bluuebunny/crossref_metadata_2025 huggingface dataset:
The N‐ and C‐termini of the tricarboxylate carrier are exposed to the cytoplasmic side of the inner mitochondrial membrane
Authors: Capobianco L. , Bisaccia F. , Michel A. , Sluse F.E. , Palmieri F.
Published: July 2002

Polyclonal antibodies were raised in rabbits against two synthetic peptides corresponding to the N‐ and C‐terminal regions of the rat‐liver mitochondria) tricarboxylate carrier. ELISA tests performed with intact and permeabilized rat‐liver mitoplasts showed that both anti‐N‐terminal and anti‐C‐terminal antibodies bind only to the cytoplasmic surface of the inner membrane, indicating that both termini of the membrane‐bound tricarboxylate carrier are exposed to the mitochondria) intermembrane space. Furthermore, tryptic digestion of intact mitoplasts markedly decreased the binding of anti‐N‐terminal and anti‐C‐terminal antibodies to the tricarboxylate carrier. These results are consistent with an arrangement of the tricarboxylate carrier monomer into an even number of transmembrane segments, with the N‐ and C‐termini protruding toward the cytosol.
Phosphate‐activated glutaminase expression during tumor development
Authors: Aledo Juan C. , Segura Juan A. , Medina Miguel A. , Alonso Francisco J. , de Castro Ignacio Núñez , Marquez Javier
Published: July 2002

Changes in phosphate‐activated glutaminase activities determined in intact cells and isolated mitochondria have been followed during mouse Ehrlich ascites carcinoma development. Glutaminase activities parallel the levels of poly(A)+ RNAs encoding for the mitochondrial phosphate activated glutaminase. During the exponential growth phase, maximum activity was observed and the relative abundance of glutaminase mRNA significantly increased with regard to the stationary growth phase. The presented results show that tumor phosphate‐activated glutaminase is subject to long‐term regulation by differential gene expression.
W‐7, a calmodulin antagonist, primes the stimulation of human neutrophil respiratory burst by formyl peptides and platelet‐activating factor
Authors: Périanin Axel , Pedruzzi Eric , Hakim Jacques
Published: July 2002

Low concentrations of the calmodulin antagonist W‐7 (1‐1μM) enhanced the respiratory burst (RB) of human polymorphonuclear leukocytes (PMN) stimulated by N‐formyl‐methionyl‐leucyl‐phenylalanine, whereas high drug concentrations (above 20 μM) depressed it. The maximal increase obtained with 5–10μM W‐7 affected both initial rate (50%) and total Superoxide anion production (150%). W‐7 also primed both parameters of the RB mediated by platelet‐activating factor, although higher drug concentrations were required (15–50 μM). By contrast, W‐7 depressed the RB induced by the calcium ionophore A23187 and by a protein kinase C activator, phorbol myristate acetate, with an 1C50 of approximately 20 and 8 μM, respectively. These data show the enhancing effect of W‐7 on chemoattractant‐mediated RB and suggest that RB priming may involve calmodulindependent regulation of chemoattractant‐mediated early signalling events.
Tyrosine‐194 of glycogenin undergoes autocatalytic glucosylation but is not essential for catalytic function and activity
Authors: Alonso Miriam D. , Lomako Joseph , Lomako Wieslawa M. , Whelan William J.
Published: July 2002

Glycogenin is the protein primer for glycogen synthesis. By autocatalytic transglucosylation from UDPglucose, it creates a malto‐octaose chain attached to its Tyr‐194. It has been uncertain whether the autocatalysis includes the addition of the first glucose residue to Tyr‐194. We now show this to be the case. However, we also demonstrate, contrary to a claim by others, that Tyr‐194 is not necessary for the catalytic function and activity of glycogenin.
Isolation and characterization of a gene for a ryanodine receptor/calcium release channel in Drosophila melanogaster
Authors: Takeshima Hiroshi , Nishi Miyuki , Iwabe Naoyuki , Miyata Takashi , Hosoya Toshihiko , Masai Ichiro , Hotta Yoshiki
Published: July 2002

The nucleotide sequence of a 25.7 kilobase Drosophila melanogaster genomic DNA segment containing a gene for a ryanodine receptor/calcium release channel homologue has been determined. Computer analysis and partial cDNA cloning revealed 26 exons comprising the protein‐coding sequence in this gene. The predicted protein is homologous in amino acid sequence and shares characteristic structural features with the mammalian ryanodine receptors. In blot hybridization analysis, a ~16 kilobase RNA species was identified abundantly in a 6–12 h embryo as the transcript from this gene. In situ hybridization to polytene chromosomes indicated that this gene locates at band position 44F on the second chromosome.
Bifurcated ubihydroquinone oxidation in the cytochrome bc1 complex by proton‐gated charge transfer
Authors: Brandt Ulrich
Published: July 2002

The unique bifurcation of electron flow at the ubihydroquinone‐oxidation center of the cytochrome bc
1 complex is the energy‐conserving reaction of the protonmotive Q‐cycle and is prerequisite to vectorial proton translocation. The widely accepted Q‐cycle reaction scheme describes the overall electron and proton pathways, but does not address the detailed chemistry of this central step. Based on a model of the ubihydroquinone‐oxidation pocket containing two ubiquinone molecules in a stacked configuration, a detailed model for the reactions during steady‐state catalysis is proposed. In this proton‐gated charge‐transfer mechanism the reaction is controlled by the deprotonation of the substrate ubihydroquinone.
Divinyl ether synthase from garlic (Allium sativum L.) bulbs: sub‐cellular localization and substrate regio‐ and stereospecificity
Authors: Grechkin Alexander N. , Hamberg Mats
Published: July 2002

Sub‐cellular localization and some properties of 13‐hydroperoxide‐specific divinyl ether synthase from garlic bulbs were studied. Sub‐cellular fractions from garlic bulbs were incubated with [1−14C](9Z,11E,13S)‐13‐hydroperoxy‐9,11‐octa‐decadienoic acid (13‐HPOD). The predominant part of divinyl ether synthase activity from garlic bulbs was found in the microsomal fraction. The enzyme utilizes 13(S)‐HPOD as its preferential substrate. Other hydroperoxides, including 9(S)‐HPOD, gave much poorer yields of divinyl ethers. Unreacted hydroperoxide after incubation of 13(R,S)‐HPOD with enzyme was composed of up to 94% 13(R)‐HPOD. Thus, divinyl ether synthase possesses stereoselectivity, utilizing preferentially the (S)‐enantiomer.
X‐ray crystal structure of the YM210W mutant reaction centre from Rhodobacter sphaeroides
Authors: McAuley Katherine E. , Fyfe Paul K. , Cogdell Richard J. , Isaacs Neil W. , Jones Michael R.
Published: July 2002

The X‐ray crystal structure of a reaction centre from Rhodobacter sphaeroides with a mutation of tyrosine M210 to tryptophan (YM210W) has been determined to a resolution of 2.5 Å. Structural conservation is very good throughout the body of the protein, with the tryptophan side chain adopting a position in the mutant complex closely resembling that of the tyrosine in the wild‐type complex. The spectroscopic properties of the YM210W reaction centre are discussed with reference to the structural data, with particular focus on evidence that the introduction of the bulkier tryptophan in place of the native tyrosine may cause a small tilt of the macrocycle of the BL monomeric bacteriochlorophyll.
Green tea compounds inhibit tyrosine phosphorylation of PDGF β‐receptor and transformation of A172 human glioblastoma
Authors: Sachinidis Agapios , Seul Claudia , Seewald Stefan , Ahn Hee-Yul , Ko Yon , Vetter Hans
Published: July 2002

The effect of the green tea compounds 2‐(3,4‐dihydroxyphenyl)‐3,4‐dihydro‐2H‐1‐benzopyran‐3,5,7‐triol (catechin), epicathechin (EC), epigallocathechin‐3 gallate (EGCG), epicathechin‐3 gallate (ECG) and catechin‐3 gallate (CG) on the tyrosine phosphorylation of PDGF β‐receptor (PDGF‐Rβ) and on the anchorage‐independent growth of A172 glioblastoma cells in semisolid agar has been investigated. Treatment of A172 glioblastoma with 50 μM CG, ECG, EGCG and 25 μM Tyrphostin 1296 resulted in an 82±17%, 77±21%, 75±8% and 55±11%, respectively (mean±S.D., n=3) inhibition of the PDGF‐BB‐induced tyrosine phosphorylation of PDGF‐Rβ. The PDGF‐Rβ downstream intracellular transduction pathway including tyrosine phosphorylation of phospholipase C‐γ1 (PLC‐γ1) and phosphatidylinositol 3′‐kinase (PI 3′‐K) was also inhibited. Spheroid formation was completely inhibited by 50 μM ECG, CG, EGCG and by 25 μM Tyrphostin 1296. We conclude that catechins of the green tea possessing the gallate group in their chemical structure act as anticancer agents probably partly via their ability to suppress the tyrosine kinase activity of the PDGF‐Rβ.
Identification and characterization of a eukaryotically encoded rubredoxin in a cryptomonad alga1
Authors: Wastl Jürgen , Sticht Heinrich , Maier Uwe-G. , Rösch Paul , Hoffmann Silke
Published: July 2002

We have identified an open reading frame with homology to prokaryotic rubredoxins (rds) on a nucleomorph chromosome of the cryptomonad alga Guillardia theta. cDNA analysis let us propose that the rd preprotein has an NH2‐terminal extension that functions as a transit peptide for import into the plastid. Compared to rds found in non‐photosynthetic prokaryotes or found in bacteria that exhibit an anoxigenic photosynthesis apparatus, nucleomorph rd has a COOH‐terminal extension, which shows high homology exclusively to the COOH‐termini of cyanobacterial rds as well as to a hypothetical rd in the Arabidopsis genome. This extension can be divided into a putative membrane anchor and a stretch of about 20 amino acids with unknown function linking the common rd fold to this anchor. Overexpression of nucleomorph rd in Escherichia coli using a T7 RNA polymerase/promotor system resulted in a mixture of iron‐containing holorubredoxin and zinc‐substituted protein. Preliminary spectroscopic studies of the iron form of nucleomorph rd suggest the existence of a native rd‐type iron site. One‐dimensional nuclear magnetic resonance spectroscopy of recombinant Zn‐rd suggests the presence of a stable tertiary fold similar to that of other rd structures determined previously.
Bgs2p, a 1,3‐β‐glucan synthase subunit, is essential for maturation of ascospore wall in Schizosaccharomyces pombe
Authors: Liu Jianhua , Tang Xie , Wang Hongyan , Balasubramanian Mohan
Published: July 2002

Previously we have reported that Drc1p/Cps1p, a 1,3‐β‐glucan synthase subunit, is essential for division septum assembly in Schizosaccharomyces pombe. In this report, we present evidence that S. pombe Bgs2p, a 1,3‐β‐glucan synthase that shows 56% identity to Drc1p/Cps1p, is essential for maturation of ascospore wall in S. pombe, but is not required for vegetative growth. Diploid cells homozygous for the bgs2‐null mutation, as well as homothallic bgs2‐null mutant haploids undergo meiosis normally. However, a 1,3‐β‐glucan containing spore wall is not assembled in these cells. The spores resulting from meiosis of a bgs2‐null mutant lyse upon release from the ascus and are therefore inviable. Using a green fluorescent protein‐tagged Bgs2p, we demonstrate that Bgs2p is localized at the periphery of the ascospores during meiosis and sporulation. However, Bgs2p is not detected in vegetative cells. We conclude that Bgs2p is required for 1,3‐β‐glucan synthesis during ascospore wall maturation.
New sequence data enable modelling of the fungal alternative oxidase and explain an absence of regulation by pyruvate
Authors: Joseph-Horne Tim , Babij Joanna , Wood Paul M , Hollomon Derek , Sessions Richard B
Published: July 2002

Respiratory rates involving the alternative oxidase (AO) were studied in mitochondria from Tapesia acuformis. There was no evidence for regulation by pyruvate, in contrast with plant AO. The site of interaction of pyruvate with the plant AO is a conserved cysteine. The primary sequence was obtained for AO from Magnaporthe grisea and compared with four published sequences for fungal AO. In all cases this cysteine was absent. Sequence data were obtained for the C‐terminal domain of a further five fungal AOs. In this region the fungal sequences were all consistent with a four‐helix, di‐iron binding structure as in the ferritin‐fold family. A molecular model of this domain was deduced from the structure of Δ‐9 desaturase. This is in general agreement with that developed for plant AOs, despite very low sequence identity between the two kingdoms. Further modelling indicated an appropriate active site for binding of ubiquinol, required in the AO redox reaction.
Membrane interactions and alignment of structures within the HIV‐1 Vpu cytoplasmic domain: effect of phosphorylation of serines 52 and 56
Authors: Henklein Peter , Kinder Rudolf , Schubert Ulrich , Bechinger Burkhard
Published: July 2002

The cytoplasmic domain of the HIV‐1 accessory protein Vpu is involved in the binding and degradation of the viral receptor CD4. In order to analyze previous structural models in the context of membrane environments, regions of VpuCYTO incorporating particular conformational features have been synthesized and labelled with 15N at selected backbone amides. Well‐oriented proton‐decoupled 15N solid‐state NMR spectra with 15N chemical shifts at the most upfield position indicate that the amphipathic helix within [15N‐Leu 45]‐Vpu27–57 strongly interacts with mechanically aligned POPC bilayers and adopts an orientation parallel to the membrane surface. No major changes in the topology of this membrane‐associated amphipathic helix were observed upon phosphorylation of serine residues 52 and 56, although this modification regulates biological function of Vpu. In contrast, [15N‐Ala 62]‐Vpu51–81 exhibits a pronounced 15N chemical shift anisotropy.
Identification of Asp197 as the catalytic nucleophile in the family 38 α‐mannosidase from bovine kidney lysosomes
Authors: Numao Shin , He Shouming , Evjen Gry , Howard Steven , Tollersrud Ole K. , Withers Stephen G.
Published: July 2002

Bovine kidney lysosomal α‐mannosidase is a family 38 α‐mannosidase involved in the degradation of glycoproteins. The mechanism‐based reagent, 5‐fluoro‐β‐L‐gulosyl fluoride, was used to trap a glycosyl–enzyme intermediate, thereby labelling the catalytic nucleophile of this enzyme. After proteolytic digestion and high performance liquid chromatography/tandem mass spectrometry (MS) analysis, a labelled peptide was localised, and the sequence: HIDPFGHSRE determined by fragmentation tandem MS analysis. Taking into consideration sequence alignments of this region with those of other α‐mannosidases of the same family, this result strongly suggests that the catalytic nucleophile in this enzyme is Asp197.
Oncostatin M and hepatocyte growth factor induce hepatic maturation via distinct signaling pathways
Authors: Kamiya Akihide , Kinoshita Taisei , Miyajima Atsushi
Published: July 2002

Liver development is regulated by soluble factors as well as cell–cell contacts. We previously reported that oncostatin M (OSM) induced hepatic maturation in a primary culture of embryonic day 14 liver cells. While OSM expression in the liver starts in mid gestation and decreases in postnatal stages, hepatocyte growth factor (HGF) is mainly expressed in the liver in the first few days after birth. In this study, we compared the effect of OSM and HGF on the differentiation of fetal hepatic cells in vitro. Like OSM, HGF in the presence of dexamethasone induced expression of glucose‐6‐phosphatase, tyrosine amino transferase and carbamoyl‐phosphate synthase, and accumulation of glycogen in fetal hepatic cells, although to a lesser extent than OSM. Interestingly, while both OSM and HGF up‐regulated production of albumin, secretion of albumin occurred only in response to OSM. In addition, although hepatic maturation induced by OSM depends on STAT3, HGF failed to activate STAT3 and HGF‐induced differentiation was independent of STAT3. These results indicate that OSM and HGF induce hepatic maturation through different signaling pathways.
Lactoferricin B causes depolarization of the cytoplasmic membrane of Escherichia coli ATCC 25922 and fusion of negatively charged liposomes
Authors: Ulvatne H , Haukland H.H , Olsvik Ø , Vorland L.H
Published: July 2002

Antimicrobial peptides have been extensively studied in order to elucidate their mode of action. Most of these peptides have been shown to exert a bactericidal effect on the cytoplasmic membrane of bacteria. Lactoferricin is an antimicrobial peptide with a net positive charge and an amphipatic structure. In this study we examine the effect of bovine lactoferricin (lactoferricin B; Lfcin B) on bacterial membranes. We show that Lfcin B neither lyses bacteria, nor causes a major leakage from liposomes. Lfcin B depolarizes the membrane of susceptible bacteria, and induces fusion of negatively charged liposomes. Hence, Lfcin B may have additional targets responsible for the antibacterial effect.
TNF‐α inhibits UCP‐1 expression in brown adipocytes via ERKs
Authors: Valladares Amparo , Roncero Cesar , Benito Manuel , Porras Almudena
Published: July 2002

Tumor necrosis factor‐α (TNF‐α) activates extracellular‐regulated kinases (ERKs) and p38 mitogen‐activated protein kinase (p38MAPK), and inhibits the expression of uncoupling protein‐1 (UCP‐1) and adipocyte‐specific genes in rat fetal brown adipocytes. MEK inhibition with PD98059 abolished the inhibitory effect of TNF‐α on UCP‐1, but not on adipogenic genes. In contrast, inhibition of p38MAPK with SB203580 potentiated the negative effect of TNF‐α on UCP‐1 and adipogenic genes. The inhibitory action of TNF‐α was partially correlated with changes in C/EBPα and β protein levels and in their DNA binding activity, suggesting a role for these transcription factors. However, other transcription factors might explain the different regulation of UCP‐1 and adipogenic genes by ERKs.
The rotor in the membrane of the ATP synthase and relatives
Authors: Arechaga Ignacio , Jones Phil C
Published: July 2002

In recent years, structural information on the F1 sector of the ATP synthase has provided an insight into the molecular mechanism of ATP catalysis. The structure strongly supports the proposal that the ATP synthase works as a rotary molecular motor. Insights into the membrane domain have just started to emerge but more detailed structural information is needed if the molecular mechanism of proton translocation coupled to ATP synthesis is to be understood. This review will focus mainly on the ion translocating rotor in the membrane domain of the F‐type ATPase, and the related vacuolar and archaeal relatives.
YidC/Oxa1p/Alb3: evolutionarily conserved mediators of membrane protein assembly
Authors: Luirink Joen , Samuelsson Tore , de Gier Jan-Willem
Published: July 2002

This review focuses on a novel, evolutionarily conserved mediator of membrane protein assembly in bacteria, mitochondria and chloroplasts. This factor is designated YidC in Escherichia coli, and is localized in the inner membrane. YidC is homologous to Oxa1p in the mitochondrial inner membrane and Alb3 in the chloroplast thylakoid membrane, but does not seem to have a homologue in the endoplasmic reticulum membrane. It has been suggested that YidC operates both as a separate unit and in connection with the SecYEG‐translocon depending on the substrate membrane protein that is integrated into the membrane. Mitochondria do not possess a SecYEG‐like complex and Oxa1p is thought to form, or to contribute to the formation of, a novel translocon in the mitochondrial inner membrane. Alb3 in the chloroplast thylakoid membrane is, just like YidC and Oxa1p, involved in membrane protein assembly, but only few details are known.
Lipobeads: a hydrogen anchored lipid vesicle system
Authors: Jin T. , Pennefather P. , Lee P.I.
Published: July 2002

A new vesicle system is described that combines complementary properties of liposomes and polymeric beads. ‘Lipobeads’ consist of a lipid bilayer shell anchored on the surface of a hydrogel polymer cores which acts like a cytoskeleton. Anchoring is provided by fatty acids covalently attached to the surface of the hydrogel. These hydrophobic chains drive spontaneous assembly of a lipid bilayer shell around the modified hydrogel bead when exposed to a suspension of liposomes. The bilayer is stable and acts as a permeability barrier to compound loaded by prior absorption into the polymer core. Lipid mobility in the shell is similar to that found in other unanchored lipid bilayers. The system has potential application in drug delivery and for functional reconstitution of membrane proteins.
Long‐chain fatty acids suppress the induction of urea cycle enzyme genes by glucocorticoid action
Authors: Tomomura Mineko , Tomomura Akito , Abdullah Abu Musa Dewan Md. , Saheki Takeyori
Published: July 2002

In order to test the possibility that free fatty acids are the mediator of the abnormal expression of urea cycle enzyme genes in carnitine‐deficient juvenile visceral steatosis (JVS) mice, the effects of fatty acids on urea cycle enzyme, carbamoylphosphate synthetase (CPS) and argininosuccinate synthetase (ASS), mRNA levels were examined in rat primary cultured hepatocytes. Addition of a synthetic glucocorticoid hormone, dexamethasone, caused increases in CPS and ASS mRNAs. Further addition of oleic acid suppressed the induction of CPS and ASS mRNAs by dexamethasone. In contrast, the phosphoenolpyruvate carboxykinase (PEPCK) mRNA level induced by dexamethasone was enhanced in the presence of oleic acid. The effects were reversed on further addition of carnitine. The mRNA levels of these enzymes induced by dibutyryl cAMP were not affected by the addition of oleic acid. A study of the specificity of fatty acids revealed that long‐chain fatty acids of more than 16 carbons chain length had a suppressive effect on the CPS mRNA level induced by dexamethasone and that the presence of double bonds enhanced the effect. The changes in gene expression of CPS, ASS and PEPCK caused by the fatty acids in the cultured hepatocytes were very similar to those observed in the liver of JVS mice. The AP‐1 DNA binding activity in the presence of dexamethasone was slightly enhanced by the addition of oleic acid. These results suggest that the long‐chain fatty acids not metabolized in JVS mice are mediators of the abnormal gene expression in the liver which results in hyperammonemia.
On the involvement of calpains in the degradation of the tumor suppressor protein p53
Authors: Gonen Hedva , Shkedy Dganit , Barnoy Sivia , Kosower Nechama S. , Ciechanover Aaron
Published: July 2002

A crude fraction that contains ubiquitin–protein ligases contains also a proteolytic activity of ∼100 kDa that cleaves p53 to several fragments. The protease does not require ATP and is inhibited in the crude extract by an endogenous ∼250 kDa inhibitor. The proteinase can be inhibited by chelating the Ca2+ ions, by specific cysteine proteinase inhibitors and by peptide aldehyde derivatives that inhibit calpains. Purified calpain demonstrates an identical activity that can be inhibited by calpastatin, the specific protein inhibitor of the enzyme. Thus, it appears that the activity we have identified in the extract is catalyzed by calpain. The calpain in the extract degrades also N‐myc, c‐Fos and c‐Jun, but not lysozyme. In crude extract, the calpain activity can be demonstrated only when the molar ratio of the calpain exceeds that of its native inhibitor. Recent experimental evidence implicates both the ubiquitin proteasome pathway and calpain in the degradation of the tumor suppressor, and it was proposed that the two pathways may play a role in targeting the protein under various conditions. The potential role of the two systems in this important metabolic process is discussed.
Interaction of annexins IV and VI with ATP
Authors: Bandorowicz-Pikuła Joanna , Awasthi Yogesh C
Published: July 2002

Annexin VI from porcine liver can be photoaffinity‐labeled with 8‐azido‐[γ‐32P]ATP in a concentration‐dependent, saturable manner. The extent of labeling varied with the concentration of calcium. The dissociation constant for the nucleotide was found to be in the range reported for ATP‐binding proteins. The ATP analog, 2′‐(or 3′)‐O‐(2,4,6‐trinitrophenyl)adenosine 5′‐triphosphate, also bound to AnxVI, as indicated by shift in its fluorescence spectra in the presence of protein. Any significant 8‐azido‐ATP or TNP‐ATP binding was not observed with AnxIV. ATP modulated the binding of AnxVI to erythrocyte membrane and increased the Ca2+ concentration required for half‐maximal binding of AnxVI to F‐actin.
Sequence and characterization of twoArabidopsis thalianacDNAs isolated by functional complementation of a yeastgln3 gdh1mutant
Authors: Truong Hoai-Nam , Caboche Michel , Daniel-Vedele Françoise
Published: July 2002

We have isolated twoArabidopsis thalianacDNAs by complementation of a yeastgln3 gdh1strain that is affected in the regulation of nitrogen metabolism. The two clones (RGA1andRGA2) are homologous to each other and to theSCARECROW(SCR) gene that is involved in regulating an asymmetric cell division in plants. RGA1, RGA2 and SCR share several structural features and may define a new family of genes.RGA1andRGA2have been mapped, respectively, to chromosome II and I, and their expression in plant is constitutive.
Light‐harvesting in Acaryochloris marina – spectroscopic characterization of a chlorophyll d‐dominated photosynthetic antenna system
Authors: Schiller Hilmar , Senger Horst , Miyashita Hideaki , Miyachi Shigetoh , Dau Holger
Published: July 2002

Oxygenic photosynthesis of the prokaryote Acaryochloris marina involves chlorophyll d (Chl d) as the major pigment [Miyashita et al. (1996) Nature 383, 402]. Four spectral forms of Chl d (peak wavelengths: 694, 714, 726 and 740 nm) are resolvable by low‐temperature absorption spectroscopy on intact cells. Based on fluorescence spectra (at 290 K and 77 K) and on analysis of fluorescence induction curves we conclude: (1) excitation energy is efficiently transferred between the various spectral forms of Chl d and the PS II reaction center; (2) Chl d serves as a light‐harvesting pigment for both, Photosystem II (PS II) and PS I; (3) excitation energy transfer between PS II units occurs.
A new family of K+ transporters from Arabidopsis that are conserved across phyla
Authors: Quintero Francisco J , Blatt Michael R
Published: July 2002

Transport of K+ in higher plants, as in bacteria and fungi, is mediated by two broad classes of transport proteins that operate in the millimolar and micromolar K+ concentration ranges. A search of the Expressed Sequence Tag database using amino acid consensus sequences for the K+ transporters HAK1 from Schwanniomyces and Kup of Escherichia coli yielded two homologous sequences for Arabidopsis. Cloning and sequencing of these genes gave single open reading frames for the putative transporters, AtKT1 and AtKT2, with predicted molecular weights of 79 and 88 kDa. The predicted gene products showed a high degree of homology at the amino acid level (56% identity) and exhibited significant hydrophobic stretches in their N‐terminal halves, consistent with 12 membrane‐spanning, α‐helical domains. Database searches using AtKT1 and AtKT2 identified 10 additional sequences in Arabidopsis as well as additional homologous sequences in the plant species Oryza and Allium, the bacterium Lactococcus lactis, and in Homo sapiens. Expression of AtKT2 rescued growth on low millimolar [K+] in Saccharomyces cerevisiae carrying deletions for the genes encoding the K+ transporters TRK1 and TRK2. Rescue was associated with a 2‐fold stimulation of Rb+ uptake and was sensitive to competition with external Na+ but not to extracellular pH, indicating that the gene encodes a low‐affinity K+ transporter. These and additional results suggest that AtKT1 and AtKT2 belong to a superfamily of cation transporters that have been conserved through evolution.
The fungal elicitor cryptogein is a sterol carrier protein
Authors: Mikes Vladimir , Milat Marie-Louise , Ponchet Michel , Ricci Pierre , Blein Jean-Pierre
Published: July 2002

Cryptogein is a protein secreted by the phytopathogenic pseudo‐fungus, Phytophthora cryptogea. It is a basic 10 kDa hydrophilic protein having a hydrophobic pocket and three disulfide bridges. These common features with sterol carrier proteins led us to investigate its possible sterol transfer activity using the fluorescent sterol, dehydroergosterol. The results show that cryptogein has one binding site with strong affinity for dehydroergosterol. Moreover, this protein catalyzes the transfer of sterols between phospholipidic artificial membranes. This is the first evidence for the existence of an extracellular sterol carrier protein and for a molecular activity of cryptogein. This property should contribute to an understanding of the role of cryptogein in plant‐microorganism interactions.
Enhanced expression of uncoupling protein 2 gene in rat white adipose tissue and skeletal muscle following chronic treatment with thyroid hormone
Authors: Masaki Takayuki , Yoshimatsu Hironobu , Kakuma Tetsuya , Hidaka Shuji , Kurokawa Mamoru , Sakata Toshiie
Published: July 2002

Evidence is rapidly emerging which suggests that uncoupling protein 2 (UCP2), by virtue of its ubiquitous expression, may be important for determining basal metabolic rate. To assess the functional modulation of UCP2 gene expression in relation to body weight control, we examined the effects of hyperthyroid state induced by chronic treatment with triiodothyronine (T3) on UCP2 mRNA expression in male rats. Daily subcutaneous injection of T3 (37 pmol/100 g body weight) for 7 days increased UCP2 mRNA expression in brown adipose tissue (BAT), white adipose tissue (WAT) and the soleus muscle 1.6‐, 1.6‐ and 1.7‐fold compared to the controls, respectively, and increased UCP1 mRNA expression in BAT 1.2‐fold. In contrast, the same treatment with T3 decreased both ob mRNA expression in WAT and plasma leptin level 0.5‐fold for each. The present results suggest that T3 may directly increase UCP2 expression independently of leptin action.
Structure and expression of mitsugumin29 gene1
Authors: Shimuta Misa , Komazaki Shinji , Nishi Miyuki , Iino Masamitsu , Nakagawara Kan-ichi , Takeshima Hiroshi
Published: July 2002

Recently mitsugumin29 unique to the triad junction in skeletal muscle was identified as a novel member of the synaptophysin family; the members of this family have four transmembrane segments and are distributed on intracellular vesicles. In this study, we isolated and analyzed mouse mitsugumin29 cDNA and genomic DNA containing the gene. The mitsugumin29 gene mapped to the mouse chromosome 3 F3‐H2 is closely related to the synaptophysin gene in exon‐intron organization, which indicates their intimate relationship in molecular evolution. RNA blot hybridization and immunoblot analysis revealed that mitsugumin29 is expressed abundantly in skeletal muscle and at lower levels in the kidney. Immunofluorescence microscopy demonstrated that mitsugumin29 exists specifically in cytoplasmic regions of the proximal and distal tubule cells in the kidney. The results obtained may suggest that mitsugumin29 is involved in the formation of specialized endoplasmic reticulum systems in skeletal muscle and renal tubule cells.
Fe65 and the protein network centered around the cytosolic domain of the Alzheimer's β‐amyloid precursor protein
Authors: Russo Tommaso , Faraonio Raffaella , Minopoli Giuseppina , De Candia Paola , De Renzis Stefano , Zambrano Nicola
Published: July 2002

A distinctive tract of all the forms of Alzheimer's disease is the extracellular deposition of a 40–42/43 amino acid‐long peptide derived from the so‐called β‐amyloid precursor protein (APP). This is a membrane protein of unknown function, whose short cytosolic domain has been recently demonstrated to interact with several proteins. One of these proteins, named Fe65, has the characteristics of an adaptor protein; in fact, it possesses three protein‐protein interaction domains: a WW domain and two PID/PTB domains. The interaction with APP requires the most C‐terminal PID/PTB domain, whereas the WW domain is responsible for the interaction with various proteins, one of which was demonstrated to be the mammalian homolog of the Drosophila enabled protein (Mena), which in turn interacts with the cytoskeleton. The second PID/PTB domain of Fe65 binds to the CP2/LSF/LBP1 protein, which is an already known transcription factor. The other proteins interacting with the cytosolic domain of APP are the Go heterotrimeric protein, APP‐BP1 and X11. The latter interacts with APP through a PID/PTB domain and possesses two other protein‐protein interaction domains. The small size of the APP cytodomain and the overlapping of its regions involved in the binding of Fe65 and X11 suggest the existence of competitive mechanisms regulating the binding of the various ligands to this cytosolic domain. In this short review the possible functional roles of this complex protein network and its involvement in the generation of Alzheimer's phenotype are discussed.
Stress‐induced expression of transcriptional repressor ICER in the adrenal gland
Authors: Della Fazia Maria Agnese , Servillo Giuseppe , Foulkes Nicholas S , Sassone-Corsi Paolo
Published: July 2002

Second messenger cyclic AMP plays a central role in signalling within the hypothalamo‐pituitary‐adrenal (HPA) axis. Changes in gene expression are central to long‐term adaptations made in response to stress in the adrenal gland. Here we demonstrate that expression of the cAMP inducible transcriptional repressor, ICER (Inducible cAMP Early Repressor), is rapidly and powerfully induced in response to surgical stress in the rat adrenal gland. Hypophysectomisation blocks stress‐induced ICER expression. Finally we demonstrate that injection of the pituitary hormone ACTH (Adrenocorticotropin Hormone) induces robust ICER expression in the adrenal cortex. Thus, induction of the transcriptional repressor ICER is coupled to the HPA axis response to stress.
Inhibition of NADPH supply by 6‐aminonicotinamide: effect on glutathione, nitric oxide and superoxide in J774 cells
Authors: Hothersall John S , Gordge Michael , Noronha-Dutra Alberto A
Published: July 2002

We have examined the integrity of J774 cell nitric oxide (NO) production and glutathione maintenance, whilst NADPH supply was compromised by inhibition of the pentose pathway with 6‐aminonicotinamide. In resting cells 6‐phosphogluconate accumulation began after 4 h and glutathione depletion after 24 h of 6‐aminonicotinamide treatment. Cellular activation by lipopolysaccharide/interferon‐λ decreased glutathione by ∼50% and synchronous 6‐aminonicotinamide treatment exacerbated this to 31.2% of control (P<0.05). In activated cells NO−
2 production was inhibited by 60% with 6‐aminonicotinamide (P<0.01), and superoxide production by 50% (P<0.01) in zymosan‐activated cells. NADPH production via the pentose pathway is therefore important to sustain macrophage NO production whilst maintaining protective levels of glutathione.
Distribution of orexin receptor mRNA in the rat brain
Authors: Trivedi Prashant , Yu Hong , MacNeil Douglas J , Van der Ploeg L.H.T , Guan Xiao-Ming
Published: July 2002

The expression pattern of mRNA encoding two orexin receptors (OX1R and OX2R) in the rat brain was examined. OX1R and OX2R exhibited marked differential distribution. Within the hypothalamus, OX1R mRNA is most abundant in the ventromedial hypothalamic nucleus whereas OX2R is predominantly expressed in the paraventricular nucleus. High levels of OX1R mRNA were also detected in tenia tecta, the hippocampal formation, dorsal raphe, and locus coeruleus. OX2R mRNA is mainly expressed in cerebral cortex, nucleus accumbens, subthalamic and paraventricular thalamic nuclei, anterior pretectal nucleus. The presence of orexin receptor mRNA in the hypothalamus is in support of its proposed role in feeding regulation. Broad central distribution of orexin receptors may indicate additional functions for orexins.
Immunohistochemical detection of dityrosine in lipofuscin pigments in the aged human brain
Authors: Kato Yoji , Maruyama Wakako , Naoi Makoto , Hashizume Yoshio , Osawa Toshihiko
Published: July 2002

Lipofuscin is a yellowish brown fluorescent pigment which is sequestered within cytoplasmic granules during aging. To examine the contribution of protein oxidation to lipofuscin accumulation, we performed immunohistochemical detection of dityrosine, which is considered one of the specific markers for protein oxidation, in lipofuscin in the aged human brain using an antibody specific to dityrosine. By characterization using competitive enzyme‐linked immunosorbent assay, the specificity of the antibody to dityrosine was confirmed. None of the other tyrosine‐related compounds such as l‐tyrosine, 3‐nitrotyrosine, 3‐chlorotyrosine, or 3,4‐dihydroxyphenylalanine cross‐reacted with the antibody. The anti‐dityrosine antibody reacted with lipofuscin granules in the pyramidal neurons of the aged human brain. The results suggest that protein oxidation by free radicals and/or peroxidases may play an important role in lipofuscin accumulation.
Balanced regulation of expression of the gene for cytochrome cM and that of genes for plastocyanin and cytochrome c6 in Synechocystis
Authors: Malakhov Michael P. , Malakhova Oksana A. , Murata Norio
Published: July 2002

The cytM gene for cytochrome c
M was previously found in Synechocystis sp. PCC 6803. Northern blotting analysis revealed that the cytM gene was scarcely expressed under normal growth conditions but its expression was enhanced when cells were exposed to low temperature or high‐intensity light. By contrast, the expression of the genes for cytochrome c
6 and plastocyanin was suppressed at low temperature or under high‐intensity light. These observations suggest that plastocyanin and/or cytochrome c
6, which are dominant under non‐stressed conditions, are replaced by cytochrome c
M under the stress conditions.
Anoxic function for the Escherichia coli flavohaemoglobin (Hmp): reversible binding of nitric oxide and reduction to nitrous oxide
Authors: Kim Sung Oog , Orii Yutaka , Lloyd David , Hughes Martin N. , Poole Robert K.
Published: July 2002

The flavohaemoglobin Hmp of Escherichia coli is inducible by nitric oxide (NO) and provides protection both aerobically and anaerobically from inhibition of growth by NO and agents that cause nitrosative stress. Here we report rapid kinetic studies of NO binding to Fe(III) Hmp with a second order rate constant of 7.5×105 M−1 s−1 to generate a nitrosyl adduct that was stable anoxically but decayed in the presence of air to reform the Fe(III) protein. NO displaced CO bound to dithionite‐reduced Hmp but, remarkably, CO recombined after only 2 s at room temperature indicative of NO reduction and dissociation from the haem. Addition of NO to anoxic NADH‐reduced Hmp also generated a nitrosyl species which persisted while NADH was oxidised. These results are consistent with direct demonstration by membrane‐inlet mass spectrometry of NO consumption and nitrous oxide production during anoxic incubation of NADH‐reduced Hmp. The results demonstrate a new mechanism by which Hmp may eliminate NO under anoxic growth conditions.
Oxidized LDL activates STAT1 and STAT3 transcription factors: possible involvement of reactive oxygen species
Authors: Mazière Cécile , Alimardani Gulie , Dantin Françoise , Dubois Françoise , Conte Marie-Alix , Mazière Jean-Claude
Published: July 2002

The effect of cupric ion‐oxidized low density lipoprotein (Cu‐LDL) or endothelial cell‐oxidized LDL (E‐LDL) on STAT1 and STAT3 (signal transducers and activators of transcription) DNA binding activity was investigated by electrophoretic mobility shift assay in human endothelial cells. Both oxidized LDL enhanced STAT1 and STAT3 binding to their respective consensus binding sites. Furthermore, the activation of STATs was proportional to the oxidation degree of LDL in that the highly oxidized Cu‐LDL exhibited a more marked effect than E‐LDL. Oxidized LDL induced an intracellular oxidative stress, as shown by the increase in the intracellular level of lipid peroxidation products (thiobarbituric acid‐reactive substances) and in the level of reactive oxygen species, measured by the fluorescence of dichlorofluorescein diacetate. The binding activity of STAT1 and STAT3 paralleled these two parameters, which suggests that it is dependent upon the redox state of the cell. The activation of STATs by oxidized LDL was almost completely inhibited by the lipophilic antioxidant vitamin E, and partially antagonized by the hydrophilic thiol‐containing compound N‐acetylcysteine, suggesting that the oxidative stress induced by oxidized LDL is involved in the observed phenomenon. Furthermore, the lipid extract of Cu‐LDL also activated STAT1 and STAT3. Since the STAT pathway plays a key role in cytokine and growth factor signal transduction, the activation of STATs by oxidized LDL might be related to their proinflammatory and fibroproliferative effect in the atherosclerotic plaque.
Significant enhancement in the binding of p‐nitrophenyl‐β‐d‐xylobioside by the E128H mutant F/10 xylanase from Streptomyces olivaceoviridis E‐86
Authors: Kuno Atsushi , Shimizu Daisuke , Kaneko Satoshi , Hasegawa Tsunemi , Gama Yasuo , Hayashi Kiyoshi , Kusakabe Isao , Taira Kazunari
Published: July 2002

Mutagenesis studies were carried out to examine the effects of replacement of either the nucleophile Glu‐236 or the acid/base Glu‐128 residue of the F/10 xylanase by a His residue. To our surprise, the affinity for the p‐nitrophenyl‐β‐D‐xylobioside substrate was increased by 103‐fold in the case of the mutant E128H enzyme compared with that of the wild‐type F/10 xylanase. The catalytic activity of the mutant enzymes was low, despite the fact that the distance between the nucleophilic atom (an oxygen in the native xylanase and a nitrogen in the mutant) and the α‐carbon was barely changed. Thus, the alteration of the acid/base functionality (Glu‐128 to His mutation) provided a significantly favorable interaction within the E128H enzyme/substrate complex in the ground state, accompanying a reduction in the stabilization effect in the transition state.
The structure of neuromelanin and its iron binding site studied by infrared spectroscopy
Authors: Bridelli M.G. , Tampellini D. , Zecca L.
Published: July 2002

The binding of neuromelanin (NM) to iron is of interest due to its role in brain aging and Parkinson's disease. In the present work, infrared spectra of both NM isolated from human brain and of synthetic NM analogues are reported with the aim of identifying the main functional groups and their chelating ability for iron. It is observed that a peptide and an aliphatic chain are present in the NM structure. The coordination of iron in NM occurs through –OH phenolic units. In synthetic melanin samples, the preferred sites for iron binding are –OH phenolic and >NH indolic groups. Amino acid analysis confirmed the presence of a peptide component in NM and synthetic melanin incubated in putamen homogenate. In addition, the elemental analysis demonstrated the presence of an aliphatic component specific of NM.
Phospholipin, a novel heterodimeric phospholipase A2 from Pandinus imperator scorpion venom
Authors: Conde Renaud , Zamudio Fernando Z , Becerril Baltazar , Possani Lourival D
Published: July 2002

The primary structure of a phospholipase A2, with unique structural and functional characteristics, was determined. The large subunit has 108 amino acid residues, linked by a disulfide bridge to the small subunit, which contains 17 residues. Its gene was cloned from a cDNA library. The nucleotide sequence showed that the same RNA messenger encodes both subunits, separated only by a pentapeptide, that is processed during maturation.
Anthrax lethal factor cleaves MKK3 in macrophages and inhibits the LPS/IFNγ‐induced release of NO and TNFα
Authors: Pellizzari Rossella , Guidi-Rontani Chantal , Vitale Gaetano , Mock Michèle , Montecucco Cesare
Published: July 2002

The lethal toxin ofBacillus anthracisconsists of two proteins, PA and LF, which together induce lethal effects in animals and cause macrophage lysis. LF is a zinc‐endopeptidase which cleaves two mitogen‐activated proten kinase kinases (MAPKKs), Mek1 and Mek2, within the cytosol. Here, we show that also MKK3, another dual‐specificity kinase that phosphorylates and activates p38 MAP kinase, is cleaved by LF in macrophages. No direct correlation between LF‐induced cell death and cleavage of these MAPKKs was found in macrophage cell lines and primary peritoneal cells exhibiting different sensitivity to LF. However, we present the first evidence that sublytic doses of LF cleave Meks and cause a substantial reduction in the production of NO and tumour necrosis factor‐α induced by lipopolysaccharide/interferonγ. We suggest that this effect of LF is relevant during the first stages ofB. anthracisinfection, when a reduction of the inflammatory response would permit growth and diffusion of the bacterium.
Cytochrome c release and caspase activation during menadione‐induced apoptosis in plants
Authors: Sun Ying-Li , Zhao Yun , Hong Xia , Zhai Zhong-He
Published: July 2002

We report here the detection of the release of cytochrome c from mitochondria into the cytosol during menadione‐induced apoptosis in tobacco protoplasts. Western blot analysis indicated that the caspase specific inhibitors AC‐DEVD‐CHO (Ac‐Asp‐Glu‐Val‐Asp‐aldehyde) and AC‐YVAD‐CHO (N‐acetyl‐Try‐Val‐Ala‐aspartinal) inhibited the degradation of a caspase 3 specific substrate PARP (poly(ADP‐ribose) polymerase), and they had no effect on the release of cytochrome c. Further study showed that menadione could not induce apoptosis of mouse liver nuclei in tobacco cytosol extract containing no mitochondria. However, when cytochrome c or mitochondria was added into the cytosol extract, apoptosis of mouse liver nuclei and the degradation of PARP could both be detected. The results provide strong evidence that menadione can induce apoptosis in tobacco protoplasts via the release of cytochrome c from mitochondria into the cytosol.
Molecular cloning and functional expression of Cav3.1c, a T‐type calcium channel from human brain
Authors: Cribbs Leanne L. , Gomora Juan Carlos , Daud Asif N. , Lee Jung-Ha , Perez-Reyes Edward
Published: July 2002

Low voltage‐activated T‐type calcium channels are encoded by a family of at least three genes, with additional diversity created by alternative splicing. This study describes the cloning of the human brain α1G, which is a novel isoform, Cav3.1c. Comparison of this sequence to genomic sequences deposited in the GenBank allowed us to identify the intron/exon boundaries of the human CACNA1G gene. A full‐length cDNA was constructed, then used to generate a stably‐transfected mammalian cell line. The resulting currents were analyzed for their voltage‐ and time‐dependent properties. These properties identify this gene as encoding a T‐type Ca2+ channel.
Sexually transmitted diseases and reproductive tract infections among contraceptive users
Authors: Guerreiro D , Gigante M.A.M , Teles L.C
Published: July 2002

Objective: To establish the association between the use of contraceptives and the prevalence of sexually transmitted diseases (STD) and other reproductive tract infections (RTI). Methods: 840 women who used contraception and who attended the Fundação Nossa Senhora do Bom Sucesso's Family Planning Clinic (FNSBS‐FPC) over a 2‐year period (1993–1994) were studied. All women answered a standard epidemiological survey and underwent a clinical laboratory assessment. The Chi‐square test and a logistic regression model were used for statistical analysis. Results: The majority of women were young (58.7%); low educational achievers (59.0%); middle or working class (85.0%); had a fixed partner (79.8%), and were of non‐African origin (95.6%). Of the 627 women who responded, 4.1% stated that their sexual behavior was of high risk. The prevalence of any infection and particularly of bacterial vaginosis (BV), was highest among users of the intra‐uterine device (IUD). Use of an IUD (P=0.001) and high risk behavior (P=0.04) were the variables associated to presence of a STD/RTI in multiple regression analysis. Conclusion: Results suggest that STD/RTIs basically occur due to high risk behavior and that, with the exception of an association between use of IUD and BV, the method of contraception does not affect the incidence of these infections.