bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cholesterol efflux from and high‐density‐lipoproteins binding to cultured bovine vascular endothelial cells are higher than with vascular smooth muscle cells
Authors: SAVION Naphtali , KOTEV‐EMETH Shlomo 
Published: March 2005

Cholesterol metabolism was studied and compared in confluent cultures of adult bovine aortic endothelial and bovine aortic smooth muscle cells which were grown under similar conditions. The total cholesterol content/mg protein was only slightly higher in smooth muscle cells than in endothelial cells and upon exposure to [3H]cholesterol the maximal specific activity/mg protein obtained was similar in both cell types. Most (98%) of the incorporated [3H]cholesterol remained in the form of free cholesterol in both cell types, and provided a system for the study of cholesterol efflux. The role of high‐density lipoproteins (HDL) and human serum in cholesterol influx and efflux, in both endothelial and smooth muscle cells, was studied. Net cholesterol transport in the cultures was calculated and net efflux was observed in both cell types. This was higher in endothelial than in smooth muscle cells and HDL was more efficient than human serum in promoting net cholesterol efflux.During the influx experiments, no conversion of [3H]cholesterol to [3H]cholesteryl ester was observed either in the cell layer or in the incubation medium. On the other hand, during efflux experiments when HDL but not human serum was the acceptor, some (about 6%) conversion of [3H]cholesterol to [3H]cholesteryl ester occurred in the incubation medium. 125I‐HDL3 binding to endothelial and smooth muscle cells was studied and demonstrated saturation at a concentration of about 100 μg protein ml for both cell types. However, endothelial cells bound about six times more 125I‐HDL3 than smooth muscle cells.These studies indicate that vascular endothelial cells are more protected against cholesterol accumulation than vascular smooth muscle cells. The greater ability of endothelial cells to bind HDL complexes when compared with smooth muscle cells, and thereby to be more susceptible to HDL induced cholesterol efflux, may add a new mechanism through which endothelial cells protect the blood vessel from cholesterol accumulation.
Structural analysis of the carbohydrate chains of a mouse monoclonal IgM antibody
Authors: KROTKIEWSKI Hubert , NILSSON Bo , SVENSSON Sigfrid 
Published: March 2005

A mouse monoclonal IgM antibody, directed against human blood group B determinant, was isolated from hybridoma culture growth medium. Chemical analysis indicated presence of N‐ and O‐linked oligosaccharides. The N‐ and O‐linked carbohydrate chains were liberated using two different conditions of reductive alkaline degradation. Structural analysis was carried out on the isolated chains using chemical analysis, 500‐MHz 1H‐NMR spectroscopy and fast‐atom‐bombardment mass spectrometry. The following composite structures of the N‐linked chains were found:

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where R = OH for biantennary structures and R = Neu5Acα2 – 3Galβ1 – 4GlcNAcβ1 – or Neu5Acα2 – 3Galβ1 – 3[Neu5Acα2 – 6]GlcNAcβ1 – for triantennary structures.The O‐linked oligosaccharides, found in the light chains. were shown to have the structure Neu5Acα2 – 3Galβ1 – 3GalNAc. The native IgM antibody could be separated on a concanavalin‐A – Sepharose column into two subfractions, differing in the presence of a high‐mannose‐type oligosaccharide.
O‐Linked GlcNAc in serotype‐2 adenovirus fibre
Authors: CAILLET‐BOUDIN Marie‐Laure , STRECKER Gérard , MICHALSKI Jean‐Claude 
Published: March 2005

Serotype‐2 adenovirus fibre is shown to possess an O‐linked GlcNAc residue and to have affinity for wheat germ agglutinin. The cytoplasmic and nuclear fibres are both glycosylated. Glycosylation seems to take place in the cytoplasm since most of the [14C]GlcN‐labelled fibre is found in this compartment, little label being associated with the microsomes. Glycosylation of the fibre was not affected by inhibitors of N‐ and O‐glycosylation.A variation in fibre glycosylation is observed among adenovirus. Among the serotypes tested, only serotype‐5 adenovirus (another subgroup C virus) also incorporated [14C]GlcN into its fibre, but did not possess affinity for wheat‐germ agglutinin.The GlcNAc is located in the N‐terminal two‐thirds of the fibre and more probably in the N‐terminal one‐third. The free or penton‐base‐associated fibres are similarly glycosylated. These results suggest that glycosylation is not involved in viral adsorption and in assembly with the capsid penton base. Thus, glycosylation might be a characteristic feature of subgroup C viruses.
The role of sodium ions in methanogenesis
Authors: KAESLER Bruno , SCHÖNHEIT Peter 
Published: March 2005

Cell suspensions of Methanosarcina barkeri were found to oxidize formaldehyde to CO2 and 2H2 (ΔG0′= ‐ 27 kJ/mol CO2), when methanogenesis was inhibited by 2‐bromoethanesulfonate. We report here that this reaction is coupled with (a) primary electrogenic Na+ translocation at a stoichiometry of 2–3 Na+/CO2, (b) with secondary H+ translocation via a Na+/H+ antiporter and (c) with ATP synthesis driven by an electrochemical proton potential. This is concluded from the following findings.Formaldehyde oxidation to CO2 and 2H2 was dependent on Na+ ions, 2–3 mol Na+/mol formaldehyde oxidized were extruded. Na+ translocation was inhibited by Na+ ionophores, but not affected by protonophores or Na+/H+ antiport inhibitors.Formaldehyde oxidation was associated with the build up of a membrane potential in the order of 100 mV (inside negative), which could be dissipated by sodium ionophores rather than by protonophores.Formaldehyde oxidation was coupled with ATP synthesis, which could be inhibited by Na+ ionophores, Na+/H+ antiport inhibitors, by protonophores and by the H+ ‐translocating‐ATP‐synthase inhibitor, dicyclohexyl‐carbodiimide.With cell suspensions of Methanobacterium thermoautotrophicum similar results were obtained.
Effect of evolution on the kinetic properties of enzymes
Authors: PETTERSSON Gösta 
Published: March 2005

The likely effect of a selective pressure in the direction of higher reaction fluxes on rate parameters for enzyme reactions confirming to Michaelis‐Menten kinetics has been analyzed on the basis of relationships which take into account the changes in metabolite concentrations that must be associated with mutational changes of the kinetic properties of enzymes participating in metabolic pathways.
Arguments are presented to show that such a pressure should tend to increase kcat, whereas Km may decrease or increase depending on what stage of evolutionary development the enzyme has reached. While the early evolution of enzymes must have been associated with decreasing Km values, an increase of both kcat and Km is mandatory for enhancement of the rate performance of extensively developed enzymes which exhibit kcat/Km ratios approaching the diffusion‐control limit. The latter limit is dependent on the equilibrium constant for the catalysed reaction.
Enzymes which have reached the diffusion‐control limit for their second‐order rate performance cannot be considered as perfectly evolved catalysts, but may well undergo further development towards a higher catalytic efficiency in response to the improvement of other enzymes in the metabolic pathway with regard to the criterion of an enhanced reaction flux. Such evolution is associated with an increase of the metabolite levels in the pathway, and a simple model system is examined in order to illustrate the ultimate limits for the metabolite levels and reaction flux that may obtain.
The theoretical evidence presented lends no support to previous proposals that certain enzymes (e.g. triosephosphate isomerase), or enzymes showing certain kinetic characteristics (e.g. kcat/Km quotients approaching 109 s–1 M–1), have reached the end of their evolutionary development. A claim that any specific enzyme has reached catalytic perfection would provide the unreasonable inference that all enzymes participating in intermediary metabolism have reached catalytic perfection.
The free radical of pyruvate formate‐lyase
Authors: UNKRIG Volker , NEUGEBAUER Franz A. , KNAPPE Joachim 
Published: March 2005

The first‐derivative EPR spectrum of the active from of Escherichia coli pyruvate formate‐lyase shows an asymmetric doublet with partially resolved hyperfine splittings (g= 2.0037). Isotope substitution studies demonstrated couplings of a carbon‐centered unpaired electron to a solvent‐exchangeable proton (a= 1.5 mT) and to further hydrogen nuclei (a= 0.36 and 0.57 mT). By selective incorporation of unlabelled tyrosine into 2H‐labelled enzyme protein, a tyrosyl radical structure has been ruled out. Circumstantial evidence indicates that the organic free radical, which also displays an ultraviolet absorption signal at 365 nm, is located on a standard amino acid residue of the polypeptide chain. EPR signal quantification found a stoichiometry of 1 spin per active site.The formate analogue hypophosphite has been characterized as a specific kcat inhibitor of pyruvate formatelyase which destroys the enzyme radical. Protein‐linked 1‐hydroxyethylphosphonate was previously described as the dead‐end product after reaction of the analogue with the intermediary acetyl‐enzyme form of the catalytic cycle [W. Plaga et al. (1988) Eur. J. Biochem. 178, 445–450]. EPR spectroscopy of this system has now identified the corresponding α‐phosphoryl radical as a reaction intermediate [g= 2.0032; a(P) = 2.72 mT, a(3H) = 1.96 mT]; it showed a half‐life of about 20 min at 0°C. This finding proves that the enzyme radical is a hydrogen‐atom‐transferring coenzymic element.
Purification and characterization of a fatty‐acid‐binding protein from the gastric mucosa of rats
Authors: KANDA Tatsuo , ISEKI Shoichi , HITOMI Masahiro , KIMURA Hideki , ODANI Shoji , KONDO Hisatake , MATSUBARA Yoichi , MUTO Terukazu , ONO Teruo 
Published: March 2005

Fatty acid‐binding protein (FABP) was purified from rat gastric mucosa by successive Sephadex G‐75 chromatography, DEAE‐cellulose chromatography and HPLC on an RP‐2 (Merck) reversed‐phase column. The purified stomach FABP migrated as a single band corresponding to an apparent molecular mass of 15 kDa on SDS/PAGE.Stomach FABP appeared to be identical with rat heart FABP, as judged from its electrophoretic mobility, amino acid composition and tryptic peptide map. In addition, the amino acid sequences of two selected tryptic peptides coincided completely with the rat heart FABP sequence deduced from that of cDNA. Stomach FABP showed immunochemical identity with rat heart FABP when tested with an antiserum against rat heart FABP. Immunohistochemically, stomach FABP was specifically stained with anti‐(rat heart FABP) serum in parietal cells of the gastric mucosa.The results suggested that the primary structure of stomach FABP is identical with that of rat heart FABP, and showed that stomach FABP is localized in parietal cells of the gastric mucosa.
Amino acid sequence of the monomer subunit of the giant multisubunit hemoglobin from the earthworm Pheretima sieboldi
Authors: SUZUKI Tomohiko 
Published: March 2005

The giant extracellular hemoglobin of the earthworm Pheretima sieboldi is mainly composed of two hemecontaining subunits: a monomer; chain I and a disulfide‐bonded trimer of chains II, III and IV. Both subunits can be separated easily by gel filtration under alkaline conditions. The amino acid sequence of chain I has been determined. It is composed of 141 residues, has two half‐cystine residues forming a intrachain disulfide bridge, and has a molecular mass of 16911 Da including a heme group. Heterogeneity was found at position 37 (His or Ser).The amino acid sequence of Pheretima chain I showed 30–50% identity with those of eight heme‐containing chains of Lumbricus and Tylorrhynchus hemoglobins. The sequences of nine chains of annelid giant hemoglobins were comparéd separately in the functionally essential central exonic region and structurally essential side exonic regions, and a phylogenetic tree was constructed. The amino acid substitution rate for the central exon was found to be about 1.5 times slower than that for the side exons.
Inhibition of transmitter release by botulinum neurotoxin A
Authors: POULAIN Bernard , WADSWORTH Jonathan D. F. , MAISEY E. Anne , SHONE Clifford C. , MELLING Jack , TAUC Ladislav , DOLLY J. Oliver 
Published: March 2005

The contribution of a proteolytic fragment (H2L) of botulinum neurotoxin type A (comprised of the amino‐terminal region of the heavy‐chain disulphide‐linked to the light chain) to inhibition of neurotransmitter release was investigated, using central cholinergic synapses of Aplysia, rodent nerve‐diaphragm preparations and cerebrocortical synaptosomes.
No reduction in neurotransmitter release was observed following external application to these preparations of highly purified H2L or after intracellular injection into Aplysia neurons.
The lack of activity was not the result of alteration in the light chain of H2L during preparation of the latter because (a) renaturation of this light chain with intact heavy chain produced a toxic di‐chain form and (b) simultaneous application of heavy chain and light chain from H2L inhibited transmitter release in Aplysia.
Bath application of H2L and heavy chain together inhibited release of transmitter; however, at the neuromuscular junction the potency of this mixture was much lower than that of native toxin. A similar blockade resulted when heavy chain was applied intracellularly and H2L added to the bath, demonstrating that H2L is taken up into cholinergic neurons of Aplysia. This uptake is shown to be mediated by the amino‐terminal moiety of heavy chain (H2), because bath application of light chain plus H2 led to a decrease in acetylcholine release from a neuron that had been injected with heavy chain.
A role within the neuron is implicated for a carboxy‐terminal portion of heavy chain (H1) since intracellular injection of light chain and H2 did not affect transmitter release. Although the situation is unclear in mammalian nerves, these collective findings indicate that blockade of transmitter release in Aplysia neurons requires the intracellular presence of light chain and H1 (by inference), whilst H2 contributes to the internalization step.
Molecular organization and clustering of cell‐wall‐bound enzymes as a source of kinetic apparent co‐operativity
Authors: DUSSERT Christophe , MULLIERT Guillermo , KELLERSHOHN Nicolas , RICARD Jacques , GIORDANI Roger , NOAT Georges , PALMARI Jacqueline , RASIGNI Monique , LLEBARIA Antoine , RASIGNI Georges 
Published: March 2005

When fixed charges and enzyme molecules are not homogeneously distributed in a matrix, the degree of organization of charges, of enzyme molecules and of charges with respect to enzyme molecules modulate the enzyme reaction rate. The overall reaction velocity of the bound enzyme system may be expressed in terms of monovariate moments of the charge density distribution and of the bivariate moments of the charge and enzyme density distributions. With respect to the situation where fixed charges and enzyme molecules are randomly distributed in the matrix, the molecular organization, as expressed by the monovariate and bivariate moments results in an increase or a decrease, of the overall reaction rate, as well as in the appearance of a kinetic co‐operativity.The degree of spatial organization of objects may be expressed quantitatively through the concept of minimal spanning tree. This concept may thus be applied to the quantification of the degree of order that may exist in the bidimensional distribution of enzyme molecules in a charged matrix.Primary walls of isolated plant cells in sterile culture behave as a polyanion and contain different enzymes. The spatial distribution in sycamore cell walls of an acid phosphatase has been studied through the concept of minimal spanning tree and shown to be non‐randomly distributed in the polyanionic matrix, but clustered in that matrix. This spatial organization results in a modulation of the reaction rate of the cell‐wall‐bound phosphatase reaction.Both the theoretical and experimental results presented in this study leave little doubt as to the validity of the idea thatin situthe organization of fixed charges and enzyme molecules modulate the overall dynamics of enzyme reactions.
α‐Actinin expression during avian myogenesis in vivo
Authors: KOBAYASHI Ryoji , ITOH Hideaki , TASHIMA Yohtalou 
Published: March 2005

The isoforms of skeletal muscle α‐actinin present during chick embryogenesis were analyzed by two‐dimensional electrophoresis in combination with the immunoblot technique. Chicken embryonic muscles at 8–15 days contain an embryo‐specific isoform of α‐actinin. The embryonic α‐actinin isoform has a molecular mass of 112 kDa and an isoelectric point of 5.8, whereas the values for the adult isoform of α‐actinin were 100 kDa and 5.85, respectively. To characterize the two classes of α‐actinin polypeptides we have compared the two proteins by 125I‐labeled two‐dimensional peptide mapping. The embryonic isoform is highly similar to, but exhibited extensive peptide differences to, the adult isoform of α‐actinin.The developmental sequence of the expression of the α‐actinins was also studied. In extracts of skeletal muscle from 8–10‐day‐old embryos, only the embryonic isoform was detected. In extracts from 15‐day‐old embryos, both the embryonic and the adult isoforms coexisted. However by 21 days, the embryonic isoform had disappeared and only the adult isoform was detected.These data suggested that the embryonic and the adult isoform of α‐actinins are distinct proteins and that during skeletal myogenesis in ovo one class of α‐actinin is replaced by a new class of α‐actinin polypeptides, and that the latter is maintained into adulthood.
Conformation of two somatostatin analogues in aqueous solution
Authors: WYNANTS Chantal , TOURWE Dirk , KAZMIERSKI Wieslaw , HRUBY Victor J. , VAN BINST Georges 
Published: March 2005

Cyclic‐disulfide‐containing analogues of somatostatin, [Xaa1= H or DPhe; Xaa3= Phe or Tyr; Xaa7= Cys, Me2Cys or Me2DCys; Xaa8= OH, Thr8 (OH) or Thr8NH2], were examined in aqueous solution by 1H‐NMR spectroscopy and circular dichroism.The influence of the helical nature of the disulfide bridge and the presence of exocyclic residues on biological activity were investigated with particular care.
Characterization of a high‐affinity monoclonal antibody to calcineurin whose epitope defines a new structural domain of calcineurin A
Authors: HUBBARD Michael J. , KLEE Claude B. 
Published: March 2005

Monoclonal antibodies have been raised against native calcineurin using conventional in vivo immunization and hybridoma procedures. The relatively high affinity of nonimmune IgG for the two subunits of calcineurin resulted in large nonspecific binding values for immunoassays of native, dissociated and denatured calcineurin, which complicated the antibody screening.Monoclonal aCn5, a high‐affinity IgG1 that exhibits specific binding, was characterized. Other calmodulin‐binding proteins tested were not recognized by aCn5. Simple binding properties were exhibited in solid‐phase experiments, Kd= 26 (±4) pM, but the stoichiometry was low. The loss of immunoreactivity after denaturation of calcineurin indicated that the aCn5 epitope is of the assembled topographic, not segmental, type. The epitope was located to the A subunit and affinity was unaffected by the presence of calcineurin B. The epitope remained intact after proteolytic removal of the amino‐terminal 20 residues of calcineurin A essential for phosphatase activity, and the carboxyl‐terminal inhibitory and calmodulin‐binding domains. The calmodulin‐binding peptide derived from calcineurin, cA8, was not recognized by aCn5. Addition of Ca2+, Mn2+, Ni2+, chelators or dithiothreitol did not influence the affinity of aCn5 for the holoenzyme. Phosphatase activity of calcineurin, in the presence and absence of calmodulin and after removal of the inhibitory domain, was little affected by aCn5. Thus, the aCn5 epitope defines a previously unidentified structural domain of calcineurin A located in a region of the proteolytically resistant core that is topologically distinct from the catalytic, inhibitory, calmodulin‐binding and calcineurin‐B‐binding domains, and not functionally connected with calcineurin B or the putative metal‐binding domain(s).
Isolation, characterization and immunocytochemical localization of caldesmon‐like protein from molluscan striated muscle
Authors: BARTEGI Aghleb , FATTOUM Abdellatif , DAGORN Caroline , GABRION Jacqueline , KASSAB Ridha 
Published: March 2005

A 140‐kDa polypeptide present in the striated muscle of Pecten maximus and Sepia officinalis was purified to homogeneity and its main properties were investigated using biochemical and cytochemical approaches. The protein was found to be similar to chicken gizzard caldesmon. It is a heat‐stable protein. It cross‐reacts immuno‐logically with anti‐(gizzard caldesmon) antibody, binds to calmodulin‐Sepharose in a Ca2+‐dependent manner, cosediments with F‐actin filaments and acts in the absence and presence of tropomyosin as a potent inhibitor of rabbit skeletal actomyosin Mg2+‐ATPase.The immunocytochemistry of ultrathin sections revealed, at the light microscopy resolution level, that caldesmon‐like protein is present in all types of muscles hitherto examined from invertebrates and vertebrates. However, according to the distribution and the intensity of the fluorescent reaction, we concluded that, under our experimental conditions, caldesmon is not homogeneously distributed and not located in the myofibrillar bands of striated muscles but rather in the sarcoplasmic elements, at the periphery of the fibres.
The stoichiometry of the tightly bound NAD+in urocanase
Authors: KLEPP Jürgen , RÉTEY János 
Published: March 2005

Urocanase, purified by classical methods [Keul, V., Kaeppeli, F., Ghosh, C., Krebs, T., Robinson, J. A. and Rétey, J. (1979)J. Biol. Chem. 254, 843–851] fromPseudomonas putidawas submitted to high‐performance liquid chromatography on a TSK‐DEAE column. The enzyme was eluted in three resolved peaks (A, B and C) exhibiting specific activities of 3.4 U/mg, 1.85 U/mg and 0.4 U/mg, respectively.The difference spectra of peaks B and A as well as of C and A showed maxima at 330 nm.Irradiation of peaks B and C at 320 nm resulted in an increase of urocanase activity by 45% and 400%, respectively. Peak A could not be photoactivated. Rechromatography of the photoactivated peaks B and C on the TSK‐DEAE column confirmed their partial transformation into peak A.Spectroscopic methods for quantitative protein determination were adapted to urocanase. The stoichiometry of bound NAD+/urocanase (form A) was determined to be 1.75 by enzymic analysis of the free NAD+released upon acid denaturation of the holoenzyme. A similar stoichiometry (1.8–1.9) was found for all three forms (A, B and C) by biosynthetic incorporation of [7‐14C]nicotinate into urocanase using a nicotinate auxotrophic mutant ofP. putida.Form A of urocanase showed, after treatment with NaBH4up to 50% inhibition, an elution pattern (TSK‐DEAE column) similar to a mixture of forms A, B and C in the approximate ratio of 1:2:1. None of these forms could be photoactivated.We conclude that form A of the urocanase dimer contains two intact NAD+molecules. In form B one of the two subunits contains an NAD+‐nucleophile adduct which is present in both subunits of form C. Full urocanase activity requires intact NAD+in both subunits. Intact NAD+can be regenerated from the adduct but not from the reduced form by photolysis. The two subunits of urocanase are independent both in their catalytic activity and in modification reactions.