bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of the alpha subunit of PABA peptide hydrolase (EC 3.4.24.18) in MDCK cells
Authors: Grünberg Jürgen , Dumermuth Eric , Eldering Joyce A. , Sterchi Erwin E.
Published: July 2002

In this paper, we report the expression of PPHα in the polarized cell line MDCK (Madin Darby canine kidney). In these cells, the enzyme was synthesized m an inactive profonn, which upon treatment with trypsin was activated. The enzyme isolated from cell extracts was core‐glycosylated and appeared to be retained in the ER as a homodimer. No PPHα was detectable on the surface of intact cells by immunofluoreseence. However, a complex glycosylated soluble but inactive form was present in the culture medium, suggesting that proteolytic removal of the C‐terminal membrane anchoring peptide leads to the secretion of PPHα.
Effects of anions on the Ca2+, H+ and electrical gradients formed by the sarcoplasmic reticulum ATPase in reconstituted proteoliposomes
Authors: Yu Xiang , Inesi Giuseppe
Published: July 2002

The chaotropic character of several ions determines their partition on membrane interfaces with aqueous media as predicted by the Hofmeister series. However, specific characteristics of each individual ion determine its ability to cross the membrane and to influence Ca2+, H+ and electrical gradients produced by the sarcoplasmic reticulum (SR) Ca2+ pump in reconstituted proteoliposomal vesicles. Specific effects of this kind may be relevant to a variety of biological systems, including the excitation‐contraction coupling of muscle fibers in which SR plays a prominent role.
How is leghemoglobin involved in peribacteroid membrane degradation during nodule senescence?
Authors: Herrada Gilles , Puppo Alain , Moreau Sophie , Day David A. , Rigaud Jean
Published: July 2002

An increase in the rate of succinate and glutamate uptake by isolated symbiosomes from French bean nodules was observed in the presence of iron plus H2O2. The lipid bilayer, and not proteins involved in transport, seems to be the major target of radical attack. Leghemoglobin in the presence of a 6‐fold excess of H2O2 (where heme breakdown and iron release occurred) provoked also an increase in peribacteroid membrane permeability. In contrast, this hemoprotein in the presence of a 2‐fold excess of H2O2 (where a protein radical was generated) was without effect. We suggest that in vivo the release of heme iron may constitute the major process concerning the involvement of leghemoglobin in the degradation of the peribacteroid membrane during nodule senescence.
Prostaglandin H synthase: Implications for membrane structure
Authors: Picot Daniel , Garavito R.Michael
Published: July 2002

The crystal structure of the membrane protein prostaglandin H synthase (PGHS) provides strong evidence for the existence of monotopic membrane proteins: PGHS seems to interact with the membrane via a motif of amphipathic helices positioned parallel to the plane of the membrane. The orientation of this unique membrane binding motif is fixed in space by an epidermial growth factor(EGF)‐like module on its amino‐terminal end and by the catalytic domain at its carboxy‐terminal end. The catalytic domain of PGHS has a high structural homology to other mammalian heme peroxidases.
Cloning and correct expression in E. coli of the petJ gene encoding cytochrome c6 from Synechocystis 6803
Authors: Díaz Antonio , Navarro Francisco , Hervás Manuel , Navarro José A. , Chávez Sebastián , Florencio Francisco J. , De la Rosa Miguel A.
Published: July 2002

Cytochrome c
6 from the cyanobacterium Synechocystis 6803 has been isolated and purified to electrophoretic homogeneity. The gene coding for such a heme protein (petJ) has been cloned and properly expressed in E. coli. This procedure yields a protein preparation completely identical to that obtained from the cyanobacterial cells. The N‐terminal amino acid sequences of cytochrome c
6 synthesized in both organisms are the same, thus allowing us to conclude that the petJ gene product is correctly processed in E. coli. To the best of our knowledge, this is the first time that any cytochrome C
6 is produced in the enterobacterium. The identical physicochemical and kinetic properties of the proteins isolated from both sources confirm that expression of the petJ gene in E. coli is an adequate tool to address the study of Synechocystis cytochrome c
6, by site‐directed mutagenesis in a parallel way to that carried out with plastocyanin from the same organism.
Lipofectin‐aided cell delivery of ribozyme targeted to human urokinase receptor mRNA
Authors: Karikó Katalin , Megyeri Katalin , Xiao Qi , Barnathan Elliot S.
Published: July 2002

A 37‐mer hammerhead ribozyme has been designed to efficiently cleave the 1.4 kb mRNA of the urokinase plasminogen activator receptor (uPAR). Under in vitro conditions, the chemically synthesized ribozyme cleaved uPAR mRNA and inhibited its translation in a concentration‐dependent fashion. The ribozymes were 5′‐[35S]thiophosphorylated and used as a model to analyze conditions for RNA delivery in a cultured human osteosarcoma cell system. Ribozymes degraded immediately in cell‐conditioned medium but ribozymes complexed with lipofectin were protected from RNases for up to 22 h. Lipofectin rapidly transported ribozyme into the cell, where it accumulated almost exclusively in the cytoplasm. Thus, lipofectin dramatically enhances stability and cytoplasmic delivery of ribozymes, potentially enabling targeting of mRNA in vivo.
Observation of the Fe–O2 and FeIV=O stretching Raman bands for dioxygen reduction intermediates of cytochrome bo isolated from Escherichia coli
Authors: Hirota Shun , Mogi Tatsushi , Ogura Takashi , Hirano Tomoyasu , Anraku Yasuhiro , Kitagawa Teizo
Published: July 2002

Reaction intermediates in dioxygen reduction by the E. coli cytochrome bo‐type ubiquinol oxidase were studied by time‐resolved resonance Raman spectroscopy using the artificial cardiovascular system. At 0–20 μs following photolysis of the enzyme—CO adduct in the presence of O2, we observed the Fe–O2 stretching Raman band at 568 cm−1 which shifted to 535 cm−1 with the 18O2 derivative. These frequencies are remarkably close to those of other oxyhemoproteins including dioxygen‐bound hemoglobin and aa
3‐type cytochrome c oxidase. In the later time range (20–40 μs), other oxygen‐isotope‐sensitive Raman bands were observed at 788 and 361 cm−1. Since the 781 cm−1 band exhibited a downshift by 37 cm−1 upon 18O2 substitution, we assigned it to the FeIV=O stretching mode. This band is considered to arise from the ferryl intermediate, but its appearance was much earlier than the corresponding intermediate of bovine cytochrome c oxidase (> 100 μs). The 361 cm−1 band showed the 16O/18O isotopic frequency shift of 14 cm−1 similar to the case of bovine cytochrome c oxidase reaction.
The fusicoccin receptor of plants is a member of the 14‐3‐3 superfamily of eukaryotic regulatory proteins
Authors: Oecking C. , Eckerskorn C. , Weiler E.W.
Published: July 2002

The receptor for the wilt‐inducing phytotoxin fusicoccin was purified to homogeneity from plasma membranes ofCommelina communisas a complex with the radioligand [3H]9′‐nor‐8′‐hydroxyfusicoccin. The preparation consisted of two polypeptides with apparent molecular masses of 30.5 kDa and 31.5 kDa and with isoelectric points of around pH 5.2 and 5.3, respectively. The proteins were N‐terminally blocked. Internal amino acid sequences were obtained for both polypeptides of the fusicoccin‐binding complex. Sequence information, as well as subsequent immunological analysis, proved that both polypeptides are members of the eukaryotic 14‐3‐3 family, which comprises structurally conserved regulatory proteins of widespread occurrence and a wide range of functions. 14‐3‐3 isoform(s) constituting the fusicoccin receptor are distinguishable from other cellular 14‐3‐3 proteins by their tight association with the plasma membrane. Applying temperature‐induced Triton X‐114 phase separation experiments, they, as well as the target enzyme of fusicoccin action, the H+‐ATPase, partitioned into the phospholipid‐rich fraction which contains the most hydrophobic proteins. The results discussed herein provide a basis for the elucidation of the molecular mechanism of fusicoccin action.
Phosphorylation of a proline‐directed kinase motif is responsible for structural changes in myogenin
Authors: Hashimotoa Naohiro , Ogashiwa Masayo , Okumura Eiichi , Endo Takeshi , Iwashita Shintaro , Kishimoto Takeo
Published: July 2002

Myogenin, a member of the MyoD family which governs skeletal muscle differentiation, was identified as a pair of phosphorylated bands on SDS‐PAGE during myogenesis. The slow migrating form was found to be hyperphosphorylated myogenin. In vitro phosphorylation by CDC2 kinase caused a prominent reduction in electrophoretic mobility of myogenin. Furthermore, we demonstrated that phosphorylation of the serine residue at position 43 contributes to the modification of myogenin in vivo and in vitro resulting in the reduction in electrophoretic mobility. We propose here that a CDC2‐like proline‐directed kinase regulates myogenin activity through its phosphorylation.
A 14 kDa release factor is involved in GTP‐dependent β‐tubulin folding
Authors: Campo Rafael , Fontalba Ana , Sanchez Luis M. , Zabala Juan C.
Published: July 2002

The tubulin folding pathway is a model system to understand protein folding in the cell. It involves the interaction of several chaperones, including TCP‐1 and other as yet uncharacterized factors. Release of tubulin monomers from folding intermediates (C900 and C300) and their incorporation into tubulin dimers is dependent on GTP hydrolysis, magnesium ions and release factors. In this work, we have purified to homogeneity the protein factor responsible for the release of β‐tubulin monomers from C300 complexes. It has an apparent molecular mass of 14 kDa (p14) as judged by SDS electrophoresis. The protein behaved as a dimer of about 28 kDa when analyzed by gel filtration chromatography. Furthermore, the p14‐dependent release of β‐tubulin monomers from C300 complexes takes place in the presence of GTP. These results suggest that p14 is a new chaperone that assists in tubulin folding by facilitating the acquisition of the native conformation.
Water‐soluble GTP‐binding protein from rat olfactory epithelium
Authors: Novoselov V.I. , Peshenko I.V. , Evdokimov V.J. , Nikolaev J.V. , Matveeva E.A. , Fesenko E.E.
Published: July 2002

Rat olfactory epithelium and ciliar cytosol of olfactory cells contained the water‐soluble 45 kDa protein which was revealed by antibodies against a peptide fragment of the α‐subunits common to the G‐proteins. No analogous proteins were found in other rat tissues. According to the photo‐affinity labeling, the 45 kDa protein possessed a high affinity to GTP; it also exhibited a low GTP hydrolytic activity.
Cholinergic differentiation of cultured sympathetic neurons induced by retinoic acid
Authors: Kobayashi Miwako , Matsuoka Ichiro , Kurihara Kenzo
Published: July 2002

Here we show that retinoie acid (RA) has the ability to alter the transmitter phenotype of cultured sympathetic neurons from newborn rats superior cervical ganglia (SCG). In the presence of RA, the level of choline acetyltransferase (ChAT) mRNA was increased, while the level of tyrosine hydroxylase (TH) mRNA was reduced in the cultured SCG neurons. Selective PCR amplification of different upstream regions of the ChAT‐mRNA indicates that RA promotes the transcription of ChAT gene from R and M exons. The RA‐induced upregulation of ChAT‐mRNA level was significantly diminished by the chronic treatment with phorbol ester, suggesting that PKC has an important role in the induction of ChAT‐mRNA in this system.
Aliphatic and aromatic inhibitors binding to the active site of catechol 2,3‐dioxygenase from Pseudomonas putida mt‐2
Authors: Bertini Ivano , Briganti Fabrizio , Scozzafava Andrea
Published: July 2002

The interaction of different classes of inhibitors with the extradiol cleaving catechol 2,3‐dioxygenase from Pseudomonas putida mt‐2 was monitored by longitudinal and transverse proton relaxation measurements as well as by kinetic activity studies in order to characterize the type of interaction of such inhibitors with the active site of the enzyme. The average distances of the inhibitors from the catalytic iron(II) ion have been estimated from the 1H longitudinal relaxation rates. Phenols and aliphatic ketones appear to be coordinated to the iron(II) ion in the active site.
Critical effects on catalytic function produced by amino acid substitutions at Asp804 and Asp808 of the al isoform of Na,K‐ATPase
Authors: Van Huysse James W. , Kuntzweiler Theresa A. , Lingrel Jerry B.
Published: July 2002

At two intramembrane carboxyl‐containing amino acids of the sheep al isoform of Na,K‐ATPase (Asp 804 and Asp808) both charge‐conserving (Asp to Glu) and charge‐deleting (Asp to Asn, Leu and Ala) replacements were made and the altered enzymes studied. Nucleotide changes encoding the amino acid substitutions were placed in a cDNA encoding a ouabainresistant enzyme (sheep α1 RD) and the encoded enzymes were expressed in ouabain‐sensitive HeLa cells. Transfections with cDNAs carrying all Asp 804 substitutions, along with those carrying As808Ala, Asp808 Asn, and Asps808Leu replacements failed to confer ouabain resistance to the cells, indicating critical roles for Asp804 and Asps808. Only the expression of the Asp 808Glu enzyme produced ouabain‐resistant HeLa cells, demonstrating that the altered protein was functional. When the inactive proteins Asp804Ala and Asp080Ala were expressed using an alternative selection system (the protein carrying the amino acid substitution was the ouabain‐sensitive wild‐type sheep αl Na,K‐ATPase, which was expressed in ouabain‐resistant 3T3 cells), intact cells were able to bind extracellular ouabain with high affinity (K
d =1–30 nM), indicating that the inactive proteins were synthesized and folded properly in the plasma membrane. The results demonstrate that carboxyl side chains at positions 804 and 808 are critical for enzyme catalytic function.
Restoration of phosphorylation capacity to the dormant half of the α‐subunits of Na+, K+‐ATPase
Authors: Liu Guoquan , Xie Zijian , Modyanov Nikolai N. , Askari Amir
Published: July 2002

Purified kidney Na+,K+‐ATPase whose α‐subunit is cleaved by chymotrypsin at Leu266‐Ala267, loses ATPase activity but forms the phosphoenzyme intermediate (EP) from ATP. When EP formation was correlated with extent of α‐cleavage in the course of proteolysis, total EP increased with time before it declined. The magnitude of this rise indicated doubling of the number of phosphorylation sites after cleavage. Together with previous findings, these data establish that half of the α‐subunits of oligomeric membrane‐bound enzyme are dormant and that interaction of the N‐terminal domain of α‐subunit with its phosphorylation domain causes this half‐site reactivity. Evidently, disruption of this interaction by proteolysis abolishes overall activity while it opens access to phosphorylation sites of all α‐subunits.
Efficient introduction of aryl bromide functionality into proteins in vivo
Authors: Sharma Nandita , Furter Rolf , Kast Peter , Tirrell David A.
Published: July 2002

Artificial proteins can be engineered to exhibit interesting solid state, liquid crystal or interfacial properties and may ultimately serve as important alternatives to conventional polymeric materials. The utility of protein‐based materials is limited, however, by the availability of just the 20 amino acids that are normally recognized and utilized by biological systems; many desirable functional groups cannot be incorporated directly into proteins by biosynthetic means. In this study, we incorporate para‐bromophenylalanine (p‐Br‐phe) into a model target protein, mouse dihydrofolate reductase (DHFR), by using a bacterial phenylalanyl‐tRNA synthetase (PheRS) variant with relaxed substrate specificity. Coexpression of the mutant PheRS and DHFR in a phenylalanine auxotrophic Escherichia coli host strain grown in p‐Br‐phe‐supplemented minimal medium resulted in 88% replacement of phenylalanine residues by p‐Br‐phe; variation in the relative amounts of phe and p‐Br‐phe in the medium allows control of the degree of substitution by the analog. Protein expression yields of 20–25 mg/l were obtained from cultures supplemented with p‐Br‐phe; this corresponds to about two‐thirds of the expression levels characteristic of cultures supplemented with phe. The aryl bromide function is stable under the conditions used to purify DHFR and creates new opportunities for post‐translational derivatization of brominated proteins via metal‐catalyzed coupling reactions. In addition, bromination may be useful in X‐ray studies of proteins via the multiwavelength anomalous diffraction (MAD) technique.
A peptide from the adenovirus fiber shaft forms amyloid‐type fibrils
Authors: Luckey Mary , Hernandez Jean-François , Arlaud Gérard , Forsyth V.Trevor , Ruigrok Rob W.H. , Mitraki Anna
Published: July 2002

The fiber protein of adenovirus consists of a C‐terminal globular head, a shaft and a short N‐terminal tail. The crystal structure of a stable domain comprising the head plus a part of the shaft of human adenovirus type 2 fiber has recently been solved at 2.4 Å resolution [van Raaij et al. (1999) Nature 401, 935–938]. A peptide corresponding to the portion of the shaft immediately adjacent to the head (residues 355–396) has been synthesized chemically. The peptide failed to assemble correctly and instead formed amyloid‐type fibrils as assessed by electron microscopy, Congo red binding and X‐ray diffraction. Peptides corresponding to the fiber shaft could provide a model system to study mechanisms of amyloid fibril formation.
Protein kinase C phosphorylates and regulates UDP‐N‐acetylglucosamine‐2‐epimerase/N‐acetylmannosamine kinase
Authors: Horstkorte Rüdiger , Nöhring Sabine , Danker Kerstin , Effertz Karin , Reutter Werner , Lucka Lothar
Published: July 2002

UDP‐N‐acetylglucosamine‐2‐epimerase/N‐acetylmannosamine kinase (UDP‐GlcNAc 2‐epimerase) is the key enzyme in the de novo synthesis pathway of neuraminic acid, which is widely expressed as a terminal carbohydrate residue on glycoconjugates. UDP‐GlcNAc 2‐epimerase is a bifunctional enzyme and catalyzes the first two steps of neuraminic acid synthesis in the cytosol, the conversion of UDP‐N‐acetylglucosamine to ManAc and the phosphorylation to ManAc‐6‐phosphate. So far, regulation of this essential enzyme by posttranslational modification has not been shown. Since UDP‐N‐acetylglucosamine is a cytosolic protein containing eight conserved motifs for protein kinase C (PKC), we investigated whether its enzymatic activity might be regulated by phosphorylation by PKC. We showed that UDP‐GlcNAc 2‐epimerase interacts with several isoforms of PKC in mouse liver and is phosphorylated in vivo. Furthermore, PKC phosphorylates UDP‐GlcNAc 2‐epimerase and this phosphorylation results in an upregulation of the UDP‐GlcNAc 2‐epimerase enzyme activity.
Accumulation of 210 kDa microtubule‐interacting protein in differentiating P19 embryonal carcinoma cells
Authors: Zı́ková Martina , Sulimenko Vadym , Dráber Pavel , Dráberová Eduarda
Published: July 2002

The MA‐01 antigen, a thermolabile 210 kDa microtubule‐interacting protein, is present in P19 embryonal carcinoma cells on microtubular structures as well as in cytosol. After aggregation of the cells and subsequent incubation with all‐trans‐retinoic acid (RA), the level of MA‐01 expression increased approximately 10 times during 15 days. The increase started after 2 days of incubation with RA and preceded the appearance of neuron‐specific tubulin βIII, MAP2C and neurofilaments. Such elevated expression of MA‐01 antigen was not detected in P19 cells treated with dimethylsulfoxide. These data indicate that enhanced expression of MA‐01 antigen is one of the earliest events occurring in P19 cells during neuronal differentiation.
Peroxisome biogenesis and peroxisome biogenesis disorders
Authors: Fujiki Yukio
Published: July 2002

Peroxisome assembly in mammals requires more than 15 genes. Two isoforms of the peroxisome targeting signal type 1 (PTS1) receptor, Pex5pS and Pex5pL, are identified in mammals. Pex5pS and Pex5pL bind PTS1 proteins. Pex5pL, but not Pex5pS, directly interacts with the PTS2 receptor, Pex7p, carrying its cargo PTS2 protein in the cytosol. Pex5p carrying the cargos, PTS1 and PTS2, docks with the initial site Pex14p in a putative import machinery, subsequently translocating to other components such as Pex13p, Pex2p, Pex10p and Pex12p, whereby the matrix proteins are imported. The peroxins, Pex3p, Pex16p and Pex19p, function in the assembly of peroxisomal membrane vesicles that precedes the import of matrix proteins. Hence, peroxisomes may form de novo and do not have to arise from pre‐existing, morphologically recognizable peroxisomes. Impaired peroxisome assembly causes peroxisome biogenesis disorders such as Zellweger syndrome.
Annexin‐I inhibits PMA‐induced c‐fos SRE activation by suppressing cytosolic phospholipase A2 signal
Authors: Oh Jiyoung , Rhee Hae Jin , Kim Seung-Wook , Kim Soon Bae , You Hye-Jin , Kim Jae Hong , Na Doe Sun
Published: July 2002

Annexin‐I (ANX‐I) is a 37‐kDa protein with a calcium‐dependent phospholipid‐binding property. Previously we have observed the inhibition of cytosolic phospholipase A2 (cPLA2) by ANX‐I in the studies using purified recombinant ANX‐I, and proposed a specific interaction model for the mechanism of cPLA2 inhibition by ANX‐I [Kim et al. (1994) FEBS Lett. 343, 251–255]. Here we have studied the role of ANX‐I in the cPLA2 signaling pathway by transient transfection assay. The stimulation of Rat2 fibroblast cells with phorbol 12‐myristate 13‐acetate (PMA) induced the c‐fos serum response element (SRE). The SRE stimulation by PMA was dramatically reduced by (1) pretreatment with a cPLA2‐specific inhibitor, arachidonyltrifluoromethyl ketone, or (2) co‐transfection with antisense cPLA2 oligonucleotide, indicating that the SRE activation was through cPLA2 activation. Co‐transfection with an ANX‐I expression vector also reduced the SRE stimulation by PMA, suggesting the inhibition of cPLA2 by ANX‐I. The active domain of ANX‐I was mapped using various deletion mutants. ANX‐I(1–113) and ANX‐I(34–346) were fully active, whereas ANX‐I(114–346) abolished the activity. Therefore the activity was in the amino acid 34 to 113 region, which corresponds to the conserved domain I of ANX‐I.
The DNA replication‐related element (DRE)–DRE‐binding factor (DREF) system may be involved in the expression of theDrosophila melanogaster TBPgene
Authors: Choi Tae-Yeong , Cho Nam-Young , Oh Younsang , Yoo Mi-Ae , Matsukage Akio , Ryu Yoonseok , Han Kyuhyung , Yoon Jaeseung , Baek Kwanghee
Published: July 2002

The TATA box binding protein (TBP) is a general transcription factor required for initiation by all three eukaryotic RNA polymerases. Previously, we found that the promoter region of theDrosophila melanogasterTBPgene contains three sequences similar to the DNA replication‐related element (DRE) (5′‐TATCGATA). In the present study, we found that the DRE‐like sequences are also present in the promoter of theDrosophila virilisTBPgene, suggesting a role for these sequences in TBP expression. Band mobility shift assays revealed that oligonucleotides containing sequences similar to the DRE ofD. melanogaster TBPgene promoter form specific complexes with a factor in a Kc cell nuclear extract and with recombinant DRE‐binding factor (DREF). Furthermore, these complexes were either supershifted or diminished by monoclonal antibodies to DREF. Transient luciferase assays demonstrated that induction of mutations in two DRE‐related sequences at positions −223 and −63 resulted in an extensive reduction of promoter activity. Thus, the DRE–DREF system appears to be involved in the expression of theD. melanogaster TBPgene.
Analysis of FAK‐associated signaling pathways in the regulation of cell cycle progression
Authors: Reiske Heinz R , Zhao JiHe , Han Dong Cho , Cooper Lee Ann , Guan Jun-Lin
Published: July 2002

Focal adhesion kinase (FAK) is an important mediator of signal transduction pathways initiated by integrins in cell migration, survival and cell cycle regulation. The ability of FAK to mediate integrin signaling in the regulation of cell cycle progression depends on the phosphorylation of Tyr397, which implies a functional significance for the formation of FAK signaling complexes with Src, phosphatidylinositol‐3‐kinase (PI3K) and Grb7. We have previously described a FAK mutant, D395A, that selectively disrupts FAK binding to PI3K, but allows FAK association with Src. Using this mutation in a mislocalized FAK mutant background, we show here that formation of a FAK/PI3K complex is not sufficient for cell cycle progression but the formation of a FAK/Src complex plays an essential role. We also show that mutation of D395 to A disrupted FAK association with Grb7. This suggests that a FAK/Grb7 complex is not involved in the cell cycle regulation either, which is supported by direct analysis of cells expressing a dominant negative Grb7 construct. Finally, we provide evidence that the Src‐dependent association of FAK with Grb2 and p130Cas are both required for the regulation of cell cycle progression by FAK. Together, these studies identify important FAK downstream signaling pathways in cell cycle regulation.
Surprisingly high stability of barley lipid transfer protein, LTP1, towards denaturant, heat and proteases
Authors: Lindorff-Larsen Kresten , Winther Jakob R
Published: July 2002

Barley LTP1 belongs to a large family of plant proteins termed non‐specific lipid transfer proteins. The in vivo function of these proteins is unknown, but it has been suggested that they are involved in responses towards stresses such as pathogens, drought, heat, cold and salt. Also, the proteins have been suggested as transporters of monomers for cutin synthesis. We have analysed the stability of LTP1 towards denaturant, heat and proteases and found it to be a highly stable protein, which apparently does not denature at temperatures up to 100°C. This high stability may be important for the biological function of LTP1.
An Arabidopsis T‐DNA mutant affected in Nrt2 genes is impaired in nitrate uptake
Authors: Filleur Sophie , Dorbe Marie-France , Cerezo Miguel , Orsel Mathilde , Granier Fabienne , Gojon Alain , Daniel-Vedele Françoise
Published: July 2002

Expression analyses of Nrt2 plant genes have shown a strict correlation with root nitrate influx mediated by the high‐affinity transport system (HATS). The precise assignment of NRT2 protein function has not yet been possible due to the absence of heterologous expression studies as well as loss of function mutants in higher plants. Using a reverse genetic approach, we isolated an Arabidopsis thaliana knock‐out mutant where the T‐DNA insertion led to the complete deletion of the AtNrt2.1 gene together with the deletion of the 3′ region of the AtNrt2.2 gene. This mutant is impaired in the HATS, without being modified in the low‐affinity system. Moreover, the de‐regulated expression of a Nicotiana plumbaginifolia Nrt2 gene restored the mutant nitrate influx to that of the wild‐type. These results demonstrate that plant NRT2 proteins do have a role in HATS.
Isoform‐specific inhibition of voltage‐sensitive Ca2+ channels by protein kinase C in adrenal chromaffin cells
Authors: Sena Cristina M. , Santos Rosa M. , Standen Nick B. , Boarder Michael R. , Rosário Luı́s M.
Published: July 2002

Selective protein kinase C (PKC) activators and inhibitors were used to investigate the involvement of specific PKC isoforms in the modulation of voltage‐sensitive Ca2+ channels (VSCCs) in bovine adrenal chromaffin cells. Exposure to the phorbol ester phorbol‐12,13‐dibutyrate (PDBu) inhibited the Ca2+ currents elicited by depolarizing voltage steps. This inhibition was occluded by the PKC‐specific inhibitor Ro 31‐8220 but remained unaffected by Gö 6976, a selective inhibitor of conventional PKC isoforms. PDBu treatment caused the translocation of PKC‐α and ‐ϵ isoforms from cytosol to membranes. PKC‐ι and ‐ζ showed no signs of translocation. It is concluded that VSCCs are specifically inhibited by the activation of PKC‐ϵ in chromaffin cells. This may be relevant to the action of phospholipase‐linked receptors involved in the control of Ca2+ influx, both in catecholaminergic cells and other cell types.
Histone deacetylase complexes: functional entities or molecular reservoirs
Authors: Khochbin Saadi , Kao Hung-Ying
Published: July 2002

Recent investigations have allowed the identification of an increasing number of distinct nuclear multi‐component complexes containing several types of enzymatic activity. Many of the complexes containing histone deacetylases are believed to control transcription and chromatin remodeling. We suggest here that at least some of these abundant complexes are likely to be ‘molecular reservoirs’ of dynamic composition that exchange factors with other less abundant and functional complexes, according to specific required activities.
A role for Cajal bodies in assembly of the nuclear transcription machinery
Authors: Gall Joseph G
Published: July 2002

Cajal bodies (CBs) are small nuclear organelles that contain the three eukaryotic RNA polymerases and a variety of factors involved in transcription and processing of all types of RNA. A number of these factors, as well as subunits of polymerase (pol) II itself, are rapidly and specifically targeted to CBs when injected into the cell. It is suggested that pol I, pol II, and pol III transcription and processing complexes are preassembled in the CBs before transport to the sites of transcription on the chromosomes and in the nucleoli.
Identification and tissue distribution of novel KET/p63 splice variants
Authors: Bamberger Casimir , Schmale Hartwig
Published: July 2002

The human p53 protein family comprises three members – p53, p63 and p73. Whereas only one p53 variant is known multiple isoforms of p63 and p73 have been described. Depending on the isoform p63 influences p53‐responsive genes in a p53‐like or ‐distinct manner. We have cloned multiple splice variants of keratinocyte transcription factor (KET), the rat ortholog of human p63. Several tissue specific variations of exon 1 resulting in different amino‐terminal ends were identified. Transactivation properties of the splice variants inversely correlated with the length of the N‐termini as determined by activation of the p53‐responsive p21 promotor. Multiple KET isoforms are colocalized in different rat tissues. The amino‐terminal truncated form ΔNKETα is expressed in epithelial tissues, while expression of the most p53‐like KET isotype TAKETγ was detected in skeletal muscle. Expression of a major KET variant appears to be a cell‐type specific rather than a differentiation specific phenomenon.
Kininogen binding protein p33/gC1qR is localized in the vesicular fraction of endothelial cells
Authors: Dedio Jürgen , Müller-Esterl Werner
Published: July 2002

The endothelial protein p33/gC1qR is thought to mediate the assembly of components of the kinin‐forming and complement‐activating pathways on the surface of cardiovascular cells. FACS analysis of intact human umbilical vein endothelial cells using specific antibodies to p33 revealed a minor fluorescence on the cell surface whereas permeabilized cells showed a bright fluorescence indicative of an intracellular localization of p33. Immunostaining of fixed cells confirmed the predominant intracellular localization of p33. Fractionation studies demonstrated that the vesicular but not the membrane fraction of EA.hy926 cells is rich in p33. We conclude that externalization of p33 must precede its complex formation with target proteins on the endothelial cell surface.
Nitric oxide induces apoptosis via triggering mitochondrial permeability transition
Authors: Hortelano Sonsoles , Dallaporta Bruno , Zamzami Naoufal , Hirsch Tamara , Susin Santos A , Marzo Isabel , Boscá Lisardo , Kroemer Guido
Published: July 2002

Nitric oxide (NO) induces apoptosis in thymocytes, peripheral T cells, myeloid cells and neurons. Here we show that NO is highly efficient in inducing mitochondrial permeability transition, thereby causing the liberation of apoptogenic factors from mitochondria which can induce nuclear apoptosis (DNA condensation and DNA fragmentation) in isolated nuclei in vitro. In intact thymocytes, NO triggers disruption of the mitochondrial transmembrane potential, followed by hypergeneration of reactive oxygen species, exposure of phosphatidyl serine on the outer plasma membrane leaflet, and nuclear apoptosis. Inhibitors of mitochondrial permeability transition such as bongkrekic acid and a cyclophilin D‐binding cyclosporin A derivative, N‐methyl‐Val‐4‐cyclosporin A, prevent the mitochondrial as well as all post‐mitochondrial signs of apoptosis induced by NO including nuclear DNA fragmentation and exposure of phosphatidylserine residues on the cell surface. These findings indicate that NO can cause apoptosis via triggering of permeability transition.
Mapping the ubiquitin‐binding domains in the p54 regulatory complex subunit of the Drosophila 26S protease
Authors: Haracska Lajos , Udvardy Andor
Published: July 2002

Short‐lived intracellular proteins, after being marked by multiubiquitination, are degraded by the 26S protease. This large ATP‐dependent protease is composed of two multiprotein complexes: the regulatory complex and the 20S proteosome. The selective recognition of ubiquitinated proteins is ensured by the regulatory complex. Using an overlay assay a single 54‐kDa multiubiquitin‐chain‐binding subunit was detected in the regulatory complex of the Drosophila 26S protease. Overlay assay with the recombinant p54 subunit confirmed its ubiquitin‐binding property. The recombinant protein showed pronounced preference for higher ubiquitin multimers, in agreement with the known preference of the 26S protease for multiubiquitinated proteins as substrates. To map the ubiquitin‐binding domain of the p54 subunit different segments of the recombinant protein were expressed in E. coli and tested by the overlay assay. The p54 subunit carries two independent ubiquitin‐binding domains. The central domain carries two highly conserved sequence blocks: the FGVDP sequence (at position 207), which is 100% conserved from yeast till human, and the DPELALALRVSMEE sequence (at position 214), which is 100% conserved in higher eukaryotes with two amino acid changes in yeast. In the C‐terminal ubiquitin‐binding domain the GVDP sequence motif is repeated and 100% conserved in higher eukaryotes. This domain, however, due to the shorter size of the yeast multiubiquitin‐binding subunit, is present only in higher eukaryotes.
Vinculin gene is non‐essential in Drosophila melanogaster
Authors: Alatortsev Vladimir E , Kramerova Irina A , Frolov Maxim V , Lavrov Sergey A , Westphal Elena D
Published: July 2002

Vinculin is thought to be an important cytoskeletal protein in the linkage between actin cytoskeleton and integrin transmembrane receptors. We identified Vinculin (Vinc) gene in the X chromosome of D. melanogaster. Drosophila vinculin is highly homologous in its N‐ and C‐terminal domains both to mammalian and nematode vinculins, and contains internal repeats and proline‐rich region typical for vinculins. The X chromosome rearrangement In(1LR)pn2a was found to disrupt Vinc so that the coding sequence is interrupted by the (AAGAG)
n
satellite DNA. Northern analysis revealed that the Vinc transcript is completely absent in the In(1LR)pn2a homozygous flies. Surprisingly, these Vinc flies are viable and fertile. This finding highlights plasticity and adaptive capacity of cellular cytoskeletal and anchorage system.
The cytochrome oxidase subunit 1 gene (cox1) from the dinoflagellate, Crypthecodinium cohnii
Authors: Norman John E , Gray Michael W
Published: July 2002

To date, no genes have been characterized from dinoflagellate mitochondrial DNA. Here we present the complete sequence of the gene (cox1) encoding subunit 1 of cytochrome c oxidase in the dinoflagellate, Crypthecodinium cohnii. Analysis of nucleotide and deduced amino acid sequences predicts a protein of 523 amino acids that is translated using universal initiation, stop and tryptophan codons. COX1 amino acid identity and phylogenetic tree analyses strongly support a close evolutionary relationship between dinoflagellates and apicomplexans; however, inclusion of the ciliates in this clade is less well supported, a result likely due to the highly derived nature of ciliate COX1 sequences.
α‐Tocopherol does not inhibit hypochlorite‐induced oxidation of apolipoprotein B‐100 of low‐density lipoprotein
Authors: Hazell Linda J , Stocker Roland
Published: July 2002

The amount of α‐tocopherol (α‐TOH) can dramatically alter the extent of radical‐induced oxidation of low density lipoprotein (LDL) lipids, a process generally thought to be important in atherogenesis. However, LDL with atherogenic features can also be formed in vitro by exposure to the strong non‐radical oxidant hypochlorite (HOCl), which preferentially oxidises LDL apolipoprotein B‐100. Here we show that varying LDL content of α‐TOH by vitamin supplementation or depletion has no effect on the extent of HOCl‐induced oxidation of apolipoprotein B‐100 as measured by the loss of lysine and tryptophan residues, and the alteration in relative electrophoretic mobility of the lipoprotein particle.
The effect of hydroxylation of linoleoyl amides on their cannabinomimetic properties
Authors: van der Stelt Marcelis , Paoletti Anna Maria , Maccarrone Mauro , Nieuwenhuizen Willem F , Bagetta Giacinto , Veldink Gerrit A , Finazzi Agrò Alessandro , Vliegenthart Johannes F.G
Published: July 2002

As yet, the physiological significance of hydroxylation of anandamide and linoleoyl amides is unknown. Therefore, we investigated whether hydroxylation of ODNHEtOH and ODNH2 influences their binding abilities to the CB‐1 receptor and whether it alters their reactivity towards a fatty acid amide hydrolase (FAAH) from rat brain. Neither the fatty acid amides nor their hydroxylated derivatives were able to displace the potent cannabinoid [3H]CP 55.940 from the CB‐1 receptor (K
i>1 μM). Hydroxylation of ODNHEtOH resulted in a strong reduction of the maximum rate of hydrolysis by a FAAH, but the affinity of FAAH for the substrate remained of the same order of magnitude. Hydroxylation of ODNH2 led to a decrease in the affinity of FAAH for the substrate, but its maximum rate of conversion was unaffected. Furthermore, hydroxylation of ODNHEtOH enhanced its capacity to inhibit competitively the hydrolysis of anandamide. The resulting prolonged lifetime of anandamide and other fatty acid amide derivatives may have a considerable impact on cellular signal transduction.
Comparison of the structure‐activity relationships of nociceptin and dynorphin A using chimeric peptides
Authors: Lapalu Sophie , Moisand Christiane , Mazarguil Honoré , Cambois Gilles , Mollereau Catherine , Meunier Jean-Claude
Published: July 2002

The aim of the present study was to delineate the functional domains of nociceptin (noc), a neuropeptide which is structurally related to dynorphin A (dyn). The binding and biological potencies towards the nociceptin (ORL1) and dynorphin A (κ‐opioid) receptors of twenty dyn/noc and noc/dyn hybrid peptides were compared with those of the parent heptadecapeptides. Replacement of as many as eleven residues in the C‐terminus of dynorphin by the corresponding nociceptin sequence has no significant effect on binding and biological activity towards the κ‐opioid receptor. In marked contrast, replacement of as few as six residues (RKLANQ) in the C‐terminus of nociceptin by the corresponding dynorphin sequence (LKWDNQ) dramatically impairs both affinity and activity towards the ORL1 receptor. This clearly indicates that the two neuropeptides have different functional architectures, despite the dual structural homology of both ligands and receptors. Moreover, the recombinant peptide approach led us to identify hybrids whose sequences differ only at positions 5 and 6 and displaying opposite or no receptor selectivity. One contains the dynorphin Leu5‐Arg6 sequence and prefers the κ‐opioid receptor, whereas the other comprises the nociceptin Thr5‐Gly6 sequence and prefers the ORL1 receptor. A third, containing the mixed dynorphin/nociceptin Leu5‐Gly6 sequence, does not discriminate between the two types of receptor.
A RIPE3b1‐like factor binds to a novel site in the human insulin promoter in a redox‐dependent manner
Authors: Read Martin L , Masson Margaret R , Docherty Kevin
Published: July 2002

In the human insulin gene, a regulatory sequence upstream of the transcription start site at −229 to −258 (the E2 element) binds a ubiquitous factor USF. The present study led to the identification of a second factor, D0, that binds to an adjacent upstream site, the C2 element, that has previously not been described. The results demonstrate that D0 exhibits similar properties to RIPE3b1, a factor shown to be an important determinant of insulin gene β‐cell‐specific expression. Binding of D0 to the C2 element was abolished by the oxidising agent diamide, and the alkylating agent N‐ethylmaleimide. The results indicate that expression of the insulin gene may be regulated by a redox‐dependent pathway involving RIPE3b1 or a RIPE3b1‐like factor.
A retinoblastoma susceptibility gene product, RB, targeting protease is regulated through the cell cycle
Authors: Fu Yu-Hsieh Florence , Nishinaka Toru , Yokoyama Kazunari , Chiu Robert
Published: July 2002

Degradation of cyclin B and cyclin‐dependent kinase inhibitor, p27, at a specific time has been shown to play a critical role in regulating the cell cycle. SPase, a nuclear and cytosol protease with cathepsin B‐ and L‐like proteolytic activity, has been identified in several cell lines. This proteolytic enzyme selectively degraded nuclear proteins such as retinoblastoma susceptibility gene product, RB, and transcription factor, SP‐1. High levels of SPase activity were detected at the G1/S, moderate levels at the G1 and S phases, and undetectable activity at the M phase of synchronized CV‐1 cells, suggesting that SPase activity is regulated through the cell cycle. Degradation of RB correlated with SPase activity throughout the cell cycle, suggesting that SPase regulates RB, which has a functional role in regulating cell cycle. These results demonstrated that SPase plays an integral role in regulating the nuclear regulator, RB, in controlling cell cycle progression.
Expression of murine novel zinc finger proteins highly homologous to Drosophila ovo gene product in testis
Authors: Masu Yasuo , Ikeda Sakahiro , Okuda-Ashitaka Emiko , Sato Eimei , Ito Seiji
Published: July 2002

We have cloned two isoforms of cDNAs encoding novel zinc finger proteins. One form encodes a 274‐amino acid protein containing an acidic amino acid and serine‐rich domain and a zinc finger domain which shows high sequence homology to that of Drosophila Ovo protein. The other form encodes a 179‐amino acid protein containing only the zinc finger domain. Expression of both proteins possessing an antigenic epitope in COS cells revealed that they are localized in the nucleus. The 1.3‐kbp mRNAs are predominantly expressed in testis, and the expression increases from 3 weeks postnatal, implying that these proteins may play important roles in the development of the testes.
Slipped loop structure of DNA: a specific nucleotide sequence forms only one unique conformer
Authors: Khomyakova E.B , Petrova M.V , Minyat E.E , Ivanov V.I
Published: July 2002

Earlier with some DNA sequences we were able to prove the existence of a new polynucleotide chain folding named slipped loop structure, or SLS [1, 2]. However, the possibility of the presence of two SLS isomers in equilibrium was not excluded in the experiments. Here we are dealing with a specially designed structure formed by two short oligonucleotides intended for avoiding such a situation. To minimize the possibility of alternative structure formation and stabilize the conformation under investigation, the oligonucleotide sequences were designed in such a way that the bimolecular structure SLS31 would have two binding sites for antibiotic distamycin A. The sample was exposed to chemical probing both in the presence of distamycin A and without the ligand and the accessible nucleotides were mapped. The results do not suggest the presence in the solution of two isomers with different types of loop slippage without interloop interactions and strongly support the formation of a unique slipped loop conformation stabilized by an additional interloop helix, or slipped loop structure.
Identification of tryptophan 55 as the primary site of [3H]nicotine photoincorporation in the γ‐subunit of the Torpedo nicotinic acetylcholine receptor
Authors: Chiara David C , Middleton Richard E , Cohen Jonathan B
Published: July 2002

[3H]nicotine has been used as a photoaffinity agonist to identify amino acids within the Torpedo nicotinic acetylcholine receptor (nAChR) γ‐subunit that contributes to the structure of the agonist binding site. UV irradiation (254 nm) of nAChR‐rich membranes equilibrated with [3H]nicotine results in covalent incorporation into α‐ and γ‐subunits that is inhibitable by agonists and competitive antagonists, but not by non‐competitive antagonists (Middleton, R.E. and Cohen, J.B. (1991) Biochemistry 30, 6887–6897). To identify sites of specific incorporation, SDS‐PAGE and reversed‐phase HPLC were used to isolate proteolytic fragments of [3H]nicotine‐labeled γ‐subunit. Amino‐terminal sequence analysis identified γTrp‐55 as the major site of [3H]nicotine photoincorporation in γ‐subunit. Thus γTrp‐55 is the first amino acid within a non‐α‐subunit to be identified by affinity labeling in direct contact with a bound agonist.
HAP1‐huntingtin interactions do not contribute to the molecular pathology in Huntington's disease transgenic mice
Authors: Bertaux Fabien , Sharp Alan H , Ross Christopher A , Lehrach Hans , Bates Gillian P , Wanker Erich
Published: July 2002

HAP1 (huntingtin associated protein) has previously been found to interact with huntingtin (htt) in a glutamine length dependent manner and has been proposed to play a role in the cell specific neurodegeneration observed in Huntington's disease (HD). We have isolated mouse HAP1 (hap1) and have shown that expression is not enriched in areas specifically affected in HD. We have used the yeast two hybrid system to demonstrate that htt amino acids 171–230 are necessary for the hap1‐htt binding and that hap1 does not interact with the transgene exon 1 protein in a transgenic model of HD.
Lack of β‐amyloid production in M19 cells deficient in site 2 processing of the sterol regulatory element binding proteins
Authors: Manni Mara E , Cescato Renzo , Paganetti Paolo A
Published: July 2002

The processing of the amyloid precursor protein (APP) and the sterol regulatory element binding protein show remarkable analogies. Following a first lumenal cleavage, both proteins undergo a cleavage within the transmembrane domain by enzymatic activities named γ‐secretase and S2P, respectively. We analyzed the processing of APP in the mutant Chinese hamster ovary (CHO) cell line M19 which lacks the S2P gene encoding for a putative metalloprotease. In these cells, we were not able to detect any β‐amyloid production from endogenous or transiently overexpressed APP, although the transport of APP along the secretory pathway, its processing by α‐ and β‐secretase, as well as its secretion were normal. This strongly suggests that the γ‐secretase cleavage in M19 cells is severely impaired.