bluuebunny/crossref_metadata_2025 huggingface dataset:
Sphingosine stimulates calcium mobilization in rat parotid acinar cells
Authors: Sugiya Hiroshi , Furuyama Shunsuke
Published: July 2002

In fura‐2‐loaded parotid acinar cells, 50–200 μM sphingosine induced an increase in cytosolic Ca2+ ([Ca2+]i). When extracellular Ca2+ was chelated by EGTA, 50 μM sphingosine failed to increase [Ca2+]i, but 100 or 200 μM sphingosine induced a slight and transient increase in [Ca2+]i. The addition of LaCl3 to the medium resulted in the same effect as chelation of extracellular Ca2+. When cells were incubated in low Ca2+ medium containing sphingosine, and extracellular Ca2+ was subsequently added, a rapid increase in [Ca2+]i depending on the concentration of sphingosine was shown. In low Ca2+ medium, a slight increase in [Ca2+]i induced by high concentrations of sphingosine was not shown after the transient increase in [Ca2+]i elicited by methacholine. Inhibitors of protein kinase C, H‐7 and K252a, did not mimic the effect of sphingosine on [Ca2+]i. These results suggest that sphingosine stimulates Ca2+‐influx and further stimulates the release of Ca2+ from agonist‐sensitive intracellular pools by a mechanism that is independent of protein kinase C.
Cell adhesive activity of two animal lectins through different recognition mechanisms
Authors: Ozeki Yasuhiro , Matsui Taei , Titani Koiti
Published: July 2002

Cell adhesive activity of two animal lectins. frog (Rama catesbeiana) S‐type 14K lectin and echinoidin (a C‐type lectin from sea urchin plasma), was studied with human rhabdomyosarcoma (RD) cells. RD cells attached to and spread on plastic plates coatce with each lectin. Cell adhesion by the frog lectin was completely inhibited by the addition of lactose or asialofetuin glycopeptide. Echinoidin‐induced cell adhesion was only inhibited by peptide GRGDS. Since echinoidin is known to contain an RGD‐sequence, our results clearly indicate that this sequence is active as the cell adhesive signal. These results suggest that some of the animal lectins may function as a cell adhesive molecule rather than using the carbohydrate‐recognition mechanism.
Effects of cholesterol‐ or 7‐ketocholesterol‐containing liposomes on colony‐forming ability of cultured cells
Authors: Khokhlov Alexander N. , Prokhorov Leonid Yu. , Ivanov Aleksey S. , Archakov Alexander I.
Published: July 2002

Experiments with cultured Chinese hamster cells showed that incubation of the cells with (phosphatidylcholine + cholesterol + 7‐ketocholesterol)‐containing liposomes (4:3:1 by weight) during two hours led to a decrease in the colony‐forming ability of cells down to zero, while (phosphatidylcholine + cholesterol)‐containing liposomes (1:1 by weight) reduce this parameter by 90%. Furthermore, the cholesterol‐containing liposomes (without 7‐ketocholesterol) induce a decrease in the number of the maximal‐site colonies accompanied by the corresponding increase in the number of the middle‐size colonies.
Subunit communication in the tryptophan synthase α2β2 complex Effects of β subunit ligands on proteolytic cleavage of a flexible loop in the α subunit
Authors: Ruvinov Sergei B. , Miles Edith Wilson
Published: July 2002

To probe the structural basis for ligand‐mediated communication between the α and β subunits in the tryptophan synthase α2β2 complex, we have determined the effects of ligands of the α and β subunits on proteolysis of a flexible loop in the α subunit. We find that addition of a ligand of the β subunit (l‐serine, d‐tryptophan, or l‐tryptophan) in combination with a ligand of the α subunit (α‐glycerol 3‐phosphate) almost completely prevents the tryptic cleavage of the α subunit loop. Thus, the binding of a ligand to the β‐site affects the conformation of the α subunit 25–30 Å distant.
Recognition of an antiparallel β‐sheet structure of human epidermal growth factor by its receptor Site‐directed mutagenesis studies of Ala‐30 and Asn‐32
Authors: Koide Hiroshi , Muto Yutaka , Kasai Hidefumi , Hoshi Kumiko , Takusari Hiromi , Kohri Kaoru , Takahashi Seizo , Sasaki Tetsuyuki , Tsukumo Ken-ichi , Miyake Tetsuo , Fuwa Tohru , Miyazawa Tatsuo , Yokoyama Shigeyuki
Published: July 2002

The Ala‐30 and Asn‐32 residues involved in the major antiparallel β‐sheet structure of human epidermal growth factor (hEGF) were substituted with various amino acid residues, and the receptor‐binding affinities of the nine variant hEGFs were determined by the use of human KB cells. The Ala‐30→Arg. Ala‐30→His and Ala‐30→Phe substitutions drastically reduced the binding affinity, suggesting that the side chain in position 30 of Ala‐30 of hEGF is required to be small for the receptor binding. The Asn‐32→Asp substitution significantly reduced the binding affinity, while the Asn‐32→His variant could bind to the receptor as well as to the wild‐type hEGF. Therefore, it seems to be important for receptor binding that the side chain in position 32 does not have a negative charve but does have an NH group. Thus, we propose that, in the ligand‐receptor complex, the receptor recognizes, on one side of the antiparallel β‐sheet structure of hEGF, a wider contact area than previously suggested.
The influence of the target structure on the efficiency of alkylation of single‐stranded DNA with the reactive derivatives of antisense oligonucleotides
Authors: Fedorova O.S. , Podust L.M. , Maksakova G.A. , Gorn V.V. , Knorre D.G.
Published: July 2002

Site‐directed alkylation of three oligonucleotide targets: 41‐mer (hairpin structure), 22‐mer (loop part of this hairpin) and 10‐mer (part of the loop) with 5′‐p‐(N‐2‐chloroethyl‐N‐methylamino)benzylamides of oligonucleotides complementary to the loop region was studied. Thermodynamic parameters of the interaction were estimated using the dependence of the limit modification extent on the reagent concentration at several temperatures. The stability of the complex increases significantly in the set: 302‐mer carrying above hairpin, 41‐mer, 22‐mer, the data for 22‐mer and 10‐mer being nearly identical. This indicates significant influence of the loop supporting structure on the interaction with antisense reagents.
Localization of proteins forming the outer surface of isolated metaphase chromosomes
Authors: Belyaev Nickolaj D. , Budker Vladimir G. , Dubrovskaya Veronika A. , Kim Andrej A. , Kiseleva Elena V. , Sidorov Victor N.
Published: July 2002

The outer surface of isolated metaphase chromosomes has been investigated by a method of thermally activated tritium labelling. We show that both chromosomal proteins and DNA are tritium‐labelled. Fractionation of the chromosomal proteins reveals that scaffold proteins are the most labelled in condensed and EDTA‐decondensed chromosomes. Exposition of some scaffold proteins on the outer surface of metaphase chromosomes is suggested.
α‐Bungarotoxin binding to two acetylcholine receptor α‐peptides and their methylmercury‐modified analogs: Intrinsic phosphorescence and optically detected magnetic resonance studies
Authors: Schlyer Bruce D. , Maki August H. , Hawrot Edward
Published: July 2002

Phosphorescence and optically detected magnetic resonance (ODMR) have been used to characterize two synthetic peptides, α181‐198 and α185‐196, of the major binding determinant of the α‐acetylcholine receptor (AChR) of Torpedo californica and its interaction with α‐bungarotoxin (BgTX) using Trp as an intrinsic probe. BgTX conformational changes are suggested upon complexation with the peptides. Methylmercury‐ modified peptides show conformational heterogeneity which brings some of the modified Cys residues into proximity of peptide Trp(s). These modified peptides, when bound to BgTX, undergo structural changes which remove the tagged Cys from its close contact with the Trp residue(s) of the peptide.
Compartmentation of dCTP pools disappears after hydroxyurea or araC treatment in lymphocytes
Authors: Spasokukotskaja T. , Sasvári-Székely M. , Taljanidisz J. , Staub M.
Published: July 2002

The calculated rate of DNA synthesis using [5‐3H]TdR was about 4 times higher than in the case of [5‐3H]CdR labeling, even after correction for the specific radioactivities of the intracellular pools. These data show a compartmentation of dCTP pools in lymphocytes. Hydroxyurea increased the specific activities of both dTTP and dCTP pools so that the calculated rate of DNA synthesis became equal. The same effect was found for araC treatment, but not for fluorodeoxyuridine. dCTP was supplied from CTP which is the lowest ribonucleotide pool in lymphocytes. Different functions of the two dCTP pools are proposed: one serving DNA replication; the other one supplies phospholipid precursors and DNA repair.
Cloning and characteristics of a positive regulatory gene, THI2 (PHO6), of thiamin biosynthesis in Saccharomyces cerevisiae
Authors: Nishimura Hiroshi , Kawasaki Yuko , Kaneko Yoshinobu , Nosaka Kazuto , Iwashima Akio
Published: July 2002

A thi2(pho6) mutant of Saccharomyces cerevisiae, defective in the expression of structural genes for thiamin‐repressible acid phosphatase and enzymes involved in thiamin biosynthesis, was found to retain sufficient thiamin transport activity. The transport activity was repressed by thiamin in growth medium. We isolated from a S. cerevisiae genomic library two hybrid plasmids, pTSR 1 and pTSR2, containing 10.2‐ and 12.0‐kilobase (kb) DNA fragments, respectively, which complement the thi2(pho6) mutation of S. cerevisiae. This gene was localized on a 6.0‐kb ClaI‐ClaI fragment in the subclone pTSR3. Complementation of the enzyme activities for thiamin metabolism in the thi2(pho6) mutant transformed by some plasmids with the THI2(PHO6) gene was also examined.
Introduction of a free cysteinyl residue at position 68 in the subtilisin Savinase, based on homology with proteinase K
Authors: Bech L.M. , Branner S. , Hastrup S. , Breddam K.
Published: July 2002

Two subfamilies of the subtilisins, distinguished by the presence or absence of a free cysteinyl residue near the essential histidyl residue of the catalytic triad, are known. In order to evaluate the significance of the presence of this ‐SH group a cysteinyl residue has been introduced by site‐directed mutagenesis into the cysteine‐free subtilisin‐like enzyme from Bacillus lentus, i.e. Savinase. The free cysteine affects the enzyme activity only slightly but renders it sensitive to mercurials presumably due to an indirect effect. The results indicate that the ‐SH group is not involved in catalysis.
The precursor form of the rat liver non‐specific lipid‐transfer protein, expressed in Escherichia coli, has lipid transfer activity
Authors: Ossendorp Bernadette C. , Geijtenbeek Teunis B.H. , Wirtz Karel W.A.
Published: July 2002

The cDNA encoding the precursor form or nonspecific lipid‐transfer protein (pre‐nsL‐TP) from rat liver was cloned into the expression vector pET3d. The resulting plasmid was transformed to the Escherichia coli strain BL21(DE3). After induction or the bacteria with isopropyl‐β‐D‐thiogalactopyranoside (IPTG) pre‐nsL‐TP was purified from the bacterial lysate by anion exchange chromatography followed by gelfiltration. From 1 1 or culture, 6–7 mg of pre‐nsL‐TP was obtained, equal to approximately 7% of the cytoplasmic protein. By use of a fluorescence lipid transfer assay, pre‐nsL‐TP was found to have lipid transfer activity identical to mature nsL‐TP.
Fidelity of the reverse transcriptase of human immunodeficiency virus type 2
Authors: Bakhanashvili Mary , Hizi Amnon
Published: July 2002

The relatively low fidelity of human immunodeficiency virus type 1 reverse transcriptase (HIV‐1 RT) was implicated as a major factor that contributes to the genetic variability of the virus. Extension of mismatched 3′ termini of the primer DNA was shown to be a major determinant of the infidelity of HIV‐1 RT. Human immunodeficiency virus type 2 (HIV‐2) also shows extensive genetic variations. Therefore, we have analyzed the fidelity of the DNA‐dependent DNA polymerase activity of HIV‐2 RT and compared it with those of RTs of HIV‐1 and murine leukemia virus (MLV). Like other retroviral RTs, the HIV‐2 RT was shown to lack a 3′→5′ exonuclease activity. The ability of HIV‐2 RT to extend preformed 3′‐terminal A:A, A:C and A:G mispairs was examined by quantitating the amount and length of extended primers. The results demonstrate a relatively efficient mispair extension by HIV‐2 RT with a specificity of A:C⪢A:A>A:G. The mispair extension appears to be affected mainly by the increase of apparent K
m values rather than by the change in V
max values. The relative extension frequencies from all mispairs with HIV‐1 and HIV‐2 RTs was 6‐ to 9‐fold greater than that of MLV RT, suggesting that the HIV enzymes are substantially more error‐prone than MLV RT.
Identification of nuclear factors which interact with the 5′ flanking region of the EF‐1αO gene in Xenopus laevis
Authors: Olesen Ole F. , Frydenberg Jane
Published: July 2002

The EF‐1αO gene of Xenopus laevis is a stage‐specific gene, being transcribed in oogonia and oocytes, but not in postmeiotic germ cells and terminally differentiated cells. We found that two trans‐acting factors from oocyte nuclear extract are able to interact with a DNA sequence in the 5′‐upstream region of the EF‐1αO gene. Methylation interference experiments suggested that the two factors recognised the same DNA element. Gel retardation assays indicated that part of the protein binding site could be confined to a 21 bp sequence, located between −51 and −72, relative to the cap site. Interestingly, this region shares great homology to a negative regulatory segment in the promoter of the TFIIIA gene, another developmentally regulated gene.
Interrelations of M‐intermediates in bacteriorhodopsin photocycle
Authors: Drachev L.A. , Kaulen A.D. , Komrakov A.Yu.
Published: July 2002

The photocycles of the wild‐type bacteriorhodopsin and the D96N mutant were investigated by the flash‐photolysis technique. The M‐intermediate formation (400 nm) and the L‐intermediate decay (520 nm) were found to be well described by a sum of two exponents (time constants, τ1 = 65 and τ2 = 250 μs) for the wild‐type bR and three exponents (τ1 = 55 μs, τ2 = 220 μs and τ3 = 1 ms) for the D96N mutant of bR. A component with τ = 1 ms was found to be present in the photocycle of the wild‐type bacteriorhodopsin as a lag‐phase in the relaxation of photoresponses at 400 and 520 nm. In the presence of Lu3+ ions or 80% glycerol this component was clearly seen as an additional phase of M‐formation. The azide effect on the D96N mutant of bR suggests that the 1‐ms component is associated with an irreversible conformational change switching the Schiff base from the outward to the inward proton channel. The maximum of the difference spectrum of the 1‐ms component of D96N bR is located at 404 nm as compared to 412 nm for the first two components. We suggest that this effect is a result of the alteration of the inward proton channel due to the Asp96→Asn substitution. Proton release measured with pyranine in the absence of pH buffers was identical for the wild‐type bR and D96N mutant and matched the M→M′ conformational transition. A model for M rise in the bR photocycle is proposed.
Ganglio‐N‐tetraosylceramide (GA1) of bovine and human brain Molecular characterization and presence in myelin
Authors: Dasgupta S. , van Halbeek H. , Hogan E.L.
Published: July 2002

During our studies of bovine brain neutral glycosphingolipids (Ngsl's), we have purified a compound that co‐migrates on thin‐layer chromatogram with standard GA1 (purified by acid hydrolysis of GM1) and close to penta‐ (nLc5Cer) glycosylceramide from bovine erythrocytes. The structure of the purified Ngsl from brain has been established by permethylation and by stepwise exoglycosidase hydrolysis. 600 MHz 1H NMR spectroscopy of the oligosaccharide obtained from the Ngsl after endoglycoceramidase hydrolysis confirms the structure as ganglio‐N‐tetraosylceramide (GgOse4Cer or GA1) as Ga1β1→3Ga1NAcβ1‐4Galβ1→4G1cβ1→1Cer. We have identified GA1 in bovine, rat and human brain and myelin by TLC‐immunostaining with monospecific anti‐GA1 antiserum.
Half‐life of ubiquinone‐9 in rat tissues
Authors: Thelin Anders , Schedin Sophia , Dallner Gustav
Published: July 2002

The half‐life of ubiquinone‐9 in various rat tissues was determined. Rats were injected intraperitoneally with [3H]mevalonate and the decay of radioactivity incorporated into ubiquinone‐9 was followed using reverse‐phase HPLC. The half‐life varied between 49 h (testis) and 125 h (kidney).
Domain of E. coli translational initiation factor IF2 homologous to lambda cI repressor and displaying DNA binding activity
Authors: Vachon Gilles , Raingeaud Joel , Dérijard Benoît , Julien Raymond , Cenatiempo Yves
Published: July 2002

The carboxy‐terminal region of translational initiation factor IF2 is a common region to the three active forms of the factor (α,β and γ) but its function is still unknown. We report here that this region of IF2 carries at least one domain which is homologous to the N‐terminal and middle part of the cI repressor of lambda phage. The IF2 homologous domain harbors functionally important features of the lambda represser, e.g. the helix‐tum‐helix motif and some of the residues essential for the structure of the hydrophobic core of the represser. This homologous domain of IF2 was fused to the β‐galactosidase protein. The hybrid protein, as well as IF2 itself, shows a consistent DNA binding activity in nitrocellulose filtration assays but does not display the specificity of the cI represser for the Pr operator. The implication of this domain in the transcriptional activity of IF2, reported by others, is discussed.
High molecular weight aspartic endopeptidase generates a coronaro‐constrictory peptide from the β‐chain of hemoglobin
Authors: Barkhudaryan Nina , Kellermann Josef , Galoyan Armen , Lottspeich Friedrich
Published: July 2002

Studying the influence of brain cathepsin D (EC 3.4.23.5) and high molecular weight (HMW) aspartic endopeptidase (EC 3.4.23.‐) on the processing of hypothalamic calmodulin‐binding coronaro‐constrictory peptide factors from the β‐chain of globin it was found that only HMW aspartic endopeptidase generates the fragment 31–40 of the β‐chain of bovine hemoglobin (Hb) by cleavage of the Leu30‐Leu31 and Phe40‐Phe41 bonds. Digestion of the β‐chain of globin was performed at 37°C at an enzyme/substrate ratio of 1:80 at pH 3.5 using different times of incubation (from 4 h to 10 h). The resulting peptides were separated by reversed‐phase high‐performance liquid chromatography (HPLC) and then identified by amino acid analysis and Edman degradation. The differences in specificity and activity of these two brain aspartic proteinases could be explained by their different structural features. Our finding provides evidence for a different biological function of these two enzymes. Data obtained give us reason to suppose that HMW aspartic proteinase probably can participate in the processing of the coronaro‐constrictory peptide in vivo by limited proteolysis of Hb or Hb‐like protein.
Transcription in vitro promoted by the Agrobacterium VirG protein
Authors: Endoh Hideki , Aoyama Takashi , Oka Atsuhiro
Published: July 2002

Expression of the virulence genes (vir) on the hairy‐root‐inducing plasmid pRiA4 is induced by plant signals in Agrobacterium cells through a two‐component regulatory system, the VirA‐VirG system. We constructed an in vitro transcription system that consisted of the purified VirG protein and the Agrobacterium RNA polymerase holoenzyme. Both versions of VirG, the non‐phosphorylated form and the VirA‐phosphorylated form, were active but showed different patterns of the pH‐dependency for transcriptional activation.
Conformational junctions between left‐handed DNA in (dA‐dT)16 and contiguous B‐DNA in a supercoiled plasmid contain chemically reactive bases
Authors: Nejedlý K. , Lilley D.M.J. , Paleček E.
Published: July 2002

Alternating adenine‐thymine sequences in supercoiled DNA may undergo a transition to the left‐handed Z‐conformation in the presence of Ni2+ ions and high Na+ concentrations [(1989) FEBS Lett. 243, 313‐317]. In this work we have studied the junctions between B‐ and Z‐conformations in a supercoiled plasmid containing a (dA‐dT)16 insert, by means of chemical probing. We observed enhanced reactivity of bases at both ends of the alternating tract to chloro‐ and bromoacetaldehyde. The degree of chemical reactivity was found to increase with the level of negative supercoiling. Only individual bases were observed to be reactive in the B‐Z junctions, consistent with tightly localized interfacial regions.
β‐Oxidation of unsaturated fatty acids in humans Isoforms of Δ3, Δ2‐enoyl‐CoA isomerase
Authors: Kilponen Johanna M. , Hiltunen J.Kalervo
Published: July 2002

This investigation was undertaken in order to elucidate the human enzymes which participate in metabolism of the double bonds of unsaturated fatty acids during, β‐oxidation. The results indicate that the human monofunctional Δ3, Δ2‐enoyl‐CoA isomerase (EC 5.3.3.8) with the native M
r of 70,000 differed significantly from its rat counterpart [Palosaari et al. (1990) J. Biol. Chem. 265, 3347–3353]; the isoelectric point of the human isoform was over three pH‐units more acidic, it showed different chromatographic behaviour, the human enzyme did not show any clear‐cut substrate chain‐length specificity and only a weak immunological cross‐reactivity was detected with the antibody to rat liver mitochondrial short‐chain enzyme. This explains the failure of attempts to apply the rat data directly to human beings. Another isomerase activity from human liver was found to be a part of the isomerase‐hydratase‐dehydrogenase polypeptide showing immunological cross‐reactivity with the previously characterized peroxisomal multifunctional enzyme (MFE) from rat liver.
The VIP2 receptor: Molecular characterisation of a cDNA encoding a novel receptor for vasoactive intestinal peptide
Authors: Lutz E.M. , Sheward W.J. , West K.M. , Morrow J.A. , Fink G. , Harmar A.J.
Published: July 2002

We have cloned and sequenced a cDNA (RPR4) encoding a new member of the secretin/calcitonin/parathyroid hormone (PTH) receptor family. RPR4 was identified by PCR of rat pituitary cDNA, and a full‐length clone was isolated from a rat olfactory bulb cDNA library. When RPR4 was functionally expressed in COS 7 cells, cyclic adenosine monophosphate (cAMP) production was stimulated by vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating peptides (PACAP‐38 and PACAP‐27) and helodermin, with equal potency. Peptide histidine isoleucine (PHI) and rat growth hormone releasing hormone (rGHRH) also stimulated cAMP production at lower potency. This suggests that RPR4 encodes a novel VIP receptor which we have designated the VIP2 receptor. In situ hybridisation showed that mRNA for this receptor was present mainly in the thalamus, hippocampus and in the suprachiasmatic nucleus.
Trimeric forms of the photosystem I reaction center complex pre‐exist in the membranes of the cyanobacterium Spirulina platensis
Authors: Shubin V.V. , Tsuprun V.L. , Bezsmertnaya I.N. , Karapetyan N.V.
Published: July 2002

Oligomeric and monomeric forms of chlorophyll—protein complexes of photosystem I (PSI) have been isolated from the mesophilic cyanobacterium Spirulina [(1992) FEBS Lett. 309, 340–342]. Electron microscopic analysis of the complexes showed that the oligomeric form is a trimer of the shape and dimensions similar to those of the trimer from thermophilic cyanobacteria. The chlorophyl ratio in the isolated trimer and monomer was found to be 7:3. The trimeric form of PSI complex in contrast to the monomeric one contains the chlorophyll emitting at 760 nm (77K), which is also found in Spirulina membranes and therefore could be used as an intrinsic probe for the trimeric complex. The 77K circular dichroism spectrum of the trimeric form is much more similar to that of Spirulina membranes than the spectrum of the monomer. Thus, the trimeric PSI complexes exist and dominate in the Spirulina membranes.
The regulation of sodium‐dependent transport of anionic amino acids in cultured human fibroblasts
Authors: Franchi-Gazzola Renata , Visigalli Rossana , Bussolati Ovidio , Gazzola Gian C.
Published: July 2002

In cultured human fibroblasts the transport of anionic amino acids through the sodium‐dependent system X−
AG is stimulated rapidly and transiently by phorbol 12,13‐dibutyrate. Transport stimulation is consistent with an effect due to the activation of protein kinase C. Bradykinin (1 μM) and PDGF‐AA (100 ng/ml) also stimulate the activity of system X−
AG. The bradykinin effect appears to be fully dependent upon PKC activation whereas the stimulation of aspartate transport by PDGF‐AA is also due to PKC‐independent mechanisms.
Similarity of Ca2+‐bound conformations of morphine and Met‐enkephalin: A computational study
Authors: Zhorov Boris S. , Ananthanarayanan Vettai S.
Published: July 2002

The conformations of the free and Ca2+‐bound forms of morphine and Met‐enkephalin were compared based on an earlier proposal that extracellular Ca2+ may dictate the bioactive conformations of peptide hormones and drugs. A Monte Carlo with energy minimization method was used to calculate Met‐enkephalin in the absence and presence of Ca2+. The Ca2+‐bound conformation of Met‐enkephalin was found to have an overall shape that matched well with that of morphine. In contrast, the uncomplexed Met‐enkephalin did not have such a match. The data suggest that a ternary association of μ‐receptor, its ligands and Ca2+ may be an initial process in the signal transduction mechanism of opioid peptides.
Expression, purification and characterization of a Kunitz‐type protease inhibitor domain from human amyloid precursor protein homolog
Authors: Petersen Lars C. , Bjørn Søren E. , Norris Fanny , Norris Kjeld , Sprecher Cindy , Foster Donald C.
Published: July 2002

The Kunitz‐type protease inhibitor domain from a recently identified homolog of the Alzheimer amyloid precursor protein (APPH KPI) was expressed in yeast, purified and characterized. Its inhibition profile towards several serine proteases was studied and compared to that of APP KPI, the Kunitz domain from the Alzheimer amyloid precursor protein. APPH KPI was shown to inhibit proteases with trypsin‐like specificity with an inhibitor profile resembling that of the APP KPI domain. The KPI domains from APP and APPH inhibited trypsin (K
i = 0.02 nM), and plasma kallikrein (K
i = 86 nM) with approximal equal affinity. In comparison to APP KPI (K
i = 82 nM) the KPI domain of the homolog, APPH KPI, (K
i = 8.8 nM) was a more potent inhibitor of glandular kallikrein. APPH KPI was a less potent inhibitor of chymotrypsin than APP KPI (K
i = 78 nM as compared to K
i = 6 nM), plasmin (K
i = 81 nM as compared to 42 nM), and factor XIa (K
i = 14 nM as compared to K
i = 0.7 nM). The affinity of factor XIa for APPH KPI is sufficiently high to allow for an interaction in the blood. It is, however, well possible that the physiological protease ligand for the receptor‐like APPH protein has yet to be identified.
Calcium sensing by human medullary thyroid carcinoma cells
Authors: Ridefelt Peter , Liu Yi-Jia , Rastad Jonas , Åkerström Göran , Gylfe Erik
Published: July 2002

Regulation of the cytoplasmic calcium concentration ([Ca2+]i) was studied in fura‐2‐loaded C‐cells from two human medullary thyroid carcinomas (MTC). K+ depolarization induced sustained rise of [Ca2+]i reversed by verapamil. Elevation of external Ca2+ from 0.5 to 3.0 mM triggered regular oscillations or steady‐state increases of [Ca2+]i. In Ca2+‐deficient medium Sr2+ caused steady‐state increase or oscillations of the 340/380 nm fluorescence ratio. The Ca2+ and Sr2+ actions were partially reversible by verapamil. La3+ and Ce3+ elicited transient [Ca2+]i peaks independent of external Ca+2+, but no oscillations. The results indicate that human MTC cells express a parathyroid‐like Ca2+ sensor coupled to intracellular mobilization and influx of Ca2+. A voltage‐dependent Ca2+ influx may be of importance for the oscillations of [Ca2+]i.
Curcumin is a non‐competitive and selective inhibitor of phosphorylase kinase
Authors: Reddy Shrikanth , Aggarwal Bharat B.
Published: July 2002

Recently, we reported that curcumin (diferuloylmethane) inhibits the growth of several different kinds of tumor cells. In order to investigate the mechanism of this inhibition, we examined the effects of curcumin on different protein kinases: highly purified protein kinase A (PkA), protein kinase C (PkC), protamine kinase (cPK), phosphorylase kinase (PhK), autophosphorylation‐activated protein kinase (AK) and pp60c‐src tyrosine kinase. While all kinases tested were inhibited by curcumin, only PhK was completely inhibited at relatively lower concentrations. At around 0.1 mM curcumin, PhK, pp60c‐src, PkC, PkA, AK, and cPK were inhibited by 98%, 40%, 15%, 10%, 1%, and 0.5%, respectively. Lineweaver‐Burk plot analysis indicated that curcumin is a non‐competitive inhibitor of PhK with a K
i of 0.075 mM. Overall, our results indicate that curcumin is a potent and selective inhibitor of phosphorylase kinase, a key regulatory enzyme involved in the metabolism of glycogen. This has important implications for the anti‐proliferative effects of curcumin.
Specificity of protein interactions with highly related SRC homology (SH) domains of FGR and FYN protein‐tyrosine kinases
Authors: Rivero-Lezcano Octavio M. , Robbins Keith C.
Published: July 2002

As an approach toward identification and isolation of cellular proteins that may act as substrates or effectors of the SRC‐family of protein‐tyrosine kinases, fusion proteins containing noncatalytic elements of two highly related SRC‐family members were tested for their ability to recognize distinct molecules present in lysates of cells known to normally express both enzymes. Our results demonstrate differences of protein binding between the SH2 elements of FYN and FGR kinases, but do not discriminate proteins binding to their SH3 domains.
Expression of GCAP 1 and GCAP2 in the retinal degeneration (rd) mutant chicken retina
Authors: Semple-Rowland Susan L. , Gorczyca Wojciech A. , Buczylko Janina , Helekar Bharati S. , Ruiz Claudia C. , Subbaraya Iswari , Palczewski Krzysztof , Baehr Wolfgang
Published: July 2002

We cloned the guanylate cyclase activating proteins, GCAP1 and GCAP2, from chicken retina and examined their expression in normal and predegenerate rd/rd chicken retina. Northern analyses show that the amounts of the single transcripts encoding GCAPI and GCAP2 are reduced to about 70% of normal levels in rdlrd retina. Western analyses reveal that GCAP2 levels appear normal in this retina, while GCAPI levels are reduced by more than 90%. The specific downregulation of GCAPI in rd/rd retina is consistent with a model for this disease in which activation of guanylate cyclase in the photoreceptors is abnormal, resulting in low levels of cGMP and an absence of phototransduction.
Cytokine regulation of astrocyte function: in‐vitro studies using cells from the human brain
Authors: Aloisi Francesca , Borsellino Giobanna , Caré Alessandra , Testa Ugo , Gallo Paolo , Russo Giobanni , Peschle Cesare , Levi Giulio
Published: July 2002

Participation of astrocytes in central nervous system pathophysiology is likely to involve cytokines, both as stimulators and mediators of astrocyte function. We have used highly enriched human astrocyte cultures as an experimental tool to investigate the influence of cytokines on adhesion molecule expression and synthesis of mediators that are probably important in immune and inflammatory reactions involving the nervous system and in cerebral tissue repair. The response of astrocytes to interferon‐γ mainly resulted in increased expression of major histocompatibility complex antigens and co‐stimulatory molecules (intercellular adhesion molecule‐1, LFA‐1α) which mediate astrocyte‐T‐cell interactions. Another co‐stimulatory molecule, B7, was neither expressed nor inducible by IFN‐γ and other cytokines. TNF‐α and IL‐1β were more efficient in stimulating synthesis of immunoregulatory and proinflammatory cytokines (IL‐6, IL‐8 and colony‐stimulating factors), cytokine antagonists (TNF‐α soluble receptors), or cytokines with a possible neuroprotective role (leukemia inhibitory factor); they also increased expression of some co‐stimulatory molecules (intercellular adhesion molecule‐1 and vascular cell adhesion molecule‐1). Transforming growth factor‐β1 was a strong inducer of leukemia inhibitory factor, but did not affect either major histocompatibility complex/co‐stimulatory molecule expression or cytokine synthesis. Thus, different cytokines activate distinct functional programs in astrocytes, which may play a specific role in different brain diseases or at different stages of the same disease. It was additionally observed that the response of human astrocytes to cytokines (in particular the inducible synthesis of certain cytokines) varied greatly depending on the presence or absence of neurons in the culture system. This finding suggests that neuronal‐glial interactions may be implicated in determining the activation threshold of astrocytes to inflammatory cytokines.
MIGRATING FETAL ASTROCYTES DO NOT INTRAVASATE SINCE THEY ARE EXCLUDED FROM BLOOD VESSELS BY VITAL BASEMENT MEMBRANE
Authors: Bernstein Jerald J. , Karp Sean M.
Published: July 2002

Astrocytes appear to be excluded from blood vessels. In order to test this observation, pial blood vessels were seeded upon cultures of purified E14 fetal astrocytes. One to seven days later co‐cultures were immunohistochemically stained for laminin and fibronectin. Confocal microscopy revealed that laminin‐positive cultured astrocytes migrated from the coverslip to and on to the surface of the blood vessels. The fetal astrocytes migrated along the basement membrane but did not enter the blood vessel. Viable (live) basement membrane is a barrier to astrocyte intravasation and is a pathway for astrocyte migration.
The World Health Organization somatoform disorders schedule. A preliminary report on design and reliability
Authors: Janca A , Burke JD , Isaac M , Burke KC , Costa JA , Silva E , Acuda SW , Altamura AC , Chandrashekar CR , Miranda CT , Tacchini G
Published: July 2002

The World Health Organization (WHO) Somatoform Disorders Schedule (SDS) is a highly standardized instrument for the assessment of somatoform disorders according to the tenth revision of the International Classification of Diseases (ICD-10) and the fourth edition of the Diagnostic and Statistical Manual (DSM-IV). The SDS was produced in the framework of the WHO International Study of Somatoform Disorders and tested for its reliability in Brazil, India, Italy, the USA and Zimbabwe. A sample of 180 patients from general psychiatry, primary care and general medical settings were interviewed with the SDS within a three-day interval by nonclinician and clinician interviewers. The agreement between the two interviews was tested using the intraclass correlation coefficients (ICC) and kappa statistic. The test-retest reliability of the SDS was found to be very good (the ICC for all the centres was 0.76; overall kappa value for SDS questions was 0.58; one-third of SDS questions had a kappa value of 0.60 or higher). The field test results of the SDS indicated that the instrument may be administered in larger studies by non-clinician interviewers without compromising the ability to document the prevalence of somatoform disorders in different cultures.
Regulation of exocytosis in chromaffin cells by phosducin‐like protein, a protein interacting with G protein βγ subunits
Authors: Gensse Marc , Vitale Nicolas , Chasserot-Golaz Sylvette , Bader Marie-France
Published: July 2002

Phosducin and related proteins have been identified as ubiquitous regulators of signalling mediated by βγ subunits of trimeric G proteins. To explore a role for phosducin in regulated exocytosis, we have examined the distribution and putative function of phosducin‐like protein (PhLP) in adrenal medullary chromaffin cells. The full‐length cDNA encoding the short splice variant of PhLP (PhLPs) was cloned from cultured chromaffin cells. Native PhLPs was found associated with plasma membranes and detected in the subplasmalemmal area of resting chromaffin cells by confocal immunofluorescence analysis. Stimulation with secretagogues triggered a massive redistribution of PhLPs into the cytoplasm. When microinjected into individual chromaffin cells, recombinant PhLPs inhibited catecholamine secretion evoked by a depolarizing concentration of K+ without affecting calcium mobilization. Thus, PhLPs may participate directly in the regulation of calcium‐evoked exocytosis.
Inhibition of eukaryotic ribonuclease P activity by aminoglycosides: kinetic studies
Authors: Tekos Apostolos , Tsagla Antigoni , Stathopoulos Constantinos , Drainas Denis
Published: July 2002

The effect of several aminoglycoside antibiotics on ribonuclease P (RNase P) was investigated using an in vitro experimental system from Dictyostelium discoideum. Detailed kinetic analysis showed that all aminoglycosides tested (tobramycin, gentamicin, kanamycin, paromomycin, neomycin) behave as classical non‐competitive inhibitors, with neomycin being the strongest inhibitor. The inhibition effect is attributed to the electrostatic competition of the cationic aminoglycosides with magnesium ions required for catalysis. Increasing Mg2+ ion concentrations reduced the effect of aminoglycosides on RNase P activity. Detailed kinetic analysis showed that aminoglycosides compete with Mg2+ for common binding sites on RNase P holoenzyme.
Inhibition of cytokine‐induced vascular cell adhesion molecule‐1 expression; possible mechanism for anti‐atherogenic effect of Agastache rugosa
Authors: Hong Jung-Joo , Choi Jae-Hoon , Oh Sei-Ryang , Lee Hyeong-Kyu , Park Jae-Hak , Lee Kun-Yeong , Kim Jung-Jae , Jeong Tae-Sook , Oh Goo Taeg
Published: July 2002

Adhesion molecules such as vascular cell adhesion molecule‐1 (VCAM‐1) play an important role during the early stages of atherogenesis. Agastache rugosa has an anti‐atherogenic effect in low density lipoprotein receptor −/− mice. Moreover, A. rugosa reduced macrophage infiltration and VCAM‐1 expression has been localized in aortic endothelium that overlies early foam cell lesions. This study ascertained that tilianin (100 μM), a major component of A. rugosa, inhibits the tumor necrotic factor‐α (TNF‐α)‐induced expression of VCAM‐1 by 74% in cultured human umbilical vein endothelial cells (HUVECs). Also, tilianin (100 μM) reduced TNF‐α‐induced activation of nuclear factor‐κB in HUVECs.
Cleavage of mitochondria‐like transfer RNAs expressed in Escherichia coli
Authors: Bourdeau Véronique , Paquin Bruno , Cedergren Robert , Delihas Nicholas
Published: July 2002

Mitochondrial (mt) transfer RNAs (tRNAs) often harbor unusual structural features causing their secondary structure to differ from the conventional cloverleaf. tRNAs designed with such irregularities, termed mt‐like tRNAs, are active in Escherichia coli as suppressors of reporter genes, although they display low steady‐state levels. Characterization of fragments produced during mt‐like tRNA processing in vitro and in vivo suggests that these RNAs are not fully processed at their 5′ ends and are cleaved internally. These abnormal processing events may account for the low levels of mature mt‐like RNAs in vivo and are most likely related to defective processing by RNase P.
Expression of ARF6 mutants in neuroendocrine cells suggests a role for ARF6 in synaptic vesicle biogenesis
Authors: M. Powelka Aimee , Buckley Kathleen M.
Published: July 2002

ARF6 regulates membrane trafficking between the plasma membrane and endosomes. We investigated the role of ARF6 in synaptic vesicle biogenesis as this process occurs both at the plasma membrane and at endosomes. We used a synaptic vesicle marker protein, p‐selectin‐horseradish peroxidase (HRP), to follow the effects of ARF6 expression on synaptic vesicle biogenesis in PC12 neuroendocrine cells. Expression of a constitutively active ARF6 mutant increased, while expression of a nucleotide‐free ARF6 mutant decreased, p‐selectin‐HRP levels in the synaptic vesicle peak. These results provide the first direct evidence for a role for ARF6 in synaptic vesicle biogenesis.
Further evidence that inhibitor‐2 acts like a chaperone to fold PP1 into its native conformation
Authors: MacKintosh Carol , Garton Andrew J. , McDonnell Annabel , Barford David , Cohen Patricia T.W. , Tonks Nicholas K. , Cohen Philip
Published: July 2002

The γ1‐isoform of protein phosphatase‐1 expressed in Escherichia coli (PP1γ) and the native PP1 catalytic subunit (PP1C) isolated from skeletal muscle dephosphorylated Ser‐14 of glycogen phosphorylase at comparable rates. In contrast, PP1γ dephosphorylated several tyrosine‐phosphorylated proteins at similar rates to authentic protein tyrosine phosphatases (PTPases), but native PP1C was almost inactive towards these substrates. The phosphorylase phosphatase (PhP) and PTPase activities of PP1γ were inhibited by vanadate with IC50 values (30–100 μM) comparable to authentic PTPases, whereas the PhP activity of native PP1C was insensitive to vanadate. PP1γ lost its PTPase activity, and its PhP activity became insensitive to vanadate, after interaction with inhibitor‐2, followed by the reversible phosphorylation of inhibitor‐2 at Thr‐72. These findings support and extend the hypothesis that inhibitor‐2 functions like a chaperone to fold PP1 into its native conformation, and suggest that the correct folding of PP1 may be critical to prevent the uncontrolled dephosphorylation of cellular phosphotyrosine residues.
Purification of acid sphingomyelinase from human placenta: Characterization and N‐terminal sequence
Authors: Lansmann Stephanie , Ferlinz Klaus , Hurwitz Robert , Bartelsen Oliver , Glombitza Gereon , Sandhoff Konrad
Published: July 2002

Human placental acid sphingomyelinase (ASM) was purified by sequential chromatography on Con A‐Sepharose, octyl‐Sepharose and Matrex gel red A. Final purification to apparent homogeneity was achieved by immunoaffinity chromatography employing polyclonal anti‐ASM antibodies. The antibodies also allowed specific detection of ASM by Western blotting at various stages of purification. The ASM activity was enriched about 110 000‐fold over that of the crude extract, yielding an enzyme preparation with a specific activity of about 1 mmol/h per mg protein in a detergent‐containing assay system. Analysis of the final preparation by SDS‐PAGE resulted in a single protein band with a molecular mass of ∼75 kDa, which was reduced to ∼60 kDa after complete deglycosylation. Microsequencing of the purified ASM revealed the N‐terminal amino acid sequence of the mature placental enzyme.
Inhibition of nucleoside diphosphate kinase (NDPK/nm23) by cAMP analogues
Authors: Anciaux Katelijne , Van Dommelen Kristof , Willems Roel , Roymans Dirk , Slegers Herman
Published: July 2002

Nucleoside diphosphate kinase (NDPK/nm23) ATP/GDP phosphotransferase activity and serine autophosphorylation is inhibited by N
6‐mbcAMP, 8‐ClcAMP and 8‐BrcAMP. Inhibition of the enzymatic activity largely depends on the concentration of ATP and becomes significant at ATP concentrations up to 0.5 mM and at effector concentrations measured in C6 cells stimulated with 1 mM cAMP analogue. N
6‐mbcAMP is a substrate of the enzyme. DbcAMP and O′2‐mbcAMP, cAMP analogues with a modified O′2‐ribose, did not affect the NDPK activity. Cyclic AMP is only a moderate inhibitor of NDPK even at low ATP concentrations. Possible inhibitory effects of cAMP and cAMP analogues on reported extra‐ and intracellular functions of NDPK/nm23 are discussed.
Evolution of dnaQ, the gene encoding the editing 3′ to 5′ exonuclease subunit of DNA polymerase III holoenzyme in Gram‐negative bacteria
Authors: Huang Yiping , Braithwaite Dan K , Ito Junetsu
Published: July 2002

The nucleotide sequences of the dnaQ genes from Salmonella typhimurium and Buchnera aphidicola, encoding the ϵ‐subunit of the DNA polymerase III holoenzyme, have been determined. The Salmonella typhimurium dnaQ protein consists of 243 amino acid residues with a calculated molecular weight of 27 224. The Buchnera aphidicola dnaQ protein contains 233 amino acid residues with a calculated molecular weight of 27 170. A multiple sequence alignment of the amino acid sequences of the dnaQ proteins and those of DNA polymerase IIIs from Gram‐positive bacteria produced six homologous segments. These homologous segments contain highly conserved amino acid sequence motifs involved in catalytically important metal ion bindings (ligands 1, 2 and 3). However, metal ligand 4 is found to be altered in the 3′‐5′ exonuclease domain of the family C DNA polymerases and dnaQ proteins in Gram‐negative bacteria. From these results, we propose that the last common ancestor of the dnaQ gene of Gram‐negative bacteria and the DNA polymerase III gene (pol C gene) of Gram‐positive bacteria was a single gene containing both 3′‐5′ exonuclease and DNA polymerase domains and then the dnaQ gene separated from the polymerase gene in Gram‐negative bacteria.
Isolation and characterization of new C‐terminal substitution mutations affecting secretion of polygalacturonase in Erwinia carotovora ssp. carotovora
Authors: Palomäki T , Saarilahti H.T
Published: July 2002

An intact C‐terminus was previously shown to be required for stability and secretion of the polygalacturonase (PehA) in Erwinia carotovora ssp. carotovora. Here we have analyzed the effects of amino acid (aa) substitutions generated to five C‐terminal positions of PehA. Conservation of two hydrophobic and one non‐hydrophobic residue (V372, V374 and N371, respectively) was found to be essential for maintenance of the protein stability. As an exception, one of the mutants (V372G) did not show major effects on protein stability, as determined by immunoblots and enzyme activity assay, yet it prevented the secretion completely. We conclude that the C‐terminus of PehA is directly involved in the formation or stabilization of a conformation‐sensitive structure needed for recognition of the protein as secreted.
Cloning and initial characterization of human and mouse Spot 14 genes1
Authors: Grillasca Joël-Paul , Gastaldi Marguerite , Khiri Hacène , Dace Alexandra , Peyrol Nicole , Reynier Pascal , Torresani Janine , Planells Richard
Published: July 2002

The intricate regulation of Spot 14 expression in rat lipogenic tissues has provided a useful tool in studying nutritional and hormonal factors involved in transcription. To gain insight into its function and its possible involvement in human lipid disorders, we cloned human and mouse Spot 14 genes that shared with the rat gene a strong homology concerning the deduced amino acid sequence (81 and 94%, respectively) as well as the promoter region. The mouse promoter was characterized by transfection studies, while quantitative RT‐PCR and in situ hybridization experiments showed that Spot 14 is expressed in human liver and, at a high level, in multiple symmetric lipomatosis nodules.
Cloning of the human IL‐13Rα1 chain and reconstitution with the IL‐4Rα of a functional IL‐4/IL‐13 receptor complex
Authors: Miloux Brigitte , Laurent Patrick , Bonnin Odile , Lupker Jan , Caput Daniel , Vita Natalio , Ferrara Pascual
Published: July 2002

The human homologue of the recently cloned murine IL‐13 binding protein (IL‐13Rα1) was cloned from a cDNA library derived from the carcinoma cell line CAKI‐1. The cloned cDNA encodes a 427 amino acid protein with two consensus patterns characteristic of the hematopoietic cytokine receptor family and a short cytoplasmic tail. The human protein is 74% identical to the murine IL‐13Rα1, and 27% identical to the human IL‐13Rα2. CHO cells expressing recombinant hIL‐13Rα1 specifically bind IL‐13 (K
d≈4 nM) but not IL‐4. Co‐expression of the cloned cDNA with that of IL‐4Rα resulted in a receptor complex that displayed high affinity for IL‐13 (K
d≈30 pM), and that allowed cross‐competition of IL‐13 and IL‐4. Electrophoretic mobility shift assay showed that IL‐13 and IL‐4 were able to activate Stat6 in cells expressing both IL‐4Rα and IL‐13Rα1, while no activation was observed in cells expressing either one or the other alone.