bluuebunny/crossref_metadata_2025 huggingface dataset:
Contribution to first‐pass metabolism of ethanol and inhibition by ethanol for retinol oxidation in human alcohol dehydrogenase family
Authors: Han Chih‐Li , Liao Chin‐Shya , Wu Chew‐Wun , Hwong Ching‐Long , Lee An‐Rong , Yin Shih‐Jiun
Published: March 2003

The alcohol dehydrogenase (ADH) family is involved in the metabolism of both ethanol and retinoids. To quantitatively assess the potential contributions to first‐pass metabolism of ethanol and the ethanol interference with retinoid homeostasis, saturation kinetics for ethanol oxidation as well as inhibition kinetics by ethanol for all‐trans‐retinol oxidation of human class I αα, β1β1, β2β2, γ1γ1, class II ππ, class III χχ, and class IV μμ were evaluated and compared. Class I and class II ADHs exhibited substrate inhibition with inhibition constants ranging over 250−720 mM (except γ1γ1) ethanol. Class IV ADH displayed no appreciable inhibition up to 1 M ethanol. Activity of the class III enzyme (190 nM subunit) was undetectable at 250 mM ethanol. The kinetic simulations indicate that the hepatic ππ and the gastric μμ can most effectively contribute to first‐pass metabolism of alcohol. The Michaelis constant (Km), turnover number (kcat), and catalytic efficiency (kcat/Km) for retinol oxidation relative to that for ethanol oxidation in class I, class II, and class IV ADHs ranged over 0.00022−1.3, 0.071−0.48, and 0.24−650, respectively. Ethanol was a competitive inhibitor against retinol for class I, II, and IV ADHs with apparent inhibition constants ranging over 0.037−11 mM, indicating that retinoic acid synthesis through the ADH pathways can be tremendously blocked during social/heavy drinking. These findings support the notion that first‐pass metabolism of alcohol may occur mainly in the liver through class II ππ and that cellular retinoid signaling may be perturbed by ethanol via ADH pathways.
Effect of dexamethasone on interleukin‐1β‐(IL‐1β)‐induced nuclear factor‐κB (NF‐κB) and κB‐dependent transcription in epithelial cells
Authors: Newton Robert , Hart Lorraine A. , Stevens David A. , Bergmann Martin , Donnelly Louise E. , Adcock Ian M. , Barnes Peter J.
Published: March 2003

The production of inflammatory mediators by epithelial cells in inflammatory lung diseases may represent an important target for the anti‐inflammatory effects of glucocorticoids. Nuclear factor‐κB (NF‐κB) is a major activator of inflammatory genes and has been proposed as a target for inhibition by glucocorticoids. We have used human pulmonary type‐II A549 and airway epithelial BEAS‐2B cells to investigate the effect of glucocorticoids on NF‐κB regulation and κB‐dependent transcription. In A549 cells following interleukin‐1β (IL‐1β) treatment, there was no effect of dexamethasone on the disappearance of IκBα protein, its subsequent reappearance 90‐min later or the rapid induction of IκBα mRNA and transcription rate. Expression of p65 and p50/p105 proteins were also unaffected by dexamethasone. In addition, the rapid IL‐1β‐induction of NF‐κB DNA binding and p65 nuclear localisation was unaffected by short (1−6 hours) dexamethasone pre‐treatments. Similarly, BEAS‐2B cells showed no effect of dexamethasone on IL‐1β‐induced NF‐κB (p50/p65). Stable transfection of a κB‐dependent reporter in A549 cells resulted in an 8−9‐fold activation by IL‐1β or phorbol ester, that was repressed 30−40 % by dexamethasone. However, in these cells, IL‐1β induction of inducible nitric oxide synthase, granulocyte‐macrophage colony stimulating factor and cyclooxygenase‐2 mRNA showed 70−90 % repression by dexamethsone. We, therefore, conclude that in these epithelial cells, the repressive effects of glucocorticoids are not mediated by up‐regulation of IκBα, decreased p50/p65 gene expression or inhibition of NF‐κB DNA binding. Furthermore, since the maximal repression of IL‐1β or phorbol‐ester‐induced κB‐dependent transcription by dexamethasone was less than 40 %, simple inhibition of κB‐dependent transcription cannot by itself account for the full repressive effects of glucocorticoids observed in these cells.
Chimeric extracellular domain of type II transforming growth factor (TGF)‐β receptor fused to the Fc region of human immunoglobulin as a TGF‐β antagonist
Authors: Komesli Sylviane , Vivien Denis , Dutartre Patrick
Published: March 2003

Transforming growth factor‐β (TGF‐β) type‐I and type‐II receptors form a ligand‐dependent heteromeric signalling complexes, in which transforming growth factor‐β receptor type II (TβRII) trends to act as the primary receptor. In the present study, we used a chimeric soluble type‐II receptor fused with the Fc regions of human immunoglobulin (TβRIIs‐Fc) in order to obtain a putative TGF‐β antagonist. Biochemical studies revealed that TβRIIs‐Fc shared the same properties as the wild‐type receptor. The TβRIIs‐Fc receptor displayed an affinity of 1370 ± 363 pM which was similar to those of the wild‐type TβRII when expressed alone in Cos‐1 cells (1122 ± 413 pM). Furthermore, the chimeric receptor showed the same selectivity for TGF‐β isoforms as the native receptor. Although both TGF‐β1 and TGF‐β3 were able to bind TβRIIs‐Fc, TGF‐β2 could not compete with the binding of TGF‐β1 to TβRIIs‐Fc. It was noted that this type of fused Fc receptor could be used in FlashPlate screening for potent agonism and antagonism of TGFβ. Moreover, biological activities of the chimeric receptor showed it to be a potent TGF‐β1‐antiproliferative and TGF‐β1‐extracellular matrix transcriptional inhibitor on responses in Mv1Lu cells. To conclude, our results clearly show that the TβRIIs‐Fc chimeric receptor could be used as a potent TGF‐β antagonist. These data raised the possibility that this TβRIIs‐Fc construct might act successfully as an antagonist of both TGF‐β1 and TGF‐β3 in vivo.
The N‐terminus of A1‐type myosin essential light chains binds actin and modulates myosin motor function
Authors: Timson David J. , Trayer Hylary R. , Trayer Ian P.
Published: March 2003

There are two isoforms (A1 and A2) of the myosin essential light chain (ELC) and consequently two isoenzymes of myosin subfragment 1 (S1), S1(A1) and S1(A2). The two isoenzymes differ in their kinetic properties with S1(A1) having a lower apparent Km for actin and a slower turnover of MgATP (kcat) than S1(A2). The two forms of the ELC differ only at their N‐termini where A1 has an additional 40‐odd amino acids that are not present in A2.

The human atrial ELC (an A1‐type ELC) was overexpressed in Escherichia coli and purified by ammonium sulphate fractionation and ion‐exchange chromatography. The recombinant ELC had actin‐activated MgATPase kinetics similar to those for rabbit skeletal S1(A1) under the same conditions. Deletion of the first 45 amino acid residues resulted in an ELC similar to the rabbit skeletal A2 isoform and, when hybridised into S1, in S1(A2)‐like kinetic properties. Results obtained with an ELC mutant that lacks the first 11 residues were intermediate between these two extremes but tending towards the S1(A2)‐like phenotype.

The wild‐type ELC (both hybridised into S1 or free in solution) could be cross‐linked to F‐actin, whereas the deletion mutant lacking the first 45 amino acids could not. The deletion mutant lacking the first 11 amino acids cross‐linked only poorly under the same conditions, consistent with the MgATPase data. We therefore conclude that these N‐terminal eleven amino acids predominantly encode an actin‐binding site which modulates the kinetics of the myosin motor. Furthermore, while free A1‐type ELC cross‐linked to both polymeric F‐actin and the monomeric G‐actin : DNase‐I complex, the same ELC in S1(A1) could only cross‐link to F‐actin. This suggests that the light chain binds to a different actin monomer than the heavy chain.
Functional groups of sialic acids involved in binding to siglecs (sialoadhesins) deduced from interactions with synthetic analogues
Authors: Kelm Sørge , Brossmer Reinhard , Isecke Rainer , Gross Hans‐Jürgen , Strenge Karen , Schauer Roland
Published: March 2003

The siglecs, formerly called sialoadhesins, are a family of I‐type lectins binding to sialic acids on the cell surface. Five members of this family have been identified : sialoadhesin, myelin‐associated glycoprotein (MAG), Schwann cell myelin protein (SMP), CD22 and CD33. We have investigated the relevance of substituents at position C‐9 and in the N‐acetyl group of N‐acetylneuraminic acid, using a series of synthetic sialic‐acid analogues either on resialylated human erythrocytes or as free α‐glycosides in hapten inhibition. All five siglecs require the hydroxy group at C‐9 for binding, suggesting hydrogen bonding of this substituent with the binding site. Remarkable differences were found among the proteins in their specificity for modifications of the N‐acetyl group. Whereas sialoadhesin, MAG and SMP do not tolerate a hydroxy group as in N‐glycolylneuraminic acid, they bind to halogenated acetyl residues. In the case of MAG, N‐fluoroacetylneuraminic acid is bound about 17‐fold better than N‐acetylneuraminic acid. In contrast, human and murine CD22 both show good affinity for N‐glycolylneuraminic acid, but only human CD22 bound the halogenated compounds. In conclusion, our data indicate that interactions of the hydroxy group at position 9 and the N‐acyl substituent contribute significantly to the binding strength.
Induction of the heat‐shock response by antiviral prostaglandins in human cells infected with human immunodeficiency virus type 1
Authors: de Marco Alessandra , Carattoli Alessandra , Rozera Carmela , Fortini Daniela , Giorgi Colomba , Belardo Giuseppe , Amici Carla , Santoro M. Gabriella
Published: March 2003

Cyclopentenone prostaglandins inhibit the replication of several DNA and RNA viruses, including retroviruses. The antiviral activity has been associated with the induction of a 70‐kDa heat‐shock protein (HSP70), via activation of the heat‐shock transcription factor (HSF) in infected cells. In the present study we investigated the effect of prostaglandin A1 (PGA1) on the regulation of HSP70 gene expression as well as on viral RNA and protein synthesis in CEM‐SS cells during acute infection with human immunodeficiency virus type 1 (HIV‐1). We report that HIV‐1 infection does not alter HSF activation by PGA1, whereas it causes an increase in intracellular HSP70 mRNA levels, as a result of enhanced HSP70 mRNA stability. We also show that, as reported in studies of different virus/host cell models, PGA1 inhibits HIV‐1 replication by acting at multiple levels during HIV‐1 infection. In addition to the previously reported block of HIV‐1 mRNA transcription, PGA1 was also found to inhibit viral protein synthesis. These results, together with the fact that prostaglandins are used clinically in the treatment of several diseases, open new perspectives in the search for novel antiretroviral drugs.
Alliinase [S‐alk(en)yl‐	L‐cysteine sulfoxide lyase] from Allium tuberosum (Chinese chive)
Authors: Manabe Tomofumi , Hasumi Asako , Sugiyama Mitsuyo , Yamazaki Mami , Saito Kazuki
Published: March 2003

Alliinase [S‐alk(en)yl‐
L‐cysteine sulfoxide lyase], a pyridoxal‐phosphate‐(Pxy‐P)‐dependent enzyme, is responsible for the degradative conversion of S‐alk(en)yl‐
L‐cysteine sulfoxide to volatile odorous sulfur‐containing metabolites in Allium plants. We have purified alliinase from shoots of Allium tuberosum (Chinese chive) to apparent homogeneity by SDS/polyacrylamide gel electrophoresis. A cDNA clone encoding alliinase was isolated from a cDNA library constructed from whole plants of A. tuberosum by hybridization screening with a synthetic 50‐residue oligonucleotide encoding a conserved region of the alliinases from onion and garlic. The isolated cDNA encoded a protein of 476 amino acid residues with a molecular mass of 54 083 Da. The deduced amino acid sequence exhibited 66−69 % identities with those of reported alliinases from onion, garlic and shallot. The partial amino acid sequence, which was determined for a V8 protease‐digested peptide fragment of the purified alliinase, was perfectly matched with the sequence deduced from the cDNA. An expression vector of recombinant alliinase cDNA was constructed in yeast. The catalytically active protein was in the soluble fraction of transformed yeast. Site‐directed mutagenesis experiments indicated that Lys280 was essential for the catalytic activity and, thus, a possible Pxy‐P‐binding residue. The mRNA expression of the alliinase gene comprising a multi‐gene family in the shoots of green plants was twofold higher than that in the roots of green plants; however, the expression in the shoots of etiolated plants was only 13 % that in green shoots, although the expression in the roots was not remarkably different between in green and etiolated plants. Immunohistochemical investigation indicated that the alliinase protein is predominantly accumulated in the bundle sheath cells of shoots of A. tuberosum.
Molecular cloning and characterization of two related and interferon‐induced 56‐kDa and 30‐kDa proteins highly similar to 2′‐5′ oligoadenylate synthetase
Authors: Rebouillat Dominique , Marié Isabelle , Hovanessian Ara G.
Published: March 2003

The 2′‐5′ oligoadenylate synthetase (OAS) represents a family of interferon‐induced proteins which when activated by double‐stranded (ds) RNA polymerizes ATP into 2′‐5′ linked oligomers with the general formula pppA(2′p5′A)n, n  1. Three forms of human OASs have been described corresponding to proteins of 40/46, 69/71 and 100 kDa (p40/p46, p69/p71 and p100). Polyclonal antibodies raised against p100 cross reacted with the other forms p40/p46, p69/p71 but also with an interferon‐induced 56‐kDa protein (p56). By screening a cDNA expression library, these polyclonal antibodies selected a cDNA encoding p56. Further studies by the RACE procedure using primers corresponding to this cDNA, a p56‐related protein of 30 kDa (p30) was isolated. Both p56 and p30 mRNA are expressed in interferon‐treated cells as transcripts of 2 kb and 1.8 kb, respectively. The 1.8‐kb mRNA is homologous to the 2‐kb mRNA but with a 243‐nucleotide deletion at position 1011, which results in a frameshift. Consequently, the p56 and p30 have their first 219 amino acid residues identical but differ at their C‐termini. In vitro transcription‐translation of p56 and p30 cDNAs generated proteins of 56 and 30 kDa, respectively. The deduced amino acid sequence of p56 kDa shares strong similarity with the previously cloned OASs, and contains the subdomains conserved in p40/p46 and p69/p71 forms. Transient expression in HeLa cells indicated that p30 has a cytoplasmic localization, whereas p56 has cytoplasmic and nucleolar localizations. The p56 isolated from transfected cells was shown to bind dsRNA and DNA, but it was devoid of 2′‐5′OAS activity typical of the three known forms of this enzyme. Thus, p56 and p30 are two related and interferon‐induced proteins outside the family of 2′‐5′OAS, which might have as yet unidentified catalytic activities or functions.
β 2‐microglobulin can be refolded into a native state from ex vivo amyloid fibrils
Authors: Bellotti Vittorio , Stoppini Monica , Mangione Palma , Sunde Margaret , Robinson Carol , Asti Lia , Brancaccio Diego , Ferri Giuseppina
Published: March 2003

β2‐microglobulin fibrils have been extracted from the femoral head of a patient who has been under chronic haemodialysis for 11 years. The primary structure of the N‐terminal portion of the protein and mass determination by electrospray mass spectrometry demonstrate that β2‐microglobulin, extracted as fibrils by the water extraction procedure, was not glycated and that Asn17 was not deamidated. Limited proteolysis was observed in more than 20 % of β2‐microglobulin molecules and the main cleavage sites were at the C‐terminus of Lys6 and Tyr10. β2‐microglobulin from fibrils has been purified by gel filtration in 6 M Gdn/HCl and submitted to a refolding procedure. The refolding conditions have been determined through the study of the unfolding pathway of the native protein. β2‐microglobulin is stable at neutral pH where it displays a lower tendency to self‐aggregate than in acidic conditions. Pulse dilution and extensive dialysis in refolding buffer at pH 7.5 yields β2‐microglobulin with a tertiary structure identical to that of the native form. The CD spectrum in the near‐ultraviolet region and the spectrum of the intrinsic fluorescence of Trp overlap those of the native protein, but the CD spectrum in the far‐ultraviolet region is affected by the contribution of oligomers created by β2‐microglobulin fragments that reduce the positive light polarisation at 205 nm typical of native β2‐microglobulin.
Reactivity studies of the tyrosyl radical in ribonucleotide reductase from Mycobacterium tuberculosis and Arabidopsis thaliana
Authors: Elleingand Eric , Gerez Catherine , Un Sun , Knüpling Moritz , Lu Guizlen , Salem Jerome , Rubin Harvey , Sauge‐Merle Sandrine , Laulhère Jean Pierre , Fontecave Marc
Published: March 2003

Ribonucleotide reductase (RNR) is a key enzyme for DNA synthesis since it provides cells with deoxyribonucleotides, the DNA precursors. Class I α2β2 RNRs contain a dinuclear iron center and an essential tyrosyl radical in the β2 component (protein R2). This is also true for the purified protein R2 of Mycobacterium tuberculosis RNR, as shown by iron analysis, light absorption and EPR spectroscopy. EPR spectroscopy at 286 GHz revealed a high gx value, suggesting that the radical is not hydrogen bonded, as in other prokaryotic R2s and in contrast with eukaryotic R2s (from Arabidopsis thaliana and mouse). Furthermore, it proved to be very resistant to scavenging by a variety of phenols and thiols and by hydroxyurea, similar to the Escherichia coli radical. By comparison, the plant and mouse radicals are very sensitive to drugs such as resveratrol and 2‐thiophenthiol. The radical from M. tuberculosis RNR does not seem to be an appropriate target for new antituberculous agents.
Heme‐regulated eIF‐2α kinase purifies as a hemoprotein
Authors: Chefalo Peter Joseph , Oh Jihyun , Rafie‐Kolpin Maryam , Kan Beki , Chen Jane‐Jane
Published: March 2003

The regulation of protein synthesis by the availability of heme in reticulocytes is well established. However, the mechanism by which heme regulates translational initiation is not clear. In this study, we have examined the heme regulation directly on the homogeneous heme‐regulated eIF‐2α kinase (HRI), which is activated during heme deficiency. We found that HRI purified as a hemoprotein with the characteristic Soret band of hemoprotein at 424 nm. This HRI was an active autokinase and eIF‐2α kinase, and its kinase activities were inhibited by submicromolar concentrations of hemin with an apparent Ki of 0.5 μM. Homogeneous HRI was a homodimer, and its activities could not be inhibited by incubation with purified inactive K199R HRI in vitro. Our results suggest that there are two distinct types of heme‐binding sites in the HRI homodimer. The binding of heme to the first site is stable, while the binding of heme to the second site is responsible for the rapid downregulation of HRI activity by heme. These results indicate that HRI binds heme and serves as a sensor of the availability of heme to coordinate the balanced synthesis of globins and heme in erythroid cells.
Mutational analysis of chitin synthase 2 of Saccharomyces cerevisiae
Authors: Yabe Tomio , Yamada‐Okabe Toshiko , Nakajima Tasuku , Sudoh Masayuki , Arisawa Mikio , Yamada‐Okabe Hisafumi
Published: March 2003

Saccharomyces cerevisiae harbors three chitin synthases termed Chs1p, Chs2p and Chs3p. Previously, we demonstrated that con1, a region that is highly conserved among all chitin synthases, contains amino acids essential for the catalytic activity of the enzyme and that Asp562, Gln601, Arg604, and Trp605 found in con1 together with Asp441 were probable catalytic sites of the enzyme. Here we report that another region, con2, in the C‐terminal half of Chs2p is also conserved exclusively in chitin synthases that resemble S. cerevisiae Chs1p and Chs2p. Alanine substitutions for the conserved amino acids in con2 identified five amino acids, Asn797, His799, Asp800, Trp803, and Thr805, the mutation of which severely diminished enzymatic activity and the enzyme's ability to rescue the yeast chs2Δ chs3Δ null mutant strain. Although the activities of some of the mutant enzymes were too low to measure enzyme kinetics, most of the alanine mutations in con2 affected the kcat values rather than the Km values. Whereas a conservative mutation of Asn797 restored the activity, those of His799, Asp800, Trp803, and Thr805 did not. A fine alignment of the amino acid sequences of con2 and Chs3p revealed that Asp800, Trp803 and Thr805 are completely conserved near the C‐terminal ends of Chs3p and its homologs in other fungi. On the basis of these findings, we propose that Asp800, Trp803, and Thr805 in con2 are additional residues involved in catalysis, and hypothesise that Asp800 together with the previously identified Asp441 and Asp562 serve as polar residues necessary for the acid‐based catalytic reaction of chitin synthase.
Rel transcription factors contribute to elevated urokinase expression in human ovarian carcinoma cells
Authors: Reuning Ute , Guerrini Luisa , Nishiguchi Tomizo , Page Sharon , Seibold Hildegard , Magdolen Viktor , Graeff Henner , Schmitt Manfred
Published: March 2003

Elevated levels of the urokinase‐type plasminogen activator (uPA) in tumor cells are conductive to tumor cell spread and metastasis. In a previous study we observed that suppression of RelA dramatically reduced endogenous uPA synthesis in the human ovarian cancer cell line OV‐MZ‐6. Because the uPA promoter contains three potential Rel‐like protein binding motifs (RRBE, 5′‐NF‐κB, and 3′‐NF‐κB) we conducted the first thorough systematic uPA promoter analysis to examine the direct impact of Rel proteins on uPA gene transcription. Disruption of RRBE resulted in a ∼40% decrease in uPA promoter activity, mutation of the 5′‐NF‐κB motif led to an additional 20% decrease. The 3′‐NF‐κB motif was not active. Overexpression of RelA significantly enhanced uPA promoter activity, whereas IκB‐α overexpression reduced uPA promoter activity by 40%. These data were supported by the finding that endogenous uPA was also increased sixfold by overexpression of RelA and decreased by 30% upon overexpression of IκB‐α. Transfection of OV‐MZ‐6 cells with antisense deoxynucleotides directed to RelA expression reduced uPA promoter activity by at least 40%. Our data clearly suggest that by binding to uPA promoter elements, Rel transcripton factors contribute directly to elevated uPA gene expression in human ovarian cancer cells, thereby promoting the multiple functions of uPA during tumor growth and metastasis.
Evolution of thyroid hormone binding by transthyretins in birds and mammals
Authors: Chang Linus , Munro Sharon L. A. , Richardson Samantha J. , Schreiber Gerhard
Published: March 2003

Transthyretin, a protein synthesized and secreted by the choroid plexus and liver, binds thyroid hormones in extracellular compartments. This binding prevents accumulation of thyroid hormones in the lipids of membranes, establishing extracellular thyroid hormone pools for the distribution of the hormones throughout the body and brain. The N‐termini of the transthyretin subunits are longer and more hydrophobic in chicken than in eutherian transthyretins. Here, we show that this is a general structural feature of avian transthyretins. Systematic changes of protein structure during evolution result from selection pressure leading to changes in function. The evolution of transthyretin function, namely, the binding of thyroid hormones, was studied in nine vertebrate species. The affinity of thyroxine binding to transthyretin is lowest in avians (mean Kd of about 30 nm), intermediate in metatherians (mean Kd of about 17 nm) and highest in eutherians (mean Kd of about 11 nm). The affinity for 3,5,3′‐triiodothyronine shows an opposite trend, being four times higher for avian transthyretins than for mammalian transthyretins.
Activation of a cGMP‐stimulated cAMP phosphodiesterase by protein kinase C in a liver Golgi–endosomal fraction
Authors: Geoffroy Valérie , Fouque Françoise , Nivet Valérie , Clot Jean‐Pierre , Lugnier Claire , Desbuquois Bernard , Benelli Chantal
Published: March 2003

The ability of Ca2+/phospholipid‐dependent protein kinase (protein kinase C, PKC) to stimulate cAMP phosphodiesterase (PDE) activity in a liver Golgi–endosomal (GE) fraction was examined in vivo and in a cell‐free system. Injection into rats of 4β‐phorbol 12‐myristate 13‐acetate, a known activator of PKC, caused a rapid and marked increase in PKC activity (+ 325% at 10 min) in the GE fraction, along with an increase in the abundance of the PKC α‐isoform as seen on Western immunoblots. Concurrently, 4β‐phorbol 12‐myristate 13‐acetate treatment caused a time‐dependent increase in cAMP PDE activity in the GE fraction (96% at 30 min). Addition of the catalytic subunit of protein kinase A (PKA) to GE fractions from control and 4β‐phorbol 12‐myristate 13‐acetate‐treated rats led to a comparable increase (130–150%) in PDE activity, suggesting that PKA is probably not involved in the in‐vivo effect of 4β‐phorbol 12‐myristate 13‐acetate. In contrast, addition of purified PKC increased (twofold) PDE activity in GE fractions from control rats but affected only slightly the activity in GE fractions from 4β‐phorbol 12‐myristate 13‐acetate‐treated rats. About 50% of the Triton‐X‐100‐solubilized cAMP PDE activity in the GE fraction was immunoprecipitated with an anti‐PDE3 antibody. On DEAE–Sephacel chromatography, three peaks of PDE were sequentially eluted: one early peak, which was stimulated by cGMP and inhibited by erythro‐9 (2‐hydroxy‐3‐nonyl) adenine (EHNA); a selective inhibitor of type 2 PDEs; and two retarded peaks of activity, which were potently inhibited by cGMP and cilostamide, an inhibitor of type 3 PDEs. Further characterization of peak I by HPLC resolved a major peak which was activated (threefold) by 5 µm cGMP and inhibited (87%) by 25 µm EHNA, and a minor peak which was insensitive to EHNA and cilostamide. 4β‐Phorbol 12‐myristate 13‐acetate treatment caused a selective increase (2.5‐fold) in the activity associated with DEAE–Sephacel peak I, without changing the Km value. These results suggest that PKC selectively activates a PDE2, cGMP‐stimulated isoform in the GE fraction.
Screening of an intragenic second‐site suppressor of purine–cytosine permease from Saccharomyces cerevisiae
Authors: Ferreira Thierry , Chevallier Jean , Paumard Patrick , Napias Christian , Brèthes Daniel
Published: March 2003

The purine–cytosine permease from Saccharomyces cerevisiae mediates the active transport through the plasma membrane of adenine, hypoxanthine, guanine and cytosine using the proton electrochemical potential difference as an energy source. Analysis of the activity of strains mutated in a hydrophilic segment (371–377) of the polypeptidic chain has shown the involvement of this segment in the maintenance of the active three‐dimensional structure of the carrier. In an attempt to identify permease domains that could interact functionally and/or physically with this segment, we looked for second‐site mutations that could suppress the effects of amino acid changes in this region. This paper describes a positive screen that has allowed the isolation of one suppressor from a permease mutant displaying the N374I change (fcy2‐20 allele), a substitution that induces a dramatic decrease in the affinity of the carrier for adenine, cytosine and hypoxanthine. The second‐site mutation corresponds to the replacement of the Ser272 residue by Leu. Its suppressive effect is shown to be a partial restoration of the binding of cytosine and hypoxanthine to the permease. To test whether this second‐site mutation is specific for the fcy2‐20 allele, two double mutants were constructed (Fcy2pT213I, S272L and Fcy2pS272L, N377G). Results obtained with these two double mutants showed that the suppressive effect of S272 L replacement was not specific for the original N374I change. To understand the general effect of this amino acid replacement for the three distinct double mutants, a strain overexpressing Fcy2pS272L was constructed. Kinetic analysis of this strain showed that, by itself, the S272 L change induced an improvement in the base‐binding step that could account for its global suppressive effect. Moreover, S272 L induced a decrease in the turnover of the permease, thus showing the involvement of S272 in the translocation process. Taking into account the topological model of the permease proposed here, this Ser residue is probably located in a transmembrane amphipathic α‐helix (TM5). The location and the observed decrease in the turnover of the carrier observed with the S272 L change lead us to propose that S272 could be part of a hydrophilic pore involved in the translocation of the base and/or the proton.
Domain–domain interactions in high mobility group 1 protein (HMG1)
Authors: Ramstein Jean , Locker Daniel , Bianchi Marco E. , Leng Marc
Published: March 2003

The high mobility group protein HMG1 is a conserved chromosomal protein with two homologous DNA‐binding domains, A and B, and an acidic carboxy‐terminal tail, C. The structure of isolated domains A and B has been previously determined by NMR, but the interactions of the different domains within the complete protein were unknown. By means of differential scanning calorimetry and circular dichroism we have investigated the thermal stability of HMG1, of the truncated protein A‐B (HMG1 without the acidic tail C) and of the isolated domains A and B. In 3 mm sodium acetate buffer, pH 5, the thermal melting of domains A and B are identical (transition temperature tm = 43 °C and 41 °C, denaturation enthalpies ΔH = 46 kcal·mol–1). The thermal melting of protein A‐B presents two nearly identical transitions (tm = 40 °C and 41 °C, ΔH = 44 kcal·mol–1 and 46 kcal·mol–1, respectively). We conclude that the two domains A and B within protein A‐B behave as independent domains. The thermal melting of HMG1 is biphasic. The two transitions have a different value of tm (38 °C and 55 °C) and corresponding values of ΔH around 40 kcal·mol–1. We conclude that within HMG1, the acidic tail C is interacting with one of the two domains A and B, however, the two domains A and B do not interact with each other. At 37 °C, one of the two domains A and B, within HMG1, is partly unfolded, whereas the other which interacts with the acidic tail C, is fully native. The interaction free energy of the acidic tail C is estimated to be in the range of 2.5 kcal·mol–1 based on simulations of the thermograms of HMG1 as a function of the interaction free energy.
Cobalt proteins
Authors: Kobayashi Michihiko , Shimizu Sakayu
Published: March 2003

In the form of vitamin B12, cobalt plays a number of crucial roles in many biological functions. However, recent studies have provided information on the biochemistry and bioinorganic chemistry of several proteins containing cobalt in a form other than that in the corrin ring of vitamin B12. To date, eight noncorrin‐cobalt‐containing enzymes (methionine aminopeptidase, prolidase, nitrile hydratase, glucose isomerase, methylmalonyl‐CoA carboxytransferase, aldehyde decarbonylase, lysine‐2,3‐aminomutase, and bromoperoxidase) have been isolated and characterized. A cobalt transporter is involved in the metallocenter biosynthesis of the host cobalt‐containing enzyme, nitrile hydratase. Understanding the differences between cobalt and nickel transporters might lead to drug development for gastritis and peptic ulceration.
Oligonucleotide aggregates bind to the macrophage scavenger receptor
Authors: Suzuki Kazuo , Doi Takefumi , Imanishi Takeshi , Kodama Tatsuhiko , Tanaka Toshiki
Published: March 2003

We have prepared a model receptor containing a Lys cluster (320–340) in the collagen‐like domain of the bovine macrophage scavenger receptor, and have shown that it has a similar binding specificity to the native scavenger receptor. The native scavenger receptor is reported to bind the quadruplex structure of nucleotides. In this study, we analyzed the model receptor binding of nucleotides with various structures, random, parallel or antiparallel quadruplex and aggregate forms. This was carried out by direct binding assays using labeled oligonucleotides or surface plasmon resonance, and by an inhibition assay using Chinese hamster ovary (CHO) cells expressing the scavenger receptor. The results showed that the nucleotides forming the quadruplex structure did not exhibit any binding. Only the aggregate forms of the nucleotide could bind to the model receptor. They also inhibited the degradation of acetylated low density lipoprotein by CHO cells expressing the native scavenger receptor, whereas nucleotides that did not bind to the model receptor had no effect on cellular acetylated low density lipoprotein degradation. Our results suggest that the quadruplex structure is not essential but may be required for the formation of the nucleotide aggregates, which can interact with the scavenger receptor.
In Saccharomyces cerevisae, feedback inhibition of homocitrate synthase isoenzymes by lysine modulates the activation of LYS gene expression by Lys14p
Authors: Feller André , Ramos Fernando , Piérard André , Dubois Evelyne
Published: March 2003

Expression of the structural genes for lysine biosynthesis responds to an induction mechanism mediated by the transcriptional activator Lys14p in the presence of α‐aminoadipate semialdehyde (αAASA), an intermediate of the pathway acting as a coinducer. This activation is reduced by the presence of lysine in the growth medium, leading to apparent repression.

  In this report we demonstrate that Saccharomyces cerevisiae possesses two genes, LYS20 and LYS21, encoding two homocitrate synthase isoenzymes which are located in the nucleus. Each isoform is inhibited by lysine with a different sensitivity. Lysine‐overproducing mutants were isolated as resistant to aminoethylcysteine, a toxic lysine analog. Mutations, LYS20fbr and LYS21fbr, are allelic to LYS20 and LYS21, and lead to desensitization of homocitrate synthase activity towards lysine and to a loss of apparent repression by this amino acid. There is a fair correlation between the I0.5 of homocitrate synthase for lysine, the intracellular lysine pool and the levels of Lys enzymes, confirming the importance of the activity control of the first step of the pathway for the expression of LYS genes. The data are consistent with the conclusion that inhibition by lysine of Lys14p activation results from the control of αAASA production through the feedback inhibition of homocitrate synthase activity.
The superoxide dismutase activity of desulfoferrodoxin from Desulfovibrio desulfuricans ATCC 27774
Authors: Romão Célia V. , Liu Ming Y. , Gall Jean Le , Gomes Cláudio M. , Braga Vera , Pacheco Isabel , Xavier António V. , Teixeira Miguel
Published: March 2003

Desulfoferrodoxin (Dfx), a small iron protein containing two mononuclear iron centres (designated centre I and II), was shown to complement superoxide dismutase (SOD) deficient mutants of Escherichia coli[Pianzzola, M.J., Soubes M. & Touati, D. (1996) J. Bacteriol.178, 6736–6742]. Furthermore, neelaredoxin, a protein from Desulfovibrio gigas containing an iron site similar to centre II of Dfx, was recently shown to have a significant SOD activity [Silva, G., Oliveira, S., Gomes, C.M., Pacheco, I., Liu, M.Y., Xavier, A.V., Teixeira, M., Le Gall, J. & Rodrigues‐Pousada, C. (1999) Eur. J. Biochem. 259, 235–243]. Thus, the SOD activity of Dfx isolated from the sulphate‐reducing bacterium Desulfovibrio desulfuricans ATCC 27774 was studied. The protein exhibits a SOD activity of 70 U·mg−1, which increases approximately 2.5‐fold upon incubation with cyanide. Cyanide binds specifically to Dfx centre II, yielding a low‐spin iron species with g‐values at 2.27 (g⊥) and 1.96 (g∥). Upon reaction of fully oxidized Dfx with the superoxide generating system xanthine/xanthine oxidase, Dfx centres I and II become partially reduced, suggesting that Dfx operates by a redox cycling mechanism, similar to those proposed for other SODs.

 Evidence for another SOD in D. desulfuricans is also presented – this enzyme is inhibited by cyanide, and N‐terminal sequence data strongly indicates that it is an analogue to Cu,Zn‐SODs isolated from other sources. This is the first indication that a Cu‐containing protein may be present in a sulphate‐reducing bacterium.
Fluorescence studies of the carboxyl‐terminal domain of smooth muscle calponin
Authors: Bartegi Aghleb , Roustan Claude , Kassab Ridha , Fattoum Abdellatif
Published: March 2003

The fluorescence parameters of the environment‐sensitive acrylodan, selectively attached to Cys273 in the C‐terminal domain of smooth muscle calponin, were studied in the presence of F‐actin and using varying salt concentrations. The formation of the F‐actin acrylodan labeled calponin complex at 75 mm NaCl resulted in a 21‐nm blue shift of the maximum emission wavelength from 496 nm to 474 nm and a twofold increase of the fluorescent quantum yield at 460 nm. These spectral changes were observed at the low ionic strengths (< 110 mm) where the calponin : F‐actin stoichiometry is 1 : 1 as well as at the high ionic strengths (> 110 mm) where the binding stoichiometry is a 1 : 2 ratio of calponin : actin monomers. On the basis of previous three‐dimensional reconstruction and chemical crosslinking of the F‐actin–calponin complex, the actin effect is shown to derive from the low ionic strength interaction of calponin with the bottom of subdomain‐1 of an upper actin monomer in F‐actin and not from its further association with the subdomain‐1 of the adjacent lower monomer which occurs at the high ionic strength. Remarkably, the F‐actin‐dependent fluorescence change of acrylodan is qualitatively but not quantitatively similar to that earlier reported for the complexes of calponin and Ca2+‐calmodulin or Ca2+‐caltropin. As the three calponin ligands bind to the same segment of the protein, encompassing residues 145–182, the acrylodan can be considered as a sensitive probe of the functioning of this critical region. A distance of 29 Å was measured by fluorescence resonance energy transfer between Cys273 of calponin and Cys374 of actin in the 1 : 1 F‐actin–calponin complex suggesting that the F‐actin effect was allosteric reflecting a global conformational change in the C‐terminal domain of calponin.
Contribution of the carbohydrate moiety to conformational stability of the carboxypeptidase Y
Authors: Dumoulin Mireille , Ueno Hiroshi , Hayashi Rikimaru , Balny Claude
Published: March 2003

The process of pressure‐induced denaturation of carboxypeptidase Y and the role of the carbohydrate moiety in its response to pressure and low temperature were investigated by measuring in situ the catalytic activity and, the intrinsic and 8‐anilino‐1‐naphthalene sulfonic acid binding fluorescences.

 Pressure‐induced denaturation of carboxypeptidase Y is a process involving at least three transitions. Low pressures (below 150 MPa) induced slight conformational changes characterized by a slight decrease in the center of the spectral mass of intrinsic fluorescence, whereas no changes in 8‐anilino‐1‐naphthalene sulfonic acid binding fluorescence were observed and 80% of the catalytic activity remained. Higher pressure (150–500 MPa) induced further conformational changes, characterized by a large decrease in the center of the spectral mass of intrinsic fluorescence, a large increase in the 8‐anilino‐1‐naphthalene sulfonic acid binding fluorescence and the loss of all catalytic activity. Thus, this intermediate exhibited characteristics of molten globule‐like state. A further increase, in pressure (above 550 MPa) induced transition from this first molten globule‐like state to a second molten globule‐like state. This two‐stage denaturation process can be explained by assuming the existence of two independent structural domains in the carboxypeptidase molecule.

 A similar three‐transition process was found for unglycosylated carboxypeptidase Y, but, the first two transitions clearly occurred at lower pressures than those for glycosylated carboxypeptidase Y. These findings indicate that the carbohydrate moiety protects carboxypeptidase Y against pressure‐induced denaturation. The origin of the protective effects is discussed based on the known crystallographic structure of CPY.
Inhibition of calcium oxalate crystal growth and aggregation by prothrombin and its fragments in vitro
Authors: Grover Phulwinder K. , Ryall Rosemary L.
Published: March 2003

During blood coagulation, prothrombin (PT) is ultimately degraded to three fragments, thrombin, fragment 1 (F1) and fragment 2 (F2), which, collectively, contain all of the structural features of PT. One of these fragments, F1, is excreted in human urine and is the principal protein occluded into calcium oxalate (CaOx) crystals precipitated from it. This urinary form of F1, which we have named urinary prothrombin fragment 1 is present in calcium stones and is a potent inhibitor of CaOx crystallization in urine in vitro. The aim of this study was to determine whether PT itself and its other activation products, namely, thrombin, F1 and F2 also inhibit CaOx crystallization, by comparing their effects in a seeded, inorganic crystallization system. A secondary objective was to assess the relationship between the structures of the proteins and their inhibitory activities. PT was isolated from a human blood concentrate rich in vitamin K‐dependent proteins. Following initial cleavage by thrombin, the resulting fragments, F1 and F2, were purified by a combination of reversed phase HPLC and low pressure column chromatography. The purity of the proteins was confirmed by SDS/PAGE and their individual effects on CaOx crystallization were determined at the same concentration (16.13 nm) in a seeded, metastable solution of CaOx using a Coulter Counter. [14C]Oxalate was used to assess deposition of CaOx and crystals were visualized using scanning electron microscopy. The Coulter Counter data revealed that the proteins reduced the size of precipitated crystals in the order F1 > PT > F2 > thrombin. These findings were confirmed by scanning electron microscopy which showed that the reduction in particle size resulted from a decrease in the degree of crystal aggregation. [14C]Oxalate analysis demonstrated that all proteins inhibited mineral deposition, in the order F1 (44%) > PT (27.4%) > thrombin (10.2%) > F2 (6.5%). It was concluded that the γ‐carboxyglutamic acid domain of PT and F1, which is absent from thrombin and F2, is the region of the molecules which determines their potent inhibitory effects. The superior potency of F1, in comparison with PT, probably results from the molecule’s greater charge to mass ratio.
Structural analysis of 20 oligosaccharide‐alditols released from the jelly coat ofRana palustriseggs by reductive β‐elimination
Authors: Maes Emmanuel , Florea Doina , Coppin Alexandra , Strecker Gérard
Published: March 2003

The eggs of amphibians are surrounded by three to eight layers of jelly coats. This extracellular matrix is mainly composed of hydrated mucin‐type glycoproteins. These highly glycosylated molecules are synthesized by oviduct and play an important role in the fertilization process.

 Recent structural analyses have shown the strict species‐specificity of the O‐linked oligosaccharides which constitute 60–70% of these oviducal mucins. Consequently, these carbohydrate chains represent new phenotypic markers, and from a biological point of view, can influence parasite tropism or can be involved in species‐specific interaction of gametes.

 The primary structure of 20 oligosaccharide‐alditols, released by alkali/borohydride treatment from the mucin ofRana palustrisegg jelly coats, was established by1H and13C‐NMR analysis. Thirteen of these components possess new structures and the polymerization of the sequence Gal(β1–3)GalNAc(α1–4) characterizes the species‐specificity ofR. palustris.
Insulin stimulates triacylglycerol secretion by perfused livers from fed rats but inhibits it in livers from fasted or insulin‐deficient rats
Authors: Zammit Victor A. , Lankester David J. , Brown Anna M. , Park Byung‐Sung
Published: March 2003

We determined whether the direction of the acute effect of insulin on hepatic triacylglycerol secretion is dependent on the prior physiological state or on the in vitro experimental system used. The effect of insulin on triacylglycerol secretion was studied using perfused livers isolated from rats under three metabolic conditions: fed normo‐insulinaemic, 24‐h fasted and fed, streptozotocin‐diabetic (insulin‐deficient). Insulin acutely activated triacylglycerol secretion (by 43%) in organs from fed, normo‐insulinaemic animals, whereas it inhibited triacylglycerol secretion in livers isolated from fasted or insulin‐deficient rats (by 30 and 33%, respectively). By contrast, in 24‐h‐cultured hepatocytes insulin invariably acutely inhibited triacylglycerol secretion irrespective of the metabolic state of the donor animals. It is concluded that the use of perfused livers enables the observation of a switch in the direction of insulin action on hepatic triacylglycerol secretion from stimulatory, in the normo‐insulinaemic state, to inhibitory in the fasting or insulin‐deficient state. The possible implications of this switch for the relationship between hyperinsulinaemia, increased hepatic very‐low‐density lipoprotein–triacylglycerol secretion and hypertriglyceridaemia observed in vivo are discussed.
Isolation and characterization of sulphated and nonsulphated forms of cholecystokinin‐58 and their action on gallbladder contraction
Authors: Bonetto Valentina , Jörnvall Hans , Andersson Mats , Renlund Staffan , Mutt Viktor , Sillard Rannar
Published: March 2003

Cholecystokinin (CCK) exists in multiple molecular forms with different polypeptide lengths and the absence or presence of sulphation. We have isolated sulphated and nonsulphated forms of CCK‐58 from porcine intestine and have determined their bioactivities in a guinea‐pig gallbladder contraction assay. Both forms co‐eluted in cation‐exchange chromatography and in several rounds of reverse‐phase (RP)‐HPLC, but separated upon RP‐HPLC using a water/acetonitrile system with heptafluorobutyric acid as counter ion. Nonsulphated CCK‐58 was the form detected by matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry because of desulphation in that process. The biological activity of CCK‐58 and CCK‐33 is equipotent, although the kinetics of the response differ. Sulphated CCK‐58 was found to be 35 times more potent than nonsulphated CCK‐58. In contrast, sulphated CCK‐8 is 150 times more potent than nonsulphated CCK‐8, and for sulphated and nonsulphated CCK‐33, the activities differ by a factor of 100. This type of correlation indicates that the N‐terminal end of CCK‐58 partially compensates for the decrease in activity arising from the lack of sulphated tyrosine. Given its fairly high bioactivity, nonsulphated CCK‐58 may have a physiological significance.
Polypeptide neurotoxins from spider venoms
Authors: Grishin Eugene
Published: March 2003

Spider venoms contain a variety of toxic components. The polypeptide toxins are divided into low and high molecular mass types.

 Small polypeptide toxins interacting with cation channels display spatial structure homology. They can affect the functioning of calcium, sodium, or potassium channels.

 A family of high molecular mass toxic proteins was found in the venom of the spider genus Latrodectus. These neurotoxins, latrotoxins, cause a massive transmitter release from a diversity of nerve endings. The latrotoxins are proteins of about 1000 amino acid residues and share a high level of structure identity.

 The structural and functional properties of spider polypeptide toxins are reviewed in this paper.