bluuebunny/crossref_metadata_2025 huggingface dataset:
SGP28, a novel matrix glycoprotein in specific granules of human neutrophils with similarity to a human testis‐specific gene product and to a rodent sperm‐coating glycoprotein
Authors: Kjeldsen Lars , Cowland Jack B. , Johnsen Anders H. , Borregaard Niels
Published: April 2003

A novel 28 kDa glycoprotein was purified from exocytosed material from human neutrophils and its primary structure partially determined. Degenerate oligonucleotide primers were used to amplify cDNA clones from a human bone marrow cDNA library. The deduced 245 amino acid sequence of the 2124 bp full‐length cDNA showed high degrees of similarity to the deduced sequences of the human gene TPX‐1 and of sperm coating glycoprotein from rat and mouse. Subcellular fractionation of human neutrophils indicated that the protein is localized in specific granules. The protein was named SGP28 (specific granule protein of 28 kDa).
Ancient divergence of long and short isoforms of adenylate kinase molecular evolution of the nucleoside monophosphate kinase family
Authors: Fukami-Kobayashi Kaoru , Nosaka Michiko , Nakazawa Atsushi , Gō Mitiko
Published: April 2003

Adenylate kinases (AK) from vertebrates are separated into three isoforms, AK1, AK2 and AK3, based on structure, subcellular localization and substrate specificity. AK1 is the short type with the amino acid sequence being 27 residues shorter than sequences of the long types, AK2 and AK3. A phylogenetic tree prepared for the AK isozymes and other members of the nucleoside monophosphate (NMP) kinase family shows that the divergence of long and short types occurred first and then differentiation in subcellular localization or substrate specificity took place. The first step involved a drastic change in the three‐dimensional structure of the LID domain. The second step was caused mainly by smaller changes in amino acid sequences.
Noradrenaline inhibition of Ca2+ channels and secretion in single patch‐clamped insulinoma cells
Authors: Sher Emanuele , Codignola Agnese , Rogers Marc , Richmond Janet
Published: April 2003

Noradrenaline effects on voltage‐operated calcium channels and exocytosis were studied, for the first time, in single patch‐clamped RINm5F insulin‐secreting cells. Noradrenaline, despite small and variable inhibition of calcium currents, strongly inhibited the increase in membrane capacitance (a measure of exocytosis) stimulated by both step depolarizations and the calcium ionophore, ionomycin. Noradrenaline similarly inhibited KCl‐ and ionomycin‐induced [3H]serotonin release from RINm5F cell populations. Noradrenaline effects were mediated by PTX‐sensitive G proteins. Noradrenaline inhibitory effects on secretion are, therefore, mainly exerted downstream from Ca2+ influx.
Flow cytometry analysis of α1‐adrenoceptor subtypes
Authors: Hirasawa Akira , Tsumaya Keiko , Awaji Takeo , Shibata Katsushi , Homma Nobuo , Shinomiya Takahisa , Tsujimoto Gozoh
Published: April 2003

To characterize the α
1‐adrenoceptor subtypes, we developed a flow cytometry method using the fluorescent ligand BODIPY‐FL prazosin and the anti‐peptide antibody against the α
1b‐adrenoceptor amino terminus (designated 1B‐N1‐C) as probes. Three α
1‐adrenoceptors (α
1a, β
1b and α
1d) expresed in CHO cells were detected by BODIPY‐FL prazosin; however, only α
1b‐adrenoceptor subtype was detected by the anti‐peptide antibody 1B‐N1‐C. Furthermore, the flow cytometry analysis with 1B‐N1‐C specifically identified α
1b‐adrenoceptor in native cells of hamster DDT1‐MF2 cells, rat hepatocytes and1 cardiomyocytes.
Intracellular free Ca2+ dynamic changes to histamine are reduced in cystic fibrosis human tracheal gland cells
Authors: Jacquot Jacky , Maizières Michaël , Spilmont Christophe , Millot Jean-Marc , Sébille Stéphane , Merten Marc , Kammouni Wafa , Manfait Michel
Published: April 2003

This study documents a difference between cystic fibrosis human (CF‐HTG) and normal human (HTG) tracheal gland cells: the ability of histamine to induce an increase of intracellular free calcium concentration [Ca2+]i was abnormally reduced in CF‐HTG cells. The magnitude of the [Ca2+]i peak rise in response to histamine is smaller in CF‐HTG cells than in HTG cells, and the percentage of CF‐HTG cells that increase [Ca2+]i is decreased compared with HTG cells. In contrast to histamine, the human neutrophil elastase (HNE) stimulation of both CF‐HTG and HTG cells generated [Ca2+]i asynchronous oscillations and the magnitude of the peak [Ca2+]i response as well as the percentage of responding cells were similar for both groups. By videomicroscopy observations, the secretory response (exocytosis of secretion granules) of CF‐HTG cells occurred with HNE, but not with histamine, thus suggesting that [Ca2+]i asynchronous oscillations may be linked to the exocytosis process in human tracheal gland cells.
The vacuolar ATPase proton pump is required for the cytotoxicity of Bacillus anthracis lethal toxin
Authors: Ménard Armelle , Altendorf Karlheinz , Breves Daniel , Mock Michèle , Montecucco Cesare
Published: April 2003

The nature of the cytopathic effect exerted by the lethal factor toxin (LF) of Bacillus anthracis on sensitive cells is unknown. The toxin requires the passage through acidic vesicles in order to exert its effect within the cytosol. Here, we show that bafilomycins and concanamycin A, selective inhibitors of the vacuolar ATPase proton pump, are the most powerful known inhibitors of LF macrophage toxicity. These inhibitors are fully active long after LF addition to macrophages, suggesting that LF enters the cytosol after having reached a late endosomal compartment.
Molecular characterization of a new urea transporter in the human kidney
Authors: Olivès Bernadette , Martial Sonia , Mattei Marie-Geneviève , Matassi Giorgio , Rousselet Germain , Ripoche Pierre , Cartron Jean-Pierre , Bailly Pascal
Published: April 2003

A cDNA clone (HUT2) sharing 61.1% and 89.9% sequence identity with the human erythroid (HUT11) and the rabbit (UT2) urea transporters, respectively, was isolated by homology cloning from a human kidney library. HUT2 transcripts were restricted to the kidney and the HUT2 polypeptide was not immunoprecipitated with blood group Kidd‐related antibodies (anti‐Jk3) in coupled transcription‐translation assays. Functional expression studies in Xenopus oocytes demonstrated that HUT2‐mediated urea transport was not inhibited by p‐chloromercuribenzene sulfonate (pCMBS) which, however, inhibited the urea flux mediated by HUT11. These findings demonstrate that at least two distinct urea transporters are present in human tissues. By in situ hybridization, the gene encoding HUT2 has been assigned to chromosome 18q12.1‐q21‐1, as found previously for the Kidd/urea transporter HUT11, suggesting that both genes evolved from duplication of a common ancestor.
Protein kinase C and F‐actin are essential for stimulation of neuronal FAK tyrosine phosphorylation by G‐proteins and amyloid beta protein
Authors: Zhang Chi , Qiu H.Erin , Krafft Grant A. , Klein William L.
Published: April 2003

Focal adhesion kinase (FAK) is a protein tyrosine kinase implicated in signal transduction pathways for integrins, neuropeptides, and lysophosphatidic acid. FAK, first discovered in non‐neuronal cells, recently has been reported to occur in neurons, where its tyrosine phosphorylation is upregulated by fibronectin and by the Alzheimer's Aβ peptide. The current work has elucidated molecular events leading to tyrosine phosphorylation of FAK in the rat B103 CNS nerve cell line. Activation of receptor‐coupled G‐proteins by Mas‐7 was found to evoke rapid upregulation of FAK tyrosine phosphorylation (Tyr(P)). Upregulation by Mas‐7 was blocked by GF109203X, a potent inhibitor of protein kinase C (PKC). Phorbol ester also upregulated FAK‐YP, verifying a role for PKC in the transduction cascade. Upregulation of FAK‐YP by activation of G‐proteins and PKC was dependent upon intact F‐actin, as cytochalasin D abolished stimulation by Mas‐7 and by phorbol ester. The relatively slow increase in FAK‐YP evoked by chronic exposure to Aβ also was abolished by GF109203X and by cytochalasin D. The results show that tyrosine phosphorylation of FAK in neurons is regulated positively by PKC, functioning down‐stream from G‐proteins through an F‐actin‐dependent mechanism. The Alzheimer's Aβ peptide is capable of activating elements of this same signal transduction pathway, via membrane events that remain to be determined.
Cloning of a melatonin‐related receptor from human pituitary
Authors: Reppert Steven M. , Weaver David R. , Ebisawa Takashi , Mahle Cathy D. , Kolakowski Lee F.
Published: April 2003

We have cloned an orphan G protein‐coupled receptor from a human pituitary cDNA library using a probe generated by PCR. The cDNA, designated H9, encodes a protein of 613 amino acids that is 45% identical at the amino acid level to the recently cloned human Mel1a and Mel1b melatonin receptors. Structural analyses of the encoded protein and its gene, along with phylogenetic analysis, further show that H9 is closely related to the G protein‐coupled melatonin receptor family. Unusual features of the protein encoded by H9 include a lack of N‐linked glycosylation sites and a carboxyl tail >300 amino acids long. H9 transiently expressed in COS‐1 cells did not bind [125I]melatonin or [3H]melatonin. H9 mRNA is expressed in hypothalamus and pituitary, suggesting that the encoded receptor and its natural ligand are involved in neuroendocrine function.
Tyrosine phosphorylation of ACK in response to temperature shift‐down, hyperosmotic shock, and epidermal growth factor stimulation
Authors: Satoh Takaya , Kato Juran , Nishida Kazuhiko , Kaziro Yoshito
Published: April 2003

The mammalian Cdc42 protein regulates various kinds of cellular responses, including formation of filopodia, polarization of T cells, and cell cycle progression. A non‐receptor tyrosine kinase ACK, which specifically binds to the GTP‐bound form of Cdc42, was isolated as a putative target of Cdc42. Here we show the induction of tyrosine phosphorylation of ACK in response to temperature shift‐down to 25°C, and hypertonic shock, as well as stimulation with epidermal growth factor (EGF) in human embryonic kidney (HEK) 293 cells. The increased tyrosine phosphorylation level upon temperature shift‐down was sustained for at least 60 min, whereas reversion of the temperature to 37°C caused rapid tyrosine dephosphorylation to the initial level. The responses to EGF and the high osmolarity were transient. Furthermore, we observed association of ACK with an adaptor protein Grb2, which may suggest the involvement of Grb2 in EGF receptor‐mediated tyrosine phosphorylation of ACK.
ADP delivery from adenylate kinase in the mitochondrial intermembrane space to oxidative phosphorylation increases in the presence of macromolecules
Authors: Laterveer Fanny D. , Nicolay Klaas , Gellerich Frank N.
Published: April 2003

Macromolecules were added to isolated rat liver mitochondria to mimic cytosolic macromolecules and tested for their effects on the ADP delivery from adenylate kinase in the intermembrane space to oxidative phosphorylation. In the presence of 10% (w/v) dextran M20 or bovine serum albumin, approximately 60% of the maximal ADP flux from adenylate kinase to oxidative phosphorylation was not accessible to an extramitochondrial ADP scavenger. In the absence of macromolecules this was 34%. ADP determinations from incubations with macromolecules demonstrated the existence of flux‐dependent ADP concentration gradients across the outer membrane which can be as high as 12 μM.
Localization of leptin receptor mRNA and the long form splice variant (Ob‐Rb) in mouse hypothalamus and adjacent brain regions by in situ hybridization
Authors: Mercer Julian G. , Hoggard Nigel , Williams Lynda M. , Lawrence C.Bruce , Hannah Lisa T. , Trayhurn Paul
Published: April 2003

Expression of the leptin receptor gene has been examined in mouse hypothalamus and other brain regions by in situ hybridization. With a probe recognizing all the known splice variants, receptor mRNA was evident in several brain regions (cortex, hippocampus, thalamus), with strong expression in the hypothalamus (arcuate, ventromedial, paraventricular and ventral premammillary nuclei), choroid plexus and leptomeninges. A probe specific to the long splice variant of the leptin receptor (Ob‐Rb), containing the putative intracellular signaling domain, again revealed strong expression in the hypothalamus; there was, however, minimal hybridization to choroid plexus and leptomeninges. These results indicate that the hypothalamus is a key site of leptin action, although other brain regions are also targeted.
Zfp60, a mouse zinc finger gene expressed transiently during in vitro muscle differentiation
Authors: Perez Marie , Rompato Giovanni , Corbi Nicoletta , De Gregorio Laura , Dragani Tommaso A. , Passananti Claudio
Published: April 2003

The complete cDNA coding sequence of the zinc finger gene Zfp60 is reported. The predicted amino acid sequence of the Zfp60 protein has been found to contain 19 zinc finger motives clustered at the C‐terminus. At its N‐terminus, Zfp60 shares with other members of the zinc finger gene family two additional conserved amino acid modules named Kruppel Associated Boxes (KRAB). The expression patterns of Zfp60, MyoD and MHC mRNAs have been followed during in vitro myogenic differentiation of C2 cells. We show that the bacterial produced Zfp60 protein binds DNA only in presence of zinc ions. Zfp60 locus has been mapped in chromosome 7, where other Zfp loci are localised.
A model for DNA polymerase translocation: worm‐like movement of DNA within the binding cleft
Authors: Wlassoff W.A. , Dymshits G.M. , Lavrik O.I.
Published: April 2003

On the basis of recent results, we propose a model for DNA polymerase translocation along DNA. Human immunodeficiency virus reverse transcriptase is taken as an example. According to the model, movement of the enzyme is the result of transition of the enzyme‐bound DNA from the A‐ to B‐form which is accompanied by lengthening of DNA within the binding channel. The driving force of this transition is the increase in water accessibility to the DNA‐binding cleft after dNTP binding. dNTP hydrolysis proceeding during the following chemical step supplies the energy for the reverse B → A transition of DNA. Translocation is considered to be an integral part of the stage of conformational change preceding catalysis and can be described as a worm‐like movement of DNA within the DNA‐binding cleft.
Inhibitory mechanisms of H+‐ATPase inhibitor bafilomycin A1 and carbonic anhydrase II inhibitor acetazolamide on experimental bone resorption
Authors: Yasuo Ohba  , Tomoko Ohba  , Koji Sumitani  , Kahori Tagami-Kondoh  , Kenji Hiura  , Yoshiki Miki  , Hisao Kakegawa  , Teruko Takano-Yamamoto  , Nobuhiko Katunuma
Published: April 2003

The effects of the vacuolar‐type H+‐ATPase inhibitor bafilomycin A1 (baf. A1) and the carbonic anhydrase II inhibitor acetazolamide (AZ) on bone resorption and procathepsin L secretion of rat osteoclasts were investigated using the bone slice assay method, pit formation test. Baf. A1 completely suppressed osteoclastic bone resorption stimulated by parathyroid hormone (PTH), but did not affect procathepsin L secretion, while AZ suppressed both bone resorption and procathepsin L secretion. These findings suggest that bone resorption by procathepsin L secretion and its processing are regulated by proton production and proton secretion.
cDNA cloning of p42, a shared subunit of two proteasome regulatory proteins, reveals a novel member of the AAA protein family
Authors: Fujiwara Tsutomua , Watanabe Takeshi K. , Tanaka Keiji , Slaughter Clive A. , DeMartino George N.
Published: April 2003

We have employed cDNA cloning to deduce the complete primary structure of p42, a protein previously identified as a common subunit of two proteasome regulatory proteins: PA700, a 700 000‐Da multisubunit complex that binds to the proteasome and promotes the ATP‐dependent degradation of ubiquitinated proteins, and modulator, a 250 000‐Da PA700‐dependent proteasome activator. Computer analysis reveals that p42 is a novel member of a large protein family characterized by a conserved 200 amino acid domain which contains a consensus sequence for ATP binding. Five other members of this family, termed AAA proteins (TPases ssociated with a variety of cellular ctivities) are also subunits of PA700. Gel filtration chromatography was employed to determine the qualitative and quantitative distribution of p42 in crude soluble lysates of bovine red blood cells. These studies demonstrated that p42 was found in two multi‐protein complexes: the 26S proteasome (formed from the 20S proteasome and PA700) and the modulator. These results establish the identity of a new protein involved in the regulation of proteasome function and indicate that this protein is found in at least two different protein complexes.
Characterization of glucose transport in the hyperthermophilic Archaeon Sulfolobus solfataricus
Authors: Cusdin Fiona S. , Robinson Matthew J. , Holman Geoffrey D. , Hough David W. , Danson Michael J.
Published: April 2003

Sulfolobus solfataricus is a hyperthermophilic Archaeon growing at 80°C, pH 3. The glucose transport system of this organism has been characterised kinetically at this temperature and pH using 2‐deoxy‐d‐glucose: the sugar analogue is transported into the cells with a K
m=1.8±0.3μM and a V
max=3.6±0.1 nmol min−1 (mg protein)−1, with an intracellular accumulation of up to 200‐fold over the extracellular concentration. Transport was significantly reduced at pH 5. Inhibition of 2‐deoxy‐d‐glucose transport was investigated using a variety of sugars and sugar analogues; d‐glucose, d‐galactose and d‐mannose showed the highest affinity for the transporter, with d‐glucose possessing a K
i=120±20 nM.
Uncleaved env gp160 of human immunodeficiency virus type 1 is degraded within the Golgi apparatus but not lysosomes in COS‐1 cells
Authors: Kimura Tominori , Nishikawa Masao , Fujisawa Jun-ichi
Published: April 2003

The fate of newly synthesized human immunodeficiency virus type 1 env gp160 was examined in COS‐1 cells. The results of morphological chase experiments involving cycloheximide demonstrated that gp160 was retained in the Golgi apparatus for longer than the half‐life of the molecule. The degradation of gp160 was insensitive to both bafilomycin A1 and leupeptin (<0.2 mM), which block lysosomal proteolysis. However, degradation was effectively suppressed by leupeptin at higher concentrations, maximally at 1.7 mM. Furthermore, undegraded gp160 was accumulated in the Golgi apparatus, but was not detected in lysosomes. These results indicate that in COS‐1 cells gp160 is not degraded in lysosomes, but rather that degradation takes place in the Golgi apparatus.
Regulation of myosin heavy chain expression in adult rat hindlimb muscles during short‐term paralysis: comparison of denervation and tetrodotoxin‐induced neural inactivation
Authors: Michel Robin N. , Parry David J. , Dunn Shannon E.
Published: April 2003

The extent to which myosin profiles within adult fast and slow muscles are altered by short‐term paralysis remains equivocal. We used an array of specific antibodies to identify adult and developmental MHC isoforms within EDL and soleus muscle fibers, and show a marked multiple expression of MHCs with a general shift towards slower and more energy efficient MHC profiles after 2 weeks of denervation or TTX nerve conduction block. Paralysis also induced marked expression of an embryonic MHC within most EDL cell types, and a subtle, paralysis‐sensitive, expression of α‐cardiac MHC within specific EDL and soleus extrafusal fibers. Comparison of treatment groups also permitted assessment of the relative influence of neural activity versus trophic factors on these isoforms, and confirmed activity as a major, but not sole, regulator of MHC expression.
Transmembrane topology of Escherichia coli H+‐ATPase (ATP synthase) subunit a
Authors: Yamada Hiroshi , Moriyama Yoshinori , Maeda Masatomo , Futai Masamitsu
Published: April 2003

Escherichia coli H+‐ATPase subunit a is a hydrophobic F0 subunit. To investigate the topology of the subunit in the membrane, we prepared site‐specific polyclonal antibodies against amino‐terminal (Ser‐3 to Leu‐16), middle loop (Lys‐167 to Gln‐181), and carboxyl‐terminal (Thr‐259 to His‐271) peptide segments. Enzyme‐linked immunosorbent assay revealed that these antibodies specifically reacted with subunit a of inside‐out membrane vesicles, but not with that of right‐side‐out spheroplasts. Full reactivity appeared when spheroplasts were disrupted with Triton X‐100 (0.5%) or by sonication. These results suggest that at least parts of the three peptide segments of subunit a face the cytoplasm. Based on these observations, we propose a novel transmembrane topology of subunit a.
Crystallization and preliminary X‐ray analysis of human brain hexokinase
Authors: Aleshin Alexander E. , Zeng Chenbo , Fromm Herbert J. , Honzatko Richard B.
Published: April 2003

Human brain hexokinase type I, expressed in Escherichia coli, has been crystallized from polyethylene glycol 8000 in the presence of inorganic phosphate. The crystals are hexagonal needles of diameter 0.25 mm, diffracting to a resolution of 3.5 Å on a rotating‐anode/area‐detector system. The crystals belong to the space group P3121/P3221 with cell dimensions . The specific volume of the crystal is 4.2 Å3/Da, suggesting an asymmetric unit with a single 100 kDa molecule and a solvent content of 71% by volume or two molecules of hexokinase with a solvent content of 41%. The complex of hexokinase with glucose crystallizes under similar conditions, giving crystals of the same morphology.
The HMG box of SRY is a calmodulin binding domain
Authors: Harley Vincent R. , Lovell-Badge Robin , Goodfellow Peter N. , Hextall Patrick J.
Published: April 2003

The HMG box domain of the testis determining factor, SRY, includes a basic amphiphilic sequence common to calmodulin (CaM) binding proteins. By affinity chromatography, native gel electrophoresis and fluorescence spectroscopy, we show the calcium‐dependent binding of SRY to CaM. Binding occurs via the HMG box and an SRY peptide of residues 57–80 binds CaM like the intact domain. SRY/CaM complex formation is specifically inhibited by the SRY DNA binding site sequence, AACAAT, but not a mutated sequence. Fluorescence spectra of the SRY/CaM complex indicate 1:1 stoichiometry and that binding is accompanied by a conformational change in SRY. The A domain of HMG1 also binds CaM and we propose that CaM binding is a property of the wider HMG box family, including SOX and TCF/LEF proteins. These results suggest that CaM may regulate the DNA binding activity of HMG box transcription factors.
Involvement of a single‐stranded DNA binding protein, ssCRE‐BP/Purα, in morphine dependence
Authors: Osugi Takeshi , Ding Yun , Tanaka Hidekazu , Kuo Che-Hui , Do Eunju , Irie Yasuyuki , Miki Naomasa
Published: April 2003

We have purified a nuclear protein from mouse cerebella that binds to single‐stranded oligo‐DNA of cAMP response element and is modulated by morphine treatment. Isolation of the cDNA clone showed that the nuclear protein (ssCRE‐BP) was identical to Purα, a DNA binding protein for single‐stranded purine‐rich sequences that was originally isolated as a replication factor. ssCRE‐BP/Purα and mRNA were abundant in the brain. The levels of ssCRE‐BP/Purα and the transcript were not changed by chronic morphine treatment, however, the levels of an activator of ssCRE‐BP/Purα, which is necessary for the DNA binding, may be modulated by the treatment.
F protein induced fusion of Sendai viral envelopes with mouse teratocarcinoma cells through LeX‐LeX interaction
Authors: Kumar Mukesh , Sarkar Debi P.
Published: April 2003

The efficiency of membrane fusion between reconstituted Sendau viral envelopes containing only the fusion protein (F‐virosomes) and the plasma membrane of mouse teratocarcinoma cells (F9) in culture was assessed using an assay based on the relief of self‐quenching of a lipid probe incorporated in the F‐virosomes. The potential of F‐virosomes was also evaluated for a targeted cytosolic delivery of lysozyme to F9 cells. [125I]Lysozyme entrapped into F‐virosomes was taken to examine its fusion‐mediated transfer to the F9 cells. Target specificity of the F‐virosomes was confirmed by the interaction between the terminal Lex moiety (Ga1β1 → 4(Fucα1 → 3)GlcNac) of F protein and the Lex determinant on the membrane of F9 cells. Incubation of the loaded F‐virosomes with cells led to fusion‐mediated delivery, as inferred from the ability of cells to internalize lysozyme in the presence of azide (a potent inhibitor of endocytosis). These results suggest that carbohydrate‐carbohydrate interaction is strong enough for target cell recognition followed by phospholipid bilayer melding induced by fusion glycoprotein of Sendai virus.
Immunopurification and characterization of a collagenase/gelatinase domain issued from basement membrane fibronectin
Authors: Boudjennah Laziz , Dalet-Fumeron Véronique , Ylätupa Sari , Pagano Maurice
Published: April 2003

The proteolytic potential of cellular fibronectin fragments issued from a basement membrane hydrolysate was investigated. Three different gelatinase activities (47, 43 and 37 kDa), located by gelatin zymography, were isolated using successively heparin‐agarose, gelatin‐agarose and immunopurification with polyclonal antibodies directed against bovine plasma fibronectin. These fragments were also characterized using a monoclonal antibody directed against the extra‐domain EDA of cellular fibronectin as a probe. A collagenase activity, reliably indicated by the gelatin zymography pattern, was also found using MCA‐Pro‐Leu‐Gly‐Leu‐DPA‐Ala‐Arg‐NH2, the intramolecularly quenched fluorogenic substrate of collagenases. From these results, cellular fibronectin was found to be able to exhibit a proteolytic function after limited proteolysis. This MMP‐like function could be associated with tissue remodeling in both normal and pathological states, such as metastasis, angiogenesis and tissue repair.
Primary structure of a novel ABC transporter with a chromosomal localization on the band encoding the multidrug resistance‐associated protein
Authors: Klugbauer Norbert , Hofmann Franz
Published: April 2003

Complementary DNA clones encoding a novel protein, ABC‐C, with the typical structural features of the ABC transporter family were identified in a human medullary thyroid carcinoma cell line. The transporter consists of 1704 amino acid residues with two homologous repeats, each harboring six putative transmembrane helices and an ATP‐binding cassette motif. The mRNA is expressed highest in normal lung, but also in varying amounts in other tissues and in C‐cell carcinoma. The ABC‐C gene is mapped on chromosome 16p13.3, in close physical proximity to another ABC transporter, the multidrug resistance‐associated protein. This related protein is assumed to confer resistance to chemotherapeutic drugs in small cell lung carcinoma. The genomic clustering of both transporters, typical also for other members of the ABC family, supports the notion that ABC‐C may be involved in development of resistance to xenobiotics.
Differences in DNA‐binding efficiency of Sp1 to aldolase and pyruvate kinase promoter correlate with altered redox states in resting and proliferating rat thymocytes
Authors: Schäfer Doris , Hamm-Künzelmann Brigitte , Hermfisse Ulrich , Brand Karl
Published: April 2003

Thymocytes induce their glycolytic enzymes as they undergo transition from the resting to the proliferating state. Corresponding increases in mRNA levels point to a transcriptional regulation. Electrophoretic mobility shift assays revealed that the DNA‐binding efficiency of Sp1 is increased when nuclear extracts from proliferating compared to resting rat thymocytes were used. Here we demonstrate that hydrogen peroxide, added to nuclear extract from proliferating cells, decreases the Spl DNA‐binding activity, whereas in nuclear extracts from resting cells dithioerythritol fully restores DNA‐binding efficiency. Moreover we show that in contrast to resting thymocytes, production of reactive peroxide anions upon priming with phorbol 12‐myristate 13‐acetate is nearly abolished in the proliferating cells. From these results we propose that reactive oxygen intermediates affect the interaction of the Sp1 transcription factor with its consensus sequence and subsequently regulate glycolytic gene expression.
A model phage display subtraction method with potential for analysis of differential gene expression
Authors: Stausbøl-Grøn Brian , Wind Troels , Kjær Svend , Kahns Liselotte , Hansen Nils J.V. , Kristensen Peter , Clark Brian F.C.
Published: April 2003

In order to establish a subtractive procedure that makes it possible to enrich selectively phage displayed antibodies directed against proteins constituting a difference between two populations of cells, a competitive selection strategy utilising two solid phases was developed and tested. Antibodies recognising a defined difference between two otherwise identical protein mixtures were isolated and their specificity confirmed. To test further the efficacy of selection inhibition during the competitive selections, selections towards a total cell extract were performed with and without competition from the same extract. An analysis of the resulting phage antibodies confirmed the subtractive nature of the system described.
MoaA of Arthrobacter nicotinovorans pAO1 involved in Mo‐Pterin cofactor synthesis is an Fe‐S protein
Authors: Menéndez Cástor , Siebert Dieter , Brandsch Roderich
Published: April 2003

MoaA, involved in an early step in the biosynthesis of the molybdopterin cofactor (MoCo), has not yet been characterized biochemically and the reaction it catalyzes is unknown. We overexpressed MoaA from pAO1 of Arthrobacter nicotinovorans in Escherichia coli as a N‐terminal fusion with either glutathione‐S‐transferase or a 6‐histidine tag. The pAO1 encoded MoaA as well as the fusion proteins functionally complement E. coli moaA mutants. Here we show that purified MoaA contains approximately 4 μM Fe and approximately 3 μM acid‐labile S/μM protein. EPR spectroscopy revealed a predominant signal at g
av = 2.01, indicative of a [3Fe‐xS] cluster.
Identification of a novel endogenous memory facilitating cyclic dipeptide cyclo‐prolylglycine in rat brain
Authors: Gudasheva T.A , Boyko S.S , Akparov V.Kh , Ostrovskaya R.U , Skoldinov S.P , Rozantsev G.G , Voronina T.A , Zherdev V.P , Seredenin S.B
Published: April 2003

Using high‐performance liquid chromatography, gas‐chromatography and chromato‐mass spectrometry methods a novel endogenous cyclic dipeptide cyclo‐prolylglycine was identified in rat brain. Its content according to gas chromatography is 2.8 ± 0.3 nmol/g wet brain. Synthetic cyclo‐prolylglycine has demonstrated antiamnesic activity in the passive avoidance test in rats at a dose of 0.1 mg/kg i.p. Cyclic dipeptide cyclo‐prolylglycine seems to be a memory facilitating substance and its presence in rat brain suggests the existence of a new mechanism of memory regulation.
Heat shock protein hsp70 overexpression confers resistance against nitric oxide
Authors: Bellmann Kerstin , Jäättelä Marja , Wissing Dorte , Burkart Volker , Kolb Hubert
Published: April 2003

Heat stress is known to render rat islet cells resistant against the toxic effects of nitric oxide, reactive oxygen intermediates and the islet cell toxin streptozotocin. We report here for the first time that protection against nitric oxide is mediated by the major heat shock protein, hsp70, even in the absence of heat stress. The human hsp70 gene was stably transfected into the rat insulinoma cell line RINm5F. Constitutive expression of hsp70 caused protection from NO‐induced cell lysis which was of the same extent as seen after heat stressing cells. Our results identify hsp70 as a defence molecule against nitric oxide.
Up‐regulation of L‐ and non‐L, non‐N‐type Ca2+ channels by basal and stimulated protein kinase C activation in insulin‐secreting RINm5F cells
Authors: Platano D. , Pollo A. , Carbone E. , Aicardi G.
Published: April 2003

We studied the effect of protein kinase C (PKC) inhibition and activation on voltage‐dependent Ca2+ channels in rat insulinoma RINm5F cells. PKC down‐regulation by chronic (24 h) treatment with the PKC activator phorbol 12‐myristate 13‐acetate (PMA) reduced by about 60% the Ba2+ currents through L‐ and non‐L, non‐N‐type high‐voltage‐activated Ca2+ channels, indicating that PKC tonically up‐regulates the two main Ca2+ channel subtypes of RINm5F cells under basal conditions. Consistently, PKC activation by acute PMA application caused only a modest increase (average 23%) of Ba2+ currents in a minority of cells (24%). L‐ and non‐L, non‐N‐type channels were differentially up‐regulated by either basal or stimulated PKC activation. Acute up‐regulation was predominant on L‐type channels and caused an I/V shift of the Ba2+ currents in the hyperpolarizing direction. Non‐L, non‐N‐type channels were less affected by acute PMA application, possibly reflecting a more effective tonic PKC up‐regulatory action. Unexpectedly, the increase of Ba2+ currents during acute PMA application was followed by a progressive current decrease, which was also observed in isolation in another 24% of the cells and could be ascribed to PKC‐induced ATP depletion, rather than to a direct effect of PKC on Ca2+ channels. We also provide evidence that PKC‐mediated phosphorylation is not involved in the G‐protein‐mediated noradrenergic modulation of Ca2+ channels in RINm5F cells.
Activation of the lamin A gene during rat liver development
Authors: Hamid Q.Anwar , Fatima Soghra , Thanumalayan Subramonian , Parnaik Veena K.
Published: April 2003

We have studied the regulation of expression of the A‐type lamins, which are constituents of the nuclear lamina. During rat liver development, high levels of lamin A and C mRNAs were observed in 15‐day fetal liver but were barely detectable in the adult. The chromatin conformation of the lamin A gene was sensitive to DNase I in 15‐day fetal liver but became mostly insensitive in the adult. Lamin A and C proteins could be detected in fetal liver and persisted in the adult. Our evidence suggests that the lamin A gene is actively transcribed early in liver differentiation and its activity declines considerably in adult liver.
Tumor necrosis factor α (TNFα) induces pro‐matrix metalloproteinase 9 production in human uterine cervical fibroblasts but interleukin 1 α antagonizes the inductive effect of TNFα
Authors: Sato Takashi , Ito Akira , Ogata Yutaka , Nagase Hideaki , Mori Yo
Published: April 2003

We have examined the regulation of precursor of matrix metalloproteinase 9 (proMMP‐9)/progelatinase B production by tumor necrosis factor α (TNFα) and interleukin 1α (IL‐1α) using human uterine cervical fibroblasts. TNFα, but not IL‐1α, induces the production of proMMP‐9 in the cervical cells. IL‐1α, however, suppresses the TNFα‐induced proMMP‐9 production. 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) also stimulates the cervical cells to produce proMMP‐9, and IL‐1α synergistically enhances its production. TNFα‐induced proMMP‐9 production is not mediated by protein kinase C (PKC), whereas the effect of IL‐1α is through PKC. By contrast, proMMP‐3/prostromelysin 1 is up‐regulated by TNFα or TPA in the presence of IL‐1α, whose modulation is PKC‐dependent. The suppressive effect of IL‐1α on the TNFα‐induced proMMP‐9 production is a new biological effect of IL‐1 on MMP production.
Mammalian DNA cytosine‐5 methyltransferase interacts with p23 protein
Authors: Zhang Xiaoyan , Verdine Gregory L.
Published: April 2003

In higher eukaryotic genomes, methylated cytosine residues (m5C) are distributed in heritable, cell‐type‐specific patterns, which are believed to be involved in the control of gene expression, developmental regulation and genomic imprinting. These methylation patterns are established and maintained by DNA cytosine‐5 methyltransferase (MTase), a ∼1500 amino acid enzyme containing a regulatory N‐terminal domain and a catalytic C‐terminal domain. The mechanism responsible for targeting MTase to particular genes is poorly understood and might possibly involve interactions with other proteins. In an effort to identify proteins that interact with the mammalian MTase, we used the yeast two‐hybrid system with several different MTase domains as baits. Here we report an interaction between the C‐terminal catalytic domain of the MTase and p23, a protein previously reported to associate with the progesterone receptor (PR) complex.
Immunohistochemical and immunoblotting identification of protein phosphatase 1γ1 in rat salivary glands
Authors: Shirakawa Satoko , Mochizuki Hajime , Kobayashi Shigeru , Takehara Tadamichi , Shima Hiroshi , Nagao Minako , Haneji Tatsuji
Published: April 2003

We have analyzed the distribution of the γ1 isotype of rat protein phosphatase type 1 catalytic subunit in rat salivary glands. Formaldehyde‐fixed paraffin sections were reacted with the PP1γ1 antibody using an immunohistochemical method. Positive staining occurred in striated ducts of parotid gland. However, the staining reaction was less intense in submandibular gland. Proteins were also prepared from rat salivary glands and subjected to SDS‐PAGE, followed by Western blotting analysis with the PP1γ1 antibody. The antibody interacted with protein corresponding to an estimated molecular mass of 36 kDa present in the parotid gland. The staining reaction was considerably weaker with the proteins from submandibular gland.
Activation of plasma membrane voltage‐dependent calcium‐permeable channels by disruption of microtubules in carrot cells
Authors: Thion Laurence , Mazars Christian , Thuleau Patrice , Graziana Annick , Rossignol Michel , Moreau Marc , Ranjeva Raoul
Published: April 2003

Plasma membrane‐bound voltage‐dependent calcium channels may couple the perception of an initial stimulus to a regulated pathway for calcium influx. The activities of these channels have been shown to be very low and highly unstable but may be recruited by large‐predepolarizing pulses, according to a process referred to as recruitment. By combining pharmacological and electrophysiological approaches, we demonstrate in the present paper that the cytoskeleton plays an important role in the regulation of the activity and stability of voltage‐dependent calcium channels during whole‐cell patch‐clamp experiments on carrot protoplasts. Whereas drugs affecting the organization of the microfilament network have no measurable effect, the manipulation of the microtubule network elicits important changes. Thus, the addition of colchicine or oryzalin, which are known to disrupt microtubule organization, leads to a 6–10‐fold increase in calcium channel activities and half‐life. In contrast, stabilization of the microtubules by taxol has no effect on any of these parameters. The data obtained suggest that interactions of microtubules and voltage‐dependent calcium channels by either direct or indirect mechanisms inhibit channel activities and decrease their half‐life. In contrast, the disruption of the network overcomes such an inhibitory effect and allows the activation of calcium channels. It is speculated that under normal physiological conditions these protein‐protein interactions may work in a reversible manner and contribute to signal transduction in higher plants.
Polyglycylation of tubulin in the diplomonad Giardia lamblia, one of the oldest eukaryotes
Authors: Weber Klaus , Schneider André , Müller Norbert , Plessmann Uwe
Published: April 2003

We have searched for post‐translational modifications in tubulin of the diplomonad Giardia lamblia, which is a representative of the earliest branches in eukaryotic evolution. The carboxyterminal peptide of α‐tubulin was isolated and characterized by automated sequencing and mass spectrometry. Some 60% of the peptide is unmodified, while the remainder shows various degrees of polyglycylation. The number of glycyl residues in the lateral side chain ranges from 2 to 23. All peptide species encountered end with alanine‐tyrosine, indicating the absence of a detyrosination/tyrosination cycle. We conclude that tubulin‐specific polyglycylation could be as old as tubulin and axonemal structures.
The primary structure of UK 114 tumor antigen
Authors: Ceciliani Fabrizio , Faotto Ludovica , Negri Armando , Colombo Irma , Berra Bruno , Bartorelli Alberto , Ronchi Severino
Published: April 2003

UK114 is a tumor antigen expressed by various malignant neoplasms. The complete amino acid sequence of UK114 purified from goat liver has been determined by automated Edman degradation of CNBr and endoproteinase Lys‐C peptides. The protein contains 137 amino acid residues, which corresponds to a molecular mass of 14 229 Da. MALDITOF analysis resulted in a molecular weight of 14 290, suggesting that the N‐terminal Met residue is acetylated. Sequence comparison shows that UK114 from goat liver (1) has 77% identity with a previously described 23 kDa protein from rat liver (Levy‐Favatier et al. (1993) Eur. J. Biochem. 212, 665–673), (2) shares a very high degree of similarity with a family of prokaryotic and eukaryotic hypothetic proteins whose function have not yet been characterized, and (3) exhibits a significant similarity to a group of tumor‐associated antigens which belongs to a superfamily of heat shock proteins, acting as possible targets for the host's antitumor immunity.
Site‐specific cleavage reaction catalyzed by leadzyme is enhanced by combined effect of lead and rare earth ions
Authors: Sugimoto Naoki , Ohmichi Tatsuo
Published: April 2003

Lead‐dependent ribozyme (leadzyme) is a ribozyme working with Pb2+. In this paper, we have investigated the combined effect of metal ions, especially rare earth ions, on the cleavage reaction by the leadzyme. As a result, it was observed that although only a rare earth ion or another divalent ion except Pb2+ did not play a role as the catalyst, the addition of a rare earth ion in the presence of Pb2+ increased significantly the yield of the cleavage reaction. The result suggests that the complex between the leadzyme and the substrate should have two classes of metal ion binding sites.
Quantitative evaluation of the dynamics of proton transfer from photoactivated bacteriorhodopsin to the bulk
Authors: Nachliel E. , Gutman M.
Published: April 2003

It has been reported by many research groups that protons released during the photocycle of bacteriorhodopsin are detected by surface bound indicators much faster than by indicators in the bulk. In this study we used numerical simulation of chemical reaction's dynamics for analyzing the delayed appearance of protons in the bulk. The results indicate that the low pK surface groups of the membrane, which form an undilutable concentrated matrix of proton binding sites, retain the protons in this space according to the mass action law. The main sites for proton accumulation are the cluster of carboxylates on the cytoplasmic side of the membrane. The protonation of an indicator in the bulk does not proceed by its reaction with free proton, but rather through self‐diffusion of the indicator to the membrane and abstraction of proton from the protonated surface group. The detailed mechanisms which correspond with these reactions are reported.
Microtubule‐active drugs suppress the closure of the permeability transition pore in tumour mitochondria
Authors: Evtodienko Yuri V. , Teplova Vera V. , Sidash Serguei S. , Ichas François , Mazat Jean-Pierre
Published: April 2003

We report the effects of anticancer drugs, inhibitors of microtubule organisation, on the mitochondrial permeability transition pore (PTP) in Ehrlich ascites tumour cells. Taxol (5–20 μM) and colchicine (100–500 μM) prevented closing of the cyclosporin A‐sensitive PTP. No taxol or colchicine effects on oxidative phosphorylation were observed in the range of concentrations used. We suggest that either membrane‐bound tubulin per se can be part of PTP and/or the attachment of mitochondria to the microtubular network is essential for PTP regulation. The taxol inhibition of PTP closure, mediated through interaction with the cytoskeleton, sheds new light on the cytotoxic properties of this anticancer drug.
A possible tyrosine phosphorylation of phytochrome
Authors: Sommer Debbie , Wells Todd A. , Pill-Soon Song
Published: April 2003

Red/far‐red light signal transduction by the phytochrome family of photoreceptors regulates plant growth and development. We investigated the possibility that tyrosine kinases and/or phosphatases are involved in phytochrome‐mediated signal transduction using crude extracts of oat seedlings that are grown in the dark. We found that a 124 kDa protein was tyrosine‐phosphorylated as determined by Western blotting with a phosphotyrosine‐specific monoclonal antibody. The 124 kDa protein was recognized by the anti‐phosphotyrosine antibody in anti‐phytochrome A immunoprecipitates. The level of anti‐phosphotyrosine antibody binding to the 124 kDa protein(s) in phytochrome immunoprecipitates that had been treated with red light prior to immunoprecipitation decreased relative to dark controls. These results suggest that either phytochrome from dark‐grown seedlings is tyrosine phosphorylated or that it co‐immunoprecipitates with a phosphotyrosine‐containing protein of the same molecular weight. The implications of these results in the regulation of (a) the putative Ser/Thr kinase activity of the photoreceptor and (b) the binding of signaling molecules, such as phospholipase C to phytochrome, are discussed.
Dynamics of insulin‐stimulated translocation of GLUT4 in single living cells visualised using green fluorescent protein
Authors: Dobson Stephen P. , Livingstone Callum , Gould Gwyn W. , Tavaré Jeremy M.
Published: April 2003

Insulin increases glucose uptake by promoting the translocation of the GLUT4 isoform of glucose transporters to the plasma membrane. We have studied this process in living single cells by fusing green fluorescent protein (GFP) to the N‐terminus (GFP·GLUT4) or C‐terminus (GLUT4·GFP) of GLUT4. Both chimeras were expressed in a perinuclear compartment of CHO cells, and in a vesicular distribution through the cytosol. Insulin promoted an increase in plasma membrane fluorescence as a result of net translocation of the chimeras to the cell surface. GLUT4·GFP, but not GFP·GLUT4, was re‐internalised upon the removal of insulin suggesting that a critical internalisation signal sequence exists in the N‐terminus of GLUT4. The use of GFP thus allows an analysis of GLUT4 trafficking in single living cells.
Formation of haemozoin/β‐haematin under physiological conditions is not spontaneous
Authors: Pandey Amit V , Tekwani Babu L
Published: April 2003

Malaria parasite detoxifies free haem, released as a result of haemoglobin digestion, by converting it into an stable, crystalline, black brown pigment known as ‘malaria pigment’ or ‘haemozoin’. Earlier studies have demonstrated the involvement of a parasite‐specific enzyme ‘haem polymerase’ in the formation of haemozoin. However, recently it has been proposed that the polymerization of haem may be a spontaneous process that could take place by incubation of haematin with carboxylic acids (pH 4.2–5.0) even without presence of any parasitic or biological component (FEBS Letters, 352, 54–57 (1994)). Here we report that no spontaneous haem polymerization occurs at physiological conditions and the product described in the study mentioned above is not haemozoin/β‐haematin (haem polymer) as characterized by us on the basis of solubility characteristics and thin layer chromatography. The infra‐red spectroscopic analysis of the product formed though exhibits the bands corresponding to formation of iron‐carboxylate bond, similar to that in haemozoin/β‐haematin, but was identified as haem‐acid adduct. Thus polymerization of haem may not occur spontaneously under the reaction conditions corresponding to food vacuoles of the malarial parasite, the physiological site of haemozoin formation.