bluuebunny/crossref_metadata_2025 huggingface dataset:
The interaction of amiloride with acetylcholinesterase and butyrylcholinesterase
Authors: Zemach Lucie , Segal Dina , Shalitin Yechiel
Published: July 2002

The diuretic drug amiloride was found to be a powerful inhibitor of the reaction of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with their specific choline ester substrates. The inhibition constant is in the micromolar range. On the other hand, when added to a mixture of cholinesterase (AChE and BChE) and neutral substrates, amiloride, in some cases, enhanced the reaction rate. The rate of the reaction of butyrylcholinesterase with p‐nitrophenyl butyrate was increased up to 12 fold by amiloride.
Crystallization and preliminary X‐ray crystallographic study of interleukin‐8
Authors: Auer Manfred , Kallen Joerg , Schleischitz Sabine , Walkinshaw Malcolm D. , Wasserbauer Erich , Ehn Gerald , Lindley Ivan J.D.
Published: July 2002

Interleukin‐8 (neutrophil‐activating factor; NAP‐1) has been crystallized by the vapour diffusion technique to give single crystals suitable for three‐dimensional structural study at a resolution higher than 2.4 Å. The crystals belong to the space group P3121 or P3221 and have unit cell dimensions a = b = 40.9 Å, c = 90.3 Å.
Cloning and chromosomal assignment of a widely expressed human receptor‐like protein‐tyrosine phosphatase
Authors: Jirik Frank R. , Janzen Nicole M. , Melhado Ian G. , Harder Kenneth W.
Published: July 2002

Insight into the regulation of the actions of the protein‐tyrosine kinases will be greatly facilitated by the full characterization of the family of protein‐tyrosine phosphatases. A search for novel phosphatases resulted in the isolation of a cDNA, termed HLPR, encoding a member of the family of human receptor‐like protein‐tyrosine phosphatases: its cDNA sequence predicts a protein of 793 amino acids (unglycosylated M
r, 87500) and includes a 121 residue extracellular domain, a single transmembrane segment, and two tandem intra‐cytoplasmic catalytic domains. The HLPR gene is located on human chromosome 20, and the protein it encodes likely plays a fundamental role in the physiology of all cells as its expression appears to be ubiquitous.
Soybean lipoxygenase‐1 is not a quinoprotein
Authors: Veldink Gerrit A. , Boelens Harry , Maccarrone Mauro , van der Lecq Fridolin , Vliegenthart Johannes F.G. , Paz Mercedes A. , Fluc̈kiger Rudolf , Gallop Paul M.
Published: July 2002

Soybean lipoxygenase‐1 was reinvestigated with respect to its quinoprotein nature. It has been reported previously that soybean lipoxygenase‐1 contains pyrroloquinoline quinone as the organic cofactor [1]. Because spectroscopie data were found to be inconsistent [2] with the evidence presented in [1], we sought to reproduce the published data by carefully following the procedures described in [1] and supplementing them with new analytical results. The combined data lead us to conclude that soybean lipoxygenase‐1 is not a quinoprotein.
Reduced levels of ADP‐ribosylatable elongation factor‐2 in aged and SV40‐transformed human cell cultures
Authors: Riis Bent , Rattan Suresh I.S. , Derventzi Anastassia , Clark Brian F.C.
Published: July 2002

The elongation step is involved in the regulation of protein synthesis during the cell cycle, environmental stress, ageing and transformation. Using a diphtheria toxin‐mediated assay for measuring the levels of ADP‐ribosylatable elongation factor EF‐2, we have observed an irreversible decrease of up to 64% in the amount of ADP‐ribosylatable EF‐2 in normal diploid human fibroblasts MRC‐5 undergoing ageing in vitro. However, a similar decrease in low serum‐associated G0/G1‐arrested cells is reversible both in MRC‐5 cells and in their SV40‐transformed counterparts. Reduced levels of ADP‐ribosylatable EF‐2 could account for the slowing‐down of protein synthesis during cell cycle arrest and during cellular ageing in culture.
Alamethicin channel permeation by Ca2+, Mn2+ and Ni2+ in bovine chromaffin cells
Authors: Fonteriz R.I. , López M.G. , Garcia-Sancho J. , Garcia A.G.
Published: July 2002

Alamethicin causes a concentration‐dependent increase of (Ca2+), in suspensions of bovine adrenal chromaffin cells loaded with fura‐2. The basal levels of Ca2+ (234 ± 37 nM; n=4) increased to a maximum of 2347±791 nM (n=3) with 100 μg/ml alamethicin. In the presence of 1 nM Ca2+ the increase reached a plateau within about 2–5 s. This increase was due to Ca2+ entry into chromaffin cells, since in the absence of Ca2+ alamethicin did not modify [Ca2+]. This contrasts with ionomycin (1 μM) which produced a Ca2+ transient even in the absence of Ca2+, Mn2+ ions also entered chromaffin cells in the presence of alamethicin, as measured by the quenching of fura‐2‐fluorescence following excitation at 360 nm. Resting chromaffin cells had a measurable permeability to Mn2+ which was drastically increased by cell depolarization by K+ (50 nM) addition. This suggests that Mn2+ is able to permeate voltage‐dependent Ca2+ channels. Ni2+ uptake into either resting of K+‐stimulated chromaffin cells was undetectable, but addition of alamethicin induced rapid uptake of this cation. The alamethicin‐induced entry of Ni2+ was decreased by 50 mM K+. Overall, the results are compatible with the formation by alamethicin of ion channels in chromaffin cell plasma membranes.
Purification and characterization of human T‐cell leukemia virus type I protease produced in Escherichia coli
Authors: Kobayashi Makoto , Ohi Yumiko , Asano Tsuneo , Hayakawa Takaki , Kato Koichi , Kakinuma Atsushi , Hatanaka Masakazu
Published: July 2002

Human T‐cell leukemia virus type I (HTLV‐I) protease has been purified to homogeneity from a strain of recombinant Escherichia coli. The protease was expressed as a larger precursor, which was autoprocessed to form a mature protease. Protein chemical analyses revealed the coding sequence of mature protease, which agreed with the putative sequence predicted from the sequence of bovine leukemia virus protease, The purified protease processed the natural substrate gag precursor (p53) to form gag p19 and gag p24. The protease activity was inhibited by pepstatin A. These results provide direct evidence that this protease belongs to the aspartic protease family and has an activity consistent with the protease in HTLV‐I virion.
Structure of the active 27‐residue fragment of human calpastatin
Authors: Ishima Rieko , Tamura Atsuo , Akasaka Kazuyuki , Hamaguchi Kaoru , Makino Keisuke , Murachi Takashi , Hatanaka Masakazu , Maki Masatoshi
Published: July 2002

A synthetic 27‐residue peptide corresponding to exon 1B of the endogenous inhibitor calpastatin contains a well‐conserved region and has an ability to inhibit the cysteine endopeptidase calpain specifically. We examined the solution structure of this peptide in DMSO‐d6 by two‐dimensional 1H NMR spectroscopy. Although regular secondary structures such as α‐helix and β‐sheet were not found, the region from Ile18 to Arg23 formed a well‐defined structure with a type I β‐turn. This region coincided well with the highly conserved region of calpastatin. The result strongly suggests that this turn structure is essential for the inhibitory activity of calpastatin.
Immunologic and structural relatedness of the integrin β7complex and the human intraepithelial lymphocyte antigen HML‐1
Authors: Krissansen Geoffrey W. , Print Cristin G. , Prestidge Ross L. , Hollander Daniel , Yuan Qian , Jiang Wei-meng , Jenkins David R. , Leung Euphemia , Mead Paul , Yong Rita , Ameratunga Rohan V. , Cerf-Bensussan Nadine , Watson James D.
Published: July 2002

We recently cloned the newest human integrin β subunit, termed β7, from a cDNA library constructed from SEA‐activated T lymphocytes. In this communication, we report on the structure of the human integrin β7 protein complex determined using a rabbit anti‐β7 peptide antibody raised to an N‐terminal 22 amino acid residue sequence deduced from the human β7 subunit cDNA. The β7 subunit (M, 116 000) expressed on PHA lymphoblasts associates with asingle major α subunit (α11) that is distinct from the prominent T cell marker, integrin α4. The α11 subunit (M
r 180 000 nonreduced) displays a distinctive shift in size on reduction to an apparent M
r of 150 000. We show that these structural properties of the integrin β7 complex are shared with the cell surrace antigen HML‐1 found highly expressed on T cells which populate the intestinal epithelium and are proposed to be involved in mucosal immunity. Sequential immunoprecipitation and Western blotting demonstrate identity or close homology between the α11β7 and HML‐1 proteins.
Involvement of DNA polymerase β in proliferation of rat liver induced by lead nitrate or partial hepatectomy
Authors: Menegazzi Marta , de Prati Alessandra Carcereri , Ogura Tsutomu , Columbano Amedeo , Ledda-Columbano Giovanna Maria , Libonati Massimo , Esumi Hiroyasu , Suzuki Hisanori
Published: July 2002

We have studied the expression pattern of DNA polymerase β in two different models of in vivo cell proliferation. Both mRNA levels and enzyme activity of DNA polymerase β markedly increased before and/or during DNA synthesis in proliferating hepatocytes in mitogen‐treated and partially hepatectomized rats. The time—courses of the expression of the gene coding for DNA polymerase β were significantly different in the two cell systems. A 5‐fold increase in DNA polymerase β mRNA was observed 8 h after lead nitrate administration, i.e. well before the onset of DNA synthesis. In the regenerative liver cells a 3‐fold increase in the amount of mRNA was observed 24–48 h after partial hepatectomy, the event being coincident with extensive DNA synthesis. In both systems, the increase of mRNA levels was always paralleled by an increase in enzyme activity, suggesting that DNA polymerase β activity may be regulated at a pre‐translational level.
Determination of the relative configuration of 5,6,7,8‐tetrahydromethanopterin by two‐dimensional NMR spectroscopy
Authors: Schleucher J. , Schwörer B. , Zirngibl C. , Koch U. , Weber W. , Egert E. , Thauer R.K. , Griesinger C.
Published: July 2002

The relative configuration of the pterin moiety of 5,6,7,8‐tetrahydromethanopterin 1, a coenzyme isolated from methanogenic archaea, has been determined by two‐dimensional NMR spectroscopy of N
5
N
10‐methenyl‐5,6,7,8‐tetrahydromethanopterin 2 to be rel‐(6R; 7S; 11R). The complete proton resonance assignment of the pterin moiety or N
5
N
10‐methylene‐5,6,7,8‐tetrahydromethanopterin 3 is described including the relative stereospecific assignment of the C(14a) methylene protons.
A conformation‐dependent monoclonal antibody against active chicken acetylcholinesterase
Authors: Chatel Jean-Marc , Vallette François-Marie , Massoulié Jean , Grassi Jacques
Published: July 2002

We show that the C‐131 monoclonal antibody, directed against chicken AChE, recognizes active chicken AChE, but not the SDS‐denatured or heat‐inactivated protein. Previous results indicated that C‐131 only binds to the active enzyme, and not to inactive molecules which also occur in the embryonic chicken brain. In contrast with C‐131, other monoclonal antibodies obtained in the same series, such as C‐6 and C‐54, also recognize denatured or inactive AChE. It is noteworthy that these antibodies all seem to react with a trypsin‐sensitive peptide which is present in chicken but not in mammalian or Torpedo AChE, whereas the C‐131 antibody binds trypsin‐modified as well as intact molecules. These results show that C‐131 is highly conformation‐dependent, specific for active AChE. They confirm our previous conclusion that active and inactive molecules arise from different folding processes.
Detection of a 10 kDa breakdown product containing the C‐terminus of the D 1‐protein in photoinhibited wheat leaves suggests an acceptor side mechanism
Authors: Cánovas Pedro M. , Barber James
Published: July 2002

Photoinhibition of intact leaves of wheat generates a 10 kDa breakdown product which is clearly observed both at 4°C and 25°C. Selective immunoblotting has shown that the 10 kDa fragment contains the C‐terminus of the D 1‐protein and, under the conditions employed, supports an acceptor side mechanism for photoinhibition in vivo. Although a corresponding 23 kDa N‐terminal Dl‐fragment was not detected our results are consistent with the argument that the primary cleavage site is in the loop joining putative transmembrane segments IV and V.
Pseudo‐symmetrical difluoroketones
Authors: Sham Hing L. , Betebenner David A. , Wideburg Norman , Saldivar Ayda C. , Kohlbrenner William E. , Craig-Kennard Adrienne , Vasavanonda Sudthida , Kempf Dale J. , Clement Jacob J. , Erickson John E. , Plattner Jacob J. , Norbeck Daniel W.
Published: July 2002

A series of novel, pseudo‐symmetrical difluoroketones which are highly potent inhibitors of the HIV‐1 protease (IC50 = 1.55‐0.02 nM) were synthesized. These compounds also possess good antiviral activity by inhibition of the cytopathic effect of HIV‐13B in MT‐4 cells in vitro.
Ionic bases of the membrane potential and intracellular pH changes induced by speract in swollen sea urchin sperm
Authors: Reynaud Enrique , De de La Torre Lucía , Zapata Otilia , Lievano Arturo , Darszon Alberto
Published: July 2002

Signal transduction initiated by the egg peptide, speract, in sea urchin sperm is not fully understood. Hypotonically swollen sperm are a suitable model to study peptide signal transduction. Ion substitution experiments now indicate (i) that the permeability to Na+, Ca2+, and Mg2+ contributes to the sperm resting membrane potential; (ii) the repolarization induced by nM concentrations of speract is Na+ dependent and mediated by an as yet unidentified channel; (iii) the depolarization triggered by nM concentrations of speract involves Ca2+ channels since it is Ca2+‐dependent and blocked by Co2+ and Ni2+, two Ca2+ channel blockers; (iv) hyperpolarizing swollen sperm with valinomycin increases intracellular pH (pHi) in the same way as speract, thus the speract‐induced hyperpolarization may be responsible for the pHi increase.
Isolation and amino acid sequence of the 30S ribosomal protein S19 from Mycobacterium bovis BCG
Authors: Ohara Naoya , Kimura Makoto , Higashi Yoshinori , Yamada Takeshi
Published: July 2002

The 30S ribosomal proteins from Mycobacterium bovis BCG were separated by reverse phase‐high performance liquid chromatography (RP‐HPLC). The isolated proteins were analyzed by SDS‐PAGE, blotted on PVDF‐membranes and subjected to sequence analyses using a gas‐phase sequencer to correlate them to those of the well studied Escherichia coli and Bacillus stearothermophilus ribosomes. Moreover, the internal amino acid sequence of one ribosomal protein, MboS19, which is homologous to E. coli ribosomal protein S19 (EcoS19) and B. stearothermophilus ribosomal protein S19 (BstS19), was further analyzed by sequencing its internal peptides and two segments from the N‐ and C‐termini of the protein were selected to deduce the sequence of two oligonucleotide primers which were used in a polymerase chain reaction. Using the amplified DNA fragment thus obtained as a hybridization probe, the gene encoding protein S19 was identified and cloned. Sequence analysis of the DNA fragment, together with peptide sequence analysis could determine the complete amino acid sequence of MboS19. This sequence proved to be 64% and 71% identical to those of the corresponding S19 proteins from the eubacteria E. coli, and B. stearothermophilus, respectively; 33% of the residues of MboS19 were identical to those in the archaebacteral ribosomal protein HmaS19.
Direct gene transfer into mouse diaphragm
Authors: Davis Heather L. , Jasmin Bernard J.
Published: July 2002

Direct gene transfer into skeletal muscle is a potential therapeutic strategy for inherited primary myopathies such as Duchenne muscular dystrophy (DMD). In order to affect the life‐expectancy of these patients, it will be necessary to carry out gene therapy on the diaphragm. To this end, we report efficient introduction of pure recombinant plasmid DNA into the mouse diaphragm, without causing significant damage. Application of this approach to the diaphragm of the mdx mouse will provide information on the potential usefulness of gene therapy for the treatment of DMD patients.
Induction of phosphatidic acid by fibroblast growth factor in cultured baby hamster kidney fibroblasts
Authors: Motoike Toshiyuki , Bieger Sophie , Wiegandt Herbert , Unsicker Klaus
Published: July 2002

Basic fibroblast growth factor (bFGF/FGF‐2) is a strong mitogenic inducer of cultured baby hamster kidney (BHK) cells. When cultured BHK cells were stimulated with FGF‐2, phosphatidic acid (PA) was induced within 2 min, peaked at 5 min and gradually decreased. Phospholipase D (PLD) was also mitogenic for cultured BHK cells and this effect was mediated via PA. The possibility that PA induction by FGF‐2 is an essential signaling step for BHK cell proliferation is discussed.
Phosphorylation of mitochondrial proteins in bovine heart
Authors: Technikova-Dobrova Zuzana , Sardanelli Anna Maria , Papa Sergio
Published: July 2002

Protein phosphorylation by [γ‐32P]ATP in total extract and subfractions of bovine heart mitochondria has been studied. The results show that, in addition to pyruvate dehydrogenase, three mitochondrial proteins, with molecular weights of 44,000, 39,000 and 31,000 Da, are phosphorylated by a cAMP‐independent mitochondrial protein kinase. Three other proteins associated with mitochondria, with molecular weights of 125,000, 19,000 and 6,500 Da, are phosphorylated by the cytoplasmic cAMP‐dependent protein kinase (kinase A).
A rat brain cDNA encoding the neurotransmitter transporter with an unusual structure
Authors: Liu Qing-Rong , Mandiyan Sreekala , López-Corcuera Beatriz , Nelson Hannah , Nelson Nathan
Published: July 2002

A rat cDNA clone encoding the novel membrane protein of the neurotransmitter transporters family was cloned and sequenced. The cDNA was identified as a transcript of the gene NTT4 of which a partial genomic clone was previously sequenced. Alignment of the amino acid sequence of NTT4 with other members of the neurotransmitter transport family revealed a marked deviattion from the conserved structure of all other members of the family. The largest extracellular loop with a potential glycosylation site was identified between membrane segments 7 and 8. The protein retains the common glycosylated loop between transmembrane helices 3 and 4 in all members of the family. The transcript of NTT4 was found exclusively in the central nervous system and is more abundant in the cerebellum and the cerebral cortex.
Epidermal growth factor and transforming growth factor‐α can induce neuronal differentiation of rat pheochromocytoma PC 12 cells under particular culture conditions
Authors: Nakafuku Masato , Kaziro Yoshito
Published: July 2002

In rat pheochromocytoma PC12 cells, NGF induces neuronal differentiation. Upon stimulation with NGF, Ras is activated to a GTP‐bound form, and the activated Ras can induce neuronal differentiation. Recently, we and others observed that epidermal growth factor (EGF) and transforming growth factor‐α (TGF‐α) can also activate Ras in PC12 cells. This is puzzling since previous reports indicated that EGF stimulates proliferation rather than differentiation in PC12 cells. In this paper, we re‐examined the biological effect of EGF and TGF‐α, and found that these factors can also induce neuronal differentiation under particular culture conditions. Not only the outgrowth of long neurites, but the induction of neurofilament proteins and the metalloprotease transin was also observed in the EGF‐ and TGF‐α‐stimulated cells. These data clearly indicate that in addition to NGF, EGF and TGF‐α can also induce the differentiation of PC12 cells under particular conditions.
Watermelon glyoxysomal malate dehydrogenase is sorted to peroxisomes of the methylotrophic yeast, Hansenula polymorpha
Authors: van der Klei I.J. , Faber K.N. , Keizer-Gunnink I. , Gietl C. , Harder W. , Veenhuis M.
Published: July 2002

We have studied the fate of the watermelon (Citrullus vulgaris Schrad.) glyoxysomal enzyme, malate dehydrogenase (gMDH), after synthesis in the methylotrophic yeast, Hansenula polymorpha. The gene encoding the precursor form of gMDH (pre‐gMDH) was cloned in an H. polymorpha expression vector downstream of the inducible H. polymorpha alcohol oxidase promoter. During methylotrophic growth, pre‐gMDH was synthesized and imported into peroxisomes, where it was enzymatically active. The apparent molecular mass of the protein located in H. polymorpha peroxisomes was equal to that of pre‐gMDH (41 kDa), indicating that N‐terminal processing of the transit peptide had not occurred in the yeast.
Gene relaxation and aging: Changes in the abundance of rat ventral prostate SGP‐2 (clusterin) and ornithine decarboxylase mRNAs
Authors: Bettuzzi S. , Strocchi P. , Marinelli M. , Astancolle S. , Davalli P. , Corti A.
Published: July 2002

Sulfated glycoprotein 2 (SGP‐2) mRNA progressively increased in the ventral prostate of the aging rat, reaching, at 24 months, 4‐fold higher than at 3 months. Ornithine decarboxylase (ODC) mRNA peaked at 6 months (4‐fold increase), and at 12 and 24 months was maintained at higher levels than at 3 months. ODC enzymatic activity was enhanced at 6 months to a much smaller extent than its own mRNA, the values at 12 and 24 months dropping to below those at 3 months. Putrescine (Put), spermidine (Spd) and spermine (Sp) concentrations also peaked at 6 months (100% increase for Put, 50% for Sp and Spd). At 24 months, Put and Spd were diminished, and Sp was unchanged with respect to the 3‐month values. Under the same conditions, glyceraldehyde‐3‐phosphate dehydrogenase mRNA did not undergo significant alterations.
Differential inhibition of eukaryotic DNA polymerases by halenaquinol sulfate, a p‐hydroquinone sulfate obtained from a marine sponge
Authors: Shioda Masaki , Kano Kazukaka , Kobayashi Motomasa , Kitagawa Isao , Shoji Mami , Yoshida Shonen , Ikegami Susumu
Published: July 2002

Halenaquinol sulfate, a p‐hydroquinone sulfate obtained from a marine sponge, inhibited the activity of eukaryotic DNA polymerases in varying degrees; the K
i values for DNA polymerases α, β, δ and ε were 1.3, 80, 17.5 and 2.0 νM, respectively, whereas it was less effective against E. coli DNA polymerase I. The inhibition occurred competitively with each of dATP and dTTP, but non‐competitively with dCTP, dGTP and the template DNA. Thus, halenaquinol sulfate is demonstrated to be a potential inhibitor of DNA polymerases α and ε, and be a useful tool for analysing the DNTP binding sites of DNA polymerases.
Effect of single‐point mutations Phe41→ His and Phe143→ Glu on folding and catalytic properties of recombinant horseradish peroxidase expressed in E. coli
Authors: Gazaryan Irina G. , Doseeva Victoria V. , Galkin Andrey G. , Tishkov Vladimir I.
Published: July 2002

Wild‐type recombinant and Phe41→ His and Phe143→ Glu mutant forms of horseradish peroxidase have been expressed in E. coli and reactivated from inclusion bodies with a yield of about 25%. The purified homogeneous preparations have been studied in the reaction of ABTS oxidation. The effect of mutations on heme entrapment and kinetics of ABTS oxidation demonstrates the essential role of the replaced residues in providing the hydrophobic crevice for the non‐covalent heme binding.
Tyrosine phosphorylation of the MUC1 breast cancer membrane proteins Cytokine receptor‐like molecules
Authors: Zrihan-Licht Sheila , Baruch Amos , Elroy-Stein Orna , Keydar Iafa , Wreschner Daniel H.
Published: July 2002

Phosphorylation on tyrosine residues is a key step in signal transduction pathways mediated by membrane proteins. Although it is known that human breast cancer tissue expresses at least 2 MUC1 type 1 membrane proteins (a polymorphic high molecular weight MUC1 glycoprotein that contains a variable number of tandem 20 amino acid repeat units, and the MUC1/Y protein that is not polymorphic and is lacking this repeat array) their function in the development of human breast cancer has remained elusive. Here it is shown that these MUC1 proteins are extensively phosphorylated, that phosphorylation occurs primarily on tyrosine residues and that following phosphorylation the MUC1 proteins may potentially interact with SH2 domain‐containing proteins and thereby initiate a signal transduction cascade. As with cytokine receptors, the MUC1 proteins do not harbor intrinsic tyrosine kinase activity yet are tyrosine phosphorylated and the MUC1/Y protein participates in a cell surface heteromeric complex whose formation is mediated by two cytoplasmically located MUC1 cysteine residues. Furthermore, the MUC1/Y protein demonstrates sequence similarity with sequences present in cytokine receptors that are known to be involved in ligand binding. Our results demonstrate that the two MUC1 isoforms are both likely to function in signal transduction pathways and to be intimately linked to the oncogenetic process and suggest that the MUC1/Y protein may act in a similar fashion to cytokine receptors.
Characterization and determination of the redox properties of the 2[4Fe‐4S] ferredoxin from Methanosarcina barkeri strain MS
Authors: Daas Piet J.H. , Hagen Wilfred R. , Keltjens Jan T. , Vogels Godfried D.
Published: July 2002

Ferredoxin was purified from methanol‐grown Methanosarcina barkeri strain MS. It was isolated as a dimer with a subunit molecular weight of 6,200. The protein contained 7.4 mol iron and 7.2 mol acid‐labile sulfur per monomer. In the reduced state the ferredoxin exhibited an EPR spectrum characteristic of two spin‐coupled [4Fe‐4S]1+ clusters. The E
m of the [4Fe‐4S]2+;1+ couple was −322 mV ± 3 mV vs. NHE at 21°C and pH 7.0. The midpoint potential was temperature but not pH dependent. At the physiological temperature of 37°C the E
m was −340 mV.
Human complement component C8
Authors: Dewald Georg , Hemmer Susanne , Nöthen Markus M.
Published: July 2002

The β‐chain of human complement component C8 exhibits a structural genetic polymorphism: using isoelectric focusing two major allotypes can be identified (C8B B (‘basic’) and C8B A (‘acidic’)). In the present report we describe a sequence polymorphism of the C8B gene (codon 63: AGA → GGA) and demonstrate that the resulting amino acid substitution (Arg → Gly) consistently differentiates between the two common charge variants of the C8β chain; the C8B B allotype is characterized by an Arg and the C8B A allotype by a Gly residue in position 63 of the C8β polypeptide chain.
cDNA cloning, expression and primary structure of Par j I, a major allergen of Parietaria judaica pollen
Authors: Costa M.A. , Colombo P. , Izzo V. , Kennedy H. , Venturella S. , Cocchiara R. , Mistrello G. , Falagiani P. , Geraci D.
Published: July 2002

A 659 bp cDNA clone1 coding for an allergen of Pj pollen has been isolated from a lambda gt11 library, and its DNA sequence determined. The cDNA insert showed an open reading frame of 429 bp coding for an allergenic protein of 14,866 Da and a deduced amino acid sequence containing 143 residues. The expressed recombinant protein represented the major allergen Par j I since it reacted with 95% of the sera from Pj‐allergic patients (n = 22) and with two Par j I‐specific monoclonal antibodies. No similarity with other known DNA and protein sequences has been detected.
Maltokinase (ATP: maltose 1‐phosphotransferase) from Actinoplanes sp.: demonstration of enzyme activity and characterization of the reaction product
Authors: Drepper Anne , Peitzmann Reinhard , Pape Hermann
Published: July 2002

Cell free extract of the acarbose producer Actinoplanes sp. catalyzes ATP‐dependent phosphorylation of maltose. This was shown by two different assays. The product was purified and its structure determined to be α‐maltose‐1‐phosphate by chemical analysis and NMR spectroscopy.
Structure and function of PCD/DCoH, an enzyme with regulatory properties
Authors: Suck Dietrich , Ficner Ralf
Published: July 2002

The bifunctional protein PCD/DCoH is both an enzyme involved in the phenylalanine hydroxylation system and a transcription coactivator forming a 2:2 heterotetrameric complex with the nuclear transcription factor HNF1. The discovery of a bacterial homologue and the expression pattern during Xenopus embryogenesis suggest a regulatory function not only restricted to HNF1. The crystal structures of the tetrameric rat and the dimeric bacterial PCD/DCoH have led to the proposal of substrate and HNF1 binding sites. The saddle‐shaped β‐sheet surfaces of the DCoH dimers likely represent binding sites for as yet unknown macromolecular interaction partners. Possible mechanisms for DCoH‐induced transcriptional regulation are discussed in the light of the three‐dimensional structures.
The effect of chronic intestinal parasitic infection on maternal and perinatal outcome
Authors: Weigel M.M. , Calle A. , Armijos R.X. , Vega I.P. , Bayas B.V. , Montenegro C.E.
Published: July 2002

Objective: The main objective of this prospective cohort study was to investigate the prevalence, risk factors and maternal‐perinatal consequences of chronic asymptomatic intestinal parasitic infection during pregnancy. Methods: Prenatal patients (n = 91) attending a public clinic in Quito, Ecuador, were followed during the third trimester. Intestinal parasite, nutritional status, sociodemographic/sanitation indicators and fetal outcome data were collected and analyzed using multivariate ANOVA and regression techniques. Results: Most subjects (93%) were infected with at least one species of pathogenic intestinal parasite: 88% with Entamoeba histolytica. Greater parasite burdens were associated with poorer maternal iron status and reduced fetal growth. In particular, a high E. histolytica load was associated with decreased maternal serum hemoglobin (P = 0.002) and hematocrit levels (P = 0.01), iron deficiency anemia (P = 0.026), and indicators of diminished intrauterine growth including a decreased ponderal index (P = 0.04), mid‐arm circumference (P = 0.01), and mid‐arm/head circumference ratio (P = 0.003). Conclusion: Asymptomatic intestinal parasitic infection represents a hidden risk factor for maternal iron deficiency anemia and fetal growth retardation.
Identification of heterochromatin protein 1 (HP1) as a phosphorylation target by Pim‐1 kinase and the effect of phosphorylation on the transcriptional repression function of HP11
Authors: Koike Naoyuki , Maita Hiroshi , Taira Takahiro , Ariga Hiroyoshi , Iguchi-Ariga Sanae M.M.
Published: July 2002

Pim‐1, a protooncogene product, is a serine/threonine kinase and is thought to play a role in signal transduction in blood cells. Few phosphorylated target proteins for Pim‐1, however, have been identified. In the present study, two‐hybrid screening to clone cDNAs encoding proteins binding to Pim‐1 was carried out, and a cDNA for heterochromatin protein 1γ (HP1γ) was obtained. Binding assays both in yeast and in vitro pull‐down using the purified HP1γ and Pim‐1 expressed in Escherichia coli showed that Pim‐1 directly bound to the chromo shadow domain of HP1γ. HP1γ was also associated with Pim‐1 in human HeLa cells and the serine clusters located at the center of HP1γ were phosphorylated by Pim‐1 in vitro. Furthermore, a transcription repression activity of HP1γ was further stimulated by the deletion of the serine clusters targeted by Pim‐1. These results suggest that Pim‐1 affects the structure or silencing of chromatin by phosphorylating HP1.
Mammalian calcitonin receptor‐like receptor/receptor activity modifying protein complexes define calcitonin gene‐related peptide and adrenomedullin receptors in Drosophila Schneider 2 cells
Authors: Aldecoa Amaya , Gujer Remo , Fischer Jan A , Born Walter
Published: July 2002

Differential glycosylation of human and rat (r) calcitonin (CT) receptor‐like receptors (CRLR) as a result of interactions with accessory receptor activity‐modifying proteins (RAMP)1 or ‐2 was considered to define CT gene‐related peptide (CGRP) or adrenomedullin (ADM) receptors in mammalian cells. Here, Drosophila Schneider (S2) cells stably co‐expressed rCRLR and RAMP1 or ‐2 as functional CGRP or ADM receptors. Different from mammalian cells, rCRLR expressed in S2 cells are uniformly glycosylated proteins independent of RAMP1 or RAMP2. Bis(sulfosuccinimidyl)suberate cross‐linking revealed receptor components with the size of rCRLR, increased by the molecular weights of the corresponding RAMPs and [125I]CGRP or [125I]ADM. In conclusion, [125I]CGRP/rCRLR/RAMP1 and [125I]ADM/rCRLR/RAMP2 complexes have been recognized in Drosophila S2 cells.
Adenovirus encoded cyclic nucleotide‐gated channels: a new methodology for monitoring cAMP in living cells
Authors: Fagan Kent A. , Schaack Jerome , Zweifach Adam , Cooper Dermot M.F.
Published: July 2002

The current, static methodologies for measuring cyclic AMP (cAMP) may underestimate its regulatory properties. Here, we have exploited the Ca2+‐conducting properties of cyclic nucleotide‐gated (CNG) channels to measure cAMP in live cells, in response to various stimuli. We placed a mutated CNG channel with high sensitivity to cAMP in adenovirus to maximize and render facile its expression in numerous cell types. The ready, continuous nature of the readout contrasted with the traditional approach, which yielded similar static information, but lacked any continuous or interactive qualities. It seems fair to predict that this readily adopted approach will broaden the perception of cAMP signaling.
Short photoperiod reduces leptin gene expression in white and brown adipose tissue of Djungarian hamsters
Authors: Klingenspor Martin , Dickopp Alexandra , Heldmaier Gerhard , Klaus Susanne
Published: July 2002

Leptin gene expression in white (WAT) and brown adipose tissue (BAT) of the Djungarian hamster (Phodopus sungorus) was analyzed during seasonal acclimatization. Leptin gene expression in WAT was markedly reduced during winter, independent of changes in environmental temperature. Exposure to artificial short photoperiod also decreased leptin gene expression in WAT as well as in BAT. Although specific leptin gene expression was lower in BAT, total depot expression was as high as in WAT depots, due to higher RNA content of BAT. Our results indicate that there is significant leptin synthesis in brown fat and that leptin might be involved in photoperiod mediated seasonal adaptations of mammals independent of food deprivation or overfeeding.
Three different GB virus C/hepatitis G virus genotypes
Authors: Mukaide Motokazu , Mizokami Masashi , Orito Etsuro , Ohba Ken-ichi , Nakano Tatsunori , Ueda Ryuzo , Hikiji Kazumasa , Iino Shiro , Shapiro Sarah , Lahat Nitza , Park Young-Min , Kim Boo-Sung , Oyunsuren Tsendsuren , Rezieg Mohammed , Al-Ahdal Mohammed N. , Lau Johnson Y.N.
Published: July 2002

The 5′‐untranslated region (5′‐UTR) sequences of 33 GB virus C/hepatitis G virus (GBV‐C/HGV) obtained from different geographic areas were determined through reverse‐transcription polymerase chain reaction and dideoxy chain termination sequencing, the alignment of sequences, the estimation of the number of nucleotide substitution per site, and construction of phylogenetic trees. The 5′‐UTR of GBV‐HGV was found to be heterogeneous, with 70.9–99.5% homology. Three distinct phylogenetic branches were observed consistently in all phylogenetic trees. GBV‐C is the prototype for one, HGV for another, and there is a new branch which consisted of GBV‐C/HGV isolates from Asia. Genotype‐specific restriction sites for the restriction enzymes, ScrFI and BsmFI, were identified, and a simple restriction fragment polymorphism analysis was developed for genotyping. These data provide evidence that GBV‐C/HGV consists of three different genotypes. Our simple genotyping assay will also provide a tool for epidemiological studies of GBV‐C/HGV infection.
A natural motif approach to protein design: a synthetic leucine zipper peptide mimics the biological function of the platelet factor 4 protein
Authors: Butcher Daniel J , Kowalska M.Anna , Li Song , Luo Zhaowen , Shan Simei , Lu Zhixian , Niewiarowski Stefan , Huang Ziwei
Published: July 2002

The design of smaller functional mimics of large proteins has long been an important challenge. In this study we use the natural leucine zipper as a structural template to design a 31‐residue peptide analog that mimics the function of the larger platelet factor 4 (PF4) protein. The heparin binding activity of PF4 has been introduced into an unrelated leucine zipper sequence only by virtue of incorporating four lysines of PF4. Circular dichroism and binding experiments have shown that the designed leucine zipper peptide adopts a stable helical conformation and shows significant PF4‐like heparin binding activity. These results strongly suggest that the lysine residues play an important role in the binding of PF4 to heparin. The de novo generation of the PF4 function in a designed leucine zipper peptide demonstrates that the leucine zipper motif is a useful scaffold for the design of functional peptides and proteins.
The stimulatory effect of negative air ions and hydrogen peroxide on the activity of superoxide dismutase
Authors: Kosenko E.A , Kaminsky Yu.G , Stavrovskaya I.G , Sirota T.V , Kondrashova M.N
Published: July 2002

The activity of erythrocyte cytosolic superoxide dismutase from rat, bovine, man and duck was considerably increased when measured after preparation or incubation in media pretreated with negative air ions (mostly superoxide) from electroeffluvial ion generator. 0.5–1.0 μM H2O2 was found in incubation medium after treatment with air ions. The stimulatory effect of air ions on superoxide dismutase activity was mimicked by addition of 0.5–6 μM H2O2. The primary physicochemical mechanism of beneficial biological action of negative air ions is suggested to be related to the stimulation of superoxide dismutase activity by micromolar concentrations of H2O2.
Inhibition of the alternative oxidase stimulates H2O2 production in plant mitochondria
Authors: Popov V.N , Simonian R.A , Skulachev V.P , Starkov A.A
Published: July 2002

The hypothesis that a non‐coupled alternative oxidase of plant mitochondria operates as an antioxygen defence mechanism [Purvis, A.C. and Shewfelt, R.L., Physiol. Plant. 88 (1993) 712–718; Skulachev, V.P., Biochemistry (Moscow) 59 (1994) 1433–1434] has been confirmed in experiments on isolated soybean and pea cotyledon mitochondria. It is shown that inhibitors of the alternative oxidase, salicyl hydroxamate and propyl gallate strongly stimulate H2O2 production by these mitochondria oxidizing succinate. Effective concentrations of the inhibitors proved to be the same as those decreasing the cyanide‐resistant respiration. The inhibitors proved to be ineffective in stimulating H2O2 formation in rat liver mitochondria lacking the alternative oxidase.
Glycogenin, the primer of glycogen synthesis, binds to actin
Authors: Baqué Susanna , Guinovart Joan J , Ferrer Juan C
Published: July 2002

We have studied the intracellular localization of glycogenin by fusing green fluorescent protein (GFP) to the N‐terminus of rabbit muscle glycogenin and expressing the chimeric protein in C2C12, COS‐1 and rat hepatic cells. The fusion protein showed a nuclear and cytosolic distribution and partially co‐localized with actin in the cytosol. Disruption of the actin cytoskeleton with cytochalasin D led to a change in the pattern of green fluorescence, which coincided with that observed for the remaining non‐depolymerized actin. The distribution of the single point mutant K324A was completely uniform and was not affected by this drug. These findings indicate that rabbit muscle glycogenin binds to actin through the heptapeptide 321DNIKKKL327, a common motif found in other actin‐binding proteins, which is located at the C‐terminal end of this protein, and suggest that the actin cytoskeleton plays an important role in glycogen metabolism.
The yeast ATP binding cassette (ABC) protein genes PDR10 and PDR15 are novel targets for the Pdr1 and Pdr3 transcriptional regulators
Authors: Wolfger Hubert , Mahé Yannick , Parle-McDermott Anne , Delahodde Agnés , Kuchler Karl
Published: July 2002

The yeast transcription factors Pdr1 and Pdr3 control pleiotropic drug resistance (PDR) development, since they regulate expression of ATP‐binding cassette (ABC) drug efflux pumps through binding to cis‐acting sites known as PDREs (
R
esponsive
lements). In this report, we show by Northern blotting, gel shift mobility assays and DNase I footprinting that transcription of the ABC genes PDR10 and PDR15 is also controlled by Pdr1 and Pdr3. In addition, in vitro band shift assays demonstrate that a GST‐Pdr1 fusion protein can bind to the PDREs of PDR10 and PDR15. DNase I footprinting allowed the identification of the precise PDRE binding motifs, indicating the presence of a novel slightly degenerate PDRE motif in the PDR15 promoter. Finally, PDR10 and PDR15 mRNA levels vary dramatically in abundance in isogenic yeast strains carrying either Δpdr1, Δpdr3 and Δpdr1 Δpdr3 deletions or pdr1‐3 and pdr3‐2 gain‐of‐function mutations, demonstrating that both PDR10 and PDR15 are new members of the yeast PDR network.
Structure of a G48H mutant of HIV‐1 protease explains how glycine‐48 replacements produce mutants resistant to inhibitor drugs1
Authors: Hong Lin , Zhang Xue-Jun , Foundling Steve , Hartsuck Jean Ann , Tang Jordan
Published: July 2002

The crystal structure of human immunodeficiency virus type 1 (HIV‐1) protease mutant G48H with peptidic inhibitor U‐89360E is described. Comparison with wild‐type protease‐inhibitor complex shows that mutation of flap residue 48 to histidine allows stabilizing van der Waals contacts between the side chains of His48 and Phe53 as well as between His48 and the P2′ and P3′ inhibitor subsites. The flap region is less mobile than in the wild‐type enzyme. A model of saquinavir‐resistant mutant protease G48V in complex with saquinavir predicts interactions similar to those found in the G48H crystal. Energetic calculations confirm the similarity of the His48 and Val48 interactions.
The K‐ATP channel regulates the effect of Ca2+ on gap junction permeability in cultured astrocytes
Authors: Granda Begoña , Tabernero Arantxa , Sánchez-Abarca Luis Ignacio , Medina José M
Published: July 2002

Using the scrape‐loading technique we show that tolbutamide and glybenzcyclamide, two inhibitors of the K+ channel sensitive to ATP (K‐ATP channel), partially prevent the inhibition of gap junction permeability promoted by Ca2+ in cultured astrocytes. This effect was dose‐dependent, reaching a maximum at 400 μM and 1 μM of tolbutamide and glybenzcyclamide, respectively. The presence of the Ca2+ ionophore A‐23187 strongly reduced the concentration of Ca2+ required to block gap junction permeability but did not abolish the effect of tolbutamide and glybenzcyclamide. These results suggest that the effect of these two compounds are not brought about by control of the intracellular concentration of Ca2+ but probably by the promotion of plasma membrane depolarization.
Coupling of proton source and sink via H+‐migration along the membrane surface as revealed by double patch‐clamp experiments
Authors: Antonenko Yuri N , Pohl Peter
Published: July 2002

Long‐range proton transfer along the surface of black lipid bilayers was observed between two integral membrane channels (gramicidins), one operating as a proton source, the other as a sink, by patch‐clamp technique. In contrast, potassium ions were shown to equilibrate with the aqueous bulk phase before being consumed. Both channels opened and closed simultaneously only if the charge between them was carried by protons. In this case an anomalous high conductance between two patched membrane fragments was measured, each of them containing one single gramicidin channel. The coupled state disappeared when the distance between these two channels was increased above the critical value. The latter was shown to increase with the channel lifetime. Our results support the idea of the `localized' proton coupling, in which protons that have been pumped across membranes migrate along the membrane surface to reach another membrane protein that utilizes the established pH gradient.
β3‐Adrenergic relaxation of bovine iris sphincter
Authors: Geyer Orna , Bar-Ilan Ayelet , Nachman Rachel , Lazar Moshe , Oron Yoram
Published: July 2002

Bovine iris sphincter in vitro responded to β‐adrenergic stimulation with pronounced relaxation (EC50 of isoproterenol=0.3 nM), which was potentiated by the cAMP phosphodiesterase inhibitor, isobutylmethylxanthine, and mimicked by the adenylyl cyclase activator, forskolin. The β1/β2 antagonist, propranolol, exhibited low potency with calculated K
i of 200 nM. The β3‐selective antagonist, bupranolol, exhibited a biphasic inhibition profile, with calculated K
is of approximately 20–50 and 200–300 nM. The β3‐selective agonist, BRL 37344, elicited 70% of maximal relaxation (EC50=30 nM). When relaxation was induced by BRL 37344, bupranolol exhibited much higher potency (calculated K
i=1 nM). Our data suggest that the β‐adrenergic relaxation response in bovine iris sphincter is mediated by a mixed population of β‐adrenergic receptors, with a predominant contribution of atypical, most likely β3 subtype, receptors.
Rapid hyaluronan uptake is associated with enhanced motility: implications for an intracellular mode of action
Authors: Collis L , Hall C , Lange L , Ziebell M , Prestwich R , Turley E.A
Published: July 2002

Texas red‐labeled hyaluronan (TR‐HA) is rapidly taken up in a CD44 independent manner into ras‐transformed 10T1/2 fibroblasts, where it accumulates in both cell ruffles/lamellae, the perinuclear area, and the nucleus. HA does not accumulate in the cell ruffles/lamellae of parental 10T1/2 cells. Addition of HA to ras‐transformed cells promotes their random motility but has no effect on 10T1/2 cell motility. 10T1/2 cells can be modified to take up HA into cell ruffles by exposure to phorbol ester or direct microinjection of HA into cells. Both treatments significantly stimulate 10T1/2 cell motility.
Expression of a tyrosine phosphorylated, DNA binding Stat3β dimer in bacteria
Authors: Becker Stefan , Corthals Garry L. , Aebersold Ruedi , Groner Bernd , Müller Christoph W.
Published: July 2002

The signal transducer and activator of transcription (STAT) proteins deliver signals from the cell membrane to the nucleus. An N‐terminally truncated fragment of murine Stat3β, Stat3βtc (127–722), was produced in bacteria. STAT proteins must be specifically phosphorylated at a single tyrosine residue for dimerization and DNA binding. Therefore, Stat3βtc was coexpressed with the catalytic domain of the Elk receptor tyrosine kinase. Stat3βtc was quantitatively phosphorylated by this kinase domain. Gel filtration chromatography revealed a Stat3βtc dimer. Y705 was identified as the major phosphorylated residue of Stat3βtc. This corresponds to the tyrosine residue which is phosphorylated by the Janus kinase in vivo. The phosphorylated Stat3βtc specifically bound to DNA binding sites. The described protocol allows the production of large amounts of activated protein for biochemical and pharmaceutical studies.
The N54 mutant of Gαs has a conditional dominant negative phenotype which suppresses hormone‐stimulated but not basal cAMP levels
Authors: Cleator John H , Mehta Nitin D , Kurtz David T , Hildebrandt John D
Published: July 2002

The phenotype of a Ser to Asn mutation at position 54 of the α subunit of Gs (N54‐αs) was characterized in transient transfection experiments in COS and HEK293 cells. Expression of either wild type or N54‐αs increased basal cAMP levels. In contrast, expression of wild type αs potentiated agonist‐stimulated cAMP levels, while expression of N54‐αs caused a decrease. Thus, the N54‐αs mutant possesses a conditional dominant negative phenotype, suppressing preferentially hormone‐stimulated effects.