bluuebunny/crossref_metadata_2025_split huggingface dataset:
Bass hepcidin is a novel antimicrobial peptide induced by bacterial challenge
Authors: Shike Hiroko , Lauth Xavier , Westerman Mark E. , Ostland Vaughn E. , Carlberg James M. , Van Olst Jon C. , Shimizu Chisato , Bulet Philippe , Burns Jane C. 
Published: March 2003

We report the isolation of a novel antimicrobial peptide, bass hepcidin, from the gill of hybrid striped bass, white bass (Morone chrysops) × striped bass (M. saxatilis). After the intraperitoneal injection of Micrococcus luteus and Escherichia coli, the peptide was purified from HPLC fractions with antimicrobial activity against Escherichia coli. Sequencing by Edman degradation revealed a 21‐residue peptide (GCRFCCNCCPNMSGCGVCCRF) with eight putative cysteines. Molecular mass measurements of the native peptide and the reduced and alkylated peptide confirmed the sequence with four intramolecular disulfide bridges. Peptide sequence homology to human hepcidin and other predicted hepcidins, indicated that the peptide is a new member of the hepcidin family. Nucleotide sequences for cDNA and genomic DNA were determined for white bass. A predicted prepropeptide (85 amino acids) consists of three domains: a signal peptide (24 amino acids), prodomain (40 amino acids) and a mature peptide (21 amino acids). The gene has two introns and three exons. A TATA box and several consensus‐binding motifs for transcription factors including	C/EBP, nuclear factor‐κB, and hepatocyte nuclear factor were found in the region upstream of the transcriptional start site. In white bass liver, hepcidin gene expression was induced 4500‐fold following challenge with the fish pathogen, Streptococcus iniae, while expression levels remained low in all other tissues tested. A novel antimicrobial peptide from the gill, bass hepcidin, is predominantly expressed in the liver and highly inducible by bacterial exposure.
NblA from Anabaena sp. PCC 7120 is a mostly α‐helical protein undergoing reversible trimerization in solution
Authors: Strauss Holger , Misselwitz Rolf , Labudde Dirk , Nicklisch Sabine , Baier Kerstin 
Published: March 2003

The nblA family of genes encodes for small proteins necessary for the ordered degradation of phycobilisomes under certain stress conditions, a process known as chlorosis. Genes homologous to nblA seem to occur in all phycobilisome‐containing organisms. However, to date, no molecular mechanism is known for the action of NblA, nor have the gene products been characterized to understand the physical properties of the molecule and thus help elucidate the mechanism on a structural basis.In this study we report on the first characterization of an NblA‐homologous gene product. The chromosomal gene from the cyanobacterium Anabaena sp. PCC 7120 was cloned, heterologously expressed in Escherichia coli and purified to apparent homogeneity. This allowed the protein to be characterized by analytical ultracentrifugation and CD spectroscopy. These experiments show that the NblA protein has a mostly α‐helical structure, undergoing an association reaction of folded monomers to form trimers in solution. No dimers are detectable.
The β‐1,4‐endogalactanase A gene from Aspergillus niger is specifically induced on arabinose and galacturonic acid and plays an important role in the degradation of pectic hairy regions
Authors: de Vries Ronald P. , Pařenicová Lucie , Hinz Sandra W. A. , Kester Harry C. M. , Beldman Gerrit , Benen Jacques A. E. , Visser Jaap 
Published: March 2003

The Aspergillus nigerβ‐1,4‐endogalactanase encoding gene (galA) was cloned and characterized. The expression of galA in A. niger was only detected in the presence of sugar beet pectin, d‐galacturonic acid and l‐arabinose, suggesting that galA is coregulated with both the pectinolytic genes as well as the arabinanolytic genes. The corresponding enzyme, endogalactanase A (GALA), contains both active site residues identified previously for the Pseudomonas fluorescensβ‐1,4‐endogalactanase.The galA gene was overexpressed to facilitate purification of GALA. The enzyme has a molecular mass of 48.5 kDa and a pH optimum between 4 and 4.5. Incubations of arabinogalactans of potato, onion and soy with GALA resulted initially in the release of d‐galactotriose and d‐galactotetraose, whereas prolonged incubation resulted in d‐galactose and d‐galactobiose, predominantly. MALDI‐TOF analysis revealed the release of l‐arabinose substituted d‐galacto‐oligosaccharides from soy arabinogalactan. This is the first report of the ability of a β‐1,4‐endogalactanase to release substituted d‐galacto‐oligosaccharides. GALA was not active towards d‐galacto‐oligosaccharides that were substituted with d‐glucose at the reducing end.
The structures of the lipooligosaccharide and capsule polysaccharide of Campylobacter jejuni genome sequenced strain NCTC 11168
Authors: Michael Frank St. , Szymanski Christine M. , Li Jianjun , Chan Kenneth H. , Khieu Nam Huan , Larocque Suzon , Wakarchuk Warren W. , Brisson Jean‐Robert , Monteiro Mario A. 
Published: March 2003

Campylobacter jejuni infections are one of the leading causes of human gastroenteritis and are suspected of being a precursor to Guillain–Barré and Miller–Fisher syndromes. Recently, the complete genome sequence of C. jejuni NCTC 11168 was described. In this study, the molecular structure of the lipooligosaccharide and capsular polysaccharide of C. jejuni NCTC 11168 was investigated. The lipooligosaccharide was shown to exhibit carbohydrate structures analogous to the GM1a and GM2 carbohydrate epitopes of human gangliosides (shown below):The high Mr capsule polysaccharide was composed of β‐d‐Ribp, β‐d‐GalfNAc, α‐d‐GlcpA6(NGro), a uronic acid amidated with 2‐amino‐2‐deoxyglycerol at C‐6, and 6‐O‐methyl‐d‐glycero‐α‐l‐gluco‐heptopyranose as a side‐branch (shown below):The structural information presented here will aid in the identification and characterization of specific enzymes that are involved in the biosynthesis of these structures and may lead to the discovery of potential therapeutic targets. In addition, the correlation of carbohydrate structure with gene complement will aid in the elucidation of the role of these surface carbohydrates in C. jejuni pathogenesis.
The reactivity of α‐hydroxyhaem and verdohaem bound to haem oxygenase‐1 to dioxygen and sodium dithionite
Authors: Sakamoto Hiroshi , Omata Yoshiaki , Hayashi Shunsuke , Harada Saori , Palmer Graham , Noguchi Masato 
Published: March 2003

Recently we have shown that ferric α‐hydroxyhaem bound to haem oxygenase‐1 can be converted to ferrous verdohaem by approximately an equimolar amount of O2 in the absence of exogenous electrons [Sakamoto, H., Omata, Y., Palmer, G., and Noguchi, M. (1999) J. Biol. Chem.274, 18196–18200]. Contrary to those results, other studies have claimed that the conversion requires both O2 and an electron. More recently, Migita et al. have reported that the major reaction product of ferric α‐hydroxyhaem with O2 is a ferric porphyrin cation radical that can be converted to ferrous α‐hydroxyhaem with sodium dithionite [Migita, C. T., Fujii, H., Matera, K. M., Takahashi, S., Zhou, H., and Yoshida, T. (1999) Biochim. Biophys. Acta1432, 203–213]. To clarify the reason(s) for the discrepancy, we compared the reactions; i.e. α‐hydroxyhaem to verdohaem and verdohaem to biliverdin, under various conditions as well as according to the procedures of Migita. We find that complex formation of α‐hydroxyhaem with haem oxygenase may be small and a substantial amount of free α‐hydroxyhaem may remain, depending on the reconstitution conditions; this could lead to a misinterpretation of the experimental results. We also find that ferrous verdohaem appears to be air‐sensitive and is therefore easily converted to a further oxidized species with excess O2. Finally, we find that dithionite seems to be inappropriate for investigating the haem oxygenase reaction, because it reduces ferrous verdohaem to a further reduced species that has not been seen in the haem degradation system driven by NADPH‐cytochrome P450 reductase.
Role of carbohydrate and protein in the binding of tissue‐type plasminogen activator to the human mannose receptor
Authors: Noorman Femke , Barrett‐Bergshoeff Marrie M. , Rijken Dingeman C. 
Published: March 2003

The 175‐kDa mannose receptor is one of the receptors that mediates the clearance of tissue‐type plasminogen activator (t‐PA). The affinity of t‐PA for the mannose receptor is much higher than the affinity of other high‐mannose‐type oligosaccharide‐containing glycoproteins. In order to find an explanation for this high affinity, we studied the biochemical interaction of various forms of t‐PA with the isolated human mannose receptor in several in vitro binding assays.t‐PA showed a high affinity (Ki = 0.2 nM) for the mannose receptor and the interaction could be fully inhibited by mannan or polyclonal antibodies against the mannose receptor. The interaction was not affected by non‐glycosylated t‐PA. The high affinity differed slightly between t‐PAs synthesized by various cell types (range Ki 0.2−0.7 nM) and between various glycoforms of t‐PA. No statistically significant difference in affinity between t‐PA and t‐PA complexed to inhibitors was observed. In contrast to intact t‐PA, a trypsin digest of t‐PA had a low affinity (Ki = 0.5 μM) for the mannose receptor. Both intact and trypsin digests of the high‐mannose‐type oligosaccharide‐containing glycoproteins ribonuclease B and ovalbumin had a low affinity (Ki 0.5−1.5 μM) for the mannose receptor.We conclude that neither protein−protein interactions, nor the complex‐type oligosaccharides and the fucose residue on t‐PA contribute significantly to the high‐affinity binding of t‐PA. We suggest that the conformation of the high‐mannose‐type oligosaccharide on t‐PA is influenced by the protein moiety of t‐PA in such a way that the oligosaccharide has a high affinity for the mannose receptor.
Autonomous folding of the recombinant large cytoplasmic loop of sarcoplasmic reticulum Ca2+‐ATPase probed by affinity labeling and trypsin digestion
Authors: Moutin Marie‐Jo , Rapin Catherine , Miras Roger , Vinçon Mathilde , Dupont Yves , McIntosh David B. 
Published: March 2003

Recombinant large cytoplasmic loop (LCL, residues 329−740) of sarcoplasmic reticulum Ca2+‐ATPase, expressed in and purified from Escherichia coli, comprises most of the active site and binds ATP [Moutin, M.‐J., Cuillel, M., Rapin, C., Miras, R., Anger, M., Lompré, A.‐M. & Dupont, Y. (1994) J. Biol. Chem. 269, 11 147−11 154]. In this study, we show that fluorescein‐5′ isothiocyanate (FITC) specifically labels the same lysine residue as in the native Ca2+‐ATPase (Lys515), with similar kinetics and pH dependence. ATP blocks the reaction with the lysine residue, but at higher concentrations compared with those for the native pump, in agreement with the lower ATP‐binding affinity found previously. Graded tryptic digestion of LCL shows that favored cleavage is at the T1 site and that the N‐terminal 75 % of LCL are resistant to trypsin, as is native Ca2+‐ATPase.Other experiments reveal differences to the native pump. (a) FITC derivatizes some ‐SH groups of LCL. (b) The C‐terminal 25 % of the polypeptide is susceptible to end‐clipping by trypsin. (c) 2′,3′‐O‐(2,4,6‐trinitrophenyl)‐ATP fails to specifically label the LCL (on the equivalent of Lys492), although it binds tightly (KD = 1.3 μM) and (d) Glutaraldehyde does not specifically cross‐link LCL (between the equivalent of Lys492 and Arg678). These results could be explained by a flexible and loose structure of the hinge region of LCL (C‐terminal 25 %). Anchoring this region in the membrane and/or interaction with the missing β‐strand domain may be required for its compact folding and proper interaction with the rest of LCL.The results suggest that the N‐terminal 75 % of LCL expressed in E. coli folds autonomously to a fairly stable unit and native‐like structure, encompassing the phosphorylation and central ATP binding sections. The hinge region does not appear to be part of the FITC‐binding site but constitutes portions of the 2′,3′‐O‐(2,4,6‐trinitrophenyl)‐ATP and, probably, ATP‐binding site.
Structural analysis of the O‐antigenic polysaccharide from Vibrio mimicus N‐1990
Authors: Landersjö Clas , Weintraub Andrej , Ansaruzzaman Mohammed , Albert M. John , Widmalm Göran 
Published: March 2003

The O‐antigenic polysaccharide part of the lipopolysaccharide from Vibrio mimicus N‐1990 has been investigated. Sugar and methylation analysis of native and dephosphorylated polysaccharide together with NMR spectroscopy show that the polysaccharide is composed of tetrasaccharide repeating units. The structure of the repeating unit of the polysaccharide from V. mimicus N‐1990 could be determined as :←4)‐α‐ D‐GalpNAc‐(1←3)‐β‐
D‐GalpNAc‐(1←2)‐4,6‐P‐β‐
D‐Galp‐(1←3)‐α‐
D‐GalpNAc‐(1←  The V. mimicus N‐1990 strain cross‐reacts with antibodies elicited against the Vibrio cholerae O139. The nature of this cross‐reactivity resides in the partial structure comprising the galactosyl residue substituted with a cyclic phosphate. This element is present in the cell‐wall‐associated polysaccharides of both strains.
Location of N‐cyclohexyl‐N′‐(4‐dimethyl‐amino‐α‐naphthyl)carbodiimide‐binding site in sarcoplasmic reticulum Ca2+‐transporting ATPase
Authors: Velasco‐Guillén Isabel , Corbalán‐García Senena , Gómez‐Fernández Juan C. , Teruel José A. 
Published: March 2003

The Ca2+‐transporting ATPase has been labeled with N‐cyclohexyl‐N′‐(4‐dimethyl‐amino‐α‐naphthyl)carbodiimide (NCD‐4), a fluorescent carbodiimide which reacts with carboxyl groups of acidic residues. It has been reported that NCD‐4 labels a transmembrane portion of the protein at the high‐affinity calcium‐binding sites. We have determined the depth of the calcium‐sensitive probe by quenching the fluorescence by nitroxide‐substituted fatty acids with its spin probe located at different carbons of the fatty acid chain (5, 7, 10, 12 and 16‐nitroxide derivatives). We have found that all the calcium‐sensitive fluorescence is quenched and that the efficiency of quenching decreases as the n‐(4,4‐dimethyl‐3‐oxazolinyloxy) (Doxyl) group is deeper in the membrane. We conclude that the NCD‐4 label which is involved in the high‐affinity calcium‐binding site is located near the water/lipid interface.The fluorescence of the NCD‐4 bound to that site can be quenched by acrylamide and Cu2+ but not by iodide, probably due to its anionic nature which will be repulsed by the abundance of negative charges of Glu and Asp residues of NCD‐4 located at this site. The hydrophobic location of NCD‐4 was confirmed by the fact that its fluorescence could be quenched by the spin label 2,2,6,6‐tetramethyl‐1‐piperidine‐N‐oxyl but not by 4‐hydroxy‐2,2,6,6‐tetramethyl‐1‐piperidine‐N‐oxyl which is much less hydrophobic.
Identification of Manα1‐3Manα1‐2Man and Man‐linked phosphate on O‐mannosylated recombinant leech‐derived tryptase inhibitor produced by Saccharomyces cerevisiae and determination of the solution conformation of the mannosylated polypeptide
Authors: Bergwerff Aldert A. , Stark Wilhelm , Fendrich Gabriele , Knecht René , Blommers Marcel J. J. , Maerki Walter , Kragten Eddy A. , van Oostrum Jan 
Published: March 2003

The production of recombinant leech‐derived tryptase inhibitor (rLDTI) by two different strains of Saccharomyces cerevisiae resulted in the secretion of non‐glycosylated and glycosylated rLTDI. Monosaccharide analysis and α‐mannosidase treatment demonstrated that glycosylated rLDTI was exclusively αmannosylated. A trypsin digest of reduced and S‐carboxymethylated glycosylated rLDTI was separated on a reverse‐phase HPLC column. Glycopeptides identified by a combination of matrix‐assisted laser desorption mass spectrometry, amino acid sequence analysis, and monosaccharide analysis revealed the presence of different glycoforms. It was found that Ser24, Ser33 and Ser36 were partially glycosylated with a single mannose residue, whereas Thr42 in glycosylated rLDTI from both strains was fully occupied with manno‐oligosaccharides with a degree of polymerization ranging over 1−3 and 1−13 depending on the yeast strain. In phosphorylated rLDTI a single phosphate group was predominantly located at the innermost Man residue of units of mannobiose, mannotriose, mannotetraose and mannopentaose at Thr42. Oligosaccharides released by alkaline treatment were reduced by sodium borohydride and separated by high‐pH anion‐exchange chromatography on a CarboPac MA1 column, and analyzed by one‐ and two‐dimensional 1H‐NMR spectroscopy. Besides the major oligosaccharide Manα1‐2Man‐ol, the (for yeast protein O‐glycosylation) unusual Manα1‐3Manα1‐2Man‐ol was determined.The solution conformation of glycosylated rLDTI was investigated by two‐dimensional NMR spectroscopy. Structure calculations by means of distance geometry showed that glycosylated rLDTI is compactly folded and contained small secondary structure elements. Analysis of the chemical shifts showed that amino acids Val32−Ser33, Ser36−Ser39 and Thr42 were affected by the O‐mannosylation. In addition, changes in chemical shift were observed within the β‐hairpin peptide regions Val13−Ser16 and Gly18−Tyr21 attributed to direct interactions of the mannose residue at Ser36. Furthermore, the protein‐linked oligosaccharides were spatially grouped in a position opposite of the canonical binding loop.
DNA interactions of antitumor trans‐[PtCl2(NH3)(quinoline)]
Authors: Žákovská Alena , Nováková Olga , Balcarová Zdeňka , Bierbach Ulrich , Farrell Nicholas , Brabec Viktor 
Published: March 2003

Recent observations that several trans‐platinum complexes exhibit antitumor activity including activity in cisplatin‐resistant tumor cells, violates the classical structure/activity relationships of platinum(II) complexes. According to these relationships, only bifunctional platinum(II) complexes with cis‐oriented leaving ligands should be therapeutically active. In order to contribute to the understanding of mechanisms underlying the antitumor activity of these new trans‐platinum analogs, various biochemical and biophysical methods as well as molecular modeling techniques were employed to study the modifications of DNA by antitumor trans‐[PtCl2(NH3)(quinoline)]. The results indicated that trans‐[PtCl2(NH3)(quinoline)] coordinated monofunctionally to DNA with a similar rate as transplatin. The overall rate of the rearrangement to bifunctional adducts was also similar to that observed in the case of DNA modification by transplatin, i.e. it was relatively slow (after 48 h ≈34 % adducts remained monofunctional). In contrast to transplatin, however, trans‐[PtCl2(NH3)(quinoline)] formed considerably more interstrand cross‐links after 48 h (≈30 %) with a much shorter half‐time (≈5 h) (≈12 % for transplatin, t1/2 > 11 h). The results also suggested that the quinoline ligand in all or in a significant fraction of DNA adducts of trans‐[PtCl2(NH3)(quinoline)], in which platinum is coordinated to base residues, was well positioned to interact with the duplex.The adducts of trans‐[PtCl2(NH3)(quinoline)] terminated in vitro RNA synthesis preferentially at guanine residues. Surprisingly, the type and extent of conformational alterations induced in DNA indicates that trans‐[PtCl2(NH3)(quinoline)] behaves in some respects like cisplatin, as indicated by the fact that trans‐[PtCl2(NH3)(quinoline)]‐modified DNA is recognized by cisplatin‐specific antibodies. Models for both monofunctional adducts and bifunctional interstrand cross‐links are proposed. Computer‐generated AMBER models show that the combination of monofunctional covalent binding and a stacking interaction between quinoline and the DNA bases can produce a kink in the duplex which is strongly suggestive of the directed bend produced by the major cisplatin‐DNA adduct (1,2 intrastrand cross‐link). Unique DNA adducts of this type formed by trans‐[PtCl2(NH3)(quinoline)] may contribute to the antitumor efficacy of this agent.
Mechanisms of melanogenesis inhibition by tumor necrosis factor‐α in B16/F10 mouse melanoma cells
Authors: Martínez‐Esparza María , Jiménez‐Cervantes Celia , Solano Francisco , Lozano José Antonio , García‐Borrón José Carlos 
Published: March 2003

The inflammatory cytokine tumor necrosis factor‐α (TNF‐α) is present in the dermal and epidermal layers of normal skin [Kilgus, O., Payer, E., Schreiber, S., Elbe, A., Strohal, R. & Stingl, G. (1993) J. Invest. Dermatol. 100, 674−680]. Its local concentrations are modified by several stimuli, including wound healing and ultraviolet irradiation. Moreover, TNF‐α inhibits melanogenesis in normal melanocytes [Swope, V., Abdel‐Malek, Z., Kassem, L. & Norlund, J. (1991) J. Invest. Dermatol. 96, 180−185], and is, therefore, a potential autocrine/paracrine regulator of pigmentation. We have analyzed the mechanisms of this effect using B16/F10 melanoma cells as a model.Nanomolar concentrations of TNF‐α inhibit the tyrosine hydroxylase and dopa oxidase activities of B16/F10 melanocytes, to less than 30 % control levels, without effects on tyrosinase‐related protein 2/dopachrome tautomerase (TRP2/DCT). The 50 % inhibition was obtained at 1 nM TNF‐α and 48 h treatment. The effect of TNF‐α was noticeable after 6 h treatment, and maximal after 24 h. This inhibition is explained by decreased intracellular levels of tyrosinase and tyrosinase‐related protein 1 (TRP1), but not of TRP2/DCT as detected by Western blotting. Northern‐blot experiments showed that the inhibitory effect is partially explained by a reduced accumulation of the corresponding mRNAs, that dropped to about 50 % of control values (48 h treatment, 5 nM TNF‐α). Moreover, the tyrosine hydroxylase and dopa oxidase activities decreased more rapidly in TNF‐α‐treated cells than in control cells, under conditions of inhibition of protein synthesis. This suggests a TNF‐mediated reduction of tyrosinase half‐life. However, the possibility of an inhibitory post‐translational modification of the enzyme induced by TNF cannot be ruled out. Therefore, the inhibitory effect of TNF‐α on tyrosinase and TRP‐1 results from combined effect on mRNA levels and enzymatic activity or protein stability.
The propeptide of prohormone convertase PC2 acts as a transferable aggregation and membrane‐association signal
Authors: Jan Gwénaël , Taylor Neil A. , Scougall Kathleen T. , Docherty Kevin , Shennan Kathleen I. J. 
Published: March 2003

Prohormone convertase 2 (PC2) is a subtilisin‐like protease involved in the intracellular processing of prohormones and proneuropeptides. Like its substrates, it is synthesised as a prepropeptide which undergoes proteolysis during transit through the regulated secretory pathway. Previous studies have shown that aggregation and membrane association of proPC2 occurs in a calcium‐dependent and pH‐dependent manner and that the pro‐region of PC2 may be involved in this process. These events may be involved in the sorting of proteins to the regulated secretory pathway. To investigate this further, we made a chimeric protein containing both the signal peptide and pro‐region of PC2 and the N‐terminal part of α1‐antitrypsin, called pro2α1. PC2, α1‐antitrypsin and pro2α1 were compared with regard to their membrane association and aggregation properties using, respectively, sucrose gradient centrifugation after expression in Xenopus oocytes, and an in vitro aggregation assay. The chimeric protein, pro2α1, underwent low‐pH‐dependent aggregation and membrane association similar to wild‐type PC2. Membrane association occurred at pH 5.5 in the absence of calcium and at pH 6.0 in the presence of 10 mM calcium but not at pH 6.5 or 7.0. α1‐antitrypsin, as expected of a constitutively secreted protein, did not aggregate at low pH, nor associate with membranes. Pro2α1 thus exhibits the membrane association and aggregation properties of PC2, confirming the role of the pro‐region in these processes.A series of deletions were performed within the 84‐residue propeptide in order to define the sequences involved. Deletion of amino acids 52−77 reduced aggregation but large deletions in the pro‐region had only a minimal effect on membrane association. These data suggest that several regions within the pro‐peptide are important in these events.