bluuebunny/crossref_metadata_2025 huggingface dataset:
Hepatic glutaminase mRNA is confined to part of the urea cycle domain in the adult rodent liver lobule
Authors: Moorman Antoon F.M. , de Boer Pict A.J. , Watford Malcolm , Dingemanse Maria A. , Lamers Wouter H.
Published: July 2002

This in situ hybridization study describes the developmental appearance of the lobular distribution of the mRNA encoding hepatic glutaminase in normal rat liver. Glutaminase has been proposed to provide the urea cycle with ammonia [Hässinger and Gerok (1983) Eur. J. Biochem. 133, 269–275]. Hence, the (developmental) pattern of expression of the mRNA would be expected to be closely linked to that of the urea cycle enzymes. From embryonic day 20 onward, hepatic glutaminase mRNA can be detected along the entire porto‐central axis, with predominant expression in the portal area. In the adult phenotype, which is acquired at the end of the first postnatal week, glutaminase mRNA is no longer present along the entire porto‐central distance but has become confined to a relatively small periportal domain in which the expression decreases in a porto‐central direction. Thus, in contrast to the large periportal domain, in which the urea cycle enzymes are expressed, the glutaminase mRNA‐expressing domain is much smaller and not contiguous with the glutamine synthase mRNA‐expressing pericentral domain, leaving a midlobular area that is devoid of glutaminase mRNA. A similar pattern of distribution was found in adult mouse liver. The significance of these observations is that, within the liver lobules, there is an area in which glutaminase is not expressed and, hence, glutamine can not be the substrate for urea synthesis.
Localization of a regulatory region on the 5′‐untranslated region of human hepatoma HepG2 γ‐glutamyltransferase mRNA and response to dexamethasone and antisense oligonucleotide treatment
Authors: Diederich Marc , Wellman Maria , Siest Gérard
Published: July 2002

We are reporting the functional analysis of the 5′‐untranslated region (5′UTR) of human hepatoma HepG2 γ‐glutamyltransferase (GGT) mRNA. Transient expression of hybrid GGT‐luciferase reporter gene mutants in HepG2 shows that this 5′UTR acts as a tissue‐specific translational enhancer. A domain of 173 bases containing a steroid hormone response element (HRE) is responsible for the enhancing effect, which can be amplified by addition of dexamethasone at 10−6 M. Furthermore, the regulatory role of the 5′UTR is demonstrated by interaction with sense and antisense oligonucleotides.
Cooperativity‐regulated parallel pathways of the bacteriorhodopsin photocycle
Authors: Tokaji Zsolt
Published: July 2002

The paper demonstrates that the actinic light density dependence of the millisecond part of the bacteriorhodopsin (BR) photocycle at high pH predicts a model, which is the same in the sequence of the intermediates as concluded previously on the basis of double flash experiments [1992, FEBS Lett. 311, 267–270]. This model consists of the Mf→N→BR and Ms→BR parallel pathways, the relative yields of which are regulated by cooperative interaction of the BR molecules. The decay of Ms is always slower than the decay of Mf and described as a direct reprotonation of the Schiff‐base from the bulk, and the recovery of the ground‐state nearly at the same time. Ms is decomposed into M′f and M′s. The first does not reprotonate, and similarly to Mf, it is suggested to be before the conformational change (switch), which latter process would be just before the decay of Mf. A simple way for the determination of the kinetics is also used. This confirms that the amount of N decreases with increasing fraction cycling and shows that the decay rate of N is independent of the fraction cycling. The differences in the kinetics are compared to each other, and they seem to allow a new way of kinetic evaluation at least under special conditions. The aim of this paper was briefly explained in my poster presented on the VIth International Conference on Retinal Proteins (see [14]).
Differential expression of PKCα and PKCβ isozymes in CD4+, CD8+ and CD4+/CD8+ double positive human T cells
Authors: Tarantino Nadine , Debré Patrice , Körner Marie
Published: July 2002

Using specific monoclonal and polyclonal antibodies, we have analyzed protein kinase C α and β isozyme expression in human T cells from peripheral blood (PB) and from thymus. While the PKCβ isozyme was present in all T cell sub‐types isolated from both PB and thymus, the α isozyme was found only in single positive CD4+ thymocytes, in PB‐CD4+ lymphocytes and in PB‐CD8+ T cells from several donors. It was absent from both, CD8+and double positive CD4+/CD8+ thymocytes. These results show that PKCα and ‐β are differentially regulated during intra‐thymic development and suggest that PKCα plays a specific role in helper T cell function.
Insect calcium channels
Authors: Grabner Manfred , Bachmann Andreas , Rosentha Friederike , Striessnig Jörg , Schulz Cordula , Tautz Diethard , Glossmann Hartmut
Published: July 2002

The complete amino acid sequence of an invertebrate calcium channel α‐subunit from housefly (Musca domestica) larvae (designated Mdlα1) has been deduced by cDNA cloning and sequence analysis. Mdlα1 shares higher percent sequence identity with 1,4‐dihydropyridine (DHP)‐sensitive L‐type than with DHP‐insensitive calcium channels. As shown by whole mount in situ hybridization and immunostaining Mdlα, is predominantly expressed in the larval body wall musculature.
Both interleukin‐8 receptors independently mediate chemotaxis
Authors: Loetscher Pius , Seitz Michael , Clark-Lewis Ian , Baggiolini Marco , Moser Bernhard
Published: July 2002

Neutrophil leukocytes, the target cells for interleukin‐8 and related CXC chemokines, bear high numbers of two types of IL‐8 receptors (IL‐8R1 and IL‐8R2). By cDNA transfection Jurkat cell lines were generated that stably express either IL‐8R1 or IL‐8R2 (J‐I8L8R1 and J‐IL8R2). J‐IL8R1 expressed 4,000 ± 1,000 copies of IL‐8R 1, and bound IL‐8 with high affinity (K
d 1–4 nM) and GROα and NAP‐2 with low affinity (K
d 200–500 nM). J‐IL8R2 expressed 17,000 ± 3,000 copies of IL‐8R2, and bound all three chemokines with high affinity. Both transfectants showed a similar degree of chemotactic migration after stimulation with IL‐8, GROa and NAP‐2. All three chemokines were equally potent as attractants of J‐IL8R2, whereas IL‐8 was 300 to 1,000‐fold more potent than GROα or NAP‐2 as attractant of J‐IL8R1. The potencies, therefore, agree with the affinities of the ligands to IL‐8R1 and IL‐8R2. Our results demonstrate that both IL‐8 receptors function independently, and mediate chemotaxis in response to IL‐8 and other CXC chemokines.
Protein kinase‐C mediates dual modulation of L‐type Ca2+ channels in human vascular smooth muscle
Authors: Schuhmann Klaus , Groschner Klaus
Published: July 2002

The role of protein kinase C (PKC) in cellular regulation of L‐type Ca2+ channels was investigated in human umbilical vein smooth muscle. Activation of PKC, by low concentrations (< 30 nM) of 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TEA) caused inhibition of Ca2+ channels, while higher concentrations of TPA (>100 nM) elicited a transient rise, followed by sustained inhibition of Ca2+ channel activity in cell‐attached patches. Low TPA concentrations predominantly reduced channel availability, while high concentrations of TPA (100 nM) transiently increased channel availability and, in addition, prolonged mean open time. The inactive 4‐α‐phorbol‐12,13‐didecanoate failed to affect channel activity, and pretreatment of the cells with PKC inhibitors (H‐7, chelerythrine) antagonized inhibitory and stimulatory effects of TPA. Our results provide evidence for two distinct PKC‐dependent mechanisms of L‐type Ca2+ channel regulation in smooth muscle.
Evidence for Rap1 in vascular smooth muscle cells
Authors: Quarck R. , Bryckaert M. , Magmer C. , Corvazier E. , Bredoux R. , de Gunzburg J. , Fontenay M. , Tobelem G. , Enouf J.
Published: July 2002

The effect of platelet‐derived growth factor (PDGF) on Rap1 expression was investigated in rat vascular smooth muscle cells (SMC). First, evidence for Rap1 proteins was shown by their: (i) detection in membranes using a specific anti‐Rap1 antibody, (ii) typical shift in electrophoretic mobility as a consequence of reduction, and (iii) cAMP‐induced phosphorylation and immunoprecipitation. Then, the mitogenic activity of 10 PDGF AA and BB for 48 h, resulting in a 2‐ and 5‐fold increase in [3H]thymidine incorporation, was correlated with that of total Rap1 protein expression which was found to be 99% ± 36% and 260% ± 70%, respectively. Further time‐course studies established that this up‐regulation of Rap1 proteins was only observed after 48 h of PDGF BB treatment. Lastly, comparative RT‐PCR of both rap1a and rap1b mRNAs showed that PDGF BB also up‐regulated the rap1a mRNA species, which was 1.5‐fold increased in contrast with the rap1b mRNA species. It is concluded that the PDGF BB‐induced SMC proliferation is associated with an up‐regulation of Rap1a protein.
The thrombin receptor in human platelets is coupled to a GTP binding protein of the Gαq family
Authors: Benka Mary Lynn , Lee Matt , Wang Guang-Rong , Buckman ShaAvhree , Burlacu Anda , Cole Laura , DePina Ana , Dias Prabha , Granger Aaron , Grant Bob , Hayward-Lester Amanda , Karki Sher , Mann Suzanne , Marcu Oana , Nussenzweig Andre , Piepenhagen Peter , Raje Manisha , Roegiers Fabrice , Rybak Sheree , Salic Adrian , Smith-Hall Jennifer , Waters Jennifer , Yamamoto Naoyuki , Yanowitz Judith , Yeow Karen , Busa William B. , Mendelsohn Michael E.
Published: July 2002

The thrombin receptor is a G protein‐coupled receptor, but the G proteins functionally coupled to this receptor in human platelets are not yet definitively identified. Thrombin stimulation of platelets leads to phospholipase C‐mediated increases in intracellular calcium, and previous studies have suggested that the thrombin receptor is coupled to members of the Gq family. We now demonstrate direct GTPase activation by thrombin receptor activation peptide (TRAP) in human platelet membranes, and specific inhibition of TRAP‐activated GTPase by antibodies to Gq. These data demonstrate functional coupling of the thrombin receptor to a member of the Gq family.
C‐type natriuretic peptide and brain natriuretic peptide inhibit adenylyl cyclase activity: interaction with ANF‐R2/ANP‐C receptors
Authors: Savoie Pierre , de Champlain Jacques , Anand-Srivastava Madhu B.
Published: July 2002

C‐type natriuretic peptide (CNP) and brain natriuretic peptide (BNP) are members of the natriuretic peptide family, which have been shown to interact with ANP‐C/ANF‐R2 receptors in addition to ANP‐B receptor subtypes. The present study was undertaken to investigate if the interaction of CNP and BNP with ANP‐C receptors results in the inhibition of adenylyl cyclase activity. CNP and BNP inhibited adenylyl cyclase activity in heart and brain striatal membranes in a concentration dependent manner with an apparent Ki between 0.1 and 1.0 nM. Maximal inhibition observed in heart membranes were about 25% and 35% for BNP and CNP respectively, however the inhibitions in brain striatal membranes were smaller (∼2%). The inhibition was dependent on the presence of guanine nucleotides and was attenuated by pertussis toxin treatment. In addition, CNP inhibited the stimulatory effect of isoproterenol on adenylyl cyclase, whereas CNP as well as BNP showed an additive effect with the inhibitory response of angiotensin II on adenylyl cyclase activity. When the combined effect of C‐ANF4−23/BNP, C‐ANF4−23/CNP and BNP/CNP at optimal concentrations was studied together on adenylyl cyclase activity, the percent inhibition remained the same for C‐ANF4−23 and BNP or C‐ANF4−23 and CNP, however, an additive inhibitory effect was observed for BNP and CNP. These results suggest that CNP and BNP like C‐ANF4−23 interact with ANP‐C receptors and result in the inhibition of adenylyl cyclase activity. On the other hand, CNP and BNP interact with the ANP‐C receptor, however, the interaction may be at different sites or there may be two subpopulations of ANP‐C receptors specific for each of the peptides. These results indicate that BNP and CNP, like ANP and C‐ANF4−23, inhibit the adenylyl cyclase/cAMP signal transduction system through an inhibitory guanine nucleotide regulatory protein, by interacting with ANP‐C receptor subtypes.
Dephosphorylation of the focal adhesion protein VASP in vitro and in intact human platelets
Authors: Abel Kathrin , Mieskes Gottfried , Walter Ulrich
Published: July 2002

The focal adhesion protein VASP, a possible link between signal transduction pathways and the microfilament system, is phosphorylated by both cAMP‐ and cGMP‐dependent protein kinases in vitro and in intact cells. Here, the analysis of VASP dephosphorylation by the serine/threonine protein phosphatases (PP) PP1, PP2A, PP2B and PP2C in vitro is reported. The phosphatases differed in their selectivity with respect to the dephosphorylation of individual VASP phosphorylation sites. Incubation of human platelets with okadaic acid, a potent inhibitor of PP1 and PP2A, caused the accumulation of phosphorylated VASP indicating that the phosphorylation status of VASP in intact cells is regulated to a major extent by serine/ threonine protein phosphatases. Furthermore, the accumulation of phosphorylated cAMP‐dependent protein kinase substrate(s) appears to account for inhibitory effects of okadaic acid on platelet function.
Expression of human inducible nitric oxide synthase in Escherichia coli
Authors: Fossetta James D. , Niu Xiao Da , Lunn Charles A. , Zavodny Paul J. , Narula Satwant K. , Lundell Daniel
Published: July 2002

We have expressed active full‐length human inducible nitric oxide synthase (iNOS) in E. coli. Expression required co‐expression with calmodulin, a particularly tight‐binding cofactor. The extracts also required tetrahydrobiopterin to display activity. Specific activity of the purified recombinant iNOS was similar to iNOS purified from murine macrophages. This result indicates that no special processing events unique to eucaryotic cells are necessary for iNOS activity.
Molecular cloning and characterization of a novel orphan receptor (P2P) expressed in human pancreas that shows high structural homology to the P2U purinoceptor
Authors: Stam Nico J. , Klomp Jan , van de Heuvel Marcel , Olijve Wiebe
Published: July 2002

Here we report the cloning of a gene encoding a new member of the superfamily of G protein‐coupled receptors. The gene encodes a protein of 365 amino acids closely resembling two recently cloned nucleotide binding receptors, called P2U and P2Y purinoceptors (71% and 49% sequence identity within the transmembrane domains, respectively). Our studies show that this new putative purinoceptor (designated P2P) is encoded by an intronless single copy gene that is exclusively expressed in pancreas, in contrast to the P2U and the P2Y purinoceptors which are widely distributed throughout the periphery. The identification of a pancreas‐specific human putative P2 purinoceptor makes it attractive to speculate that the reported actions of ADP/ATP analogues in pancreas on insulin secretion are mediated through this receptor.
Blockade of HERG channels expressed in Xenopus oocytes by the histamine receptor antagonists terfenadine and astemizole
Authors: Suessbrich H. , Waldegger S. , Lang F. , Busch A.E.
Published: July 2002

The widely used histamine receptor antagonists terfenadine and astemizole were shown to prolong the QT interval in electrocardiographic recordings in cases of overdose or inappropriate co‐medications, indicating a possible interaction with cardiac K+ channels. Here, terfenadine and astemizole both inhibited the human ether‐a‐go‐go related gene (HERG) encoded channels expressed in Xenopus oocytes at nanomolar concentrations in a use‐ and voltage‐dependent fashion. In contrast, inhibition of other delayed rectifier (Kvl.1 and IsK) or inward rectifier K+ channels (IRK1) was much weaker and occurred only at high micromolar concentrations. These results suggest that blockade of HERG channels by terfenadine and astemizole might contribute to the cardiac side effects of these compounds.
Fetal myosin heavy chain increases in the human masseter muscle during aging
Authors: Monemi Mehrdad , Eriksson Per-Olof , Dubail Ihari , Butler-Browne Gillian S. , Thornell Lars-Erik
Published: July 2002

Biochemical, immunohistochemical and molecular biological methods were used to detect fetal myosin heavy chain (MyHC) in the human masseter of elderly and young subjects. Samples from the elderly subjects contained larger amounts of fetal MyHC than those of young adults. Only a very small amount of embryonic MyHC could be detected in both age groups. Embryonic and fetal MyHCs were never detected in the control adult orofacial, limb and trunk muscles. Polymerase chain reaction (PCR) analysis revealed the presence of fetal mRNA sequences in elderly and young masseter muscles. We conclude that fetal MyHC is present in the human masseter throughout the life span and that there is an increase in the relative amount of this protein with age.
Analysis of basic fibroblast growth factor in rats with inherited retinal degeneration
Authors: McLaren M.J. , An W. , Brown M.E. , Inana G.
Published: July 2002

In RCS rats, photoreceptors degenerate between postnatal days 20 and 60, secondary to a genetic defect expressed in the neonatal retinal pigmented epithelium (RPE). Previous work has shown delay of the photoreceptor degeneration in this model by intraocular injection of basic fibroblast growth factor (bFGF). Evidence is presented here, from bFGF immunostaining and Northern analysis of bFGF mRNA, for reduced bFGF expression in uncultured RPE of dystrophic RCS pups. It is also shown that in the mutant eyes angiogenesis in the underlying choroid, which normally occurs between postnatal days 7 and 10, is markedly delayed, with irregular distribution of vessels, consistent with a reduction in this known angiogenesis factor. Mutational analysis of the bFGF transcript and gene by denaturing gradient gel electrophoresis and Southern analysis did not, however, reveal abnormalities in the coding sequence of this gene in RCS rats.
The phosphorylation state of MAP‐kinases modulates the cytotoxic response of smooth muscle cells to hydrogen peroxide
Authors: Cantoni Orazio , Boscoboinik Daniel , Fiorani Mara , Stäuble Barbara , Azzi Angelo
Published: July 2002

Micromolar concentrations of hydrogen peroxide induced the phosphorylation of mitogen‐activated protein (MAP) kinases and a lethal response in growth‐arrested smooth muscle cells (A7r5). The H2O2‐induced phosphorylation of MAP‐kinases was markedly lower in the presence of protein tyrosine kinase (PTK) inhibitors or in protein kinase C (PKC) down‐regulated cells. Similarly, the toxicity of H2O2 was diminished by concomitant addition of either PKC or PTK inhibitors and was also lower in PKC down‐regulated cells. These results are consistent with the possibility that phosphorylation of MAP‐kinases is a critical event in the toxic response of cultured smooth muscle cells to H2O2.
Depletion of intracellular calcium stores activates an outward potassium current in mast and RBL‐1 cells that is correlated with CRAC channel activation
Authors: Hoth Markus
Published: July 2002

Highly Ca2+ selective Ca2+ channels activated by store depletion have been recently described in several cell types and have been termed CRAC channels (for calcium release‐activated calcium). The present study shows that following store depletion in mast and RBL‐1 cells, monovalent outward currents could be recorded if the internal solution contained K+ but not Cs+. The activation of the outward K+ current correlated with the activation of ICRAC, in both time and amplitude, suggesting that the K+ current might be carried by CRAC channels. The amplitude of the outward current was increased if external Ca2+ was reduced or replaced by external Ba2+. The outward K+ conductance might have a physiological role in maintaining the driving force for Ca2+ entry during the activation of CRAC channels.
DNA‐nuclease activity of the single‐chain ribosome‐inactivating proteins dianthin 30, saporin 6 and gelonin
Authors: Roncuzzi L. , Gasperi-Campani A.
Published: July 2002

The single‐chain ribosome‐inactivating proteins (sc‐RIPs) from plant origin are antiviral and antiproliferative agents employed in the preparation of immunotoxins. Similarly to the A‐chains of ricin, sc‐RIPs act as rRNA N‐glycosidases. We demonstrate here that dianthin 30, saporin 6 and gelonin exert a specific nuclease activity on supercoiled DNA. Four specific sites of cleavage introduced by dianthin 30 and by saporin 6 and two specific sites of cleavage introduced by gelonin have been identified and mapped in pBR322.
Investigations of cerebrospinal fluid in Borna disease virus seropositive psychiatric patients
Authors: Bechter K , Herzog S , Behr W , Schüttler R
Published: July 2002

Borna disease virus (BDV) appears to cause meningoencephalitis and schizophreniform psychosis in sporadic cases according to earlier cerebrospinal fluid (CSF) inoculation experiments (Rott et al, 1991). However, CSF parameters in BDV seropositive psychiatric patients proved nearly all normal; only the most sensitive CSF/serum index I-BDV for intrathecally produced BDV specific IgG was pathologic in 10.5–29.0% (according to different methodological limits) of patients. An increase in sensitivity was attempted to detect specific IgG in CSF in a part of the cases by concentration. Concentration procedure does not significantly increase methodological bias according to a statistical analysis of the results. Our findings support the hypothesis that BDV may cause or contribute to the pathogenesis of a diagnostically broad pattern of psychiatric syndromes. The occurence of a spectrum of diagnoses is expected from non-specificity of psychiatric symptoms in other infectious diseases of the brain as well as from results in experimental Borna disease (BD) in animals, when a majority of the animals showed rather unspecific symptomatology due to slight, preferentially limbic encephalitis. Slight deficiencies from an earlier BDV infection could explain continuing symptoms in a part of the cases. Recurrences years after infection are well known in experimental and natural BD in animals. It remains open, whether this mechanism could play a more prominent role in a form of “symptomatic” cyclothymia and “symptomatic” schizophrenia, although the results of CSF investigations are more clear in BDV seropositive patients with major psychoses.
Structural characterisation of the native fetuin‐binding protein Scilla campanulata agglutinin: a novel two‐domain lectin
Authors: Wright Lisa M. , Reynolds Colin D. , Rizkallah Pierre J. , Allen Anthony K. , Van Damme Els J.M. , Donovan Michael J. , Peumans Willy J.
Published: July 2002

The three‐dimensional structure of a 244‐residue, multivalent, fetuin‐binding lectin, SCAfet, isolated from bluebell (Scilla campanulata) bulbs, has been solved at 3.3 Å resolution by molecular replacement using the coordinates of the 119‐residue, mannose‐binding lectin, SCAman, also from bluebell bulbs. Unlike most monocot mannose‐binding lectins, such as Galanthus nivalis agglutinin from snowdrop bulbs, which fold into a single domain, SCAfet contains two domains with approximately 55% sequence identity, joined by a linker peptide. Both domains are made up of a 12‐stranded β‐prism II fold, with three putative carbohydrate‐binding sites, one on each subdomain. SCAfet binds to the complex saccharides of various animal glycoproteins but not to simple sugars.
First evidence and characterization of an uncoupling protein in fungi kingdom: CpUCP of Candida parapsilosis
Authors: Jarmuszkiewicz Wiesława , Milani Graziela , Fortes Fabiane , Schreiber Angelica Z. , Sluse Francis E. , Vercesi Anibal E.
Published: July 2002

An uncoupling protein (UCP) was identified in mitochondria from Candida parapsilosis (CpUCP), a non‐fermentative parasitic yeast. CpUCP was immunodetected using polyclonal antibodies raised against plant UCP. Activity of CpUCP, investigated in mitochondria depleted of free fatty acids, was stimulated by linoleic acid (LA) and inhibited by GTP. Activity of CpUCP enhanced state 4 respiration by decreasing ΔΨ and lowered the ADP/O ratio. Thus, it was able to divert energy from oxidative phosphorylation. The voltage dependence of electron flux indicated that LA had a pure protonophoretic effect. The discovery of CpUCP proves that UCP‐like proteins occur in the four eukaryotic kingdoms: animals, plants, fungi and protists.
The involvement of p38 mitogen‐activated protein kinase in the α‐melanocyte stimulating hormone (α‐MSH)‐induced melanogenic and anti‐proliferative effects in B16 murine melanoma cells
Authors: Smalley Keiran , Eisen Tim
Published: July 2002

Activation of p38 or p44/42 mitogen‐activated protein (MAP) kinases has been shown to trigger differentiation in a number of cell types. The present study has investigated the roles of these kinases in the α‐melanocyte stimulating hormone (α‐MSH)‐induced melanogenic and proliferative responses in B16 melanoma cells. Treatment of cells with α‐MSH led to the time‐dependent phosphorylation of both p38 and p44/42 MAP kinases. However, only inhibition of p38 MAP kinase activity with SB 203580 blocked both the α‐MSH‐induced melanogenic and anti‐proliferative effects. It therefore appears that activation of the p38 pathway can promote melanogenesis and inhibit growth of B16 melanoma cells.
A direct photo‐activated affinity modification of tetracycline transcription repressor protein TetR(D) with tetracycline
Authors: Beliakova Maria M. , Anokhina Maria M. , Spiridonova Vera A. , Dobrov Eugeny N. , Egorov Tsezi A. , Wittmann-Liebold Brigitte , Orth Peter , Saenger Wolfram , Kopylov Alexei M.
Published: July 2002

Results of a first successful application of a direct photo‐induced affinity modification of Tet repressor (TetR(D)) protein with tetracycline within a complex of known three‐dimensional structure are described. The conditions of the modification have provided suitable yields of the modified complex and allowed characterization of the modified segments of the protein. The potential of tetracycline as a fine modifying reagent was established. In the complex of TetR(D) protein with tetracycline, the antibiotic modifies at least two segments, Ile59–Glu73 and Ala173–Glu183, which form a binding tunnel for the drug according to the X‐ray analysis. These data open possibilities for the use of different tetracycline targets for structural studies in solution.
Systematic mapping of regions of human cardiac troponin I involved in binding to cardiac troponin C: N‐ and C‐terminal low affinity contributing regions
Authors: Ferrières Gaëlle , Pugnière Martine , Mani Jean-Claude , Villard Sylvie , Laprade Michel , Doutre Patrick , Pau Bernard , Granier Claude
Published: July 2002

The Spot method of multiple peptide synthesis was used to map in a systematic manner regions of the human cardiac troponin I sequence (hcTnI) involved in interactions with its physiological partner, troponin C (cTnC). Ninety‐six 20‐mer peptides describing the entire hcTnI sequence were chemically assembled; their reactivity with [125I]cTnC, in the presence of 3 mM Ca2+, enabled the assignment of six sites of interaction (residues 19–32, 45–54, 129–138, 145–164, 161–178 and 191–210). For several sites, a good correlation with literature data was obtained, thus validating this methodological approach. Synthetic peptides, each containing in their sequence an interaction site, were prepared. As assessed by BIACORE, all of them exhibited an affinity for cTnC in the range of 10−6–10−7 M, except for hcTnI [39–58] which showed a nanomolar affinity. This peptide was also able to block the interaction between hcTnI and cTnC. We therefore postulate that despite the existence of multiple cTnC interaction sites on the hcTnI molecule, only that region of hcTnI allows a stabilization of the complex. Residues 19–32 from the N‐terminal cardio‐specific extension of hcTnI were also found to be involved in interaction with cTnC; residues 19–32 may correspond to the minimal sequence of the extension which could switch between the N‐ and C‐terminal TnC domains, depending on its phosphorylation state. Finally, two Ca2+‐dependent cTnC binding domains within the C‐terminal part of hcTnI (residues 164–178 and 191–210) were also mapped. The latter site may be linked with the cardiac dysfunction observed in stunned myocardium.
Phosphorylation of telokin by cyclic nucleotide kinases and the identification of in vivo phosphorylation sites in smooth muscle
Authors: MacDonald Justin A. , Walker Lori A. , Nakamoto Robert K. , Gorenne Isabelle , Somlyo Avril V. , Somlyo Andrew P. , Haystead Timothy A.J.
Published: July 2002

The Ca2+‐independent acceleration of dephosphorylation of the regulatory light chain of smooth muscle myosin and relaxation of smooth muscle by telokin are enhanced by cyclic nucleotide‐activated protein kinase(s) [Wu et al. (1998) J. Biol. Chem. 273, 11362–11369]. The purpose of this study was to determine the in vivo site(s) and in vitro rates of telokin phosphorylation and to evaluate the possible effects of sequential phosphorylation by different kinases. The in vivo site(s) of phosphorylation of telokin were determined in rabbit smooth muscles of longitudinal ileum and portal vein. Following stimulation of ileum with forskolin (20 μM) the serine at position 13 was the only amino acid to exhibit increased phosphorylation. Rabbit portal vein telokin was phosphorylated on both Ser‐13 and ‐19 as a result of forskolin and GTPγS stimulation in vivo. Point mutation of Ser‐13 (to Ala or Asp) abolished in vitro phosphorylation by cyclic nucleotide‐dependent protein kinases.
Electrogenic uptake of nucleosides and nucleoside‐derived drugs by the human nucleoside transporter 1 (hCNT1) expressed in Xenopus laevis oocytes
Authors: Lostao M.Pilar , Mata Joao F. , Larrayoz Ignacio M. , Inzillo Simone M. , Casado F.Javier , Pastor-Anglada Marçal
Published: July 2002

The concentrative pyrimidine‐preferring nucleoside transporter 1 (hCNT1), cloned from human fetal liver, was expressed in Xenopus laevis oocytes. Using the two‐electrode voltage‐clamp technique, it is shown that translocation of nucleosides by this transporter generates sodium inward currents. Membrane hyperpolarization (from −50 to −150 mV) did not affect the K
0.5 for uridine, although it increased the transport current approximately 3‐fold. Gemcitabine (a pyrimidine nucleoside‐derived drug) but not fludarabine (a purine nucleoside‐derived drug) induced currents in oocytes expressing the hCNT1 transporter. The K
0.5 value for gemcitabine at −50 mV membrane potential was lower than that for natural substrates, although this drug induced a lower current than uridine and cytidine, thus suggesting that the affinity binding of the drug transporter is high but that translocation occurs more slowly. The analysis of the currents generated by the hCNT1‐mediated transport of nucleoside‐derived drugs used in anticancer and antiviral therapies will be useful in the characterization of the pharmacological profile of this family of drug transporters and will allow rapid screening for uptake of newly developed nucleoside‐derived drugs.
Control of interleukin‐18 secretion by dendritic cells: role of calcium influxes
Authors: Gardella Stefania , Andrei Cristina , Poggi Alessandro , Zocchi M.Raffaella , Rubartelli Anna
Published: July 2002

Here we show that dendritic cells accumulate the precursor form of the leaderless secretory protein interleukin‐18 (pro‐interleukin‐18) in the cell cytosol and in organelles co‐fractionating with endolysosomes. Upon antigen specific contact with T lymphocytes, particulated pro‐interleukin‐18 decreases rapidly, and the cytokine appears extracellularly, suggesting that exocytosis of pro‐interleukin‐18‐containing organelles is induced. Exocytosis of secretory lysosomes is modulated by calcium: in agreement with this, calcium influx results in secretion of pro‐interleukin‐18. In turn, pro‐interleukin‐18 secretion induced by T cells is prevented by the calcium channel blocker nifedipine. Our results demonstrate a novel, calcium‐mediated mechanism of post‐translational regulation of secretion for interleukin‐18, that allows a fast release of the cytokine.
The T cell antigen receptor activates phosphatidylinositol 3‐kinase‐regulated serine kinases protein kinase B and ribosomal S6 kinase 1
Authors: Lafont Virginie , Astoul Emmanuelle , Laurence Arian , Liautard Janny , Cantrell Doreen
Published: July 2002

The present study has explored T cell antigen receptor‐regulated serine kinases in human T cells. The results identify two phosphatidylinositol 3‐kinase (PI3K)‐controlled serine kinases operating downstream of the T cell receptor (TCR) in primary T cells: (i) protein kinase B whose activation regulates the phosphorylation of glycogen synthase kinase 3 and (ii) ribosomal S6 kinase 1, a kinase with a critical role in the regulation of protein synthesis and cell growth. T cells express two isoforms of S6k1: a 70 kDa cytoplasmic kinase and an 85 kDa isoform that has a classic nuclear localisation. TCR ligation triggers a parallel engagement of both the 70 and 85 kDa isoforms of S6k1 in a response that requires PI3K function.
Pyrimidine tract binding protein and La autoantigen interact differently with the 5′ untranslated regions of lentiviruses and oncoretrovirus mRNAs
Authors: Waysbort Axel , Bonnal Sophie , Audigier Sylvie , Estève Jean-Pierre , Prats Anne-Catherine
Published: July 2002

Retrovirus genomic mRNA exhibits a several hundred nucleotides‐long untranslated region (5′ UTR) which encloses many control elements required for retrovirus replication. In addition, this 5′ UTR contains translation regulatory elements, such as internal ribosome entry sites (IRESes) that have been described in oncoretroviruses, as well as in lentiviruses. UV cross‐linking experiments suggested that the pyrimidine tract binding protein (PTB), a cellular protein known to regulate the activity of several picornaviral IRESes, binds to human T‐cell leukemia virus (HTLV)‐I RNA but not to lentiviral human immunodeficiency virus (HIV)‐1, HIV‐2 or simian immunodeficiency virus RNAs. To calculate the affinity of such RNA–protein interactions, we developed a new method based on the BIAcore technology. The absence of affinity of PTB for lentiviral RNAs was confirmed, whereas its affinity for HTLV‐I RNAs was 1000‐fold lower than for picornaviral RNAs. The BIAcore technology also revealed a significant affinity of the La autoantigen, previously described for its involvement in translational control of viral mRNAs, for HIV‐1 and HTLV‐I RNAs. Addition of recombinant PTB to in vitro translation experiments weakly enhanced translation initiation in the presence of HTLV‐I IRES, suggesting that such an IRES requires additional trans‐acting factors.
Regulation of the activity of branched‐chain 2‐oxo acid dehydrogenase (BCODH) complex by binding BCODH kinase
Authors: Obayashi Mariko , Sato Yuzo , Harris Robert A , Shimomura Yoshiharu
Published: July 2002

Branched‐chain 2‐oxo acid dehydrogenase (BCODH) kinase is responsible for inactivation of BCODH complex by phosphorylation of the complex. Activity of the kinase towards its substrate, the E1 component of the BCODH complex, is known dependent upon binding of the kinase to the E2 component. The possible existence as well as importance of unbound mitochondrial BCODH kinase has been largely ignored in previous studies. Evidence is presented here for the existence of free and bound BCODH kinase in the matrix space of rat liver mitochondria. Furthermore, in female rats, in which diurnal variations in liver BCODH complex and kinase activities occur, the amount of the kinase bound to the complex changes between morning and evening without a change in total kinase protein. Activity of the kinase correlates with the amount of bound rather than total kinase protein, suggesting only the bound form is active. Changes in amount of kinase bound and therefore active appear responsible for diurnal variation in BCODH complex activity in the female rat. We propose that change in the amount of bound BCODH kinase is a key feature of a novel regulatory mechanism for determining the activity state of the BCODH complex.
Direct observation of two distinct affinity conformations in the T state human deoxyhemoglobin
Authors: Shibayama Naoya , Saigo Satoshi
Published: July 2002

The main features of cooperative oxygenation of human hemoglobin have been described by assuming the equilibrium between two affinity conformations of the entire molecule, T and R. However, the molecular basis for explaining the wide variation in the O2 affinities of the deoxy T state has remained obscure. We address this long‐standing issue by trapping the conformational states of deoxyhemoglobin molecules within wet porous transparent silicate sol‐gels. The equilibrium O2 binding measurements of the encapsulated deoxyhemoglobin samples showed that deoxyhemoglobin free of anions coexists in two conformations that differ in O2 affinity by 40 times or more, and addition of inositol hexaphosphate to this anion‐free deoxyhemoglobin brings about a very slow redistribution of these affinity conformations. These results are the first, direct demonstration of the existence of equilibrium between two (at least two) functionally distinguishable conformational states in the T state deoxyhemoglobin.
Akt mediates insulin induction of glucose uptake and up‐regulation of GLUT4 gene expression in brown adipocytes
Authors: Hernandez Rosario , Teruel Teresa , Lorenzo Margarita
Published: July 2002

Insulin acutely stimulated glucose uptake in rat primary brown adipocytes in a PI3‐kinase‐dependent but p70S6‐kinase‐independent manner. Since Akt represents an intermediate step between these kinases, this study investigated the contribution of Akt to insulin‐induced glucose uptake by the use of a chemical compound, ML‐9, as well as by transfection with a dominant‐negative form of Akt (ΔAkt). Pretreatment with ML‐9 for 10 min completely inhibited insulin stimulation of (1) Akt kinase activity, (2) Akt phosphorylation on the regulatory residue Ser473 but not on Thr308, and (3) mobility shift in Akt1 and Akt2. However, ML‐9 did not affect insulin‐stimulated PI3‐kinase nor PKCζ activities. In consequence, ML‐9 precluded insulin stimulation of glucose uptake and GLUT4 translocation to plasma membrane (determined by Western blot), without any effect on the basal glucose uptake. Moreover, ΔAkt impaired insulin stimulation of glucose uptake and GFP‐tagged GLUT4 translocation to plasma membrane in transiently transfected immortalised brown adipocytes and HeLa cells, respectively. Furthermore, ML‐9 treatment for 6 h down‐regulated insulin‐induced GLUT4 mRNA accumulation, without affecting GLUT1 expression, in a similar fashion as LY294002. Indeed, co‐transfection of brown adipocytes with ΔAkt precluded the transactivation of GLUT4‐CAT promoter by insulin in a similar fashion as a dominant‐negative form of PI3‐kinase. Our results indicate that activation of Akt may be an essential requirement for insulin regulation of glucose uptake and GLUT4 gene expression in brown adipocytes.
Pinopsin expressed in the retinal photoreceptors of a diurnal gecko
Authors: Taniguchi Yuki , Hisatomi Osamu , Yoshida Masao , Tokunaga Fumio
Published: July 2002

Retinal cDNAs encoding the putative opsins, dg3 and dg4, were isolated from a diurnal gecko, Phelsuma madagascariensis longinsulae. dg3 mRNA is localized in about 20% of the thin members of type C double cones, and likely encodes an opsin of the ultraviolet‐sensitive pigment. Surprisingly, dg4 is very similar to chicken pinopsin, a pineal‐specific photoreceptive molecule. An anti‐dg4 antiserum recognized a small population of photoreceptor outer segments in the retina and a large number of pinealocytes. Our results suggest that P. m. longinsulae expresses pinopsin in its retina, which usually plays a role as a photoreceptive molecule in the pineal organ.
Gastrin‐induced DNA synthesis requires p38‐MAPK activation via PKC/Ca2+ and Src‐dependent mechanisms
Authors: Dehez Stephanie , Daulhac Laurence , Kowalski-Chauvel Aline , Fourmy Daniel , Pradayrol Lucien , Seva Catherine
Published: July 2002

We present evidence that gastrin, binding to a G protein‐coupled receptor, activates the p38‐mitogen‐activated protein kinase (MAPK) pathway. Blockage of protein kinase C (PKC) by GF109203X, depletion of intracellular calcium by thapsigargin or inhibition of Src family kinases by PP2 prevented p38‐MAPK activation and the Src kinase activity stimulated by gastrin. Inhibition of the PI 3‐kinase by wortmannin or LY294002 did not affect these responses. In addition, the p38‐MAPK inhibitor, SB203580, repressed gastrin‐induced [3H]thymidine incorporation, indicating a major role of p38‐MAPK in the growth‐promoting effect of gastrin. Our results demonstrate that gastrin‐induced DNA synthesis requires p38‐MAPK activation through mechanisms that involve calcium mobilization, PKC and Src family kinases.
The role of the N‐terminal domain of photoactive yellow protein in the transient partial unfolding during signalling state formation
Authors: van der Horst Michael A , van Stokkum Ivo H , Crielaard Wim , Hellingwerf Klaas J
Published: July 2002

It is shown that the N‐terminal domain of photoactive yellow protein (PYP), which appears relatively independently folded in the ground state of the protein, plays a key role in the transient unfolding during signalling state formation: genetic truncation of the N‐terminal domain of PYP significantly decreases the extent of cooperativity of the titration curve that describes chromophore protonation in the ground state of PYP, which is in agreement with the notion that the N‐terminal domain is linked through a hydrogen‐bonding network with the chromophore‐containing domain of the protein. Furthermore, deletion of the N‐terminal domain completely abolishes the non‐linearity of the Arrhenius plot of the rate of ground state recovery.
Interaction of human recombinant αA‐ and αB‐crystallins with early and late unfolding intermediates of citrate synthase on its thermal denaturation
Authors: Rajaraman K , Raman B , Ramakrishna T , Rao Ch.Mohan
Published: July 2002

We have investigated the role of recombinant human αA‐ and αB‐crystallins in the heat‐induced inactivation and aggregation of citrate synthase. Homo‐multimers of both αA‐ and αB‐crystallins confer protection against heat‐induced inactivation in a concentration‐dependent manner and also prevent aggregation. Interaction of crystallins with early unfolding intermediates of citrate synthase reduces their partitioning into aggregation‐prone intermediates. This appears to result in enhanced population of early unfolding intermediates that can be reactivated by its substrate, oxaloacetate. Both these homo‐multimers do not form a stable complex with the early unfolding intermediates. However, they can form a soluble, stable complex with aggregation‐prone late unfolding intermediates. This soluble complex formation prevents aggregation. Thus, it appears that the chaperone activity of α‐crystallin involves both transient and stable interactions depending on the nature of intermediates on the unfolding pathway; one leads to reactivation of the enzyme activity while the other prevents aggregation.
Protein dynamics, folding and misfolding: from basic physical chemistry to human conformational diseases
Authors: Ferreira Sérgio T , De Felice Fernanda G
Published: July 2002

Proteins exhibit a variety of motions ranging from amino acid side‐chain rotations to the motions of large domains. Recognition of their conformational flexibility has led to the view that protein molecules undergo fast dynamic interconversion between different conformational substates. This proposal has received support from a wide variety of experimental techniques and from computer simulations of protein dynamics. More recently, studies of the subunit dissociation of oligomeric proteins induced by hydrostatic pressure have shown that the characteristic times for subunit exchange between oligomers and for interconversion between different conformations may be rather slow (hours or days). In such cases, proteins cannot be treated as an ensemble of rapidly interconverting conformational substates, but rather as a persistently heterogeneous population of different long‐lived conformers. This is reminiscent of the deterministic behavior exhibited by macroscopic bodies, and may have important implications for our understanding of protein folding and biological functions. Here, we propose that the deterministic behavior of proteins may be closely related to the genesis of conformational diseases, a class of pathological conditions that includes transmissible spongiform encephalopathies, Alzheimer's disease and other amyloidosis.
Model of interaction of the IL‐1 receptor accessory protein IL‐1RAcP with the IL‐1β/IL‐1RI complex
Authors: Casadio Rita , Frigimelica Elisabetta , Bossù Paola , Neumann Detlef , Martin Michael U , Tagliabue Aldo , Boraschi Diana
Published: July 2002

A preliminary model has been calculated for the activating interaction of the interleukin 1 receptor (IL‐1R) accessory protein IL‐1RAcP with the ligand/receptor complex IL‐1β/IL‐1RI. First, IL‐1RAcP was modeled on the crystal structure of IL‐1RI bound to IL‐1β. Then, the IL‐1RAcP model was docked using specific programs to the crystal structure of the IL‐1β/IL‐1RI complex. Two types of models were predicted, with comparable probability. Experimental data obtained with the use of IL‐1β peptides and antibodies, and with mutated IL‐1β proteins, support the BACK model, in which IL‐1RAcP establishes contacts with the back of IL‐1RI wrapped around IL‐1β.
Chaperonins – keeping a lid on folding proteins
Authors: Kusmierczyk Andrew R , Martin Jörg
Published: July 2002

Two classes of chaperonins are known in all groups of organisms to participate in the folding of newly synthesized proteins. Whereas bacterial type I chaperonins use a reversibly binding cofactor to temporarily sequester folding substrate proteins within the cylindrical chaperonin cavity, type II chaperonins in archaea and the eukaryotic cytosol appear to have evolved a built‐in lid for this purpose. Not entirely surprisingly, this has consequences for the folding modes of the two types of chaperonins.
The actin‐binding protein Filamin‐A interacts with the metabotropic glutamate receptor type 7
Authors: Enz Ralf
Published: July 2002

A yeast two‐hybrid screen identified Filamin‐A as a binding partner of the metabotropic glutamate receptor type 7b (mGluR7b) splice variant. In addition, Filamin‐A interacted with mGluR4a, mGluR5a, mGluR5b, mGluR7a and mGluR8a. Domain mapping revealed that alternative splicing of mGluR4, mGluR7 and mGluR8 C‐termini regulated the interaction. A conserved tyrosine within mGluR C‐termini was identified to mediate the binding to Filamin‐A. Protein interactions were verified in biochemical assays using recombinant and native proteins. Finally, co‐expression of Filamin‐A and mGluR7 splice variants was shown in brain regions. These findings suggest that Filamin‐A may physically link metabotropic glutamate receptors to the actin cytoskeleton.