bluuebunny/crossref_metadata_2025 huggingface dataset:
Bruton's tyrosine kinase regulates B cell antigen receptor‐mediated JNK1 response through Rac1 and phospholipase C‐γ2 activation
Authors: Inabe Kazunori , Miyawaki Toshio , Longnecker Richard , Matsukura Hiroyoshi , Tsukada Satoshi , Kurosaki Tomohiro
Published: July 2002

Bruton's tyrosine kinase (Btk) is essential for B cell development and B cell antigen receptor (BCR) function. Recent studies have shown that Btk plays an important role in BCR‐mediated c‐Jun NH2‐terminal kinase (JNK) 1 activation; however, the mechanism by which Btk participates in the JNK1 response remains elusive. Here we show that the BCR‐mediated Rac1 activation is significantly inhibited by loss of Btk, while this Rac1 activation is not affected by loss of phospholipase C‐γ2 (PLC‐γ2). Since PLC‐γ2 is also required for BCR‐mediated JNK1 response, our results suggest that Btk regulates Rac1 pathway as well as PLC‐γ2 pathway, both of which contribute to the BCR‐mediated JNK1 response.
Two fundamentally different types of variable chlorophyll fluorescence in vivo
Authors: Schreiber Ulrich , Krieger Anja
Published: July 2002

Upon onset of saturating continuous light only the first part of the observed polyphasic fluorescence rise follows Q
A
reduction (photochemical phase), whereas the remaining part (thermal phases) is kinetically limited by relatively slow reactions with light saturated half‐times in the order of 10–50 ms. A simple hypothesis is presented for the interpretation of these fundamentally different types of variable fluorescence. The hypothesis, which is based on the reversible radical pair model of PSII, assumes stimulation of both prompt and recombination fluorescence upon Q
A
reduction, with only recombination fluorescence being in competition with nonradiative energy loss processes at the reaction centers. It is proposed that changes in the rate constants of these processes modulate the yield of recombination fluorescence in closed centers, thus causing large variations in the maximal fluorescence yield and also giving rise to the ‘thermal phases’. This hypothesis can reconcile numerous experimental findings which so far have seemed difficult to interpret.
Identification of amino acid residues required for a specific interaction between Src‐tyrosine kinase and proline‐rich region of phosphatidylinositol‐3′ kinase
Authors: Mak Paul , He Zhiqing , Kurosaki Tomohiro
Published: July 2002

The binding of ligand to B‐cell antigen receptors (BCR) leads to the activation of receptor‐associated Src‐family kinases and phosphatidylinositol‐3′ kinase (PI‐3 kinase). Although it has been demonstrated that SH3 domains of several Src‐family kinases interact with PI‐3 kinase by binding to a proline‐rich region of PI‐3 kinase in vitro, there is no direct evidence to support their interaction in vivo. Thus, we utilized the yeast two‐hybrid assay to reconstitute this protein‐protein interaction. This genetic screen clearly indicates that the interaction between SH3 domain of Fyn and the proline‐rich region (residues: 80–104) of PI‐3 kinase is highly specific. Mutational analysis revealed that amino acid residues Asp92, Tyr93, Arg96 and Thr97 of the SH3 domain of Fyn are essential for interacting with the proline‐rich peptide of PI‐3 kinase.
Characterization of a uterine luminal fluid protein ULF‐250 using N‐terminal microsequencing and RT‐PCR identifies a novel estrogen‐regulated gene in the rat uterus
Authors: Kumar G Pradeep , Laloraya Malini , Koide Samuel S.
Published: July 2002

We had previously identified an estrogen responsive protein ULF‐250, synthesized and secreted by the estrous rat uterus, which is immunologically distinct from complement C3 and α2‐macroglobulin. The N‐terminal microsequencing of ULF250 followed by sequence homology analysis showed that this protein is a new member of a class of estrogen responsive proteins in the uterus. Polymerase chain reaction with a ULF‐250 specific primer yielded partial sequence information of its message. The observed pattern of ULF‐250 message in the uterus during the various stages of the reproductive cycle in the rat suggested a possible regulation of ULF‐250 message by 17β‐estradiol. Upstream sequencing of ULF‐250 message and its promoter domains would provide insight into the mechanism of its regulation by estradiol.
The presence of H+ and Na+‐linked Ca2+ extruding systems in Methanobacterium thermoautotrophicum
Authors: Varečka Ľudovít , Šmigáň Peter , Greksák Miloslav
Published: July 2002

The effects of monovalent cations (Na+, K+ and choline+) and the uncoupler 3,3′,4′,5‐tetrachlorosalicylanilide (TCS) were tested on 45Ca2+ uptake by non‐energized cells of Methanobacterium thermoautotrophicum. 45Ca2+ uptake was stimulated by the addition of K+ and (less) by choline+ while Na+ slowed down and even reversed it, thereby mimicking the energization of cells. The uncoupler agent, TCS, suppressed 45Ca2+ uptake in non‐energized cells in the presence or absence of Na+ but in cells energized in an atmosphere of CO2+H2 it exerted a stimulating effect. Uncoupled 45Ca2+ efflux was measured in cells pre‐loaded with 45Ca2+ by means of the divalent ionophore A23187 following its washing out by buffer containing serum albumin. The efflux was temperature‐dependent and was stimulated by external 40Ca2+ and Na+. In the absence of Na+, the uncoupled efflux was completely inhibited by TCS, whereas in the presence of Na+, TCS was without any effect. The results are in agreement with the model in which the Ca2+ influx pathway is represented by a membrane potential‐driven uniport whereas Ca2+ efflux is mediated by two transport systems ‐ and antiporters ‐ whose participation in the total efflux is dependent on the energy of the corresponding gradients of driving ions.
Clavanins, α‐helical antimicrobial peptides from tunicate hemocytes
Authors: Lee I.Hee , Zhao Chengquan , Cho Yoon , Harwig Sylvia S.L , Cooper Edwin L , Lehrer Robert I
Published: July 2002

Hemocytes from the invertebrate Styela clava, a solitary tunicate, contained a family of four α‐helical antimicrobial peptides that were purified, sequenced and named clavanins A, B, C and D. Each clavanin contained 23 amino acid residues and was C‐terminally amidated. The tunicate peptides resembled magainins in size, primary sequence and antibacterial activity. Synthetic clavanin A was prepared and displayed comparable antimicrobial activity to magainins and cecropins. The presence of α‐helical antimicrobial peptides in the hemocytes of a urochordate suggests that such peptides are primeval effectors of innate immunity in the vertebrate lineage.
Block of ShakerB K+ channels by Pi1, a novel class of scorpion toxin
Authors: Gómez-Lagunas F , Olamendi-Portugal T , Possani L.D
Published: July 2002

Here we describe the basic features of the interaction of K+ channels with Pi1, a recently described 35 amino acid scorpion toxin, which has four disulfide bridges instead of the three commonly found in all the other known scorpion toxins. We found that: (a) Pi1 blocks ShakerB from the outside with a 1:1 stoichiometry, and a K
d of 32 nM in zero external [K+]; (b) extracellular K+, Rb+ and Cs+ but not NH+
4 ions strongly impede (destabilize) the block by this toxin; interestingly (c) the destabilizing binding of K+, Rb+, and Cs+ is described by a Hill coefficient n>1; (d) external K+ is more effective than internal K+ to reduce the block by Pi1.
Protein expression of the epsilon subspecies of protein kinase C ceases as Swiss 3T6 fibroblasts increase in cell density even though message for the protein is still present
Authors: Littlebury Paul , Watson John , Williams Timothy , Beale Gary , Rumsby Martin
Published: July 2002

We have noted previously that growth of C6 glioma cells from low cell density to confluency and quiescence in serum is accompanied by changes in protein content of different protein kinase C (PKC) subspecies. Here we show that the same occurs as non‐contact‐inhibiting Swiss 3T6 fibroblasts grow to high density in the presence of serum. Protein expression of PKC subspecies α and δ increases as the cells increase in density while that of PKC‐ζ remains the same. Unusally, protein expression of PKC‐ϵ is completely down‐regulated as cells grow beyond about 50% confluency and no PKC‐ϵ protein can be detected in 3T6 fibroblasts at high density by Western blotting. However, mRNA for PKC‐ϵ is expressed at all stages of fibroblast growth as revealed by RT‐PCR. When high‐density 3T6 fibroblasts are passaged to low density in fresh medium, re‐expression of PKC‐ϵ protein is observed within 15 min and becomes down‐regulated again as cells become more dense. This very rapid synthesis of PKC‐ϵ is not blocked by the transcription inhibitor actinomycin D but is inhibited by cycloheximide. PKC‐ϵ has some characteristics of a novel ‘early response’ protein whose synthesis in newly passaged 3T6 cells is regulated at the translational level.
Rapid sequence‐independent cellular response to oligodeoxynucleotides
Authors: Balakireva Larissa A , Levashova Zoya B , Chroboczek Jadwiga , Vlassov Valentin V
Published: July 2002

The presence of receptors for oligodeoxynucleotides (OdN) on the surface of L929 cells has previously been described. To study the possible coupling of the receptor to cellular signal transducing systems, the effect of phosphodiester OdN of different sequences on cellular phospholipase C and protein kinase C (PKC) activities in L929 fibroblasts was studied. Treatment of cells with OdN induced an increase in 32P labeling of phosphatidic acid which was accompanied by a gradual decrease in diacylglycerol. These effects seem to be independent of the OdN sequence. PKC activity in membranes isolated from OdN‐treated cells was found to be lower than that in membranes of control cells. SDS‐PAGE of the 32P‐labeled cellular proteins revealed that OdN treatment caused a decrease in phosphorylation of the 26 and 73 kDa cellular proteins in the cells.
Regulation of hyaluronate metabolism by progesterone in cultured fibroblasts from the human uterine cervix
Authors: Tanaka Kanji , Nakamura Toshiya , Takagaki Keiichi , Funahashi Masaru , Saito Yoshiharu , Endo Masahiko
Published: July 2002

The effects of progesterone, dehydroepiandrosterone sulfate and 17β‐estradiol on the synthesis and degradation of hyaluronate were investigated using human uterine cervix fibroblasts. The cells were incubated with [3H]glucosamine in the presence of the hormones and then [3H]hyaluronate was isolated from the medium. The changes in the radioactivity of [3H]hyaluronate showed that progesterone suppressed hyaluronate synthesis by 22% of the control levels, while dehydroepiandrosterone sulfate and 17β‐estradiol enhanced it by 22% and 12% of the control levels, respectively. Furthermore, progesterone induced degradation of high‐molecular‐weight [14C]hyaluronate into low‐molecular‐weight hyaluronate (M
r≈40 000). These results suggest that in cultured fibroblasts from the human uterine cervix progesterone converts hyaluronate metabolism from the synthesis phase to the degradation phase. © 1997 Federation of European Biochemical Societies. All rights reserved
Expression of PC3, carboxypeptidase E and enkephalin in human monocyte‐derived macrophages as a tool for genetic studies
Authors: LaMendola Joseph , Martin Sean K. , Steiner Donald F.
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Activation of LA‐N‐2 cell phospholipases by single alanine substitution analogs of amyloid β peptide (25–35)
Authors: Singh I.N , Sato K , Takashima A , Kanfer J.N
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Bot IT2: a new scorpion toxin to study receptor site on insect sodium channels
Authors: Cestèle Sandrine , Borchani Lamia , El Ayeb Mohamed , Rochat Hervé
Published: July 2002

© 1997 Federation of European Biochemical Societies.
The role of palmitoyl–protein thioesterase in the palmitoylation of endothelial nitric oxide synthase
Authors: Michel Jeffrey B. , Michel Thomas
Published: July 2002

Palmitoylation of eNOS is required for targeting to plasmalemmal caveolae and agonist‐promoted depalmitoylation leads to eNOS translocation, modifying the agonist response. To date, one palmitoyl–protein thioesterase (PPT) has been purified and cloned. To explore the role of PPT in eNOS palmitoylation, we first established that PPT mRNA and protein are expressed in endothelial cells. Coexpression of PPT and eNOS in heterologous systems (COS and Sf‐9 cells) resulted in a marked reduction in [3H]palmitate labeling of eNOS. We found, however, that co‐expression did not alter subcellular targeting of eNOS, but that [3H]palmitate incorporation into cellular lipids, in particular palmitoyl‐CoA, was significantly reduced. These results suggest that while PPT expression can significantly alter cellular lipid metabolism, it has no effect on eNOS palmitoylation.
Functional characterization of the trout insulin promoter: implications for fish as a favorable model of pancreas development
Authors: Argenton Francesco , Walker Michael D , Colombo Lorenzo , Bortolussi Marino
Published: July 2002

The complex anatomy of the mammalian pancreas, in which the endocrine cells are grouped in islets dispersed among the predominant exocrine component, has hampered study of the molecular events governing the development of pancreatic cell lineages. To investigate whether fish may provide relevant, complementary models of pancreas development, we characterized the trout insulin (tINS) promoter and its molecular interactions with PDX1, a key transcriptional and developmental factor of the mammalian pancreas. Transfection of a luciferase reporter plasmid containing the 280 bp 5′‐flanking region of the tINS gene resulted in strong activity in mammalian pancreatic β cells but not in CHO or pituitary cells. Footprinting assays and cotransfection experiments indicated that mammalian PDX1 binds to and activates the tINS promoter. By microinjecting plasmids to fertilized zebrafish eggs, we showed that the expression of mouse PDX1 is capable of activating the co‐injected tINS promoter plasmid in most cell types of the 24‐h zebrafish embryo. The conserved role of PDX1 in vertebrate insulin gene regulation opens the possibility to exploit fish models in the study of pancreas development.
Different utrophin and dystrophin properties related to their vascular smooth muscle distributions
Authors: Rivier François , Robert Agnès , Hugon Gérald , Mornet Dominique
Published: July 2002

Monoclonal antibodies used to distinguish between dystrophin and utrophin were systematically applied to skeletal muscles containing arteries and veins. Small arteries were found to contain long forms of both utrophin and dystrophin, while small veins contained only long forms of utrophin. In addition, all sizes of vascular smooth muscles were demonstrated to contain another related M
r 80 kDa protein (possibly a short utrophin transcript). Regardless of their tissue distributions, we assumed that each of these molecules had distinct properties, i.e. dystrophin with a mechanical function and utrophin with an architectural function. This difference in the roles of dystrophin and utrophin could reduce the efficiency of protection against muscle membrane degeneration when utrophin overexpression is programmed.
Purification and characterization of a fusion protein of plant acetohydroxy acid synthase and acetohydroxy acid isomeroreductase
Authors: Dumas Renaud , Biou Valérie , Douce Roland
Published: July 2002

The nucleotide sequence coding for the Arabidopsis thaliana acetohydroxy acid synthase was genetically fused in frame with the nucleotide sequence coding for the Spinacia oleracea acetohydroxy acid isomeroreductase and expressed in Escherichia coli. This construction allowed the production of large amounts of soluble fusion protein. The pure chimeric enzyme exhibits high acetohydroxy acid synthase and acetohydroxy acid isomeroreductase specific activities. Fusion and native enzymes exhibit similar K
m values for their substrates and for most cofactors. Furthermore, whereas native plant acetohydroxy acid synthase is highly unstable, the stability of this enzyme in the fusion has been increased. Thus, the chimeric enzyme appears to be a useful tool for the determination of kinetic and structural properties of plant acetohydroxy acid synthase.
Analysis of the black‐eyed pea trypsin and chymotrypsin inhibitor‐α‐chymotrypsin complex
Authors: de Freitas Sonia M , de Mello Luciane V , da Silva Maria Cristina M , Vriend Gerrit , Neshich Goran , Ventura Manuel M
Published: July 2002

The black‐eyed pea trypsin and chymotrypsin inhibitor (BTCI) is a member of the Bowman‐Birk protease inhibitor (BBI) family. The three‐dimensional model of the BTCI‐chymotrypsin complex was built based on the homology to Bowman‐Birk inhibitors with known structures. An extensive theoretical and experimental study of these known structures has been performed. The model confirms the ideas about Bowman‐Birk inhibitor structure‐function relations and agrees well with our experimental data (circular dichroism, IR and light scattering). The electrostatic potentials at the enzyme‐inhibitor contact surface reveal a pattern of complementary electrostatic potentials from which mutations can be inferred that could give these inhibitors an altered specificity.
Conservative Val47 residue of POU homeodomain: role in DNA recognition
Authors: Stepchenko Alexander G , Luchina Nadezda N , Polanovsky Oleg L
Published: July 2002

Conservative Val47 residue, located in the third recognition helix of the Oct‐2 POU domain, was alternately substituted with other 19 amino acids. Affinity and specificity of interaction with oct‐site ATGCAAANGA and homeo‐specific site ATAANGA were determined for all mutants. The wild type protein (with Val47) has maximal affinity and specificity in POU domain interaction with octamer sequence. However, V47I mutant showed stronger interaction with homeo‐specific site. The highest specificity of interaction with homeo‐site was recorded for V47S mutant. We conclude that only Val47 provides sequence‐specific high‐affinity binding of POU proteins with octamer targets other than the homeo‐specific site. It is shown also that damages caused by point mutations may be at least partially compensated by participation in the oct‐site recognition of both POUh and POUs domains.
Identification of an intracellular domain of the EGF receptor required for high‐affinity binding of EGF
Authors: Van der Heyden Marcel A.G , Nievers Mirjam , Verkleij Arie J , Boonstra Johannes , Van Bergen en Henegouwen Paul M.P
Published: July 2002

Although all EGF receptors in EGF receptor‐expressing cells are molecularly identical, they can be subdivided in two different classes that have either a high or a low affinity for EGF. Specifically the high‐affinity class is associated with filamentous actin. To determine whether the interaction of the EGF receptor with actin induces its high‐affinity state, we studied EGF‐binding properties of an EGF receptor mutant that lacks the actin‐binding site. Interestingly, we found that cells expressing this mutant receptor still display both high‐ and low‐affinity classes of EGF receptors, indicating that the actin‐binding domain does not determine the high‐affinity binding state. By further mutational analysis we identified a receptor domain, within the tyrosine kinase domain, that regulates the affinity for EGF.
Bcl‐2 protein inhibits oxysterol‐induced apoptosis through suppressing CPP32‐mediated pathway
Authors: Harada Kenji , Ishibashi Shun , Miyashita Toshiyuki , Osuga Jun-ichi , Yagyu Hiroaki , Ohashi Ken , Yazaki Yoshio , Yamada Nobuhiro
Published: July 2002

Oxysterols are presumed to mediate cytotoxicity of oxidized LDL in atherosclerotic lesions. To elucidate its molecular mechanism, we established murine macrophage‐like P388‐D1 cells which over‐express Bcl‐2 protein by retrovirus‐mediated gene transfer. Oxysterols (7‐ketocholesterol, 25‐hydroxycholesterol) induced nuclear condensation and oligonucleosomal DNA fragmentation, which were partially inhibited by Bcl‐2 over‐expression. Though CPP32 inhibitor suppressed the cell death in control cells, it showed no additive protection in the cells over‐expressing Bcl‐2. These findings indicate that oxysterols induce apoptosis via Bcl‐2‐inhibitable and ‐uninhibitable pathways, and the former depends on CPP32 activation.
A common core for binding single‐stranded DNA: structural comparison of the single‐stranded DNA‐binding proteins (SSB) from E. coli and human mitochondria
Authors: Webster Gordon , Genschel Jochen , Curth Ute , Urbanke Claus , Kang ChulHee , Hilgenfeld Rolf
Published: July 2002

The crystal structure of the DNA‐binding domain of E. coli SSB (EcoSSB) has been determined to a resolution of 2.5 Å. This is the first reported structure of a prokaryotic SSB. The structure of the DNA‐binding domain of the E. coli protein is compared to that of the human mitochondrial SSB (HsmtSSB). In spite of the relatively low sequence identity between them, the two proteins display a high degree of structural similarity. EcoSSB crystallises with two dimers in the asymmetric unit, unlike HsmtSSB which contains only a dimer. This is probably a consequence of the different polypeptide chain lengths in the EcoSSB heterotetramer. Crucial differences in the dimer‐dimer interface of EcoSSB may account for the inability of EcoSSB and HsmtSSB to form cross‐species heterotetramers, in contrast to many bacterial SSBs.
Potential CRE suppression by familial Alzheimer's mutants of APP independent of adenylyl cyclase regulation
Authors: Giambarella Ugo , Murayama Yoshitake , Ikezu Tsuneya , Fujita Toshiro , Nishimoto Ikuo
Published: July 2002

In familial Alzheimer's disease (FAD), mutations to I, F, and G have been discovered at V642 in the neuron‐specific version of the amyloid precursor protein APP695. It has been found that expression of each FAD mutant suppresses the transcriptional activity of the cAMP response element CRE in a Gαo‐dependent manner in a COS cell clone NK1 [Ikezu et al. (1996) EMBO J. 15, 2468–2475]. Here we show that adenylyl cyclase (AC) inhibition is probably not the prerequisite for this pathway. First, expression of each FAD mutant in NK1 cells had no effect on AC activity stimulated by cholera toxin and by mutationally activated Gαs, although the same expression completely repressed the stimulated CRE. Second, a transfected activating mutant of Gαo inhibited CRE without detectable suppression of AC, whereas similarly transfected activating Gαi2 inhibited both AC and CRE. Third, FAD mutant‐induced inhibition occurred for CRE activity stimulated by dibutyryl cAMP. These data suggest that CRE suppression by FAD mutants of APP could occur independently of AC.
Selective uptake of high density lipoprotein‐associated cholesterylesters by differentiated Ob1771 adipocytes is modulated by endogenous and exogenous lipoprotein lipase
Authors: Schorsch Frida , Malle Ernst , Sattler Wolfgang
Published: July 2002

The present study aimed at investigating mechanisms of selective uptake of HDL3‐associated cholesterylesters (HDL3‐CEs) by differentiated Ob1771 adipocytes. Our findings indicate that Ob1771 cells are capable of pronounced selective uptake of HDL3‐CEs in 2.6‐fold excess of HDL3 holoparticle uptake. Selective uptake of HDL3‐CEs into a releasable pool (presumably located in the cellular plasma membrane) was temperature insensitive while prominent internalization into a non‐releasable and subsequent hydrolysis in a non‐chloroquine sensitive compartment occurred at 37°C. Release of membrane bound endogenous LPL by heparin resulted in decreased HDL3 holoparticle, total CE and selective CE uptake. Accordingly, the addition of exogenous LPL to the culture medium resulted in increased holoparticle, total CE and selective CE uptake.
Glucokinase is located in secretory granules of pancreatic D‐cells
Authors: Toyoda Yukiyasu , Yoshie Sumio , Fujita Tsuneo , Ito Yuki , Nonogaki Tsunemasa , Miwa Ichitomo
Published: July 2002

We immunohistochemically examined the distribution of glucokinase in rat pancreatic islets. Glucokinase immunoreactivity under light microscopy was detected in the cytoplasm of somatostatin cells as well as in that of insulin cells. No specific immunoreactivity was detected in glucagon and pancreatic polypeptide cells. In somatostatin cells, glucokinase immunoreactivity was located by electron microscopy exclusively within secretory granules.
Nitrogen source‐dependent expression of a 126 kDa protein in the plasma membrane of the cyanobacterium Synechococcus PCC 7942
Authors: Zinovieva Maria , Fresneau Chantal , Arrio Bernard
Published: July 2002

The expression of a 126 kDa protein in the cytoplasmic membrane of Synechococcus PCC 7942 is shown to be dependent on the nitrogen source. It is absent in ammonium‐grown cells and its quantity is inversely related to the concentration of nitrate or nitrite in the growth medium. Addition of ammonium‐grown cells to a medium containing nitrate or l‐methionine‐d
l‐sulfoximine results in the expression of this protein. It is present in the plasmalemma of the Synechococcus NC3 mutant (nrtC gene deleted) and absent in the NA3 mutant (nrtABCD genes deleted). These results may suggest involvement of the 126 kDa protein in nitrate transport through Synechococcus cytoplasmic membrane.
Chicken thyroid hormone receptor α requires the N‐terminal amino acids for exclusive nuclear localization
Authors: Andersson Monika L , Vennström Björn
Published: July 2002

The subcellular localization of natural and engineered forms of the chicken thyroid hormone receptor (cTRα) is dependent on amino acids encoded in the N‐terminal region. The full length receptor protein, cTRα‐p46, was found to localize exclusively to the nucleus, whereas the N‐terminally shorter variant, cTRα‐p40, localizes to both the nucleus and the cytoplasm. The exclusive nuclear localization of cTRα‐p46 is dependent on the presence of the first 11 N‐terminal amino acids, but independent of the phosphorylation of the serine at position 12. Our data identify a novel role for an N‐terminal domain of the full length thyroid hormone receptor.
Identification of functional synergism between monoclonal antibodies. Application to the enhancement of plasminogen activator inhibitor‐1 neutralizing effects
Authors: Ngo Thu-Hoa , Debrock Sophie , Declerck Paul J
Published: July 2002

The serpin plasminogen activator inhibitor‐1 (PAI‐1), an important risk factor for thrombotic disease can be neutralized by distinct mechanisms. We hypothesized that the combination of two compounds, with PAI‐1 neutralizing properties based on different mechanisms, may result in a synergistic effect. Therefore, seven monoclonal antibodies with PAI‐1 neutralizing properties were pairwise evaluated for the possible presence of synergistic or antagonistic effects. Out of 21 combinations, three particular combinations, i.e. MA‐33H1/MA‐33B8, MA‐33B8/MA‐7D4B7, and MA‐7D4B7/MA‐33H1 exhibited strong synergistic effects in comparison with their properties when evaluated individually. The observed synergism resulted in a maximum enhancement between 2‐ and 5‐fold (P<0.05, vs. theoretically expected effect calculated based on additive effects). Strikingly, synergism was only observed between monoclonal antibodies directed against different epitopes and with different molecular mechanisms of PAI‐1 neutralization. This phenomenon of synergism opens new perspectives in the design of therapeutic or preventive strategies aimed at enhancing endogenous fibrinolysis through modulation of PAI‐1 activity.
The `assembly‐promoting sequence region' of microtubule‐associated protein 4 failed to promote microtubule assembly
Authors: Katsuki Miho , Tokuraku Kiyotaka , Nakagawa Hiroyuki , Murofushi Hiromu , Kotani Susumu
Published: July 2002

In order to study the function of the bovine MAP4 microtubule‐binding domain (the assembly‐promoting (AP) sequence region), a fragment corresponding to the AP sequence region was prepared using an Escherichia coli expression system. When the fragment was mixed with purified tubulin at 37°C, the fragment caused a time‐ and dose‐dependent turbidity increase, and the fragment bound to tubulin. However, the products were cold‐stable, and amorphous aggregates were observed by electron microscopy. Using axonemes as the seeds for microtubule assembly, the microtubule‐elongating activity of the fragment was examined. A dose‐dependent turbidity increase of the sample was observed, and electron microscopic observation revealed that microtubules were dose‐dependently elongated from the axonemes. Consequently, the AP sequence region does not nucleate microtubules, but elongates them.
Cyclolinopeptide A (CLA) mediates its immunosuppressive activity through cyclophilin‐dependent calcineurin inactivation
Authors: Gaymes Terry J , Cebrat Marek , Siemion Ignacy Z , Kay John E
Published: July 2002

The immunosuppressive cyclic nonapeptide cyclolinopeptide A inhibits calcium‐dependent, but not calcium‐independent, activation of T lymphocytes comparably to the actions of cyclosporin A and FK506. The concentration required for complete inhibition, however, is 10 times higher than that of cyclosporin A. In addition, we demonstrate that calcineurin, a phosphatase which plays an important role in T lymphocyte signalling, is inhibited in vitro by cyclolinopeptide A by a mechanism dependent on the peptidyl‐prolyl cis‐trans isomerase (PPIase) cyclophilin A but not FKBP12. Direct binding of cyclolinopeptide A to cyclophilin A was confirmed using tryptophan fluorescence studies and PPIase assays. These results represent a third example of the production of a natural product that neutralises calcineurin by a mechanism dependent on the primary binding to a PPIase.
Identification of the transcription start site for the spinach chloroplast serine tRNA gene
Authors: Wu Chih-Yu , Lin Chi-Hui , Chen Liang-Jwu
Published: July 2002

Deleting part of the 3′ end of the spinach chloroplast serine tRNA coding region, which destroyed the proper folding of its RNA transcript and resulted in the inhibition of tRNA processing, allowed the detection of a serine tRNA primary transcript. The transcription start site for this primary transcript, synthesized from the internal promoter, was mapped to −12 upstream from the mature tRNA coding region. Transcription analysis with various 5′ deletion mutants suggested that the AT‐rich region between −31 and −11, immediately upstream of the serine tRNA transcription start site, affects the transcription efficiency, and possibly the selection of transcription start site. Identification of the transcription start site for the spinach chloroplast serine tRNA gene in this study represents the first example of 5′ end mapping of a tRNA precursor transcribed from chloroplast tRNA genes containing an internal promoter.
Structural analysis of the products of chymotryptic cleavage of the E1 form of Na,K‐ATPase α‐subunit: identification of the N‐terminal fragments containing the transmembrane H1‐H2 domain
Authors: Ivanov Alexander , Askari Amir , Modyanov Nikolai N
Published: July 2002

Chymotryptic cleavage of the Na,K‐ATPase in NaCl medium abolishes ATPase activity and alters other functional parameters. The structure of this modified enzyme is uncertain since only one product of selective proteolysis, the 83‐kDa fragment of the α‐subunit (Ala267–C‐terminus) has been identified previously. Here, we applied additional tryptic digestion followed by oxidative cross‐linking to identify the products originating from the N‐terminal part of the α‐subunit. These fragments start at Ala72 or Thr74 and contain the transmembrane H1‐H2 domain. Formation of cross‐linked product between α‐fragments containing H1‐H2 and H7‐H10 demonstrate that the structural integrity of the membrane moiety is preserved. We also determined that secondary cleavage of the 83‐kDa fragment leads to the formation of C‐terminal 48‐kDa α‐fragments with multiple N‐termini at Ile582, Ser583, Met584 and Ile585.
Secondary structure determination of 15N‐labelled human Long‐[Arg‐3]‐insulin‐like growth factor 1 by multidimensional NMR spectroscopy
Authors: Laajoki Leanne G , Le Breton Eugene , Shooter Gary K , Wallace John C , Francis Geoffrey L , Carver John A , Keniry Max A
Published: July 2002

Insulin‐like growth factors (IGFs) are a group of proteins that promote cell growth and differentiation. Long‐[Arg‐3]‐IGF‐I (Francis et al. (1992) J. Mol. Endocrinol. 8, 213–223), a potent analogue of IGF‐I, which has a Glu‐3 to Arg‐3 substitution and a hydrophobic, thirteen amino acid N‐terminal extension, has been studied by 1H,15N NMR spectroscopy. All the backbone 1H and 15N assignments and most of the 1H sidechain assignments have been completed. The secondary structure elements were identified by determining the sequential and medium range NOEs from sensitivity‐enhanced 15N‐NOESY‐HSQC and sensitivity‐enhanced 15N‐HSQC‐NOESY‐HSQC spectra. The IGF‐I domain of Long‐[Arg‐3]‐IGF‐I was found to have an almost identical structure to IGF‐I. The N‐terminal seven amino acid residues of the extension have very few medium range or long range NOEs but the next five amino acids form a turn‐like structure that is spatially close to the beginning of helix 1 in the IGF‐I domain. Hydrogen‐deuterium exchange experiments show that all the slowly exchanging backbone amide protons in the IGF‐I domain are either in the helical or the extended structural elements. Many of the amide protons in the N‐terminal extension are also protected from the solvent although the residues in this part of the extension do not have any identifiable secondary structure. The results are interpreted in terms of the increased biological potency of Long‐[Arg‐3]‐IGF‐I and the decreased binding to insulin‐like growth factor binding proteins.
Regulation of activin type I receptor function by phosphorylation of residues outside the GS domain
Authors: Willis Susan A , Mathews Lawrence S
Published: July 2002

Activin signals through a heteromeric complex of receptor serine kinases by inducing type II receptor‐mediated phosphorylation, and consequent activation, of the type I receptor. Type I receptor phosphorylation occurs at a glycine‐ and serine‐rich site in the juxtamembrane domain; phosphorylation at that site correlates with signaling. Investigation of type I activin receptor mutants impaired for GS domain phosphorylation revealed that, in the presence of elevated amounts of type II activin receptor, GS domain phosphorylation is not required for signaling. The type I receptor showed activin‐dependent phosphorylation of several tryptic phosphopeptides, suggesting that phosphorylation of receptor I at sites both within and outside the GS domain is required for full signaling.
Involvement of ecto‐ATPase and extracellular ATP in polymorphonuclear granulocyte‐endothelial interactions
Authors: Sud'ina Galina F , Mirzoeva Olga K , Galkina Svetlana I , Pushkareva Marina A , Ullrich Volker
Published: July 2002

We conclude that PMN‐endothelial adhesion is counteracted by an ecto‐ATPase or by ATP receptors with ATPase activity. Such interactions may play a role in PMN rolling and diapedesis as well as in the pathophysiology of PMN activation by an anergic endothelium.
Mild proteolysis induces a ready‐to‐fuse state on Sendai virus envelope
Authors: Tomasi Maurizio , Baiocchi Marta , Moscufo Nicola , Parolini Isabella , Squatriti Teodoro , Bellini Tiziana , Dallocchio Franco
Published: July 2002

The Sendai virus fuses with host cell membranes in a pH‐independent manner through an unknown mechanism. Here we report that mild trypsin pre‐treatments of Sendai virions, for example 15 min at 4°C, give Sendai virions the ability to fuse at a rate up to 10‐fold higher than control. By using human erythrocytes as host cell membranes, viral fusion was assessed by hemolysis as well as fluorescence dequenching of octadecyl rhodamine B chloride. The mild protease treatment strikingly shortens the lag time taken by the virus to start the fusion process. Similar data were obtained on reconstituted Sendai virus envelope. Among proteases, tested as fusion enhancer, trypsin is more effective than either endoproteinase Lys‐C, chymotrypsin, or endoproteinase Arg‐C. After removal of trypsin from treated virions the fusion rate enhancement remains for hours at room temperature. The lack of protease specificity, together with the impossibility to detect any new N‐terminal products, suggests that only a small percentage of viral envelope components are cleaved, still a large enough number to set the envelope in a ready‐to‐fuse state.
Proton exchange kinetics in [d(ACGTATACGT)]2‐echinomycin and [d(ACGTTAACGT)]2‐echinomycin complexes
Authors: Park Jin-Young , Lee Joon-Hwa , Choi Byong-Seok
Published: July 2002

Based on imino proton exchange catalysis, base‐pair lifetimes and apparent dissociation constants are reported on the complexes formed by bisintercalation of echinomycin at the CpG steps of the d(ACGTATACGT)2 and d(ACGTTAACGT)2 duplexes. The lifetimes of the four central A·T base pairs between two echinomycin binding sites are much shorter than in the free duplexes. The destabilization of base pairs adjacent to the binding sites is propagated one additional base pair away from the binding site.
Activation of bcl‐2 suppressible 40 and 44 kDa p38‐like kinases during apoptosis of early and late B lymphocytic cell lines
Authors: Maucher Cora , Weissinger Eva M , Kremmer Elisabeth , Baccarini Manuela , Procyk Kasia , Henderson Darren W , Wolff Linda , Kolch Walter , Kaspers Bernd , Mushinski J.Frederic , Mischak Harald
Published: July 2002

Activation of several different kinases characterizes the induction of apoptosis. Abelson virus transformed pre‐B lymphocytes undergo apoptosis within 24 h of serum deprivation, PKA activation or γ‐irradiation, and the activity of two kinases of ca. 40 and 44 kDa is specifically induced during this apoptotic process. Bcl‐2 expression prevents both apoptosis and the induction of these kinases. Immunologic and substrate similarities indicate that these kinases are related to the p38 family of MAP kinases. More mature cells of the B lymphocytic lineage, plasmacytomas, also exhibit induction of these kinases when apoptosis is induced by withdrawal of serum or IL‐6. Treatment of the pre‐B cells with ICE protease inhibitors when apoptotic stimuli are delivered prevents induction of the kinase activity, and partially inhibits apoptosis. These findings indicate that the induction of these 40 and 44 kDa p38 related kinases is a common feature of apoptosis in mouse B lymphocytic cells and may represent a step downstream of ICE proteases in the signal cascade that leads to programmed cell death.
Caveolin‐3 is not an integral component of the dystrophin glycoprotein complex
Authors: Crosbie Rachelle H , Yamada Hiroki , Venzke David P , Lisanti Michael P , Campbell Kevin P
Published: July 2002

The dystrophin‐glycoprotein complex is a multi‐subunit protein complex that spans the muscle plasma membrane (sarcolemma) and forms a link between the intracellular cytoskeleton and the extracellular matrix. Caveolin‐3, the muscle specific form of caveolin, is also a major structural and regulatory integral membrane protein found at the sarcolemma. Oligomers of caveolin‐3 form the structural framework for small membrane pockets known as caveolae. We directly examined whether caveolin‐3 is an integral component of the dystrophin‐glycoprotein complex by examining four common biochemical and cellular properties of proteins integrally bound to the dystrophin‐glycoprotein complex. We found that caveolin‐3 de‐enriches with partial purification of the dystrophin‐glycoprotein complex although a small amount of caveolin‐3 is present. Sucrose gradient fractionation and laminin affinity chromatography completely separate this residual caveolin‐3 from the core components of the dystrophin‐glycoprotein complex. We also show that caveolin‐3 expression at the sarcolemma is not reduced in patients with primary mutations in either dystrophin or the sarcoglycans. This data demonstrates that localization of caveolin‐3 to the sarcolemma occurs independently of the dystrophin‐glycoprotein complex and that caveolin‐3 is not an integral component of the dystrophin‐glycoprotein complex.
Human substance P receptor expressed in Chinese hamster ovary cells directly activates Gαq/11, Gαs, and Gαo
Authors: Roush Eric D , Kwatra Madan M
Published: July 2002

Substance P receptor (SPR) stably expressed in Chinese hamster ovary (CHO) cells stimulates at least three second messenger systems including phosphoinositide hydrolysis, cyclic AMP (cAMP) formation, and arachidonic acid release. Whether these second messenger systems are activated via single or multiple G proteins is not known. Therefore, in the present study we examined whether human SPR (hSPR) stably expressed in CHO cells activates multiple G proteins. This was achieved by photoaffinity labeling of Gα‐subunits with [32P]azidoanilido‐GTP ([32P]AA‐GTP) upon hSPR stimulation in CHO‐hSPR membranes followed by immunoprecipitation of the labeled Gα‐subunits with antibodies specific for various Gα‐subunits. These experiments reveal that hSPR directly activates Gαq/11, Gαs and Gαo. While hSPR is known to couple Gαq/11, the present study provides the first evidence that hSPR can also activate Gαs and Gαo in a mammalian system.
Isoform specific phosphorylation of protein phosphatase 2C expressed in COS7 cells
Authors: Kobayashi Takayasu , Kusuda Kazuyuki , Ohnishi Motoko , Wang Hong , Ikeda Shoko , Hanada Masahito , Yanagawa Yuchio , Tamura Shinri
Published: July 2002

Of the six distinct isoforms of mouse protein phosphatase 2C (PP2C) (α, β‐1, β‐2, β‐3, β‐4 and β‐5), PP2Cα was specifically phosphorylated on the serine residue(s) when expressed in COS7 cells. Analysis of phosphorylation sites using site‐directed mutagenesis demonstrated that Ser‐375 and/or Ser‐377 were phosphorylated in vivo. These serine residues were the sites of phosphorylation by casein kinase II in vitro. Phosphorylation of PP2Cα was enhanced two‐fold by the addition of okadaic acid to the culture medium, but addition of cyclosporin A had no such effect. These results suggest that the expressed PP2Cα is phosphorylated by a casein kinase II‐like protein kinase and dephosphorylated by PP1 and/or PP2A in COS7 cells.
The compactness of ribonuclease A and reduced ribonuclease A
Authors: Zhou Jun-Mei , Fan Ying-Xin , Kihara Hiroshi , Kimura Kazumoto , Amemiya Yoshiyuki
Published: July 2002

The compactness of ribonuclease A with intact disulfide bonds and reduced ribonuclease A was investigated by synchrotron small‐angle X‐ray scattering. The R
g values and the Kratky plots showed that non‐reduced ribonuclease A maintain a compact shape with a R
g value of about 17.3 Å in 8 M urea. The reduced ribonuclease A is more expanded, its R
g value is about 20 Å in 50 mM Tris‐HCl buffer at pH 8.1 containing 20 mM DTT. Further expansions of reduced ribonuclease A were observed in the presence of high concentrations of denaturants, indicating that reduced ribonuclease A is more expanded and is in neither a random coil [A. Noppert et al., FEBS Lett. 380 (1996) 179–182] nor a compact denatured state [T.R. Sosnick and J. Trewhella, Biochemistry 31 (1992) 8329–8335]. The four disulfide bonds keep ribonuclease A in a compact state in the presence of high concentrations of urea.
Identification of the tRNAs which up‐regulate agrostin, barley RIP and PAP‐S, three ribosome‐inactivating proteins of plant origin
Authors: Brigotti Maurizio , Keith Gérard , Pallanca Alessandra , Domenica Carnicelli  , Alvergna Paola , Dirheimer Guy , Montanaro Lucio , Sperti Simonetta
Published: July 2002

Ribosome‐inactivating proteins (RIP) are RNA‐N‐glycosidases widely diffused in plants which depurinate ribosomal RNA at a specific universally conserved position, A4324 in rat ribosomes. A small group of RIPs (cofactor‐dependent RIPs) require ATP and tRNA to reach maximal activity on isolated ribosomes. The tRNA which stimulates gelonin was identified as tRNATrp. The present paper reports the identification of three other tRNAs which stimulate agrostin (tRNAAla), barley RIP (tRNAAla, tRNAVal) and PAP‐S (tRNAGly), while for tritin‐S no particular stimulating tRNA emerged. The sequences of tRNAVal and tRNAGly correspond to the already known ones (rabbit and man, respectively). The tRNAAla (anticodon IGC) identifies a new isoacceptor. Only the stimulating activity of the tRNAAla for agrostin approaches the specificity previously observed for the couple gelonin‐tRNATrp.
Photovoltage evidence that Glu‐204 is the intermediate proton donor rather than the terminal proton release group in bacteriorhodopsin
Authors: Kalaidzidis Inna V , Belevich Ilya N , Kaulen Andrey D
Published: July 2002

Electrogenic events in the E204Q bacteriorhodopsin mutant have been studied. A two‐fold decrease in the magnitude of microsecond photovoltage generation coupled to M intermediate formation in the E204Q mutant is shown. This means that deprotonation of E204 is an electrogenic process and its electrogenicity is comparable to that of the proton transfer from the Schiff base to D85. pH dependence of the electrogenicity of M intermediate formation in the wild‐type bacteriorhodopsin reveals only one component corresponding to the protonation of D85 in the bacteriorhodopsin ground state and transition of the purple neutral form into the blue acid form. Thus, the pK of E204 in the M state is close to the pK of D85 in the bacteriorhodopsin ground state (<3) and far below the pK of the terminal proton release group (∼6). It is concluded that E204 functions as the intermediate proton donor rather than the terminal proton release group in the bacteriorhodopsin proton pump.
Purification and characterization of the 3‐chloro‐4‐hydroxy‐phenylacetate reductive dehalogenase of Desulfitobacterium hafniense
Authors: Christiansen Nina , Ahring Birgitte K , Wohlfarth Gert , Diekert Gabriele
Published: July 2002

The membrane‐bound 3‐chloro‐4‐hydroxyphenylacetate (Cl‐OHPA) reductive dehalogenase from the chlorophenol‐reducing anaerobe Desulfitobacterium hafniense was purified 11.3‐fold to apparent homogeneity in the presence of the detergent CHAPS. The purified dehalogenase catalyzed the reductive dechlorination of Cl‐OHPA to 4‐hydroxyphenylacetate with reduced methyl viologen as the electron donor at a specific activity of 103.2 nkat/mg protein. SDS‐PAGE revealed a single protein band with an apparent molecular mass of 46.5 kDa. The enzyme contained 0.68±0.2 mol corrinoid, 12.0±0.7 mol iron, and 13.0±0.7 mol acid‐labile sulfur per mol subunit. The N‐terminal amino acid sequence of the enzyme was determined and no significant similarity was found to any protein present in the gene bank.
Characterization of a novel structural member, LukE‐LukD, of the bi‐component staphylococcal leucotoxins family
Authors: Gravet A. , Colin D.A. , Keller D. , Giradot R. , Monteil H. , Prévost G.
Published: July 2002

A new member of the staphylococcal bi‐component leucotoxins family, LukE (32 kDa) and LukD (34.3 kDa) has been characterized from Staphylococcus aureus strain Newman. LukE was 58–68% identical with the class S proteins, whereas LukD was 71–77% identical with the class F proteins of the family. A partial immunoreactivity with the various affinity‐purified antibodies specific for the other proteins was observed. Immunoprecipitation assay and gene probing confirmed a 30% frequency among human clinical isolates, differing from the distribution of the other known leucotoxins (P<0.005). LukE+LukD was as effective as the Panton‐Valentine leucocidin for inducing dermonecrosis when injected in the rabbit skin, but not hemolytic and poorly leucotoxic compared to other leucotoxins expressed by Staphylococcus aureus.