bluuebunny/crossref_metadata_2025 huggingface dataset:
Internal amino acid sequence analysis of the 80 kDa protein kinase C substrate from rat brain: Relationship to the 87 kDa substrate from bovine brain
Authors: Erusalimsky Jorge D. , Morris Clive , Perks Kate , Brown Ron , Brooks Susan , Rozengurt Enrique
Published: July 2002

We have obtained sequence data from five proteolytic peptides totalling 102 amino acid residues of the 80 kDa protein kinase C substrate purified from rat brain. The amino acid sequences of these five peptides were compared with that deduced from a cDNA encoding the 87 kDa protein kinase C substrate from bovine brain. The overall amino acid sequence identity within the regions covered by our peptides is 54%. Two peptides aligned at the C‐ and N‐termini of the bovine protein kinase C substrate with a very high degree of homology (more than 80% identity). Two other peptides exhibited 62% and 46% identity with two regions located in the C‐terminal half of the bovine protein. The fifth peptide which contains the sequence PEQPEQPEQ did not reveal any similarity with the bovine protein. Based on the homologies of our experimentally determined sequences, which represent about 30% of the deduced sequence of the bovine protein, we suggest that although these protein kinase C substrates are not identical, they may belong to a family of related proteins.
Development of a monoclonal antibody against recombinant neuroendocrine 7B2 protein
Authors: van Duijnhoven Hans L.P. , Ayoubi Torik A.Y. , Timmera Erika D.J. , Braks Anneke A.M. , Roebroek Anton J.M. , Martens Gerard J.M. , van de Ven Wim J.M.
Published: July 2002

Mouse monoclonal antibody MON‐100 was raised against the neuroendocrine protein 7B2 using bacterially produced hybrid proteins. In Western blot analysis, MON‐100 reacted with 3 different 7B2 hybrid proteins and not with the respective carrier proteins. Furthermore, MON‐100 was reactive with recombinant 7B2 cleaved from a hybrid protein. In an immunohistochemical study, MON‐100 exhibited strong reactivity with the intermediate lobe of the Xenopus pituitary gland, a tissue previously shown to contain 7B2 mRNA. Using MON‐100, immunoprecipitation analysis of newly synthesized proteins produced by in vitro incubated Xenopus neurointermediate lobes revealed the biosynthesis of a single protein of M
r 24 kDa, the expected size of the 7B2 protein. It appears, therefore, that the anti‐7B2 monoclonal antibody MON‐100 can be successfully used for Western blot analysis and immunohistochemical analysis as well as for immunoprecipitation experiments.
Regulation of LacZ mRNA translatability in a cell‐free system at heat shock by the last four sense codons
Authors: Denisenko Oleg N. , Yarchuk Oleg B.
Published: July 2002

Under heat shock conditions translation of Xenopus laevis normal mRNAs in a rabbit reticulocyte cell‐free system is blocked whereas hsp70 mRNA is translated. mRNA for E. coli β‐galactosidase containing the last four sense codons of Drosophila hsp70 at its 3′‐end was constructed. This mRNA is efficiently translated in a rabbit reticulocyte cell‐free system at 43°C.
Possible mechanism of nuclear translocation of proteasomes
Authors: Tanaka Keiji , Yoshimura Tetsuro , Tamura Tomohiro , Fujiwara Tsutomu , Kumatori Atsushi , Ichihara Akira
Published: July 2002

Proteasomes (multicatalytic proteinase complexes), which are identical to the ubiquitous eukaryotic 20S particles, are localized in both the cytoplasm and the nucleus, but the mechanism of their co‐localization in the two compartments is unknown. On examination of the primary structures of subunits of proteasomes, a consensus sequence for nuclear translocation of proteins, X‐X‐K‐K(R)‐X‐K(R) (where X is any residue), was found to be present in some subunits and to be highly conserved in the subunits of a wide range of eukaryotes. In addition, proteasomal subunits were found to bear a cluster of acidic amino acid residues and also a potential tyrosine phosphorylation site that was located in the same polypeptide chain as the nuclear location signal. These structural properties suggest that two sets of clusters with positive and negative charges serve to regulate the translocation of proteasomes from the cytoplasm to the nucleus, and that phosphorylation of tyrosine in certain subunits may play an additional role in transfer of proteasomes into the nucleus.
Differential movement of ions in artificial phospholipid vesicles
Authors: Sedgwick Edward G. , Bragg Philip D.
Published: July 2002

Pyranine was incorporated into sonicated unilamellar vesicles of soybean phosphatidylcholine to monitor changes in the internal pH of the vesicles. Dilution of soybean phosphatidylcholine vesicles loaded with 0.3 M KCl, KNO3 or K2SO4 into salt‐free buffer resulted in rapid exchange of K+ and protons. A pseudoequilibrium distribution of ions was achieved, since addition of valinomycin, uncoupler or nigericin now caused a rapid alkalinization of the vesicle interior. Dilution into buffer containing NaCl gave a further exchange of Na+ and protons following the initial K+/proton exchange. Na+ permeation was slower than that of K+. A stable membrane potential was not generated by the ion movements. It is proposed that aqueous channels are formed through the phospholipid bilayers and that K+ and Na+ permeate through these channels as the hydrated ions.
Regulation of the hexaheme nitrite/nitric oxide reductase of Desulfovibrio desulfuricans, Wolinella succinogenes and Escherichia coli
Authors: Costa C. , Macedo A. , Moura I. , Moura J.J.G. , Le Gall J. , Berlier Y. , Liu M.-Y. , Payne W.J.
Published: July 2002

Dissimilatory nitrite reduction, carried out by hexaheme proteins, gives ammonia as the final product. Representatives of this enzyme group from 3 bacterial species can also reduce NO to either ammonia or N2O. The redox regulation of the nitrite/nitric oxide activities is discussed in the context of the denitrifying pathway.
Species‐specific Mn2+/Mg2+ antiport from Mg2+‐loaded erythrocytes
Authors: Günther T. , Vormann J. , Cragoe E.J.
Published: July 2002

Mg2+‐loaded rat erythrocytes performed Mn+/Mg2+ antiport, which was nonspecifically stimulated by anions and cations. Mn2+/Mg2+ antiport was shown to operate via the Na+Mg2+ antiporter because extracellular Na+ and Mn2+ inhibited the intracellular uptake of each other's ions competitively. Furthermore, Mn2+/Mg2+ antiport and Na+/Mg2+ antiport were identically inhibited by various amiloride derivatives. Na+/Mg2+ antiport of chicken and human erythrocytes cannot perform Mn2+/Mg2+ antiport although chicken erythrocytes took up more Mn2+ than raterythrocytes.
Immunoelectron microscopical localization of the catalytic subunit of cAMP‐dependent protein kinases in brain microtubules and neurofilaments
Authors: Trinczek Bernhard , Schwoch Gerhild
Published: July 2002

The catalytic subunit of cAMP‐dependent protein kinases was localized in microtubules and neurofilaments by immunogold electron microscopy. In microtubules, the label was similarly distributed as an immunolabel for the microtubule associated protein MAP 2. The neurofilaments showed no reaction with the MAP 2‐antiserum. Our results support the suggestion of an in vivo role of cAMP‐dependent protein kinases in the regulation of microtubules. In addition, this is the first demonstration that cAMP‐dependent protein kinase is associated with neurofilaments.
Injected inositol 1,4,5‐trisphosphate activates Ca2+ ‐sensitive K + channels in the plasmalemma of Eremosphaera viridis
Authors: Förster Birgit
Published: July 2002

InsP3, an established mediator of intracellular Ca2+ signals in animal cells, is microinjected into the cytoplasm of Eremosphaera viridis. InsP3, but not Ins, InsP1, InsP2 or F2,6‐P2 induce a transient opening of Ca2+ ‐dependent K+ channels in the plasmalemma of this alga. This effect is indicated by a transient polarization (TP) with a simultaneous increase of membrane conductance. The TP is inhibited by TMB8 (2 mM), an intracellular Ca2+ antagonist or by BAPTA (20 mM), microinjected together with InsP3. The results suggest that InsP3 initiates an increase in the cytoplasmic Ca2+ activity and an activation of Ca2+‐dependent membrane currents, hence, opening of K+ channels.
31P and 23Na nuclear magnetic resonance studies of resting and stimulated mast cells
Authors: Pilatus Ulrich , Degani Hadassa , Pecht Israel
Published: July 2002

Exocytosis induced by crosslinking the type I receptor for Fce, domains present on rat mucosal mast cells (RBL‐2H3‐line) requires the influx of Ca2+ ions and is markedly influenced by the concentration of monovalent cations (K+, Na+ and protons) in their medium. We investigated the role of these ions in coupling the immunological stimulus to secretion using NMR spectroscopy to monitor simultanously intraccllular pH, ATP and Na+ concentrations and the secretory response of living adherent mast cells. Using this methodology we observed that: (i) ATP concentration and intracellular pH are highly regulated and no changes could be resolved in them upon stimulation and during exocytosis. (ii) In the absence of potassium ions in the cells' medium, a decrease is observed in the intracellular pH and ATP concentration and an increase in the Na+ concentration. (iii) From the influx of extracellular Na+ following inhibition of the Na+, K+‐ATPase by ouabain, we estimated the inward Na+ current of resting cells to 5 × 107 ions/(cell·s). This value does not vary by more than 10% during exocytosis.
Effect of canine surfactant protein (SP‐A) on the respiratory burst of phagocytic cells
Authors: Weber Hans , Heilmann Peter , Meyer Barbara , Maier Konrad L.
Published: July 2002

Cells obtained from bronchoalveolar lavage, or neutrophils of peripheral blood of dog, were incubated with the canine surfactant‐associated protein A (SP‐A). A significant decrease of the production of Superoxide anion was observed after subsequent stimulation with phorbol‐12‐myristate‐13‐acetate (PMA) as measured by the lucigenin‐dependent chemiluminesence (CL). Several other proteins used for control experiments did not decrease lucigenin‐dependent CL, indicating a specific effect of SP‐A on phagocytes. Treatment of SP‐A with collagenase prior to incubation with neutrophils destroyed the depleting effect on oxygen radical production of PMA‐stimulated cells. We propose that SP‐A acts as a regulatory factor of the respitatory burst of alveolar macrophages and neutrophils in the lungs. The inhibitory effect of SP‐A is down‐regulated by collagenase released from stimulated alveolar macrophages.
Cloning and sequence analysis of complementary DNA encoding a precursor for chicken natriuretic peptide
Authors: Akizuki Naoya , Kangawa Kenji , Minamino Naoto , Matsou Hisayuki
Published: July 2002

Chicken α‐natriuretic peptide (α‐chNP) has been identified in chicken heart, which showed higher homology to brain natriuretic peptide (BNP) than to atrial natriuretic peptide (ANP) [1]. Complementary DNA (cDNA) clone encoding a chNP precursor (pre‐chNP) precursor (pre‐chNP) was isolated from cardiac cDNA library and sequenced. Pre‐chNP was 140‐residue signal peptide at the N‐terminus and α‐chNP at the C‐terminus, and did not exhibit high homology to poreine BNP except for the C‐terminal region. However, a characteristic AT‐rich nucleotide sequence commonly found in mammalian BNPs was also present in the 3′‐untranslated region. Thus, chNP is concluded to be classified into the BNP‐type
Comparison of the HIV‐1 and HIV‐2 proteinases using oligopeptide substrates representing cleavage sites in Gag and Gag‐Pol polyproteins
Authors: Tözsér József , Bláha Ivo , Copeland Terry D. , Wondrak Ewald M. , Oroszlan Stephen
Published: July 2002

The substrate specificity of the human immunodeficiency virus type 1 (HIV‐1) and type 2 (HIV‐2) proteinases was compared using oligopeptides corresponding to cleavage sites in the Gag and Gag‐Pol polyproteins of both viruses. All peptides mimicking cleavage sites at the junction of major functional protein domains were correctly cleaved by both enzymes. However, some other peptides thought to represent secondary cleavage sites remained intact. The kinetic parameters (K
m and k
m) obtained for the different substrates showed several hundred‐fold variation but were similar for the same substrate.
ATP‐dependent reversible association of proteasomes with multiple protein components to form 26S complexes that degrade ubiquitinated proteins in human HL‐60 cells
Authors: Orino Etsuko , Tanaka Keiji , Tamura Tomohiro , Sone Saburo , Ogura Takeshi , Ichihara Akira
Published: July 2002

The role of proteasomes in ubiquitin (Ub)‐dependent protein degradation was studied by analyzing lysates of human promyelocytic leukemia HL‐60 cells by glycerol density gradient centrifugation. High succinyl‐Leu‐Leu‐Val‐Tyr‐4‐methylcoumaryl‐7‐amide hydrolyzing activity was found in the 26S fraction, whereas the 20S fraction containing proteaomes had no activity. Addition of 0.05% sodium dodecylsulfate to the latter fraction, however, induced marked activity. The 26S, but not the 20S fraction catalyzed ATP‐dependent degradation of [125I]lysozyme‐Ub conjugate. Depletion from the lysate of ATP caused complete shift of the active 26S complex to the latent 20S form, whereas in the lysate prepared from ATP‐depleted cells, ATP converted 20S proteasomes to 26S complexes. The immunoprecipitated 26S complexes were found to consist of proteasomes and 13–15 other proteins ranging in size from 35 to 110 kDa. We conclude that in the lysate, latent proteasomes undergo reversible, ATP‐dependent association with multiple protein components to form 26S complexes that catalyze ATP‐dependent degradation of Ub‐protein conjugates.
Molecular cloning of cDNA encoding the rat neural cell adhesion molecule L1 Two L1 isoforms in the cytoplasmic region are produced by differential splicing
Authors: Miura Masayuki , Kobayashi Masaaki , Asou Hiroaki , Uyemura Keiichi
Published: July 2002

We have isolated and sequenced a full‐length cDNA encoding the rat neural cell adhesion molecule L1. The deduced amino acid sequence as a whole shows high homology to mouse L1 sequence. In addition to this complete form of L1, we found an isoform, L1cs, which lacks four animo acid residues (RSLE) in the cytoplasmic domain and probably is derived from the same single L1 gene by tissue‐specific alternative splicing. While L1 mRNA was predominantly expressed in the brain, L1cs mRNA was found exclusively in peripheral nervous tissue. Differential splicing in the highly conserved cytoplasmic domain may play an important role in modulating the function of L1 in different cells.
Co‐operative binding of hsp60 may promote transfer hsp70 and correct folding of imported proteins in mitochondria
Authors: Endo Toshiya
Published: July 2002

I propose that a molecular chaperone hsp60 binds to and dissociates from the unfolded polypeptide or folding intermediate in a positively co‐operative manner, but another chaperone hsp70 shows no such co‐operativity. This could simply explain the fact that the protein newly imported in the mitochondrial matrix is transferred from hsp70 to hsp60 and hsp6O promotes corrert folding or the substrate protein while hsp70 does not.
Okadaic acid inhibits angiotensin II stimulation of Ins(1,4,5)P3 and calcium signalling in rat hepatocytes
Authors: Mattingly Raymond R. , Garrison James C.
Published: July 2002

OKA2 and CL‐A significantly inhibit the ability of angiotensin II, ATP and vasopressin to raise [Ca2+]i in rat hepatocytes, with a partial inhibition of the initial spike, and a complete inhibition of the following plateau. In contrast, the [Ca2+]i response to thapsigargin, which releases intracellular calcium stores through a mechanism independent of inositol phosphates, is much less affected. The ability of angiotensin II to stimulate Ins(1.4.5)P3 production is also reduced by OKA, with kinetics consistent with the inhibited [Ca2+], response. Since OKA and CL‐A are potent and selective inhibitors or phosphoprotein phosphatases. these results provide further evidence that agonist‐stimulated Ins(1,4,5)P3 signalling can be inhibited by protein phosphorylation.
Tissue inhibitor of metalloproteinase‐2 (TIMP‐2) has erythroid‐potentiating activity
Authors: Stetler-Stevenson William G. , Bersch Noelle , Golde David W.
Published: July 2002

Tissue inhibitor of metalloproteinase (TIMP) was purified and molecularly cloned on the basis of its erythroid‐potentiating activity (EPA). TIMP/EPA appears to be a bifunctional molecule with both growth factor and anti‐enzymatic activity. Recently, a second TIMP‐related molecule was identified and we have investigated its possible erythroid‐potentiating activity. Native, purified human TIMP‐2 was assayed for erythroid‐potentiating activity using an in vitro erythroid burst formation assay and was compared with that of previously characterized recombinant EPA/TIMP‐1. The results demonstrate that both members of the tissue inhibitor or metalloproteinase family, TIMP‐1 and TIMP‐2, possessed erythroid potentiating activity which was inhibited by antibodies developed to neutralize EPA. These results suggest that TIMP‐2 shares a common structural domain with EPA/TIMP‐ I that is responsible for the erythroid‐potentiating activity of these inhibitors. Therefore, TIMP‐1 and TIMP‐2, with both anti‐protease activity and growth factor activity, join a family of bifunctional molecules such as Fibroblast growth factor and thrombin which have both enzymatic and growth factor activity.
Heat‐labile enterotoxin crystal forms with variable A/B5 orientation Analysis of conformational flexibility
Authors: Sixma Titia K. , Aguirre Angel , van Scheltinga Anke C.Terwisscha , Wartna Ellen S. , Kalk Kor H. , Hol Wim G.J.
Published: July 2002

A new native crystal form of heat‐labile enterotoxin (LT) has two AB5 complexes in the asymmetric unit with different orientations of the A subunit with respect to the B pentamer. Comparison with other crystal forms of LT shows that there is considerable conformational freedom for orientating the A subunit with respect to the B pentamer. The rotations of A in different crystal forms do not follow one specific axis, but most of them share a hinge point, close to the main interaction area between A and B5. Analysis of the two high‐resolution structures available shows that these rotations cause very little change in the actual interactions between A and B5.
Codon context effect in virus translational readthrough A study in vitro of the determinants of TMV and Mo‐MuLV amber suppression
Authors: Valle Rosaura P.C. , Drugeon Gabrièle , Devignes-Morch Marie-Dominique , Legocki Andrzej B. , Haenni Anne-Lise
Published: July 2002

To assess the role of codon context on the efficiency of eukaryotic suppression of termination codons, we have compared, in a rabbit cell‐free translation system, the readthrough efficiency related to two synthetic transcripts differing by the codon context around an amber codon. The codon contexts are derived from tobacco mosaic virus (TMV) and Moloney murine leukemia virus (Mo‐MuLV) RNAs. The Mo‐MuLV‐like codon context does not promote suppression. Substituting TMV‐derived triplets in the Mo‐MuLV‐like codon context shows that the two codons downstrem from the TMV UAG signal are important determinants of suppression, as recently demonstrated in vivo.
The 26 S proteasome is activated at two points in the ascidian cell cycle
Authors: Kawahara Hiroyuki , Sawada Hitoshi , Yokosawa Hideyoshi
Published: July 2002

The proteasome undergoes cell cycle‐dependent changes in its subcellular distribution during ascidian embryonic development [(1992) Dev. Biol. 151, 27–33]. In this study, we demonstrate that the 26 S proteasome is markedly activated in both prophase and metaphase of the mitotic cell cycle in the ascidian embryos in comparison with the case of the 20 S proteasome. These results suggest that the 26 S proteasome is activated and participates in proteolysis at the restricted stages of the cell cycle.
Molecular cloning of G protein α subunits from the central nervous system of the mollusc Lymnaea stagnalis
Authors: Knol Jaco C. , Weidemann Wolfgang , Planta Rudi J. , Vreugdenhil Erno , van Heerikhuizen Harm
Published: July 2002

The central nervous system of the pond snail, Lymnaea stagnalis, contains many large, identified neurons which can be easily manipulated making it an advantageous model system to elucidate in vivo the architecture of neuronal signal transduction pathways. We have isolated three cDNA clones encoding G protein α subunits that are expressed in the Lymnaea CNS, i.e. Gαo, Gα, and Gαi. The deduced proteins exhibit a very high degree of sequence identity to their vertebrate and invertebrate counterparts. The strong conservation or G protein α subunits suggests that functional insights into G protein‐mediated signalling routes obtained through the experimental amenability of the Lymnaea CNS will have relevance for similar pathways in the mammalian brain.
Primary structure determination of mono‐ and diacylglycerol lipase from Penicillium camembertii
Authors: Isobe Kimiyasu , Nokihara Kiyoshi
Published: July 2002

The complete amino acid sequence of mono‐ and diacylglycerol lipase from Penicillium camembertii was determined. This lipase has a single polypeptide chain consisting of 276 amino acid residues with two disulfide linkages. The primary structure was revealed by sequencing the digests of the intact and S‐pyridylethylated proteins by trypsin, endoproteinase Lys‐C and V8 protease. The two‐dimensional electrophoresis was also carried out to confirm the internal sequence. The catalytic triad of this lipase was Ser, Asp and His, and one potential N‐glycosylation site was also revealed.
Catabolite inactivation of the yeast maltose transporter is due to proteolysis
Authors: Lucero Pilar , Herweijer Marga , Lagunas Rosario
Published: July 2002

The maltose transport capacity of fermenting Saccharomyces cerevisiae rapidly decreases when protein synthesis is impaired. Using polyclonal antibodies against a recombinant maltose transporter‐protein we measured the cellular content of the transporter along this inactivation process. Loss of transport capacity was paralleled by a decrease of cross‐reacting material which suggests degradation of the transporter. We also show that in ammonium‐starved cells the half‐life of the maltose transporter is 1.3 h during catabolism of glucose and > 15 h during catabolism of ethanol.
Bibrotoxin, a novel member of the endothelin/sarafotoxin peptide family, from the venom of the burrowing asp Atractaspis bibroni
Authors: Becker Andreas , Dowdle Eugene B. , Hechler Ulrike , Kauser Katalin , Donner Peter , Schleuning Wolf-Dieter
Published: July 2002

A new member of the endothelin/sarafotoxin family of vasoconstrictor peptides, bibrotoxin (BTX), was isolated from the venom of the burrowing aspAtractaspis bibroni by reversed‐phase FPLC. The amino acid sequence of BTX differs from SRTX‐b in the substitution Ala4 instead of Lys4, which suggests that it represents the peptide isoform of Atractaspis bibroni corresponding to SRTX‐b. BTX competed for [125I]ET‐1 binding to human ETB‐type receptor with a Ki
of 3.2 × 10−9 M compared to 4.2 × 10−9 M for SRTX‐b. In rat thorax aorta BTX induced vasoconstrictions with a threshold concentration of 3 × 10−8 M compared to 1 × 10−9 for ET‐1.
Why do genes have introns?
Authors: Dibb N.J.
Published: July 2002

This review outlines some of the models that have been proposed to explain why genes have introns and then explores the possibility that alternative splicing might be the cause rather than a consequence of split genes.
Iron deprivation‐induced apoptosis in HL‐60 cells
Authors: Fukuchi Kunihiko , Tomoyasu Shigeru , Tsuruoka Nobuyoshi , Gomi Kunihide
Published: July 2002

Iron deprivation of HL‐60 cells with deferoxamine B mesylate (DFO) induced apoptosis. DNA fragmentation became apparent with 10−6 M DFO after 48 h treatment. The apoptosis peak according to the DNA histogram on flow cytometory and typical nuclear collapse and were observed microscopically after 48 h treatment with 10−4 M DFO. Cells treated with 10−4 M DFO for as little as 24 h were shown to be committed to apoptosis, as chromatin condensation progressed gradually thereafter.
Visualisation by electron microscopy of the unique part of the cytoplasmic domain of a desmoglein, a cadherin‐like protein of the desmosome type of cell junction
Authors: Rutman A.J. , Buxton R.S. , Burdett I.D.J.
Published: July 2002

Part of the cytoplasmic domain of a human desmoglein, Dsgl, a cadherin‐like protein found in desmosomes of epithelial cells, has been visualised by electron microscopy. The cloned fragment contains five repeats of a 29 ± 4 residue sequence unique to desmogleins, followed by a glycine‐rich region. In rotary shadowed preparations the molecule consists of a globular head attached to a thin tail, the latter perhaps corresponding to the glycine‐rich region. This portion of the molecule is thought to span the width of the inner dense plaque. The structure and dimensions concur well to the configuration deduced from the protein sequence.
Identification of a cDNA encoding a Drosophila calcium/calmodulin regulated protein phosphatase, which has its most abundant expression in the early embryo
Authors: Brown Linda , Chen Mao Xiang , Cohen Patricia T.W.
Published: July 2002

A 3.3 kb cDNA encoding the complete amino acid sequence of a calcium/calmodulin regulated protein phosphatase has been isolated from a Drosophila eye disc cDNA library. The predicted protein of 560 amino acids (molecular mass 62 kDa) is 73–78% identical to human PP2B isoforms. The cDNA hybridised to the X‐chromosome at cytological position 14D1‐4. Two transcripts of 3.5 kb and 3.0 kb were expressed during embryonic development, their levels being highest in the early embryo. The larger transcript was also clearly present in adult females. This pattern of expression indicates a role for calcium/calmodulin regulated protein phosphatase in embryonic development.
Reduction of 14–16 kDa allergenic proteins in transgenic rice plants by antisense gene
Authors: Tada Yuichi , Nakase Masayuki , Adachi Takahiro , Nakamura Ryo , Shimada Hiroaki , Takahashi Masayoshi , Fujimura Tatsuhito , Matsuda Tsukasa
Published: July 2002

An antisense gene strategy was applied to suppress the 14–16 kDa allergen gene expression in maturing rice seeds. Gene constructs producing antisense RNAs of the 16 kDa allergen under the control of some rice seed‐specific promoters were introduced into rice by electroporation. Immunoblot and RNA blot analyses of the seeds from the transgenic rice plants using the allergen‐specific monoclonal antibody and a sequence‐specific antisense RNA probe demonstrated that the 14–16 kDa allergen proteins and their transcripts of the seeds from several transgenic lines were present in much lower in amounts than those of the seeds from parental wild‐type rice. The high levels of reduction observed were stably inherited in at least three generations.
Protein stabilization through phage display
Authors: Chakravarty Suvobrata , Mitra Nivedita , Queitsch Iris , Surolia Avadhesha , Varadarajan Raghavan , Dübel Stefan
Published: July 2002

RNase S consists of two proteolytic fragments of RNase A, residues 1–20 (S20) and residues 21–124 (S pro). A 15‐mer peptide (S15p) with high affinity for S pro was selected from a phage display library. Peptide residues that are buried in the structure of the wild type complex are conserved in S15p though there are several changes at other positions. Isothermal titration calorimetry studies show that the affinity of S15p is comparable to that of the wild type peptide at 25°C. However, the magnitudes of ΔH° and ΔC
p are lower for S15p, suggesting that the thermal stability of the complex is enhanced. In agreement with this prediction, at pH 6, the T
m of the S15p complex was found to be 10°C higher than that of the wild type complex. This suggests that for proteins where fragment complementation systems exist, phage display can be used to find mutations that increase protein thermal stability.
Genetic determinants of homocysteine thiolactonase activity in humans: implications for atherosclerosis
Authors: Jakubowski Hieronim , Ambrosius Walter T , Pratt J.Howard
Published: July 2002

A metabolite of homocysteine (Hcy), the thioester Hcy thiolactone, damages proteins by modifying their lysine residues which may underlie Hcy‐associated cardiovascular disease in humans. A protein component of high density lipoprotein, Hcy thiolactonase (HTase) hydrolyzes thiolactone to Hcy. Thiolactonase is a product of the polymorphic PON1 gene, also involved in detoxification of organophospates and implicated in cardiovascular disease. Polymorphism in PON1 affects the detoxifying activity of PON1 in a substrate‐dependent manner. However, how PON1 polymorphism affects HTase activity is unknown. Here we report a strong association between the thiolactonase activity and PON1 genotype in human populations. High thiolactonase activity was associated with L55 and R192 alleles, more frequent in blacks than in whites. Low thiolactonase activity was associated with M55 and Q192 alleles, more frequent in whites than in blacks. High thiolactonase activity afforded better protection against protein homocysteinylation than low thiolactonase activity. These results suggest that variations in HTase may play a role in Hcy‐associated cardiovascular disease.
The human and murine protocadherin‐β one‐exon gene families show high evolutionary conservation, despite the difference in gene number
Authors: Vanhalst Koen , Kools Patrick , Vanden Eynde Eveline , van Roy Frans
Published: July 2002

Extensive cDNA analysis demonstrated that all human and mouse protocadherin‐β genes are one‐exon genes. The protein sequences of these genes are highly conserved, especially the three most membrane‐proximal extracellular domains. Phylogenetic analysis suggested that this unique gene family evolved by duplication of one single protocadherin‐β gene to 15 copies. The final difference in the number of protocadherin‐β genes in man (#19) and mouse (#22) is probably caused by duplications later in evolution. The complex relationship between human and mouse genes and the lack of pseudogenes in the mouse protocadherin‐β gene cluster suggest a species‐specific evolutionary pressure for maintenance of numerous protocadherin‐β genes.
1H NMR evidence that Glu‐38 interacts with the N‐terminal functional domain in interleukin‐8
Authors: Rajarathnam Krishnakumar , Clark-Lewis Ian , Dewald Beatrice , Baggiolini Marco , Sykes Brian D.
Published: July 2002

In order to assess the importance of the buried Glu‐38 observed in the structure of interleukin‐8, an analog in which Glu‐38 was replaced with Ala (E38A analog) was investigated by 1H NMR spectroscopy and neutrophil activation. Detailed analysis of the NMR NOESY data showed that the solution structure of the E38A analog is essentially the same as that for the native protein. Also, the neutrophil elastase activity of the E38A analog was similar to that of the native protein. However, the Gln‐8 and Cys‐9 amide proton chemical shifts, which are significantly downfield‐shifted in the native protein, exhibit more ‘normal’ values. This observation indicates that in the native protein, Glu‐38 side‐chain carboxylate interacts with Gln‐8 and Cys‐9 amide protons. Although the N‐terminal residues are critical for function, this interaction is not essential for neutrophil activation.
A flexible lid controls access to the active site in 1,3,8‐trihydroxynaphthalene reductase
Authors: Andersson Arnold , Jordan Douglas , Schneider Gunter , Lindqvist Ylva
Published: July 2002

The crystal structures of apo‐1,3,8‐trihydroxy‐naphthalene reductase from Magnaporthe grisea and a binary complex of the enzyme with NADPH have been determined to 2.8 Å resolution. In both cases, the overall structure is preserved compared to the structure of the ternary complex of the enzyme with NADPH and an active site inhibitor. No electron density for the helix–loop–helix region comprising residues 214–244 is observed indicating structural disorder in this part of the apoenzyme and the binary complex. In the ternary complex, this region is in contact with NADPH and the inhibitor and closes off the active site. The observed increase in flexibility in the absence of the inhibitor indicates that this region acts as a lid which closes the active site upon binding of the inhibitor and, possibly the substrate, 1,3,8‐trihydroxynaphthalene.
Human mitochondrial import receptor, Tom20p. Use of glutathione to reveal specific interactions between Tom20‐glutathione S‐transferase and mitochondrial precursor proteins
Authors: Schleiff Enrico , Shore Gordon C , Goping Ing Swie
Published: July 2002

© 1997 Federation of European Biochemical Societies.
A novel fluorescent marker for assembled mitochondria ATP synthase of yeast
Authors: Prescott Mark , Lourbakos Afrodite , Bateson Michael , Boyle Glen , Nagley Phillip , Devenish Rodney J.
Published: July 2002

We have shown that OSCP, a subunit of yeast mitochondrial ATP synthase, can be incorporated into the intact enzyme as a fusion protein representing OSCP fused at its C‐terminus to the green fluorescent protein (GFP) of Aequorea victoria. The relevant fusion OSCP‐GFP‐h6 additionally contains a hexahistidine tag at the C‐terminus. Expression of OSCP‐GFP‐h6 in yeast cells lacking endogenous OSCP led to the efficient restoration of growth of cells on the non‐fermentable substrate, ethanol. Confocal laser scanning microscopy revealed fluorescence due to GFP in mitochondria of cells expressing OSCP‐GFP‐h6. Use of immobilised metal ion affinity chromatography enabled the recovery of assembled ATP synthase complexes which contained OSCP‐GFP‐h6 identified by its mobility on SDS‐PAGE and immunoreactivity to anti‐OSCP and anti‐GFP antibodies. The successful isolation of the assembled multisubunit ATP synthase containing GFP fused to one of the essential subunits of the complex widely expands the potential applications of GFP. In principle, these include the spatial and temporal monitoring of ATP synthase complexes in vivo, and the exploration of interactions involving ATP synthase subunits by fluorescence resonance energy transfer (FRET).
Effect of chromophore exchange on the resonance Raman spectra of recombinant phytochromes
Authors: Kneip C , Mozley D , Hildebrandt P , Gärtner W , Braslavsky S.E , Schaffner K
Published: July 2002

The recombinant 65‐kDa polypeptide of phyA oat phytochrome was expressed by yeast Pichia pastoris and assembled into two chromopeptides with the chromophores phytochromobilin (PΦB) and phycocyanobilin (PCB), respectively. The Pr and Pfr states of the two protein variants were characterized by resonance Raman (RR) spectroscopy and compared with native phyA oat phytochrome demonstrating that the deletion of the C‐terminal half of phyA does not alter the structure of the chromophore site within the N‐terminal half. Most of the RR spectral changes observed upon replacing PΦB by PCB can be attributed exclusively to altered vibrational mode compositions due to the different ring D substitutions (vinyl vs. ethyl), implying that the chromophore structures are largely the same for PΦB‐ and PCB‐assembled phytochromes. Only in the Pr state may the RR spectral changes also reflect subtle differences of the PΦB and PCB conformations in the 65‐kDa phyA, presumably brought about by the specific steric requirements of the vinyl and ethyl groups.
Ro31‐8220 inhibits protein kinase C to block the phorbol ester‐stimulated release of choline‐ and ethanolamine‐metabolites from C6 glioma cells: p70 S6 kinase and MAPKAP kinase‐1β do not function downstream of PKC in activating PLD
Authors: Morreale Angela , Mallon Barbara , Beale Gary , Watson John , Rumsby Martin
Published: July 2002

The use of bisindolylmaleimide derivatives of staurosporine as selective inhibitors of protein kinase C (PKC) is in doubt following the report by Alessi [FEBS Lett. 402 (1997) 121–123] that Ro31‐8220 and GF109203X are potent in vitro inhibitors of p70 S6 kinase and mitogen‐activated protein kinase‐activated protein kinase‐1β, as well as of PKC. Here we show that the phorbol ester‐stimulated release of choline‐ and ethanolamine‐metabolites from C6 glioma cells due to phospholipid hydrolysis by phospholipase D (PLD) is not inhibited by rapamycin or PD98059, specific inhibitors respectively of p70 S6 kinase and MAPKK (MEK) and thus of MAPKAP kinase‐1β but is still completely blocked by Ro31‐8220. We conclude therefore that p70S6k and MAPKAP kinase‐1β as well as MAPK are not involved in signalling pathways downstream of PKC that regulate phorbol ester‐stimulated phospholipid turnover and that the inhibitory action of Ro31‐8220 occurs by blocking PKC which regulates at least one pathway to PLD activation. The PI‐3 kinase inhibitor, wortmannin, inhibits the phorbol ester‐stimulated release of ethanolamine‐ but not choline‐metabolites from C6 cells suggesting that different PLD isoforms regulate the turnover of PtdEth and PtdCho in C6 cells. Both PLD isoforms are activated via PKC but the PtdEth‐PLD is also regulated via a wortmannin‐sensitive pathway.
Two immunoglobulin‐like domains of the Z‐disc portion of titin interact in a conformation‐dependent way with telethonin
Authors: Mues Alexander , van der Ven Peter F.M , Young Paul , Fürst Dieter O , Gautel Mathias
Published: July 2002

The giant muscle protein titin/connectin plays a crucial role in myofibrillogenesis as a molecular ruler for sarcomeric protein sorting. We describe here that the N‐terminal titin immunoglobulin domains Z1 and Z2 interact specifically with telethonin in yeast two‐hybrid analysis and protein binding assays. Immunofluorescence with antibodies against the N‐terminal region of titin and telethonin detects both proteins at the Z‐disc of human myotubes. Longer titin fragments, comprising a serine‐proline‐rich phosphorylation site and the next domain, do not interact. The interaction of telethonin with titin is therefore conformation‐dependent, reflecting a possible phosphorylation regulation during myofibrillogenesis.
Isolation and characterization of adociavirin, a novel HIV‐inhibitory protein from the sponge Adocia sp.1
Authors: O'Keefe Barry R , Erim Tolga , Beutler John A , Cardellina John H , Gulakowski Robert J , Krepps Benjamin L , McMahon James B , Sowder Raymond C , Johnson Donald G , Buckheit Robert W , Halliday Susan , Boyd Michael R
Published: July 2002

Aqueous extracts of the New Zealand sponge Adocia sp. (Haplosclerida) displayed potent anticytopathic activity in CEM‐SS cells infected with HIV‐1. Protein fractions of the extract bound both to the viral coat protein gp120 and to the cellular receptor CD4, but not to other tested proteins. The purified active protein, named adociavirin, was characterized by isoelectric focusing, amino acid analysis, MALDI‐TOF mass spectrometry and N‐terminal sequencing. Adociavirin, a disulfide‐linked homodimer with a native molecular weight of 37 kDa, was active against diverse strains and isolates of HIV‐1, as well as HIV‐2, with EC50 values ranging from 0.4 nM to >400 nM. The anti‐HIV potency of adociavirin appears dependent on host cell type, with macrophage cultures being the most sensitive and peripheral blood lymphocytes the most resistant.
Lipophilin, a novel heterodimeric protein of human tears
Authors: Lehrer Robert I , Xu Guorong , Abduragimov Adil , Dinh Nhu Nguyen , Qu Xiao-Dan , Martin Daniel , Glasgow Ben J
Published: July 2002

We identified a novel heterodimeric protein, lipophilin AC, in human tears. One of its components, lipophilin A (69 residues; mass, 7575.1; pI, 9.47) was homologous to the C1 and C2 components of prostatein (‘estramustine‐binding protein'), the major secreted protein of rat prostate. Human lipophilin C (77 residues; mass, 8854.1; pI, 4.94) was homologous to the rat prostatein C3 component and to human mammaglobin, a protein overexpressed in some mammary carcinomas. Tear lipophilins A and C expand the roster of human uteroglobin superfamily members and provide models for exploring these typically steroid‐regulated and steroid‐binding molecules.
Upregulation of caveolin‐1 and caveolae organelles in Taxol‐resistant A549 cells
Authors: Yang Chia-Ping Huang , Galbiati Ferruccio , Volonté Daniela , Horwitz Susan Band , Lisanti Michael P
Published: July 2002

Caveolin is a principal component of caveolae membranes. It has been demonstrated that the interaction of the caveolin scaffolding domain with signaling molecules can functionally inhibit the activity of these molecules. Taxol is an antitumor agent that suppresses microtubule dynamics and binds to microtubules thereby stabilizing them against depolymerization. The drug also has been implicated in the induction of apoptosis through activation of components in signal transduction cascades. Here we have investigated the role of caveolin in the development of drug resistance by examining the expression of caveolins in low‐ and high‐level drug‐resistant cell lines. Caveolin‐1, but not caveolin‐2, was upregulated in highly multidrug resistant SKVLB1 cells that express high levels of P‐glycoprotein, and in low‐level Taxol‐resistant A549 cell lines that express low amounts of P‐glycoprotein. Two drug‐resistant A549 cell lines (one 9‐fold resistant to Taxol and the other 1.5‐fold resistant to epothilone B), both of which express no P‐glycoprotein, demonstrate a significant increase in the expression of caveolin‐1. These results indicate that in low‐level epothilone B‐ or Taxol‐resistant A549 cells, increased caveolin‐1 expression occurs independently of P‐glycoprotein expression. Electron microscopic studies clearly demonstrate the upregulation of caveolae organelles in Taxol‐resistant A549 cells. Upregulation of caveolin‐1 expression in drug‐sensitive A549 cells was observed acutely beginning 48 h after incubation with 10 nM Taxol. Thus, caveolin‐1 may play a role in the development of Taxol resistance in A549 cells.
The artificial α1β1‐contact mutant hemoglobin, Hb Phe‐35β, shows only small functional abnormalities
Authors: Nakatsukasa Takashi , Nomura Noriko , Miyazaki Gentaro , Imai Kiyohiro , Wada Yoshinao , Ishimori Koichiro , Morishima Isao , Morimoto Hideki
Published: July 2002

It was previously reported that Hb Philly with a mutation of Phe for Tyr at 35(C1)β showed non‐cooperative oxygen binding with a very high affinity and instability leading to hemolysis. Further, it lacked the 1H‐NMR signal at 13.1 ppm from 2,2‐dimethyl‐2‐silapentane‐5‐sulfonate in normal hemoglobin (Hb A), so that this signal was assigned to a hydrogen bond formed by Tyr‐35(C1)β. Surprisingly, our artificial mutant hemoglobin with the same mutation as Hb Philly showed slightly lowered oxygen affinity, almost normal cooperativity, the 1H‐NMR signal at 13.1 ppm and no sign of instability. Our results indicate that the mutation reported for Hb Philly and the assignment of the 13.1 ppm signal need reexamination.
Selective recognition of the membrane‐bound CX3C chemokine, fractalkine, by the human cytomegalovirus‐encoded broad‐spectrum receptor US28
Authors: Kledal Thomas N , Rosenkilde Mette M , Schwartz Thue W
Published: July 2002

The 7TM receptor, US28, encoded by human cytomegalovirus binds a broad spectrum of endogenous CC chemokines with sub‐nanomolar affinity as determined in homologous competition binding assays. We here find that US28 also recognizes the membrane‐associated CX3C chemokine, fractalkine, with sub‐nanomolar affinity (IC50=0.42±0.09 nM). Importantly, although fractalkine could compete with high affinity against the binding of CC chemokines, the secreted CC chemokines were only able to compete for binding against radioactive fractalkine with very low affinity. It is concluded that US28, which is known to enhance cell‐cell fusion processes through interaction with an as yet unidentified, human cell‐specific factor, has been optimized by cytomegalovirus to selectively recognize the membrane‐associated fractalkine. It is suggested that US28 expressed on the surface of infected cells and possibly on the envelope of the virion is involved in transfer of the virus from cell to cell.
Identification of a splice variant of neutrophil collagenase (MMP‐8)
Authors: Hu Shou-Ih , Klein Melissa , Carozza Marc , Rediske John , Peppard Jane , Qi Jian-Shen
Published: July 2002

We have identified a splice variant of human neutrophil collagenase (MMP‐8) transcript (MMP‐8alt) that has a 91 bp insertion between codons for amino acid residues 34 and 35 of MMP‐8 cDNA. This splice variant encodes an open reading frame for a 444 residue protein, lacking a secretory signal sequence. Our data suggested that, as opposed to the original MMP‐8, the translation product of MMP‐8alt is not a secreted protein; nevertheless, it is enzymatically active. Further studies aimed at identifying the physiological substrates of MMP‐8alt protein may lead to uncover novel roles it plays in cellular physiology.
Effects of nitric oxide on the eosinophil survival in vitro. A role for nitrosyl‐heme
Authors: Beauvais Francis , Joly Francine
Published: July 2002

In a previous paper we showed that the nitric oxide (NO) donors azide and hydroxylamine inhibited eosinophil apoptosis. Azide and hydroxylamine generate a nitrosyl‐heme complex – due to endogenous catalase activity – which activates soluble guanylate cyclase. In contrast, in the present paper, we show that NO donors (SNAP, SIN‐1, S‐nitroso‐l‐cysteine, NOC‐18) which spontaneously release NO in physiological solutions did not support the survival of eosinophils and induced apoptosis or necrosis. However, the addition of hematin (the ferric form of heme) together with low doses of NO (SNAP 10 μM) promoted eosinophil survival. In conclusion, we propose that NO and heme (e.g. from heme‐containing enzymes such as peroxidase or catalase), both released in inflammation sites, could form nitrosyl‐heme and thus promote eosinophilic inflammation.
Re‐entering the translocon from the lumenal side of the endoplasmic reticulum. Studies on mutated carboxypeptidase yscY species
Authors: Plemper Richard K. , Deak Peter M. , Otto Ralf T. , Wolf Dieter H.
Published: July 2002

Misfolded or unassembled secretory proteins are retained in the endoplasmic reticulum (ER) and subsequently degraded by the cytosolic ubiquitin‐proteasome system. This requires their retrograde transport from the ER lumen into the cytosol, which is mediated by the Sec61 translocon. It had remained a mystery whether ER‐localised soluble proteins are at all capable of re‐entering the Sec61 channel de novo or whether a permanent contact of the imported protein with the translocon is a prerequisite for retrograde transport. In this study we analysed two new variants of the mutated yeast carboxypeptidase yscY, CPY*: a carboxy‐terminal fusion protein of CPY* and pig liver esterase and a CPY* species carrying an additional glycosylation site at its carboxy‐terminus. With these constructs it can be demonstrated that the newly synthesised CPY* chain is not retained in the translocation channel but reaches its ER lumenal side completely. Our data indicate that the Sec61 channel provides the essential pore for protein transport through the ER membrane in either direction; persistent contact with the translocon after import seems not to be required for retrograde transport.
Effective expression and purification of recombinant onconase, an antitumor protein
Authors: Notomista Eugenio , Cafaro Valeria , Fusiello Rossano , Bracale Aurora , D'Alessio Giuseppe , Di Donato Alberto
Published: July 2002

Several members of the RNase A superfamily are endowed with antitumor activity, showing selective cytotoxicity toward several tumor cell lines. One of these is onconase, the smallest member of the RNase A superfamily, which is at present undergoing phase III clinical trials. We report here the expression of recombinant onconase in Escherichia coli inclusion bodies, the correct processing of the protein, followed by its purification in high yields. The recombinant protein has biological and catalytic properties identical to those of the natural enzyme.