bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytochrome P‐450 isozyme/isozyme functional interactions and NADPH‐cytochrome P‐450 reductase concentrations as factors in microsomal metabolism of warfarin
Authors: KAMINSKY Laurence S. , GUENGERICH F. Peter
Published: March 2005

The basis for our previous observations [Kaminsky, L. S., Guengerich, F. P., Dannan, G. A. & Aust, S. D. (1983) Arch. Biochem. Biophys. 225, 398–404] that rates of microsomal metabolism of warfarin were markedly less than the sum of rates of the reconstituted constituent isozymes of cytochrome P‐450 has been investigated. Metabolism of warfarin to 4′‐, 6‐, 7‐, 8‐, and 10‐hydroxywarfarin and dehydrowarfarin by highly purified rat liver cytochrome P‐450 (P‐450) isozymes reconstituted with NADPH–cytochrome P‐450 reductase and by hepatic microsomes from variously pretreated rats was used to probe functional consequences of P‐450 isozyme/isozyme interactions and of the effect of microsomal reductase concentrations. Binary mixtures of P‐450 isozymes were reconstituted and the regioselectivity and stereoselectivity were used to probe metabolism by each individual isozyme. The isozymes specifically inhibited each other to variable extents and the order of inhibitory potency was: P‐450UT‐F > P‐450PB‐D≥ P‐450UT‐A≥ P‐450BNF/ISF‐G > P‐450PB/PCN‐E > P‐450PB‐B≥ P‐450PB‐C≥ P‐450BNF‐B. The inhibition, possibly a consequence of aggregation, explains the low rate of microsomal metabolism relative to the metabolic potential of the component P‐450 isozymes. When purified reductase was added to microsomes it appeared to bind to microsomes at different sites from endogenous reductase and it enhanced warfarin hydroxylase activity only to a minor extent, thus possibly precluding low reductase concentrations from being a major factor in the relatively low rates of microsomal metabolism. Antibody to the reductase differentially inhibited microsomal metabolism of warfarin by the various P‐450 isozymes. The results suggest that the reductase and P‐450 isozymes may be located differently relative to one another in the various microsomal preparations.
Heterogeneous glycosylation of murine transferrin receptor subunits
Authors: VAN DRIEL Ian R. , GODING James W.
Published: March 2005

The N‐linked glycosylation of the murine receptor for transferrin has been investigated. Previously we have found that purified receptors appear as two bands after sodium dodecyl sulfate/polyacrylamide gel electrophoresis and Coomassie blue staining [van Driel, I. R., Stearne, P. A., Grego, B., Simpson, R. J. and Goding, J. W. (1984) J. Immunol. 133, 3220–3224]. In the current report we show that the two bands are due to different glycosylation of individual receptor molecules. The receptors have three asparagines to which N‐linked glycans can be added, but only two sites are glycosylated in all receptors. The level of glycosylation of the third site varies depending on cell line or tissue. Limited endoglycosidase digestion of mature receptors indicates that differential glycosylation probably occurs at only one particular asparagine residue. Possible mechanisms that could result in such a glycosylation pattern are discussed.
Regulation of gene expression in Tetrahymena pyriformis under heat‐shock and during recovery
Authors: GALEGO Lisete , RODRIGUES‐POUSADA Claudina
Published: March 2005

Proteins extracted from Tetrahymena pyriformis cells, which were shifted from 28°C to 34°C (heat‐shocked) and then labelled with [35S]‐methionine, were analysed by two‐dimensional gel electrophoresis according to the method of O'Farrell. In the fluorogram obtained after 2.5 min heat‐shock new polypeptide spots already appear in the region of 70–75 kDa and 25–29 kDa, indicating that the induction response is very rapidly triggered. Other newly induced proteins become more intense after 15 min and 30 min of heat‐shock. After a 60‐min treatment, the electrophoretic analysis shows the normal pattern of proteins resembling that of exponentially growing cells. Our experiments in vivo and in vitro also show that the messengers coding for stress proteins have a short lifetime, suggesting that modulation of the translation efficiency of heat‐shock protein mRNAs is accompanied by an alteration in their biological stability.

Studies using the inhibitor actinomycin D suggest that control of this response is exercise mainly at the transcriptional level for the 70–75‐kDa and at the translational level for the 25–29‐kDa heat‐shock proteins.

When T. pyriformis cells are allowed to recover at 28°C the heat‐shock proteins disappear completely and normal protein synthesis is completely re‐established. Although the level of normal protein synthesis attained is lower, cells are able to recover completely from heat‐shock when new mRNA synthesis is blocked by actinomycin D, indicating a conservation of normal pre‐existing mRNAs during heat‐shock.
Molecular cloning and analysis of cDNA sequences for two ribosomal proteins from Artemia
Authors: MAASSEN J. Antonie , SCHOP Enno N. , BRANDS Jolanda H. G. M. , VAN HEMERT Formijn J. , LENSTRA Johannes A. , MÖLLER Wim
Published: March 2005

The large subunit of eukaryotic ribosomes contains acidic phosphoproteins which are related to L7/L12 from Escherichia coli. In the brine shrimp Artemia these proteins are designated eL12 and eL12′. We have isolated cDNA clones for these proteins from a cDNA bank that was constructed by the use of size‐fractionated poly(A)‐rich RNA (8–10S fraction) from Artemia and a synthetic oligonucleotide as primer. Clones containing DNA sequences coding for eL12 and eL12′ were characterized by hybrid‐selected translation and DNA sequencing. The proteins eL12 and eL12′ share an identical peptide of 22 amino acids at their carboxy termini whereas the remaining part of the protein shows little sequence homology. The nucleotide sequences show a different codon use for the amino acids in the common carboxy terminus, thereby excluding a common exon coding for this part of both proteins. Despite the differences in amino acid sequence in the major part of eL 12 and eL 12′ the proteins have a considerable degree of homology on the basis of the distribution of hydrophobic and hydrophilic amino acids over the polypeptide chains, in agreement with a related folding and function of both proteins.

Relative levels of mRNA coding for eL12, eL12′ and elongation factor 1α were determined during the development of Artemia from a dormant cyst to a nauplius. The data show a coordinate expression of the genes for EF‐1 α and both ribosomal proteins, excluding a differential expression of the genes for these related ribosomal proteins during embryogenesis.

Analysis of the gene copy number for eL 12 and eL 12′ indicates the presence of a few genes for each protein.
The phosphoenolpyruvate‐dependent fructose‐specific phosphotransferase system in Rhodopseudomonas sphaeroides
Authors: LOLKEMA Juke S. , TEN HOEVE‐DUURKENS Ria H. , ROBILLARD George T.
Published: March 2005

The phosphoenolpyruvate‐dependent fructose‐specific phosphotransferase system in Rhodopseudomonas sphaeroides is a membrane‐bound complex of two enzymes, an integral membrane protein EII and a soluble factor SF. In media of high ionic strength the binding constant of SF to the membranes is 55 nM. Phosphorylation of SF, the first step in the reaction sequence, has no influence on the binding. The second step is the transfer of the phosphoryl group from SF to EII. The physical existence of both phosphorylated SF and EII is demonstrated and it is shown that the phosphoryl group is donated to the next species in the reaction sequence. The molecular mass of SF is 110 kDa. EII is almost completely extracted from the membrane as a dimer. The molecular mass of the monomer is 55 kDa. Both SF and EII possess thiols that are essential for catalysis. The thiol on EII is protected against inactivation by N‐ethylmaleimide in the phosphorylated state. Kinetic experiments show that the binding site for fructose on EII is induced by phosphorylation of EII (‘ping‐pong’ mechanism). The affinity constants of the phosphotransferase complex for phosphoenolpyruvate and fructose at infinite concentration of the other substrate are 25 μM and 8 μM, respectively. The fructose phosphorylation rate equation is given as a function of the concentration of the two enzymes and the two substrates.
Imino‐proton resonances of yeast tRNAPhe studied by two‐dimensional nuclear Overhauser enhancement spectroscopy
Authors: HEERSCHAP Arend , MELLEMA Jan‐Remt , JANSSEN Henny G. J. M. , WALTERS Johannes A. L. I. , HAASNOOT Cornelis A. G. , HILBERS Cornelis W.
Published: March 2005

Application of two‐dimensional nuclear Overhauser enhancement (NOE) spectroscopy to yeast tRNAPhe in H2O solution demonstrates that all imino‐proton resonances, related to the secondary structure, and nearly all imino proton resonances, originating from the tertiary structure, can be assigned efficiently by this method. The results corroborate the assignments of the imino‐proton resonances of this tRNA as established previously by one‐dimensional NOE experiments (only the assignment of base pairs G1· C72 and C2· G71 should be reversed). The advantages of two‐dimensional NOE spectroscopy over one‐dimensional NOE spectroscopy for the assignments of imino‐proton resonances and the structure elucidation of tRNA are illustrated and discussed. Furthermore, the use of non‐exchangeable proton resonances as probes of the molecular structure is explored.
Effect of single amino acid substitutions on the protease susceptibility of tryptophan synthase a subunit
Authors: OGASAHARA Kyoko , TSUNASAWA Susumu , SODA Yuji , YUTANI Katsuhide , SUGINO Yoshinobu
Published: March 2005

In order to explore the correlation between protease susceptibility and conformational stability of a protein, the proteolytic degradation by trypsin, subtilisin and pronase P of the wild‐type α subunit of tryptophan synthase from Escherichia coli and of its two mutant proteins was studied by measuring circular dichroism at 222 nm at various pH values at 37°C. The mutant proteins are substituted by Gln or Met in place of Glu at position 49. The single amino acid substitutions at position 49 significantly affected susceptibility of this protein to the three proteases. Dependence of protease susceptibility of the wild‐type and the two mutant proteins on pH was characteristic of each protein and similar for the three proteases. Comparison of the present results with the conformational stabilities of the three proteins previously measured shows that the order of resistance to the proteases among the three proteins coincides with the order of the values of unfolding Gibbs energy change, suggesting that protein degradation depends upon the conformational stability of a protein.
Prokaryotic triterpenoids
Authors: BISSERET Philippe , ZUNDEL Magali , ROHMER Michel
Published: March 2005

2β‐Methylhopanoids, a new series of triterpenoids was identified from two prokaryotes. 2β‐Methyldiplopterol was isolated from the methylotrophic bacterium Methylobacterium organophilum, and three different 2β‐methyl‐bacteriohopanepolyols from the cyanobacterium Nostoc muscorum. The structures of these compounds was deduced by direct comparison with 2β‐methyldiplopterol synthesized from 22‐hydroxyhopan‐3‐one.
Purification of and kinetic studies on a narrow specificity synaptosomal membrane pyroglutamate aminopeptidase from guinea‐pig brain
Authors: O'CONNOR Brendan , O'CUINN Gerard
Published: March 2005

A pyroglutamate aminopeptidase activity, distinct from that of cytoplasm, was released from a synaptosomal membrane preparation of guinea‐pig brain by papain treatment. This activity was further purified 3560‐fold relative to the homogenate with a yield of 17% by a procedure involving gel filtration chromatography, calcium phosphate cellulose chromatography and hydrophobic interaction chromatography on phenyl‐Sepharose CL‐4B. The purified synaptosomal pyroglutamate aminopeptidase hydrolysed only thyroliberin, acid‐thyroliberin, the luliberin N‐terminal tripeptide (Glp‐His‐Trp) and, only slightly, Glp‐His‐Gly. No hydrolysis was observed with dipeptides containing N‐terminal pyroglutamic acid (Glp) or with pyroglutamyl peptides containing more than three amino acids. A Km value of 40 μM was recorded when thyroliberin was used as substrate; however, luliberin was found to inhibit the hydrolysis of thyroliberin competitively with a Ki value of 20 μM.
Human red‐blood‐cell Ca2+‐antagonist binding sites
Authors: STRIESSNIG Jörg , ZERNIG Gerald , GLOSSMANN Hartmut
Published: March 2005

The human red blood cell ghost Ca2+‐antagonist binding sites were characterized with (±)‐[3H]nimodipine. The labelled 1,4‐dihydropyridine bound in a non‐cooperative, reversible manner with a Kd of 52 nM at 25°C to 9.65 pmol sites/mg ghost protein. The stereochemistry of the binding domain was evaluated with the optically pure enantiomers of chiral 1,4‐dihydropyridines. In contrast to the 1,4‐dihydropyridine‐selective receptors on Ca2+ channels in electrically excitable tissues, the (+) enantiomer of nimodipine and the (−) enantiomer of the benzoxadiazol 1,4‐dihydropyridine (PN 200–110) were bound with higher affinity than the respective optical antipodes. The human red blood cell ghost [3H]nimodipine‐labelled sites also interacted with the inorganic Ca2+‐antagonist La3+ (increase in the number of binding sites), and were allosterically regulated by the optical enantiomers of the phenylalkylamine‐type Ca2+‐antagonists (e.g. verapamil, desmethoxyverapamil, methoxyverapamil). The benzothiazepines d‐ or l‐cis‐diltiazem were without effect. Nucleosides (adenosine ∼ inosine > cytidine) were inhibitory at the nimodipine‐labelled site, as were the nucleoside uptake inhibitors dipyridamole, hexobendine, dilazep, nitrobenzylthioinosine and nitrobenzylthioguanosine. The binding sites have essential sulfhydryl groups, show trypsin sensitivity, but are relatively heat stable. When nitrobenzylthioinosine was employed as a covalent probe to inactivate the red blood cell ghost nucleoside carrier, [3H]nimodipine binding was irreversibly lost. (+)‐Nimodipine > (−)‐nimodipine inhibited [14C]adenosine transport into human red blood cells. A good correlation between IC50 values for inhibition of [3H]nimodipine binding and IC50 values for inhibition of [14C]adenosine uptake was found for 18 compounds. Sheep red blood cells (which lack the nucleoside transporter) had no detectable [3H]nimodipine binding sites. It is concluded that the Ca2+‐antagonist receptor sites of the human erythrocyte are coupled to the nucleoside transporter.
Investigation of the solution structure of a DNA octamer [d(GGAATTCC)]2 using two‐dimensional nuclear Overhauser enhancement spectroscopy
Authors: BROIDO Michelle S. , JAMES Thomas L. , ZON Gerald , KEEPERS Joe W.
Published: March 2005

Proton two‐dimensional nuclear Overhauser enhancement (2D NOE) spectra in the pure absorption phase were obtained at 500 MHz for [d(GGAATTCC)]2 in aqueous solution at a series of mixing times. The experimental data were analyzed by comparison with theoretical spectra calculated using the complete 70 × 70 relaxation matrix including all proton dipole‐dipole interactions and spin diffusion [Keepers, J. W. & James, T. L. (1984) J. Magn. Reson. 57, 404–426]. The theoretical spectra at each mixing time were calculated using two structures: a standard B‐form DNA structure and an energy‐minimized structure based on the similarity of the six internal residues of the title octamer with those of the dodecamer [d(CGCGAATTCGCG)]2, for which the crystal structure has been determined. Neither the standard B‐form nor the energy‐minimized structure will yield theoretical 2D NOE spectra which accurately reproduce all peak intensities in the experimental spectra. However, many features of the experimental spectra can be represented by both the B‐form and the energy‐minimized structure. Sequence‐dependent structural characteristics are manifest in the 2D NOE spectra, in particular at the purine‐pyrimidine junction as noted previously in the crystal structure. On the whole, the energy‐minimized structure appears to yield theoretical 2D NOE spectra which mimic many, if not all, aspects of the experimental spectra. All 2D NOE data were consistent with nanosecond correction times as implied by proton spin‐lattice relaxation time measurements. But better fits of some of the 2D NOE data using small variations in an effective isotropic correlation time suggest that there may be some local variations in mobility within the octamer duplex structure in solution.
Inhibition of substance P degradation in rat brain preparations by peptide hydroxamic acids
Authors: LAUFER Ralph , EWENSON Ariel , GILON Chaim , CHOREV Michael , SELINGER Zvi
Published: March 2005

A peptidase activity of rat diencephalon membranes, which acts on the C‐terminal hexapeptide sequence of substance P, was characterized using the radiolabeled substrate Nα‐([125I]iododesaminotyrosyl)‐substance P (6–11)‐hexapeptide. This activity presents certain characteristics similar to those of the substance‐P‐degrading enzyme purified from human brain by Lee et al. [Eur. J. Biochem. 114, 315–327 (1981)]. It is inhibited by metal chelators and some thiol reagents, but is insensitive to inhibitors of serine proteases and aminopeptidases. The activity is different from angiotensin‐converting enzyme and enkephalinase, since it is not affected by specific inhibitors of these enzymes. Substance P and substance P C‐terminal fragments longer than the pentapeptide inhibited the degradation of the radiolabeled substrate with inhibition constants around 200 μM. Short fragments of the substance P sequence, such as Boc‐Phe‐Phe‐OMe and Boc‐Phe‐Phe‐Gly‐OEt, were also found to inhibit the degradation of the substrate. When the metal‐chelating hydroxamic acid moiety was attached to the carboxyl terminus of these short peptides, potent inhibitors of the substance‐P‐degrading activity were obtained, with inhibition constants in the micromolar range. The most potent of these compounds, iododesaminotyrosyl‐Phe‐Phe‐Gly‐NHOH (IBH‐Phe‐Phe‐Gly‐NHOH), is a competitive inhibitor, with a Ki value of 1.9 μM. The degradation of substance P by rat diencephalon slices was inhibited to the same extent (40–50%) by IBH‐Phe‐Phe‐Gly‐NHOH (20 μM) and by phosphoramidon (1 μM). A combination of both reagents reduced the degradation rate by 75–80%, suggesting that both enkephalinase and the substance‐P‐degrading activity are involved in the metabolism of substance P in this preparation. IBH‐Phe‐Phe‐Gly‐NHOH seems to be quite specific for the latter enzyme, since at a high concentration (0.1 mM) it did not affect the degradation of the radiolabeled substrate by α‐chymotrypsin, papain, or thermolysin.
Kinetic studies on reduction of cytochromes P‐450 and b5 by dithionite
Authors: DAVYDOV Dmitry R. , KARYAKIN Alexander V. , BINAS Bert , KURGANOV Boris I. , ARCHAKOV Alexander I.
Published: March 2005

The kinetics of reduction of cytochromes P‐450 and b5 by dithionite had been studied in solution and in microsomal and proteoliposomal membranes by the stopped‐flow technique. In all the cases studied the kinetic curves of reduction of cytochrome b5 obey first‐order kinetics in relation to cytochrome with the rate constant about 14s−1 at 10.6 mM dithionite. The kinetic curves of reduction of cytochrome P‐450 fit an equation for the sum of two exponentials with the parameters varying from system to system. The simple first‐order kinetics of cytochrome P‐450 reduction had been observed in the presence of non‐ionic detergent Triton N‐101. The apparent biphasity of cytochrome P‐450 reduction by dithionite should be the result asymmetric distribution of hemoprotein in microsomal and proteoliposomal membranes as well as in cytochrome oligomers in solution.
Embryonic and adult forms of two galactosyltransferases differ in their degrees of sialylation
Authors: FURUKAWA Kiyoshi , ROTH Stephen
Published: March 2005

Embryonic and adult chicken liver galactosyltransferases behave differently on DEAE‐Sepharose chromatography. After solubilization, two embryonic activities (one transfers galactose in a β1–4 linkage to asialo‐aglactoα1‐acid glycoprotein and to N‐acetylglucosamine; the other transfers galactose in a β1–3 linkage to asialo‐ovine submaxillary mucin) elute after the bulk of the protein, and after free glucose. The same two enzymes in adults elute more rapidly, almost coincident with the bulk of the protein, and before free glucose. The difference in elution patterns occurs when the column buffer contains 0.1 M NaCl. Without salt, both embryonic and adult transferases bind to the column, but with 0.5 M NaCl, the embryonic and adult transferases elute identically, and with the bulk of the protein. After treatment with neuraminidase, the embryonic transferase activities elute significantly earlier on a DEAE‐Sepharose column in the presence of 0.1 M NaCl. The embryonic forms migrate more rapidly than do the adult forms on cellulose acetalte electrophoresis, but neuraminidase treatment renders both enzyme forms immobile in this system. Neuraminidase treatment also inhibits the binding of the embryonic transferases to a wheat‐germ‐agglutinin–Sepharose column. Kinetically, the embryonic and adult transferases are indistinguishable.
Mechanism of activation of bovine kidney pyruvate dehydrogenase a kinase by malonyl‐CoA and enzyme‐catalyzed decarboxylation of malonyl‐CoA
Authors: RAHMATULLAH Mohammed , ROCHE Thomas E. , JILKA Joseph M. , KAZEMI Mohammad
Published: March 2005

The activity of the pyruvate dhydrogenase kinase, which phosphorylates and therby inactivates the pyruvate dehydrogenase complex, was stimulated by malonyl‐CoA. Treatment with [2‐14C]malonyl‐CoA resulted in acylation of sites in the complex. Both acylation and activation of kinase activity increased in a time‐dependent manner with a parallel increase in those activities when the malonul‐CoA:CoA ratio was varied. Protein‐bound acyl groups were labilized by performic acid treatment indicating their attachment to protein at thiol residues; however, the product released was volatile, which is not characteristic of malonic acid. While malonyl‐CoA was initially free of acetyl‐CoA, stimulation of kinase activity and acylation of sites in the complex by malonyl‐CoA were shown to be contingent upon enzyme‐catalyzed decarboxylation. Decarboxylation appeared to be catlayzed by a trace contaminant present in highly purified preparations of both the pyruvate and 2‐oxoglutarate dehydrogenase complexes. Under conditions in which both free CoA was removed (by conversion to succinyl‐CoA) and then, after various periods, free acetyl‐CoA was removed (by enzymic conversion to acetyl phosphate), both aceylation of sites in the complex and activation of kinase activity increased in a time‐dependent manner. Concomitantly there was a decrease in the concentration dependence for activation of the kinase by malonyl‐CoA. Our results strongly support the conclusion that activation of kinase activity is associated with acylation of sites in the complex, and that, in the case of malonyl‐CoA, those processes depend on enzyme‐catalyzed decarboxyzed decarboxylation.
Hepatic carbamoyl phosphate metabolism
Authors: PAUSCH Jürgen , RASENACK Jens , HÄUSSINGER Dieter , GEROK Wolfgang
Published: March 2005

1. The interrelationship between the two carbamoyl phosphate pools in intact hepatocytes and intact liver was studied with respect to de novo pyrimidine synthesis by use of selective inhibitors of the mitochonodiral and the cytosolic carbamoyl‐phosphate synthetase.

2. Inhibition of mitochondrial carbamoyl phosphate synthesis by 4‐pentenoate was without effect on galactosamine‐stimulated pyrimidine synthesis. Conditions favouring mitochondrial carbamoyl phosphate accumulation, like excess ammonium ions or L‐norvaline, led to an increase in pyrimidine synthesis bypassing the feedback inhibition of cytosolic carbamoyl‐phosphate synthetase by UTP.

3. A stimulation of pyrimidine synthesis was not observed when the carbamoyl phosphate accumulation was due to aspartate deficiency in the presence of aminooxyacetate. The full response of pyrimidine synthesis to excess ammonium ions was restored, even in the presence of aminooxyacetate, when aspartate was substituted. This is explained by an inhibition of aspartate carbamoyltransferase flux [in view of the Km (aspartate = 0.7 mmol/1) of this enzyme] resulting from a 90% decrease in aspartate tissue levels.

4. Acivicin, the inhibitor of cytosolic carbamoyl‐phosphate synthetase, completely abolished the galactosamine‐induced stimulation of pyrimidine synthesis, but was without effect on the stimulation of pyrimidine synthesis by ammonium ions and L‐norvaline.

5. It is concluded that experimental changes in mitochondrial carbamoyl phosphate content exert effects on de novo pyrimidine synthesis; however, it is considered unlikely that relevant amounts of mitochondrial carbamoyl phosphate are used for pyrimidine synthesis under physiological conditions. In addition the data point to a potential regulatory role of aspartate in hepatic pyrimidine synthesis.
Cholecalcin (a 9‐kDa cholecalciferol‐induced calcium‐binding protein) messenger RNA
Authors: PERRET Christine , DESPLAN Claude , THOMASSET Monique
Published: March 2005

In view of the possible physiological importance of the 9‐kDa cholecalcin (a 9000‐Mr cholecalciferol‐induced calcium‐binding protein) in the intestinal transport of calcium in mammals, the gene expression of this protein has been analysed. Its regulation in the digestive tract of the growing rat by calcitriol (1,25‐dihydroxycholecalciferol) was studied using a specific cloned [32P]cDNA to 9‐kDa cholecalcin. Northern hybridisation studies show that the cDNA sequence hybridises to a single 500–600‐nucleotide species throughout the digestive tract and therefore demonstrate identical 9‐kDa‐cholecalin mRNA processing in the whole of the intestine and caecum. The highest concentrations of cholecalcin mRNA occur in the duodenum, proximal jejunum and caecum. The observed differences in 9‐kDa‐cholecalcin mRNA levels correlate well with both the in vivo variations in cholecalcin itself and with the known intestinal sites of calcium absorption. The whole intestine is able to respond to exogenous calcitriol but the response of the distal intestine and caecum, as measured by the increase in cholecalcin mRNA and corresponding protein, was proportionally higher than in the duodenum. The rapid production of fully functional cholecalcin mRNA, which was detectable as early as 1 h after a single dose of calcitriol to vitamin‐D‐deficient rats, provides convincing evidence that calcitriol increases 9‐kDa cholecalcin production by increasing cholecalcin gene expression at the transcriptional level.
Activation of protein kinase isozymes by cyclic nucleotide analogs used singly or in combination
Authors: ØGREID Dagfinn , EKANGER Roald , SUVA Robert H. , MILLER Jon P. , STURM Priscilla , CORBIN Jackie D. , DØSKELAND Stein Ove
Published: March 2005

104 cAMP analogs, most of them modified in the adenine moiety, were tested as activators of cAMP‐dependent protein kinase I (from rabbit or rat skeletal muscle) and kinase II (from bovine heart or rat skeletal muscle).

When tested singly, only 2‐phenyl‐1, N6‐etheno‐cAMP showed a considerably (sevenfold) higher potency as an activator of kinase II than of kinase I. Analogs containing an 8‐amino modification preferentially activated kinase I, some being more than 10‐fold more potent as activators of kinase I than kinase II.

When two analogs were combined, the concentration of one (complementary) analog required to half‐maximally activate each isozyme was determined in the presence of a fixed concentration of another (priming) analog. Analogs tested in combination had been analyzed for their affinity for the intrasubunit binding sites (A, B) of isozyme I and II. The degree to which complementary analogs preferentially activated one isozyme was plotted against the mean site selectivity, i.e. (affinity A/B isozyme I · affinity A/B isozyme II)1 /2. This plot produced a straight line, the slope of which reflected the ability of the priming analog to discriminate homologous sites on the isozymes. This means that the isozyme discriminating power of an analog pair can be quantitatively predicted from the affinity of the analogs for site A and B of the two enzymes. It also means that a systematic analysis of those features of analogs imparting a high mean site selectivity or the ability to discriminate between homologous isozyme sites will facilitate the synthesis of new even more isozyme‐selective analogs.
Interaction between glucocorticoids, 8‐bromoadenosine 3′,5′‐monophosphate, and insulin in regulation of synthesis of carbamoyl‐phosphate synthetase I in Reuber hepatoma H‐35
Authors: KITAGAWA Yasuo , SUGIMOTO Etsuro
Published: March 2005

Regulation of synthesis of carbamoyl‐phosphate synthetase I by glococorticoids, 8‐bromoadenosine 3′,5′‐monophosphate (8‐bromo‐cAMP), and insulin was investigated in Reuber hepatoma H‐35. By measuring the incorporation of [35S]methionine into carbamoyl‐phosphate synthetase I and its precursor, we showed that dexamethasone stimulates the enzyme synthesis approximately fivefold. A detectable stimulation was observed at 1 nM of dexamethasone, half‐maximal stimulation at 4 nM, and maximal stimulation above 40 nM. Corticosterone was more effective than dexamethasone both for the minimal concentration needed and for the extent of the stimulation. Hydrocortisone was less effective than dexamethasone. 8‐Bromo‐cAMP also stimulated the enzyme synthesis at a concentration of 3 mM. The effect of 8‐bromo‐cAMP was suggested to be additive to the effect of dexamethasone. Physiological concentrations of insulin strongly supressed the stimulatory effect of dexamethasone on the enzyme synthesis but could not completely counteract the effect of dexamethasone. The half‐maximal and maximal effects of insulin were observed at 0.5 nM and 5 nM, respectively. Insuln also counteracted the effect of 8‐bromo‐cAMP on the enzyme synthesis.
Differentiation of rat myoblasts
Authors: JACOBS Frederik A. , BIRD R. Curtis , SELLS Bruce H.
Published: March 2005

The reulation of ribosomal proteins (r‐proteins) and their mRNAs (rp‐mRNAs) was studied in the L6 myoblast, a mammalian cell line which can undergo myogenesis. Upon terminal differentiation, the rate of accumulation of mature ribosomes dropped to approximately 25% of the rate found in undifferentiated myoblasts. Despite the drop in the rate of ribosome accumulation and the rate of rRNA synthesis following terminal differentiation, the rate of r‐protein synthesis remained constant. The excess r‐protein synthesized in myotubes was quickly degraded. The levels of rp‐mRNAs were assessed before and after differentiation. Over 90% of the rp‐mRNAs were found on polysomes in both myoblasts and myotubes and represented similar fractions of total poly(A)‐rich mRNA. The half‐lives of the rp‐mRNAs averaged approximately 11 h in both myoblasts and myotubes. In vitro nuclear transcription measurements of a representative rp‐mRNA (L32 mRNA) revealed that following differentiation, its rate of synthesis relative to the overall transcription rate dropped by approximately 26% in myotubes while the rate of transcription of rRNA dropped by approximately 77%. These results indicate that the coordination of r‐protein and rRNA synthesis observed in myoblasts was uncoupled in myotubes at the level of transcription.
Binding of histidinal to histidinol dehydrogenase
Authors: GÖRISH Helmut , HÖLKE Werner
Published: March 2005

One molecule of the enzymatic intermediate histidinal is firmly bound per subunit of histidinol dehydrogenase (EC 1.1.1.23) and protected against decomposition. The dissociation rate constant of the histidinal–histidinol dehydrogenase complex is estimated as 2.5 × 10–5 S–1. Steady‐state kinetic measurements studying the oxidation of histidinol to histidine and the reduction of histidinal to histidinol allow to calculate the association rate constants for histidinal. For both reactions the association rate constant is found as 1.9 × 106 M–1 S–1. Thus the dissociation constant of the histidinal–histidinol dehydrogenase complex is estimated to be of the order of 1.4 × 10–11 M.
Steady‐state and time‐resolved fluorescence anisotropy study of phospholipid molecular motion in the gel phase using 1‐palmitoyl‐2[9‐(2‐anthryl)‐nonanoyl]‐sn‐glycero‐3‐phosphocholine as probe
Authors: VINCENT Michel , GALLAY Jacques , de BONY Jacqueline , TOCANNE Jean‐François
Published: March 2005

1‐Palmitoyl‐2‐[9‐(2‐anthryl)‐nonanoyl]‐sn‐glycero‐3‐phosphocholine (Anthr‐PC), a non‐perturbing phospholipid probe [de Bony, J. and Tocane, J. F. (1983) Chem. Phys. Lipids 32, 105–121], has been designed in order to obtain insight into the membrane lipid organization at a ‘microscopic’ level, in terms of lateral distribution both in model and in natural membranes [de Bony, J. et al. (1984) Eur. J. Biochem. 143, 373–379; FEBS Lett. 174, 1–6]. In the present study, the molecular motions of this new fluorescent probe embedded in a lipid matrix have been investigated by fluorescence anisotropy techniques in steady‐state and time‐resolved modes. The results indicate that long axis rotation, monitored by the out‐of‐plane mode of rotation of the fluorophore, is fast even in the phospholipid gel state. It is moderately sensitive to the phase transition. The data suggest that this rotation is anisotropic. Cholesterol exhibits little effect on this rotation. The rotation of the long axis itself is sensitive to the transition. It is hindered as inferred from measurements at wavelength where both the in‐plane and out‐of‐plane motions contribute to the depolarization of the emitted fluorescence light. Cholesterol restricts this motion. The behaviour of the free 9‐(2‐anthryl)‐nonanoic acid is not significantly different from that of Anthr‐PC. These results are discussed with respect to the influence of orientational constraints on the photodimerization process when this lipid probe is used to monitor phospholipid lateral distribution.
Evidence for two oxygen‐linked binding sites for polyanions in dromedary hemoglobin
Authors: AMICONI Gino , BERTOLLINI Alberto , BELLELLI Andrea , COLETTA Massimo , CONDÒ Saverio G. , BRUNORI Maurizio
Published: March 2005

The functional properties of dromedary hemoglobin have been studied as a function of chloride, polyphosphates and pH and compared with those of human hemoglobin. The two proteins have the same amino acid residues at the anion‐binding sites as well as at the level of the groups responsible for the alkaline Bohr effect. Analysis of the experimental data reveals that: (a) intrinsic oxygen affinity and the Bohr effect are very similar for the two proteins; (b) the association equilibrium constants of chloride are substantially higher in the dromedary system, both in the unligated and ligated state; (c) two polyanion‐binding sites occur in dromedary oxy and deoxyhemoglobin; (d) association constants of polyphosphates for the higher‐affinity binding site (probably in the cavity between β chains) are comparable for the two proteins under physiological conditions; association constants for the second binding site in dromedary hemoglobin are not affected by pH changes; (e) the dependence of oxygen affinity in dromedary hemoglobin upon chloride concentration is complex, this anion at relatively low concentrations reverses the effect of millimolar polyphosphate; (f) both in stopped‐flow and flash photolysis experiments the kinetic behaviour of dromedary hemoglobin is consistent with the equilibrium results.

The pronounced sensitivity to solvent composition of the functional properties of dromedary hemoglobin even in the oxy state stresses the potential relevance of this conformation for regulating the oxygen transport in vivo.
Insulin receptors and bioresponses in a human liver cell line (Hep G‐2)
Authors: PODSKALNY Judith M. , TAKEDA Shigemasa , SILVERMAN Robert E. , TRAN Dien , CARPENTIER Jean‐Louis , ORCI Lelio , GORDEN Phillip
Published: March 2005

A newly developed human hepatoma cell line, designated Hep G‐2, expresses high‐affinity insulin receptors meeting all the expected criteria for classic insulin receptors. 125I‐insulin binding is time‐dependent and temperature‐dependent and unlabeled insulin competes for the labeled hormone with a half‐maximal displacement of 1–3 ng/ml. This indicates a Kd of about 10−10 M. Since Scatchard analysis of the binding data results in a curvilinear plot and unlabeled insulin accelerates the dissociation of bound hormone, these receptors exhibit the negative cooperative interactions characteristic of insulin receptors in many other cell and tissue types. Proinsulin and des(Ala, Asp)‐insulin compete for 125I‐insulin binding with 4% and 2%, respectively, of the potency of insulin. Anti‐(insulin receptor) antibody competes fully for insulin binding. The two insulin‐like growth factors, multiplication‐stimulating activity and IGF‐I are 2% as potent as insulin against the Hep G‐2 insulin receptor. Furthermore, Hep G‐2 cells respond to insulin in several bioassays. Glucose uptake, glycogen synthase, uridine incorporation into RNA and acetate incorporation into lipid are all stimulated to varying degrees by physiological concentrations of insulin. In addition, these cells ‘down‐regulate’ their insulin receptor, internalize 125I‐insulin and degrade insulin in a manner similar to freshly isolated rodent hepatocytes.

This is the first available human liver cell line in permanent culture in which both insulin receptors and biological responses have been carefully examined.
Novel phosphonoglycosphingolipids containing pyruvylated galactose from the nervous system of Aplysia kurodai
Authors: ARAKI Shigeko , ABE Sachiko , SATAKE Mei , HAYASHI Akira , KON Kazuo , ANDO Susumu
Published: March 2005

We have reported the existence of a phosphonoglycosphingolipid containing a pyruvylated galactose, FGL‐IIb, in nerve fibers of Aplysia kurodai (Araki, S., Abe, S., Ando, S., Kon, K., Fujiwara, N. & Satake, M. (1989) J. Biol. Chem. 264, 19 922–19 927).

We have now isolated two other pyruvylated galactose‐containing phosphonoglycosphingolipids, named FGL‐V and FGL‐IIa, from the nervous tissue of Aplysia, and characterized them as [3,4‐O‐(S‐1‐carboxy‐ethylidene)]Galβ1 → 3GalNAcα1 → 3[6′‐O‐(2‐aminoethylphosphonyl)Galα1 → 2] (2‐aminoethylphosphonyl → 6) Galβ1 → 4Glcβ1 → 1 ceramide and [3,4,O‐(S‐1‐carboxyethylidene)]Galβ1 → 3GalNAc α1 → 3[6′‐O‐(2‐aminoethylphosphonyl)Galα1 → 2]Galβ1 → 4Glcβ11 → ceramide, respectively.

Their major aliphatic components are palmitic acid, octadeca‐4‐sphingenine and anteisononadeca‐4‐sphingenine.

Thus, the nervous system of Aplysia contains several pyruvylated phosphonoglycolipids.
Clayey injection veins and pseudotachylyte from two boreholes penetrating the Chelungpu Fault, Taiwan: Their implications for the contrastive seismic slip behaviors during the 1999 Chi‐Chi earthquake
Authors: Otsuki Kenshiro , Uduki Takayuki , Monzawa Nobuaki , Tanaka Hidemi
Published: March 2005

Abstract  The seismic slip that occurred during the 1999 Chi‐Chi earthquake in Taiwan showed contrastive behaviors in different regions along the Chelungpu Fault: A large and smooth slip occurred in the north, while a relatively small slip associated with high‐frequency seismic wave radiation occurred in the south. The core samples from shallow boreholes at northern (Fengyuan) and southern (Nantou) sites penetrating the seismic Chelungpu Fault were analyzed. The fault zones at the northern site are characterized by soft clayey material associated with clayey injection veins. This suggests that the fault zones were pressurized during ancient seismic slip events, and hence the elastohydrodynamic lubrication occurred effectively. In contrast, the fault rock from the southern site is old pseudotachylyte that has been shattered by repeated ancient seismic slip events. Statistical analysis of many pseudotachylyte fragments reveals that the degree of frictional melting tended to be low. In this case, the seismic slip is restrained by the mechanical barrier of a highly viscous melt layer. These contrastive fault rocks were produced by repeated ancient seismic slip events, but the two corresponding mechanisms of friction are likely to have also occurred during the 1999 Chi‐Chi earthquake, thus causing the contrastive slip behaviors in the north and south.
Malodour in denture wearers: an ill‐defined problem
Authors: Verran J
Published: March 2005

Denture plaque has not been studied to the same extent as dental plaque, and although there are many similarities in microbial composition, there are some significant differences. Denture‐induced stomatitis is associated with poor denture hygiene, a more acidogenic plaque and the presence of Candida albicans. Obligate Gram‐negative anaerobic micro‐organisms, although present in denture plaque, have rarely been specifically investigated. Opportunist pathogens including coliforms and staphylococci have been isolated from dentures. Teeth adjacent to partial dentures are more susceptible to caries and periodontal diseases, perhaps due to an increased plaque buildup at the prosthesis/tooth interface. Little work has been published on malodour associated with dentures. The inert material provides a substratum for the plaque biofilm, which encompasses a range of odour‐producing species. The microbiology of the tongue in denture wearers has not been specifically studied. Thus the nature, origin and extent of malodour in denture wearers is ill‐defined, but many species capable of producing malodorous compounds are present. The wide age and health range presented by denture wearers further confounds investigation. There is a need for further work in the area, both for cosmetic‐ and health‐associated reasons in the increasing elderly population.
The rationale and potential for the reduction of oral malodour using Streptococcus salivarius probiotics
Authors: Burton JP , Chilcott CN , Tagg JR
Published: March 2005

The primary treatment for oral malodour is the reduction of bacterial populations, especially those present on the tongue, by use of a variety of antimicrobial agents or mechanical devices. However, shortly after treatment the problematic bacteria quickly repopulate the tongue and the malodour returns. In our studies, we have used a broadly‐active antimicrobial (chlorhexidine) to effect temporary depletion of the oral microbiota and then have attempted to repopulate the tongue surface with Streptococcus salivarius K12, a benign commensal probiotic. The objective of this is to prevent re‐establishment of non‐desirable bacterial populations and thus help limit the re‐occurrence of oral malodour over a prolonged period. In this paper, we discuss why contemporary probiotics are inadequate for treatment of oral malodour and examine the rationale for selection of particular bacterial species for future use in the treatment of this condition. In our preliminary trials of the use of a chlorhexidine rinse followed by strain K12 lozenges, the majority (8/13) of subjects with confirmed halitosis maintained reduced breath levels of volatile sulphur compounds for at least 2 weeks. We conclude that probiotic bacterial strains originally sourced from the indigenous oral microbiotas of healthy humans may have potential application as adjuncts for the prevention and treatment of halitosis.
Pilot study of a breath test for volatile organic compounds associated with oral malodor: evidence for the role of oxidative stress
Authors: Phillips M , Cataneo RN , Greenberg J , Munawar MI , Nachnani S , Samtani S
Published: March 2005

Background:  We performed a pilot study of a new method to identify the volatile organic compounds (VOCs) in breath associated with oral malodor, using gas chromatography and mass spectroscopy (GC/MS).

Methods:  Oral cavity breath was collected from seven patients with oral malodor. Breath samples (150 ml) were concentrated onto sorbent traps and analyzed by GC/MS.

Results:  Organoleptic scores ranged from 3.0 to 4.0 (mean = 3.3) on a scale of 0–5. Twenty‐four of 30 (80.0%) of the most abundant oral malodor volatile organic compounds (OMVOCs) were alkanes and methylated alkanes. These VOCs are products of oxidative stress, generated by lipid peroxidation of polyunsaturated fatty acids in cell membranes.

Conclusions:  Increased oxidative stress in the oral cavity of patients with oral malodor may account for the increased risk of atherosclerosis, coronary heart disease and stroke associated with periodontal disease. The breath test for OMVOCs could potentially provide an objective new test for the assessment of oral malodor.
Quantitative detection of volatile sulfur compound‐ producing microorganisms in oral specimens using real‐time PCR
Authors: Kato H , Yoshida A , Awano S , Ansai T , Takehara T
Published: March 2005

Objective:  It is well‐known that some periodontopathic bacteria, especially Porphyromonas gingivalis, Fusobacterium nucleatum, Tannerella forsythia (formerly Bacteroides forsythus or Tan. forsythensis), and Treponema denticola, actively produce volatile sulfur compounds (VSCs), such as H2S and CH3SH. We previously reported a qualitative relationship between periodontopathic bacteria and VSCs; however, a quantitative analysis of periodontopathic bacteria in oral specimens is required for further characterization of the relationship between oral bacteria and VSCs. In this study, we report a real‐time polymerase chain reaction (PCR) method for the quantitative analysis of VSC‐producing bacteria in oral specimens.

Subjects and methods:  Specimens were collected from 22 patients who visited the Preventive Dentistry and Breath Odor Clinic of Kyushu Dental College. A real‐time PCR assay using the TaqMan system, based on the 5′–3′ exonuclease activity of Taq polymerase, was employed for the quantitative analysis of periodontopathic bacteria that produce VSCs.

Results:  Using real‐time PCR, we performed a quantitative analysis of P. gingivalis, F. nucleatum, Tan. forsythia, and T. denticola in the saliva, on the tongue coat, and in the subgingival plaque of patients with oral malodor.

Conclusion:  Real‐time PCR using the TaqMan system can be used for the quantitative analysis of VSC‐producing oral bacteria.
P25  Clinical assessment of oral malodor intensity expressed as absolute value using electronic nose
Authors: Nonaka A , Tanaka M , Anguri H , Nagata H , Kita J , Shizukuishi S
Published: March 2005

We have previously reported that the score as determined under top‐note mode with an electronic nose provides objective halitosis‐related measurements, but those obtained by the multiple linear regression method (EM‐MLR) show only relative and not absolute values. The purpose of this investigation was to assess clinically oral malodor intensity expressed as an absolute value using an electronic nose. Sixty‐six patients who complained of oral malodor participated. Malodor intensity (EN‐MI) calculated by totaling the contributions based on the measurement of multiple category gases and EM‐MLR were measured by analyzing mouth air samples with an electronic nose system (FF‐1, Shimadzu). Oral malodor was also assessed by an organoleptic test and volatile sulfur compound levels (VSC) using gas chromatography. Probing depth (PD), tongue coating score (TCS), and plaque control record (PCR) were used as oral health parameters. Receiver operating characteristics (ROC) procedures were applied to analyze the diagnostic accuracy of EM‐MI using an organoleptic test as a gold standard. The area under the ROC plots for EN‐MI (0.975) was significantly larger than that for logVSC (0.896) (P = 0.036), but did not differ significantly from that for EN‐MLR (0.932). In logistic regression analyses of the possibility of subjects being in the upper 25th percentile of EN‐MI distribution as a dependent variable, percentage of teeth with PD.4 mm, TCS, and PCR were each significantly associated with EN‐MI and respective odds ratios were 13.0, 7.1, and 28.2. These results suggest that oral malodor intensity expressed as an absolute value using the electronic nose may be useful for evaluating clinically oral malodor.