bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of mouse kidney tubular epithelial cell‐specific expression of core 2 GlcNAc transferase
Authors: Sekine Michiko , Taya Choji , Kikkawa Yoshiaki , Yonekawa Hiromichi , Takenaka Masaru , Matsuoka Yasuko , Imai Enyu , Izawa Mineko , Kannagi Reiji , Suzuki Akemi
Published: March 2003

A mouse gene, Gsl5, controls the expression of Galβ1–4(Fucα1–3)GlcNAcβ1–6(Galβ1–3)Gb4Cer and its precursor glycolipids in the kidney by regulating transcription of β‐1,6‐GlcNAc transferase. Here we report that Gsl5 controls the expression of the core 2 structure [GlcNAcβ1–6(Galβ1–3)GalNAcα1‐Ser/Thr] of glycoproteins as well as the glycolipid, GlcNAcβ1–6(Galβ1–3)GalNAcβ1–3Galα1–4Galβ1–4Glcβ1‐ceramide. Immunohistochemical studies using an anti‐(core 2‐Lex) monoclonal antibody demonstrated that lysosome‐like vesicles of proximal tubule cells were clearly stained in a Gsl5 wild type mouse, but not in a Gsl5 mutant strain of mice. Western blotting of microsomal fractions of kidney tissue with the same antibody confirmed the histological findings. In situ hybridyzation with an antisense probe to the kidney‐specific mRNA demonstrated that the mRNA is localized at proximal tubule‐cells in the cortex adjacent to the medulla, but not detected in glomeruli nor in collecting duct cells in the medulla. The results obtained by immunohistological staining and in situ hybridyzation are compatible and lead to the conclusion that the kidney specific mRNA is expressed in a proximal tubular cell specific manner and produces core 2 GlcNAc transferase responsible for the production of glycoproteins localized at vesicles in the proximal tubular cells. Glycosylation regulated by Gsl5 gene may modify functions of membrane glycoproteins in proximal tubular cells.
Ectoenzymatic breakdown of diadenosine polyphosphates by Xenopus laevis oocytes
Authors: Aguilar José S. , Reyes Rosalina , Asensio Aaron C. , Oaknin Sol , Rotllán Pedro , Miledi Ricardo
Published: March 2003

Xenopus laevis oocytes exhibit ectoenzymatic activity able to hydrolytically cleave extracellular diadenosine polyphosphates (ApnA). The basic properties of this ectoenzyme were investigated using as substrates di‐(1,N6‐ethenoadenosine) 5′,5′′′‐P1,P4‐tetraphospate [ε‐(Ap4A)] and di‐(1,N6‐ethenoadenosine) 5′,5′′′‐P1,P5‐pentaphospate [ε‐(Ap5A)], fluorogenic derivatives of Ap4A and Ap5A, respectively. ε‐(Ap4A) and ε‐(Ap5A) are hydrolysed by folliculated oocytes according to hyperbolic kinetics with Km values of 13.4 and 12.0 µm and Vmax values of 4.8 and 5.5 pmol per oocyte per min, respectively. The ectoenzyme is activated by Ca2+ and Mg2+, reaches maximal activity at pH 8–9 and is inhibited by suramin. Defolliculated oocytes also hydrolyse both substrates with similar Km values but Vmax values are approximately doubled with respect to folliculated controls. Chromatographic analysis indicates that extracellular ε‐(Ap4A) and ε‐(Ap5A) are first cleaved into 1,N6‐ethenoAMP (ε‐AMP) + 1,N6‐ethenoATP (ε‐ATP) and ε‐AMP + 1,N6‐ethenoadenosine tetraphosphate (ε‐Ap4), respectively, which are catabolized to 1,N6‐ethenoadenosine (ε‐Ado) as the end product by folliculated oocytes. Denuded oocytes, however, show a drastically reduced rate of ε‐Ado production, ε‐AMP being the main end‐product of extracellular ε‐(ApnA) catabolism. Results indicate that, whereas the ApnA‐cleaving ectoenzyme appears to be located mainly in the oocyte, ectoenzymes involved in the dephosphorylation of mononucleotide moieties are located mainly in the follicular cell layer.
Cloning and expression analysis of rainbow trout Oncorhynchus mykiss tumour necrosis factor‐α
Authors: Laing Kerry J. , Wang Tiehui , Zou Jun , Holland Jason , Hong Suhee , Bols Niels , Hirono Ikuo , Aoki Takashi , Secombes Christopher J.
Published: March 2003

A rainbow trout (Oncorhynchus mykiss) gene for tumor necrosis factor (TNF) has been cloned and sequenced. The cDNA contains an open reading frame of 738 nucleotides that translate into a 246 amino‐acid putative peptide, with a 5′ untranslated region (UTR) of 140 bp and a 3′ UTR of 506 bp. Two potential N‐linked glycosylation sites exist in the translation. The genomic sequence measures 2007 bp and contains three introns that intercept four coding exons. Expression studies using RT‐PCR have shown that the trout TNF gene is constitutively expressed in the gill and kidney of unstimulated fish. Trout TNF expression could be up‐regulated by stimulation of isolated head kidney leucocytes with lipopolysaccharide (LPS). Similarly, stimulation of a trout macrophage cell line (RTS11) with LPS resulted in an increased transcript level, as did incubation with recombinant trout interleukin (IL)‐1β. The optimal timing for induction of TNF expression in trout macrophages was determined using recombinant trout IL‐1β, where a clear induction was apparent by 2 h and peaked at 4 h. Evidence that this TNF gene is equivalent to mammalian TNF‐α is discussed.
Heat capacity analysis of oxidized Escherichia coli thioredoxin fragments (1–73, 74–108) and their noncovalent complex
Authors: Georgescu Roxana E. , M. Garcia‐Mira Maria , Tasayco Maria L. , Sanchez‐Ruiz Jose M.
Published: March 2003

We have calculated the absolute heat capacities of fragments 1–73 (N fragment) and 74–108 (C fragment) from thioredoxin, their complex and the uncleaved protein, from the concentration dependence of the apparent heat capacities of the solutions determined by differential scanning calorimetry. We find that, while the absolute heat capacities of uncleaved, unfolded thioredoxin and the C fragment are in good agreement with the theoretical values expected for fully solvated chains (calculated as the sum of the contributions of the constituent amino acids), the absolute heat capacities of the N fragment and the unfolded complex are about 2 kJ·K−1·mol−1 lower than the fully solvated‐chain values. We attribute this discrepancy to burial of the apolar surface in the N fragment (as burial of the polar area is expected to lead to an increase in heat capacity). Illustrative calculations suggest that burial of about 1000–1600 Å2 of apolar surface takes place in the N fragment (probably accompanied by the burial of a smaller amount of polar surface). In general, this work is supportive of heat capacity measurements on protein fragments being useful as probes of surface burial in studies to characterize protein unfolded states and the high regions of protein folding landscapes.
Structure and promoter analysis of the mouse membrane‐bound transferrin‐like protein (MTf) gene
Authors: Nakamasu Kazuko , Kawamoto Takeshi , Yoshida Eri , Noshiro Mitsuhide , Matsuda Yoichi , Kato Yukio
Published: March 2003

Recently, we purified membrane‐bound transferrin‐like protein (MTf) from the plasma membrane of rabbit chondrocytes and showed that the expression levels of MTf protein and mRNA were much higher in cartilage than in other tissues [Kawamoto T, Pan, H., Yan, W., Ishida, H., Usui, E., Oda, R., Nakamasu, K., Noshiro, M., Kawashima‐Ohya, Y., Fujii, M., Shintani, H., Okada, Y. & Kato, Y. (1998) Eur. J. Biochem.256, 503–509]. In this study, we isolated the MTf gene from a constructed mouse genomic library. The mouse MTf gene was encoded by a single‐copy gene spanning ≈ 26 kb and consisting of 16 exons. The transcription‐initiation site was located 157 bp upstream from the translation‐start codon, and a TATA box was not found in the 5′ flanking region. The mouse MTf gene was mapped on the B3 band of chromosome 16 by fluorescence in situ hybridization. Using primary chondrocytes, SK‐MEL‐28 (melanoma cell line), ATDC5 (chondrogenic cell line) and NIH3T3 (fibroblast cell line) cells, we carried out transient expression studies on various lengths of the 5′ flanking region of the MTf gene fused to the luciferase reporter gene. Luciferase activity in SK‐MEL‐28 cells was higher than in primary chondrocytes. Although no luciferase activity was detectable in NIH3T3 cells, it was higher in chondrocytes than in ATDC5 chondrogenic cells. These findings were consistent with the levels of expression of MTf mRNA in these cells cultured under similar conditions. The patterns of increase and decrease in the luciferase activity in chondrocytes transfected with various 5′ deleted constructs of the MTf promoter were similar to that in ATDC5 cells, but differed from that in SK‐MEL‐28 cells. The findings obtained with primary chondrocytes suggest that the regions between −693 and −444 and between −1635 and −1213 contain positive and negative cis‐acting elements, respectively. The chondrocyte‐specific expression of the MTf gene could be regulated via these regulatory elements in the promoter region.
Molecular cloning and biochemical characterization of a lipoxygenase in almond (Prunus dulcis) seed
Authors: Mita Giovanni , Gallo Antonia , Greco Valentina , Zasiura Colette , Casey Rod , Zacheo Giuseppe , Santino Angelo
Published: March 2003

We have characterized an almond (Prunus dulcis) lipoxygenase (LOX) that is expressed early in seed development. The presence of an active lipoxygenase was confirmed by western blot analysis and by measuring the enzymatic activity in microsomal and soluble protein samples purified from almond seeds at this stage of development. The almond lipoxygenase, which had a pH optimum around 6, was identified as a 9‐LOX on the basis of the isomers of linoleic acid hydroperoxides produced in the enzymatic reaction. A genomic clone containing a complete lipoxygenase gene was isolated from an almond DNA library. The 6776‐bp sequence reported includes an open reading frame of 4667 bp encoding a putative polypeptide of 862 amino acids with a calculated molecular mass of 98.0 kDa and a predicted pI of 5.61. Almond seed lipoxygenase shows 71% identity with an Arabidopsis LOX1 gene and is closely related to tomato fruit and potato tuber lipoxygenases. The sequence of the active site was consistent with the isolated gene encoding a 9‐LOX.
A ‘select and swap’ strategy for the isolation of clones with tightly regulated transgenes
Authors: Sullivan Matthew J. , Carpenter Adam J. , Porter Andrew C. G.
Published: March 2003

Increasing numbers of biological problems are being addressed by genetic approaches that rely on inducible expression of transgenes. It is desirable that expression of such a transgene is tightly regulated, from close to zero expression in the ‘off’ state, to appreciable (at least physiological) expression in the ‘on’ state. Although there are many examples where tight regulation has been achieved, certain factors, including chromosomal position effects due to random integration of the transgene, often cause suboptimal inducibility and make the isolation of tightly regulated clones difficult and/or laborious. Here we describe a ‘select and swap’ strategy for the isolation, from a population of stable transfectants, of clones with tightly regulated transgenes. In this approach, a positively and negatively selectable, inducible marker gene is used to select for clones with optimal transgene regulation. After isolation of such clones, the marker gene is swapped with a linked gene of interest by the use of site‐specific recombination. To test this strategy we introduced into human cells a plasmid with a tetracycline‐inducible bacterial gpt gene linked to a promoterless luciferase gene, isolated clones with tight gpt expression and used the Cre/loxP site‐specific recombination system to swap the gpt gene with the luciferase gene. We discuss ways for refining and developing the system and widening its applicability.
Organization and regulation of the rat Cx31 gene
Authors: Gabriel Heinz‐Dieter , Ströbl Barabara , Hellmann Peter , Buettner Reinhard , Winterhager Elke
Published: March 2003

The connexin31 (Cx31) gene, a member of the connexin multigene family, is expressed in a characteristic spatiotemporal pattern during placental development in rodents. To elucidate the trophoblast‐specific regulation of Cx31, we have isolated the rat Cx31 gene and performed structural and functional promoter analysis. The isolated Cx31 gene contains two exons separated by an intron of 2.6 kb. The first exon of the Cx31 gene is preceded by a TATA‐less promoter region. Transcription is initiated in exon 1 from two transcription start sites producting transcripts of 105 and 139 bp. The 935 bp of the 5′ flanking region of exon 1 comprises five putative binding sites for the GATA transcription factors as well as a NF‐κB element, a CAAT‐box and E‐box/E‐box‐related sequences. For functional promoter analysis, the rat choriocarcinoma cell line Rcho‐1 and the mouse keratinocyte cell line Hel37, which both express Cx31, were chosen. Only constructs including exon 1 and the complete intron showed high activity in transient transfection experiments in both cell lines. All deletion fragments of the putative promoter region, but which contain the entire intron sequence, did not reveal any obvious changes in luciferase activity. However, deletion of 1.1 kb of the intron sequence downstream of the splice donor site resulted in the loss of promoter activity. The intron exhibits no enhancer activity for the gene; however, the mRNA stability was increased in the presence of the intron sequence. These results indicate that parts of the intron sequence are critical for basic promoter function of the Cx31 gene.
Analysis of KdpC of the K+‐transporting KdpFABC complex of Escherichia coli
Authors: Gaßel Michael , Altendorf Karlheinz
Published: March 2003

The Kdp complex, a high affinity ATP‐driven K+ transport system of Escherichia coli, is composed of the four membrane‐bound subunits KdpF, KdpA, KdpB and KdpC. Whereas the role of KdpB (catalytical subunit), KdpA (K+‐translocating subunit) and KdpF (stabilizing peptide) is well understood, the function of KdpC is still unknown. Therefore, a kdpC deletion strain was constructed and complementation experiments were performed using different kdpC constructs. Truncations of the kdpC gene revealed that only one derivative, which lacks base pairs coding for the four C‐terminal amino acids, was able to complement the chromosomal deletion of kdpC. Furthermore, complementation was also observed with kdpC of Mycobacterium tuberculosis, but not with kdpC from Clostridium acetobutylicum or Synechocystis sp. PCC6803. Sequence alignment of 17 different KdpC proteins led to the construction of hybrids between kdpC of E. coli and that of C. acetobutylicum. Complementation revealed that the N‐terminal transmembrane segment as well as the C‐terminal‐third of the protein can be exchanged between both species, but only one after the other. A simultaneous substitution of both regions was not possible.
A third crystal form of Wolinella succinogenes quinol:fumarate reductase reveals domain closure at the site of fumarate reduction
Authors: Lancaster C. Roy D. , Groß Roland , Simon Jörg
Published: March 2003

Quinol:fumarate reductase (QFR) is a membrane protein complex that couples the reduction of fumarate to succinate to the oxidation of quinol to quinone. Previously, the crystal structure of QFR from Wolinella succinogenes was determined based on two different crystal forms, and the site of fumarate binding in the flavoprotein subunit A of the enzyme was located between the FAD‐binding domain and the capping domain [Lancaster, C.R.D., Kröger, A., Auer, M., & Michel, H. (1999) Nature402, 377–385]. Here we describe the structure of W. succinogenes QFR based on a third crystal form and refined at 3.1 Å resolution. Compared with the previous crystal forms, the capping domain is rotated in this structure by approximately 14° relative to the FAD‐binding domain. As a consequence, the topology of the dicarboxylate binding site is much more similar to those of membrane‐bound and soluble fumarate reductase enzymes from other organisms than to that found in the previous crystal forms of W. succinogenes QFR. This and the effects of the replacement of Arg A301 by Glu or Lys by site‐directed mutagenesis strongly support a common mechanism for fumarate reduction in this superfamily of enzymes.
Characterization of the Cph1 holo‐phytochrome from Synechocystis sp. PCC 6803
Authors: Hübschmann Thomas , Börner Thomas , Hartmann Elmar , Lamparter Tilman
Published: March 2003

The cph1 gene from the unicellular cyanobacterium Synechoycstis sp. PCC 6803 encodes a protein with the characteristics of plant phytochromes and histidine kinases of two‐component phospho‐relay systems. Spectral and biochemical properties of Cph1 have been intensely studied in vitro using protein from recombinant systems, but virtually nothing is known about the situation in the natural host. In the present study, His6‐tagged Cph1 was isolated from Synechocystis cells. The cph1–his gene was expressed either under the control of the natural cph1 promoter or over‐expressed using the strong promoter of the psbA2 gene. Upon purification with nickel affinity chromatography, the presence of Cph1 in extracts was confirmed by immunoblotting and Zn2+‐induced fluorescence. The Cph1 extracts exhibited a red/far‐red photoactivity characteristic of phytochromes. Difference spectra were identical with those of the phycocyanobilin adduct of recombinant Cph1, implying that phycocyanobilin is the chromophore of Cph1 in Synechocystis.
Solution structure of microcin J25, the single macrocyclic antimicrobial peptide from Escherichia coli
Authors: Blond Alain , Cheminant Michel , Ségalas‐Milazzo Isabelle , Péduzzi Jean , Barthélémy Michel , Goulard Christophe , Salomón Raúl , Moreno Felipe , Farías Ricardo , Rebuffat Sylvie
Published: March 2003

The three‐dimensional solution structure of microcin J25, the single cyclic representative of the microcin antimicrobial peptide class produced by enteric bacteria, was determined using two‐dimensional 1H NMR spectroscopy and molecular modeling. This hydrophobic 21‐residue peptide exhibits potent activity directed to Gram‐negative bacteria. Its primary structure, cyclo(‐V1GIGTPISFY10GGGAGHVPEY20F‐), has been determined previously [Blond, A., Péduzzi, J., Goulard, C., Chiuchiolo, M. J., Barthélémy, M., Prigent, Y., Salomón, R.A., Farías, R.N., Moreno, F. & Rebuffat, S. (1999) Eur. J. Biochem., 259, 747–755]. Conformational parameters (3JNHCαH coupling constants, quantitative nuclear Overhauser enhancement data, chemical shift deviations, temperature coefficients of amide protons, NH–ND exchange rates) were obtained in methanol solution. Structural restraints consisting of 190 interproton distances inferred from NOE data, 11 φ backbone dihedral angle and 9 χ1 angle restraints derived from the coupling constants and three hydrogen bonds in agreement with the amide exchange rates were used as input for simulated annealing calculations and energy minimization in the program xplor. Microcin J25 adopts a well‐defined compact structure consisting of a distorted antiparallel β sheet, which is twisted and folded back on itself, thus resulting in three loops. Residues 7–10 and 17–20 form the more regular part of the β sheet. The region encompassing residues Gly11–His16 consists of a distorted β hairpin, which divides into two small loops and is stabilized by an inverse γ turn and a type I′β turn. The reversal of the chain leading to the Phe21–Pro6 loop results from a mixed β/γ turn. A cavity, in which the hydrophilic Ser8 side‐chain is confined, is delimited by two crab pincer‐like regions that comprise residues 6–8 and 18–1.
Nucleotide sequence analysis of 5′‐flanking region of salicylate hydroxylase gene, and identification and purification of a LysR‐type regulator, SalR
Authors: Sato Hiroyuki , Kudo Sayumi , Ohnishi Kuniharu , Mizuguchi Mitsuo , Goto Eiji , Suzuki Kenzi
Published: March 2003

The sal gene comprised of 1266 nucleotides encoding salicylate hydroxylase was cloned from the chromosomal DNA of Pseudomonas putida S‐1 and sequenced [Suzuki, K., Mizuguchi, M., Ohnishi, K. and Itagaki, E. (1996) Biochim. Biophys. Acta1275, 154–156]. Here, we describe the nucleotide sequences of the regulatory region of the sal gene and an ORF (salR gene) divergently oriented from the sal gene, which encodes the protein SalR. This gene product positively controls sal gene expression at the transcriptional level. The salR gene consists of 930 base pairs starting from a GTG codon and encodes a protein of 309 amino acids with a molecular mass of 34 542 Da. The amino‐acid sequence is homologous to LysR‐family regulatory proteins such as CatR of P. putida RB1 and has helix‐turn‐helix DNA binding motif near its N‐terminal. Transcription start sites of sal and salR genes were determined to lie 30‐ and 24‐bp upstream of the respective initiation codons and separated from each other by 78 nucleotides. A Shine–Dalgarno sequence and the putative promoter sequences containing −10 and −35 sequences were seen in the sal and salR genes. Expression of the salR gene on a plasmid in Escherichia coli cells was confirmed by DNA mobility shift assay. For the overexpression of the salR gene, it was cloned to pET28a (pSAHR) which was transferred to E. coli BL21 (E. coli BL21/pSAHR), and expressed by an inducer, isopropyl thio‐β‐d‐galactoside. SalR was further purified to homogeneity from the cell‐free extracts in yields of approximately 3 mg·L−1 culture volume. The molecular mass was determined to be 33 kDa and the N‐terminal amino‐acid sequence was the same as that deduced from the nucleotide sequence of salR gene. Native SalR was also purified to homogeneity from P. putida S‐1 with very low contents. The properties of the protein were similar to those of SalR expressed in E. coli.
Structural consequences of site‐directed mutagenesis in flexible protein domains
Authors: Golovanov Alexander P. , Hawkins Dawn , Barsukov Igor , Badii Ramin , Bokoch Gary M. , Lian Lu‐Yun , Roberts Gordon C. K.
Published: March 2003

The guanine dissociation inhibitor RhoGDI consists of a folded C‐terminal domain and a highly flexible N‐terminal region, both of which are essential for biological activity, that is, inhibition of GDP dissociation from Rho GTPases, and regulation of their partitioning between membrane and cytosol. It was shown previously that the double mutation L55S/L56S in the flexible region of RhoGDI drastically decreases its affinity for Rac1. In the present work we study the effect of this double mutation on the conformational and dynamic properties of RhoGDI, and describe the weak interaction of the mutant with Rac1 using chemical shift mapping. We show that the helical content of the region 45–56 of RhoGDI is greatly reduced upon mutation, thus increasing the entropic penalty for the immobilization of the helix, and contributing to the loss of binding. In contrast to wild‐type RhoGDI, no interaction with Rac1 could be identified for amino‐acid residues of the flexible domain of the mutant RhoGDI and only very weak binding was observed for the folded domain of the mutant. The origins of the effect of the L55S/L56S mutation on the binding constant (decreased by at least three orders of magnitude relative to wild‐type) are discussed with particular reference to the flexibility of this part of the protein.
Insight into thyrotropin receptor cleavage by engineering the single polypeptide chain luteinizing hormone receptor into a cleaving, two subunit receptor
Authors: Chazenbalk Gregorio D. , McLachlan Sandra M. , Chen Chun‐Rong , Rapoport Basil
Published: March 2003

To gain insight into the thyrotropin hormone (TSH) receptor (TSHR) cleavage, we sought to convert the noncleaving luteinizing hormone (LH) receptor (LHR) into a cleaved, two‐subunit molecule. For this purpose, we generated a series of LHR mutants and chimeric LH‐TSH receptors. Cleavage of mature, ligand binding receptors on the cell surface was determined by covalent 125I‐labeled hCG crosslinking to intact, stably transfected mammalian cells. We first targeted a cluster of three N‐linked glycans in the LHR (N295, N303, N317) in a region corresponding to the primary TSHR cleavage site, which has only one N‐linked glycan. Elimination by mutagenesis of the most strategic N‐linked glycan (LHR‐N317Q) generated only a trace amount of LHR cleavage. Removal of the other N‐linked glycans had no additive effect. A much greater degree of cleavage (≈ 50%) was evident in a chimeric LH‐TSHR in which the juxtamembrane segment of the LHR (domain E; amino acids 317–367) was replaced with the corresponding domain of the TSHR (residues 363–418). Similarly cleaving LHR were created using a much smaller component within this region, namely LHR‐NET317–319 replaced with TSHR‐GQE367–369, or by substitution of the same three amino‐acid residues with AAA (LHR‐NET317–319AAA). In summary, our data alter current concepts regarding TSHR cleavage by suggesting limited (not absent) amino‐acid specificity in a region important for TSHR cleavage (GQE367–369). The data also support the concept of a separate and distinct downstream cleavage site 2 in the TSHR.
Low and high density lipoprotein metabolism in primary cultures of hepatic cells from normal and apolipoprotein E knockout mice
Authors: Auger Anick , Truong To‐Quyen , Rhainds David , Lapointe Jany , Letarte Fréderic , Brissette Louise
Published: March 2003

Apolipoprotein E (apoE) plays a major role in lipoprotein metabolism by mediating the binding of apoE‐containing lipoproteins to receptors. The role of hepatic apoE in the catabolism of apoE‐free lipoproteins such as low density lipoprotein (LDL) and high density lipoprotein‐3 (HDL3) is however, unclear. We analyzed the importance of hepatic apoE by comparing human LDL and HDL3 metabolism in primary cultures of hepatic cells from control C57BL/6J and apoE knockout (KO) mice. Binding analysis showed that the maximal binding capacity (Bmax) of LDL, but not of HDL3, is increased by twofold in the absence of apoE synthesis/secretion. Compared to control hepatic cells, LDL and HDL3 holoparticle uptake by apoE KO hepatic cells, as monitored by protein degradation, is reduced by 54 and 77%, respectively. Cleavage of heparan sulfate proteoglycans (HSPG) by treatment with heparinase I reduces LDL association by 21% in control hepatic cells. Thus, HSPG alone or a hepatic apoE‐HSPG complex is partially involved in LDL association with mouse hepatic cells. In apoE KO, but not in normal hepatic cells, the same treatment increases LDL uptake/degradation by 2.4‐fold suggesting that in normal hepatic cells, hepatic apoE increases LDL degradation by masking apoB‐100 binding sites on proteoglycans. Cholesteryl ester (CE) association and CE selective uptake (CE/protein association ratio) from LDL and HDL3 by mouse hepatic cells were not affected by the absence of apoE expression. We also show that 69 and 72% of LDL‐CE hydrolysis in control and apoE KO hepatic cells, respectively, is sensitive to chloroquine revealing the importance of a pathway linked to lysosomes. In contrast, HDL3‐CE hydrolysis is only mediated by a nonlysosomal pathway in both control and apoE KO hepatic cells. Overall, our results indicate that hepatic apoE increases the holoparticle uptake pathway of LDL and HDL3 by mouse hepatic cells, that HSPG devoid of apoE favors LDL binding/association but impairs LDL uptake/degradation and that apoE plays no significant role in CE selective uptake from either human LDL or HDL3 lipoproteins.
Conformational model for the consensus V3 loop of the envelope protein gp120 of HIV‐1 in a 20% trifluoroethanol/water solution
Authors: Vranken Wim F. , Fant Franky , Budesinsky Milos , Borremans Frans A. M.
Published: March 2003

Based on experimental NMR data, a model was generated for the conformation of the disulfide‐bond‐closed cyclic peptide corresponding to the whole V3 loop of the consensus HIV‐1 strain in a 20% trifluoroethanol/water solution. The obtained family of structures shows a prominent and well‐defined amphipathic α helix at the C‐terminal end of the peptide from Thr23 to Gln32. A series of turns characterizes the central Gly15–Tyr21 region, while the N‐terminal region is poorly defined. Independent experimental data confirms the features of this model, and suggests that this type of conformation can be readily adopted when the V3 loop is in contact with a membrane. The examined V3 loop belongs to a macrophage tropic strain, and using the model, a structural explanation is proposed for the different requirements of V3 loops belonging to macrophage and T‐cell line tropic HIV‐1 strains.
Carbohydrate binding properties of banana (Musa acuminata) lectin
Authors: Mo Hanqing , Winter Harry C. , Van Damme Els J. M. , Peumans Willy J. , Misaki Akira , Goldstein Irwin J.
Published: March 2003

Examination of lectins of banana (Musa acuminata) and the closely related plantain (Musa spp.) by the techniques of quantitative precipitation, hapten inhibition of precipitation, and isothermal titration calorimetry showed that they are mannose/glucose binding proteins with a preference for the α‐anomeric form of these sugars. Both generate precipitin curves with branched chain α‐mannans (yeast mannans) and α‐glucans (glycogens, dextrans, and starches), but not with linear α‐glucans containing only α1,4‐ and α1,6‐glucosidic bonds (isolichenan and pullulan). The novel observation was made that banana and plantain lectins recognize internal α1,3‐linked glucosyl residues, which occur in the linear polysaccharides elsinan and nigeran. Concanavalin A and lectins from pea and lentil, also mannose/glucose binding lectins, did not precipitate with any of these linear α‐glucans. This is, the authors believe, the first report of the recogniton of internal α1,3–glucosidic bonds by a plant lectin. It is possible that these lectins are present in the pulp of their respective fruit, complexed with starch.
Secreted aspartic proteases of Candida albicans, Candida tropicalis, Candida parapsilosis and Candida lusitaniae
Authors: Pichová Iva , Pavlíčková Libuše , Dostál Jiří , Dolejší Elena , Hrušková‐Heidingsfeldová Olga , Weber Jan , Ruml Tomáš , Souček Milan
Published: March 2003

The frequency of Candida infections has increased in recent years and it has been accompanied by a significant rise in morbidity and mortality. The secretion of aspartic proteases by Candida spp. was demonstrated to be one of the virulence determinants. Candida albicans is classified as the major human pathogen in the genus Candida. However, other species of this genus have been found to cause an increasing number of candidiases. We isolated secreted aspartic proteases (Saps) of C. albicans (Sap2p), C. tropicalis (Sapt1p), C. parapsilosis (Sapp1p), and C. lusitaniae (Saplp) from culture media. All the isolated proteases were N‐terminally sequenced. Their specific proteolytic activities and sensitivity to series of peptidomimetic inhibitors modified in the type of scissile bond replacement as well as in the N‐ and C‐termini were analyzed. The most divergent substrate specificity was observed for the Sap of C. tropicalis. The specificity of Sap of C. lusitaniae is most closely related to that of Sap of C. parapsilosis. We designed and prepared an inhibitor containing phenylstatine isoster that was equipotent towards all four proteases within the range of 10−10−10−9 m. The HIV‐1 protease inhibitors ritonavir, saquinavir, indinavir, and nelfinavir were also tested for the inhibition of four Saps. Only ritonavir and saquinavir inhibited Sap2p, Sapt1p, Sapp1p, and Saplp in micromolar concentrations.
Conformation and polarity of the active site of xylanase I from Thermomonospora sp. as deduced by fluorescent chemoaffinity labeling
Authors: George Sudeep P. , Rao Mala B.
Published: March 2003

A fluorescent chemoaffinity label o‐phthalaldehyde (OPTA) was used to ascertain the conformational flexibility and polarity at the active site of xylanase I (Xyl I). The kinetics of inactivation of Xyl I with OPTA revealed that complete inactivation occurred due to the binding of one molecule of OPTA to the active site of Xyl I. The formation of a single fluorescent isoindole derivative corroborated these findings. OPTA has been known to form a fluorescent isoindole derivative by crosslinking the proximal thiol and amino groups of cysteine and lysine. The involvement of cysteine in the formation of a Xyl I‐isoindole derivative has been negated by fluorometric and chemical modification studies on Xyl I with group‐specific reagents and by amino‐acid analysis. The kinetic analysis of diethylpyrocarbonate‐modified Xyl I established the presence of an essential histidine at or near the catalytic site of Xyl I. Modification of histidine and lysine residues by diethylpyrocarbonate and 2,4,6‐trinitrobenzenesulfonic acid, respectively, abolished the ability of the enzyme to form an isoindole derivative with OPTA, indicating that histidine and lysine participate in the formation of the isoindole complex. A mechanism for the reaction of OPTA with histidine and lysine residues present in the protein structure has been proposed. Experimental evidence presented here suggests for the first time that the active site of Xyl I is conformationally more flexible and more easily perturbed in the presence of denaturants than the molecule as a whole. The changes in the fluorescence emission maxima of a model compound (isoindole adduct) in solvents of different polarity were compared with the fluorescence behaviour of the Xyl I‐isoindole derivative, leading to the conclusion that the active site is located in a microenvironment of low polarity.
Expression, crystallization and derivatization of the complete extracellular domain of the βc subunit of the human IL‐5, IL‐3 and GM‐CSF receptors
Authors: Gustin Sonja E. , Church Alice P. , Ford Sally C. , Mann David A. , Carr Paul D. , Ollis David L. , Young Ian G.
Published: March 2003

The major signalling entity of the receptors for the haemopoietic cytokines granulocyte‐macrophage colony stimulating factor (GM‐CSF), interleukin‐3 (IL‐3) and interleukin‐5 (IL‐5) is the shared βc receptor, which is activated by ligand‐specific α receptors. The βc subunit is a stable homodimer whose extracellular region consists of four fibronectin domains and appears to be a duplication of the cytokine receptor homology module. No four domain structure has been determined for this receptor family and the structure of the βc subunit remains unknown. We have expressed the extracellular domain in insect cells using the baculovirus system, purified it to homogeneity and determined its N‐terminal sequence. N‐glycosylation at two sites was demonstrated. Crystals of the complete domain have been obtained that are suitable for X‐ray crystallographic studies, following mutagenesis to remove one of the N‐glycosylation sites. The rhombohedral crystals of space group R3, with unit cell dimensions 186.1 Å and 103.5 Å, diffracted to a resolution of 2.9 Å using synchrotron radiation. Mutagenesis was also used to engineer cysteine substitution mutants which formed isomorphous Hg derivatives in order to solve the crystallographic phase problem. The crystal structure will help to elucidate how the βc receptor is activated by heterodimerization with the respective α/ligand complexes.
Folding and activity of recombinant human procollagen C‐proteinase enhancer
Authors: Moschcovich Laura , Bernocco Simonetta , Font Bernard , Rivkin Hadassah , Eichenberger Denise , Chejanovsky Nor , Hulmes David J. S. , Kessler Efrat
Published: March 2003

Recombinant human procollagen C‐proteinase enhancer (rPCPE) was expressed using a baculovirus system and purified to homogeneity using a three‐step procedure including heparin affinity chromatography. Heparin binding was dependent on the C‐terminal netrin‐like domain. The recombinant protein was found to be active, increasing the activity of procollagen C‐proteinase/bone morphogenetic protein‐1 on type I procollagen in a manner comparable to the native protein. Enhancing activity was dependent on intact disulfide bonding within the protein. By circular dichroism, the observed secondary structure of rPCPE was consistent with the known three‐dimensional structures of proteins containing homologous domains.
CYP94A5, a new cytochrome P450 from Nicotiana tabacum is able to catalyze the oxidation of fatty acids to the ω‐alcohol and to the corresponding diacid
Authors: Le Bouquin Renaud , Skrabs Manuela , Kahn Rachel , Benveniste Irène , Salaün Jean‐Pierre , Schreiber Lukas , Durst Francis , Pinot Franck
Published: March 2003

A full length cDNA encoding a new cytochrome P450‐dependent fatty acid hydroxylase (CYP94A5) was isolated from a tobacco cDNA library. CYP94A5 was expressed in S. cerevisiae strain WAT11 containing a P450 reductase from Arabidopsis thaliana necessary for catalytic activity of cytochrome P450 enzymes. When incubated for 10 min in presence of NADPH with microsomes of recombinant yeast, 9,10‐epoxystearic acid was converted into one major metabolite identified by GC/MS as 18‐hydroxy‐9,10‐epoxystearic acid. The kinetic parameters of the reaction were Km,app = 0.9 ± 0.2 µm and Vmax,app = 27 ± 1 nmol·min−1·nmol−1 P450. Increasing the incubation time to 1 h led to the formation of a compound identified by GC/MS as 9,10‐epoxy‐octadecan‐1,18‐dioic acid. The diacid was also produced in microsomal incubations of 18‐hydroxy‐9,10‐epoxystearic acid. Metabolites were not produced in incubations with microsomes of yeast transformed with a control plasmid lacking CYP94A5 and their production was inhibited by antibodies raised against the P450 reductase, demonstrating the involvement of CYP94A5 in the reactions. The present study describes a cytochrome P450 able to catalyze the complete set of reactions oxidizing a terminal methyl group to the corresponding carboxyl. This new fatty acid hydroxylase is enantioselective: after incubation of a synthetic racemic mixture of 9,10‐epoxystearic acid, the chirality of the residual epoxide was 40/60 in favor of 9R,10S enantiomer. CYP94A5 also catalyzed the ω‐hydroxylation of saturated and unsaturated fatty acids with aliphatic chain ranging from C12 to C18.
Detection of a tryptophan radical in the reaction of ascorbate peroxidase with hydrogen peroxide
Authors: Hiner Alexander N. P. , Martínez Jesús I. , Arnao Marino B. , Acosta Manuel , Turner Daniel D. , Lloyd Raven Emma , Rodríguez‐López José Neptuno
Published: March 2003

The reactivity of recombinant pea cytosolic ascorbate peroxidase (rAPX) towards H2O2, the nature of the intermediates and the products of the reaction have been examined using UV/visible and EPR spectroscopies together with HPLC. Compound I of rAPX, generated by reaction of rAPX with 1 molar equivalent of H2O2, contains a porphyrin π‐cation radical. This species is unstable and, in the absence of reducing substrate, decays within 60 s to a second species, compound I*, that has a UV/visible spectrum [λmax (nm) = 414, 527, 558 and 350 (sh)] similar, but not identical, to those of both horseradish peroxidase compound II and cytochrome c peroxidase compound I. Small but systematic differences were observed in the UV/visible spectra of compound I* and authentic rAPX compound II, generated by reaction of rAPX with 1 molar equivalent H2O2 in the presence of 1 molar equivalent of ascorbate [λmax (nm) = 416, 527, 554, 350 (sh) and 628 (sh)]. Compound I* decays to give a ‘ferric‐like’ species (λmax = 406 nm) that is not spectroscopically identical to ferric rAPX (λmax = 403 nm) with a first order rate constant, kdecay′ = (2.7 ± 0.3) × 10−4 s−1. Authentic samples of compound II evolve to ferric rAPX [kdecay = (1.1 ± 0.2) × 10−3 s−1]. Low temperature (10 K) EPR spectra are consistent with the formation of a protein‐based radical, with g values for compound I* (g∥ = 2.038, g⊥ = 2.008) close to those previously reported for the Trp191 radical in cytochrome c peroxidase (g∥ = 2.037, g⊥ = 2.005). The EPR spectrum of rAPX compound II was essentially silent in the g = 2 region. Tryptic digestion of the ‘ferric‐like’ rAPX followed by RP‐HPLC revealed a fragment with a new absorption peak near 330 nm, consistent with the formation of a hydroxylated tryptophan residue. The results show, for the first time, that rAPX can, under certain conditions, form a protein‐based radical analogous to that found in cytochrome c peroxidase. The implications of these data are discussed in the wider context of both APX catalysis and radical formation and stability in haem peroxidases.
Molecular cloning, characterization and regulation by cadmium of a superoxide dismutase from the ectomycorrhizal fungus Paxillus involutus
Authors: Jacob Christophe , Courbot Mikaël , Brun Annick , Steinman Howard M. , Jacquot Jean‐Pierre , Botton Bernard , Chalot Michel
Published: March 2003

The gene encoding a superoxide dismutase (PiSOD) was cloned by suppressive subtractive hybridization from cDNA library of the ectomycorrhizal fungus, Paxillus involutus, grown under cadmium‐stress conditions. The encoded protein was presumed to be localized in the peroxisomes because it contained a C‐terminal peroxisomal localization peptide (SKL) and lacked an N‐terminal mitochondrial transit peptide. Complementation of an Escherichia coli SOD null strain that is unable to grow in the presence of paraquat or cadmium indicated that cloned Pisod encoded a functional superoxide dismutase. Sensitivity of PiSOD activity to H2O2 but not KCN, and sequence homologies to other SODs strongly suggest that it is a manganese‐containing superoxide dismutase. Monitoring PiSOD transcript, immunoreactive polypeptide and superoxide dismutase activity following cadmium stress suggests that the principal level of control is post‐translational. This is, to our knowledge, the first insight in the characterization of molecular events that take place in an ectomycorrhizal fungus during exposure to heavy metals.
Regulation of cellular senescence by p53
Authors: Itahana Koji , Dimri Goberdhan , Campisi Judith
Published: March 2003

Many normal cells respond to potentially oncogenic stimuli by undergoing cellular senescence, a state of irreversibly arrested proliferation and altered differentiated function. Cellular senescence very likely evolved to suppress tumorigenesis. In support of this idea, it is regulated by several tumor suppressor genes. At the heart of this regulation is p53. p53 is essential for the senescence response to short telomeres, DNA damage, oncogenes and supraphysiological mitogenic signals, and overexpression of certain tumor suppressor genes. Despite the well‐documented central role for p53 in the senescence response, many questions remain regarding how p53 senses senescence‐inducing stimuli and how it elicits the senescent phenotype.
Allosteric sites of phosphodiesterase‐5 (PDE5)
Authors: Gopal Venkatesh K. , Francis Sharron H. , Corbin Jackie D.
Published: March 2003

To date, relative cellular levels of cGMP and cGMP‐binding proteins have not been considered important in the regulation of smooth muscle or any other tissue. In rabbit penile corpus cavernosum, intracellular cGMP was determined to be 18 ± 4 nm, whereas the cGMP‐binding sites of types Iα and Iβ cGMP‐dependent protein kinase (PKG) and cGMP‐binding cGMP‐specific phosphodiesterase (PDE5) were 58 ± 14 nm and 188 ± 6 nm, respectively, as estimated by two different methods for each protein. Thus, total cGMP‐binding sites (246 nm) greatly exceed total cGMP. Given this excess of cGMP‐binding sites and the high affinities of PKG and PDE5 for cGMP, it is likely that a large portion of intracellular cGMP is associated with these proteins, which could provide a dynamic reservoir for cGMP. Phosphorylation of PDE5 by PKG is known to increase the affinity of PDE5 allosteric sites for cGMP, suggesting the potential for regulation of a reservoir of cGMP bound to this protein. Enhanced binding of cGMP by phosphorylated PDE5 could reduce the amount of cGMP available for activation of PKG, contributing to feedback inhibition of smooth muscle relaxation or other processes. This introduces a new concept for cyclic nucleotide signaling.
Methionine oxidation and its effect on the stability of a reconstituted subunit of the light‐harvesting complex from Rhodospirillum rubrum
Authors: Wang Zheng‐Yu , Shimonaga Masahiro , Muraoka Yoshiyuki , Kobayashi Masayuki , Nozawa Tsunenori
Published: March 2003

An additional component in the purified core light‐harvesting complex (LH1) from wild‐type purple photosynthetic bacterium Rhodospirillum rubrum has been identified as an oxidized species of α‐polypeptide by MALDI‐TOF mass spectrometry. This component appears as a slightly earlier‐eluting peak in the RP‐HPLC chromatogram compared with the authentic α‐polypeptide. The oxidation site has been determined to be the N‐terminal methionine residue by high‐resolution NMR spectroscopy, where the methionine is oxidized to methionine sulfoxide in a diastereoisomeric form. Interconversion between the oxidized and authentic α‐polypeptides has been confirmed by selective oxidation and reduction. The oxidative modification of methionine is shown to have discernible effects on the ability to form B820 subunit with β‐polypeptide and bacteriochlorophyll a, and on the stability of the reconstituted B820 subunit. Both the ability and the stability for the samples using the oxidized α‐polypeptide are moderately reduced, indicating that the oxidation‐induced conformational change in the N‐terminal domain of α‐polypeptide may affect the pigment‐binding environment through a long‐range interaction. The MALDI‐TOF mass results also reveal that the N‐terminus of α‐polypeptide is formylated and no phosphorylation has occurred in this polypeptide.
Oxidative folding of murine prion mPrP(23–231)
Authors: Lu Bao‐Yuan , Beck Pamela J. , Chang Jui‐Yoa
Published: March 2003

A systematic study of the oxidative folding of murine prion protein mPrP(23–231) is reported here. Folding of mPrP(23–231) involves formation of a single disulfide bond, Cys179–Cys214. Despite this simplicity, reduced mPrP(23–231) exhibits numerous unusual folding properties. In the absence of denaturant, folding of mPrP(23–231) is extremely sluggish, regardless of pH. The optimal pH for mPrP(23–231) folding was found to be 4–5. At pH 8.0, a condition that typically favors disulfide formation, folding of mPrP(23–231) hardly occurs, and it not facilitated by inclusion of redox agent. In the presence of denaturant (4 m urea or 2 m guanidine hydrochloride) and basic pH (8.0), reduced mPrP(23–231) refolds to the native structure quantitatively. The efficiency of folding can be further promoted by the presence of oxidized glutathione. At pH 4.0 and in the presence of 4 m urea, reduced mPrP(23–231) converts to three distinctive conformational isomers, unable to form the native structure. These unusual properties lead us to the following conclusions. The reduced mPrP(23–231) adopts a highly rigid structure with the two cysteines buried or situated apart. The presence of denaturant or low pH disrupts this rigid structure and lowers the energy barrier, which permits oxidation and refolding of the reduced mPrP(23–231). Under selected conditions, reduced mPrP(23–231) is capable of taking on multiple forms of stable conformational isomer that are segregated by energy barriers.
Allosteric activation of pyruvate kinase via NAD+ in rat liver cells
Authors: Devin Anne , Nogueira Véronique , Leverve Xavier , Guérin Bernard , Rigoulet Michel
Published: March 2003

In isolated rat hepatocytes, it has previously been reported that a rise in the ATP content induces a proportional increase in cytosolic NAD+ concentration [Devin, A., Guérin, B. & Rigoulet, M. (1997) FEBS Lett.410, 329–332]. This occurs under physiological conditions such as various substrates or different energetic states. To investigate the effect of a physiological rise in cytosolic [NAD+] per se on glycolysis and gluconeogenesis, an increase in [NAD+] induced by exogenous nicotinamide addition was obtained without a change in redox potential, ATP/ADP ratio and ATP concentration. Using dihydroxyacetone as substrate, we found that an increase in cytosolic [NAD+] decreases gluconeogenesis and enhances glycolysis without significant alteration of dihydroxyacetone consumption rate. These modifications are the consequence of an allosteric activation of pyruvate kinase via cytosolic NAD+ content. Thus, in addition to the well‐known thermodynamic control of glycolysis by pyridine‐nucleotide redox status, our study points to a new mechanism of glycolytic flux regulation by NAD+ concentration at the level of pyruvate kinase activity.