bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of κ‐ and δ‐opioid receptor transcripts in immune cells
Authors: Gavériaux Claire , Peluso Jean , Simonin Fréderic , Laforet Jean , Kieffer Brigitte
Published: April 2003

To investigate the role of opioids as direct modulators of the immune response, we have searched for expression of the recently cloned δ, μ and κ opioid receptors in immune cells. We have devised a reverse transcriptase‐polymerase chain reaction strategy which specifically detects a region spanning putative transmembrane regions 2 to 7 for each transcript in both human and mouse immune cells. In human peripheral blood lymphocyte and monocyte preparations, δ was undetectable while the κ transcript was present. The analysis of human cell lines revealed low but significant levels of δ opioid receptor transcripts in T, B or monocyte cell lines while the κ transcript was found in B cell lines only. Investigation of murine cells showed the presence of transcript for the δ receptor in splenocytes and in some T and B cell lines. Unexpectedly, no expression of the μ receptor was detected. Sequence analysis of PCR products demonstrated nucleotide identity between immune and neuronal transcripts, indicating that they derive from the same genes. In conclusion, our results lead to the identification of κ and δ opioid receptor transcripts in immune cells.
The modulation and characterisation of the Ca2+‐induced Ca2+ release mechanism in cultured human myometrial smooth muscle cells
Authors: Morgan J.M. , Gillespie J.I.
Published: April 2003

The process of Ca2+‐induced Ca2+ release (CICR) was studied in saponin permeabilised human myometrial smooth muscle cells in which the sarcoplasmic reticulum (SR) was pre‐loaded with 45Ca2+. A rise in the free Ca2+ concentration of the bathing solution from 100 nM tot 10 μM increased the rate of 45Ca2+ loss, while a reduction to 10 nM decreased the rate of 45Ca2+ loss. Ruthenium red (20 μM) lowered the basal rate of 45Ca2+ loss and reduced CICR. Caffeine did not activate 45Ca2+ release although ryanodine induced 45Ca2+ release and CICR was augmented in the presence of caffeine. These data suggest the operation of a Ca2+‐activated 45Ca2+ release process which is similar in many of its properties to the CICR process described in many cells. The basic properties of the CICR mechanism in myometrium differs in many respects from the CICR process recently described in human vascular smooth muscle.
Differential expression of voltage‐activated Na+ currents in two prostatic tumour cell lines: contribution to invasiveness in vitro
Authors: Grimes J.A. , Fraser S.P. , Stephens G.J. , Downing J.E.G. , Laniado M.E. , Foster C.S. , Abel P.D. , Djamgoz M.B.A.
Published: April 2003

The voltage‐gated ionic currents of two rodent prostatic cancer cell lines were investigated using the whole‐cell patch clamp technique. The highly metastatic Mat‐Ly‐Lu cells expressed a transient, inward Na+ current (blocked by 600 nM tetrodotoxin), which was not found in any of the weakly metastatic AT‐2 cells. Although both cell lines expressed a sustained, outward K+ current, this occurred at a significantly higher density in the AT‐2 than in the Mat‐Ly‐Lu cells. Incubation of the Mat‐Ly‐Lu cell line with 600 nM tetrodotoxin significantly reduced the invasive capacity of the cells in vitro. Under identical conditions, tetrodotoxin had no effect on the invasiveness of the AT‐2 cells.
Dissociation between exocytosis and Ca2+‐channel activity in mouse pancreatic β‐cells stimulated with calmidazolium (compound R24571)
Authors: Kindmark Henrik , Köhler Martin , Larsson Olof , Khan Akhtar , Berggren Per-Olof
Published: April 2003

Calmidazolium, a calmodulin inhibitor, suppressed influx of Ca2+ through voltage‐gated Ca2+ channels in mouse pancreatic β‐cells. Despite this fact, calmidazolium stimulated insulin release from β‐cells at basal glucose concentration. This effect was not mediated by protein kinase C (PKC), since it persisted in PKC‐depleted cells. RpcAMPS significantly attenuated tha calmidazolium‐stimulated insulin secretion, indicating that calmidazolium acts, at least partly, through PKA. The compound also stimulated insulin secretion from electropermeabilized β‐cells, indicating effects on distal steps in the stimulus‐secretion coupling. The use of calmidazolium offers possibilities to investigate the mechanisms activating exocytosis under conditions where the cytoplasmic‐free Ca2+ concentration does not increase.
Temperature‐induced exposure of hydrophobic surfaces and its effect on the chaperone activity of α‐crystallin
Authors: Das Kali P. , Surewicz Witold K.
Published: April 2003

α‐Crystallin, the major protein of the ocular lens, is known to have extensive similarities to small heat shock proteins and to act as a molecular chaperone. The exposure of hydrophobic surfaces on α‐crystallin was studied by fluorescence spectroscopy using the hydrophobic probe bis‐ANS. Upon heating the protein undergoes a conformational transition which is associated with a marked increase in surface hydrophobicity. This transition, which occurs between approximately 38 and 5‐°C, lacks reversibility. The increase in surface hydrophobicity correlates with the increased chaperone activity of the protein. These results indicate that hydrophobic interactions play a major role in the chaperone action of α‐crystallin.
Non‐replicating vaccinia vector efficiently expresses bacteriophage T7 RNA polymerase
Authors: Sutter Gerd , Ohlmann Marion , Erfle Volker
Published: April 2003

Modified vaccinia virus Ankara (MVA), a host range restricted and highly attenuated vaccinia virus strain, is unable to multiply in human and most other mammalian cell lines. Since viral gene expression is unimpaired in non‐permissive cells recombinant MVA viruses are efficient as well as exceptionally safe expression vectors. We constructed a recombinant MVA that expresses the bacteriophage T7 RNA polymerase and tested its usefulness for transient expression of recombinant genes under the control of a T7 promoter. Using the chloramphenicol acetyltransferase (CAT) gene as a reporter gene, infection with MVA‐T7pol allowed efficient synthesis of recombinant enzyme in mammalian cells. Despite the severe host restriction of MVA, enzyme activities induced by infection with MVA‐T7pol were similar to those determined after infection with a replication‐competent vaccinia‐T7pol recombinant virus. Thus, MVA‐T7pol may be used as a novel vaccinia vector to achieve T7 RNA polymerase‐specific recombinant gene expression in the absence of productive vaccinia virus replication.
Non‐enzymatic glycosylation of the dipeptide l‐carnosine, a potential anti‐protein‐cross‐linking agent
Authors: Hipkiss Alan R. , Michaelis Jürgen , Syrris Petros
Published: April 2003

The dipeptide carnosine (β‐alanyl‐l‐histidine) was readily glycosylated non‐enzymatically upon incubation with the sugars glucose, galactose, deoxyribose and the triose dihydroxyacetone. Carnosine inhibited glycation of actyl‐Lys‐His‐amide by dihydroxyacetone and it protected α‐crystallin, superoxide dismutase and catalise against glycation and cross‐linking mediated by ribose, deoxyribose, dihydroxyacetone, dihydroxyacetone phosphate and fructose. Unlike certain glycated amino acids, glycated carnosine was non‐mutagenic. The potential biological and therapeutic significance of these observations are discussed.
Glutamate‐101 is critical for the function of the sodium and chloride‐coupled GABA transporter GAT‐1
Authors: Keshet Gilmor I. , Bendahan Annie , Su Hailing , Mager Sela , Lester Henry A. , Kanner Baruch I.
Published: April 2003

We have investigated the possible role of selected negatively‐charged amino acids of the sodium and chloride‐coupled GABA transporter GAT‐1 on sodium binding. These residues located adjacent to putative transmembrane domains and which are conserved throughout the large superfamily of neurotransmitter transporters were changed by site‐directed mutagenesis. The functional consequences were that one of the residues, glutamate‐101, was critical for transport. Its replacement by aspartate left only 1% of the activity, and no activity could be detected when it was replaced by other residues. Expression levels and targeting to the plasma membrane of the mutant transporters appeared normal. Transient sodium currents were not observed in the mutants, and increased sodium concentrations did not affect the percentage of wild type transport of the E101D mutant. It is concluded that residue glutamate‐101 is critical for one or more of the conformational changes of GAT‐1 during its transport cycle.
rap1 p21 regulates the interaction of ras p21 with RGL, a new effector protein of ras p21
Authors: Ikeda Masahiro , Koyama Shinya , Okazaki Michiko , Dohi Kiyohiko , Kikuchi Akira
Published: April 2003

We have recently found that ralGDS family members (RGL and ralGDS) are putative effector proteins of ras p21. rap1 p21 is a small GTP‐binding protein which has the same amino acid sequence as the effector loop of ras p21. We examined the effect of rap1 p21 on the interaction of ras p21 with RGL. The GTP‐bound form of rap1 p21 interacted with RGL as well as did ras p21. rap1 p21 inhibited the interaction of ras p21 with RGL. RGL was phosphorylated by cyclic AMP‐dependent protein kinase (protein kinase A). Phosphorylation of RGL did not affect its binding to ras p21 and rap1 p21 under the conditions that phosphorylation of Raf‐1 reduced its affinity for ras p21. These results demonstrate that rap1 p21 but not protein kinase A regulates the interaction of ras p21 with RGL and suggest that rap1 p21 and protein kinase A may cooperate to distinguish the signal of ras p21 to RGL from that to Raf‐1.
On the role of extracellular loops of opioid receptors in conferring ligand selectivity
Authors: Metzger Thomas G. , Ferguson David M.
Published: April 2003

Based on an analysis of results taken from site‐directed mutagenesis studies performed on opioid receptors, a role for the extracellular loops in conferring opioid subtype selectivity is proposed. It is suggested that the extracellular loop regions (which represent the region of highest sequence variability among opioid subtypes) interact with opioid ligands in a primarily non‐specific fashion. Although these interactions are non‐specific, they appear to play a discriminatory role in ligand binding and, in certain cases, prevent particular ligands from binding among receptor subtypes. We propose that selectivity may be imparted through a mechanism of exclusion, rather than specific pharmacophore recognition within the extracellular loops and N‐terminal domain. This hypothesis is supported by a careful analysis of the binding profiles of several selective and non‐selective ligands to a variety of chimeric mutants. These results, when combined with results taken from single‐point mutation experiments point to the existence of a high affinity binding pocket within the transmembrane region which may be common among the opioid subtypes.
Crystal structures of factor Xa specific inhibitors in complex with trypsin: structural grounds for inhibition of factor Xa and selectivity against thrombin
Authors: Stubbs Milton T. , Huber Robert , Bode Wolfram
Published: April 2003

Crystal structures of DX9065a and a related bisamidino‐aryl inhibitor specific for the blood‐clotting factor Xa have been solved in complex with bovine β‐trypsin to a resolution of 1.9 Å. Each inhibitor exhibits an extended conformation along the active site, in contrast to the compact folded structures observed for thrombin specific inhibitors. Few direct contacts (predominantly in the S1 pocket) are made between trypsin and the inhibitors. Transfer of the inhibitors to the active site of factor Xa suggests a three‐site interaction: salt bridge formation at the base of the primary specificity pocket, extensive hydrophobic surface burial and a weak electrostatic interaction between the distal basic component of the inhibitor and an electronegative cavity of factor Xa formed by three backbone carbonyl oxygens. Additivity of these three interactions is the basis for the observed strong inhibition of factor Xa and provides a framework for the design of novel factor Xa inhibitors. A propionic acid group of the inhibitor would clash with the thrombin specific ‘60‐insertion loop’, thus conferring selectivity against thrombin.
Cloning, sequencing and tissue distribution of two related G protein‐coupled receptor candidates expressed prominently in human lung tissue
Authors: An Songzhu , Tsai Cannit , Goetzl Edward J.
Published: April 2003

A novel G protein‐coupled receptor, named GPR12A, was cloned by a PCR strategy using degenerate primers designed from sequences conserved among receptors for inflammatory mediators. Screening of a human lung cDNA library with GPR12A as a probe also identified a closely‐related cDNA (GPR6C.1) that has been previously reported as GPR4 [13]. GPR12A and GPR6C.1 are 46.1% identical in amino acid sequence, but are less than 33% identical to any other known receptors. Northern analysis revealed that they are expressed prominently in the lung. Although the ligands for GPR12A and GPR6C.1 are unknown, their similarity suggests that they are receptors for ligands of similar or identical chemical nature.
Self‐association of CPV3, an avian thymic parvalbumin
Authors: Henzl Michael T. , Zhao Hongmei , Saez Cristian T.
Published: April 2003

The avian parvalbumin called CPV3 readily forms disulfide‐linked oligomers. Sedimentation data presented herein reveal that CPV3 also undergoes noncovalent self‐association. Interestingly, the noncovalent interaction is promoted by either Ca2+ or Mg2+, whereas covalent complex formation displays an absolute requirement for the Ca2+‐bound protein. Apo‐CPV3 exhibits an apparent sedimentation coefficient of 2.08 S at 20°C, in 0.15 M NaCl, 0.025 M HEPES‐NaOH, pH 7.4. This value increases to 2.85 S or 3.16 S with addition of 1.0 mM Ca2+ or 5.0 mM Mg2+, respectively. Least‐squares analysis of sedimentation equilibrium data suggests that 100 μM apo‐CPV3 is primarily a mixture of monomeric and dimeric forms. With the addition of Ca2+, the equilibrium becomes exclusively monomer‐trimer, with negligible amounts of dimer. A comparable distribution is observed in the presence of Mg2+.
SnoRNA U21 is also intron‐encoded in Drosophila melanogaster but in a different host‐gene as compared to warm‐blooded vertebrates
Authors: Renalier Marie-Hélène , Nicoloso Monique , Qu Liang-Hu , Bachellerie Jean-Pierre
Published: April 2003

U21 is an intron‐encoded snoRNA in vertebrates which contains a 13‐nt tract of complementarity to an invariant sequence in eukaryotic 28S rRNA. Here, we report the characterization of its Drosophila melanogaster homolog which is the first case of an intron‐encoded snoRNA in an invertebrate metazoan. In D. melanogaster, U21 is encoded within the ARF‐1 (ADP ribosylation factor 1) gene, whereas in chicken and mammals it is found in the ribosomal protein L5 gene. In D. melanogaster, like in vertebrates, U21 is devoid of a 5′ trimethylguanosine cap, thus, likely resulting from processing of intronic RNA. The only portion of U21 sequence preserved between D. melanogaster and vertebrates, in addition to the hallmark box C and box D motifs, corresponds precisely to the 13‐nt complementary to rRNA, pointing to an important role of the pairing of U21 to pre‐rRNA.
Effects of beta cell granule components on human islet amyloid polypeptide fibril formation
Authors: Westermark Per , Li Zhan-Chun , Westermark Gunilla T. , Leckström Arnold , Steiner Donald F.
Published: April 2003

Formation of amyloid‐like fibrils in a solution of human islet amyloid polypeptide (hIAPP) with and without the presence of other β‐cell granule components was studied in vitro. Insulin at less than equimolar concentration strongly inhibited hIAPP fibrillogenesis. Proinsulin had a weaker inhibitory effect while C‐peptide, Ca2+ and Zn2+ each individually enhanced fibril formation. C‐peptide combined with Ca2+ had an inhibitory effect. Since IAPP was found almost exclusively in the halo fractions of isolated islet secretory granules, primarily the concentrations of C‐peptide, Ca2+ and possibly proinsulin may be crucial for the native state of IAPP. It is concluded that an imbalance between fibril formation enhancers and inhibitors may be of importance in the pathogenesis of amyloid in the islets of Langerhans.
Deletion of the ABL SH3 domain reactivates de‐oligomerized BCR‐ABL for growth factor independence
Authors: Maru Yoshiro , Witte Owen N. , Shibuya Masabumi
Published: April 2003

Biological activities of BCR‐ABL, an activated tyrosine kinase oncogene responsible for pathogenesis of human leukemias, can be completely inactivated by a deletion of the BCR aminoterminal sequence with a tetramerizing property (BCR‐ABL Δ1–40). We attempted several ways to restore the ability to induce growth factor independence to the de‐oligomerized BCR‐ABL Δ1–40 and found that an additional deletion of the ABL SH3 domain could. In BCR‐ABL Δ1–40 reactivated by the SH3 deletion, transphosphoryation of other cellular proteins like p62 or SHC in vivo and autophosphorylation with recruitment of GRB‐2 were also recovered.
Accumulation of nitrotyrosine on the SERCA2a isoform of SR Ca‐ATPase of rat skeletal muscle during aging: a peroxynitrite‐mediated process?
Authors: Viner Rosa I. , Ferrington Deborah A. , Hühmer Andreas F.R. , Bigelow Diana J. , Schöneich Christian
Published: April 2003

The SR Ca‐ATPase in skeletal muscle SR vesicles isolated from young adult (5 months) and aged (28 months) rats was analyzed for nitrotyrosine. Only the SERCA2a isoform contained significant amounts with approximately one and four nitrotyrosine residues per young and old Ca‐ATPase, respectively. The in vitro exposure of SR vesicles of young rats to peroxynitrite yielded selective nitration of the SERCA2a Ca‐ATPase even in the presence of excess SERCA1a. No nitration was observed during the exposure of SR vesicles to nitric oxide in the presence of O2. These data suggest the in vivo presence of peroxynitrite in skeletal muscle. The greater nitrotyrosine content of SERCA2a from aged tissue imolies an age‐associated increase in susceptibility to oxidation by this species.
A photo‐reactive derivative of ganglioside GM1 specifically cross‐links VIP21‐caveolin on the cell surface
Authors: Fra Anna M. , Masserini Massimo , Palestini Paola , Sonnino Sandro , Simons Kai
Published: April 2003

Previous studies have shown that sphingolipids may be enriched in caveolae, plasmalemmal invaginations implicated in endocytosis and signal transduction. We synthesised a radiolabeled derivative of ganglioside GM1 bearing a photo‐reactive cross‐linker at the end of its acyl chain. After insertion in the plasma membrane of cultured A431 or MDCK cells and photoactivation, the main protein cross‐linked by the GM1 derivative was VIP21‐caveolin, an essential structural component of caveolae. This result shows close proximity between GM1 molecules and VIP21‐caveolin in the caveolar membrane and strongly implicates sphingolipid segregation in the biogenesis of caveolae.
Broad spectrum antibiotic activity of skin‐PYY
Authors: Vouldoukis Ionnis , Shai Yechiel , Nicolas Pierre , Mor Amram
Published: April 2003

Neuropeptide Y (NPY) and polypeptide YY (PYY) are two ubiquitous neuropeptides, found in brain and intestines, respectively, where they exert important regulatory functions. In this study, a new member of the YY family recently isolated from amphibian skin, skin‐PYY (SPYY), is reported to inhibit irreversibly the proliferation of a broad spectrum of pathogenic microorganisms. NPY and PYY are shown to be endowed with the same activity. Their potency is similar to that of other antibacterial peptides which have been shown to exert their function by disintegrating the bacterial membrane. These findings and the fact that the C‐terminal alpha‐helical domain SPYY14–36, which is highly conserved among the family members, was responsible for killing microorganisms and for permeation of phospholipid vesicles, suggested that the antibiotic activity may emerge via a membrane permeation mechanism. These findings also raise the question whether NPY and PYY exert in vivo a similar function in mammals.
Molecular mechanism of pyruvate‐ferredoxin oxidoreductases based on data obtained with the Clostridium pasteurianum enzyme
Authors: Moulis Jean-Marc , Davasse Valérie , Meyer Jacques , Gaillard Jacques
Published: April 2003

Pyruvate‐ferredoxin oxidoreductase oxidises pyruvate in many fermentative microorganisms. The enzyme from Clostridium pasteurianum is an air‐sensitive homodimer of 2×120000 daltons, for which pyruvate is the best substrate found among several α‐ketoacids. Each subunit contains eight iron atoms in two [4Fe‐4S] clusters. Two distinct EPR signals, possibly associated with two ligand environments, arise from one of these clusters. Binding of pyruvate does not generate a radical. The results reported suggest a scheme for the electron flow in pyruvate ferredoxin oxidoreductases according to which the detailed reaction mechanism depends on the number (even or odd) of [4Fe‐4S] clusters present in a given enzyme.
Direct measurement of oscillatory generation of superoxide anions by single phagocytes
Authors: Isogai Yasuhiro , Tsuyama Tomomi , Osada Hiroyuki , Iizuka Tetsutaro , Tanaka Kazuko
Published: April 2003

Phagocytic cells such as neutrophils generate superoxide anions (O2
−) within phagocytic vacuoles for killing and digesting microorganisms. Here we report the simultaneous observation of morphological changes and O2
− generation in single phagocytic cells during phagocytosis. Point stimulation of a cell by contact with an opsonized microelectrode at the cell surface induced significant deformation to engulf the electrode, and also induced the O2
− generation which was measured by the electrode. Periodic fluctuations in the magnitude of the O2
− generation were observed in the time course. These oscillations may be caused by metabolic regulation of the formation of NADPH, which is the substrate for the O2
− generation.
Primary structure of desulfoferrodoxin from Desulfovibrio desulfuricans ATCC 27774, a new class of non‐heme iron proteins
Authors: Devreese Bart , Tavares Pedro , Lampreia Jorge , Van Damme Nancy , Le Gall Jean , Moura JoséJ.G. , Van Beeumen Jozef , Moura Isabel
Published: April 2003

The primary structure of desulfoferrodoxin from Desulfovibrio desulfuricans ATCC 27774, a redox protein with two mononuclear iron sites, was determined by automatic Edman degradation and mass spectrometry of the composing peptides. It contains 125 amino acid residues of which five are cysteines. The first four, Cys‐9, Cys‐12, Cys‐28 and Cys‐29, are responsible for the binding of Center I which has a distorted tetrahedral sulfur coordination similar to that found in desulforedoxin from D. gigas. The remaining Cys‐115 is proposed to be involved in the coordination of Center II, which is probably octahedrally coordinated with predominantly nitrogen/oxygen containing ligands as previously suggested by Mössbauer and Raman spectroscopy.
Characterization of the interaction between RhoA and the amino‐terminal region of PKN
Authors: Shibata Hideki , Mukai Hideyuki , Inagaki Yoshimasa , Homma Yoshimi , Kimura Kazushi , Kaibuchi Kozo , Narumiya Shuh , Ono Yoshitaka
Published: April 2003

The yeast two‐hybrid system and in vitro binding assay were carried out to characterize the interaction between PKN and a small GTP‐binding protein, RhoA. It was revealed that the region corresponding to the amino acid residues 33–111 in the amino‐terminal region of PKN was sufficient to confer the ability to associate with RhoA. Each synthetic peptide fragment corresponding to the amino acid residues 74–93 and 94–113 of PKN inhibited the interaction between PKN and RhoA in the in vitro binding assay, suggesting that this region is important in the association with RhoA. The endogenous and the GAP‐stimulated GTPase activity of RhoA was inhibited by the interaction with PKN, suggesting the presence of a regulatory mechanism that sustains the GTP‐bound active form of RhoA.
Identification and immunohistochemical localization of macrophage migration inhibitory factor in human cornea
Authors: Matsuda Akira , Tagawa Yoshitsugu , Matsuda Hidehiko , Nishihira Jun
Published: April 2003

We identified macrophage migration inhibitory factor (MIF) mRNA expression in human cornea, and demonstrated its immunohistological localization. Reverse transcription‐polymerase chain reaction analysis revealed that MIF mRNA was expressed in both the corneal epithelial and endothelial cells. Immunohistochemical study using the polyclonal antibody prepared from immunizing a rabbit with human recombinant MIF showed that MIF was present in the basal cells of corneal epithelium and endothelial cells. The fact that MIF exists in those cells of the cornea indicates that MIF may play an important role in corneal cell immunity and cellular differentiation.
An SH3 domain‐mediated interaction between the phagocyte NADPH oxidase factors p40phox and p47phox
Authors: Ito Takashi , Nakamura Rika , Sumimoto Hideki , Takeshige Koichiro , Sakaki Yoshiyuki
Published: April 2003

The phagocyte NADPH oxidase is activated during phagocytosis to produce superoxide, following assembly of a membrane‐integrated cytochrome b
558 with cytosolic proteins, p47
phox
, p67
phox
and p40
phox, each containing Src homology 3 (SH3) domains. While both p47
phox
and p67
phox
are indispensable for the oxidase activity, role of p40
phox
remains obscure. Here we study interaction between p40
phox
and p47
phox
by two independent methods, a two‐hybrid system in the yeast and an in vitro binding assay using purified proteins. The present results show that the interaction is mediated via binding of the SH3 domain of p40
phox
to a C‐terminal proline‐rich region of p47
phox
. This proline‐rich region is also the target for binding of p67
phox
, and the SH3 domain of p40
phox
can inhibit the binding of the C‐terminal one of p67
phox
to p47
phox
.
Glucose catabolism in cancer cells: regulation of the type II hexokinase promoter by glucose and cyclic AMP
Authors: Rempel Annette , Mathupala Saroj P. , Pedersen Peter L.
Published: April 2003

The increased glucose consumption of many tumor cells depends to a large extent on the overexpression of hexokinase Type II. In a previous study we isolated and sequenced the hepatoma Type II hexokinase promoter and showed that it is activated by glucose in the highly glycolytic AS‐30D hepatoma cell line under study, but not activated in control hepatocytes [Mathupala, S.P., Rempel, A. and Pedersen, P.L. (1995) J. Biol. Chem. 270, 16918–16925]. Here we report that the promoter of the hexokinase Type II gene is maximally activated by glucose at concentrations above 5 mM. Moreover, the data strongly suggest that glucose can act alone without requirement for metabolism. Also, glucose‐mediated promoter activation is markedly potentiated by cAMP. This response may serve as a strategy for cancer cells to maintain the hexokinase transcription rate high to ensure an efficient glucose utilization even under conditions where carbohydrates are limiting.
Differential expression of cGMP‐inhibited cyclic nucleotide phosphodiesterases in human hepatoma cell lines
Authors: Murata Taku , Taira Masato , Manganiello Vincent C.
Published: April 2003

PDE3 or cGMP‐inhibited cyclic nucleotide phosphodiesterase (cGI PDE) activity was detected in homogenates of HepG2, Hep3B and HuH7, but not SK‐Hep‐1, human hepatoma cells. In HepG2 and Hep3B cells PDE3 activity was found predominantly in particulate fractions; in HuH7, in both particulate and supernatant fractions. cDNAs encoding two human PDE3s (an ‘adipocyte’ type, HcGIP1),and a ‘cardiovascular’ type, HcGIP2) have been cloned. HcGIP1 cDNA hybridized strongly with poly(A)+ RNA species from HepG2 and Hep3B. Both HcGIPI and HcGIP2 mRNAs were expressed in Hep3B and HuH7 cells. The nucleotide sequence of an ∼300‐bp cDNA fragment, isolated after RT‐PCR cloning from HepG2 RNA, was identical to a sequence within the conserved domain of HcGIP1 cDNA, consistent with the presence of HcGIPI mRNA in HepG2 cells.
Fibrillin: evidence that chondroitin sulphate proteoglycans are components of microfibrils and associate with newly synthesised monomers
Authors: Kielty Cay M. , Whittaker Stephen P. , Shuttleworth C.Adrian
Published: April 2003

We have investigated the potential association of proteoglycans with intact fibrillin‐containing microfibrils from foetal bovine elastic tissues and with newly synthesised fibrillin in human and bovine cell cultures. Microfbril integrity was disrupted by chondroitinase ABC lyase and chondroitinase AC lyase, but not by keratanase or hyaluronidase. Following chondroitinase treatment, beads were disrupted but the underlying fibrillar scaffold appeared intact. Cuprolinic blue was prominently associated with beaded domains at a critical electrolyte concentration. Electron‐dense rods were often associated with cuprolinic blue‐treated microfbrils isolated from fixed tissues. Positive staining revealed charged foci at the beads. Newly synthesised fibrillin could be labelled with 35S TransLabel, [3H]glucosamine or 35SO4 but its electrophoretic mobility was not influenced by treatment with chondroitinase ABC or AC lyase. A diffuse 35SO4‐labelled chondroitinase‐sensitive component with a resistant band (M
r 35000) co‐immunoprecipitated with fibrillin. These experiments indicate that chondroitin sulphate proteoglycans associate with fibrillin and contribute to microfibril assembly. This association has major implications for microfibril function in health and disease.
Limulus kexin: a new type of Kex2‐like endoprotease specifically expressed in hemocytes of the horseshoe crab
Authors: Kawabata Shun-ichiro , Saeki Kazuko , Iwanaga Sadaaki
Published: April 2003

A Kex2‐like protease was identified in hemocytes of the horseshoe crab (Tachypleus tridentatus), named limulus kexin, and a full‐length cDNA was obtained from a hemocyte cDNA library. The deduced amino acid sequence contains 752 residues, composed of five domains with a signal sequence, a propeptide, a catalytic domain, a Ser/Thr‐rich domain, and a transmembrane domain. The domain organization is very similar to that of the yeast Kex2 except that limulus kexin does not have a cytoplasmic tail. The catalytic domain exhibits striking sequence identities with those of furins, especially Drosophila furin1 (79%). Northern blotting showed specific expression of limulus kexin in hemocytes, suggesting the involvement in proteolytic processing of the granule components of hemocytes.
Kir2.2v: a possible negative regulator of the inwardly rectifying K+ channel Kir2.2
Authors: Namba Noriyuki , Inagaki Nobuya , Gonoi Tohru , Seino Yoshiki , Seino Susumu
Published: April 2003

We have cloned the human genes encoding the inwardly rectifying K+ (Kir) channel subunits, Kir2.2 (hKir2.2) and its variant, termed hKir2.2v. When expressed in Xenopus oocytes, hKir2.2 produced strong inwardly rectifying K+ currents, whereas the expression of hKir2.2v did not elicit significant currents. Coexpression of hKir2.2v with hKir2.2 showed an hKir2.2v inhibition of hKir2.2 K+ currents, indicating that it acts as a negative regulator of hKir2.2 channel activity. Mutational analysis of hKir2.2v and studies of chimeras between hKir2.2 and hKir2.2v suggest that the intracellular C‐terminal region of hKir2.2v participates in the negative regulation of the hKir2.2v channel activity.
Deletion of the ATH1 gene in Saccharomyces cerevisiae prevents growth on trehalose
Authors: Nwaka Solomon , Mechler Bernd , Holzer Helmut
Published: April 2003

The biological function of the yeast trehalases (EC 3.2.1.28) consists of down‐regulation of the concentration of trehalose via glucose formation by trehalose hydrolysis. While it is generally accepted that the cytosolic neutral trehalase (encoded by the NTH1 gene) is responsible for trehalose hydrolysis in intact cells, very little is known about a role of the vacuolar acid trehalase and the product of the recently described neutral trehalase gene YBR0106 (NTH2). We have analyzed the role of the acid trehalase in trehalose hydrolysis using the ATH1 deletion mutant (Δathl) of Saccharomyces cerevisiae [M. Destruelle et al. (1995) Yeast 11, 1015–1025] deficient in acid trehalase activity under various nutritional conditions. In contrast to wild‐type and a mutant deficient in the neutral trehalase (Δathl ), the Aathl mutant does not grow on trehalose as a carbon source. Experiments with diploid strains heterozygous for Δathl show a gene dosage effect for the ATH1 gene for growth on trehalose. The need for acid trehalase for growth on trehalose is supported by the finding that acid trehalase activity is induced during exponential growth of cells on trehalose while no such induction is measurable during growth on glucose. Our results show that the vacuolar acid trehalase Ath1p is necessary for the phenotype of growth on trehalose, i.e. trehalose utilization, in contrast to cytosolic neutral trehalase Nth1p which is necessary for intracellular degradation of trehalose. For explanation of the need for vacuolar acid trehalase and not cytosolic neutral trehalase for growth on trehalose, the participation of endocytosis for uptake of trehalose from medium to the vacuoles is discussed.
Insertion of poly(ethylene glycol) derivatized phospholipid into pre‐formed liposomes results in prolonged in vivo circulation time
Authors: Uster Paul S. , Allen Theresa M. , Daniel Barbra E. , Mendez Cecilia J. , Newman Mary S. , Zhu George Z.
Published: April 2003

Transfer of MPEG1900‐DSPE from micellar phase to pre‐formed liposomes imparts long in vivo circulation half‐life to an otherwise rapidly cleared lipid composition. MPEG1900‐DSPE transfers efficiently and quickly in a time and temperature dependent manner. There is negligible content leakage and a strong correlation between assayed mol% MPEG1900‐DSPE, liposome diameter increase, and pharmacokinetic parameters such as distribution phase half‐life. Since a biological attribute (liposome clearance rate) can be modified by the insertion process, it suggests a simple and economical way to impart site‐specific targeting to a variety of liposome delivery systems. This method is also a convenient way to measure the ‘brush’ thickness of such conjugates directly.
Combined effects of ATP and its analogs on the membrane permeability in transformed mouse fibroblasts
Authors: Arav Ruth , Friedberg Ilan
Published: April 2003

Extracellular ATP (0.6 mM) induces a marked decrease in the membrane potential, followed by an increase in cell membrane permeability in transformed mouse fibroblasts. The effects of the ATP analogs, p[CH2]ppA and p[NH]ppA (0.6 mM), on the membrane potential and permeability are much less pronounced. ATP at 0.05 mM has no effect by itself, but markedly increases the analog‐induced membrane potential dissipation and permeability. The data suggest that ATP‐induced membrane permeation is composed of two processes: One is common to ATP and its analogs and appears to be a receptormediated process. The second is unique for ATP, effective even at low concentration (0.05 mM), and might be mediated by cell surface enzymes, for which ATP, but not its analogs, serves as a substrate.
The inhibitor protein of phosphorylated nitrate reductase from spinach (Spinacia oleracea) leaves is a 14‐3‐3 protein
Authors: Bachmann Markus , Huber Joan L. , Liao Pao-Chi , Gage Douglas A. , Huber Steven C.
Published: April 2003

The inhibitor protein (IP) that inactivates spinach leaf NADH:nitrate reductase (NR) has been identified for the first time as a member of the eukaryotic 14‐3‐3 protein family based on three lines of evidence. First, the sequence of an eight amino acid tryptic peptide, obtained from immunopurified IP, matched that of a highly conserved region of the 14‐3‐3 proteins. Second, an authentic member of the 14‐3‐3 family, recombinant Arabidopsis GF14ω, caused inactivation of phospho‐NR in a magnesium‐dependent manner identical to IP. Third, an anti‐GF14 monoclonal antibody cross‐reacted with IP and anti‐IP monoclonal antibodies cross‐reacted with GF14ω.
Drosophila melanogaster SL2 cells contain a hypoxically inducible DNA binding complex which recognises mammalian HIF‐1 binding sites
Authors: Nagao Masaya , Ebert Benjamin L. , Ratcliffe Peter J. , Pugh Christopher W.
Published: April 2003

Nuclear extracts from Drosophila SL2 cells were found to contain a hypoxically inducible complex capable of binding to hypoxia response elements from mammalian genes. This complex (HIF‐D) resembled mammalian hypoxia inducible factor (HIF‐1) in DNA sequence specificity, abrogation of induction by cycloheximide, induction by desferrioxamine and redox sensitivity of DNA binding. However, HIF‐D was not induced by cobalt and was less sensitive to phosphatase than HIF‐1. Endogenous phosphoglycerate kinase mRNA in SL2 cells showed similar inducible characteristics to HIF‐D. These findings are evidence that the mammalian HIF‐1 dependent system of oxygen regulated gene expression has a functional homologue in Drosophila.
The ternary complex of DNase I, actin and thymosin β4
Authors: Reichert Andreas , Heintz Daniela , Echner Hartmut , Voelter Wolfgang , Faulstich Heinz
Published: April 2003

We have recently described a method for identifying contact sites between actin and thymosin β4 (Tβ4) by following spectrophotometrically the extent and kinetics of distinct, thiolspecific crosslinking reactions between appropriate derivatives of the two proteins [Reichert et al. (1996) J. Biol. Chem. 271, 1301–1308]. In the present study this method was used to show that such crosslinking, which is indicative of complex formation, occurs to the same extent with the actin‐DNase I complex as with pure actin, although at a somewhat lower rate. Further evidence for the formation of the ternary complex was given by gel electrophoresis. From fluorescence spectroscopy the K
D value of Tβ4 from the actin‐DNase I complex was found to be identical to that from pure actin. In line with these data, the capacity of actin for inhibiting DNase I was not affected by the addition of Tβ4. In conclusion, DNase I and Tβ4 are independent of each other in their interaction with actin, suggesting that the binding sites of thymosin β4 and DNase I on actin do not overlap. A ternary complex of DNase I, actin and Tβ4, if obtained in crystalline form, could thus provide an approach for studying the interface of Tβ4 and actin by X‐ray analysis.
X‐ray structure analysis of an engineered Fe‐superoxide dismutase Gly‐Ala mutant with significantly reduced stability to denaturant
Authors: Cooper J.B. , Saward S. , Erskine P.T. , Badasso M.O. , Wood S.P. , Zhang Y. , Young D.
Published: April 2003

We have refined the X‐ray structure of a site‐directed G152A mutant of the iron‐dependent superoxide dismutase from Mycobacterium tuberculosis at 2.9 Å resolution. The mutation which replaces a glycine residue in a surface loop with alanine was designed to alter the conformation of this loop region which has previously been shown to play a crucial structural role in quaternary interactions within the SOD tetramer. Gly‐152 was targeted as it has dihedral angles (φ=83.1°, Ψ=−0.3°) close to the left‐handed α‐helical conformation which is rarely adopted by other amino acids except asparagine. Gly‐152 was replaced by alanine as it has similar size and polarity, yet has a very low tendency to adopt similar conformations. X‐ray data collection on crystals of this mutant at 2.9 Å resolution and subsequent least‐squares refinement to an R‐value of 0.169 clearly establish that the loop conformation is unaffected. Fluorescence studies of guanidine hydrochloride denaturation establish that the mutant is 4 kcal/mol less stable than the wild‐type enzyme. Our results indicate that strict conformational constraints imposed upon a region of polypeptide, due for example to interactions with a neighbouring subunit, may force an alanine residue to adopt this sterically hindered conformation with a consequent reduction in stability of the folded conformation.
Palmitoylcarnitine, a surface‐active metabolite
Authors: Goñi Félix M. , Requero M.Asun , Alonso Alicia
Published: April 2003

Palmitoylcarnitine is a well‐known intermediate in mitochondrial fatty acid oxidation. Less known are its properties as a surfactant, with a capacity to solubilize biological membranes similar to that of many synthetic detergents used in the biochemical laboratory. Some of the physico‐chemical properties of palmitoylcarnitine may help to explain the need for coenzyme A‐carnitine‐coenzyme A acyl exchange during mitochondrial fatty acid import. The amphiphilic character of palmitoylcarnitine may also explain its proposed involvement in the pathogenesis of myocardial ischemia.
High‐molecular‐weight kininogen binds two molecules of cysteine proteinases with different rate constants
Authors: Turk Boris , Stoka Veronika , Turk Vito , Johansson Gunnar , Cazzulo Juan Jose , Björk Ingemar
Published: April 2003

Fluorescence titrations showed that high‐molecular‐weight kininogen binds two molecules of papain, cruzipain and cathepsin S with high affinity. The 2:1 binding stoichiometry was confirmed by stopped‐flow kinetic measurements of papain binding, which also revealed that the two sites bind the enzyme with different association rate constants (k
ass,1 = 23.0 × 106 M− s−1 and k
ass2, = 3.4 × 106 M−1s−1. As for low‐molecular‐weight kininogen, comparison of these kinetic constants with previous data for intact low‐ and high‐molecular‐weight kininogen and the separated domains indicated that the faster‐binding site is also the tighter‐binding site and is that of domain 3, whereas the slower‐binding, lower‐affinity site is on domain 2. The results further demonstrate that there is no appreciable steric interference between the two domains or by the kininogen light chain in the binding of proteinases. Similarly, the binding of kininogen via its light chain to a surface, as indicated by the binding to the model surface, heparin, did not affect the inhibitory properties of kininogen. The M
r of high‐molecular‐weight kininogen was determined to be 83 500 by sedimentation equilibrium measurements, in agreement with the value calculated from amino acid sequence and carbohydrate analysis.
Identification of DNA binding‐site preferences for nuclear factor I‐A
Authors: Osada Shigehiro , Daimon Shoko , Nishihara Tsutomu , Imagawa Masayoshi
Published: April 2003

Nuclear factor I (NFI) proteins constitute a large family of DNA binding proteins. These proteins promote the initiation of adenovirus replication and regulate the transcription of viral and cellular genes. The binding sites for NFI have been reported in a wide variety of promoters, and they exhibit flexibility in their sequences. To clarify the DNA binding site of NFI‐A, one of the NFI proteins, we performed a polymerase chain reaction‐mediated random site selection, and determined the optimal sequence as 5′‐TTGGCANNNN(G/T)CCA(G/A)‐3′.
Role of the N‐ and C‐termini of porin in import into the outer membrane of Neurospora mitochondria
Authors: Court Deborah A. , Kleene Ralf , Neupert Walter , Lill Roland
Published: April 2003

The signals for targeting and assembly of porin, a protein of the mitochondrial outer membrane, have not been clearly defined. Targeting information has been hypothesized to be contained in the N‐terminus, which may form an amphipathic α‐helix, and in the C‐terminal portion of the protein. Here, the role of the extreme N‐ and C‐termini of porin from Neurospora crussa in its import into the mitochondrial outer membrane was investigated. Deletion mutants were constructed which lacked the N‐terminal 12 or 20 residues or the C‐terminal 15 residues. The porins truncated at their N‐termini were imported in a receptor‐dependent manner into the outer membrane of isolated mitochondria. When integrated into the outer membrane, these preproteins displayed an increased sensitivity to protease as compared to wild‐type porin. In contrast, mutant porin truncated at its C‐terminus did not acquire protease resistance upon incubation with mitochondria. Thus, unlike most other mitochondrial preproteins, porin appears to contain important targeting and/or assembly information at its C‐terminus, rather than at the N‐terminus.
The size differences among mammalian introns are due to the accumulation of small deletions
Authors: Ogata Hiroyuki , Fujibuchi Wataru , Kanehisa Minoru
Published: April 2003

In order to investigate the molecular mechanisms that alter intron size, we conducted an extensive interspecies comparison of homologous introns among three mammalian groups: human, artiodactyls, and rodents. The size differences of introns were statistically significant among all three groups (longest intron was for human and shortest for rodents), and appear to be due to the accumulation of small deletions, according to the separate count of insertion and deletion frequencies. The distribution of intron size differences also has a shape similar to that for the distribution of insertion/deletion sizes found in pseudogenes. It is suggested that introns are selectively neutral to small‐scale changes of the genome size, which inherently contain the bias of favoring short deletions against short insertions.
Characterization of recombinant human HBP/CAP37/azurocidin, a pleiotropic mediator of inflammation‐enhancing LPS‐induced cytokine release from monocytes
Authors: Rasmussen Poul B. , Bjørn Søren , Hastrup Sven , Nielsen Per F. , Norris Kjeld , Thim Lars , Wiberg Finn C. , Flodgaard Hans
Published: April 2003

Neutrophil‐derived heparin‐binding protein (HBP) is a strong chemoattractant for monocytes. We report here for the first time the expression of recombinant HBP. A baculovirus containing the human HBP cDNA mediated in insect cells the secretion of a 7‐residue N‐terminally extended HBP form (pro‐HBP). Deletion of the pro‐peptide‐encoding cDNA sequence resulted in correctly processed HBP at the N‐terminus. Electrospray mass spectrum analysis of recombinant HBP yielded a molecular weight of 27.237 ± 3 amu. Consistent with this mass is a HBP form of 225 amino acids (mature part +3 amino acid C‐terminal extension). The biological activity of recombinant HBP was confirmed by its chemotactic action towards monocytes. Furthermore, we have shown that recombinant HBP stimulates in a dose‐dependent manner the lipopolysaccharide (LPS)‐induced cytokine release from human monocytes.
Dexamethasone enhanced by insulin, but not by thyroid hormones stimulates apolipoprotein B mRNA editing in cultured rat hepatocytes depending on the developmental stage
Authors: Lorentz Axel , Plonné Dietmar , Schulze Hans-Peter , Dargel Rolf
Published: April 2003

The increase of hepatic apolipoprotein B (apoB) mRNA editing during rat development was not affected by hypothyroidism. Furthermore, the addition of 3,3′,5‐triiodothyronine (T3) to cultured hepatocytes taken from fetal, neonatal and adult rats had no effect on apoB mRNA editing. In contrast, dexamethasone markedly stimulated apoB mRNA editing in hepatocytes taken from neonates. This effect was enhanced by the addition of insulin. For the first time our data provide evidence that glucocorticoids together with insulin are important for the regulation of apoB mRNA editing during postnatal development, whereas thyroid hormones are not critical for this process.
Basic helix‐loop‐helix protein MyoD displays modest DNA binding specificity
Authors: Künne Andreas G.E. , Meierhans Daniel , Allemann Rudolf K.
Published: April 2003

The expression of MyoD can activate muscle specific genes and myogenic differentiation in many cell types. The hypothesis that the DNA binding specificity of MyoD is responsible for its biological specificity was tested. Homodimers of MyoD bind to E‐box containing DNA with high affinity, but do not form stable and well defined complexes with heterologous DNA sequences. The physiologically active heterodimer of MyoD and E12 binds an oligonucleotide containing an E‐box sequence with an affinity only two orders of magnitude higher than a completely unrelated DNA sequence, stressing the importance of cooperative interactions with other proteins of the transcriptional machinery for specific gene activation.