bluuebunny/crossref_metadata_2025 huggingface dataset:
Hsp25, a member of the Hsp30 family, promotes inclusion formation in response to stress
Authors: Katoh Yumiko , Fujimoto Mitsuaki , Nakamura Kosuke , Inouye Sachiye , Sugahara Kazuma , Izu Hanae , Nakai Akira
Published: April 2004

Protein aggregates are oligomeric complexes of misfolded proteins, and serve as the seeds of inclusion bodies termed aggresomes in the cells. Heat shock proteins (Hsps) prevent misfolding and aggregate formation. Here, we found that only avian Hsp25 dominantly accumulated in the aggresomes induced by proteasome inhibition. Molecular cloning of chicken Hsp25 (cHsp25) revealed that it belongs to the Hsp30 family, which is a subfamily of the α‐crystallin/small Hsp gene family. Unexpectedly, overexpression of cHsp25 into HeLa cells promoted inclusion formation whereas overexpression of mouse Hsp27 and its chicken homologue did not. These results suggest that cHsp25 acts differently from other small Hsps on protein aggregates.
Calcium and mitochondria
Authors: Gunter Thomas E. , Yule David I. , Gunter Karlene K. , Eliseev Roman A. , Salter Jason D.
Published: April 2004

The literature suggests that the physiological functions for which mitochondria sequester Ca2+ are (1) to stimulate and control the rate of oxidative phosphorylation, (2) to induce the mitochondrial permeability transition (MPT) and perhaps apoptotic cell death, and (3) to modify the shape of cytosolic Ca2+ pulses or transients. There is strong evidence that intramitochondrial Ca2+ controls both the rate of ATP production by oxidative phosphorylation and induction of the MPT. Since the results of these processes are so divergent, the signals inducing them must not be ambiguous. Furthermore, as pointed out by Balaban [J. Mol. Cell. Cardiol. 34 (2002 ) 11259–11271], for any repetitive physiological process dependent on intramitochondrial free Ca2+ concentration ([Ca2+]m), a kind of intramitochondrial homeostasis must exist so that Ca2+ influx during the pulse is matched by Ca2+ efflux during the period between pulses to avoid either Ca2+ buildup or depletion. In addition, mitochondrial Ca2+ transport modifies both spatial and temporal aspects of cytosolic Ca2+ signaling. Here, we look at the amounts of Ca2+ necessary to mediate the functions of mitochondrial Ca2+ transport and at the mechanisms of transport themselves in order to set up a hypothesis about how the mechanisms carry out their roles. The emphasis here is on isolated mitochondria and on general mitochondrial properties in order to focus on how mitochondria alone may function to fulfill their physiological roles even though the interactions of mitochondria with other organelles, particularly with endoplasmic and sarcoplasmic reticulum [Sci. STKE re1 (2004) 1–9], may also influence this story.
The insect antimicrobial peptide, l‐pyrrhocoricin, binds to and stimulates the ATPase activity of both wild‐type and lidless DnaK
Authors: Chesnokova Liudmila S. , Slepenkov Sergey V. , Witt Stephan N.
Published: April 2004

Recent reports have indicated that insect antimicrobial peptides kill bacteria by inhibiting the molecular chaperone DnaK. It was proposed that the antimicrobial peptide, all‐l‐pyrrhocoricin (l‐PYR), binds to two sites on DnaK, the conventional substrate‐binding site and the multi‐helical C‐terminal lid, and that inhibition of DnaK comes about from the lid mode of binding. In this report, we show using two different assays that l‐PYR binds to and stimulates the ATPase activity of both wild‐type and a lidless variant of DnaK. Our study shows that l‐PYR interacts with DnaK much like the all‐l NR (NRLLLTG) peptide, which is known to bind in the conventional substrate‐binding site of DnaK. l‐PYR antimicrobial activity is thus a consequence of the competitive inhibition of bacterial DnaK.
Rrs1p, a ribosomal protein L11‐binding protein, is required for nuclear export of the 60S pre‐ribosomal subunit in Saccharomyces cerevisiae
Authors: Miyoshi Keita , Shirai Chiharu , Horigome Chihiro , Takenami Kazuhiko , Kawasaki Junko , Mizuta Keiko
Published: April 2004

Rrs1p is a ribosomal protein L11‐binding protein in Saccharomyces cerevisiae. We have obtained temperature‐sensitive rrs1 mutants by random PCR mutagenesis. [3H]Methionine pulse‐chase analysis reveals that the rrs1 mutations cause a defect in maturation of 25S rRNA. Ribosomal protein L25‐enhanced green fluorescent protein, a reporter of the 60S ribosomal subunit, concentrates in the nucleus with enrichment in the nucleolus when the rrs1 mutants are shifted to the restrictive temperature. These results suggest that Rrs1p stays on the pre‐60S particle from the early stage to very late stage of the large‐subunit maturation and is required for export of 60S subunits from the nucleolus to the cytoplasm.
Reducing friction by chemically modifying the surface of extrasynovial tendon grafts
Authors: Sun Yu‐Long , Yang Chao , Amadio Peter C. , Zhao Chunfeng , Zobitz Mark E. , An Kai‐Nan
Published: April 2004

In this study we evaluated methods to reduce the friction of an extrasynovial tendon under a pulley in vitro. The surface of canine peroneus longus (PL) tendons was modified with gelatin and/or hyaluronic acid (HA) under different conditions. The gliding resistance between treated or control tendons and the canine digital flexor pulley was measured during 500 cycles of simulated flexion/extension. After 500 cycles, the gliding resistance of normal PL tendon increased 10‐fold (p < 0.05), while the gliding resistance of tendons coated with carbodiimide derivatized gelatin (cd‐gelatin) or carbodiimide derivatized gelatin/HA (cd‐gelatin–HA) did not increase significantly. The PL tendon treated with cd‐gelatin–HA had a similar friction response during the repetitive motion as the intrasynovial flexor digitorum profundus (FDP) tendon. Scanning electron microscopy after 500 cycles of motion showed that the tendon surface in the group treated with cd‐gelatin–HA appeared smoother than tendons in the other groups. © 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.
High spatial resolution radon measurements reveal hidden active faults on Mt. Etna
Authors: Burton M. , Neri M. , Condarelli D.
Published: April 2004

Radon emissions are frequently monitored in volcanically and tectonically active areas in order to reveal changes in soil degassing, as radon acts a tracer for the more abundant CO2 degassing commonly observed in such areas. Between July 2002 and May 2003 a series of discrete measurements of radon concentrations in soil were made with high spatial resolution (∼5–100 m) in the Santa Venerina area on Mt. Etna. These measurements revealed well‐defined linear anomalies that we interpret as being caused by active faults whose higher porosity than surrounding soils allows an increased CO2 flux, carrying radon from beneath. These faults were not visible at the surface and were therefore revealed at high spatial resolution by our radon survey. Our hypothesis that the positive anomalies are attributable to active faults was strengthened by the observation of concentrated damage along this geometry during the earthquakes that struck this area in late October 2003.
Evidence of a recent input of magmatic gases into the quiescent volcanic edifice of Panarea, Aeolian Islands, Italy
Authors: Caliro S. , Caracausi A. , Chiodini G. , Ditta M. , Italiano F. , Longo M. , Minopoli C. , Nuccio P. M. , Paonita A. , Rizzo A.
Published: April 2004

On 2nd/3rd November 2002, a huge amount of gas, mainly composed of CO2, was suddenly released from the sea bottom off the coast of Panarea, producing a “crater” 20 by 10 meters wide and 7 meters deep. The gas output was estimated to be 109 l/d, two orders of magnitude higher than that measured in the 1980s. The anomalous degassing rate lasted for some weeks, slowly decreasing to an almost constant rate of about 4 × 107 l/d after two months. The geo‐thermo‐barometric estimations revealed an increase of both the temperature and pressure in the geothermal system feeding the sampled vents. The 3He/4He ratios were similar to those measured in nearby Stromboli. We have monitored the area for the last two decades, and based on our intensive and extensive geochemical measurements, have ascertained that the geothermal reservoir has lost its steady state. We maintain that a new magmatic input caused these phenomena.
Paleogeodetic records of seismic and aseismic subduction from central Sumatran microatolls, Indonesia
Authors: Natawidjaja Danny Hilman , Sieh Kerry , Ward Steven N. , Cheng Hai , Edwards R. Lawrence , Galetzka John , Suwargadi Bambang W.
Published: April 2004

We utilize coral microatolls in western Sumatra to document vertical deformation associated with subduction. Microatolls are very sensitive to fluctuations in sea level and thus act as natural tide gauges. They record not only the magnitude of vertical deformation associated with earthquakes (paleoseismic data), but also continuously track the long‐term aseismic deformation that occurs during the intervals between earthquakes (paleogeodetic data). This paper focuses on the twentieth century paleogeodetic history of the equatorial region. Our coral paleogeodetic record of the 1935 event reveals a classical example of deformations produced by seismic rupture of a shallow subduction interface. The site closest to the trench rose 90 cm, whereas sites further east sank by as much as 35 cm. Our model reproduces these paleogeodetic data with a 2.3 m slip event on the interface 88 to 125 km from the trench axis. Our coral paleogeodetic data reveal slow submergence during the decades before and after the event in the areas of coseismic emergence. Likewise, interseismic emergence occurred before and after the 1935 event in areas of coseismic submergence. Among the interesting phenomenon we have discovered in the coral record is evidence of a large aseismic slip or “silent event” in 1962, 27 years after the 1935 event. Paleogeodetic deformation rates in the decades before, after, and between the 1935 and 1962 events have varied both temporally and spatially. During the 25 years following the 1935 event, submergence rates were dramatically greater than in prior decades. During the past four decades, however, rates have been lower than in the preceding decades, but are still higher than they were prior to 1935. These paleogeodetic records enable us to model the kinematics of the subduction interface throughout the twentieth century.
Seismicity quiescence and activation in western Japan associated with the 1944 and 1946 great earthquakes near the Nankai trough
Authors: Ogata Yosihiko
Published: April 2004

Significant changes in seismicity (quiescence and activation) took place during the period of 1944–1946 in some regions in western Japan. They are well explained by the corresponding changes in Coulomb failure stress caused by the 1944 Tonankai earthquake of M7.9 and the 1946 Nankai earthquake of M8.0, both of which occurred in the Nankai trough. From the seismicity changes I try to identify precursory anomalies such as the quiescence in several regions before the Tonankai event and the enhanced activity in southern Kii peninsula before the Nankai event. These quiescence and enhanced activity may be due to precursory aseismic slips in an area on the plate interface downdip of the Tonankai rupture and slips transferring from the Tonankai to Nankai rupture zone. The records of felt shocks at the Wakayama Observatory during 1900–1995 provide support for the quiescence in the Wakayama District during 1944–1946, as well as a scenario of seismicity cycle till the next great event(s) in the Nankai trough to occur sometime this century.
Reference frequency of teleseismic body waves
Authors: Oki S. , Fukao Y. , Obayashi M.
Published: April 2004

Anelasticity of the Earth causes physical dispersion of seismic waves. The appropriate choice of reference frequency is important to determine arrival times of body waves, especially later arrival times, from broadband seismograms by fitting observed to synthetic waveforms. Our question is how to choose the reference frequency of physical dispersion, f0, consistent with the extensive data sets of hand‐picked body wave arrival times compiled through the long history of seismology. Setting the reference frequency to be the conventional 1 Hz, we measured S‐P differential travel times by waveform fitting. We found a systematic discrepancy between the measured and hand‐picked S‐P times that can be removed by changing f0 from 1 to 2 Hz. This change of f0 requires a correction in Q of the PREM model: (1) if Q is strictly frequency‐independent, we need a 15% increase in Q value or (2) if Q is fixed to the value given by PREM at a period of 200 s, we need to introduce a small frequency dependence in Q, where the dependence is expressed by the power law with an exponent of 0.04.
A representative curve for lightning current waveshape of first negative stroke
Authors: Visacro Silvério
Published: April 2004

A new approach is proposed for determining a representative lightning current waveshape for first negative strokes. This approach is applied to current wave registers obtained by direct measurement at Morro do Cachimbo Station, Brazil. The resulting curve is able to represent actual patterns of lightning current waves better than the traditional Berger's curve and therefore seems to be more appropriate for application in lightning studies.
Poly(l‐lysine)‐modified silica nanoparticles for the delivery of antisense oligonucleotides
Authors: Zhu Shi‐Guo , Xiang Juan‐Juan , Li Xiao‐Ling , Shen Shou‐Rong , Lu Hong‐bin , Zhou Jie , Xiong Wei , Zhang Bi‐Cheng , Nie Xin‐Min , Zhou Ming , Tang Ke , Li Gui‐Yuan
Published: April 2004

Silica nanoparticles were prepared in a microemulsion system, using polyoxyethylene nonylphenyl ether/cyclohexane/ammonium hydroxide. The surface charge of the particle was modified with PLL [poly(l‐lysine)]. PAGE demonstrated the ability of PMS‐NP (PLL‐modified silica nanoparticles) to bind and protect antisense ODNs (oligonucleotides). The intracellular localization of FITC‐labelled ODN was investigated by fluorescence microscopy. The results demonstrated that ODN could be delivered to cytoplasm. Flow‐cytometry analysis showed a 20‐fold enhancement of ODN delivered by PMS‐NP compared with free ODN for a serum‐free medium. Blocking efficacy of c‐myc antisense ODN, delivered by PMS‐NP, was examined in HNE1 and HeLa cell lines. Significant down‐regulation of c‐myc mRNA levels was observed in both the cell lines. However, the cellular uptake efficiency and antisense effects on target gene decreased in the presence of serum‐containing medium. The analysis of the filtration assay showed that PMS‐NP interacted with serum proteins. These results indicated that PMS‐NP was a suitable delivery vector for antisense ODN, although its delivery efficiency decreased in the presence of a serum‐containing medium.
Preparation of a highly stable, very active and high‐yield multilayered assembly of glucose oxidase using carbohydrate‐specific polyclonal antibodies
Authors: Jan Ulfat , Husain Qayyum
Published: April 2004

The purified oligosaccharide chains of Aspergillus niger glucose oxidase were coupled to BSA with the help of the cross‐linking reagent glutaraldehyde. The neoglycoconjugate thus obtained was purified by concanavalin A–Sepharose chromatography and characterized by SDS/PAGE. Immunization of rabbits with the neoglycoprotein raised the glycosyl‐specific anti‐(glucose oxidase) polyclonal antibodies. Antibodies were purified by (NH4)2SO4 precipitation, followed by DEAE‐cellulose chromatography. The IgG–Sepharose was prepared by covalently coupling the purified polyclonal antibodies to the CNBr‐activated Sepharose 4B. The large assembly of glucose oxidase was made on the IgG–Sepharose by alternate incubation of glucose oxidase and glycosyl‐specific anti‐(glucose oxidase) polyclonal IgG. The immunoaffinity‐layered assembled preparations were highly active and, after six alternate binding cycles with enzyme and glycosyl‐specific IgG, the amount of enzyme immobilized could be raised 30‐fold. The Km value of immunoaffinity‐layered glucose oxidase preparations remained unaltered, while the Vmax slightly decreased as compared with the soluble enzyme. A layer‐by‐layer immobilization of glucose oxidase resulted in significant improvement in stability against high temperature, 4.0 M urea and very high concentrations of water‐miscible organic solvents such as acetone, dimethylformamide, dioxan and tetrahydrofuran.
Conversion of 7‐deoxy‐10‐deacetylbaccatin‐III into 6‐α‐hydroxy‐7‐deoxy‐10‐deacetylbaccatin‐III byNocardioides luteus
Authors: Hanson Ronald L. , Kant Joydeep , Patel Ramesh N.
Published: April 2004

6‐α‐Hydroxy‐7‐deoxy‐10‐deacetylbaccatin‐III is an intermediate that is potentially useful for synthesis of analogues of paclitaxel. Screening of microbial strains identified an enzyme activity inNocardioides luteusSC 13912 (A.T.C.C. 55426) which converted 7‐deoxy‐10‐deacetylbaccatin‐III into 6‐hydroxy‐7‐deoxy‐10‐de‐acetylbaccatin‐III with a maximum yield of 44%.
A kinetic study of bovine haemoglobin hydrolysis by pepsin immobilized on a functionalized alumina to prepare hydrolysates containing bioactive peptides
Authors: Ticu Elena‐Loredana , Vercaigne‐Marko Dominique , Huma Anca , Artenie Vlad , Toma Ovidiu , Guillochon Didier
Published: April 2004

The hydrolysis kinetics of native and denatured haemoglobin, using pepsin immobilized on aluminium oxide, was studied in order to produce hydrolysates containing bioactive peptides. Pepsin was immobilized on acidic alumina and on 2‐ethanolamine‐O‐phosphate (2‐EAOP)‐modified acidic alumina. Surface charge of the two supports was determined as a function of pH. Kinetic studies were performed at 23 °C in 0.1 M acetate buffer, pH 4.5. At this pH, the surface charge of the two supports was almost the same. The coating of alumina by 2‐EAOP only introduced a two carbon spacer between alumina surface and the reaction medium. Adsorption on the two supports of haemoglobin, haem and peptides produced in the course of hydrolyses were compared. Fixation of 2‐EAOP on a pepsin–alumina complex gave hydrolysis kinetics of urea‐denatured haemoglobin close to that obtained with the same amount of pepsin in solution, but with comparatively less adsorption of peptides and complete adsorption of haem. Heterogeneous hydrolyses of haemoglobin with pepsin, immobilized on functionalized alumina, resulted in the presence of VV‐haemorphin‐4, VV‐haemorphin‐7 and neokyotorphin in the supernatants without haem, the presence of which makes further purification difficult.
One‐step purification and kinetic properties of the recombinant l‐asparaginase from Erwinia carotovora
Authors: Krasotkina Julya , Borisova Anna A. , Gervaziev Yuri V. , Sokolov Nikolay N.
Published: April 2004

ECAR‐LANS, the recombinant l‐asparaginase from Erwinia carotovora, is a prospective therapeutic enzyme for leukaemia treatment. An efficient and economical scheme was developed for the purification, cloning and expression in Eschericha coli of ECAR‐LANS. More than 90% purity, complemented with 72% active enzyme recovery, was achieved with a single chromatographic purification step. The activity of purified l‐asparaginase was 630 i.u./mg. The ECAR‐LANS Km value was 98×10−6 M for the main physiological substrate l‐Asn and 3400×10−6 M for l‐Gln. ECAR‐LANS was found to have low relative glutaminase activity (1.2%) at physiological concentrations of l‐Asn and l‐Gln in blood. Kinetic studies of ECAR‐LANS showed that the recombinant asparaginase combined the main advantages of Erw. chrysanthemi and E. coli l‐asparaginases II, currently used in the treatment of acute lymphoblastic leukaemia.
Palmitoylation is not required for trafficking of human anion exchanger 1 to the cell surface
Authors: CHEUNG Joanne C. , REITHMEIER Reinhart A. F.
Published: April 2004

AE1 (anion exchanger 1) is a glycoprotein found in the plasma membrane of erythrocytes, where it mediates the electroneutral exchange of chloride and bicarbonate, a process important in CO2 removal from tissues. It had been previously shown that human AE1 purified from erythrocytes is covalently modified at Cys-843 in the membrane domain with palmitic acid. In this study, the role of Cys-843 in human AE1 trafficking was investigated by expressing various AE1 and Cys-843Ala (C843A) mutant constructs in transiently transfected HEK-293 cells. The AE1 C843A mutant was expressed to a similar level to AE1. The rate of N-glycan conversion from high-mannose into complex form in a glycosylation mutant (N555) of AE1 C843A, and thus the rate of trafficking from the endoplasmic reticulum to the Golgi, were comparable with that of AE1 (N555). Like AE1, AE1 C843A could be biotinylated at the cell surface, indicating that a cysteine residue at position 843 is not required for cell-surface expression of the protein. The turnover rate of AE1 C843A was not significantly different from AE1. While other proteins could be palmitoylated, labelling of transiently transfected HEK-293 cells or COS7 cells with [3H]palmitic acid failed to produce any detectable AE1 palmitoylation. These results suggest that AE1 is not palmitoylated in HEK-293 or COS7 cells and can traffic to the plasma membrane.
Expression of YAP4 in Saccharomyces cerevisiae under osmotic stress
Authors: NEVITT Tracy , PEREIRA Jorge , AZEVEDO Dulce , GUERREIRO Paulo , RODRIGUES-POUSADA Claudina
Published: April 2004

YAP4, a member of the yeast activator protein (YAP) gene family, is induced in response to osmotic shock in the yeast Saccharomyces cerevisiae. The null mutant displays mild and moderate growth sensitivity at 0.4 M and 0.8 M NaCl respectively, a fact that led us to analyse YAP4 mRNA levels in the hog1 (high osmolarity glycerol) mutant. The data obtained show a complete abolition of YAP4 gene expression in this mutant, placing YAP4 under the HOG response pathway. YAP4 overexpression not only suppresses the osmosensitivity phenotype of the yap4 mutant but also relieves that of the hog1 mutant. Induction, under the conditions tested so far, requires the presence of the transcription factor Msn2p, but not of Msn4p, as YAP4 mRNA levels are depleted by at least 75% in the msn2 mutant. This result was further substantiated by the fact that full YAP4 induction requires the two more proximal stress response elements. Furthermore we find that GCY1, encoding a putative glycerol dehydrogenase, GPP2, encoding a NAD-dependent glycerol-3-phosphate phosphatase, and DCS2, a homologue to a decapping enzyme, have decreased mRNA levels in the yap4-deleted strain. Our data point to a possible, as yet not entirely understood, role of the YAP4 in osmotic stress response.
Enhancing effects of intracellular ascorbic acid on peroxynitrite-induced U937 cell death are mediated by mitochondrial events resulting in enhanced sensitivity to peroxynitrite-dependent inhibition of complex III and formation of hydrogen peroxide
Authors: GUIDARELLI Andrea , FIORANI Mara , CANTONI Orazio
Published: April 2004

A short-term pre-exposure to dehydroascorbic acid (DHA) promotes U937 cell death upon exposure to otherwise non-toxic levels of peroxynitrite (ONOO−). Toxicity is mediated by a saturable mechanism and cell death takes place as a consequence of mitochondrial permeability transition. The following lines of evidence are consistent with the notion that the enhancing effects of DHA were related to mitochondrial events resulting in inhibition of complex III upon exposure to otherwise inactive concentrations of ONOO−. First, DHA, as well as bona fide complex III inhibitors, similarly enhanced toxicity and subsequent formation of H2O2 induced by ONOO− via a rotenone- or catalase-sensitive mechanism. Secondly, bona fide complex III inhibitors were ineffective in DHA-pre-loaded cells. In addition, respiration-deficient cells were resistant to toxicity elicited by ONOO− and their supplementation with increasing concentrations of DHA, although resulting in the accumulation of vitamin C levels identical with those observed in respiration-proficient cells, failed to affect ONOO− toxicity. Finally, oxygen-consumption experiments demonstrated that pre-exposure to DHA promotes the ONOO−-dependent inhibition of complex III. In conclusion, the above results collectively demonstrate that increasing the intracellular accumulation of vitamin C promotes mitochondrial events leading to ONOO−-dependent formation of H2O2 and resulting in a rapid necrotic response.
Role of transient receptor potential canonical 6 (TRPC6) in non-transferrin-bound iron uptake in neuronal phenotype PC12 cells
Authors: MWANJEWE James , GROVER Ashok K.
Published: April 2004

Cells take up transferrin-bound iron or NTBI (non-transferrin-bound iron). After treatment with NGF (nerve growth factor), PC12 cells exhibited a neuronal phenotype and an increase in the NTBI uptake (55Fe2+ or 55Fe3+). We loaded the cells with the dye calcein, whose fluorescence increases in the presence of Ca2+ but is quenched with Fe2+ or Fe3+. When examined using calcein fluorescence or radioactive iron, DAG (diacylglycerol)-stimulated NTBI entry was more in NGF-treated PC12 cells compared with untreated cells. All experiments were performed at 1.5 mM extracellular Ca2+. Nramp2 (natural-resistance-associated macrophage protein 2) mRNA expression did not change after the NGF treatment. Expression of the bivalent cation entry protein TRPC6 (transient receptor potential canonical 6) was detected only in the NGF-treated cells. To verify that increased NTBI uptake depended on TRPC6, we examined whether transfecting HEK-293 (human embryonic kidney 293) cells with TRPC6 also increased the NTBI (55Fe) uptake. We also cotransfected HEK-293 cells with two plasmids, one expressing TRPC6 and the other expressing the fluorescent protein DsRED2 to identify the transfected cells. Challenging the calcein-loaded HEK-293 cells (which intrinsically express the α1-adrenergic receptors) with phenylephrine or a cell-permeant DAG increased the fluorescence signal more rapidly in transfected cells compared with untransfected cells. However, when iron (Fe2+ and Fe3+) was added before adding phenylephrine or DAG, the fluorescence intensity decreased more rapidly in transfected cells compared with untransfected cells, thereby indicating a greater stimulation of the NTBI uptake in cells expressing TRPC6. We postulate that the increase in the NTBI entry into neuronal PC12 cells is through TRPC6, a pathway that is unique since it is receptor-stimulated. Since neuronal cells express TRPC6, this pathway may have a role in neurotoxicity.
Oxidative-stress-related proteome changes in Helicobacter pylori-infected human gastric mucosa
Authors: BAEK Hye Yeon , LIM Joo Weon , KIM Hyeyoung , KIM Jung Mogg , KIM Joo Sung , JUNG Hyun Chae , KIM Kyung Hwan
Published: April 2004

Helicobacter pylori infection leads to gastroduodenal inflammation, peptic ulceration and gastric carcinoma. Proteomic analysis of the human gastric mucosa from the patients with erosive gastritis, peptic ulcer or gastric cancer, which were either infected or not with H. pylori, was used to determine the differentially expressed proteins by H. pylori in the human gastric mucosa in order to investigate the pathogenic mechanism of H. pylori-induced gastric diseases. Prior to the experiment, the expression of the main 18 proteins were identified in the gastric mucosa and used for a proteome map of the human gastric mucosa. Using two-dimensional electrophoresis of the protein isolated from the H. pylori-infected tissues, Coomassie Brilliant Blue staining and computerized analysis of the stained gel, the expression of eight proteins were altered in the H. pylori-infected tissues compared with the non-infected tissues. MS analysis (matrix-assisted laser desorption/ionization–time of flight MS) of the tryptic fragment and a data search allowed the the identification of the four increased proteins (78 kDa glucose-regulated protein precursor, endoplasmin precursor, aldehyde dehydrogenase 2 and l-lactate dehydrogenase B chain) and the four decreased proteins (intracellular chloride channel protein 1, glutathione S-transferase, heat-shock protein 60 and cytokeratin 8) caused by H. pylori infection in the gastric mucosa. These proteins are related to cell proliferation, carcinogenesis, cytoskeletal function and cellular defence mechanism. The common feature is that these proteins are related to oxidative-stress-mediated cell damage. In conclusion, the established gastric mucosal proteome map might be useful for detecting the disease-related protein changes. The H. pylori-induced alterations in protein expression demonstrate the involvement of oxidative stress in the pathogenesis of H. pylori-induced gastric diseases, including inflammation, ulceration and carcinogenesis.
Intracellular proteoglycans
Authors: KOLSET Svein Olav , PRYDZ Kristian , PEJLER Gunnar
Published: April 2004

Proteoglycans (PGs) are proteins with glycosaminoglycan chains, are ubiquitously expressed and have a wide range of functions. PGs in the extracellular matrix and on the cell surface have been the subject of extensive structural and functional studies. Less attention has so far been given to PGs located in intracellular compartments, although several reports suggest that these have biological functions in storage granules, the nucleus and other intracellular organelles. The purpose of this review is, therefore, to present some of these studies and to discuss possible functions linked to PGs located in different intracellular compartments. Reference will be made to publications relevant for the topics we present. It is beyond the scope of this review to cover all publications on PGs in intracellular locations.
The nuclear tRNA aminoacylation-dependent pathway may be the principal route used to export tRNA from the nucleus in Saccharomyces cerevisiae
Authors: STEINER-MOSONYI Marta , MANGROO Dev
Published: April 2004

Nuclear tRNA export in Saccharomyces cerevisiae has been proposed to involve three pathways, designated Los1p-dependent, Los1p-independent nuclear aminoacylation-dependent, and Los1p- and nuclear aminoacylation-independent. Here, a comprehensive biochemical analysis was performed to identify tRNAs exported by the aminoacylation-dependent and -independent pathways of S. cerevisiae. Interestingly, the major tRNA species of at least 19 families were found in the aminoacylated form in the nucleus. tRNAs known to be exported by the export receptor Los1p were also aminoacylated in the nucleus of both wild-type and mutant Los1p strains. FISH (fluorescence in situ hybridization) analyses showed that tRNATyr co-localizes with the U18 small nucleolar RNA in the nucleolus of a tyrosyl-tRNA synthetase mutant strain defective in nuclear tRNATyr export because of a block in nuclear tRNATyr aminoacylation. tRNATyr was also found in the nucleolus of a utp8 mutant strain defective in nuclear tRNA export but not nuclear tRNA aminoacylation. These results strongly suggest that the nuclear aminoacylation-dependent pathway is principally responsible for tRNA export in S. cerevisiae and that Los1p is an export receptor of this pathway. It is also likely that in mammalian cells tRNAs are mainly exported from the nucleus by the nuclear aminoacylation-dependent pathway. In addition, the data are consistent with the idea that nuclear aminoacylation is used as a quality control mechanism for ensuring nuclear export of only mature and functional tRNAs, and that this quality assurance step occurs in the nucleolus.
BmTx3, a scorpion toxin with two putative functional faces separately active on A-type K+ and HERG currents
Authors: HUYS Isabelle , XU Chen-Qi , WANG Cheng-Zhong , VACHER Hélène , MARTIN-EAUCLAIRE Marie-France , CHI Cheng-Wu , TYTGAT Jan
Published: April 2004

A novel HERG channel blocker was isolated from the venom of the scorpion Buthus martensi Karsch, sequenced and characterized at the pharmacological level after chemical synthesis. According to the determined amino acid sequence, the cDNA and genomic genes were then cloned. The genomic gene consists of two exons interrupted by an intron of 65 bp at position −6 upstream from the mature toxin. The protein sequence of this toxin was completely identical with that of a known A-type K+ current blocker BmTx3, belonging to scorpion α-KTx subfamily 15. Thus BmTx3 is the first reported α-KTx peptide also showing HERG-blocking activity, like γ-KTx peptides. Moreover, different from classical α-KTx peptides, such as charybdotoxin, BmTx3 cannot block Shaker-type K+ channels. Phylogenetic tree analysis reveals that this toxin takes an intermediate position between classical α-KTx and γ-KTx toxins. From a structural point of view, we propose that two separate functional faces might exist on the BmTx3 molecule, responsible for the two different K+-current-blocking functions. Face A, composed of Arg18 and Lys19 in the α-helix side, might correspond to HERG blocking activity, whereas Face B, containing a putative functional dyad (Lys27 and Tyr36) in the β-sheet side, might correspond to A-type blocking activity. A specific deletion mutant with the disrupted Face B, BmTx3-Y36P37del, loses the A-type current-blocking activity, but keeps a similar HERG-blocking activity, as seen with the wild-type toxin.
Induction of fibroblast apolipoprotein E expression during apoptosis, starvation-induced growth arrest and mitosis
Authors: QUINN Carmel M. , KÅGEDAL Katarina , TERMAN Alexei , STROIKIN Uri , BRUNK Ulf T. , JESSUP Wendy , GARNER Brett
Published: April 2004

Apolipoprotein E (apoE) mediates the hepatic clearance of plasma lipoproteins, facilitates cholesterol efflux from macrophages and aids neuronal lipid transport. ApoE is expressed at high levels in hepatocytes, macrophages and astrocytes. In the present study, we identify nuclear and cytosolic pools of apoE in human fibroblasts. Fibroblast apoE mRNA and protein levels were up-regulated during staurosporine-induced apoptosis and this was correlated with increased caspase-3 activity and apoptotic morphological alterations. Because the transcription of apoE and specific pro-apoptotic genes is regulated by the nuclear receptor LXR (liver X receptor) α, we analysed LXRα mRNA expression by quantitative real-time PCR and found it to be increased before apoE mRNA induction. The expression of ABCA1 (ATP-binding cassette transporter A1) mRNA, which is also regulated by LXRα, was increased in parallel with apoE mRNA, indicating that LXRα probably promotes apoE and ABCA1 transcription during apoptosis. Fibroblast apoE levels were increased under conditions of serum-starvation-induced growth arrest and hyperoxia-induced senescence. In both cases, an increased nuclear apoE level was observed, particularly in cells that accumulated lipofuscin. Nuclear apoE was translocated to the cytosol when mitotic nuclear disassembly occurred and this was associated with an increase in total cellular apoE levels. ApoE amino acid sequence analysis indicated several potential sites for phosphorylation. In vivo studies, using 32P-labelling and immunoprecipitation, revealed that fibroblast apoE can be phosphorylated. These studies reveal novel associations and potential roles for apoE in fundamental cellular processes.
Zinc-finger protein A20, a regulator of inflammation and cell survival, has de-ubiquitinating activity
Authors: EVANS Paul C. , OVAA Huib , HAMON Maureen , KILSHAW Peter J. , HAMM Svetlana , BAUER Stefan , PLOEGH Hidde L. , SMITH Trevor S.
Published: April 2004

Ubiquitination regulates the stability and/or activity of numerous cellular proteins. The corollary is that de-ubiquitinating enzymes, which ‘trim’ polyubiquitin chains from specific substrate proteins, play key roles in controlling fundamental cellular activities. Ubiquitin is essential at several stages during the activation of NF-κB (nuclear factor κB), a central co-ordinator of inflammation and other immune processes. Ubiquitination is known to cause degradation of the inhibitory molecule IκBα (inhibitor of κB). In addition, activation of TRAF (tumour-necrosis-factor-receptor-associated factor) and IKKγ (IκB kinase γ)/NEMO (NF-κB essential modifier) signal adaptors relies on their modification with ‘nonclassical’ forms of polyubiquitin chains. Ubiquitin also plays a key role in determining cell fate by modulating the stability of numerous pro-apoptotic or anti-apoptotic proteins. The zinc-finger protein A20 has dual functions in inhibiting NF-κB activation and suppressing apoptosis. The molecular mechanisms of these anti-inflammatory and cytoprotective effects are unknown. Here we demonstrate that A20 is a de-ubiquitinating enzyme. It contains an N-terminal catalytic domain that belongs to the ovarian-tumour superfamily of cysteine proteases. A20 cleaved ubiquitin monomers from branched polyubiquitin chains linked through Lys48 or Lys63 and bound covalently to a thiol-group-reactive, ubiquitin-derived probe. Mutation of a conserved cysteine residue in the catalytic site (Cys103) abolished these activities. A20 did not have a global effect on ubiquitinated cellular proteins, which indicates that its activity is target-specific. The biological significance of the catalytic domain is unknown.
Induction of p21 and p27 expression by amino acid deprivation of HepG2 human hepatoma cells involves mRNA stabilization
Authors: LEUNG-PINEDA Van , PAN YuanXiang , CHEN Hong , KILBERG Michael S.
Published: April 2004

mRNA abundance for a number of genes is increased by amino acid limitation. From an array screening study in HepG2 human hepatoma cells, it was established that one set of genes affected by amino acid availability is the set associated with cell-cycle control. The present study describes the increased expression of both mRNA and protein for the cyclin-dependent kinase inhibitors p21 and p27 in response to deprivation of HepG2 cells for a single essential amino acid, histidine. The increase in p21 and p27 mRNA content depended on de novo protein synthesis and involved a post-transcriptional mRNA stabilization component. For p21, increase in mRNA by histidine depletion appeared to be independent of p53 transactivation, and the absolute level of p53 protein was unaffected by this treatment. Histidine limitation caused an increase in the phosphorylation of ERK1/ERK2 (extracellular-signal-regulated kinase), and inhibition of the ERK signal transduction pathway resulted in a reduction in the starvation-dependent increase in p21 mRNA. Blockade of the phosphoinositide 3-kinase and mTOR (mammalian target of rapamycin) pathways also blunted the increase in p21 mRNA content. These results document the amino acid-dependent control of the synthesis of specific cell-cycle regulators and help to explain the block at G1 phase after amino acid limitation.
beta-Glucosidase, exo-beta-glucanase and pyridoxine transglucosylase activities of rice BGlu1
Authors: OPASSIRI Rodjana , HUA Yanling , WARA-ASWAPATI Onnop , AKIYAMA Takashi , SVASTI Jisnuson , ESEN Asim , CAIRNS James R. KETUDAT
Published: April 2004

The bglu1 cDNA for a β-glucosidase cloned from rice (Oryza sativa L.) seedlings was expressed as a soluble and active protein in Escherichia coli and designated BGlu1. This enzyme hydrolysed β-1,4-linked oligosaccharides with increasing catalytic efficiency (kcat/Km) values as the DP (degree of polymerization) increased from 2 to 6. In contrast, hydrolysis of β-1,3-linked oligosaccharides decreased from DP 2 to 3, and polymers with a DP greater than 3 were not hydrolysed. The enzyme also hydrolysed p-nitrophenyl β-d-glycosides and some natural glucosides but with lower catalytic efficiency than β-linked oligosaccharides. Pyridoxine 5´-O-β-d-glucoside was the most efficiently hydrolysed natural glycoside tested. BGlu1 also had high transglucosylation activity towards pyridoxine, producing pyridoxine 5´-O-β-d-glucopyranoside in the presence of the glucose donor p-nitrophenyl β-d-glucoside.
PRELI (protein of relevant evolutionary and lymphoid interest) is located within an evolutionarily conserved gene cluster on chromosome 5q34-q35 and encodes a novel mitochondrial protein
Authors: FOX Elizabeth J. , STUBBS Sally A. , TUN Jimmy KYAW , LEEK Jack P. , MARKHAM Alexander F. , WRIGHT Stephanie C.
Published: April 2004

The characterization of mitochondrial proteins is important for the understanding of both normal cellular function and mitochondrial disease. In the present study we identify a novel mitochondrial protein, PRELI (protein of relevant evolutionary and lymphoid interest), that is encoded within the evolutionarily conserved MAD3/PRELI/RAB24 gene cluster located at chromosome 5q34–q35. Mouse Preli is expressed at high levels in all settings analysed; it is co-expressed with Rab24 from a strong bi-directional promoter, and is regulated independently from the S-phase-specific Mad3 gene located at its 3´ end. PRELI contains a stand-alone 170 amino acid PRELI/MSF1p´ motif at its N-terminus. This domain is found in a variety of proteins from diverse eukaryotes including yeast, Drosophila and mammals, but its function is unknown, and the subcellular location of higher eukaryotic PRELI/MSF1P´ proteins has not been determined previously. We show here that PRELI is located in the mitochondria, and by using green-fluorescent-protein fusion proteins we identify a mitochondrial targeting signal at its N-terminus.
Mapping of catalytically important residues in the rat l-histidine decarboxylase enzyme using bioinformatic and site-directed mutagenesis approaches
Authors: FLEMING John V. , SÁNCHEZ-JIMÉNEZ Francisca , MOYA-GARCÍA Aurelio A. , LANGLOIS Michael R. , WANG Timothy C.
Published: April 2004

HDC (l-histidine decarboxylase), the enzyme responsible for the catalytic production of histamine from l-histidine, belongs to an evolutionarily conserved family of vitamin B6-dependent enzymes known as the group II decarboxylases. Yet despite the obvious importance of histamine, mammalian HDC enzymes remain poorly characterized at both the biochemical and structural levels. By comparison with the recently described crystal structure of the homologous enzyme l-DOPA decarboxylase, we have been able to identify a number of conserved domains and motifs that are important also for HDC catalysis. This includes residues that were proposed to mediate events within the active site, and HDC proteins carrying mutations in these residues were inactive when expressed in reticulocyte cell lysates reactions. Our studies also suggest that a significant change in quartenary structure occurs during catalysis. This involves a protease sensitive loop, and incubating recombinant HDC with an l-histidine substrate analogue altered enzyme structure so that the loop was no longer exposed for tryptic proteolysis. In total, 27 mutant proteins were used to test the proposed importance of 34 different amino acid residues. This is the most extensive mutagenesis study yet to identify catalytically important residues in a mammalian HDC protein sequence and it provides a number of novel insights into the mechanism of histamine biosynthesis.
Peroxisomal-proliferator-activated receptor alpha activates transcription of the rat hepatic malonyl-CoA decarboxylase gene: a key regulation of malonyl-CoA level
Authors: LEE Gha Young , KIM Nam Hee , ZHAO Zheng-Shan , CHA Bong Soo , KIM Y. Sam
Published: April 2004

MCD (malonyl-CoA decarboxylase), which catalyses decarboxylation of malonyl-CoA, is known to play an important role in the regulation of malonyl-CoA concentration. Recently, it has been observed that the expression of MCD is significantly decreased in the hearts of the PPARα (peroxisome-proliferator-activated receptor α) (−/−) mice, where the rate of fatty-acid oxidation is decreased by the increased malonyl-CoA level [Campbell, Kozak, Wagner, Altarejos, Dyck, Belke, Severson, Kelly and Lopaschuk (2002) J. Biol. Chem. 277, 4098–4103]. This suggests that MCD may be transcriptionally regulated by PPARα. To investigate whether PPARα is truly responsible for transcriptional regulation of the rat MCD gene, transient reporter assay was performed in CV-1 cells. The promoter activity was increased by 17-fold in CV-1 cells co-transfected with PPARα/retinoid X receptor α expression plasmid. In sequence analysis of the promoter region, three putative PPREs (PPAR response elements) were identified, and promoter deletion analysis showed that PPRE2 and PPRE3 were functional. Electrophoretic mobility-shift assays revealed that PPARα/retinoid X receptor α heterodimer indeed bound to the two PPREs, and the binding specificity of PPARα on PPRE was also confirmed by experiments with mutated oligonucleotides. These results indicate that the elements behaved as a responsive site to PPARα activation. MCD mRNA levels in WY14643-treated rat hepatoma cells as well as in the liver of fenofibrate-fed Otsuka Long-Evans Tokushima fatty rats were also found to be increased, suggesting that PPARα can activate the rat hepatic MCD transcription by binding to the PPREs in the promoter. We propose that MCD performs an important role in understanding the regulatory mechanism between activated PPARα and fatty-acid oxidation by altering the malonyl-CoA concentration.
Effect of dicarbonyl-induced browning on alpha-crystallin chaperone-like activity: physiological significance and caveats of in vitro aggregation assays
Authors: KUMAR M. Satish , REDDY P. Yadagiri , KUMAR P. Anil , SUROLIA Ira , REDDY G. Bhanuprakash
Published: April 2004

α-Crystallin is a member of the small heat-shock protein family and functions like a molecular chaperone, and may thus help in maintaining the transparency of the eye lens by protecting the lens proteins from various stress conditions. Non-enzymic glycation of long-lived proteins has been implicated in several age- and diabetes-related complications, including cataract. Dicarbonyl compounds such as methylglyoxal and glyoxal have been identified as the predominant source for the formation of advanced glycation end-products in various tissues including the lens. We have investigated the effect of non-enzymic browning of α-crystallin by reactive dicarbonyls on its molecular chaperone-like function. Non-enzymic browning of bovine α-crystallin in vitro caused, along with altered secondary and tertiary structures, cross-linking and high-molecular-mass aggregation. Notwithstanding these structural changes, methylglyoxal- and glyoxal-modified α-crystallin showed enhanced anti-aggregation activity in various in vitro aggregation assays. Paradoxically, increased chaperone-like activity of modified α-crystallin was not associated with increased surface hydrophobicity and rather showed less 8-anilinonaphthalene-l-sulphonic acid binding. In contrast, the chaperone-like function of modified α-crystallin was found to be reduced in assays that monitor the prevention of enzyme inactivation by UV-B and heat. Moreover, incubation of bovine lens with methylglyoxal in organ culture resulted in cataract formation with accumulation of advanced glycation end-products and recovery of α-crystallin in high proportions in the insoluble fraction. Furthermore, soluble α-crystallin from methylglyoxal-treated lenses showed decreased chaperone-like activity. Thus, in addition to describing the effects of methylglyoxal and glyoxal on structure and chaperone-like activity, our studies also bring out an important caveat of aggregation assays in the context of the chaperone function of α-crystallin.