bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for a hydroxy‐aluminium polymer (Al13) in synaptosomes
Authors: Rao Gazula Valeswara , Rao K.S.Jagannatha
Published: July 2002

The presence of the hydroxy‐aluminium polymer (Al13‐(OH)24O4(H2O)7+
12) was noticed inside synaptosomes when synaptosomes were incubated with Al(NO3)3 at neutral pH values. Dysprosium nitrate (Dy(NO3)3) ‐ a shift reagent ‐ facilitates the identification of the Al13 species distinctly inside the synaptosomes. 27Al NMR was used as a tool to detect the Al13 complex inside and outside the synaptosomes.
Regulation of 1α,25‐dihydroxyvitamin D3 synthesis in macrophages from arthritic joints by phorbol ester, dibutyryl‐cAMP and calcium ionophore (A23187)
Authors: Yuan J.Y. , Freemont A.J. , Mawer E.B. , Hayes M.E.
Published: July 2002

Phorbol 12‐myristate 13‐acetate (100 nM), a potent protein kinase C and macrophage activator, has a biphasic affect on 25(OH)D3‐1α‐hydroxylase activity in synovial fluid macrophages from arthritis patients, After 5 h, 1α,25(OH)D3 synthesis fell from 5.2 ± 0.1 to 1.6 ± 0.2 pmol/h per 106 cells, however, after 24 h and 48 h, synthesis increased to 17.4 ± 0.3 and 22.3 ± 1.4 pmol/h per 106 cells, respectively. Although an independent short‐term mechanism is suggested, protein kinase C may promote macrophage activation, thus increasing long‐term 25(OH)D3‐1α‐hydroxylase expression. Intracellular calcium and cAMP are unlikely to activate the enzyme, since 0.1 μM of the calcium ionophore, A23187, and 1 mM dibutyryl‐cAMP inhibited synthesis by 87% and 79%, respectively, after 24 h.
The PSI‐E subunit of photosystem I binds ferredoxin:NADP+ oxidoreductase
Authors: Andersen Birgitte , Scheller Henrik Vibe , Møller Birger Lindberg
Published: July 2002

A photosystem I complex containing the polypeptides PSI‐A to PSI‐L, light‐harvesting complex I and ferredoxin:NADP+ oxidoreductase has been isolated from barley using the non‐ionic detergent n‐decyl‐β‐d‐maltopyranoside. The ratio between bound forredoxin:NADP+ oxidoreductase and P700 is 0.4 ± 0.2. The complex is highly active in catalyzing light‐induced transfer of electrons from plastocyanin to NADP+ at rates of 280±150 and 1800 ± 800, μmol NADPH/(mg chl h), without and in the presence of saturating amounts of exogenously added ferredoxin:NADP+ oxidoreductase, respectively. Endogenously bound ferredoxin:NADP+ oxidoreductase interacts with the PSI‐E subunit as demonstrated by cross‐linking experiments using two different types of cross‐linkers and identification of the products by Western blotting and the use of monospecific antibodies.
Interferon‐γ induces cell surface expression for both types of tumor necrosis factor receptors
Authors: Pandita Raj , Pocsik Eva , Aggarwal Bharat B.
Published: July 2002

Interferons are known to potentiate various biological effects of tumor necrosis factor (TNF). Recently, two different types of TNF receptors with molecular masses of 60 kDa (p60) and 80 kDa (p80), primarily expressed by epithelial cells and myeloid cells, respectively, have been identified. In the present report, we examined the effect of interferon‐γ (IFN‐γ) on each type of TNF receptor. Our results indicate that IFN‐γ induces TNF receptors on both myeloid (e.g. HL‐60) and epithelial cells (e.g. HeLa). Furthermore, by using antibodies specific to each type of receptor, we demonstrate that both TNF receptors are equally inducible by IFN‐α, IFN‐β and IFN‐γ. Thus, the increase in TNF receptors by interferons may play a role in their synergistic cellular response.
Inactivation of the Na,K‐ATPase by modification of Lys‐501 with 4‐acetamido‐4′‐isothiocyanatostilbene‐2,2′‐disulfonic acid (SITS)
Authors: Pedemonte Carlos H. , Kirley Torence L. , Treuheit Michael J. , Kaplan Jack H.
Published: July 2002

The sodium pump or Na,K‐ATPase, maintains the Na+ and K+ gradients across eukaryotic cell membranes at the expense of ATP. Incubation of purified canine renal Na,K‐ATPase with 4‐acetamido‐4′‐isothiocyanatostilbene‐2,2′‐disulfonic acid (SITS) inhibited the ATPase activity. Both the labeling of the protein and the loss or ATPase activity were prevented by co‐incubation with ADP (acting as an ATP analog) or KCI. Only the α‐subunit was labeled by SITS. The α‐subunit from the inhibited enzyme was extensively digested with trypsin, and SITS‐labeled peptides were purified by reverse‐phase HPLC and sequenced. The amino acid sequence determined, His‐Leu‐Leu‐Val‐Met‐X‐Gly‐Ala‐Pro‐Glu, indicated that SITS modifies Lys‐501 (X) on the α‐subunit of Na,K‐ATPase.
Correlation of cofactor binding and the quaternary structure of pyruvate decarboxylase as revealed by 31P NMR spectroscopy
Authors: Hübner Gerhard , König Stephan , Schnackerz Klaus D.
Published: July 2002

The pH dependence of the quaternary structure of pyruvate decarboxylase (EC 4.1.1.1) has recently been discovered [(1990) FEBS Lett. 266, 17–20; (1992) Biochemistry (in press)]. In the present study we have investigated the change in quaternary structure by observing the binding of the cofactor, thiamine pyrophosphate, using 31P NMR spectroscopy. The dissociation of the native tetramers into dimers when increasing the pH coincides with a weaker binding of the cofactor and loss of enzyme activity. The results provide further evidence that thiamine pyrophosphate is bound primarily via the β‐phosphate moiety. In addition, a phosphoserine has been discovered in two of the four subunits.
Fusidic acid‐dependent ribosomal complexes protect Escherichia coli ribosomes from the action of the type 1 ribosome‐inactivating protein crotin 2
Authors: Iglesias Rosario , Escarmis Cristina , Alegre Carlos , Ferreras J.Miguel , Girbés Tomás
Published: July 2002

The type 1 ribosome‐inactivating protein crotin 2 depurinated Escherichia coli ribosomes which, upon treatment of the isolated rRNA with acid aniline, released a fragment of around 240 nucleotides whose 5'‐end sequence was 5'‐GAGGACCGGAGUGGAC‐3'. The formation of fusidic acid‐dependent ribosomal complexes completely prevented release of the fragment. Ribosomes from crotin 2‐pretreated fusidic acid complexes were insensitive to acid aniline. They released the RNA fragment only after a second treatment with crotin 2 and acid aniline whereas unprotected ribosomes released the fragment directly after acid aniline.
Cell cycle phase‐dependent effect of retinoic acid on the induction of granulocytic differentiation in HL‐60 promyelocytic leukemia cells
Authors: Hui Eric Ka-Wai , Yung Benjamin Yat-Ming
Published: July 2002

The efficiency of retinoic acid (RA)‐induced differentiation was dependent on the position of HL‐60 cells in the cell cycle. Our results demonstrated that cells at the G1/S border were more efficiently induced to differentiate by short exposure to RA than cells at other phases of the cell cycle. Synchronization of cells in G1/S phase by aphidicolin (APH) or mimosine (MIMO) increased the sensitivity of cells to RA short exposure treatment. Pretreatment with sphinganine (SP), a protein kinase C (PKC) inhibitor, potentiated RA‐induced cell differentiation. By cell cycle analysis, SP was found to block the cell progression through the G1/S phase. Consequently, cells accumulated in the G1/S phase of the cell cycle. The present data therefore suggest a possible mechanism of action of SP to enhance RA‐induced differentiation.
Cleavage of human MDR1 mRNA by a hammerhead ribozyme
Authors: Kobayashi Hiroyuki , Dorai Thambi , Holland James F. , Ohnuma Takao
Published: July 2002

We designed a hammerhead ribozyme which site‐specifically cleaved the GUC sequence in codon 179 of MDR1 mRNA. The cleavage site was 6 amino acids upstream from the drug binding site and was considered sufficiently close to the essential locus for P‐glycoprotein function. The ribozyme cleaved the MDR1 mRNA under physiological conditions in vitro. The cleavage was dependent on ribozyme concentration and on incubation time. Mg2+ ion was essential for the cleavage. These results show that a potentially useful tool is at hand which may inactivate MDR1 mRNA and revert the multidrug resistance phenotype.
Participation of cathepsin L on bone resorption
Authors: Kakegawa Hisao , Nikawa Takeshi , Tagami Kahori , Kamioka Hiroshi , Sumitani Koji , Kawata Terushige , Drobnič-Kosorok Marinka , Lenarčič Brigita , Turk Vito , Katunuma Nobuhiko
Published: July 2002

The proteinase responsible for bone collagen degradation in osteo‐resorption was examined. The bone pit formation induced by parathyroid hormone (PTH) was markedly suppressed by leupeptin, E‐64 and cystatin A, while no inhibition was observed by CA‐074, a specific inhibitor of cathepsin B. Pig leucocyte cysteine proteinase inhibitor (PLCPI), a specific inhibitor of cathepsin L, and chymostatin, a selective inhibitor of cathepsin L, completely inhibited the pit formation. Cathepsin L activity in osteoclasts was much higher than the other cathepsin activities. Serum calcium in rats placed on a low calcium diet was decreased by treatment of E‐64 or cystatin A, but not by CA‐074. These findings suggest that cathepsin L is the main proteinase responsible for bone collagen degradation.
Inhibition of uncoupled respiration in tumor cells
Authors: Gabai Vladimir L.
Published: July 2002

Uncouplers CCCP (2–4 μM) or DNP (200–400 μM) when added to EL‐4 thymoma or Ehrlich carcinoma ascites cells initially stimulated endogenous respiration about 2‐fold but then inhibited it to a first‐order rate 20–25% of controls. This inhibition was accelerated by intracellular acidification or by A23187, a Ca2+/H+‐antiporter (i.e. when mitochondrial Ca2+ efflux was stimulated) whereas Ruthenium red, an inhibitor of uniporter‐driven Ca2+ efflux, significantly slowed down the effect of uncouplers. The respiratory inhibition was associated with NAD(P)H oxidation and was partially reversed by exogenous substrates (glutamine or glucose). In the permeabilized cells, endogenous and glutamine‐supported respiration was inhibited by EGTA, while succinate‐supported respiration was Ca2+ independent. It is suggested that mitochondrial Ca2+ is necessary for NADH‐dependent respiration of tumor cells, and uncouplers inhibit it by activation of mitochondrial Ca2+ efflux.
Interaction of GTP‐binding protein Gq with photoactivated rhodopsin in the photoreceptor membranes of crayfish
Authors: Terakita Akihisa , Hariyama Takahiko , Tsukahara Yasuo , Katsukura Yuki , Tashiro Hideo
Published: July 2002

Interaction of G‐protein with photoactivated rhodopsin (Rh∗) in crayfish photoreceptor membranes was investigated by immunoprecipitation using an antibody against rhodopsin. Two kinds of protein were co‐precipitated with rhodopsin. One is an α subunit of class‐q G‐protein (42 kDa, CGqα) which showed light‐induced, dose‐dependent binding to rhodopsin, and the other is an actin‐like protein (44 kDa) with light‐independent binding. Most of the CGqα was available for binding to Rh∗ but was dissociated from Rh∗ in the presence of GTPγS. These findings demonstrate that, in the crayfish photoreceptor, a Gq class of G‐protein is activated by Rh∗.
Excitatory amino acid‐induced slow biphasic responses of free intracellular calcium in human neuroblastoma cells
Authors: Naarala Jonne , Nykvist Petri , Tuomala Marjo , Savolainen Kai
Published: July 2002

Effects of an excitatory amino acid, glutamate, and of ionotropic and metabotropic glutamate receptor agonists on the levels of free intracellular calcium, and their specific receptor binding in human SH‐SY5Y neuroblastoma cells were studied. The calcium response was always biphasic, except for AMPA, suggesting both stimulatory and inhibitory effects on free intracellular calcium upon glutamate receptor stimulation, both with ionotropic and metabotropic glutamate receptor agonists. Specific binding of glutamate and other glutamate receptor agonists, together with the biphasic calcium response, suggests that human SH‐SY5Y neuroblastoma cells express both ionotropic and metabotropic glutamate receptors. These findings shed new light on the use of human SH‐SY5Y neuroblastoma cells as a human neuronal tumor cell model.
A novel ryanodine sensitive calcium release mechanism in cultured human myometrial smooth‐muscle cells
Authors: Lynn Stephen , Morgan Joanna M. , Gillespie James I. , Greenwell John R.
Published: July 2002

In cultured human myometrial cells application of caffeine (1–30 mM) did not result in an elevation of intracellular Ca2+ ([Ca2+]i). Caffeine was found to reversibly inhibit both spontaneous and agonist‐induced repetitive rises in [Ca2+]i possibly as a consequence of its ability to interfere with the binding of inositol trisphosphate (IP3) to the receptor on the sarcoplasmic reticulum. Brief applications of ryanodine (1–10μM) were observed to elevate [Ca2+]i and repeated exposures to ryanodine could elicit Ca2+ transients of similar magnitude. Ryanodine was also observed to mobilise Ca2+ in cells bathed in nominally Ca2+‐free solution. These observations suggest the presence of a novel type of ryanodine‐sensitive Ca2+‐induced Ca2+ release (R‐CICR) system in human myometrial cells.
Calcitonin gene expression in normal human liver
Authors: Bracq S. , Machairas M. , Clement B. , Pidoux E. , Andreoletti M. , Moukhtar M.S. , Jullienne A.
Published: July 2002

Immunoreactive calcitonin (CT) is present in liver. This could represent hormone synthesized by liver cells, degraded or bound to specific receptors reported in this organ. We report here that the calcitonin gene is expressed in liver. We proved this by demonstrating, by PCR amplification using specific primers, the presence of calcitonin messenger in human liver and in primary cultures of human hepatocytes and detected by radioimmunoassay CT in hepatic tissues and cells. The synthesis of hormone by liver that also possesses specific receptors for CT favors the presence of an autocrine or paracrine system involving calcitonin in this organ.
pH dependence of light‐induced proton release by bacteriorhodopsin
Authors: Kono Masahiro , Misra Saurav , Ebrey Thomas G.
Published: July 2002

We have measured the current generated by light‐activated proton release from bacteriorhodopsin into solution as a function of both pH and ionic strength. We find that proton release into solution decreases with increasing pH with an intrinsic pK
a of 8.2 ± 0.2. This pH dependence indicates that the deprotonation of a certain group inhibits or abolishes proton release. Under physiological conditions, this group either releases a proton directly into solution or interacts with the site of proton release. The most immediate candidates for this protonatable species are tyrosine‐57, tyrosine‐185, arginine‐82, and water; acting individually or cooperatively. The salt dependence of the apparent pK
a of this group also allows us to calculate the surface charge density of about −5 charges per bacteriorhodopsin, compatible with previous estimates.
Essential role of the Arg112 residue of cytochrome P450cam for electron transfer from reduced putidaredoxin
Authors: Koga Hideo , Sagara Yasuhiro , Yaoi Tsuyoshi , Tsujimura Mitsushi , Nakamura Kazuhide , Sekimizu Kazuhisa , Makino Ryu , Shimada Hideo , Ishimura Yuzuru , Yura Kei , Go Mitiko , Ikeguchi Masamichi , Horiuchi Tadao
Published: July 2002

Cytochrome P450cam (CYP101) of Pseudomonas
putida PpGl in which Arg112 is substituted by Cys was isolated by in vitro random mutagenesis of the camC gene DNA coding for P450cam. The absorption spectra of the purified mutant enzyme were similar to those of the wild type enzyme, but its substrate‐dependent NADH oxidation activity in the presence of putidaredoxin (Pd) and putidaredoxin reductase (PdR) was extremely low. The rate constant of electron transfer from reduced Pd to the heme of the mutant P450cam, measured on an anaerobic stopped flow apparatus, was 1/400 of that of the wild type enzyme and the dissociation constant of the mutant P450cam for oxidized Pd was several fold higher than that of the wild type enzyme. A considerable decrease in mid‐point potential of the mutant enzyme was also noted. We conclude that Arg112, which is located on the surface of the P450cam molecule and hydrogen‐bonded to one of the heme propionate chains, plays an essential role in the electron transfer from Pd.
Cloning and characterization of the rat 5‐HT5Breceptor
Authors: Wisden William , Parker Eric M. , Mahle Cathy D. , Grisel Darcy A. , Nowak Henry P. , Yocca Frank D. , Felder Christian C. , Seeburg Peter H. , Voigt Mark M.
Published: July 2002

A gene encoding a novel G protein‐coupled 5‐hydroxytryptamine (5‐HT) receptor, termed 5‐HT5B, was cloned. The ligand binding profile of this receptor is distinct from that of other cloned 5‐HT receptors. The 5‐HT5Breceptor couples to a G protein in COS1 cell membranes; however, activation of the 5‐HT5Breceptor does not appear to alter either cAMP accumulation or phosphoinositide turnover in a variety of fibroblast cell lines. In the rat brain, 5‐HT5Bgene expression occurs predominantly in the medial habenulae and hippocampal CA1 cells of the adult. Little expression is seen during embryonic development.
Derivatives of thapsigargin as probes of its binding site on endoplasmic reticulum Ca2+ ATPase
Authors: Christensen Søren Brøgger , Andersen Annette , Poulsen Jens-Christian J. , Treiman Marek
Published: July 2002

The naturally occurring sesquiterpene lactone thapsigargin is a potent and selective inhibitor of SERCA ATPases, a family of Ca2+‐pumping ATPases present in the endoplasmic reticulum of all mammalian cells. We have studied some of the molecular features of thapsigargin responsible for its inhibitory action towards these Ca2+ ATPases. A series of thapsigargin analogues were synthesised and their inhibitory potencies determined using the uptake of 45Ca2+ in bovine cerebellar microsomes as a sensitive marker of Ca2+ ATPase activity. An attenuation of the inhibitory potency relative to the parent compound was found ranging from slight to over 3 orders of magnitude. The inhibitory activity showed a very strong configuration dependence, a major contribution from the ester groups at C3 and C10, and an apparently minor contribution from the lactone ring substituents. The data are consistent with thapsigargin fitting to a sterically discriminating cleft involving the hydrophobic transmembrane region of the ATPase, and is compatible with available kinetic evidence of thapsigargin‐mediated inhibition.
Differential shedding of the two subunits of the interleukin‐6 receptor
Authors: Müllberg Jürgen , Dittrich Elke , Graeve Lutz , Gerhartz Claudia , Yasukawa Kiyoshi , Taga Tetsuya , Kishimoto Tadamitsu , Heinrich Peter C. , Rose-John Stefan
Published: July 2002

cDNAs coding for the two receptor subunits of the interleukin‐6 receptor have been stably expressed in Madine Darby canine kidney (MDCK) cells. The fate of the IL‐6 binding protein (IL‐6R) and of the signal transducing protein gp130 was studied independently. Both proteins were proteolytically cleaved from cells metabolically labeled with [35S]methionine/cysteine leading to the release of soluble receptor proteins of 55 kDa and 100 kDa, respectively. In contrast to the shedding of the IL‐6R gp 130 was inefficiently released from the cells and the process was not significantly stimulated by the phorbolester PMA. In addition we show that the soluble forms of the IL‐6R and gp 130 released by transfected cells can form a ternary complexe with interleukin‐6 indicating that such complexes also may occur in vivo. gp 130; Interleukin‐6; Interleukin‐6‐receptor; Protein kinase C; Shedding
Identification and molecular characterization of a high‐affinity cardiomyocyte transforming growth factor‐β2 receptor
Authors: Ross Julie , Janero David R. , Hreniuk David
Published: July 2002

Rat neonatal heart muscle cells (cardiomyocytes) were found to express a high‐affinity surface receptor for transforming growth factor‐β2 (TGF‐β2). Specific binding was rapid, saturable, ligand‐selective, and reversible. Equilibrium binding analyses revealed that the cardiomyocyte had one class of specific binding sites with a K
d ⩽ 26 pM TGF‐β2, a B
max of ~9 fmol/106
cells, and ~5,000 binding sites/cardiomyocyte. Binding was selective for TGF‐β2 in comparison to other TGF‐β isoforms and to unrelated growth factors. Affinity‐binding experiments revealed three types of cardiomyocyte TGF‐β2 binding proteins, the most prominent of which corresponded to the high‐molecular mass proteoglycan. These data raise the possibility that the anti‐ischemic cardioprotective effects ofTGF‐β may reflect receptor‐mediated signal transduction at the cardiomyocyte level.
Reaction of nitric oxide with hydrogen peroxide to produce potentially cytotoxic singlet oxygen as a model for nitric oxide‐mediated killing
Authors: Noronha-Dutra Alberto A. , Epperlein Monica M. , Woolf Neville
Published: July 2002

Nitric oxide, as well as being a major regulator of vascular reactivity, has been shown to be one of the mediators of cytotoxicity in macrophages. This cytotoxic effect seems to be due to the interaction between nitric oxide and oxygen‐related free radicals. This study shows that, in vitro, nitric oxide reacts with hydrogen peroxide to release large amounts of chemiluminescence with the characteristics of the highly cytotoxic species, singlet oxygen. This is supported by the observation that when nitric oxide was added to a Superoxide generating system, catalase inhibited the production of singlet oxygen while Superoxide dismutase enhanced it.
Enhancement of Ca2+ release channel activity by phosphorylation of the skeletal muscle ryanodine receptor
Authors: Herrmann-Frank Annegret , Varsányi Magdolna
Published: July 2002

The Ca2+ release channel of rabbit skeletal muscle sarcoplasmic reticulum (SR) can be phosphorylated by membrane associated protein kinase(s) utilizing endogenously synthesized or exogenously added ATP. The channel protein has been enriched in non‐phosphorylated and phosphorylated form from heavy SR following solubilization with CHAPS (3‐[(3‐cholamidopropyl)dimethylammonio‐1‐propane‐sulfonate) and ultracentrifugation on a linear sucrose/CHAPS gradient. Reconstitution of the isolated channels into planar bilayers shows that phosphorylation enhances the open probability by increasing the sensitivity towards micromolar Ca2+ and ATP. The phosphorylation induced enhancement of the channel activity can be reversed by purified protein phosphatase 2A.
Rapid pressure driven exocytosis‐endocytosis cycle in a single plant cell
Authors: Zorec Robert , Tester Mark
Published: July 2002

The whole‐cell patch‐clamp technique has been used to increase intracellular pressure via the pipette while monitoring changes in membrane capacitance (related to membrane area) in single aleurone protoplasts. Increased pressure increased membrane capacitance and diameter, upon the release of pressure both parameters returned to resting levels. Pressure also reversibly increased membrane conductance. Comparison between diameter and capacitance shows that the capacitance changes are due to changes in cell surface area. These results show that pressure can rapidly alter the balance between exo‐ and endocytosis. Pressure‐induced changes in conductance are suggested to be due to insertion/withdrawal of channels with the exo‐/endocytosed membrane.
Experimental evidence for 60 Hz magnetic fields operating through the signal transduction cascade
Authors: Liburdy R.P. , Callahan D.E. , Harland J. , Dunham E. , Sloma T.R. , Yaswen P.
Published: July 2002

We tested the hypothesis that early alterations in calcium influx induced by an imposed 60 Hz magnetic field are propagated down the signal transduction (ST) cascade to alter c‐MYC mRNA induction. To test this we measured both ST parameters in the same cells following 60 Hz magnetic field exposures in a specialized annular ring device (220 G (22 mT), 1.7 maximal E
induced, 37°C, 60 min). Ca2+ influx is a very early ST marker that precedes the specific induction of mRNA transcripts for the proto‐oncogene c‐MYC, an immediate early response gene. In three experiments influx of 45Ca2+ in the absence of mitogen was similar to that in cells treated with suboptimal levels of Con‐A (1 ). However, calcium influx was elevated 1.5‐fold when lymphocytes were exposed to Con‐A plus magnetic fields; this co‐stimulatory effect is consistent with previous reports from our laboratory [FEBS Lett. 301 (1992) 53‐59; FEBS Lett. 271 (1990) 157‐160; Ann. N.Y. Acad. Sci. 649 (1992) 74‐95]. The level of c‐MYC mRNA transcript copies in non‐activated cells and in suboptimally‐activated cells was also similar, which is consistent with the above calcium influx findings. Significantly, lymphocytes exposed to the combination of magnetic fields plus suboptimal Con‐A responded with an approximate 3.0‐fold increase in band intensity of c‐MYC mRNA transcripts. Importantly, transcripts for the housekeeping gene GAPDH were not influenced by mitogen or magnetic fields. We also observed that lymphocytes that failed to exhibit increased calcium influx in response to magnetic fields plus Con‐A, also failed to exhibit an increase in total copies of c‐MYC mRNA. Thus, calcium influx and c‐MYC mRNA expression, which are sequentially linked via the signal transduction cascade in contrast to GAPDH, were both increased by magnetic fields. These findings support the above ST hypothesis and provide experimental evidence for a general biological framework for understanding magnetic field interactions with the cell through signal transduction. In addition, these findings indicate that magnetic fields can act as a co‐stimulus at suboptimal levels of mitogen; pronounced physiological changes in lymphocytes such as calcium influx and c‐MYC mRNA induction were not triggered by a weak mitogenic signal unless accompanied by a magnetic field. Magnetic fields, thus, have the ability to potentiate or amplify cell signaling.
Functional expression of human P‐glycoprotein in Schizosaccharomyces pombe
Authors: Ueda Kazumitsu , Shimabuku Alfredo M. , Konishi Haruko , Fujii Yuko , Takebe So , Nishi Kazunori , Yoshida Minoru , Beppu Teruhiko , Komano Tohru
Published: July 2002

Human MDR1 cDNA was introduced into the human cultured cells KB‐3‐1 and Schizosaccharomyces pombe pmdI null mutant KN3. The drug sensitivity of KB‐G2 and KN3/pgp, expressing human P‐glycoprotein, was examined. KB‐G2 was resistant to the peptide antibiotics valinomycin and gramicidin D as well as having a typical multidrug resistance (MDR) phenotype. KN3/pgp was resistant to valinomycin and actinomycin D, but not to adriamycin. The ATP‐hydrolysis‐deficient mutant did not confer KN3 resistance to these antibiotics. Human P‐glycoprotein expressed in S. pombe seemed to lack N‐glycosylation. The N‐glycosylation‐deficient mutant, however, conferred a typical MDR phenotype on KB‐3‐1. These results suggest that human P‐glycoprotein functions as an efflux pump of valinomycin and actinomycin D in the membrane of S. pombe.
Glut 4 content in the plasma membrane of rat skeletal muscle: comparative studies of the subcellular fractionation method and the exofacial photolabelling technique using ATB‐BMPA
Authors: Lund S. , Holman G.D. , Schmitz O. , Pedersen O.
Published: July 2002

Employing subcellular membrane fractionation methods it has been shown that insulin induces a 2‐fold increase in the Glut 4 protein content in the plasma membrane of skeletal muscle from rats. Data based upon this technique are, however, impeded by poor plasma membrane recovery and cross‐contamination with intracellular membrane vesicles. The present study was undertaken to compare the subcellular fractionation technique with the technique using [3H]ATB‐BMPA exofacial photolabelling and immunoprecipitation of Glut 4 on soleus muscles from 3‐week‐old Wistar rats. Maximal insulin stimulation resulted in a 6‐fold increase in 3‐O‐methylglucose uptake, and studies based on the subcellular fractionation method showed a 2‐fold increase in Glut 4 content in the plasma membrane, whereas the exofacial photolabelling demonstrated a 6‐ to 7‐fold rise in cell surface associated Glut 4 protein. Glucose transport activity was positively correlated with cell surface Glut 4 content as estimated by exofacial labelling. In conclusion: (1) the increase in glucose uptake in muscle after insulin exposure is caused by an augmented concentration of Glut 4 protein on the cell surface membrane, (2) at maximal insulin stimulation (20 mU/ml) approximately 40% of the muscle cell content of Glut 4 is at the cell surface, and (3) the exofacial labelling technique is more sensitive than the subcellular fractionation technique in measuring the amount of glucose transporters on muscle cell surface.
Biochemical events in naturally occurring forms of cell death
Authors: Fesus Laszlo
Published: July 2002

Several molecular elements of programmed cell death and apoptosis have recently been revealed. The function of gene products which deliver the lethal ‘hit’ is still not known. Well‐characterized and newly discovered cell surface structures (e.g. antigen receptors, FAS/APO‐1), as well as transcriptional factors (steroid receptor, c‐myc, P53, retinoblastoma protein and others), have been implicated in the initiation of the death pathway. Negative regulators of the process (ced‐9 gene product in programmed death of cells in Chaenorhabditis elegans and bcl‐2 protein in apoptosis) have been described. Biochemical mechanisms responsible for the silent nature of natural deaths of cells include their rapid engulfment (mainly through integrin receptors), transglutaminase‐catalyzed cross‐linking of cellular proteins, and fragmentation of DNA. Several lines of evidence suggest that distinct molecular mechanisms may operate in various forms of natural cell death.
Drastic reduction of the zinc‐ and magnesium‐stimulated protein tyrosine kinase activities in Alzheimer's disease hippocampus
Authors: Vener Alexander V. , Aksenova Marina V. , Burbaeva Gulnur Sh.
Published: July 2002

Tyrosine phosphorylation of proteins from postmortem hippocampi of five Alzheimer's disease and five control cases have been compared. It was found that addition of Zn2+ or Mg2+ to membrane fractions of control hippocampi caused the phosphorylation of 32‐, 40‐, 55‐, 60‐, 80‐ and 100‐kDa proteins or 43‐, 55‐, 60‐ and 90‐KdA proteinsrespectively. The phosphorylation of all these proteins is shown to be drastically reduced in Alzheimer's disease hippocampi. Vanadate an inhibitor of protein tyrosine phosphataseshad no influence on the level of protein phosphorylation. Western blot analysis did not reveal any differences in the anti‐phosphotyrosine immunoreactive membrane proteins from Alzheimer's disease and control hippocampi. Tyrosine kinase activity of immunoprecipitated p60c‐src from Alzheimer's disease and control hippocampi were the same. In conclusion, the Zn2+‐ and Mg2+ ‐stimulated tyrosine kinase activities distinct from activity of p60c‐src are decreased in Alzheimer's disease hippocampus.
Spleen‐derived growth factor, SDGF‐3, is identified as keratinocyte growth factor (KGF)
Authors: Suzuki Masashi , Itoh Takehiro , Osada Hiroyuki , Rubin Jeffrey S. , Aaronson Stuart A. , Suzuki Tohru , Koga Nobumitsu , Saito Takao , Mitsui Youji
Published: July 2002

A heparin‐binding mitogen rat for rat hepatocytes was partially purified from bovine spleen by a combination of heparin‐affinity, cation‐exchange and gel‐filtration chromatography. Besides stimulating rat hepatocytes, this factor, which was designated spleen‐derived growth factor‐3 (SDGF‐3), exhibited mitogenic activity for mouse epidermal keratinocytes but not mouse fibroblasts. Its apparent epithelial specificity and heparin‐binding properties corresponded to those of keratinocyte growth factor (KGF). These findings, together with the fact that the mitogenic activity of SDGF‐3 was abolished by a neutralizing monoclonal antibody specific for KGF, identify this bovine spleen‐derived hepatocyte mitogen as KGF.
The pde2 gene of Saccharomyces cerevisiae is allelic to rcal and encodes a phosphodiesterase which protects the cell from extracellullar cAMP
Authors: Wilson Robert B. , Renault Georges , Jacquet Michel , Tatchell Kelly
Published: July 2002

The high affinity cAMP phosphodiesterase, encoded by PDE2, is an important component of the cAMP‐dependent protein kinase signaling system in Saccharomyces cerevisiae. An unexpected phenotype of pde2 mutants is sensitivity to external cAMP. This trait has been found independently for rca1 mutants and has been used to monitor the effects of cAMP on several biological processes. We demonstrate here that RCA1 is identical to PDE2. Further analysis of the phenotype of pde2 deletions reveal that exogenously added cAMP results in an increase in the internal level of cAMP. This increase slows down the rate of cell division by increasing the length of the G1 phase of the cell cycle and leads to increased cell volume. Also, cells with a disrupted PDE2 gene previously arrested by nutrient starvation rapidly lose thermotolerance when incubated with exogenous cAMP. From these observations we propose that a role of the PDE2‐encoded phosphodiesterase may be to help insulate the internal cAMP pools from the external environment. This protective role might also be important in other eukaryotic organisms where cAMP is a key second messenger.
Interactions of the HIV‐1 reverse transcriptase ‘AZT‐resistant’ mutant with substrates and AZT‐TP
Authors: Pokholok Dmitry K. , Gudima Severin O. , Yesipov Dmitry S. , Dobrynin Vladimir N. , Rechinsky Vladimir O. , Kochetkov Sergey N.
Published: July 2002

To investigate the biochemical basis of the HIV‐1 resistance to AZT we obtained the RT mutant containing four amino acid substitutions by an oligonucleotide‐directed mutagenesis technique. Enzymatic properties of the wild type and mutant RTs were compared. ‘AZT‐resistant’ mutations in RT were shown to be associated with the reduced capability of AZT‐TP to block the DNA‐ but not RNA‐directed DNA synthesis.
Interaction of urokinase‐type plasminogenactivator (u‐PA) with its cellular receptor (u‐PAR) induces phosphorylation on tyrosine of a 38 kDa protein
Authors: Dumler I. , Petri T. , Schleuning W.-D.
Published: July 2002

We demonstrate by immunoprecipitation that u‐PAR is associated with a 38 kDa protein that is phosphorylated on tyrosine after u‐PA treatment of cells. As tyrosine phosphorylation is the hallmark of many signal transduction pathways that promote growth and differentiation, these data suggest that u‐PA, besides its role as a regulatory protease, might act as a para‐ or autocrine hormone.
The ovalbumin family of serpin proteins
Authors: Remold-O'Donnell Eileen
Published: July 2002

A protein family, the ‘Ov‐serpins’ has been identified by comparing amino acid sequence, protein characteristics and gene organization. The Ov‐serpins would not be recognized as a family based on sequence identity alone. This example suggests that combinations of characteristics may need to be examined to identify family groupings within the serpin superfamily.
Membrane topology of the 22 kDa integral peroxisomal membrane protein
Authors: Kaldi K. , Diestelkötter P. , Stenbeck G. , Auerbach S. , Jäkle U. , Mägert H.-J. , Wieland F.T. , Just W.W.
Published: July 2002

In order to study the membrane topology and the possible function of the rat liver 22 kDa integral peroxisomal membrane protein (PMP 22) at a molecular level, we have cloned PMP 22 from a λgt11 expression library and sequenced its cDNA. Hydropathy analysis of the deduced primary structure indicates 4 putative transmembrane segments. The accessibility to exogenous aminopeptidase of PMP 22 in intact peroxisomes suggests that the N‐terminus faces the cytosol. A model of the topology of PMP 22 in the peroxisomal membrane is discussed. Homology studies revealed a striking similarity with the Mpv 17 gene product. Lack of this membrane protein causes nephrotic syndrome in mice.
Lipid transfer proteins (nsLTPs) from barley and maize leaves are potent inhibitors of bacterial and fungal plant pathogens
Authors: Molina Antonio , Segura Ana , García-Olmedo Francisco
Published: July 2002

Four homogeneous proteins (Cw18, Cw20, Cw21, Cw22) were isolated from etiolated barley leaves by extraction of the insoluble pellet from a Tris‐HCl (pH 7.5) homogenate with 1.5 M LiCl and fractionation by reverse‐phase high‐performance liquid chromatography. All 4 proteins inhibited growth of the pathogen Clavibacter michiganensis subsp. sepedonicus (EC50S = 1−3 × 10−7 M) and had closely related N‐terminal amino acid sequences. The complete amino acid sequences of proteins Cw18 and Cw21 were determined and found to be homologous to previously described, non‐specific lipid transfer proteins from plants (32–62% identical positions). The proteins also inhibited growth of the bacterial pathogen Pseudomonas solanacearum (EC50s = 3–6 × 10−7 M) and the fungus Furium solani (EC50s = 3–20 × 10−6 M). A homologous protein from maize leaves (Cw41) was purified in a similar manner and also found to have inhibitory properties, A synergistic effect against the fungus was observed when protein Cw21, was combined with thionins. A defense role for non‐specific lipid transfer proteins from plants is proposed.
Structure of the proteinase inhibitor eglin c with hydrolysed reactive centre at 2.0 Å resolution
Authors: Betzel Christian , Dauter Zbigniew , Genov Nicolay , Lamzin Victor , Navaza Jorge , Schnebli Hans Peter , Visan Marcia , Wilson Keith S.
Published: July 2002

The inhibition of serine proteinases by both synthetic and natural inhibitors has been widely studied. Eglin c is a small thermostable protein isolated from the leech, Hirudo medicinalis. Eglin c is a potent serine proteinase inhibitor. The three‐dimensional structure of native eglin and of its complexes with a number of proteinases are known. We here describe the crystal structure of hydrolysed eglin not bound to a proteinase. The body of the eglin has a conformation remarkably similar to that in the known complexes with proteinases. However, the peptide chain has been cut at the ‘scissile’ bond between residues 45 and 46, presumed to result from the presence of subtilisin DY in the crystallisation sample. The residues usually making up the inhibiting loop of eglin take up a quite different conformation in the nicked inhibitor leading to stabilising contacts between neighbouring molecules in the crystal. The structure was solved by molecular replacement techniques and refined to a final R‐factor of 14.5%.
The rat insulin‐degrading enzyme
Authors: Baumeister Hans , Müller Dieter , Rehbein Monika , Richter Dietmar
Published: July 2002

The primary structure of the rat insulin‐degrading enzyme (IDE) was determined by cDNA analysis. Rat IDE, as well as the previously characterized homologs from human and Drosophila, contain the carboxyl‐terminal consensus sequence A/S‐K‐L for peroxisome targeting. A stretch of 43 bp surrounding an alternatively used polyadenylation site is highly conserved between rat and human, suggesting that it may contain important regulatory information. Northern blot analysis revealed two IDE transcripts of 3.7 and 5.5 kb in various tissues. Testis was found to be exceptional in having three different RNAs (3.7, 4.1 and 6.1 kb) at a relatively high abundance. The expression of the IDE gene in testis is correlated with sexual maturation.
Chemotactic and protease‐inhibiting activities of antibiotic peptide precursors
Authors: Verbanac Donatella , Zanetti Margherita , Romeo Domenico
Published: July 2002

We have recently shown that two antimicrobial peptides (Bac5 and Bac7) and/or their immature forms (proBac5 and proBac7) can be released extracellularly from activated neutrophils. In the present study we have investigated the biological activities of the immature forms, which do not exhibit antimicrobial effects. We show that proBac7 is a monocyte‐selective chemoattractant, potentially contributing to the recruitment of these cells to infection sites, whereas proBac5 efficiently inhibits the in vitro activity of cathepsin L, a cysteine proteinase thought to contribute to tissue injury in inflammation.
Effects of tyrosine kinase inhibitors on protein kinase‐independent systems
Authors: Young Stephen W. , Poole Robert C. , Hudson Alan T. , Halestrap Andrew P. , Denton Richard M. , Tavaré Jeremy M.
Published: July 2002

Tyrosine kinase inhibitors have been widely used to probe the role of tyrosine phosphorylation in cellular signalling. These inhibitors exhibit an apparent specificity for tyrosine kinases over the serine/threonine kinases but little is known about their effects on other enzymes or biological systems. We demonstrate that genistein, erbstatin and α‐cyanoeinnamamides (tyrphostins) have inhibitory effects on fatty acid synthesis, lactate transport, mitochondrial oxidative phosphorylation and aldehyde dehydrogenase. We propose, therefore, that results obtained using tyrosine kinase inhibitors should be interpreted with caution, particularly if used at concentrations sufficient to inhibit these non‐protein kinase‐dependent events.
Around the growth phase transition S. cerevisiae's make‐up favours sustained oscillations of intracellular metabolites
Authors: Richard Peter , Teusink Bas , Westerhoff Hans V. , van Dam Karel
Published: July 2002

Under a limited set of hitherto incompletely defined conditions, inhibition of respiration has been shown to cause transient oscillations in NAD(P)H fluorescence of yeast cells. In this paper, we apply a new method [1992, Anal. Biochem. 204, 118‐132] for extraction of intracellular metabolites. This method involves spraying the cells into −40°C methanol; the neutral pH allows extraction of nearly all intracellular metabolites, including NADH. Close to the shift from glucose to ethanol as a growth substrate, the cells acquire a make‐up amenable to sustained oscillations in intracellular concentrations of NADH and glycolytic intermediates such as glucose‐6‐phosphate. NADH was found to oscillate between 200 μM and 400 μM intracellular concentration. The cellular make‐up determining the tendency to oscillate is ‘remembered’ by the cells after three hours of starvation.
Activation of two angiotensin‐generating systems in the balloon‐injured artery
Authors: Shiota Naotaka , Okunishi Hideki , Fukamizu Akiyoshi , Sakonjo Hiroshi , Kikumori Mikito , Nishimura Takayoshi , Nakagawa Toshimasa , Murakami Kazuo , Miyazaki Mizuo
Published: July 2002

Participation of angiotensin II in the myointimal proliferation following a vascular injury was postulated. This study assessed the potential involvement of the local angiotensin II‐forming enzymes in injured arteries of dogs. The potential angiotensin II‐forming enzymes are angiotensin‐converting enzyme (ACE) and chymostatin‐sensitive angiotensin II‐generating enzyme (CAGE) which is highly homologous to or could be identical to the mast cell chymase. Both ACE and CAGE catalyze the conversion of angiotensin I to angiotensin II. We found that the enzymatic activities of ACE and CAGE, and the mRNA levels of ACE and chymase were increased in the injury‐induced hypertrophied vessels. The results suggest that ACE and CAGE participate in the hypertrophy through the production of angiotensin II which is a growth promoter for vascular smooth muscle cells.
Neurotrophin induced cAMP and IP3 responses in PC12 cells
Authors: Knipper Marlies , Beck Anja , Rylett Jane , Breer Heinz
Published: July 2002

NGF and BDNF elevate cAMP and IP3 levels in membranes of PC12 cells within a subsecond time period. The cAM P formation induced by NGF and BDNF pretreatment for 2 s was reduced by GDP‐β‐S and PTX, but not the trkNGFR inhibitor K 252a. NGF, but not BDNF, induced IP3 formation. IP3 formation was reduced by K 252a, but not by GDP‐β‐S and PTX. Using p75NGFR expressing, but trkNGFR‐deficient PCNA cell membranes, NGF and BDNF induced cAMP formation, but not IP3 formation. We suggest that NGF and BDNF induced cAMP formation is mediated via a p75NGFR/G‐protein mediated mechanism, and IP3 formation via a K 252a sensitive pathway.
Glutamate receptors of Drosophila melanogaster
Authors: Ultsch Andreas , Schuster Christoph M. , Laube Bodo , Betz Heinrich , Schmitt Bertram
Published: July 2002

The NMDA subtype of ionotropic glutamate receptors has been implicated in the activity‐dependent modification of synaptic efficacy in the mammalian brain. Here we describe a cDNA isolated from Drosophila melanogaster which encodes a putative invertebrate NMDA receptor protein (DNMDAR‐I). The deduced amino acid sequence of DNMDAR‐I displays 46% amino acid identity to the rat NMDAR1 polypeptide and shows significant homology (16–23%) to other vertebrate and invertebrate glutamate receptor proteins. The DNMDAR‐I gene maps to position 83AB of chromosome 3R and is highly expressed in the head of adult flies. Our data indicate that the NMDA subtype of glutamate receptors evolved early during phylogeny and suggest the existence of activity‐dependent synaptic plasticity in the insect brain.
Engineering type 1 copper sites in proteins
Authors: Canters G.W. , Gilardi G.
Published: July 2002

The use of site‐directed mutagenesis methods has revolutionalized the study of the so‐called type 1 and type 2 copper sites in proteins. In particular our understanding of the relation between the structure, and the mechanistic and spectroscopic features of these sites is benefitting from the application of these techniques. Recent progress in the field is reviewed with emphasis on the study of type 1 sites. Topics covered comprise the characteristics of the natural type 1 and type 2 sites, the genetics of blue copper proteins, the modification of Cu sites, the spectroscopy of natural and engineered type 1 and type 2 sites, the effect of mutations on midpoint potentials and the mechanism of electron transfer as carried out by the blue copper proteins.
The mitochondrial genome on its way to the nucleus: different stages of gene transfer in higher plants
Authors: Brennicke Axel , Grohmann Lutz , Hiesel Rudolf , Knoop Volker , Schuster Wolfgang
Published: July 2002

The vast majority of mitochondrial proteins are in all eukaryotes encoded in the nuclear genomes by genes which have been transferred from the original endosymbiont. DNA as well as RNA was and is exchanged between organelies. A functionally successful information transfer, however, requires complex structural and regulatory alterations of the concerned gene. The recently identified variations of the information content in mitochondrial genomes of different plant species represent different stages of the transfer process. These evolutionary intermediates allow a definition of requirements and chances of successful gene transfers.
New model for 70 kDa heat‐shock proteins' potential mechanisms of function
Authors: Tsang Tom C.
Published: July 2002

The 70 kDa family of heat‐shock proteins (hsp 70s and hsc 70s) can facilitate protein transport to several organelles. This process is thought to involve electrostatic interactions between hsp 70s and the cellular protein targeting sequences. Analysis of the highly conserved structural and functional properties of the hsp 70 family indicated that they may cross‐link cellular proteins to the actin microfilament network. Direct experimental support for this hypothesis was provided by the finding that hsp 70 is constitutively bound to actin through hydrophobic interactions. The cross‐linker model may provide an explanation for the mechanism by which the cytoskeletal matrix could mediate various cellular processes.
Derivation of the formyl‐group oxygen of chlorophyll b from molecular oxygen in greening leaves of a higher plant (Zea mays)
Authors: Porra R.J. , Schäfer W. , Cmiel E. , Katheder Ingrid , Scheer H.
Published: July 2002

Using mass spectroscopy, we demonstrate as much as 93% enrichment of the 7‐formyl group oxygen of chlorophyll b when dark‐grown, etiolated maize leaves are greened under white light in the presence of 18O2. This suggests that a mono‐oxygenase is involved in the oxidation of its methyl group precursor. The concomitant enrichment of about 75% of the 131‐oxygen confirms the well‐documented finding that this oxo group, in both chlorophyll a and b, also arises from O2. High 18O enrichment into the 7‐formyl oxygen relative to the substrate 18O2 was achieved by optimization of the greening conditions in combination with a reductive extraction procedure. It indicates not only a single pathway for Chl b formyl group formation, but also unequivocally demonstrates that molecular oxygen is the sole precursor of the 7‐formyl oxygen.