bluuebunny/crossref_metadata_2025 huggingface dataset:
An exact mathematical expression for describing competitive binding of two different ligands to a protein molecule
Authors: Wang Zhi-Xin
Published: July 2002

The dissociation constant for the binding of a spectroscopically invisible or non‐radioactive ligand to its protein receptor can be determined in a competition experiment by using a structural analog that contains a reporter group. Many plotting and numerical analysis methods have been developed to calculate the binding constant of unlabeled ligand from the displacement experiments. However, a common problem with these plotting methods is that the equation transformations inevitably result in non‐standard error distribution, and thus simple linear regression can not be used to extract correct values for the parameters. In the case of the numerical analysis methods, one would be faced with the possible existence of multiple solutions. In this paper, the exact mathematical expression for describing competitive binding of two different ligands to a protein molecule is presented in terms of the total concentrations of species in the system. Thus, using a commercially available non‐linear regression program, all unknown parameters for describing this system can be determined by fitting the experimental data to the algebraically explicit equation without any data transformations. The distribution curves of all the species in the system can also be constructed with this equation. It is particularly useful for the cases in which the concentrations of all the species in the system are comparable to each other.
1α,25‐Dihydroxyvitamin D3 increases IGF binding protein‐5 expression in cultured osteoblasts
Authors: Schmid C. , Schläpfer I. , Gosteli-Peter M.A. , Hauri C. , Froesch E.R. , Zapf J.
Published: July 2002

Rat osteoblasts produce insulin‐like growth factors (IGFs) and IGF binding proteins (IGFBPs). Expression of IGFBP‐5, an IGFBP which stimulates DNA synthesis of osteoblasts, was studied in vitro under the influence of 1α,25‐dihydroxyvitamin D3 (1,25(OH)2D3) and parathyroid hormone (PTH). These two calcium‐regulating hormones stimulated the expression of IGFBP‐5 mRNA in cultured rat osteoblasts at low concentrations (10 pM) and in a dose‐ and time‐dependent manner. Intact IGFBP‐5 was not detected in the culture medium, but was found attached to the cell layer. IGFBP‐5 may thus direct IGFs to the sites of bone remodeling.
GTP and guanosine synergistically enhance NGF‐induced neurite outgrowth from PC12 cells
Authors: Gysbers J.W. , Rathbone M.P.
Published: July 2002

Six per cent of rat pheochromocytoma (PC12) cells extended neurites (processes greater than one cell diameter in length) in the presence of 300 μM extracellular GTP or 300 μM guanosine for 48 hr, compared to only 2.5% of cells in control cultures. In the presence of 40 ng/ml of 2.5S NGF, about 20–35% of PC12 cells had neurites after 48 hr, and the addition of 300 μM guanosine or GTP together with NGF synergistically increased the proportion of cells with neurites to 40–65%. GTP and guanosine also increased the average number of branches per neurite, from 0.6 in NGF‐treated cultures to 1.2 (guanosine) or 1.5 (GTP). Neurites formed after exposure to NGF alone had axonal characteristics as determined by immunocytochemistry with antibody, SMI‐31, against axonal‐specific polyphosphorylated neurofilament epitopes. Neurites generated with the addition of both guanosine or GTP had the same characteristics.

GTP probably did not exert its effects via the P2X or P2Y purinoceptors because the adenine nucleotides ATP, ATPγS, ADPβS, and ADP, which are all agonists of these receptors, inhibited rather than enhanced, NGF‐induced neurite outgrowth. UTP also enhanced the proportion of cells with neurites, although not to the same degree as did GTP. This may indicate activity through a P2U‐like nucleotide receptor. However, the response profile obtained, GTP > UTP ≫ ATP, does not fit the profile of any known P2Y, P2X or P2U receptor. The poorly hydrolyzable GTP analogues, GTPγS and GDPβs were also unable to enhance the proportion of cells with neurites. This implied that GTP may produce its effects through a GTP‐specific ectoenzyme or kinase. This idea was supported by results showing that another poorly hydrolyzable analogue, GMP‐PCP, competitively inhibited the effects of GTP on neurite outgrowth. GTP did not exert its effects after hydrolysis to guanosine since the metabolic intermediates GDP and GMP were also ineffective in enhancing the proportion of cells with neurites. Moreover, the effects of GTP and guanosine were mutually additive, implying that these two purines utilized different signal transduction mechanisms.

The effects of guanosine were not affected by the nucleoside uptake inhibitors nitrobenzylthioinosine (NBTI) and dipyridamole, indicating that a transport mechanism was not involved. Guanosine also did not activate the purinergic P1 receptors, because the A2 receptor antagonists, 1, 3‐dipropyl‐7‐methylxanthine (DPMX) or CGS15943, and the At receptor antagonist, 1, 3‐dipropyl‐8‐(2‐amino‐4‐chloro)xanthine (PACPX) did not inhibit its reaction. Therefore guanosine enhanced neurite outgrowth by a signal transduction mechanism that does not include the activation of the Pt purinoceptors.

The enhancement of the neuritogenic effects of NGF by GTP and guanosine may have physiological implications in sprouting and functional recovery after neuronal injury in the CNS, due to the high levels of nucleosides and nucleotides released from dead or injured cells.
Antidepressant effect of 5-methoxypsoralen: the melatonin synchronizer hypothesis
Authors: Darcourt G , Feuillade P , Bistagnin Y , Robert P , Pringuey D , Touari M , Merdji Y , Bensmaïl B
Published: July 2002

5-methoxypsoralen (5-MOP) stimulates pineal melatonin secretion, and a decrease in dark phase melatonin levels has been described in major depression. As exogenous melatonin has shown synchronizer properties, authors hypothesized that giving 5-MOP would have antidepressant properties.

Twenty-six inpatients meeting the criteria of major depressive disorders were enrolled in a four-week, double blind trial of 5-MOP versus amitriptyline. Clinical improvement was identical in both treatment groups but biological changes were different in each group: 5-MOP patients showed an early nocturnal surge of melatonin levels that was maintained at the fourth treatment week, while melatonin levels remained unchanged in patients treated with amitriptyline.
Inversion of the anomeric configuration of the transferred sugar during inactivation of the macrolide antibiotic oleandomycin catalyzed by a macrolide glycosyltransferase
Authors: Quirós Luis M. , Carbajo Rodrigo J. , Salas José A.
Published: July 2002

Macrolides are a group of antibiotics structurally characterized by a macrocyclic lactone to which one or several deoxy‐sugar moieties are attached. The sugar moieties are transferred to the different aglycones by glycosyltransferases (GTF). The OleI GTF of an oleandomycin producer, Streptomyces antibioticus, catalyzes the inactivation of this macrolide by glycosylation. The product of this reaction was isolated and its structure elucidated. The donor substrate of the reaction was UDP‐α‐D‐glucose, but the reaction product showed a β‐glycosidic linkage. The inversion of the anomeric configuration of the transferred sugar and other data about the kinetics of the reaction and primary structure analysis of several GTFs are compatible with a reaction mechanism involving a single nucleophilic substitution at the sugar anomeric carbon in the catalytic center of the enzyme.
Recent developments and future directions in computational genomics
Authors: Tsoka Sophia , Ouzounis Christos A
Published: July 2002

Computational genomics is a subfield of computational biology that deals with the analysis of entire genome sequences. Transcending the boundaries of classical sequence analysis, computational genomics exploits the inherent properties of entire genomes by modelling them as systems. We review recent developments in the field, discuss in some detail a number of novel approaches that take into account the genomic context and argue that progress will be made by novel knowledge representation and simulation technologies.
Characteristics of super αA‐crystallin, a product of in vitro exon shuffling
Authors: van Rijk Anke F. , van den Hurk Maarten J.J. , Renkema Wouter , Boelens Wilbert C. , de Jong Wilfried W. , Bloemendal Hans
Published: July 2002

αA‐Crystallin, a small heat shock protein with chaperone‐like activity, forms dynamic multimeric complexes. Recently we described the spontaneous generation of a mutant protein (super αA‐crystallin) by exon duplication arisen via exon shuffling confirming a classic hypothesis by Gilbert [Nature 271 (1978) 501]. Comparison of super αA‐crystallin, which is viable in a mouse skeletal muscle cell line, with normal αA‐crystallin shows that it has diminished thermostability, increased exposure of hydrophobic patches, a larger complex size and lost its chaperone activity. However, super αA‐crystallin subunits exchange as readily between complexes as does normal αA‐crystallin. These data indicate that chaperone‐like activity may vanish independent of subunit hydrophobicity and exchangeability.
AMP‐activated protein kinase counteracted the inhibitory effect of glucose on the phosphoenolpyruvate carboxykinase gene expression in rat hepatocytes
Authors: Hubert Aurélie , Husson Annie , Chédeville Arlette , Lavoinne Alain
Published: July 2002

The effect of AMP‐activated protein kinase (AMPK) in the regulation of the phosphoenolpyruvate carboxykinase (PEPCK) gene expression was studied in isolated rat hepatocytes. Activation of AMPK by AICAR counteracted the inhibitory effect of glucose on the PEPCK gene expression, both at the mRNA and the transcriptional levels. It is proposed that a target for AMPK is involved in the inhibitory effect of glucose on PEPCK gene transcription.
Intracellular Ca2+ channel immunoreactivity in neuroendocrine axon terminals
Authors: Fisher Thomas E , Carrion-Vazquez Mariano , Fernandez Julio M
Published: July 2002

The concentration of neuroendocrine terminals in the neurohypophysis facilitates the identification and localization of Ca2+ channel subtypes near neuroendocrine release sites. Immunoblots of rat neurohypophysial tissue identified the α11.3, α12.1, α12.2, and α12.3 Ca2+ channel subunits. Immunofluorescence staining of axon terminal plasma membranes was weak, suggesting that Ca2+ channels are dispersed. This contrasts with the highly punctate α12.2 immunoreactivity in bovine chromaffin cells; the neurohypophysial terminals may therefore lack the specialized release zones found in those cells. Immunofluorescence and immunogold labeling identify dense core granule‐like structures in the terminal cytoplasm containing multiple Ca2+ channel types. Ca2+ channels in internal membranes may play an important role in channel targeting and distribution in neuroendocrine cells.
Ganoderma extract activates MAP kinases and induces the neuronal differentiation of rat pheochromocytoma PC12 cells
Authors: Cheung William M.W. , Hui Wing S. , Chu Patrick W.K. , Chiu Siu W. , Ip Nancy Y.
Published: July 2002

The pharmacology and clinical application of traditional Chinese medicine has been extensively documented. We have used an in vitro model system, PC12 cells, to demonstrate the presence of neuroactive compounds inGanoderma lucidum(lingzhi). Ganoderma extract induced the neuronal differentiation of PC12 cells and prevented nerve growth factor‐dependent PC12 neurons from apoptosis. Moreover, these effects of ganoderma might be mediated via the ras/extracellular signal‐regulated kinase (Erk) and cAMP‐response element binding protein (CREB) signaling pathways, as demonstrated by the phosphorylation of Erk1, Erk2 and CREB. Thus, our data not only present the first evidence of the presence of neuroactive compounds that mediate the neuronal differentiation and neuroprotection of the PC12 cells, but also reveal the potential signaling molecules involved in its action.
Glutamic acid 160 is the acid‐base catalyst of β‐xylosidase from Bacillus stearothermophilus T‐6: a family 39 glycoside hydrolase
Authors: Bravman Tsafrir , Mechaly Adva , Shulami Smadar , Belakhov Valery , Baasov Timor , Shoham Gil , Shoham Yuval
Published: July 2002

A β‐xylosidase from Bacillus stearothermophilus T‐6 was cloned, overexpressed in Escherichia coli and purified to homogeneity. Based on sequence alignment, the enzyme belongs to family 39 glycoside hydrolases, which itself forms part of the wider GH‐A clan. The conserved Glu160 was proposed as the acid‐base catalyst. An E160A mutant was constructed and subjected to steady state and pre‐steady state kinetic analysis together with azide rescue and pH activity profiles. The observed results support the assignment of Glu160 as the acid‐base catalytic residue.
Comparative EPR and fluorescence conformational studies of fully active spin‐labeled melanotropic peptides
Authors: Nakaie Clovis R. , Barbosa Simone R. , Vieira Renata F.F. , Fernandez Roberto M. , Cilli Eduardo M. , Castrucci Ana M.L. , Visconti Maria A. , Ito Amando S. , Lamy-Freund M.Teresa
Published: July 2002

Similar to melanocyte stimulating hormone (α‐MSH), its potent and long‐acting analogue, [Nle4, D‐Phe7]α‐MSH, when labeled with the paramagnetic amino acid probe 2,2,6,6‐tetramethylpiperidine‐N‐oxyl‐4‐amino‐4‐carboxylic acid (Toac), maintains its full biological potency, thus validating any comparative structural investigations between the two labeled peptides. Correlation times, calculated from the electron paramagnetic resonance signal of Toac bound to the peptides, and Toac–Trp distances, estimated from the Toac fluorescence quenching of the Trp residue present in the peptides, indicate a more rigid and folded structure for the potent analogue as compared to the hormone, in aqueous medium.
L273S missense substitution in human lysosomal acid lipase creates a new N‐glycosylation site
Authors: Pariyarath Rajalakshmi , Pagani Franco , Stuani Cristiana , Garcia Rodolfo , Baralle Francisco E.
Published: July 2002

Human lysosomal acid lipase (LAL), when expressed in Hela cells using the Vaccinia T7 expression system, showed four major molecular forms ranging from 42 to 54 kDa. Treatment with endoglycosidase H resulted in a 42 kDa protein, indicating that the molecular weight variations were due to N‐glycosylation. A missense substitution, L273S, previously detected in a patient with cholesteryl ester storage disease (CESD), produced catalytically inactive LAL showing a largest molecular mass form of 56 kDa instead of 54 kDa. Analysis of the amino acid sequence in the close proximity of the mutation (NMS → NML) indicated that the L273S mutation creates an additional N‐glycosylation consensus (N‐X‐S/T) in this region. Two site directed mutants disrupting this consensus, QMS and QML, when expressed in HeLa cells, did not show the 56 kDa form but the normal 54 kDa band whereas deglycosylation always resulted in the major 42 kDa form, as observed with normal LAL and the L273S mutant. These data confirmed that an additional N‐glycosylation at N271 was responsible for the 56 kDa form of the protein produced from the L273S allele. Furthermore, deglycosylation of normal LAL reduced the acid hydrolase activity towards both tri‐oleyl glycerol and cholesteryl oleate by 50%, strongly suggesting that N‐linked carbohydrate residues are important for optimal catalytic activity.
Biochemical characterization of Pkn2, a protein Ser/Thr kinase from Myxococcus xanthus, a Gram‐negative developmental bacterium
Authors: Udo Hiroshi , Inouye Masayori , Inouye Sumiko
Published: July 2002

Pkn2, a protein Ser/Thr kinase, from the developmental bacterium Myxococcus xanthus was expressed under a T7 promoter in Escherichia coli and purified. Purified Pkn2 retained the autophosphorylation activity with the K
m value of 177 μM for ATP and 73 nmol/min/mg for V
max. The optimum pH and temperature were determined to be 7.5 and 35°C, respectively. The autophosphorylation activity was inhibited by staurosporine with the IC50 value of 400 nM while H‐7 and genistein had little effect on this kinase. Pkn2 appears to be unique for its higher manganese dependence. This is the first biochemical characterization of the prokaryotic protein Ser/Thr kinase.
Anaerobic carotenoid biosynthesis in Rhodobacter sphaeroides 2.4.1: H2O is a source of oxygen for the 1‐methoxy group of spheroidene but not for the 2‐oxo group of spheroidenone
Authors: Yeliseev Alexei A , Kaplan Samuel
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Activation of the p21‐activated protein kinases from neutrophils with an antibody that reacts with the N‐terminal region of Pak 1
Authors: Lian Jian P , Badwey John A
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Molecular characterization of PadA, a phenylacetaldehyde dehydrogenase from Escherichia coli
Authors: Ferrández Abel , Prieto Marı́a A. , Garcı́a José L. , Dı́az Eduardo
Published: July 2002

The padA gene encoding the phenylacetaldehyde dehydrogenase involved in the catabolism of 2‐phenylethylamine in Escherichia coli has been cloned, sequenced, and located at 31.0 min on the chromosome. The deduced PadA polypeptide contains 499 amino acid residues with a predicted molecular mass of 53.7 kDa, and its primary structure reveals significant similarity with that of members of the aldehyde dehydrogenase superfamily. By engineering optimal transcription and translation elements, a high expression of the padA gene has been achieved. The active PadA enzyme is a homodimer that prefers NAD+ over NADP+ as coenzyme. The enzyme efficiently oxidizes only phenylacetaldehyde‐like aromatic aldehydes, and has a weak esterase activity with p‐nitrophenol. The padA gene constitutes a new catabolic tool for designing DNA cassettes to expand the abilities of microorganisms to degrade toxic aromatic compounds.
Cloning, expression and characterisation of murine procathepsin E
Authors: Tatnell Peter J , Lees Wendy E , Kay John
Published: July 2002

The cDNA encoding murine procathepsin E was isolated and sequenced and recombinant enzyme was produced in Escherichia coli. The activity of the purified recombinant mouse cathepsin E was characterised quantitatively using two synthetic peptide substrates and naturally occurring inhibitors. The majority of the recombinant enzyme was present as a homodimer (Mr ∼80) in which the two monomers were linked by an intermolecular disulfide bond. By analogy to previous studies with human cathepsin E, this is most likely a consequence of the presence of a unique cysteine residue near the N‐terminus of the mature proteinase. The availability of (i) recombinant murine enzyme in reasonable quantities and (ii) a full‐length cDNA now enables structural investigations and attempts to generate ‘knock‐out’ mice deficient in this important aspartic proteinase to be undertaken.
SKK4, a novel activator of stress‐activated protein kinase‐1 (SAPK1/JNK)
Authors: Lawler Sean , Cuenda Ana , Goedert Michel , Cohen Philip
Published: July 2002

A cDNA was cloned and expressed that encodes human stress‐activated protein kinase kinase‐4 (SKK4), a novel MAP kinase kinase family member whose mRNA is widely expressed in human tissues. SKK4 activated SAPK1/JNK in vitro, but not SAPK2a/p38, SAPK2b/p38β, SAPK3/ERK6 or SAPK4. It appears to be the mammalian homologue of HEP, an activator of SAPK1/JNK in Drosophila. In human epithelial KB cells SKK4 and SKK1/MKK4 (another activator of SAPK1/JNK) were both activated by stressful stimuli, but only SKK4 was activated by proinflammatory cytokines. The identification of SKK4 explains why the major SAPK1/JNK activator detected in many mammalian cell extracts is chromatographically separable from SKK1/MKK4.
Identification of cDNAs encoding two G protein‐coupled receptors for lysosphingolipids1
Authors: An Songzhu , Bleu Thieu , Huang Wei , Hallmark Olivia G , Coughlin Shaun R , Goetzl Edward J
Published: July 2002

The structural similarity of lysosphingolipids to lysophosphatidic acid (LPA) prompted a sequence‐based search for lysosphingolipid receptors using cDNA sequence of the Edg2 human LPA receptor. Two closely related G protein‐coupled receptors, rat H218 and human Edg3, are highly similar to Edg2. When overexpressed in Jurkat cells, H218 and Edg3 activated serum response element‐driven transcriptional reporter gene in response to sphingosine 1‐phosphate (S1P), dihydro‐S1P and sphingosylphosphorylcholine, but not to LPA. H218 and Edg3 expressed in Xenopus oocytes conferred responsiveness to S1P and dihydro‐S1P in agonist‐triggered 45Ca2+ efflux. Therefore, H218 and Edg3 are functional receptors for S1P and perhaps other closely related lysosphingolipids.
Characterization of room temperature induced apoptosis in HL‐60
Authors: Shimura Mari , Ishizaka Yukihito , Yuo Akira , Hatake Kiyohiko , Oshima Mieko , Sasaki Takehito , Takaku Fumimaro
Published: July 2002

We found that exposure to room temperature (RT/21°C) causes apoptosis in HL‐60 cells. Here we characterized RT‐induced apoptosis in HL‐60. After exposure to RT, apoptosis starts within 6 h and more than 80% of the cells underwent apoptosis within 20 h. All cells, however, were committed to apoptosis after 16 h and no viable cells could be recovered. The caspase‐1 inhibitor (YVAD‐CHO) effectively blocked apoptosis, whereas the caspase‐3 inhibitor (DEVD‐CHO) did not. About 20% of newly obtained early passage HL‐60 cells (passage 10) also underwent apoptosis by RT treatment. These data suggest that some population in HL‐60 which responds to RT with apoptosis became dominant during passaging.
Adrenomedullin has mitogenic effects on human oral keratinocytes: involvement of cyclic AMP
Authors: Kapas Supriya , Brown Dean W , Farthing Paula M , Hagi-Pavli Eleni
Published: July 2002

The effects of the novel vasoactive regulatory peptide, adrenomedullin, on human oral keratinocytes was investigated. Adrenomedullin, acting via its specific receptor, stimulated a dose‐dependent increase in DNA synthesis, and, in addition, stimulated further changes in the cell cycle resulting in the proliferation of keratinocytes. When cells were incubated in the presence of increasing concentrations of adrenomedullin, there was a rapid and dose‐dependent rise in intracellular cyclic AMP levels. Stimulation of mitogenesis and cell proliferation in these cells were mimicked by the cell permeable cAMP analogue, di‐butyryl cAMP. Adrenomedullin‐stimulated mitogenesis was attenuated by the adenylyl cyclase inhibitor SQ22,536, but was unaffected by inhibitors of PKC, tyrosine kinase or the CGRP receptor antagonist, CGRP(8–37). These data identify adrenomedullin as a new mitogenic regulatory peptide of keratinocytes acting via the cAMP cascade.
Existence of four acetylcholinesterase genes in the nematodes Caenorhabditis elegans and Caenorhabditis briggsae1
Authors: Grauso Marta , Culetto Emmanuel , Combes Didier , Fedon Yann , Toutant Jean-Pierre , Arpagaus Martine
Published: July 2002

Three genes, ace‐1, ace‐2 and ace‐3, respectively located on chromosomes X, I and II, were reported to encode acetylcholinesterases (AChEs) of classes A, B and C in the nematode Caenorhabditis elegans. We have previously cloned and sequenced ace‐1 in the two related species C. elegans and C. briggsae. We report here partial sequences of ace‐2 (encoding class B) and of two other ace sequences located in close proximity on chromosome II in C. elegans and C. briggsae. These two sequences are provisionally named ace‐x and ace‐y, because it is not possible at the moment to establish which of these two genes corresponds to ace‐3. Ace‐x and ace‐y are transcribed in vivo as shown by RT‐PCR and they are likely to be included in a single operon.
Immunoglobulin superfamily members gp65 and gp55: tissue distribution of glycoforms
Authors: Langnaese Kristina , Mummery Rosemary , Gundelfinger Eckart D. , Beesley Philip W.
Published: July 2002

Gp65 and gp55 are immunoglobulin superfamily members produced by alternative splicing of the same gene transcript, and originally identified as components of synaptic membranes. A monoclonal antibody specific for gp65 and gp55 has been used to detect immunoreactive species in a wide range of tissues. All immunoreactive species bind to concanavalin A and deglycosylation studies show that in all tissues tested other than brain the immunoreactive species are derived from gp55. HEK cells transfected with gp65 or gp55 express different glycoforms from brain showing that the pattern of glycosylation of these molecules is dependent upon the cell type in which they are expressed.
Structure, function and regulation of the vacuolar (H+)‐ATPases
Authors: Forgac Michael
Published: July 2002

The vacuolar (H+)‐ATPases (or V‐ATPases) function to acidify intracellular compartments in eukaryotic cells, playing an important role in such processes as receptor‐mediated endocytosis, intracellular membrane traffic, protein degradation and coupled transport. V‐ATPases in the plasma membrane of specialized cells also function in renal acidification, bone resorption and cytosolic pH maintenance. The V‐ATPases are composed of two domains. The V1 domain is a 570‐kDa peripheral complex composed of 8 subunits (subunits A–H) of molecular weight 70–13 kDa which is responsible for ATP hydrolysis. The V0 domain is a 260‐kDa integral complex composed of 5 subunits (subunits a–d) which is responsible for proton translocation. The V‐ATPases are structurally related to the F‐ATPases which function in ATP synthesis. Biochemical and mutational studies have begun to reveal the function of individual subunits and residues in V‐ATPase activity. A central question in this field is the mechanism of regulation of vacuolar acidification in vivo. Evidence has been obtained suggesting a number of possible mechanisms of regulating V‐ATPase activity, including reversible dissociation of V1 and V0 domains, disulfide bond formation at the catalytic site and differential targeting of V‐ATPases. Control of anion conductance may also function to regulate vacuolar pH. Because of the diversity of functions of V‐ATPases, cells most likely employ multiple mechanisms for controlling their activity.
Role of sympathetic activity in controlling the expression of vascular endothelial growth factor in brown fat cells of lean and genetically obese rats
Authors: Tonello Cristina , Giordano Antonio , Cozzi Valeria , Cinti Saverio , Stock Michael J , Carruba Michele O , Nisoli Enzo
Published: July 2002

The thermogenic activity of brown adipose tissue (BAT) is heavily dependent on high perfusion, through its dense vascular system. Angiogenesis must go hand‐in‐hand with BAT functions, but little is known about the factors controlling it. In the present study we demonstrate that: (a) vascular endothelial growth factor (VEGF) is synthesised and released in brown adipocytes in culture; (b) VEGF mRNA isoforms and protein appear in dispersed mature brown adipocytes and whole tissue; (c) VEGF expression is increased in BAT from cold‐exposed rats, and in cultured brown adipocytes exposed to noradrenaline and the β3‐adrenoceptor agonists; (e) BAT from genetically obese (fa/fa) rats exhibits reduced expression of VEGF as well as a change in the ratio of mRNA isoforms. It is concluded that sympathetic control of VEGF expression via noradrenaline acting on β3‐adrenoceptors plays a major role in developmental and adaptive angiogenesis, and defects in this contribute to the reduced thermogenic capacity of BAT in genetic obesity.
p42‐MAP kinase is activated in EGF‐stimulated interphase but not in metaphase‐arrested HeLa cells
Authors: Gomez-Cambronero Julian
Published: July 2002

It is known that cellular signals produced in response to an inappropriate spindle formation cause the cell to be arrested at metaphase (M) in the cell cycle. We report here that the 42‐kDa isoform of MAPK (ERK2) was tyrosyl‐phosphorylated and activated in response to epidermal growth factor (EGF) in interphase but not in M‐arrested HeLa cells. However, the basal level of activity of M‐arrested cells was higher than that of interphase, although the overall tyrosyl phosphorylation content was small. Further, the EGF receptor and its associated proteins GTPase‐activating protein and phospholipase C were phosphorylated in M‐arrested cells to a lower extent than they were in interphase. This implies that in spite of its high level of basal activity, the scarcity of MAPK activation in mitosis in response to EGF stems from an early impairment of phosphorylation of the receptor and neighboring proteins. The biological significance of these results underlies the importance of keeping the cell sheltered from extracellular signals when it undergoes division.
Insulin‐like growth factors I and II are unable to form and maintain their native disulfides under in vivo redox conditions1
Authors: Hober Sophia , Lundström Ljung Johanna , Uhlén Mathias , Nilsson Björn
Published: July 2002

Insulin‐like growth factor (IGF) I does not quantitatively form its three native disulfide bonds in the presence of 10 mM reduced and 1 mM oxidized glutathione in vitro [Hober, S. et al. (1992) Biochemistry 31, 1749–1756]. In this paper, we show (i) that both IGF‐I and IGF‐II are unable to form and maintain their native disulfide bonds at redox conditions that are similar to the situation in the secretory vesicles in vivo and (ii) that the presence of protein disulfide isomerase does not overcome this problem. The results indicate that the previously described thermodynamic disulfide exchange folding problem of IGF‐I in vitro is also present in vivo. Speculatively, we suggest that the thermodynamic disulfide exchange properties of IGF‐I and II are biologically significant for inactivation of the unbound growth factors by disulfide exchange reactions to generate variants destined for rapid clearance.
Cross‐linking of the B cell receptor induces activation of phospholipase D through Syk, Btk and phospholipase C‐γ2
Authors: Hitomi Tomohiro , Yanagi Shigeru , Inatome Ryoko , Yamamura Hirohei
Published: July 2002

Phospholipase D (PLD) has been proposed to play a key role in the signal transduction of cellular responses to various extracellular signals. Herein we provide biochemical and genetic evidence that cross‐linking of the B cell receptor (BCR) induces rapid activation of PLD through a Syk‐, Btk‐ and phospholipase C (PLC)‐γ2‐dependent pathway in DT40 cells. Activation of PLD upon BCR engagement is completely blocked in Syk‐ or Btk‐deficient cells, but unaffected in Lyn‐deficient cells. Furthermore, in PLC‐γ2‐deficient cells, BCR engagement failed to activate PLD. These results demonstrate that Syk, Btk and PLC‐γ2 are essential for BCR‐induced PLD activation.
Amber mutations in ribosome recycling factors of Escherichia coli and Thermus thermophilus: evidence for C‐terminal modulator element
Authors: Fujiwara Toshinobu , Ito Koichi , Nakayashiki Toru , Nakamura Yoshikazu
Published: July 2002

Ribosome recycling factor, referred to as RRF, is essential for bacterial growth because of its activity of decomposition of the post‐termination complex of the ribosome after release of polypeptides. In this study, we isolated a conditionally lethal amber mutation, named frr‐3, in the Escherichia coli RRF gene at amino acid position 161, showing that the truncation of the C‐terminal 25 amino acids of RRF is lethal to E. coli. An RRF gene cloned from Thermus thermophilus, whose protein is 44% identical and 68% similar to E. coli RRF, failed to complement the frr‐3(Am) allele. However, truncation of the C‐terminal five amino acids conferred intergeneric complementation activity on T. thermophilus RRF, demonstrating the modulator activity of the C‐terminal tail. Rapid purification of T. thermophilus RRF was achieved by T7‐RNA polymerase‐driven overexpression for crystallography.
Angiopoietin‐3, a novel member of the angiopoietin family
Authors: Nishimura Motoi , Miki Takashi , Yashima Rei , Yokoi Norihide , Yano Hideki , Sato Yasufumi , Seino Susumu
Published: July 2002

A cDNA clone encoding angiopoietin‐3 protein (Ang3), a novel member of the angiopoietin family, was identified. Ang3 cDNA was cloned from a human aorta cDNA library. Ang3 is a 503 amino acid protein having 45.1% and 44.7% identity with human angiopoietin‐1 and human angiopoietin‐2, respectively. Ang3 mRNA is expressed in lung and cultured human umbilical vein endothelial cells (HUVECs). Ang3 mRNA expression in HUVECs was slightly decreased by vascular endothelial cell growth factor treatment, suggesting that the regulation of Ang3 mRNA expression is different from that of Ang2.
NMR assignments and secondary structure of the UvrC binding domain of UvrB
Authors: Alexandrovich Alexander , Sanderson Mark R. , Moolenaar Geri F. , Goosen Nora , Lane Andrew N.
Published: July 2002

The 55 residue C‐terminal domain of UvrB that interacts with UvrC during excision repair in Escherichia coli has been expressed and purified as a (His)6 fusion construct. The fragment forms a stable folded domain in solution. Heteronuclear NMR experiments were used to obtain extensive 15N, 13C and 1H NMR assignments. NOESY and chemical shift data showed that the protein comprises two helices from residues 630 to 648 and from 652 to 670. 15N relaxation data also show that the first 11 and last three residues are unstructured. The effective rotational correlation time within the structured region is not consistent with a monomer. This oligomerisation may be relevant to the mode of dimerisation of UvrB with the homologous domain of UvrC.
The Drosophila gene 2A5 complements the defect in mitochondrial F1‐ATPase assembly in yeast lacking the molecular chaperone Atp11p
Authors: Wang Zhen-Guo , Schmid Karl J. , Ackerman Sharon H.
Published: July 2002

Assembly of mitochondrial F1‐ATPase in Saccharomyces cerevisiae requires the molecular chaperone, Atp11p. Database searches have identified protein sequences from Schizosaccharomyces pombe and two species of Drosophila that are homologous to S. cerevisiae Atp11p. A cDNA encoding the putative Atp11p from Drosophila yakuba was shown to complement the respiratory deficient phenotype of yeast harboring an atp11::HIS3 disruption allele. Furthermore, the product of this Drosophila gene was shown to interact with the S. cerevisiae F1 β subunit in the yeast two‐hybrid assay. These results indicate that Atp11p function is conserved in higher eukaryotes.
Regulation of Fas antibody induced neutrophil apoptosis is both caspase and mitochondrial dependent
Authors: Watson R.William G. , O'Neill Amanda , Brannigen Ann E. , Coffey Ronan , Marshall John C. , Brady Hugh R. , Fitzpatrick John M.
Published: July 2002

Resolution of neutrophil mediated inflammation is achieved, in part, through induction of neutrophil apoptosis. This constitutively expressed programme can be delayed by inflammatory mediators and induced by ligation of the Fas receptor. However, functional activation of the neutrophil results in resistance to Fas signalled death. We evaluated the effects of Fas antibody engagement on caspase activation and mitochondrial permeability, and the impact of co‐stimulation by lipopolysaccharide (LPS) or granulocyte macrophage‐colony stimulating factor (GM‐CSF) on these events. Fas engagement by an agonistic anti‐Fas antibody resulted in enhanced caspase 3 and 8 activity and increased mitochondrial permeability. Studies with pharmacological inhibitors of caspase activity showed that activation of caspase 8 occurred before, and activation of caspase 3 occurred after mitochondrial disruption. The mitochondrial stabilising agent bongkrekic acid also inhibited caspase activation and apoptosis. LPS, GM‐CSF and increased glutathione stabilised the mitochondria and inhibited caspase 3. Caspase 8 activity was also inhibited by co‐stimulation through a mechanism independent of mitochondrial stabilisation. Glutathione directly inhibited caspase 3 and 8 activity. We conclude inhibition of Fas antibody induced apoptosis by inflammatory proteins is associated with augmented mitochondrial stability and reduced caspase 3 activity that may be glutathione mediated.
Molten globule versus variety of intermediates: influence of anions on pH‐denatured apomyoglobin
Authors: Tcherkasskaya Olga , Ptitsyn Oleg B.
Published: July 2002

The molten globule state was shown to be the third thermodynamic state of protein molecules in addition to their native and unfolded states. On the other hand, it was reported that optical and hydrodynamic properties of pH‐denatured apomyoglobin depend on the nature of anions added to the protein solution. This observation was used to conclude that there are many ‘partly folded’ intermediates between the native and unfolded states rather than one distinct molten globule state. However, little is known on the structures of pH‐denatured apomyoglobin in the presence of different anions. Two tyrosine residues in horse apomyoglobin have been successively modified by the reaction with tetranitromethane. This approach was employed to measure the distances between tryptophans and modified tyrosines in different states of apomyoglobin by the method of direct energy transfer. Experimental data show that the distance between the middle of the A‐helix and the beginning of the G‐helix and/or the end of the H‐helix in ‘anion‐induced’ states are very close to those in the native holo‐ and apomyoglobins. This suggests that the AGH helical complex, being the most structured part of apomyoglobin in the molten globule state, exists also in pH‐denatured apomyoglobin in the presence of different anions. Consequently, all non‐native forms of apomyoglobin studied so far share the common important feature of its native structure.
H3‐rules: identification of CDR‐H3 structures in antibodies
Authors: Shirai Hiroki , Kidera Akinori , Nakamura Haruki
Published: July 2002

For the third complementarity determining region of the antibody heavy chain (CDR‐H3), we propose the ‘H3‐rules’, which should identify the tertiary structure from the amino acid sequence of the CDR‐H3 segment. A total of 100 CDR‐H3 segments from well‐determined crystal structures were analyzed. Distinctive relationships between the structures and the sequences were revealed from 55 segments, and the rules were examined for the other 45 segments and were verified. In some antibodies, basic residues at specific positions were revealed to be notable signals, with their ability to form salt bridges and to assume conformations inconsistent with the rules.
Identification of MYPT1 and NIPP1 as subunits of protein phosphatase 1 in rat liver cytosol
Authors: Boudrez An , Evens Kristien , Beullens Monique , Waelkens Etienne , Stalmans Willy , Bollen Mathieu
Published: July 2002

Various studies have provided evidence for the existence of spontaneously active cytosolic species of protein phosphatase 1, but these enzymes have never been purified and characterized. We have used chromatography on microcystin‐Sepharose and Resource Q to purify cytosolic protein phosphatases from rat liver. Two of the isolated enzymes were identified by Western analysis and peptide sequencing as complexes of the catalytic subunit of protein phosphatase 1 and either the inhibitor NIPP1 or the myosin‐binding subunit MYPT1, which reportedly is not present in chicken liver. In contrast, PCR cloning revealed the expression of two MYPT1 splice variants in rat liver.
Co‐introduction of an antisense gene for an endogenous seed storage protein can increase expression of a transgene in Arabidopsis thaliana seeds
Authors: Goossens Alain , Van Montagu Marc , Angenon Geert
Published: July 2002

We have investigated whether the expression in Arabidopsis thaliana seeds of a transgene (the Phaseolus vulgaris arcelin (arc)5‐I gene) could be enhanced by the simultaneous introduction of an antisense gene for an endogenous seed storage protein (2S albumin). Seeds of plants transformed with both the arc5‐I gene and a 2S albumin antisense gene contained reduced amounts of 2S albumins and increased arcelin‐5 (Arc5) accumulation levels compared to lines harboring the arc5‐I gene only. Arc5 production could be enhanced to more than 24% of the total seed protein content, suggesting that antisense technology could be of great utility to favor high expression of transgenes.
Photoinduced electron transfer in singly labeled thiouredopyrenetrisulfonate azurin derivatives
Authors: Borovok N. , Kotlyar A.B. , Pecht I. , Skov L.K. , Farver O.
Published: July 2002

A novel method for the initiation of intramolecular electron transfer reactions in azurin is reported. The method is based on laser photoexcitation of covalently attached thiouredopyrenetrisulfonate (TUPS), the reaction that generates the low potential triplet state of the dye with high quantum efficiency. TUPS derivatives of azurin, singly labeled at specific lysine residues, were prepared and purified to homogeneity by ion exchange HPLC. Transient absorption spectroscopy was used to directly monitor the rates of the electron transfer reaction from the photoexcited triplet state of TUPS to Cu(II) and the back reaction from Cu(I) to the oxidized dye. For all singly labeled derivatives, the rate constants of copper ion reduction were one or two orders of magnitude larger than for its reoxidation, consistent with the larger thermodynamic driving force for the former process. Using 3‐D coordinates of the crystal structure of Pseudomonas aeruginosa azurin and molecular structure calculation of the TUPS modified proteins, electron transfer pathways were calculated. Analysis of the results revealed a good correlation between separation distance from donor to Cu ligating atom (His‐N or Cys‐S) and the observed rate constants of Cu(II) reduction.
The small intestine can both absorb and glucuronidate luminal flavonoids
Authors: Spencer Jeremy P.E , Chowrimootoo George , Choudhury Ruksana , Debnam Edward S , Srai S.Kaila , Rice-Evans Catherine
Published: July 2002

We have studied the perfusion of the jejunum and ileum in an isolated rat intestine model with flavonoids and hydroxycinnamates and the influence of glycosylation on the subsequent metabolism. Flavone and flavonol glucosides and their corresponding aglycones are glucuronidated during transfer across the rat jejunum and ileum and this glucuronidation occurs without the need for gut microflora. Furthermore, this suggests the presence of glycosidases as well as UDP‐glucuronyl transferase in the jejunum. In contrast, quercetin‐3‐glucoside and rutin are mainly absorbed unmetabolised. The results suggest that the more highly reducing phenolics are absorbed predominantly as glucuronides (96.5%±4.6) of the amount absorbed, whereas monophenolic hydroxycinnamates and monophenolic B‐ring flavonoids are less predisposed to glucuronidation and higher levels of aglycone (88.1%±10.1) are detected on absorption through both the jejunum and ileum.
Hormonal control of protein expression and mRNA levels of the MaxiK channel α subunit in myometrium
Authors: Song Min , Zhu Ning , Olcese Riccardo , Barila Bruna , Toro Ligia , Stefani Enrico
Published: July 2002

Large conductance voltage‐dependent and Ca2+‐modulated K+ channels play a crucial role in myometrium contractility. Western blots and immunocytochemistry of rat uterine sections or isolated cells show that MaxiK channel protein signals drastically decrease towards the end of pregnancy. Consistent with a transcriptional regulation of channel expression, mRNA levels quantified with the ribonuclease protection assay correlated well with MaxiK protein levels. As a control, Na+/K+‐ATPase protein and RNA levels do not significantly change at different stages of pregnancy. The low numbers of MaxiK channels at the end of pregnancy may facilitate uterine contraction needed for parturition.
Does diphenylene iodonium chloride have any effect on the O2−‐generating step of plant peroxidases?
Authors: Barceló A.Ros , Ferrer Marı́a A.
Published: July 2002

The O2

−‐generating step of plant peroxidases during their catalytic cycle is represented by the decay of compound III (CoIII) into ferriperoxidase, which most likely involves the dissociation of a ferric‐O2

− complex to yield the ferric form of the enzyme and O2

−. Diphenylene iodonium chloride (DPI), at 50–100 μM, does not significantly enhance the stability of CoIII of peroxidase, as judged by the values of k
decay, and therefore, DPI appears to have no effect on the O2

−‐generating step of plant peroxidases. From these results, it is concluded that caution should be exercized when considering peroxidase as a possible enzyme target of O2

−‐mediated plant physiological processes sensitive to DPI inhibition.
Electrogenicity of the Na+‐ATPase from the marine microalga Tetraselmis (Platymonas) viridis and associated H+ countertransport
Authors: Balnokin Yurii V. , Popova Larissa G. , Andreev Igor M.
Published: July 2002

Sodium accumulation by the Na+‐ATPase in the plasma membrane (PM) vesicles isolated from the marine alga Tetraselmis (Platymonas) viridis was shown to be accompanied by ΔΨ generation across the vesicle membrane (positive inside) and H+ efflux from the vesicle lumen. Na+ accumulation was assayed with 22Na+; ΔΨ generation was detected by recording absorption changes of oxonol VI; H+ efflux was monitored as an increase in fluorescence intensity of the pH indicator pyranine loaded into the vesicles. Both ATP‐dependent Na+ uptake and H+ ejection were increased by the H+ ionophore carbonyl cyanide m‐chlorophenylhydrazone (ClCCP) while ΔΨ was collapsed. The lipophilic anion tetraphenylboron ion (TPB−) inhibited H+ ejection from the vesicles and abolished ΔΨ. Based on the effects of ClCCP and TPB− on H+ ejection and ΔΨ generation, the conclusion was drawn that H+ countertransport observed during Na+‐ATPase operation is a secondary event energized by the electric potential which is generated in the course of Na+ translocation across the vesicle membrane. Increasing Na+ concentrations stimulated H+ efflux and caused the decrease in the ΔΨ observed, thus indicating that Na+ is likely a factor controlling H+ permeability of the vesicle membrane.
Properties of the human long and short isoforms of the uncoupling protein‐3 expressed in yeast cells
Authors: Hinz W. , Grüninger S. , De Pover A. , Chiesi M.
Published: July 2002

Two splice variants of the human uncoupling protein‐3 (UCP3L and UCP3S) are highly expressed in skeletal muscle. The properties of UCP3L and S have been compared to those of UCP1 in a heterologous yeast expression system under the control of the galactose promoter. Both UCP3 isoforms were found to strongly impair the coupling efficiency of respiring cells thus resulting in increased thermogenesis. The uncoupling properties of both UCP3L and S could be clearly demonstrated also in isolated yeast mitochondria both in terms of coupled respiration and in the capacity to polarize the inner membrane in conditions of limited substrate availability. Contrary to what was observed with mitochondria containing UCP1, millimolar GDP and ATP had little if any effect on the uncoupling activity of UCP3. A very marked uncoupling of whole cells and isolated mitochondria was observed at very low expression levels of UCP3S indicating that the short isoform is more active than the long one.