bluuebunny/crossref_metadata_2025 huggingface dataset:
Neopterin activates transcription factor nuclear factor‐κB in vascular smooth muscle cells
Authors: Hoffmann Georg , Schobersberger Wolfgang , Frede Stilla , Pelzer Larissa , Fandrey Joachim , Wachter Helmut , Fuchs Dietmar , Grote Jürgen
Published: April 2003

We have previously shown that the pteridine compound neopterin stimulates inducible nitric oxide synthase (iNOS) gene expression in vascular smooth muscle cells in vitro. The mechanisms whereby neopterin exhibits these effects remained unclear. The present study demonstrates that neopterin induces the translocation of the transcription factor nuclear factor‐κB (NF‐κB) to the nucleus. Pretreatment of cells with the antioxidant pyrrolidine dithiocarbamate completely suppressed the effects of neopterin on NF‐κB activation, iNOS gene expression, and nitric oxide release. From these data we conclude that neopterin activates the translocation of NF‐κB subunits to the nucleus by modulating the intracellular redox state. This is one possible explanation for the impact of neopterin on iNOS gene expression.
NMR structures of a mitochondrial transit peptide from the green alga Chlamydomonas reinhardtii
Authors: Lancelin Jean-Marc , Gans Pierre , Bouchayer Eftychia , Bally Isabelle , Arlaud Gérard J. , Jacquot Jean-Pierre
Published: April 2003

The 26‐amino‐acid pre‐sequence of the ATP synthase βs subunit that directs the protein from the cytosol to mitochondria in the unicellular green alga Chlamydomonas reinhardtii has been synthesised and analysed using NMR spectroscopy/circular dichroism and compared to a chloroplast transit peptide from the same organism. The results demonstrate that the peptide, though mainly unstructured in water, undergoes a strong conformational change in a 36% water/64% 2,2,2‐trifluoroethanol mixture. In this solvent condition, an α‐helix was characterised by NMR from residue 2 to 26. Structure calculations under NMR restraints lead to a population of models of which 60% are kinked at position 9–10. Structural analysis indicates two hydrophobic sectors on the models with a discontinuity at the 9–10 kink level. The structures suggest a different interaction mode with the mitochondrial membrane compared to the chloroplast transit peptide.
Requirement for nickel of the transmembrane translocation of NiFe‐hydrogenase 2 in Escherichia coli
Authors: Rodrigue A. , Boxer D.H. , Mandrand-Berthelot M.A. , Wu L.-F.
Published: April 2003

The cellular location of membrane‐bound NiFe‐hydrogenase 2 (HYD2) from Escherichia coli was studied by immunoblot analysis and by activity staining. Treatment of spheroplasts with trypsin was able to release active HYD2 into the soluble fraction, indicating that HYD2 is attached to the periplasmic side of the cytoplasmic membrane and that HYD2 undergoes a trans‐membrane translocation during its biosynthesis. By using a nik mutant deficient in the high affinity specific nickel transport system, we show that the intracellular availability of nickel is essential for the processing of the large subunit and for the transmembrane translocation of HYD2. We also demonstrate that the processing of the precursor, which is related with nickel incorporation, can occur in the membrane‐depleted soluble fraction and that it is associated with the increase in resistance to proteolysis of the processed form of the large subunit. The mechanism of the transmembrane translocation of HYD2 is discussed.
Hybridization of two oligodeoxynucleotides to both strands of an RNA hairpin structure increases the efficiency of RNA‐DNA duplex formation
Authors: Malygin Alexey , Karpova Galina , Westermann Peter
Published: April 2003

Hybridization of two oligodeoxyribonucleotides (ON1 and ON2), complementary to opposite strands of the apical domain of Escherichia coli 4.5S RNA, was studied. ON1, complementary to bases 58–71, was not able to form a stable RNA‐DNA hybrid whereas ON2, complementary to bases 38–53, was. Addition of both oligonucleotides at the same time resulted in the formation of a ternary complex permitting hybridization of ON1 and increasing hybridization of ON2. Under this condition, binary complexes of ON1 or ON2 with 4.5S RNA were not observed. The data demonstrate that hybridization of oligonucleotides to both strands of an RNA hairpin structure increases the efficiency of hybridization of either oligonucleotide.
Direct evidence for the different roles of the N‐ and C‐terminal regulatory disulfides of sorghum leaf NADP‐malate dehydrogenase in its activation by reduced thioredoxin
Authors: Issakidis Emmanuelle , Lemaire Martine , Decottignies Paulette , Jacquot Jean-Pierre , Miginiac-Maslow Myroslawa
Published: April 2003

Plant NADP‐dependent malate dehydrogenase is activated through thiol/disulfide interchange with reduced thioredoxin. Previous studies showed that this process involves the reduction of two different disulfides per subunit: one N‐terminal, the other C‐terminal. Substitution of regulatory cysteines at each end by site‐directed mutagenesis and comparison of activation kinetics of the mutants led us to propose a model for the activation mechanism where the C‐terminal end shielded the access to the catalytic residues, whereas the N‐terminal end was involved in the slow conformational change of the active site. In the present study, we took advantage of the previous identification of the catalytic histidine residue which can be specifically derivatized by diethyl pyrocarbonate to test the accessibility of the active site. The results clearly show that in the mutants where the C‐terminal bridge is open the active site histidine is freely accessible to the reagent, whereas in the mutants where the N‐terminal bridge is open, the active site cannot be reached without activation, thus demonstrating the validity of the model.
Immunotoxins containing A‐chain of mistletoe lectin I are more active than immunotoxins with ricin A‐chain
Authors: Tonevitsky Alexander G. , Agapov Igor I. , Shamshiev Abdijapar T. , Temyakov Dmitryi E. , Pohl Peter , Kirpichnikov Michail P.
Published: April 2003

Conjugates of anti‐CD25 monoclonal antibodies against cell surface IL‐2 receptor with MLIA and RTA were prepared and investigated. Both of the immunotoxins had high specific cytotoxic activity on target cells. The IC50 value of the anti‐CD25/MLIA immunotoxin was 15‐fold greater than that of the anti‐CD25/RTA. Previous studies of the anti‐CD5 immunotoxins with MLIA and RTA showed that the anti‐CD5/MLIA IT was 80‐fold more active than anti‐CD5/RTA IT [Tonevitsky et al. (1991) Int. J. Immunopharmacol. 13, 1037–1041]. The surface hydrophobicity of the MLI A‐chain was 4‐fold higher than that of the ricin A‐chain as estimated by binding with ANS. In model experiments with small unilamellar DMPC liposomes, MLIA but not RTA increased the turbidity of liposome suspensions at pH 4.5. Our results indicate that the greater cytotoxic activity of the MLI A‐chain immunotoxin probably provided a higher surface hydrophobicity of the protein and the ability to interact with phospholipid membranes.
Purification and characterization of phosphoenolpyruvate carboxylase from the hyperthermophilic archaeon Methanothermus sociabilis
Authors: Sako Yoshihiko , Takai Ken , Uchida Aritsune , Ishida Yuzaburo
Published: April 2003

Phosphoenolpyruvate carboxylase (PEPC) was purified for the first time from hyperthermophilic archaeon Methanothermus sociabilis, growing autotrophically with an optimum at 88°C. The optimum temperature for enzyme activity was similar to that for growth and was 85°C. The native enzyme was a homotetramer of 240 kDa molecular mass and the subunit displayed an apparent molecular mass of 60 kDa. The archaeal PEPC was insensitive to various metabolites which are known as allosteric effectors for most bacterial and eucaryal counterparts. The enzyme showed extreme thermostability such that there remained 80% of the enzyme activity after incubation for 2 h at 80°C. These results implied that archaeal PEPC was significantly different from bacterial and eucaryal entities.
Positive and negative regulation of a sterol biosynthetic gene (ERG3) in the post‐squalene portion of the yeast ergosterol pathway
Authors: Arthington-Skaggs B.A. , Crowell D.N. , Yang H. , Sturley S.L. , Bard M.
Published: April 2003

Regulation of sterol biosynthesis in the terminal portion of the pathway represents an efficient mechanism by which the cell can control the production of sterol without disturbing the production of other essential mevalonate pathway products. We demonstrate that mutations affecting early and late steps in sterol homeostasis modulate the expression of the ERG3 gene: a late step in sterol biosynthesis in yeast. Expression of ERG3 is increased in response to a mutation in the major isoform of HMG CoA reductase which catalyzes the rate‐limiting step of sterol biosynthesis. Likewise, mutations in non‐auxotrophic ergosterol biosynthetic genes downstream of squalene production (erg2, erg3, erg4, erg5, and erg6) result in an up‐regulation of ERG3 expression. Deletion analysis of the ERG3 promoter identified two upstream activation sequences: UAS1, which when deleted reduces ERG3 gene expression 3–4‐fold but maintains sterol regulation and UAS2, which when deleted further reduces ERG3 expression and abolishes sterol regulation. The recent isolation of two yeast genes responsible for the esterification of intracellular sterol (ARE1 and ARE2) has enabled us to directly analyze the relationship between sterol esterification and de novo biosynthesis. Our results demonstrate that the absence of sterol esterification leads to a decrease in total intracellular sterol and ERG3 is a target of this negative regulation.
Genetic inactivation of the H+‐translocating NADH:ubiquinone oxidoreductase of Paracoccus denitrificans is facilitated by insertion of the ndh gene from Escherichia coli
Authors: Finel Moshe
Published: April 2003

The H+‐translocating NADH:ubiquinone oxidoreductase (NDH1) is probably an obligatory enzyme in Paracoccus denitrificans and disruption of its genes may be lethal to this organism. In order to overcome this problem and delete the nqo8 and nqo9 genes of NDH1, it was necessary to render the enzyme non‐essential. This was achieved by constructing a deletion plasmid in which most of the coding regions of nqo8 and nqo9 were replaced by the ndh gene of Escherichia coli that encodes an alternative NADH:ubiquinone oxidoreductase (NDH2), and a kanamycin resistance gene. Subsequent homologous recombination gave rise to a mutant the membranes of which catalyzed rotenone‐insensitive NADH oxidation, but which did not oxidize deamino‐NADH. Hence, this mutant expressed active and membrane‐bound NDH2, and lacked NDH1 activity.
Overexpression of either liver type or pancreatic β cell type glucokinase via recombinant adenovirus enhances glucose oxidation in isolated rat hepatocytes
Authors: Takeuchi Hideo , Inoue Yasushi , Ishihara Hisamitsu , Oka Yoshitomo
Published: April 2003

To elucidate a role of glucokinase in hepatic glucose metabolism, we overexpressed hexokinase I (HKI), liver type glucokinase (LGK), or β cell type glucokinase (βGK) in primary rat hepatocytes using a recombinant adenovirus vector system. Overexpression of HKI and LGK induced a 34‐ and 25‐fold increase, respectively, in glucose phosphorylation activity measured in cell homogenates. While HKI overexpression induced only a 1.3‐fold increase in glucose oxidation, LGK overexpression increased glucose oxidation by 2.9‐fold. Overexpression of βGK had essentially the same effect as LGK. The results indicate that glucokinase does indeed regulate the rate of hepatic glucose oxidation and that the liver‐specific sequence of this enzyme is not essential for this function.
Localisation of the major reactive lysine residue involved in the selfcrosslinking of proteinase‐activated Limulusα2‐macroglobulin
Authors: Dolmer Klavs , Husted Lise B. , Armstrong Peter B. , Sottrup-Jensen Lars
Published: April 2003

When α
2‐macroglobulin (α
2M) from the American horseshoe crab, Limulus polyphemus, reacts with proteinases, its thiol esters, like those of other α‐macroglobulins, become activated, leading to the formation of covalently crosslinked species that can be detected as high molecular weight bands in reducing SDS‐PAGE. While other α‐macroglobulins extensively form crosslinks to the reacting proteinase, Limulus α
2M does not. It rather becomes internally crosslinked. It was found from N‐terminal sequence analysis of purified [14C]carboxymethylated peptides from Limulus α
2M‐trypsin complexes that an isopeptide bond formed in approx. 60% yield from the thiol esterified Gln‐1002 specifically to Lys‐254 in the opposing monomer of the disulphide bridged dimer is the main cause of the internal crosslinking.
Molecular cloning of rat phosphatidylinositol synthase cDNA by functional complementation of the yeast Saccharomyces cerevisiae pis mutation
Authors: Tanaka Susumu , Nikawa Jun-ichi , Imai Hideaki , Yamashita Satoshi , Hosaka Kohei
Published: April 2003

Phosphatidylinositol synthase (CDP‐1,2‐diacyl‐sn‐glycerol: 3‐phosphatidyltransferase, EC 2.7.8.11) catalyzes the formation of phosphatidylinositol and CMP from CDP‐diacyl‐glycerol and myo‐inositol. We have cloned a phosphatidylinositol synthase cDNA from rat brain by functional complementation of the yeast pis mutation, which is defective in phosphatidylinositol synthase. The deduced protein comprised 213 amino acids with a calculated molecular mass of 23 613 Da. The predicted protein sequence is highly homologous to the previously determined yeast phosphatidylinositol synthase sequence. The cDNA hybridized to a 1.7‐kb mRNA that was abundantly expressed in rat brain and kidney.
Human glioma U‐251 cells contain type 1 plasminogen activator inhibitor in a rapidly releasable form
Authors: Salonen Eeva-Marjatta , Gombau Lourdes , Engvall Eva , Schleef Raymond R.
Published: April 2003

Because recent information suggests that the localized deposition of protease inhibitors is one mechanism by which cells regulate pericellular proteolysis during tissue invasion, the distribution of type 1 plasminogen activator inhibitor (PAI‐1) associated with the invasive human glioma cell line U‐251 was investigated. Direct and reverse fibrin zymography indicated the presence of urokinase‐like plasminogen activator (u‐PA) and PAI‐1 in U‐251 conditioned media and cell lysates. PAI‐1 antigen was detected immunologically in cytoplasmic granules present within cellular processes of U‐251 cells and these organelles could be isolated on Percoll density gradients in a high density band. In contrast, u‐PA activity and another secreted protein, amyloid β‐protein precursor, were only present in the low density region of the gradients. Functional analysis of PAI‐1 in the granules contained within the high density fractions revealed the presence of active PAI‐1. Incubation of U‐251 cells with the secretagogue, 8‐bromoadenosine 3′:5′‐cyclic monophosphate, resulted in a 3‐fold increase in the release of PAI‐1 in the media conditioned by these cells. These data suggest that the human glioma cell line U‐251 contains PAI‐1 in a rapidly releasable form, which may provide another mechanism by which these tumors could regulate proteolytic activity in a localized manner.
Probing native‐like orientation of pigments in modified reaction centers from Rhodobacter sphaeroides R26 by linear dichroism
Authors: Meyer Michaela , Scheer Hugo , Breton Jacques
Published: April 2003

Site‐specific pigment modifications are useful to investigate structure‐function relationships in photosynthesis. In reaction centers bearing modified (bacterio)pheophytins, changed electron transfer kinetics have been related to the changed redox potentials of the pigments introduced (Huber, H. et al. (1995) Chemical Physics, Special Issue, vol 197 (Hochstrasser, R.M. and Hofacker, G.L. eds.) pp. 297–305; [1]). In order to analyze potentially interfering structural changes induced in these reaction centers by the exchange procedure, in particular mispositioning or misorientation of the pigments, low‐temperature linear dichroism spectra have been measured for reaction centers from Rhodobacter sphaeroides containing modified bacteriopheophytins and bacteriochlorophylls at the sites HAB and BA,B, respectively. They show that all modified pigments are oriented similar to the native ones, and that they do not affect significantly the linear dichroism of the monomeric bacteriochlorophylls and bacteriopheophytins or of the primary donor.
5S rRNA sugar‐phosphate backbone protection in complexes with specific ribosomal proteins
Authors: Shpanchenko Olga V. , Zvereva Maria I. , Dontsova Olga A. , Nierhaus Knud H. , Bogdanov Alexey A.
Published: April 2003

5S ribosomal RNA forms stable specific complexes with ribosomal proteins L18, L25 and L5. In this work, interaction of phosphate residues of E. coli 5S rRNA within 5S rRNA‐protein complexes has been studied. For this purpose 5S rRNA with statistically distributed phosphorothioate residues has been used for complex formation and the accessibility of phosphorothioates to iodine cleavage in the complex and in the free state has been studied. In free 5S rRNA, the phosphate residue at A73 was partially protected, probably due to being involved in the organization of the spatial structure of 5S rRNA. This protection is stronger in the complex with three proteins when the 5S rRNA structure is stabilized. In the 5S rRNA‐L18 complex only two phosphate groups, G7 and A34, were protected. L25 in a complex with 5S rRNA protects large numbers of phosphorothioate groups concentrating in two clusters, indicating the possibility of two binding sites for this protein on 5S rRNA. The protection pattern differs from that for individual proteins because of the possible rearrangement of the structure.
Molecular cloning and tissue‐specific expression of mouse kidney 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase
Authors: Batra Ritu Sonia , Brown Ruth , Brown Garry K. , Craig Ian W.
Published: April 2003

A 1932 bp cDNA clone encoding a novel isozyme of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase (PFK‐2/FBPase‐2) was isolated from a mouse kidney cDNA library. The sequence encodes 519 amino acids and, based on homology to rat heart genomic sequence, appears to be the product of alternative splicing from PFK‐2/FBPase‐2 gene B with an extended version of exon 15. Northern blot analysis indicated that this clone corresponds to an 8 kb mRNA expressed in multiple tissues, with the strongest signal in kidney, and detects several additional transcripts which may be alternatively spliced from gene B.
Osteotropic factor‐stimulated synthesis of thrombospondin in rat dental pulp cells
Authors: Islam Md.Rokibul , Ueno Akemichi , Nishikawa Hiroyuki , Nagata Toshihiko , Inoue Hideo
Published: April 2003

The amount of thrombospondin (TSP) mRNA in confluent clonal rat dental pulp cells was increased by transforming growth factor‐β (TGF‐β), 1α,25‐dihydroxyvitamin D3 (1,25(OH)2D3), and phorbol 12,13‐didecanoate (PDD), with maximal levels 6, 36 and 2 h, respectively, after stimulation. These increases were accompanied by enhanced syntheses of TSP proteins which were found in the different forms in cell layer/matrix fraction (198 and 165 kDa TSP) and the culture medium (180 kDa TSP). These three factors also raised the mRNA level of osteopontin, which is thought to play an important role in mineralization in dentin and bone. The order of effectiveness of these factors was PDD > TGF‐β > 1,25(OH)2D3 for all the stimulations described above. These results suggest that the osteotropic factors enhance TSP synthesis at the pretranslational level and that TSP produced by dental pulp cells participates in formation of reparative dentin.
Hormone‐ and endotoxin‐modulated gene expression of a long‐term organ culture system of adult rat liver
Authors: Sultan K. , Hartung J. , Bade E.G.
Published: April 2003

Precision‐cut slices of normal adult rat liver maintained in serum‐free medium remain hormone‐ and endotoxin‐responsive for at least 48 h. They respond to glucocorticoid (dexamethasone) with the induction of the gluconeogenic enzyme tyrosine aminotransferase (TAT), as determined by enzymatic activity and by the increase in enzyme protein. Furthermore, endotoxin (LPS) induced nitric oxide synthase II (i‐NOS), and this induction is repressed, similarly to the in vivo situation, by dexamethasone (DEX). All increases are inhibited by cycloheximide (CHX). The length of the period of responsiveness suggests that this organ culture system might be generally useful for studying the modulation of liver gene expression by physiological and pathological influences.
Small‐angle X‐ray scattering reveals differences between the quaternary structures of oxygenated and deoxygenated tarantula hemocyanin
Authors: Decker Heinz , Hartmann Hermann , Sterner Reinhard , Schwarz Erika , Pilz Ingrid
Published: April 2003

Small‐angle X‐ray scattering (SAXS) curves have been recorded for the oxygenated and deoxygenated states of the 4 × 6‐meric hemocyanin from the tarantula Eurypelma californicum. A comparison of the curves shows that the quaternary structures of the two states are different by three criteria, which all indicate that the hemocyanin is less compact in the oxygenated compared to the deoxygenated form: (a) The radius of gyration is 8.65 ± 0.05 nm for the deoxy‐ and 8.80 ± 0.05 nm for the oxy‐form. (b) The maximum particle dimension amounts to 25.0 ± 0.5 nm for the deoxy‐ and to 27.0 ± 0.5 nm for the oxyform. (c) A dip in the intramolecular distance distribution function p(r) is more pronounced and shifted to larger distances in the oxy‐form. The p(r) functions based on SAXS measurements were compared to p(r) functions deduced from published electron microscopical images of three different 4 × 6‐meric hemocyanins from closely related species. The p(r) functions of SAXS and electron microscopy were similar in one case, whereas in the other two cases the distance between the two 12‐meric half‐molecules had to be changed by 1–1.5 nm to obtain good agreement. The differences between the p(r) functions of oxygenated and deoxygenated 4 × 6‐meric tarantula hemocyanin are much larger than one would expect from a comparison of X‐ray structures of the oxygenated and deoxygenated states of a closely related 6‐meric hemocyanin. Thus, the conformational changes upon oxygenation occur at various levels of the quaternary structure, as postulated by hierarchical theories of allosteric interactions.
Thymosin β10 mRNA expression during early postimplantation mouse development
Authors: Carpintero Pablo , Franco del Amo Francisco , Anadón Ramón , Gómez-Márquez Jaime
Published: April 2003

The β‐thymosins are a family of monomeric actin sequestering peptides that regulate actin dynamics within the cells. During embryogenesis the control of actin polymerization is essential in processes such as cell migration, angiogenesis and neurogenesis. Here we report that the levels of thymosin β
10 (Tβ
10) mRNA strongly increase during early postimplantation mouse embryogenesis as well as during in vitro P19 cell differentiation, indicating that this peptide plays an important role in early development. Moreover, analysis of the spatial distribution of Tβ
10 mRNA in 9.5–12.5 days postcoitum mouse embryos showed a remarkable presence of this transcript in mesenchymal structures as well as in the mantle layer of spinal cord. Interestingly, we observed differences in the distribution of the mRNAs encoding Tβ
10 and Tβ
4, another member of the β‐thymosin family, suggesting different roles for these peptides during mouse embryogenesis.
Expression of glucagon‐like peptide 1 receptor in a murine C cell line Regulation of calcitonin gene by glucagon‐like peptide 1
Authors: Lamari Y. , Boissard C. , Moukhtar M.S. , Jullienne A. , Rosselin G. , Garel J.-M.
Published: April 2003

We have characterized, by RT‐PCR amplification using specific primers, the presence of glucagon‐like peptide‐1 (GLP‐1) receptor mRNA in CA‐77 cells, a C cell line derived from a rat medullary thyroid carcinoma. Down‐regulation of the GLP‐1 receptor mRNA was observed after exposure of CA‐77 C cells with GLP‐1 (7–37). Increased secretion of both calcitonin gene‐related peptide (CGRP) and calcitonin (CT) occurred after treatment with GLP‐1 (7–37) associated with elevated steady‐state levels of CGRP and CT mRNA. GLP‐1 (7–37) increased cAMP formation in CA‐77 cells in a dose‐dependent manner; exendin (9–39), a GLP‐1 receptor antagonist, inhibited cAMP production. The GLP‐1 peptide which is produced by intestinal cells could be involved in the control of CT secretion through an entero‐thyroidal axis implying GLP‐1 receptor and increased CT gene expression.
Structural comparison of a 15 residue peptide from the V3 loop of HIV‐1IIIb and an O‐glycosylated analogue
Authors: Huang Xiaolin , Charles Smith M. , Berzofsky Jay A. , Barchi Joseph J.
Published: April 2003

As part of a program to study the effect of glycosylation on the three‐dimensional structures of HIV‐1
IIIB
V3 peptide constructs, we have examined the solution structures of a 15 residue peptide (RIQRGPGRAFVTIGK, P18
IIIB
), originally mapped as an epitope recognized by CD8+ D
d
class I MHC‐restricted murine cytotoxic T‐lymphocytes (CTL), and an analogue (P18
IIIB
‐g), O‐glycosylated with an α‐galactosamine on Thr‐12, using NMR, circular dichroism and molecular modeling methods. Our studies show that the peptides sample mainly random conformations in aqueous solution near 25°C and become more ordered by the addition of trifluoroethanol. Upon decreasing the temperature to 5°C, a reverse turn is formed around the immunodominant tip (G5−R8). Glycosylation on T12 ‘tightens’ the turn slightly as suggested by NOE and CD analysis. In addition, the sugar has a defined conformation with respect to the peptide backbone and influences the local peptide conformation. These data suggest that simple glycosylation may influence the conformational equilibrium of a V3 peptide which contains a domain critical for antibody recognition and virus neutralization. We also show that the ability of cytotoxic T‐lymphocytes (CTL) to lyse tumor cells presenting P18
IIIB
was completely abrogated by threonine glycosylation.
Affinity and kinetic analysis of the bovine plasma C‐type lectin collectin‐43 (CL‐43) interacting with mannan
Authors: Holmskov Uffe , Fischer Per B. , Rothmann Anette , Højrup Peter
Published: April 2003

Collectins are C‐type lectins which have been implied to play an important role in the innate immune defence against microorganisms. The critical discriminatory event in the opsonization of microorganisms by collectins is the interaction of the C‐type lectin domain with microbial carbohydrates. Surface plasmon resonance measurements allow for quantitative real‐time measurements of binding interaction between immobilized carbohydrate and unlabelled lectin in solution. Binding analysis were carried out with purified collectin‐43 (CL‐43) which structurally is the simplest collectin consisting of only three polypeptides each terminating in a C‐type lectin domain. The target was immobilized yeast mannan. The molecular mass of native CL‐43 was determinated by mass spectroscopy to 99.8 kDa. The dissociation rate (k
diss) of the C‐type lectin‐carbohydrate binding was fast (1.19–1.36 × 10−2 second−1), and the association rate (k
ass) was 4.37–5.07 × 105 M−1] second−1
. The equilibrium constant for dissociation (K
d) was 2.68–2.72 × 10−8 M.
Tissue‐specific molecular heterogeneity of human growth hormone‐releasing hormone receptor protein
Authors: Fujinaka Yuichi , Yokogoshi Yutaka , Zhang Chen-Yu , Okura Toshihiro , Kitagawa Kouki , Saito Shiro
Published: April 2003

A site‐directed anti‐peptide antibody (anti‐hGHRHRc18) was generated against the cytoplasmic tail of human GHRH receptor. The dissociation constant (K

d
) and the antibody binding site (AbT) of anti‐hGHRHRc18 were 2.5 nmol/l and 0.54 nmol/l, respectively. In an immunoblotting experiment, affinity‐purified anti‐hGHRHRc18 specifically recognized a single 50‐kDa protein in human pituitary. In a screening of the expression of GHRH receptor protein in extra‐pituitary tissues, only human kidney showed a single 52‐kDa protein. Our results suggest that the GHRH receptor protein exhibits tissue‐specific molecular heterogeneity.
Direct thyroid hormone signalling via ADP‐ribosylation controls mitochondrial nucleotide transport and membrane leakiness by changing the conformation of the adenine nucleotide transporter
Authors: Mowbray John , Hardy D.Louise
Published: April 2003

Addition of triiodothyronine at 10 pM in vitro to hypothyroid rat liver mitochondria doubles the rate of the adenine nucleotide transporter at low ADP concentrations. Nicotinamide abolishes this effect in parallel with its inhibition of the ADP‐ribosylation of an inner membrane protein identical in size to the transporter. Nicotinamide also renders euthyroid preparations indistinguishable from hypothyroid ones. A mechanism is offered to explain these findings in which it is proposed that the adenine nucleotide transporter is a true allosteric protein and that its covalent modification by ADP‐ribosylation increases the stability of the less favoured externally‐facing C‐conformation and thus increases the proportion of transporters in this orientation: although the C‐conformation is significantly more leaky to cations than the tight matrix‐facing M‐conformation, this enhances ADP import. This model is shown to offer an explanation not only for the transport effects of T3 but also for those of oxidative stress and ADP‐ribosylation inhibitors on Ca2+, H+ and K+ transfer across the mitochondrial inner membrane. Ca2+ at 30 nM appears to stabilize the M‐conformation of the transporter by a mechanism other than ADP‐ribosylation.
Site‐directed mutagenesis of azurin from Pseudomonas aeruginosa enhances the formation of an electron‐transfer complex with a copper‐containing nitrite reductase from Alcaligenes faecalis S‐6
Authors: Kukimoto Mutsuko , Nishiyama Makoto , Tanokura Masaru , Murphy Michael E.P. , Adman Elinor T. , Horinouchi Sueharu
Published: April 2003

Kinetic analysis of electron transfer between azurin from Pseudomonas aeruginosa and copper‐containing nitrite reductase (NIR) from Alcaligenes faecalis S‐6 was carried out to investigate the specificity of electron transfer between copper‐containing proteins. Apparent values of k

cat
and K

m
of NIR for azurin were 300‐fold smaller and 172‐fold larger than those for the physiological redox partner, pseudoazurin from A. faecalis S‐6, respectively, suggesting that the electron transfer between azurin and NIR was less specific than that between pseudoazurin and NIR. One of the major differences in 3‐D structure between these redox proteins, azurin and pseudoazurin, is the absence and presence of lysine residues near their type 1 copper sites, respectively. Three mutated azurins, D11K, P36K, and D11K/P36K, were constructed to evaluate the importance of lysine residues in the interaction with NIR. The redox potentials of D11K, P36K, and D11K/P36K azurins were higher than that of wild‐type azurin by 48, 7, and 55 mV, respectively. As suggested by the increase in the redox potential, kinetic analysis of electron transfer revealed reduced ability of electron transfer in the mutated azurins. On the other hand, although each of the single mutations caused modest effects on the decrease in the K

m
value, the simultaneous mutations of D11K and P36K caused significant decrease in the K

m
value when compared to that for wild‐type azurin. These results suggest that the introduction of two lysine residues into azurin facilitated docking to NIR.
cDNA cloning reveals a tissue specific expression of alternatively spliced transcripts of the ryanodine receptor type 3 (RyR3) calcium release channel
Authors: Marziali Giovanna , Rossi Daniela , Giannini Giuseppe , Charlesworth Alexandra , Sorrentino Vincenzo
Published: April 2003

Ryanodine receptors (RyRs) are a family of intracellular calcium release channels. Three cDNAs encoding different isoforms of RyR have been identified and cloned. We report the complete sequence of the mink RyR3 cDNA and the characterization of three alternative spliced regions. The first two splicing sites are represented by insertions of five and six amino acids, respectively. The third site is represented by a mutually exclusive splicing. The tissue distribution of the alternatively spliced transcripts revealed a ubiquitóus expression of splicing site I and a differential distribution of sites II and III, indicating that a further level of complexity in RyR3 expression may result from alternative splicings in this gene.
Comparative analysis of mouse hepcidin 1 and 2 genes: evidence for different patterns of expression and co‐inducibility during iron overload1
Authors: Ilyin Gennady , Courselaud Brice , Troadec Marie-Bérengère , Pigeon Christelle , Alizadeh Mehdi , Leroyer Patricia , Brissot Pierre , Loréal Olivier
Published: April 2003

In contrast to the human genome, the mouse genome contains two HEPC genes encoding hepcidin, a key regulator of iron homeostasis. Here we report a comparative analysis of sequence, genomic structure, expression and iron regulation of mouse HEPC genes. The predicted processed 25 amino acid hepcidin 2 peptide share 68% identity with hepcidin 1 with perfect conservation of eight cysteine residues. Both HEPC1 and HEPC2 genes have similar genomic organization and have probably arisen from a recent duplication of chromosome 7 region, including the HEPC ancestral gene and a part of the adjacent USF2 gene. Insertion of a retroviral intracisternal A‐particle element was found upstream of the HEPC1 gene. Both genes are highly expressed in the liver and to a much lesser extent in the heart. In contrast to HEPC1, a high amount of HEPC2 transcripts was detected in the pancreas. Expression of both genes was increased in the liver during carbonyl–iron and iron–dextran overload. Overall our data suggest that both HEPC1 and HEPC2 genes are involved in iron metabolism regulation but could exhibit different activities and/or play distinct roles.
Lysenin, a unique sphingomyelin‐binding protein
Authors: Shakor Abo-Bakr Abdel , Czuryło Edward A. , Sobota Andrzej
Published: April 2003

Sphingomyelin plays complex structural and signaling functions in the plasma membrane. Of special interest is that hydrolysis of sphingomyelin to ceramide can modulate dynamics of membrane rafts, which serve as signaling platforms for various receptors. This review is focused on a recently discovered sphingomyelin‐binding protein, lysenin, which can be used as a unique probe to trace distribution and turnover of sphingomyelin in cellular membranes. We analyze the primary and secondary structures of lysenin with respect to its interaction with the plasma membrane. The specificity of lysenin binding to sphingomyelin, revealed by both biochemical and cytochemical approaches, is discussed.
Preliminary characterisation of the promoter of the human p22phox gene: identification of a new polymorphism associated with hypertension
Authors: Moreno Marı́a U , San José Gorka , Orbe Josune , Páramo José A , Beloqui Oscar , Dı́ez Javier , Zalba Guillermo
Published: April 2003

The p22phox subunit is an essential protein in the activation of NAD(P)H oxidase. Here we report the preliminary characterisation of the human p22phox gene promoter. The p22phox promoter contains TATA and CCAC boxes and Sp1, γ‐interferon and nuclear factor κB sites. We screened for mutations in the p22phox promoter and identified a new polymorphism, localised at position −930 from the ATG codon, which was associated with hypertension. Mutagenesis experiments showed that the G allele had higher promoter activity than the A allele. These results suggest that the −930A/G polymorphism in the p22phox promoter may be a novel genetic marker associated with hypertension.
Conserved arginine and aspartate residues are critical for function of MjNhaP1, a Na+/H+ antiporter of M. jannaschii
Authors: Hellmer Jens , Teubner Andreas , Zeilinger Carsten
Published: April 2003

Recently MjNhaP1 was identified as a pH‐regulated Na+/H+ antiporter of Methanococcus jannaschii [Hellmer, J. et al. (2002) FEBS Lett. 527, 245–249]. The antiporter is active at pH 6.0 and displays continuously decreasing activity towards alkaline pH. We have performed a site‐directed mutagenesis study on all histidines as well as on conserved Asp, Glu and Arg residues of MjNhaP1, and analyzed the mutated proteins for activity. The mutants fall into three classes, i.e. normally active mutants, mutants with intermediate activity and mutants which are completely inactive. None of the histidine residues appears to be essential unlike in the bacterial proteins. The results point at an important role of a number of aspartate and arginine residues.
The poplar root transcriptome: analysis of 7000 expressed sequence tags
Authors: Kohler Annegret , Delaruelle Christine , Martin David , Encelot Nathalie , Martin Francis
Published: April 2003

To date, most poplar expressed sequence tags (ESTs) are from above‐ground tissues such as wood, leaf and buds. Here, we present a large‐scale production of ESTs from roots of the hybrid cottonwood, Populus trichocarpa×deltoides. cDNA libraries were generated from the root system of 2‐month‐old rooted cuttings, and roots of 2.5‐month‐old cuttings water‐stressed for 19 days. Partial sequences obtained from 7013 clones were assembled into 1347 clusters and 3527 singletons. This set of ESTs represents 4874 unique transcripts expressed in roots. Putative functions could be assigned to 3021 (62%) of the transcripts. A significant portion of the ESTs encode proteins of common metabolic pathways; energy and metabolism represented 5% and 8% of total transcripts, respectively. Of specific interest to root functions are the 6% of ESTs involved in signalling pathways and hormone metabolism, and 4% encoding transporters and channels. The current poplar root ESTs and the aspen root ESTs present in public databases represent 6700 unique transcripts. The Unigene set was selected from the ESTs and used to generate nylon microarrays. Changes in aquaporins and transporter transcripts were then studied during adventitious root development.
Identification of Primula fatty acid Δ6‐desaturases with n‐3 substrate preferences1
Authors: Sayanova Olga V. , Beaudoin Frédéric , Michaelson Louise V. , Shewry Peter R. , Napier Johnathan A.
Published: April 2003

Fatty acid Δ6‐desaturation, the first committed step in C20 polyunsaturated fatty acid biosynthesis, is generally considered not to discriminate between n‐3 and n‐6 substrates. We previously identified higher plant species that showed preferential Δ6‐desaturation of n‐3 C18 fatty acid substrates. A polymerase chain reaction‐based approach was used to isolate ‘front‐end’ cytochrome b
5 fusion desaturases from Primula vialii Franchet and Primula farinosa L. Functional analysis in Saccharomyces cerevisiae identified fatty acid Δ6‐desaturases with a strong specificity for the n‐3 substrate α‐linolenic acid (18:3Δ9,12,15). These results indicate that the accumulation of octadecatetraenoic acid (18:4Δ6,9,12,15) in planta is due to the activity of a novel n‐3‐specific fatty acid Δ6‐desaturase.
Glutamate substitutions at a PKA consensus site are consistent with inactivation of calpain by phosphorylation
Authors: Smith Scott D , Jia Zongchao , Huynh Kassidy K , Wells Alan , Elce John S
Published: April 2003

Regulation of calpain by phosphorylation has often been suggested, but has proved difficult to detect. Calpains extracted from mammalian tissue are reported to contain 2–4 mol phosphate/mol of enzyme distributed over multiple sites, but phosphate groups are not detectable in the X‐ray structures of recombinant calpain. Some serine and threonine residues in the large subunit of rat m‐calpain were converted to aspartic or glutamic acid residues, at sites suggested by previous studies, to assess the probable effects of phosphate groups on the enzyme. Expression of the mutant calpains in Escherichia coli, and their heat stabilities, did not differ from those of the wild‐type enzyme. m‐Calpains with the mutations Ser50Asp, Ser50Glu, Ser67Glu, and Thr70Glu had the same specific activity and Ca2+ requirement as the wild‐type enzyme. In contrast, Ser369Asp‐, Ser369Glu‐, and Thr370Glu‐m‐calpain were inactive. This result is consistent with the recent report that phosphorylation at position 369 or 370 in vivo reduced m‐calpain activation.
Protonmotive force generation by a redox loop mechanism
Authors: Jormakka Mika , Byrne Bernadette , Iwata So
Published: April 2003

Respiration involves the oxidation and reduction of substrate for the redox‐linked formation of a protonmotive force (PMF) across the inner membrane of mitochondria or the plasma membrane of bacteria. A mechanism for PMF generation was first suggested by Mitchell in his chemiosmotic theory. In the original formulations of the theory, Mitchell envisaged that proton translocation was driven by a ‘redox loop’ between two catalytically distinct enzyme complexes. Experimental data have shown that this redox loop does not operate in mitochondria, but has been confirmed as an important mechanism in bacteria. The nitrate respiratory pathway in Escherichia coli is a paradigm for a protonmotive redox loop. The structure of one of the enzymes in this two‐component system, formate dehydrogenase‐N, has revealed the structural basis for the PMF generation by the redox loop mechanism and this forms the basis of this review.
Evolutionary conservancy of the endocytic and trafficking machinery in the unicellular eukaryote Paramecium
Authors: Surmacz Liliana , Wiejak Jolanta , Wyroba Elzbieta
Published: April 2003

Abstract Molecular search for the homologues of the mammalian proteins in the unicellular eukaryote Paramecium involved in endocytosis and membrane trafficking is discussed. We cloned and sequenced the gene fragments encoding the following components participating in endosome formation, sorting and maturation of the proprotein precursors, respectively, dynamin 2, Rab7 and furin. There is a proof that all these genes are expressed in this unicellular organism. The function of the identified immunoanalogues of the above described components of Paramecium endocytic machinery as well as a high degree of sequence homology to the respective human counterparts points to the evolutionary conservancy of these pathways.
Experience of the Community-Residing Individual with Self-Induced Water Intoxication and the Family/Caregiver
Authors: May Deborah L.
Published: April 2003

BACKGROUND: With long-term psychiatric hospitals closing, the severely mentally ill with self-induced water intoxication (SIWI) are residing in the community. Knowledge about the experience of the community-residing individuals with SIWI and their family/caregiver is needed to develop community programs of care.

OBJECTIVES: The purpose of this multi-site study was to understand the experience of the community-residing individual with SIWI and of the family.

STUDY DESIGN: Thirty community-residing individuals with SIWI and at least one of their family members/caregivers were interviewed. Semi-structured open-ended questions were used. Urine specific gravity testing was conducted to assure that the individual met the criteria of SIWI.

RESULTS: Individuals reported that they felt better when they drank excess fluids, experienced anger when they drank, and believed that staying busy through work helped them control excess drinking. Individuals with SIWI and their families experienced negative feelings including fear of harm and helplessness and distanced themselves from the individual. Families agreed that distress triggered excess fluid drinking. Families also identified boredom as a trigger and felt helpless in being able to help the individual decrease fluid drinking.

CONCLUSIONS: Excess fluid drinking is viewed as beneficial by individuals with SIWI, is triggered by distress and boredom, and creates fear and distancing when the family feels threatened by the intoxicated individual. Distress and boredom reduction need to be addressed in programs of care before the community-residing individual with SIWI will be motivated to participate in fluid management.
Colored pain drawings: preliminary observations in a neurosurgical practice
Authors: Masferrer Roberto , Prendergast Virginia , Hagell Peter
Published: April 2003

Background: Black and white pain drawings were introduced as a proposed means to identify patients, presenting with low back pain, who demonstrated functional overlay upon neurological testing. The use of color may enhance the usefulness of such pain drawings, but has not been described for adult patients.

Aims: To retrospectively explore the use of colored pain drawings in patients with neck, low back, or radicular pain.

Methods: Patients with neck, low back, or radicular pain referred to a community‐based neurosurgical practice for evaluation during 1 year (n=359) depicted their pain on anatomical drawings using colored pencils representing different pain characteristics. Patients with abnormal (n=55) and normal (n=54) pain drawings were selected for this study. Use of medications, findings on physical examination, radiographic findings, activity levels, Waddell signs, and pending litigation were recorded and compared between patients with normal and abnormal pain drawings, as assessed according to the Ransford penalty point system.

Results: Patients whose colored pain drawings were abnormal, demonstrated a greater use of medications, more non‐focal clinical findings, Waddell signs, impaired activity levels, involvement in pending litigation, and significantly fewer pathological radiographic findings than patients with normal pain drawings.

Conclusions: Our findings agree with previous observations using black and white pain drawings, indicating that colored pain drawings are no less useful than the black and white approach. Further research is necessary to examine the psychometric properties and clinical usefulness of colored pain drawings to predict outcomes and/or determine treatment.
Robust cluster analysis of microarray gene expression data
with the number of clusters determined biologically
Authors: Bickel David R.
Published: April 2003

Motivation: The success of each method of cluster analysis depends on how well its underlying model describes the patterns of expression. Outlier-resistant and distribution-insensitive clustering of genes are robust against violations of model assumptions.

Results: A measure of dissimilarity that combines advantages of the Euclidean distance and the correlation coefficient is introduced. The measure can be made robust using a rank order correlation coefficient. A robust graphical method of summarizing the results of cluster analysis and a biological method of determining the number of clusters are also presented. These methods are applied to a public data set, showing that rank-based methods perform better than log-based methods.

Availability: Software is available from http://www.davidbickel.com.

Contact: dbickel@mail.mcg.edu

Supplementary Information: http://www.davidbickel.com will have updates and related articles

* To whom correspondence should be addressed.