, maximum velocities and time courses of hydrolysis of several substrates, in particular α‐limit dextrins, were used to characterize this complex of α‐glucosidases.4. The participation of the enzyme complex in the hydrolysis of α‐limit dextrins and more generally in pathways for starch breakdown in the pig digestive tract is examined and discussed.
Single‐step elongation of oligodeoxynucleotides using terminal deoxynucleotidyl transferase
Authors: SCHOTT Herbert , SCHRADE Herbert 
Published: March 2005

Procedures for the stepwise addition of one or more deoxyribonucleotide residues to the 3′ end of an oligodeoxyribonucleoside phosphate acceptor using commercially available terminal deoxynucleotidyl transferase is described. 2–80 nmol of acceptors with a chain length of four, five or nine monomer units were elongated with a single 2′‐deoxyribonucleoside 5′‐triphosphate in yields of 20–30%. The monomers carried no protecting groups and were used both radioactively labelled and unlabelled.The elongated oligodeoxynucleoside phosphates were isolated by reverse‐phase (Nucleosil C18) high‐performance liquid chromatography or paper chromatography. The isolated products were sequenced by the fingerprint method. Advantages and disadvantages of this new methodology for the enzymatic synthesis of defined oligodeoxynucleotides are discussed.
Acetylcholine‐like effects of 1‐O‐alkyl‐2–acetyl‐sn‐glycero‐3‐phosphocholine (‘platelet‐activating factor’) and its analogues in exocrine secretory glands
Authors: SÖLING Hans‐Dieter , EIBL Hansjörg , FEST Werner 
Published: March 2005

The effect of 1‐O‐hexadecyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine (1), 1‐O‐octadecyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine (2), 1‐O–hexadecyl‐sn‐glycero‐3‐phosphocholine (3), 1‐O‐octadecyl‐2‐O‐methyl‐sn‐glycero‐3‐phosphocholine (4) and its enantiomer 3‐O‐octadecyl‐2‐O‐methyl‐sn‐glycero‐1‐phosphocholine (5) on the secretion of amylase from guinea pig isolated parotid gland and exocrine pancreatic lobules was examined. Compounds 1, 2 and 4 led to a significant stimulation of amylase release in both systems, effects being already visible between 10–100pM. Maximal stimulation with compounds 1 and 2 occurred at 5nM, with compound 4 at 1 nM. Higher concentrations were less effective and at 0.1 μM stimulation was very low. In contrast, compound 5 showed a continuous increase in activity up to 0.01–0.1 μM without a decrease at higher concentrations. Compound 3 had no effect.For compound 1, its effects on calcium and lipid metabolism have been analyzed and compared with those of the acetylcholine analogue carbamoylcholine. Compound 1 mimicked in every respect the effects of carbamoylcholine. It stimulated the uptake of 45Ca by isolated parotid gland lobules in a non‐ionophoretic way. In isolated pancreatic lobules it enhanced the incorporation of [32P]phosphate into phosphatidic acid, phosphatidylinositol and poly(phosphoinositide), increased the formation of diacylglycerols and triacylglycerol, led to the same two‐phasic responses of myo‐[3H]inositol‐labeled polyphosphoinositides, and initiated a rapid short‐lasting formation of free inositol triphosphate.Accordingly, ‘platelet activating factor(s)’ can affect the function of exocrine glands at low concentrations. The effects observed resemble those produced by acetylcholine and result most likely from the interaction of platelet‐activating factor with plasma membrane receptors.
Molecular basis of the ‘box effect’
Authors: LABOUESSE Michel , NETTER Pierre , SCHROEDER Renee 
Published: March 2005

In the mitochondrial DNA of Saccharoimyces cerevisiae, the genes cob‐box and oxi3, coding for apocytochrome b and cytochrome oxidase subunit I respectively, are split. Several mutations located in the introns of the cob‐box gene s of cytochrome b and cytochrome oxidase subunit I (this is known as the ‘box effect’).‐We have elucidated the molecular basis of this phenomenon: these mutants are unable to excise the fourth intron of oxi3 from the cytochrome oxidase subunit I pre‐mRNA; the absence of a functional bI4 mRNA maturase, a trans‐acting factor encoded by the fourth intron of the cob‐box gene explains this phenomenon. This maturase was already known to control the excision of the bI4 intron; consequently we have demonstrated that it is necessary for the processing of two introns located in two different genes.Mutations altering this maturase can be corrected, but only partially, by extragenic suppressors located in the mitochondrial (mim2) or in the nuclear (NAM2) genome. The gene product of these two suppressors should, therefore, control (directly or indirectly) the excision of the two introns as the bI4 mRNA maturase normally does.
Encephalomyocarditis virus replication complexes preferentially utilizing nucleoside diphosphates as substrates for viral RNA synthesis
Authors: KOONIN Eugene V. , AGOL Vadim I. 
Published: March 2005

Replication complexes (RC) of the encephalomyocarditis (EMC) virus were shown previously to contain components that exhibit marked preference for nucleoside diphosphates over nucleoside triphosphates (NTP) as substrates for viral RNA synthesis [Koonin and Agol (1983), Virology 129, 309–318]. These NDP‐preferring components have now been found to posses the following properties.

When RC preparations were fractionated by sucrose density gradient centrifugation, the fractions containing NDP‐preferring components exhibited a considerably higher nucleotide kinase activity as compared to either the fractions containing NTP‐preferring components or corresponding fractions from mock‐infected cells.
When NDP‐preferring RC were incubated with ADP and three other NTP, very low concentrations of endogenously generated ATP ensured a greater rate of RNA synthesis than did much higher concentrations of exogenous ATP.
When an equimolar mixture of differently labelled UDP and UTP was used as a substrate for NDP‐preferring RC, the label from UDP predominated in the newly synthesized RNA, even though the UDP‐derived UTP constituted a minor portion of the total UTP pool.
When labelled UDP was diluted with unlabelled uridine nucleotides, unlabelled UTP proved to be far less efficient than unlabelled UDP in diminishing the specific radioactivity of UMP incorporated into RNA by NDP‐preferring RC. These data are interpreted in the sense that the NTP generated by the built‐in nucleotide kinase system are not freed into the external milieu but rather form a separate pool preferentially used for synthesis of viral RNA by NDP‐preferring RC. It is suggested that this functional compartmentation of NTP may be significant for the replication of viral RNA in vivo.