bluuebunny/crossref_metadata_2025 huggingface dataset:
Analyses of the CYP11B gene family in the guinea pig suggest the existence of a primordial CYP11B gene with aldosterone synthase activity
Authors: Bülow Hannes E. , Bernhardt Rita
Published: March 2003

In this study we describe the isolation of three genes of the CYP11B family of the guinea pig. CYP11B1 codes for the previously described 11β‐hydroxylase [Bülow, H.E.,Möbius, K., Bähr, V. & Bernhardt, R. (1996) Biochem. Biophys. Res. Commun. 221, 304–312] while CYP11B2 represents the aldosterone synthase gene. As no expression for CYP11B3 was detected this gene might represent a pseudogene. Transient transfection assays show higher substrate specificity for its proper substrate for CYP11B1 as compared to CYP11B2, which could account for the zone‐specific synthesis of mineralocorticoids and glucocorticoids, respectively. Thus, CYP11B2 displayed a fourfold higher ability to perform 11β‐hydroxylation of androstenedione than CYP11B1, while this difference is diminished with the size of the C17 substituent of the substrate. Furthermore, analyses with the electron transfer protein adrenodoxin indicate differential sensitivity of CYP11B1 and CYP11B2 as well as the three hydroxylation steps catalysed by CYP11B2 to the availability of reducing equivalents. Together, both mechanisms point to novel protein intrinsic modalities to achieve tissue‐specific production of mineralocorticoids and glucocorticoids in the guinea pig. In addition, we conducted phylogenetic analyses. These experiments suggest that a common CYP11B ancestor gene that possessed both 11β‐hydroxylase and aldosterone synthase activity underwent a gene duplication event before or shortly after the mammalian radiation with subsequent independent evolution of the system in different lines. Thus, a differential mineralocorticoid and glucocorticoid synthesis might be an exclusive achievement of mammals.
Presence and regulation of the endocannabinoid system in human dendritic cells
Authors: Matias Isabel , Pochard Pierre , Orlando Pierangelo , Salzet Michel , Pestel Joel , Di Marzo Vincenzo
Published: March 2003

Cannabinoid receptors and their endogenous ligands, the endocannabinoids, have been detected in several blood immune cells, including monocytes/macrophages, basophils and lymphocytes. However, their presence in dendritic cells, which play a key role in the initiation and development of the immune response, has never been investigated. Here we have analyzed human dendritic cells for the presence of the endocannabinoids, anandamide and 2‐arachidonoylglycerol (2‐AG), the cannabinoid CB1 and CB2 receptors, and one of the enzymes mostly responsible for endocannabinoid hydrolysis, the fatty acid amide hydrolase (FAAH). By using a very sensitive liquid chromatography‐atmospheric pressure chemical ionization‐mass spectrometric (LC‐APCI‐MS) method, lipids extracted from immature dendritic cells were shown to contain 2‐AG, anandamide and the anti‐inflammatory anandamide congener, N‐palmitoylethanolamine (PalEtn) (2.1 ± 1.0, 0.14 ± 0.02 and 8.2 ± 3.9 pmol·10−7 cells, respectively). The amounts of 2‐AG, but not anandamide or PalEtn, were significantly increased following cell maturation induced by bacterial lipopolysaccharide (LPS) or the allergen Der p 1 (2.8‐ and 1.9‐fold, respectively). By using both RT‐PCR and Western immunoblotting, dendritic cells were also found to express measurable amounts of CB1 and CB2 receptors and of FAAH. Cell maturation did not consistently modify the expression of these proteins, although in some cell preparations a decrease of the levels of both CB1 and CB2 mRNA transcripts was observed after LPS stimulation. These findings demonstrate for the first time that the endogenous cannabinoid system is present in human dendritic cells and can be regulated by cell activation.
Functional characterization of the maltose ATP‐binding‐cassette transporter of Salmonella typhimurium by means of monoclonal antibodies directed against the MalK subunit
Authors: Stein Anke , Seifert Martina , Volkmer‐Engert Rudolf , Siepelmeyer Jörg , Jahreis Knut , Schneider Erwin
Published: March 2003

The maltose ATP‐binding cassette transporter of Salmonella typhimurium is composed of a membrane‐associated complex (MalFGK2) and a periplasmic receptor (MalE). In addition to its role in transport, the complex acts as a repressor of maltose‐regulated gene expression and is subject to inhibition in the process of inducer exclusion. These activities are thought to be mediated by interactions of the ATPase subunit, MalK, with the transcriptional activator, MalT, and nonphosphorylated enzyme IIA of the glucose phosphotransferase system, respectively. To gain further insight in protein regions that are critical for these functions, we have generated nine MalK‐specific monoclonal antibodies. These bind to four nonoverlapping linear epitopes: 60‐LFig‐63 (5B5), 113‐RVNQVAEVLQL‐123 (represented by 4H12), 309‐GHETQI‐314 (2F9) and 352‐LFREDGSACR‐361 (represented by 4B3). All mAbs recognize their epitopes in soluble MalK and in the MalFGK2 complex with Kd values ranging from 10−6 to 10−8 m. ATP reduced the affinity of the mAbs for soluble MalK, indicating a conformational change that renders the epitopes less accessible. 4H12 and 5B5 inhibit the ATPase activity of MalK and the MalE/maltose‐stimulated ATPase activity of proteoliposomes, while their Fab fragments displayed no significant effect. The results suggest a similar solvent‐exposed position of helix 3 in the MalK dimer and in the intact complex and might argue against a direct role in the catalytic process. 4B3 and 2F9 exhibit reduced binding to the MalFGK2 complex in the presence of MalT and enzyme IIAGlc, respectively, thereby providing the first direct evidence for the C‐terminal domain of MalK being the site of interaction with the regulatory proteins.
Enhancement of oxidative stress‐induced apoptosis by Hsp105α in mouse embryonal F9 cells
Authors: Yamagishi Nobuyuki , Saito Youhei , Ishihara Keiichi , Hatayama Takumi
Published: March 2003

Hsp105α is one of the major mammalian heat shock proteins that belongs to the HSP105/110 family, and is expressed at especially high levels in the brain as compared with other tissues in mammals. Previously, we showed that Hsp105α prevents stress‐induced apoptosis in neuronal PC12 cells, and is a novel anti‐apoptotic neuroprotective factor in themammalian brain. On the other hand, we have also demonstrated that Hsp105α is expressed transiently at highlevels during mouse embryogenesis and is found not only invarious tissues but also in apoptotic cells. In the presentstudy, to elucidate the role of Hsp105α during mouseembryogenesis, we established mouse embryonal F9 cell lines that constitutively over‐express Hsp105α. Over‐expression of Hsp105α enhanced hydrogen peroxide‐induced apoptosis by enhancing the activation of caspase‐3, poly(ADP‐ribose)polymerase cleavage, cytochrome crelease and activation of p38 mitogen‐activated protein kinase (p38). Furthermore, oxidative stress‐induced apoptosis was suppressed by SB202190, a potent inhibitor of p38, in F9 cells. These findings indicated that the activation of p38 is an essential step for apoptosis in F9 cells and that Hsp105α enhances activation of p38, release of cytochrome c and caspase activation. Hsp105α may play important roles in organogenesis, during which marked apoptosis occurs, by enhancing apoptosis during mouse embryogenesis.
Identification and localization of glycine in the inner core lipopolysaccharide of Neisseria meningitidis
Authors: Cox Andrew D. , Li Jianjun , Richards James C.
Published: March 2003

The amino acid glycine is identified as a component of the inner core oligosaccharide in meningococcal lipopolysaccharide (LPS). Ester‐linked glycine residues were consistently found by mass spectrometry experiments to be located on the distal heptose residue (HepII) in LPS from several strains of Neisseria meningitidis. Nuclear magnetic resonance studies confirmed and extended this observation locating the glycine residue at the 7‐position of the HepII molecule in L3 and L4 immunotype strains.
The outer membrane component of the multidrug efflux pump from Pseudomonas aeruginosa may be a gated channel
Authors: Yoshihara Eisaku , Maseda Hideaki , Saito Kohjiro
Published: March 2003

OprM, the outer membrane component of the MexAB‐OprM multidrug efflux pump of Pseudomonas aeruginosa, has been assumed to facilitate the export of antibiotics across the outer membrane of this organism. Here we purified to homogeneity the OprM protein, reconstituted it into liposome membranes, and tested its channel activity by using the liposome swelling assay. It was demonstrated that OprM is a channel‐forming protein and exhibits the channel property that amino acids diffuse more efficiently than saccharides. However, antibiotics showed no significant diffusion through the OprM channel in the liposome membrane, suggesting that OprM functions as a gated channel. We reasoned that the protease treatment may cause the disturbance of the gate structure of OprM. Hence, we treated OprM reconstituted in the membranes with α‐chymotrypsin and examined its solute permeability. The results demonstrated that the protease treatment caused the opening of an OprM channel through which antibiotics were able to diffuse. To elucidate which cleavage is intimately related to the opening, we constructed mutant OprM proteins where the amino acid at the cleavage site was replaced with another amino acid. By examining the channel activity of these mutant proteins, it was shown that the proteolysis at tyrosine 185 and tyrosine 196 of OprM caused the channel opening. Furthermore, these residues were shown to face into the periplasmic space and interact with other component(s). We considered the possible opening mechanism of the OprM channel based on the structure of TolC, a homologue of OprM.
Inhibition of the NF‐κB transcriptional activity by protein kinase A
Authors: Takahashi Naoko , Tetsuka Toshifumi , Uranishi Hiroaki , Okamoto Takashi
Published: March 2003

The cAMP‐dependent protein kinase (PKA) signaling pathway plays a major role in a number of pathophysiological conditions. However, there have been conflicting evidences regarding the action of cAMP/PKA on nuclear factor‐ κB (NF‐κB). In this study, we have explored the effect of cAMP/PKA on NF‐κΒ activity and determined its molecular mechanism. PKA activating agents or expression of the catalytic subunit of PKA (PKAc) inhibited the NF‐κΒ‐dependent reporter gene expression induced by tumor necrosis factor α (TNFα). PKA activators affected neither IκBα phosphorylation, IκΒα degradation, nor the NF‐κΒ/DNA binding. Expression of PKAc inhibited the transactivation potential of Gal4‐p65 (286–551) suggesting that the inhibitory action of PKA is through the C‐terminal transactivation domain of p65 but not by phosphorylation of the consensus PKA recognition site containing serine at position 276. Overexpression of coactivators, CBP (CREB‐binding protein) and p300, failed to reverse the PKA‐mediated inhibition of p65 transactivation. Thus, the inhibitory action of the cAMP/PKA pathway on the transcriptional activity of NF‐κB appears to be exhibited by modifying the C‐terminal transactivation domain of p65, either directly or indirectly.
Kinetics of violaxanthin de‐epoxidation by violaxanthin de‐epoxidase, a xanthophyll cycle enzyme, is regulated by membrane fluidity in model lipid bilayers
Authors: Latowski Dariusz , Kruk Jerzy , Burda Kvetoslava , Skrzynecka‐Jaskier Marta , Kostecka‐Gugała Anna , Strzałka Kazimierz
Published: March 2003

This paper describes violaxanthin de‐epoxidation in model lipid bilayers. Unilamellar egg yolk phosphatidylcholine (PtdCho) vesicles supplemented with monogalactosyldiacylglycerol were found to be a suitable system for studying this reaction. Such a system resembles more the native thylakoid membrane and offers better possibilities for studying kinetics and factors controlling de‐epoxidation of violaxanthin than a system composed only ofmonogalactosyldiacylglycerol and is commonly used in xanthophyll cycle studies. The activity of violaxanthin de‐epoxidase (VDE) strongly depended on the ratio of monogalactosyldiacylglycerol to PtdCho in liposomes. The mathematical model of violaxanthin de‐epoxidation was applied to calculate the probability of violaxanthin to zeaxanthin conversion at different phases of de‐epoxidation reactions. Measurements of deepoxidation rate and EPR‐spin label study at different temperatures revealed that dynamic properties of the membrane are important factors that might control conversion of violaxanthin to antheraxanthin. A model of the molecular mechanism of violaxanthin de‐epoxidation where the reversed hexagonal structures (mainly created by monogalactosyldiacylglycerol) are assumed to be required for violaxanthin conversion to zeaxanthin is proposed. The presence of monogalactosyldiacylglycerol reversed hexagonal phase was detected in the PtdCho/monogalactosyldiacylglycerol liposomes membrane by 31P‐NMR studies. The availability of violaxanthin for de‐epoxidation is a diffusion‐dependent process controlled by membrane fluidity. The significance of the presented results for understanding themechanism of violaxanthin de‐epoxidation in native thylakoid membranes is discussed.
Solution structure of the HIV gp120 C5 Domain
Authors: Guilhaudis Laure , Jacobs Amy , Caffrey Michael
Published: March 2003

In HIV the viral envelope protein is processed by a host cell protease to form gp120 and gp41. The C1 and C5 domains of gp120 are thought to directly interact with gp41 but are largely missing from the available X‐ray structure. Biophysical studies of the HIV gp120 C5 domain (residues 489–511 of HIV‐1 strain HXB2), which corresponds to the carboxy terminal region of gp120, have been undertaken. CD studies of the C5 domain suggest that it is unstructured in aqueous solutions but partially helical in trifluoroethanol/aqueous and hexafluoroisopropanol/aqueous buffers. The solution structure of the C5 peptide in 40% trifluoroethanol/aqueous buffer was determined by NMR spectroscopy. The resulting structure is a turn helix structural motif, consistent with the CD results. Fluorescence titration experiments suggest that HIV C5 forms a 1 : 1 complex with the HIV gp41 ectodomain in the presence of cosolvent with an apparent Kd of ≈ 1.0 µm. The absence of complex formation in the absence of cosolvent indicates that formation of the turn‐helix structural motif of C5 is necessary for complex formation. Examination of the C5 structure provides insight into the interaction between gp120 and gp41 and provides a possible target site for future drug therapies designed to disrupt the gp120/gp41 complex. In addition, the C5 structure lends insight into the site of HIV envelope protein maturation by the host enzymes furin and PC7, which provides other possible targets for drug therapies.
BIGH3 (TGFBI) Arg124 mutations influence the amyloid conversion of related peptides in vitro
Authors: Schmitt‐Bernard Clair‐Florent , Chavanieu Alain , Herrada Gudrun , Subra Guy , Arnaud Bernard , Demaille Jacques G. , Calas Bernard , Argilés Ángel
Published: March 2003

Amyloid deposits with Arg124 mutated TGFBI protein have been identified in autosomal dominant blinding corneal dystrophies. We assessed in vitro the mechanisms determining TGFBI protein amyloid transformation involving mutations of Arg124. Eight peptides synthesized following the TGFBI protein sequence, centered on codon Arg124 holding the previously reported amyloidogenic mutations and the respective controls were studied. Cys124 and His124 mutated peptide preparations contained significantly higher amounts of amyloid than the native peptide. Blocking the SH group of Cys124 and deleting the first four NH2‐terminal amino acids including Val112‐Val113 resulted in a decrease in amyloid fibril formation while deletion of the nine CONH2‐terminal residues increased amyloid fibril concentration. Fourrier transformed‐infrared spectroscopy analysis of the different peptide solutions showed an increase in β‐pleated sheet structures in those with enhanced amyloid yielding. We designed a peptide (BB1) likely to counteract the role of Val112‐Val113 in amyloid fibril formation. Incubation of Cys124 peptide with BB1 indeed resulted in a 35% inhibition of amyloid fibril formation.

Our results are in keeping with the clinical observations of Arg124 mutation‐linked amyloidosis and show the importance of Val112–Val113, disulfide and hydrogen bonding in increasing the β‐pleated conformation and amyloid formation. These findings shed new light on the molecular mechanisms of TGFBI protein amyloidogenesis and encourage further research on the use of specifically designed peptides as putative therapeutic agents for these disabling diseases.
Tag‐mediated isolation of yeast mitochondrial ribosome and mass spectrometric identification of its new components
Authors: Gan Xiang , Kitakawa Madoka , Yoshino Ken‐ichi , Oshiro Noriko , Yonezawa Kazuyoshi , Isono Katsumi
Published: March 2003

Mitochondrial ribosomal proteins (mrps) of the budding yeast, Saccharomyces cerevisiae, have been extensively characterized genetically and biochemically. However, the list of the genes encoding individual mrps is still not complete and quite a few of the mrps are only predicted from their similarity to bacterial ribosomal proteins. We have constructed a yeast strain in which one of the small subunit proteins, termed Mrp4, was tagged with S‐peptide and used for affinity purification of mitochondrial ribosome. Mass spectrometric analysis of the isolated proteins detected most of the small subunit mrps which were previously identified or predicted and about half of the large subunit mrps. In addition, several proteins of unknown function were identified. To confirm their identity further, we added tags to these proteins and analyzed their localization in subcellular fractions. Thus, we have newly established Ymr158w (MrpS8), Ypl013c (MrpS16), Ymr188c (MrpS17) and Ygr165w (MrpS35) as small subunit mrps and Img1, Img2, Ydr116c (MrpL1), Ynl177c (MrpL22), Ynr022c (MrpL50) and Ypr100w (MrpL51) as large subunit mrps.
The import of ferredoxin–NADP+ reductase precursor into chloroplasts is modulated by the region between the transit peptide and the mature core of the protein
Authors: Rial Daniela V. , Lombardo Verónica A. , Ceccarelli Eduardo A. , Ottado Jorgelina
Published: March 2003

Protein transport across organelles' membranes requires that precursor proteins adopt an unfolded structure in order to be translocated by the import machinery. Ferredoxin–NADP+ reductase precursor, as well as many others, acquires a tightly folded structure that needs to be unfolded before or during its import. Several steps of chloroplast protein import are not fully understood. In particular, the role of different regions of the precursor protein has not been completely elucidated. In this work, we have studied the import into chloroplasts of precursor proteins with inclusions of amino acid spacers between the transit peptide and the mature protein, and with deletions in the N‐terminal region of the mature enzyme. We measured the import rate constants for these precursors and the results indicate that the distance between the transit peptide and the core of the mature protein determines the import kinetics. The longer precursors were imported into the organelle faster than the wild type form. Precursors with deletions in the N‐terminal region of the mature protein also showed increased import rates compared to the wild type. Homology studies amongst all family members reveal that only chloroplastic proteins possess this region. We suggest that even if the first amino acids of the mature protein do not contribute to its overall structural stability, they condition the kinetic parameters of the import reaction. Besides, the distance between the transit peptide and the mature protein core may be modulating the import rate at which the chloroplast incorporates this protein from the cytosol.
Oxidation of phenols by laccase and laccase‐mediator systems
Authors: d'Acunzo Francesca , Galli Carlo , Masci Bernardo
Published: March 2003

To investigate how solubility and steric issues affect the laccase‐catalysed oxidation of phenols, a series of oligomeric polyphenol compounds, having increasing size and decreasing solubility in water, was incubated with laccase. The extent of substrate conversion, and the nature of the products formed in buffered aqueous solutions, were compared to those obtained in the presence of an organic cosolvent, and also in the presence of two mediating species, i.e. N‐hydroxyphthalimide (HPI) and 2,2,6,6‐tetramethylpiperidin‐1‐yloxy (TEMPO). This approach showed not only an obvious role of solubility, but also a significant role of the dimension of the substrate upon the enzymatic reactivity. In fact, reactivity decreases as substrate size increases even when solubility is enhanced by a cosolvent. This effect may be ascribed to limited accessibility of encumbered substrates to the enzyme active site, and can be compensated through the use of the appropriate mediator. While TEMPO was highly efficient at enhancing the reactivity of large, less soluble substrates, HPI proved less effective. In addition, whereas the laccase/HPI system afforded the same products as laccase alone, the use of TEMPO provided a different product with high specificity. These results offer the first evidence of the role of ‘oxidation shuttles’ that the mediators of laccase may have, but also suggest two promising routes towards an environmentally friendly process for kraft pulp bleaching: (a) the identification of mediators which, once oxidized by laccase, are able to target strategic functional groups present in lignin, and (b) the introduction of those strategic functional groups in an appropriate pretreatment.
Characterization of a Saccharomyces cerevisiae NADP(H)‐dependent alcohol dehydrogenase (ADHVII), a member of the cinnamyl alcohol dehydrogenase family
Authors: Larroy Carol , Parés Xavier , Biosca Josep A
Published: March 2003

A new NADP(H)‐dependent alcohol dehydrogenase (the YCR105W gene product, ADHVII) has been identified in Saccharomyces cerevisiae. The enzyme has been purified to homogeneity and found to be a homodimer of 40 kDa subunits and a pI of 6.2–6.4. ADHVII shows a broad substrate specificity similar to the recently characterized ADHVI (64% identity), although they show some differences in kinetic properties. ADHVI and ADHVII are the only members of the cinnamyl alcohol dehydrogenase family in yeast. Simultaneous deletion of ADH6 and ADH7 was not lethal for the yeast. Both enzymes could participate in the synthesis of fusel alcohols, ligninolysis and NADP(H) homeostasis.
The effects of ring‐size analogs of the antimicrobial peptide gramicidin S on phospholipid bilayer model membranes and on the growth of Acholeplasma laidlawii B
Authors: Kiricsi Monika , Prenner Elmar J. , Jelokhani‐Niaraki Masood , Lewis Ruthven N. A. H. , Hodges Robert S. , McElhaney Ronald N.
Published: March 2003

We have examined the effects of three ring‐size analogs of the cyclic β‐sheet antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior and permeability of phospholipid model membranes and on the growth of the cell wall‐less Gram‐positive bacteria Acholeplasma laidlawii B. These three analogs have ring sizes of 10 (GS10), 12 (GS12) or 14 (GS14) amino acids, respectively. Our high‐sensitivity differential scanning calorimetric studies indicate that all three of these GS analogs perturb the gel/liquid‐crystalline phase transition of zwitterionic phosphatidylcholine (PtdCho) vesicles to a greater extent than of zwitterionic phosphatidylethanolamine (PtdEtn) or of anionic phosphatidylglycerol (PtdGro) vesicles, in contrast to GS itself, which interacts more strongly with PtdGro than with PtdCho and PtdEtn bilayers. However, the relative potency of the perturbation of phospholipid phase behavior varies markedly between the three peptides, generally decreasing in the order GS14 > GS10 > GS12. Similarly, these three GS ring‐size analogs also differ considerably in their ability to cause fluorescence dye leakage from phospholipid vesicles, with the potency of permeabilization also generally decreasing in the order GS14 > GS10 > GS12. Finally, these GS ring‐size analogs also differentially inhibit the growth of A. laidlawii with growth inhibition also decreasing in the order GS14 > GS10 > GS12. These results indicate that the relative potencies of GS and its ring‐size analogs in perturbing the organization and increasing the permeability of phospholipid bilayer model membranes, and of inhibiting the growth of A. laidlawii B cells, are at least qualitatively correlated, and provide further support for the hypothesis that the primary target of these antimicrobial peptides is the lipid bilayer of the bacterial membrane. The very high antimicrobial activity of GS14 against the cell wall‐less bacteria A. laidlawii as compared to various conventional bacteria confirms our earlier suggestion that the avid binding of this peptide to the bacterial cell wall is primarily responsible for its reduced antimicrobial activity against such organisms. The relative magnitude of the effects of GS itself, and of the three ring‐size GS analogs, on phospholipid bilayer organization and cell growth correlate relatively well with the effective hydrophobicities and amphiphilicities of these peptides but less well with their relative charge density, intrinsic hydrophobicities or conformational flexibilities. Nevertheless, all of these parameters, as well as others, may influence the antimicrobial potency and hemolytic activity of GS analogs.
Loss‐of‐function variants of the human melanocortin‐1 receptor gene in melanoma cells define structural determinants of receptor function
Authors: Más Jesús Sánchez , Sánchez Concepción Olivares , Ghanem Ghanem , Haycock John , Teruel José Antonio Lozano , García‐Borrón José Carlos , Jiménez‐Cervantes Celia
Published: March 2003

The α‐melanocyte‐stimulating hormone (αMSH) receptor (MC1R) is a major determinant of mammalian skin and hair pigmentation. Binding of αMSH to MC1R in human melanocytes stimulates cell proliferation and synthesis of photoprotective eumelanin pigments. Certain MC1R alleles have been associated with increased risk of melanoma. This can be theoretically considered on two grounds. First, gain‐of‐function mutations may stimulate proliferation, thus promoting dysplastic lesions. Second, and opposite, loss‐of‐function mutations may decrease eumelanin contents, and impair protection against the carcinogenic effects of UV light, thus predisposing to skin cancers. To test these possibilities, we sequenced the MC1R gene from seven human melanoma cell (HMC) lines and three giant congenital nevus cell (GCNC) cultures. Four HMC lines and two GCNC cultures contained MC1R allelic variants. These were the known loss‐of‐function Arg142His and Arg151Cys alleles and a new variant, Leu93Arg. Moreover, impaired response to a superpotent αMSH analog was demonstrated for the cell line carrying the Leu93Arg allele and for a HMC line homozygous for wild‐type MC1R. Functional analysis in heterologous cells stably or transiently expressing this variant demonstrated that Leu93Arg is a loss‐of‐function mutation abolishing agonist binding. These results, together with site‐directed mutagenesis of the vicinal Glu94, demonstrate that the MC1R second transmembrane fragment is critical for agonist binding and maintenance of a resting conformation, whereas the second intracellular loop is essential for coupling to the cAMP system. Therefore, loss‐of‐function, but not activating MC1R mutations are common in HMC. Their study provides important clues to understand MC1R structure‐function relationships.
Purification and characterization of two secreted purple acid phosphatase isozymes from phosphate‐starved tomato (Lycopersicon esculentum) cell cultures
Authors: Bozzo Gale G. , Raghothama Kashchandra G. , Plaxton William C.
Published: March 2003

Two secreted acid phosphatases (SAP1 and SAP2) were markedly up‐regulated during Pi‐starvation of tomato suspension cells. SAP1 and SAP2 were resolved during cation‐exchange FPLC of culture media proteins from 8‐day‐old Pi‐starved cells, and purified to homogeneity and final p‐nitrophenylphosphate hydrolyzing specific activities of 246 and 940 µmol Pi produced·min−1 mg·protein−1, respectively. SDS/PAGE, periodic acid‐Schiff staining and analytical gel filtration demonstrated that SAP1 and SAP2, respectively, exist as 84 and 57 kDa glycosylated monomers. SAP1 and SAP2 are purple acid phosphatases (PAPs) as they displayed an absorption maximum at 518 and 538 nm, respectively, and were not inhibited by l‐tartrate. The respective sequence of a SAP1 and SAP2 tryptic peptide was very similar to a portion of the deduced sequence of several putative Arabidopsis thaliana PAPs. CNBr peptide mapping indicated that SAP1 and SAP2 are structurally distinct. Both isozymes displayed a pH optimum of approximately pH 5.3 and were heat stable. Although they exhibited wide substrate specificities, the Vmax of SAP2 with various phosphate‐esters was significantly greater than that of SAP1. SAP1 and SAP2 were activated by up to 80% by 5 mm Mg2+, and demonstrated potent competitive inhibition by molybdate, but mixed and competitive inhibition by Pi, respectively. Interestingly, both SAPs exhibited significant peroxidase activity, which was optimal at approximately pH 8.4 and insensitive to Mg2+ or molybdate. This suggests that SAP1 and SAP2 may be multifunctional proteins that operate: (a) PAPs that scavenge Pi from extracellular phosphate‐esters during Pi deprivation, or (b) alkaline peroxidases that participate in the production of extracellular reactive oxygen species during the oxidative burst associated with the defense response of plants to pathogen infection.
Structure of the O polysaccharides and serological classification of Pseudomonas syringae pv. porri from genomospecies 4
Authors: Zdorovenko Evelina L. , Zatonskii George V. , Kocharova Nina A. , Shashkov Aleksander S. , Knirel Yuriy A. , Ovod Vladimir V.
Published: March 2003

Strains of Pseudomonas syringae pv. porri are characterized by a number of pathovar‐specific phenotypic and genomic characters and constitute a highly homogeneous group. Using monoclonal antibodies, they all were classified in a novel P. syringae serogroup O9. The O polysaccharides (OPS) isolated from the lipopolysaccharides (LPS) of P. syringae pv. porri NCPPB 3365 and NCPPB 3364T possess multiple oligosaccharide O repeats, some of which are linear and composed of l‐rhamnose (l‐Rha), whereas the major O repeats are branched with l‐rhamnose in the main chain and GlcNAc in side chains (structures 1 and 2). Both branched O repeats, which differ in the position of substitution of one of the Rha residues and in the site of attachment of GlcNAc, were found in the two strains studied, O repeat 1 being major in strain NCPPB 3365 and 2 in strain NCPPB 3364T.

The relationship between OPS chemotype and serotype on one hand and the genomic characters of P. syringae pv. porri and other pathovars delineated in genomospecies 4 on the other hand is fdiscussed.
Microperoxidase 8 catalysed nitrogen oxides formation from oxidation of N‐hydroxyguanidines by hydrogen peroxide
Authors: Ricoux Rémy , Boucher Jean‐Luc , Mandon Dominique , Frapart Yves‐Michel , Henry Yann , Mansuy Daniel , Mahy Jean‐Pierre
Published: March 2003

Nitric oxide (NO) is a potent intra‐ and intercellular messenger involved in the control of vascular tone, neuronal signalling and host response to infection. In mammals, NO is synthesized by oxidation of l‐arginine catalysed by hemeproteins called NO‐synthases with intermediate formation of Nω‐hydroxy‐l‐arginine (NOHA). NOHA and some hydroxyguanidines have been shown to be able to deliver nitrogen oxides including NO in the presence of various oxidative systems. In this study, NOHA and a model compound, N‐(4‐chlorophenyl)‐N′‐hydroxyguanidine, were tested for their ability to generate NO in the presence of a haemprotein model, microperoxidase 8 (MP8), and hydrogen peroxide. Nitrite and nitrate production along with selective formation of 4‐chlorophenylcyanamide was observed from incubations of N‐(4‐chlorophenyl)‐N′‐hydroxyguanidine in the presence of MP8 and hydrogen peroxide. In the case of NOHA, the corresponding cyanamide, Nδ‐cyano‐L‐ornithine, was too unstable under the conditions used and l‐citrulline was the only product identified. A NO‐specific conversion of 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl 3‐oxide to 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl and formation of MP8–Fe–NO complexes were observed by EPR spectroscopy and low‐temperature UV/visible spectroscopy, respectively. These results clearly demonstrate the formation of nitrogen oxides including NO from the oxidation of exogenous hydroxyguanidines by hydrogen peroxide in the presence of a minienzyme such as MP8. The importance of the bioactivation of endogenous (NOHA) or exogenous N‐hydroxyguanidines by peroxidases of physiological interest remains to be established in vivo.
Thermal behaviour of cubic phases rich in 1‐monooleoyl‐rac‐glycerol in the ternary system
Authors: Persson Gerd , Edlund Håkan , Lindblom Göran
Published: March 2003

Using synchrotron X‐ray diffraction the thermal behaviour was studied of the cubic phases in the 1‐monooleoyl‐rac‐glycerol (MO)/n‐octyl‐β‐d‐glucopyranoside (OG)/2H2O system with 58 or 45 wt % MO concentration and varying OG/2H2O contents. These MO contents correspond to a Pn3m cubic single‐phase or a Pn3m cubic phase in excess water on the binary MO/water axis of the ternary phase diagram. The cubic liquid crystalline phases are stable with small fractions of OG, while higher OG concentrations trigger a cubic‐to‐lamellar phase transition. Moreover, with increasing OG concentration the initial Pn3m structure is completely converted to an Ia3d structure prior to the Lα phase being formed. Upon heating this effect is reversed, resulting in an Ia3d‐to‐Pn3m phase transition. For some samples additional peaks were observed in the diffractograms upon heating, resulting from the metastability notoriously shown by bicontinuous cubic phases. This judgement is supported by the fact that upon cooling these peaks were absent. Remarkably, both the Ia3d and the Pn3m cubic structures could be in equilibrium with excess water in this ternary system. A comparison is made with previous results on n‐dodecyl‐β‐d‐maltoside (DM), showing that cubic phases with OG have higher thermal and compositional stability than with DM.
Bioenergetics of the formyl‐methanofuran dehydrogenase and heterodisulfide reductase reactions in Methanothermobacter thermautotrophicus
Authors: de Poorter Linda M. I. , Geerts Wim G. , Theuvenet Alexander P. R. , Keltjens Jan T.
Published: March 2003

The synthesis of formyl‐methanofuran and the reduction of the heterodisulfide (CoM‐S‐S‐CoB) of coenzyme M (HS‐CoM) and coenzyme B (HS‐CoB) are two crucial, H2‐dependent reactions in the energy metabolism of methanogenic archaea. The bioenergetics of the reactions in vivo were studied in chemostat cultures and in cell suspensions of Methanothermobacter thermautotrophicus metabolizing at defined dissolved hydrogen partial pressures ( pH2). Formyl‐methanofuran synthesis is an endergonic reaction (ΔG°′ = +16 kJ·mol−1). By analyzing the concentration ratios between formyl‐methanofuran and methanofuran in the cells, free energy changes under experimental conditions (ΔG′) were found to range between +10 and +35 kJ·mol−1 depending on the pH2 applied. The comparison with the sodium motive force indicated that the reaction should be driven by the import of a variable number of two to four sodium ions.

Heterodisulfide reduction (ΔG°′ = −40 kJ·mol−1) was associated with free energy changes as high as −55 to −80 kJ·mol−1. The values were determined by analyzing the concentrations of CoM‐S‐S‐CoB, HS‐CoM and HS‐CoB in methane‐forming cells operating under a variety of hydrogen partial pressures. Free energy changes were in equilibrium with the proton motive force to the extent that three to four protons could be translocated out of the cells per reaction. Remarkably, an apparent proton translocation stoichiometry of three held for cells that had been grown at pH2<0.12 bar, whilst the number was four for cells grown above that concentration. The shift occurred within a narrow pH2 span around 0.12 bar. The findings suggest that the methanogens regulate the bioenergetic machinery involved in CoM‐S‐S‐CoB reduction and proton pumping in response to the environmental hydrogen concentrations.
Properties of purified gut trypsin from Helicoverpa zea, adapted to proteinase inhibitors
Authors: Volpicella Mariateresa , Ceci Luigi R. , Cordewener Jan , America Twan , Gallerani Raffaele , Bode Wolfram , Jongsma Maarten A. , Beekwilder Jules
Published: March 2003

Pest insects such as Helicoverpa spp. frequently feed on plants expressing protease inhibitors. Apparently, their digestive system can adapt to the presence of protease inhibitors. To study this, a trypsin enzyme was purified from the gut of insects that were raised on an inhibitor‐containing diet. The amino‐acid sequence of this enzyme was analysed by tandem MS, which allowed assignment of 66% of the mature protein amino acid sequence. This trypsin, called HzTrypsin‐S, corresponded to a known cDNA sequence from Helicoverpa. The amino acid sequence is closely related (76% identical) to that of a trypsin, HzTrypsin‐C, which was purified and identified in a similar way from insects raised on a diet without additional inhibitor. The digestive properties of HzTrypsin‐S and HzTrypsin‐C were compared. Both trypsins appeared to be equally efficient in degrading protein. Four typical plant inhibitors were tested in enzymatic measurements. HzTrypsin‐S could not be inhibited by > 1000‐fold molar excess of any of these. The same inhibitors inhibited HzTrypsin‐C with apparent equilibrium dissociation constants ranging from 1 nm to 30 nm. Thus, HzTrypsin‐S seems to allow the insect to overcome different defensive proteinase inhibitors in plants.
A functional polymorphism at the transcriptional initiation site in β2‐glycoprotein I (apolipoprotein H) associated with reduced gene expression and lower plasma levels of β2‐glycoprotein I
Authors: Mehdi Haider , Manzi Susan , Desai Purnima , Chen Qi , Nestlerode Cara , Bontempo Franklin , Strom Stephen C. , Zarnegar Reza , Kamboh M. Ilyas
Published: March 2003

Human β2‐glycoprotein I (β2GPI), also known as apolipoprotein H, has been implicated in haemostasis and the production of anti‐phospholipid antibodies. There is a wide range of interindividual variation in β2GPI plasma levels that is thought to be under genetic control, but its molecular basis remains unknown. To understand the genetic basis of β2GPI variation, we analyzed the 5′ flanking region of the β2GPI gene for mutation detection by DHPLC and identified a point mutation at the transcriptional initiation site (−1C→A) with a carrier frequency of 12.1%. The mutation was associated with significantly lower β2GPI plasma levels (P < 0.0001) and low occurrence of anti‐phospholipid antibodies in lupus patients (4.8% antibody‐positive group vs. 16.6% in the antibody‐negative group; P = 0.019). Northern blot analysis confirmed that the −1C→A mutation was associated with lower mRNA levels and it reduced the reporter (luciferase) gene expression by twofold. Electrophoretic gel mobility shift assay (EMSA) revealed that the −1C→A mutation disrupts the binding for crude hepatic nuclear extracts and purified TFIID. These results suggest that the substitution of C with A at the β2GPI transcriptional initiation site is a causative mutation that affects its gene expression at the transcriptional level and ultimately β2GPI plasma levels and the occurrence of anti‐phospholipid antibodies.
The heterogeneity of mast cell tryptase from human lung and skin
Authors: Peng Qi , McEuen Alan R. , Benyon R. Christopher , Walls Andrew F.
Published: March 2003

There has long been conjecture over the degree to which there may be structural and functional heterogeneity in the tetrameric serine protease tryptase (EC 3.4.21.59), a major mediator of allergic inflammation. We have applied 2D gel electrophoresis to analyze the extent, nature, and variability of this heterogeneity in lysates of mast cells isolated from lung and skin, and in preparations of purified tryptase. Gels were silver stained, or the proteins transferred to nitrocellulose blots and probed with either tryptase‐specific monoclonal antibodies or various lectins. Tryptase was the major protein constituent in mast cell lysates, and presented as an array of 9–12 diffuse immunoreactive spots with molecular masses ranging from 29 to 40 kDa, and pI values from 5.1 to 6.3. Although the patterns obtained for lung and skin tryptase were broadly similar, differences were observed between tissues and between individual donors. Lectin binding studies indicated the presence of mono‐antennary or bi‐antennary complex‐type oligosaccharide with varying degrees of sialylation. Deglycosylation with protein‐N‐glycosidase F (PNGase F) reduced the size of both lung and skin tryptase, while incubation with PNGase F or neuraminidase narrowed the pI range, indicating variable degrees of glycosylation as a major contributor to the size and charge heterogeneity. Comparison of different purified preparations of lung and skin tryptase revealed no significant difference in pH profiles, but differences were seen in reactivity towards a range of chromogenic substrates, with substantial differences in Km, kcat and degree of cooperativity. Mathematical modeling indicated that the variety in kinetics parameters could not result solely from the sum of varying amounts of isoforms obeying Michaelis–Menten kinetics but with different values of Km and kcat. The heterogeneity demonstrated for tryptase in these studies suggests that there are important differences in tryptase function in different tissues.
The 3′‐UTR of the mRNA coding for the major protein kinase C substrate MARCKS contains a novel CU‐rich element interacting with the mRNA stabilizing factors HuD and HuR
Authors: Wein Georg , Rössler Marek , Klug Roland , Herget Thomas
Published: March 2003

The expression of the major protein kinase C substrate MARCKS (myristoylated alanine‐rich C kinase substrate) is controlled by the stability of its mRNA. While the MARCKS mRNA is long living in quiescent fibroblasts (t1/2 = 14 h), its half‐life time is drastically reduced (t1/2 = 2 h) in cells treated with phorbol esters to activate protein kinase C (PKC) or treated with growth factors. In a first step to study the underlying mechanism we identified both a cis‐element on the MARCKS mRNA and the corresponding trans‐acting factors. Fusing the complete 3′‐UTR or specific regions of the 3′‐UTR of the MARCKS gene to a luciferase reporter gene caused a drastic decrease in luciferase expression to as low as 5–10% of controls. This down‐regulation was a result of destabilization of the chimeric transcript as shown by RNA run‐off and Northern blot‐assays. By RNase/EMSA and UV‐cross‐linking experiments, we identified a stretch of 52 nucleotides [(CUUU)11(U)8] in the 3′‐UTR of the MARCKS mRNA specifically recognized by two RNA‐binding proteins, HuD and HuR. These trans‐acting factors are members of the ELAV gene family and bind the MARCKS CU‐rich sequence with high affinity. Overexpression of HuD and HuR in murine fibroblasts caused a striking stabilization of the endogenous MARCKS mRNA even under conditions when the MARCKS mRNA is normally actively degraded, i.e. after treating cells with phorbol ester.

These data imply, that the identified CU‐rich cis‐element of the MARCKS 3′‐UTR is involved in conferring instability to mRNAs and that members of the ELAV gene family oppose this effect. Based on its structural and functional properties, the (CUUU)11(U)8 sequence described here can be grouped into class III of AU‐rich elements.
Identification and characterization of B′′‐subunits of protein phosphatase 2 A in Xenopus laevis oocytes and adult tissues
Authors: Stevens Ilse , Janssens Veerle , Martens Ellen , Dilworth Stephen , Goris Jozef , Van Hoof Christine
Published: March 2003

Protein phosphatase 2A is a phosphoserine/threonine phosphatase implicated in many cellular processes. The core enzyme comprises a catalytic and a PR65/A‐subunit. The substrate specificity and subcellular localization are determined by a third regulatory B‐subunit (PR55/B, PR61/B′ and PR72/130/B′′).

To identify the proteins of the B′′ family in Xenopus laevis oocytes, a prophase Xenopus oocyte cDNA library was screened using human PR130 cDNA as a probe. Three different classes of cDNAs were isolated. One class is very similar to human PR130 and is probably the Xenopus orthologue of PR130 (XPR130). A second class of clones (XN73) is identical to the N‐terminal part of XPR130 but ends a few amino acids downstream of the putative splicing site of PR130. To investigate how this occurs, the genomic structure of the human PR130 gene was determined. This novel protein does not act as a PP2A subunit but might compete with the function of PR130. The third set of clones (XPR70) is very similar to human PR48 but has an N‐terminal extension. Further analysis of the human EST‐database and the human PR48 gene structure, revealed that the human PR48 clone published is incomplete. The Xenopus orthologue of PR48 encodes a protein of 70 kDa which like the XPR130, interacts with the A‐subunit in GST pull‐down assays. XPR70 is ubiquitously expressed in adult tissues and oocytes whereas expression of XPR130 is very low in brain and oocytes. Expression of XN73 mainly parallels XPR130 with the exception of the brain.
A simple protocol to study blue copper proteins by NMR
Authors: Gelis Ioannis , Katsaros Nikolaos , Luchinat Claudio , Piccioli Mario , Poggi Luisa
Published: March 2003

In the case of oxidized plastocyanin from Synechocystis sp. PCC6803, an NMR approach based on classical two and three dimensional experiments for sequential assignment leaves unobserved 14 out of 98 amino acids. A protocol which simply makes use of tailored versions of 2D HSQC and 3D CBCA(CO)NH and CBCANH leads to the identification of nine of the above 14 residues. The proposed protocol differs from previous aproaches in that it does not involve the use of unconventional experiments designed specifically for paramagnetic systems, and does not exploit the occurrence of a corresponding diamagnetic species in chemical exchange with the blue copper form. This protocol is expected to extend the popularity of NMR in the structural studies of copper (II) proteins, allowing researchers to increase the amount of information available via NMR on the neighborhood of a paramagnetic center without requiring a specific expertise in the field. The resulting 3D spectra are standard spectra that can be handled by any standard software for protein NMR data analysis.