bluuebunny/crossref_metadata_2025 huggingface dataset:
Cysteine proteases of positive strand RNA viruses and chymotrypsin‐like serine proteases
Authors: Gorbalenya Alexander E. , Donchenko Alexei P. , Blinov Vladimir M. , Koonin Eugene V.
Published: July 2002

Evidence is presented, based on sequence comparison and secondary structure prediction, of structural and evolutionary relationship between chymotrypsin‐like serine proteases, cysteine proteases of positive strand RNA viruses (3C proteases of picornaviruses and related enzymes of como‐, nepo‐ and potyviruses) and putative serine protease of a sobemovirus. These observations lead to re‐identification of principal catalytic residues of viral proteases. Instead of the pair of Cys and His, both located in the C‐terminal part of 3C proteases, a triad of conserved His, Asp(Glu) and Cys(Ser) has been identified, the first two residues resident in the N‐terminal, and Cys in the C‐terminal β‐barrel domain. These residues are suggested to form a charge‐transfer system similar to that formed by the catalytic triad of chymotrypsin‐like proteases. Based on the structural analogy with chymotrypsin‐like proteases, the His residue previously implicated in catalysis, together with two partially conserved Gly residues, is predicted to constitute part of the substrate‐binding pocket of 3C proteases. A partially conserved ThrLys/Arg dipeptide located in the loop preceding the catalytic Cys is suggested to confer the primary cleavage specificity of 3C toward Glx/Gly(Ser) sites. These observations provide the first example of relatedness between proteases belonging, by definition, to different classes.
Interleukin‐6 is the major regulator of acute phase protein synthesis in adult human hepatocytes
Authors: Castell José V. , Gómez-Lechón Maria J. , David Martina , Andus Tilo , Geiger Thomas , Trullenque Ramón , Fabra Ricardo , Heinrich Peter C.
Published: July 2002

The three monokines interleukin‐1β (IL‐1β), tumor necrosis factor α (TNFα), and interleukin‐6 (IL‐6) modulate acute phase plasma protein synthesis in adult human hepatocytes. Only IL‐6 stimulates the synthesis of the full spectrum of acute phase proteins as seen in inflammatory states in humans, i.e. synthesis and secretion of C‐reactive protein, serum amyloid A, fibrinogen, α1‐antitrypsin, α1‐antichymotrypsin and haptoglobin are increased while albumin, transferrin and fibronectin are decreased. IL‐1β as well as TNFα, although having a moderate effect on the positive acute phase proteins and inhibiting the synthesis of fibrinogen, albumin and transferrin, fail to induce serum amyloid A and C‐reactive protein. These data suggest that IL‐6 plays the key role in the regulation of acute phase protein synthesis in human hepatocytes.
CD3/T‐cell receptor coupling to a pertussis and cholera toxin‐insensitive G‐protein
Authors: Kvanta Anders , Nordstedt Christer , van der Ploeg Ingeborg , Jondal Mikael , Fredholm Bertil B.
Published: July 2002

We have analyzed the effect of CD3/T‐cell receptor stimulation on GTP hydrolysis and GTP binding. We show that stimulation of Jurkat, T‐cell, membranes withT3 results in a 50% increase in GTP hydrolysis which is specifically inhibited by GDP. Pretreatment of the membranes with neither pertussis toxin nor cholera toxin inhibited the GTP hydrolysis. We also show that stimulation withT3 increases the binding of GTPγS to Jurkat membranes. These data strongly implicate the involvement of a G‐protein in CD3/T‐cell receptor signalling.
Specific binding ofadaic acid, a new tumor promoter in mouse skin
Authors: Suganuma Masami , Suttajit Maitree , Suguri Hiroko , Ojika Makoto , Yamada Kiyoyuki , Fujiki Hirota
Published: July 2002

The tumor promoteradaic acid binds specifically to a particulate as well as a cytosolic fraction of various mouse tissues, e.g., skin, brain, lung and colon. The K
D value was 21.7 nM for receptors in the particulate fraction and 1.0 nM for those in the cytosolic fraction of mouse skin. The specific binding of [3H]okadaic acid to the particulate fraction of mouse skin was inhibited dose‐dependently byadaic acid, but notaidaic acid tetramethyl ether, an inactive compound, or by other tumor promoters, such as 12‐O‐tetradecanoylphorbol‐13‐acetate and teleocidin. The results suggest a new pathway of tumor promotion mediated through theadaic acid receptor(s).
Analysis of free sulfhydryl groups and disulfide bonds in Na+,K+‐ATPase
Authors: Gevondyan N.M. , Gevondyan V.S. , Gavrilyeva E.E. , Modyanov N.N.
Published: July 2002

The content of free SH groups and disulfide bonds in the purified pig kidney Na+,K+‐ATPase was determined by ammetric titration with silver nitrate. In the native enzyme, most of the free SH groups are masked due to their location in the polypeptide chain regions poorly accessible to SH reagents. Denaturation with 5% SDS and 8 M urea makes these regions accessible thus revealing 22 free SH groups/mol of the protein. After complete blocking of free SH groups with silver ions, 8 SH groups/mol of the protein are being released upon sulfitolysis which indicates the presence of four disulfide bonds in the enzyme. At least one disulfide bridge is located in the α‐subunit whereas the β‐subunit contains three disulfide bonds.
Sequence analysis of the cloned streptococcal cell surface antigen I/II
Authors: Kelly C. , Evans P. , Bergmeier L. , Lee S.F. , Progulske-Fox A. , Hams A.C. , Aitken A. , Bleiweis A.S. , Lehner T.
Published: July 2002

The gene spa P (formerly designated as spa P1) encoding the M
r 185,000 surface antigen (I/II) of Streptococcus mutons, serotype c (NG5), has been sequenced. The gene (4683 bp) encodes a protein of 1561 amino acid residues including putative signal peptide (residues 1–38) and transmembrane (residues 1537–1556) sequences. The N‐terminal region (60–550) has alanine‐rich repeats and is predicted to be α‐helical. However, the C‐ternunal region (800–1540) is proline‐rich and favours an extended structure. Except for a short central variable region the sequences appear to be highly conserved for S. mutans serotype c. N‐Tenninal sequencing of separated antigen I and antigen II polypeptides suggests that the former represents the N‐terminal and the latter the C‐terminal portions of the intact antigen.
Sheep neuropeptide Y
Authors: Sillard Rannar , Agerberth Birgitta , Mutt Viktor , Jörnvall Hans
Published: July 2002

Mammalian forms of neuropeptide Y (NPY) for which the amino acid sequences have previously been determined, are the human, pig, ox, rabbit, rat, and guinea‐pig polypeptides. The only difference among these forms is at position 17, where pig and ox NPY have Leu and the others Met. We now show that sheep NPY differs from all the earlier characterized mammalian forms of NPY by having Asp instead of Glu at position 10. At position 17 it has Leu as do both pig and ox NPY. Consequently, 3 different structural types of mammalian NPY are now known.
Cyanobacterial microcystin‐LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants
Authors: MacKintosh Carol , Beattie Kenneth A. , Klumpp Susanne , Cohen Philip , Codd Geoffrey A.
Published: July 2002

The cyclic heptapeptide, microcystin‐LR, inhibits protein phosphatases 1 (PP1) and 2A (PP2A) with K
i, values below 0.1 nM. Protein phosphatase 2B is inhibited 1000‐fold less potently, while six other phosphatases and eight protein kinases tested are unaffected. These results are strikingly similar to those obtained with the tumour promoter okadaic acid. We establish that okadaic acid prevents the binding of microcystin‐LR to PP2A, and that protein inhibitors 1 and 2 prevent the binding of microcystin‐LR to PP1. We discuss the possibility that inhibition of PP1 and PP2A accounts for the extreme toxicity of microcystin‐LR, and indicate its potential value in the detection and analysis of protein kinases and phosphatases.
Role of butyrylcholinesterase in canine tracheal smooth muscle function
Authors: Adler Michael , Filbert Margaret G.
Published: July 2002

The role of butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) in regulating acetylcholine (ACh) lifetime was investigated by use of selective cholinesterase (ChE) inhibitors. Addition of 1 μM tetraisopropylpyrophosphoramide (iso‐OMPA) led to a 98% inhibition of BuChE activity with little or no effect on AChE activity. This inhibition wa accompanied by a 26% increase in the amplitude and a 43% prolongation in the half‐relaxation time of contractions elicited by electric field stimulation (EFS). Coapplication of BW 284C51 (a selective AChE inhibitor) and 1 μM iso‐OMPA resulted in increases of 2‐fold in the amplitude and 10‐fold in the half‐relaxation time of EFS‐induced contractions. These alterations were accompanied by small but sustained baseline contractures that were antagonized completely by incubation with exogenous BuChE (2.5 ). The results suggest that BuChE serves to coregulate the lifetime of ACh in canine tracheal smooth muscle.
Uni‐site ATP synthesis in thylakoids
Authors: Labahn Andreas , Fromme Petra , Gräber Peter
Published: July 2002

Uni‐site ATP synthesis was measured with thylakoids. The membrane‐bound ATP‐synthase, CF0F1 was brought into the active, reduced state by illumination in the presence of thioredoxin, dithiothreitol and phosphate. This enzyme contains two tightly bound ATP per CFoF1. ATP was released from the enzyme when ADP was added in substoichiometric amounts during illumination. Experiments with [14C]ADP indicated that after binding the same nucleotide was phosphorylated and released as [14C]ATP, i.e. only one site is involved in ATP‐synthesis (uni‐site ATP‐synthesis'). The two tightly bound ATP are not involved in the catalytic turnover. The rate constant for ADP binding was (4 ± 2) × 106 M−1s−1. Compared to deenergized conditions the rate constant for ADP binding and that for ATP‐release were drastically increased, i.e. membrane energization increased the rate constants for the ATP‐synthesis direction.
Reduction of nerve growth factor level in the brain of genetically ataxic mice (weaver, reeler)
Authors: Matsui Kyoko , Furukawa Shoei , Shibasaki Hiroshi , Kikuchi Tateki
Published: July 2002

By a highly sensitive enzyme immunoassay we measured the level of nerve growth factor (NGF) in the cerebellum and cerebrum of the neurologically mutant mice, weaver, reeler and Purkinje cell degeneration (PCD). A significant decrease in NGF level was observed in both cerebellum and cerebrum of weaver and reeler mutants of either sex. However, there was no such difference between normals and mutants in the case of the PCD mice. These results show that weaver and reeler mice have abnormalities of NGF synthesis and/or degradation not only in the cerebellum but also in the cerebrum.
Modified reaction centers from Rhodobacter sphaeroides R26
Authors: Struck A. , Scheer H.
Published: July 2002

Incubation of photosynthetic reaction centers from Rhodobacter sphaeroides R26 with exogenous 132‐OH‐bacteriochlorophyll ap or aGG according to Scheer et al. (1987) results in the exchange of endogenous bacteriochlorophyll ap. The exchange amounts to ⩽ 50% according to HPLC analysis, corresponding to a complete replacement of the ‘monomeric’ bacteriochlorophylls, bm and bl, by exogenous pigment. The absorption spectra show small, but distinct changes in the Qx‐region of the bacteriochlorophylls, and bleaching of the modified reaction centers is retained. The corresponding binding sites must be accessible from the exterior, and allow for the introduction of a polar residue at C‐132. This is supported by the observation of side reactions of the endogenous ‘monomeric’ bacteriochlorophylls within the reaction center pigments, e.g. epimerization and hydroxylation at C‐132.
Enzymatic synthesis of dihydrosirohydrochlorin (precorrin‐2) and of a novel pyrrocorphin by uroporphyrinogen III methylase
Authors: Warren Martin J. , Stolowich Neal J. , Santander Patricio J. , Roessner Charles A. , Sowa Blair A. , Scott A.Ian
Published: July 2002

Uroporphyrinogen III methylase was purified from a recombinant hemB− strain of E. coli harbouring a plasmid containing the cysG gene. N‐terminal analysis of this purified protein gave an amino acid sequence corresponding to that predicted from the genetic code. From the u.v./visible spectrum of the reaction catalysed by this SAM dependent methylase it was possible to observe the sequential appearance of the chromophores of a dipyrrocorphin and subsequently of a pyrrocorphin. Confirmation of this transformation was obtained from 13C‐NMR studies when it was demonstrated, for the first time directly, that uroporphyrinogen is initially converted into dihydrosirohydrochlorin (precorrin‐2) and then, by further methylation, into a novel trimethylpyrrocorphin.
(+)‐Tubocurarine is a potent inhibitor of cation channels in the vacuolar membrane of Chenopodium rubrum L
Authors: Weiser T. , Bentrup F.-W.
Published: July 2002

The effect of the acetylcholine antagonist and channel blocker (+)‐tubocurarine on the calcium‐dependent slow vacuolar (SV) cation channels in the tonoplast of suspension‐cultured cells of Chenopodium rubrum L. was examined using the patch‐clamp technique. In whole‐vacuolar recordings the drug strongly suppressed the potassium conductance (EC50: 6 μM) and altered the kinetics of channel inactivation. In excised membrane patches (+)‐tuboeurarine evokes channel‐‘flickering’ without affecting the single‐channel conductance (approx. 80 pS).
Intracellular aluminium inhibits acetylcholine‐ and caffeine‐evoked Ca2+ mobilization
Authors: Wakui M. , Itaya K. , Birchall D. , Petersen O.H.
Published: July 2002

The effect of in tracellular aluminium on Ca2+ signalling in single internally perfused mouse pancreatic acinar cells was investigated by measurement of the Ca2+‐dependent Cl− current using the patch‐clamp whole‐cell recording configuration. Acetylcholine (ACh) normally evoked a pulsatile Ca2+‐dependent Cl− current, but when A1C13 (1 mM) was present in the internal perfusion solution the ACh responses were virtually absent. When aluminium was acutely infused into the internal perfusion solution, the ACh‐evoked Ca2+ signals and also the caffeine‐evoked responses quickly disappeared, but the Ca2+ ionophore, ionomycin (100 nM), could still induce a large increase in the Cl− current. It is concluded that intracellular aluminium can abolish receptor‐activated intracellular Ca2+ release probably by inhibition of Ca2+‐induced Ca2+ release
Multifrequency EPR evidence for a bimetallic center at the CuA site in cytochrome c oxidase
Authors: Kroneck Peter M.H. , Antholine William E. , Kastrau Dieter H.W. , Buse Gerhard , Steffens Guy C.M. , Zumft Walter G.
Published: July 2002

Multifrequency electron paramagnetic resonance (EPR) spectra of the Cu(II) site in bovine heart cytochrome c oxidase (COX) and nitrous oxide reductase (N2OR) from Pseudomonas stutzeri confirm the existence of Cu‐Cu interaction in both enzymes. C‐band (4.5 GHz) proves to be a particularly good frequency complementing the spectra of COX and N2OR recorded at 2.4 and 3.5 GHz. Both the high and low field region of the EPR spectra show the presence of a well‐resolved 7‐line pattern consistent with the idea of a binuclear Cu center in COX and N2OR. Based on this assumption consistent g‐values are calculated for gz and gx at four frequencies. No consistent g‐values are obtained with the assumption of a 4‐line pattern indicative for a mononuclear Cu site.
K+ channel expression in primary cell cultures mediated by vaccinia virus
Authors: Karschin Andreas , Alyar Jayashree , Gouin Annie , Davidson Norman , Lester Henry A.
Published: July 2002

A recombinant vaccinia virus (VV) was used to express functional Drosophila Shaker H4 K+ channels in primary cell cultures from rat heart (atrial and ventricular myocytes, fibroblasts), autonomic ganglia (SCG neurons) and CNS (hippocampal neurons, cerebral astroglia). In most cells the expressed currents possessed the typical characteristics of the native Drosophila muscle A currents; a few cells showed evidence of hetero‐oligomers with new properties. The maximum current density corresponded to a channel density of 2–3/μm3. Voltage recordings in heart cells showed altered action potential waveforms after successful infection. VV vectors thus are useful for studying altered excitability and cell‐specific processing of ion channel proteins.
Rapid purification of protein phosphatase 2A from mouse brain by microcystin‐affinity chromatography
Authors: Nishiwaki Shinji , Fujiki Hirota , Suganuma Masami , Nishiwaki-Matsushima Ric , Sugimura Takashi
Published: July 2002

Microcystin LR. which is a monocyclic heptapeptide containing two L‐amino acids, and leucine and arginine, is a new inhibitor of protein phosphatases 1 and 2A. Microcystin LR‐affinity chromatography was used to purify protein phosphatase 2A as a holoenzyme. Five mg of microcystin LR were immobilized to ECH Sepharose 4B by the carbodiimide coupling reaction. Following DEAE‐cellulose column chromatography, microcystin‐affinity chromatography, as the second step in the procedure, resulted in purification of protein phosphatase 2A in a pure form. The enzyme isolated from mouse brain consisted of two regulatory subunits of 67 kDa and 58 kDa and a catalytic subunit of 41 kDa. Microcystin‐affinity chromatography is useful for isolation of protein phosphatase 2A.
Influence of the H‐subunit and Fe2+ on electron transport from I− to QA in Fe2+‐free and/or H‐free reaction centers from Rhodobacter sphaeroides R‐26
Authors: Liu Ben-li , van Kan Paul J.M. , Hoff Arnold J.
Published: July 2002

From reaction centres (RC) of Rhodobacter sphaeroides R‐26 two LM preparations with 0.90 Fe2+/RC (LM) and 0.10 Fe2+/RC (LM/dFe) were prepared. Reconstitution of LM/dFe with the H‐subunit and subsequently with Zn2+ yielded LMH/dFe and LMH/dFe+Zn preparations, respectively. In these four samples the decay of the primary radical pair P+I− was studied by means of transient absorption spectroscopy and compared with that in native RC. In LMH/dFe the reduction of QA by Bpheo a occurred in 5 ns, with concomitant increase in the yield of PT, the triplet state of the primary donor. In the LM/dFe, LM and LMH/dFe+Zn preparations the decay of I− had the same rate (200 ps)−1 as in native RC. Thus, neither the H‐subunit in the RC nor a divalent metal as Fe2+ or Zn2+ are necessary per se for fast reduction of QA. Only demetallation in the presence of the H‐subunit slows down the reduction of QA.
The gene organisation of the human β2 integrin subunit (CD18)
Authors: Weitzman Jonathan B. , Wells Charmian E. , Wright Ann H. , Clark Paula A. , Law S.K.Alex
Published: July 2002

We have studied the gene of the human β2 integrin subunit (CD18) and found it to be organised into 16 exons spanning a region of about 40 kb. All exon/intron boundaries conform to the GT/AG splicing consensus. The exons coding for the cysteine‐rich region, which has been postulated to consist of 3 or 4 repeating elements, are not organised correspondingly. Transcription of the gene initiates from multiple sites which may be due to the absence of an upstream TATA box. The polyadenylation site is also heterogeneous. Five different sites were identified over a stretch of 10 bases.
Isolation and characterization of a bradykinin‐potentiating peptide from a bovine peptic hemoglobin hydrolysate
Authors: Piot Jean-Marie , Zhao Qiuyu , Guillochon Didier , Ricart Guy , Thomas Daniel
Published: July 2002

A bradykinin potentiating peptide was isolated from a peptic bovine hemoglobin hydrolysate, by the use of reversed‐phase high‐performance liquid chromatography (RP‐HPLC). Its primary structure, determined by fast atom bombardment (FAB) and tandem mass spectrometry (MS/MS), was identical to fragment 129–134 of the α‐chain of bovine hemoglobin. The bradykinin potency of this peptide, as exhibited by the guinea‐pis ileum contraction, was significant and comparable with some others previously described.
Up‐regulation of interleukin (IL)‐6 receptor gene expression in vitro and in vivo in IL‐6 deprived myeloma cells
Authors: Portier Marielle , Lees Delphine , Caron Emmanuelle , Jourdan Michel , Boiron Jean-Michel , Bataille Regis , Klein Bernard
Published: July 2002

Mycloma cells absolutely require interleukin‐6 (IL‐6) for growing in vivo in patients with multiple mycloma and exogenous IL‐6‐dependentmycloma cell lines have been reproducibly obtained. In this study we show a dramatic up‐regulation of the IL‐6 receptor (gp80 chain) gene expression in mycloma cell lines following the removal of exogenous IL‐6. Such a regulation was also known to occur in IL‐6‐deprived mycloma cells in vivo in three patients who were treated with optimal doses of anti‐IL‐6 monoclonal antibodies. The direct effect of IL‐6 on IL‐6 receptor gene expression in mycloma cells was further confirmed by adding IL‐6 to an autonomously growing mycloma cell line.
Murine ST2 gene is a member of the primary response gene family induced by growth factors
Authors: Yanagisawa Ken , Tsukamoto Toshihiko , Takagi Toshimitsu , Tominaga Shin-ichi
Published: July 2002

The murine ST2 gene, which encodes a protein remarkably similar to the extracellular portion of murine interleukin 1 receptor types 1 and 2, is expressed in growth‐stimulated BALB/c‐3T3 cells in the presence of 50 μg/ml of cycloheximide. The treatment with 1,000 U/ml of purified native murine β‐interferon superinduced, rather than suppressed, the ST2 mRNA expression as in the cases or c‐myc and JE mRNAs. These results suggested that the murine ST2 gene belongs to the family of primary response genes induced by growth factors. Furthermore, a longer ST2‐related mRNA was found in BALB/c‐3T3 cells that were stimulated to proliferate in the presence of cycloheximide.
Intracellular delivery of protein kinase C‐α or ε isoform‐specific antibodies promotes acquisition of a morphologically differentiated phenotype in neuroblastoma cells
Authors: Leli Ubaldo , Parker Peter J. , Shea Thomas B.
Published: July 2002

The protein kinase C (PKC) family participates in a ubiquitous cell signalling system utilizing increased turnover of phosphoinositides. Because down‐regulation of total PKC activity has been implicated in the acquisition of a morphologically differentiated phenotype in SH‐SY5Y neuroblastoma cells, we aimed to identify the specific PKC isoforms in this process. Here we report that intracellular delivery of PKC‐α and ‐ε, but not ‐β|4‐γ or ‐δ isoform‐specific antibodies is sufficient to induce acquisition of a morphologically differentiated phenotype in SH‐SY5Y neuroblastoma cells.
The presence of free D‐serine in rat brain
Authors: Hashimoto Atsushi , Nishikawa Toru , Hayashi Tohiski , Fujii Noriko , Harada Kaoru , Oka Takae , Takahashi Kiyohisa
Published: July 2002

Free amino acid enantiomers in adult rat brain extracts were analysed as their N,O‐pentafluoropropionyl isopropyl derivatives by gas chromatography on a capillary column of Chirasil‐L‐Val. A peak X, which exhibited the same retention time as the N,O‐pentafluoropropionyl isopropyl derivative of authentic D‐serine, was detected in the brain extracts. Electron impact and positive chemical ionization mass spectra of the peak X of the brain extracts were identical to those of authentic D‐serine. The concentration of free D‐serine and the ratio of D‐serine/total serine in the brain were estimated to be 0.27 and 0.23, υmol/g of wet weight, respectively. These data provide the first evidence that substantial quantities of free D‐serine are present in mammalian brain tissues.
Retinoic acid‐induced changes in differentiation‐defective embryonal carcinoma RAC65 cells
Authors: Malý Petr , Dráber Petr
Published: July 2002

RAC65 is a mutant clone of mouse embryonal carcinoma cells, P19, which does not undergo terminal differentiation upon treatment with retinoic acid (RA), RAC65 cells express a truncated RA receptor α (RARα) which, however, does not fully explain their defect. Here we show that RAC65 cells exhibit an additional defect in RARα mRNA which may reflect a defect in RNA splicing. The parental and mutant cells also differ in their capacities to bind [3H]RA into nuclear fractions and in expression of cellular RA binding protein (CRABP) mRNA after treatment with RA. The combined data suggest that the deffect in RAC65 RARα results in reduced expression of the CRABP gene after RA treatment and, therefore, increased flow of RA into the nucleus.
Transport protons do not participate in ATP synthesis/hydrolysis at the nucleotide binding site of the H+‐ATPase from chloroplasts
Authors: Labahn Andreas , Gräber Peter
Published: July 2002

The H+‐ATPase from chloroplasts, CF0P1, was brought into the active, reduced state by illumination of thylakoids in the presence of thioredoxin and dithiothreitol. Uni‐site ATP synthesis was initiated by the addition of 20 nM [α‐32P]ADP, and enzyme‐bound and free nucleotides were separated by a pressure column. The ratio of enzyme‐bound ADP to ATP was 0.55 ± 0.05. In a second experiment, uni‐site ATP hydrolysis under energized conditions was initiated by the addition of 36 nM [α‐32P]ATP; enzyme‐bound and free nucleotides were separated by a pressure column. Both procedures were carried out under continuous illumination. The ratio of enzyme‐bound ADP to ATP was 0.46 ± 0.04. In a third experiment, uni‐site ATP hydrolysis under de‐energized conditions was initiated by the addition of 39 nM [α‐32P]ATP and NH4Cl/valinomycin in the absence of illumination. Free and enzyme‐bound nucleotides were separated also by a pressure column. The ratio of enzyme‐bound ADP to ATP was 0.43 ± 0.02. This ratio was always the same irrespective of whether the reaction runs in the synthesis or the hydrolysis direction. Furthermore, the ratio does not depend on the membrane energization. We conclude, therefore, that the protons are not directly involved in the reaction at the catalytic site.
Diethyldithiocarbamate inhibits induction of macrophage NO synthase
Authors: Mülsch Alexander , Schray-Utz Beate , Mordvintcev Peter I. , Hauschildt Sunna , Busse Rudi
Published: July 2002

We investigated whether sodium diethyldithiocarbamate (DETC), an inhibitor of the nuclear transcription factor kappa B (NFkappaB), modulates induction of NO synthase (NOS) in murine bone marrow‐derived macrophages. A short exposure (between 1 and 16 h) of L929‐cell medium‐preconditioned macrophages to E. coli lipopolysaccharide (LPS) significantly increased the level of NOS mRNA, and elicited NO formation as detected by electron spin resonance spectroscopy and by the release of nitrite. DETC (0.1–1 mM) present during stimulation with LPS prevented the increase in NOS mRNA and the expression of NOS activity. These findings suggest that NFkappaB is involved in the signal transduction pathway linking stimulation of macrophages by LPS with transcription of, the gene encoding inducible NOS.
Staurosporine‐related compounds, K252a and UCN‐01, inhibit both cPKC and nPKC
Authors: Mizuno Keiko , Saido Takaomi C. , Ohno Shigeo , Tamaoki Tatsuya , Suzuki Koichi
Published: July 2002

The potent inhibitors of protein kinase C (PKC), H7, staurosporine, and staurosporine derivatives, were examined for their inhibitory effects on novel PKC (nPKC) isozymes δ and ε. H7 and staurosporine, usually used as selective inhibitors of PKC, showed similar inhibitory effects on cPKC (a mixture of cPKCα,β, and γ) and nPKCδ and ε. The inhibitory effects of K252a, a non‐selective protein kinase inhibitor, on cPKC was 3.2‐ and 22‐fold higher than those on nPKCε and δ, respectively. The staurosporine derivatives UCN‐01 and UCN‐01‐Me also showed selective inhibition of cPKC.
Evidence for calmodulin binding to the cytoplasmic domains of two C‐CAM isoforms
Authors: Edlund Magnus , Öbrink Björn
Published: July 2002

C‐CAM (cell‐CAM 105) is a transmembrane cell adhesion molecule, belonging to the immunoglobulin superfamily. It is expressed in epithelia, vessel endothelia and leukocytes, and mediates intercellular adhesion in rat hepatocytes by homophilic binding. Two major isoforms (C‐CAM1 and C‐CAM2) that differ in their cytoplasmic domains occur. A previous study demonstrated that C‐CAM can bind calmodulin in a Ca2+‐dependent manner. In this study we have expressed the cytoplasmic domains of C‐CAM1 and C‐CAM2 in fusion proteins and measured calmodulin binding by a gel overlay assay, using 125I‐labelled calmodulin. Our results indicate that the cytoplasmic domains of both C‐CAM1 and C‐CAM2 can bind calmodulin.
An improved retroviral vector for assaying promoter activity
Authors: Nakajima Kazunori , Ikenaka Kazuhiro , Nakahira Kensuke , Morita Noriyuki , Mikoshiba Katsuhiko
Published: July 2002

We recently developed a novel promoter assay system using a retroviral vector (pIP200 series). Transcription from the internal promoter, which had been inserted for the promoter assay, was shown to be interfered with by transcription from the upstream long terminal repeat (LTR). Here we report a new high‐titer ‘self‐inactivating’ vector, in which transcription interference was virtually eliminated. This new vector was constructed by introducing only a very minor mutation into the ‘TATA box’ in the 3'‐LTR. This mutation was successfully transferred to the 5'‐LTR after reverse transcription, yielding a provirus incapable of transcribing viral RNA. The viral titer was not reduced by the mutation, permitting general application of this virus.
A hybrid protein kinase‐RNase in an interferon‐induced pathway?
Authors: Bork Peer , Sander Chris
Published: July 2002

The sequence of RNase L has been re‐examined by computer analysis. We propose a molecular architecture of RNase L, with an unusual combination, in one protein chain, of 9 ankyrin‐like repeats, a functional active protein kinase and a C‐terminal catalytic RNase similar to the yeast protein, IRE1. The protein kinase may be involved in a new signal transduction pathway which remains to be discovered.
Identification of two distinct synucleins from human brain
Authors: Jakes Ross , Spillantini Maria Grazia , Goedert Michel
Published: July 2002

Two abundant proteins of 140 and 134 amino acids were purified and sequenced from human brain. They were identified through their reactivity on immunoblots with a partially characterised monoclonal antibody that recognises tau protein in a phosphorylation‐dependent manner. The 140 amino acid protein is identical with the precursor of the non‐Aβ component of Alzheimer's disease amyloid which in turn is highly homologous to synuclein from Torpedo electroplaques and rat brain. The 134 amino acid protein is the human homologue of bovine phosphoneuroprotein 14; it is 61% identical in sequence to the 140 amino acid protein. The previously unrecognised homology between these two proteins defines a family of human brain synucleins. We refer to the 140 and 134 amino acid proteins as α‐synuclein and β‐synuclein, respectively. Both synucleins are expressed predominantly in brain, where they are concentrated in presynaptic nerve terminals.
The calpain—calpastatin system is regulated differently during human neuroblastoma cell differentiation to Schwannian and neuronal cells
Authors: Saito Yumiko , Saido Takaomi C. , Sano Kimihiko , Kawashima Seiichi
Published: July 2002

Changes in expression of calpains and calpastatin during differentiation in GOTO cells were examined using antibodies specific to calpains and calpastatin. Neuronal differentiation induced by dibutyryl cyclic AMP elicited a remarkable decrease in m‐calpain. No marked changes in the levels of calpains were found in bromodeoxyuridine‐induced Schwannian differentiation. Calpastatin was detected as a single band of 110k in undifferentiated and in neuronally differentiated cells by Western blot analysis. However, the appearance of a 120k species was detected in Schwannian differentiation associated with morphological change. The data show that marked changes in m‐calpain and calpastatin occur in a differentiation‐specific manner.
The thrombospondin‐like chains of cartilage oligomeric matrix protein are assembled by a five‐stranded α‐helical bundle between residues 20 and 83
Authors: Efimov Vladimir P. , Lustig Ariel , Engel Jürgen
Published: July 2002

The N‐terminal fragment of rat cartilage oligomeric matrix protein (COMP), comprising residues 20–83, was over‐expressed in E. coli and purified under non‐denaturing conditions. The fragment forms pentamers similar to the assembly domain of the native protein. Its five chains can be covalently linked in vitro by oxidation of cysteines 68 and 71. The fragment adopts a predominantly α‐helical structure as judged by circular dichroism spectroscopy. On the basis of these findings we propose the model of a five‐stranded α‐helical bundle for the assembly domain of COMP. The studied sequence is conserved in thrombospondins 3 and 4 thus raising the possibility that these proteins are also pentamers.
Bacterial protein translocase: a unique molecular machine with an army of substrates
Authors: Economou Anastassios
Published: July 2002

Secretion of most polypeptides across the bacterial plasma membrane is catalyzed by the Sec protein translocase. This complex molecular machine comprises a flexible transmembrane conduit coupled to a motor‐like component and displays four activities: (a) it is a specific receptor at its cytoplasmic side for all secretory polypeptides, (b) it converts metabolic energy from ATP and proton gradients into mechanical motion, (c) it prevents substrates from folding in statu translocanti and (d) it binds and releases short segments of the polymeric substrate sequentially. Combination of these activities allows translocase to move processively along the length of the substrate. Substrates are thus gradually expelled from the membrane and are released for subsequent extracytoplasmic folding.
Intracellular compartmentalization of leukotriene synthesis: unexpected nuclear secrets
Authors: Peters-Golden Marc , Brock Thomas G
Published: July 2002

Leukotrienes are important lipid mediators implicated in the regulation of various cellular processes and in disease states as well as homeostasis. Regulation of leukotriene biosynthesis is therefore of considerable interest. Although the levels of expression and catalytic activity of leukotriene‐forming proteins have long been recognized as important determinants of leukotriene biosynthesis, it has recently become apparent that their intracellular compartmentalization also affects the integrated output of this biosynthetic pathway. In this minireview, we focus on the unexpected discovery that the nucleus is the key intracellular site for leukotriene biosynthesis and discuss the mechanisms that regulate protein localization and the potential implications of these findings.
IGF‐1 receptor as an alternative receptor for metabolic signaling in insulin receptor‐deficient muscle cells
Authors: Baudry A. , Lamothe B. , Bucchini D. , Jami J. , Montarras D. , Pinset C. , Joshi R.L.
Published: July 2002

We have derived skeletal muscle cell lines from wild‐type (wt) and insulin receptor (IR) knockout mice to unravel the metabolic potential of IGF‐1 receptor (IGF‐1R). Both wt and IR−/− myoblasts differentiated into myotubes with similar patterns of expression of muscle‐specific genes such as MyoD, myogenin and MLC1A indicating that IR is not required for this process. Binding of 125I‐IGF‐1 on wt and IR−/− myotubes was similar showing that IGF‐1R was not upregulated in the absence of IR. Stimulation of IR−/− myotubes with IGF‐1 (10−10 to 10−7 M) increased glucose uptake and incorporation into glycogen, induced IRS‐1 phosphorylation and activated PI 3‐kinase and MAP kinase, two enzymes of major signaling pathways. These effects were comparable to those obtained with wt myotubes using insulin or IGF‐1 or with IR−/− myotubes using insulin at higher concentrations. This study provides a direct evidence that IGF‐1R can represent an alternative receptor for metabolic signaling in muscle cells.
Electrostatic effects play a central role in cold adaptation of trypsin
Authors: Brandsdal Bjørn Olav , Smalås Arne O. , Åqvist Johan
Published: July 2002

Organisms that live in constantly cold environments have to adapt their metabolism to low temperatures, but mechanisms of enzymatic adaptation to cold environments are not fully understood. Cold active trypsin catalyses reactions more efficiently and binds ligands more strongly in comparison to warm active trypsin. We have addressed this issue by means of comparative free energy calculations studying the binding of positively charged ligands to two trypsin homologues. Stronger inhibition of the cold active trypsin by benzamidine and positively charged P1‐variants of BPTI is caused by rather subtle electrostatic effects. The different affinity of benzamidine originates solely from long range interactions, while the increased binding of P1–Lys and –Arg variants of BPTI is attributed to both long and short range effects that are enhanced in the cold active trypsin compared to the warm active counterpart. Electrostatic interactions thus provide an efficient strategy for cold adaptation of trypsin.
Secondary structure and limited proteolysis give experimental evidence that the precursor of pulmonary surfactant protein B contains three saposin‐like domains
Authors: Zaltash Shahparak , Johansson Jan
Published: July 2002

The 42 kDa precursor of surfactant protein B generates the 79 residue mature SP‐B polypeptide, which belongs to the family of saposin‐like proteins and has unique functional roles in pulmonary surfactant. From sequence comparisons it has been suggested that proSP‐B, in addition to SP‐B, contains two saposin‐like domains, but their existence has until now not been experimentally verified. The 381 residue human proSP‐B was now fused to an N‐terminal poly‐His tag, expressed in Escherichia coli, and purified from inclusion bodies by resolubilisation with 2.5% (w/v) SDS and, after removal of SDS, subsequent metal affinity chromatography. Recombinant proSP‐B thus obtained exhibits about 35% α‐helical structure in sodium phosphate buffer and is proteolytically cleaved preferentially between the three saposin‐like domains. These results experimentally support that proSP contains, in addition to SP‐B, two further saposin‐like domains.
Effects of the Kupffer cell inactivator gadolinium chloride on rat liver oxygen uptake and content of mitochondrial cytochromes
Authors: Ferreira Jorge , Tapia Gladys , Videla Luis A
Published: July 2002

The effect of gadolinium chloride (GdCl3) on the content of rat liver mitochondrial cytochromes was investigated in relation to the basal rate of O2 uptake and Kupffer cell functioning, assessed in liver perfusion studies. (1) A single dose of GdCl3 (10 mg/kg) produced a significant diminution in Kupffer cell functioning, evidenced by the decreases in colloidal carbon uptake and in carbon‐induced O2 uptake observed at 6–24 h after treatment, without changes in the sinusoidal lactate dehydrogenase efflux as index of tissue viability; at 48 h after GdCl3 administration, carbon phagocytosis was recovered to control values, whereas carbon‐induced O2 uptake remained lower than control values. (2) GdCl3 also caused a 34% decrease in the basal rate of O2 consumption of the liver at 24 h after treatment, which returned towards control values at 48 h. (3) The content of mitochondrial cytochromes c1 and c at 24 h after GdCl3 treatment was significantly reduced by 40 and 32%, respectively, which returned to control values at 48 h, without changes in that of cytochromes b and a+a3. It is concluded that GdCl3‐induced decrease in liver O2 consumption is a reversible phenomenon that seems to be due to a diminution in the content of mitochondrial cytochromes c1 and c.
Association of β1 integrin with protein kinase activity in large detergent resistant complexes
Authors: Skubitz Amy P.N , Campbell Kenneth D , Goueli Said , Skubitz Keith M
Published: July 2002

Integrins play a critical role in cell adhesion and mediate cell signaling. This report identifies the association of serine protein kinase activity with the β1 integrin by immunoprecipitation and phosphoamino acid analysis techniques. Reprecipitation techniques suggested that the serine kinase activity was not a member of the protein kinase C family. By gel filtration, most of the protein kinase activity associated with β1 integrin as well as most of the cell‐surface β1 integrin was present in large detergent resistant complexes. These results suggest that serine protein kinase activity associated with the β1 integrin may play a role in signaling via the β1 integrin.
Total chemical synthesis of bovine pancreatic trypsin inhibitor by native chemical ligation
Authors: Lu Wuyuan , Starovasnik Melissa A , Kent Stephen B.H
Published: July 2002

Bovine pancreatic trypsin inhibitor (BPTI) is an important model for the study of protein folding. Herein we describe a robust approach to the total chemical synthesis of BPTI using native chemical ligation of unprotected peptide segments in aqueous solution. After refolding and oxidative formation of disulfides, the target protein was purified by affinity chromatography. The synthetic BPTI was characterized by mass spectrometry, inhibition assay, thermal denaturation and 2D NMR spectroscopy, and was shown to be structurally and functionally identical to natural BPTI. The synthetic strategy presented in this paper has enabled us to establish rapid access to novel analogues of BPTI.
Characterization of three episodic ataxia mutations in the human Kv1.1 potassium channel
Authors: Zerr Patricia , Adelman John P , Maylie James
Published: July 2002

Episodic ataxia (EA) is a rare inherited neurological disorder due to mutation in the voltage‐dependent Kv1.1 potassium channel. In nine unrelated families, a different missense point mutation at highly conserved positions has been reported. We have previously characterized six of the EA mutants. In this study, three recently identified mutations were introduced into the human Kv1.1 cDNA and expressed in Xenopus oocytes. Compared to wild type, T226A and T226M reduced the current amplitude by >95%, shifted the voltage dependence by 15 mV, and slowed activation and deactivation kinetics. Currents from G311S were ∼25% of wild type, less steeply voltage‐dependent and had more pronounced C‐type inactivation. These altered gating properties will reduce the delayed‐rectifier potassium current which may underlie the symptoms of EA.
Determinants of plasma coenzyme Q10 in humans
Authors: Kaikkonen Jari , Nyyssönen Kristiina , Tuomainen Tomi-Pekka , Ristonmaa Ulla , Salonen Jukka T
Published: July 2002

In the present study, we assessed the strongest determinants of plasma coenzyme Q10 (Q10) in 518 men and women (aged 45–70 years) with a stepwise multivariate regression model. Male gender (P<0.001), serum cholesterol (P<0.001), serum γ‐glutamyltransferase (P<0.001), serum triglycerides (P<0.001), age (P=0.017) and 4‐day alcohol consumption (P=0.03) were the most important factors which were directly associated with plasma Q10. The intensity of conditioning exercise (P=0.03) and use of statins (P<0.05) showed an inverse association with plasma Q10. None of the assessed nutrients increased plasma Q10 levels significantly. Our results suggest that many confounding factors, in addition to serum cholesterol and triglycerides, should be taken into account when the role of plasma Q10 is examined in epidemiological research.
Substrate specificity of catechol oxidase from Lycopus europaeus and characterization of the bioproducts of enzymic caffeic acid oxidation1
Authors: Rompel Annette , Fischer Helmut , Meiwes Dirk , Büldt-Karentzopoulos Klaudia , Magrini Annette , Eicken Christoph , Gerdemann Carsten , Krebs Bernt
Published: July 2002

The substrate specificity of catechol oxidase from Lycopus europaeus towards phenols is examined. The enzyme catalyzes the oxidation of o‐diphenols to o‐quinones without hydroxylating monophenols, the additional activity of tyrosinase. Substrates containing a ‐COOH group are inhibitors for catechol oxidase. The products of enzymic oxidation of caffeic acid were analyzed and isolated by HPLC with diode array detection. The neolignans of the 2,3‐dihydro‐1,4‐benzodioxin type (3, 6–8), 6,7‐dihydroxy‐1‐(3,4‐dihydroxyphenyl)‐2,3‐dicarboxy‐1,2‐dihydronaphthaline (1) 6,7‐dihydroxy‐1‐(3,4‐dihydroxyphenyl)‐3‐carboxynaphthaline (5) and 2,6‐bis‐(3′,4′‐dihydroxyphenyl)‐1‐carboxy‐3‐oxacyclo‐(3,0)‐pentan‐2‐on‐1‐ene (4) were formed. A reaction mechanism for the formation of (1, 4 and 5) is discussed.
Hypoxia‐induced activation of HIF‐1: role of HIF‐1α‐Hsp90 interaction
Authors: Minet E , Mottet D , Michel G , Roland I , Raes M , Remacle J , Michiels C
Published: July 2002

The protein chaperone heat shock protein 90 (Hsp90) is a major regulator of different transcription factors such as MyoD, a basic helix loop helix (bHLH) protein, and the bHLH‐Per‐aryl hydrocarbon nuclear translocator (ARNT)‐Sim (PAS) factors Sim and aryl hydrocarbon receptor (Ahr). The transcription factor hypoxia‐inducible factor‐1α (HIF‐1α), involved in the response to hypoxia, also belongs to the bHLH‐PAS family. This work was aimed to investigate the putative role of Hsp90 in HIF‐1 activation by hypoxia. Using a EGFP‐HIF‐1α fusion protein, co‐immunoprecipitation experiments evidenced that the chimeric protein expressed in COS‐7 cells interacts with Hsp90 in normoxia but not in hypoxia. We also demonstrated that Hsp90 interacts with the bHLH‐PAS domain of HIF‐1α. Moreover, Hsp90 is not co‐translocated with HIF‐1α into the nucleus. At last, we showed that Hsp90 activity is essential for HIF‐1 activation in hypoxia since it is inhibited in the presence of geldanamycin. These results indicate that Hsp90 is a major regulator in HIF‐1α activation.
A soluble NH2‐terminally truncated catalytically active form of rat cytochrome P450 2E1 targeted to liver mitochondria
Authors: Neve Etienne P.A. , Ingelman-Sundberg Magnus
Published: July 2002

The role of the NH2‐terminus of cytochrome P450 2E1 (CYP2E1) in intracellular targeting was investigated. Two NH2‐terminal CYP2E1 mutants, Δ(2–29)2E1, lacking the transmembrane domain, and N++2E1, having Ala2Lys and Val3Arg substitutions, were generated and expressed in the H2.35 mouse hepatoma cell line. In cells transfected with both constructs, a 40 kDa fragment of CYP2E1 (Δ2E1) was found to be localized to mitochondria as evidenced from immunofluorescence microscopy and subcellular fractionation studies. Δ2E1 was shown to be a soluble protein localized inside the mitochondria, displayed catalytic activity when reconstituted with adrenodoxin and adrenodoxin reductase, and was also present in mitochondria isolated from rat liver. It is concluded that in the absence of the hydrophobic NH2‐terminal sequence, a putative mitochondrial import signal is exposed which targets CYP2E1 to this organelle where it is further processed.
Global importance of infertility and its treatment: role of fertility technologies
Authors: Hamberger L. , Janson P.O.
Published: July 2002

Understanding the reasons for infertility and the development of new techniques for its treatment is one of the fields within medicine which has undergone a dramatic development during the last two decades. In the past, gynecologists seemed more successful in the treatment of female problems than urologists and andrologists on the male side, while in the last couple of years this scenario has changed due to the development of both operative procedures to harvest spermatozoa from the epididymis and the testis and the ability to micro‐inject single spermatozoa (ICSI) or even spermatides directly into metaphase II oocytes. It should, however, be emphasized that the successful development of in vitro fertilization (IVF) and other assisted reproduction technologies (ART), could not have taken place without the development of diagnostic innovations, such as immunoassays of hormones, high resolution ultrasound and various endoscopic techniques. Today, treatment of the infertile couple is less of a medico‐technical problem and more a socio‐economic dilemma. This communication is an attempt to summarize the more prominent steps in reproductive medicine during the last decades and one more general conclusion of global relevance which can be drawn, is that less money must be spent on diagnosis and more resources transferred to treatment options.