bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of a protein that appears in the nervous system of the moth Manduca sexta coincident with neuronal death
Authors: Montemayor Michelle E. , Fahrbach Susan E. , Giometti Carol S. , Roy Edward J.
Published: July 2002

Two‐dimensional gel electrophoresis was used to locate potential neuronal death‐related proteins in the moth Manduca sexta. Protein patterns of ganglia of pharate adult moths (taken prior to adult ecdysis) compared with protein patterns of one‐day‐old adults revealed reproducible changes in protein patterns. An acidic protein of approximately 40000 Da was present in all samples from adult moths undergoing neuronal death and essentially absent from pharate adult samples.
Coupling of early response gene expression to distinct regulatory pathways during α‐interferon and phorbol ester‐induced plasmacytoid differentiation of B chronic lymphocytic leukaemia cells
Authors: Murphy John J. , Norton John D.
Published: July 2002

Phorbol esters (phorbol 12‐myristate 13‐acetate; PMA) and α‐interferon (α‐IFN) act through divergent signal transduction pathways to induce B chronic lymphocytic leukaemia cells (B‐CLL) to undergo plasmacytoid differentiation in vitro. By using a panel of PMA‐inducible early response gene probes we show that these two different effectors are coupled to second messenger pathways that do not converge on a common gene regulatory programme in differentiation of B‐CLL cells. Moreover, using the calcium ionophore, A23187, four distinct regulatory classes of early response gene could be defined implying that multiple regulatory pathways may mediate the process of terminal differentiation in B lymphocytes.
The primary structure of a triple‐helical domain of collagen type VIII from bovine Descemet's membrane
Authors: Mann Karlheinz , Jander Renate , Korsching Eberhard , Kühn Klaus , Rauterberg Jürgen
Published: July 2002

We have isolated and sequenced a fragment of 469 amino acid residues from bovine type VIII collagen. The sequence was composed of a series of Gly‐X‐Y repeats which was interrupted 8 times by short imperfections. The number and relative location of these interruptions were similar to those of chicken α1(X) and rabbit αl(VIII) chain triple‐helical domains. Comparison to published N‐terminal sequences to two triple‐helical fragments of bovine type VIII collagen and to the cDNA derived sequence of the rabbit αl(VIII) chain showed that this fragment was the triple‐helical domain of a second type VIII collagen chain which we designate α2(VIII).
Co‐expression of slow‐twitch/cardiac muscle Ca2+‐ATPase (SERCA2) and phospholamban
Authors: Fujii Junichi , Maruyama Kei , Tada Michihiko , MacLennan David H.
Published: July 2002

Full length cDNAs encoding both slow‐twitch/cardiac (SERCA2) and fast‐twitch skeletal muscle (SERCA1) Ca2+‐ATPases were expressed by transient transfection of COS‐1 cells. Studies of the Ca2+‐dependency of Ca2+‐transport in microsomes isolated from these cells showed that both isoforms had an affinity for Ca2+ of about 0.2 μM. The Ca2+‐affinity of SERCA2 was lowered when phospholamban was co‐expressed with it, demonstrating that the two proteins interact in this expression system. These studies support the view that phospholamban inhibition accounts for the low Ca2+‐amnity and low activity of SERCA2 in cardiac muscle sarcoplasmic reticulum.
Membrane elasticity of mouse dorsal root ganglion neurons decreases with aging
Authors: Horie Hidenori , Ikuta Shinichiro , Takenaka Toshifumi
Published: July 2002

Elastic area compressibility modulus (EACM) method was applied to measure the membrane elasticity of cultured dorsal root ganglion neurons from fetal and 3‐month‐old mice. The values of the EACM were 21.3 in 3‐month‐old neurons and 2.8 in fetal neurons. These results indicate that neural cell membrane elasticity decreases with aging.
Inactivation of human plasma α1‐proteinase inhibitor by human PMN leucocyte collagenase
Authors: Knäuper Vera , Reinke Heinz , Tschesche Harald
Published: July 2002

Highly purified human polymorphonuelear leucocyte collagenase cleaved human α‐1‐proteinase inhibitor (α1‐PI) at the carboxyl site of Phe352 (P7). The inhibitor was thereby rapidly inactivated and generated a primary degradation product as shown by reverse‐phase HPLC and N‐terminal sequencing. Prolonged incubation of the modified inhibitor with polymorphonuclear leucocyte collagenase led to the generation of a secondary degradation product with additional cleavage at the carboxyl site of Pro357 (P2).
Three phosphorylation sites in elongation factor 2
Authors: Ovchinnikov Lev P. , Motuz Lyudmila P. , Natapov Pavel G. , Averbuch Lidiya J. , Wettenhall Richard E.H. , Szyszka Ryszard , Kramer Gisela , Hardesty Boyd
Published: July 2002

Elongation factor 2 (EF‐2) of rabbit reticulocytes was phosphorylated in vitro by incubation with partially purified EF‐2 kinase and (γ32P)ATP. After exhaustive tryptic hydrolysis 4 phosphopeptides were revealed by two‐dimensional peptide mapping. The phosphopeptides were isolated by high performance liquid chromatography and sequenced. A comparison of the primary structure of the phosphopeptides with that of EF‐2 showed that all 4 phosphopeptides originated from one region of EF‐2 located near the N‐terminus that contains 3 threonine residues: Thr‐53, Thr‐56, Thr‐58. A direct estimation of localization of radioactive phosphate in the phosphopeptides demonstrated that all the enumerated threonine residues in EF‐2 can be phosphorylated in vitro.
Uptake of endocytic markers into mitotic yeast cells
Authors: Nevalainen Leena T. , Makarow Marja
Published: July 2002

[3H]α‐Factor and Lucifer yellow were used to measure receptor mediated and fluid‐phase endocytosis in the yeast Saccharomyces cerevisiae, arrested in mitosis by depolymerization of the microtubules or due to a mutation preventing nuclear division (cdc16. Both processes continued at roughly the same level as during interphase. This shows that in yeast endocytosis is not interrupted during mitosis like in mammalian cells.
The binding site of the strongly bound Eu3+ in Eu3+‐regenerated bacteriorhodopsin
Authors: Sweetman L.L. , El-Sayed M.A.
Published: July 2002

A Scatchard plot for the strongly bound Eu3+ to deionized bacteriorhodopsin (bR) was made using a method based on measuring the concentration of unbound Eu3+ from its fluorescence intensity. The results suggest that the first mole of Eu3+ added to a mole of bR is strongly bound by displacing 2–3 protons. In order to reconcile this result with the previous time‐resolved fluorescence studies on Eu3+‐regenerated bR, which showed the pressence of 3 sites of comparable binding constants, one is forced to conclude that the emission from the strongly bound Eu3+ is completely quenched, e.g. by energy transfer to the retinal. For this to take place, the Eu3+ must be within a few A from the retinal, i.e. within the retinal pocket (the active site). The possible importance of this conclusion to the deprotonation mechanism of the protonated Schiff base, the switch of the proton pump in bR, is discussed.
The Na+/H+ exchanger is phosphorylated in human platelets in response to activating agents
Authors: Livne Avinoam A. , Sardet Claude , Pouyssegur Jacques
Published: July 2002

α‐Thrombin, phorbol esters (PMA) and 1,2‐diacylglycerol (DAG), three activators of the amiloride‐sensitive Na+/H+ exchange in human platelets, rapidly increase the intracellular pH and the level of phosphorylation of the Na+/H+ exchanger protein (NHE1). This stimulatory effect is suppressed by staurosporine, a potent kinase inhibitor, and increased by okadaic acid, a potent inhibitor of phosphatase 1 and 2A. The modulations of NHE1 phosphorylation by these factors correlate well with their effects on platelet pH. Thus, we conclude that in platelets (i) Na+/H+ exchange is mediated by NHE1. and (ii) platelet activating agents stimulate NHE1 via the modulation of the kinase/phosphatase equilibrium.
Rat platelets are deficient in internal Ca2+ release and require influx of extracellular Ca2+ for activation
Authors: Heemskerk Johan W.M. , Feijge Marion A.H. , Rietman Ellen , Hornstra Gerard
Published: July 2002

Calcium fluxes were studied in fura‐2‐labeled rat platelets. Thrombin, ADP and ionomycin induced rapid mobilization of internally stored Ca2+, which resulted in only a moderate increase of cytosolic [Ca2+]i. Thrombin and ADP stimulated influx of extracellular Ca2+, which was monitored as uptake of 45Ca2+ and of Mn2+. With either agonist, the influx of Ca2+ magnified the initial increase of [Ca2+]i. Since responses of rat platelets were dependent on external [Ca2+], we conclude that Ca2+ influx complements the mobilization of internal stores to reach sufficiently high [Ca2+]i for full activation. A regulatory effect of protein kinase C modulators was observed on both agonist‐induced elevation of [Ca2+]i and receptor‐mediated Ca2+ entry.
Functional differences in human neutrophils isolated pre‐ and post‐prandially
Authors: Uhlinger David J. , Burnham David N. , Mullins Richard E. , Kalmar John R. , Cutler Christopher W. , Arnold Roland R. , Lambeth J.David , Merrill Alfred H.
Published: July 2002

Activated polymorphonuclear leukocytes have been associated with neoplasia, atherogenesis and reperfusion injury. Since some of these conditions are also correlated with dietary fat, we examined the functional characteristics of leukocytes isolated from subjects before and after consumption of a lipid‐rich meal. There was up to 2‐fold greater superoxide generation in response to agonists in leukocytes obtained post‐prandially; the maximum increase was observed about 4 h after eating and followed the peak (2–4 h) in serum triglycerides. Neutrophils isolated post‐prandially also exhibited impaired chemotaxis and defective bacterial killing, but normal phagocytosis. These findings provide a new variable that should be considered in studies of leukocytes.
Presence of an acyl carrier protein in NADH:ubiquinone oxidoreductase from bovine heart mitochondria
Authors: Runswick Michael J. , Fearnley Ian M. , Skehel J.Mark , Walker John E.
Published: July 2002

The amino‐acid sequence of a subunit of NADH:ubiquinone oxidoreductase from bovine heart mitochondria has been determined and is closely related to those of acyl carrier proteins that are involved in fatty acid biosynthesis in Escherichia coli and plants. Evidence for the presence of covalently attached pantetheine‐4′‐phosphate in the bovine protein has been obtained by determination of the molecular mass of the isolated subunit by electrospray mass spectrometry, before and after incubation of the protein at alkaline pH under reducing conditions. This decreased the molecular mass from 10751.6 to 10449.4, a difference of 302.2 mass units; the value calculated from the protein sequence with one covalently attached pantetheine‐4′‐phosphate is 10449.8. The acyl group which is removed by alkaline reduction, appears to be attached via a thioester linkage, By analogy with the bacterial protein it is likely that the attachment site of the pantetheine‐4‐phosphate is serine‐44, which is found in a highly conserved region of the sequence. At present the function of the acyl carrier protein in mitochondrial complex I is not understood.
Sphingosine and unsaturated fatty acids modulate the base exchange enzyme activities of rat brain membranes
Authors: Kanfer J.N. , McCartney D.
Published: July 2002

The base exchange enzymes catalyze the incorporation of L‐serine, ethanolamine and choline into their corresponding phospholipids. The L‐serine base enzyme activity was increased 120% by 0.1 mM sphingosine. There was a modest increase of the ethanolamine base exchange enzyme activity but the choline base exchange enzyme activity was unaffected. Na‐arachadonate, Na‐oleate and Na‐linolenate at 0.2 mM concentration increased the activity of the L‐serine and ethanolamine base exchange enzymes but inhibited the choline base exchange enzyme activity. A model is proposed suggesting that modulations of the L‐serine base exchange enzyme may participate in the regulation of the calcium phospholipid‐dependent protein kinase C.
Synchronous free Ca2+ changes in individual neutrophils stimulated by leukotriene B4
Authors: Davies E.V. , Campbell A.K. , Hallett M.B.
Published: July 2002

Calcium (Ca2+) signals were monitored in individual neutrophils using ratio imaging of fura‐2. In contrast to N‐formyl‐L‐leucyl‐L‐phenylaianine (f‐met‐leu‐phe), which produced grossly asynchronous Ca2+ signals with delays in response (up to 60 s), leukotriene B4 (LTB4) provoked synchronous and immediate elevations in cytosolic free Ca2+. Some individual neutrophils which responded immediately to LTB4, subsequently displayed delayed Ca2+ signals in response to f‐met‐leu‐phe. A sub‐population of neutrophils failed to respond to both LTB4 and f‐met‐leu‐phe. The asynchrony of the Ca2+ signalling to f‐met‐leu‐phe is not, therefore, an obligatory property of signal transduction in neutraphils.
Conformation of wheat gluten proteins Comparison between functional and solution states as determined by infrared spectroscopy
Authors: Pézolet Michel , Bonenfant Sacha , Dousseau Françoise , Popineau Yves
Published: July 2002

The conformation of wheat gluten proteins in their functional hydrated solid state (doughy state) has been studied for the first time using attenuated total reflection infrared spectroscopy. The amide I band of functional gluten proteins reveals that, in addition to β‐turns and α‐helices, these proteins contain a significant amount of intra‐ and intermolecular extended β‐sheet structures. It appears that the solubilization of gluten proteins results in a major decrease of the amount of β‐sheet structures accompanied by an increase of the content of the β‐turn and α‐helical conformations. In addition, the α‐helices appears to be more distorded in solution than in the functional state. Furthermore, spectra of ω‐ and γ‐gliadins, which are two types of prolamins of differing amino acid sequence and conformation, confirm the results obtained on the functional protein system. These results suggest that viscoelastic gluten proteins may interact through aligned β‐sheets corresponding to their repetitive domains.
Caf1R gene and its role in the regulation of capsule formation of Y. pestis
Authors: Karlyshev A.V. , Galyov E.E. , Abramov V.M. , Zav'yalov V.P.
Published: July 2002

A new transcription unit of the f1 gene cluster was found. The DNA sequencing revealed one long open reading frame. Deletion and frame shift mutation analyses have demonstrated the importance of a corresponding gene product for the F1 antigen biosynthesis. A homology of the deduced amino acid sequence with that of AraC family DNA‐binding regulators was shown. A potential regulatory DNA region is discussed.
Antithrombin Budapest 3 An antithrombin variant with reduced heparin affinity resulting from the substitution L99F
Authors: Olds R.J. , Lane D.A. , Boisclair M. , Sas G. , Bock S.C. , Thein S.L.
Published: July 2002

The molecular basis and functional properties of a variant antithrombin (AT) protein. AT Budapest 3, were studied. A single base substitution was identified in codon 99, TC→TC, altering the normal leucine to phenylalanine. The proband presented with a history of venous thrombotic disease and was found to be homozygous for the mutation. The variant protein demonstrated reduced heparin affinity and reduced antiproteinase activity in the presence of either unfractionated heparin or the AT‐binding heparin pentasaccharide, when compared to normal AT. A small change in the isoelectric point was also identified. The substituted amino acid residue of AT Budapest 3 is located near to the proposed AT heparin binding site, and it is suggested that reduced heparin affinity of the variant protein may result from substitution‐induced distortion of positive charge geometry in the binding site and/or changes in its position relative to the rest of the inhibitor molecule.
High level expression of transfected G protein αi3 subunit is required for plasma membrane targeting and adenylyl cyclase inhibition in NIH 3T3 fibroblasts
Authors: Hermouet Sylvie , de Mazancourt Philippe , Spiegel Allen M. , Farquhar Marilyn Gist , Wilson Bridget S.
Published: July 2002

The α subunits of pertussis toxin‐sensitive G proteins Gi1, Gi2 and Gi3 have been shown to inhibit adenylyl cyclase in transfected cells. However, Gi3 has recently been associated with protein transport and localized to the Golgi apparatus in a number of cell lines, rather than to the plasma membrane. We studied NIH 3T3 clones stably expressing different levels of a constitutively activated mutant of the α subunit of Gi3 (αi3‐Q204L). Transfected αi3 subunits were localized to the Golgi apparatus in all NIH 3T3 clones. In clones expressing αi3‐Q204L at high levels, αi3 subunits were also localized to the plasma membrane. Those clones which demonstrated expression of αi3 at the plasma membrane showed a 40% to 60% inhibition of forskolin‐induced cAMP accumulation. Transfected NIH 3T3 clones in which plasma membrane αi3 was undetectable, did not show inhibition of forskolin‐induced cAMP accumulation. These data suggest that, unless high expression is achieved in transfected cells, αi3 is targeted predominantly to the Golgi, not to the plasma membrane, and does not control adenylyl cyclase activity in NIH 3T3 fibroblasts.
Over‐expression of human phospholipase C‐γ2 enhances platelet‐derived growth factor‐induced mobilization of intracellular Ca2+ and the release of arachidonic acid and prostaglandins in NIH 3T3 fibroblasts
Authors: Totzke Frank , Hug Hubert , Fitzke Edith , Marmé Dieter , Dieter Peter
Published: July 2002

Over‐expression of human phospholipase C‐γ2 in murine NIH 3T3 fibroblasis has been shown to result in an increased platelet‐derived growth factor‐mediated formation of inositol phosphates. Here we show that phospholipase C‐γ2 over‐expression is associated with an increased platelet‐derived growth factor‐mediated release of arachidonic acid, prostaglandin E2, 6‐keto prostaglandin F10 and prostaglandin F20. The phorbol ester, calcium ionophore‐ and fluoride‐induced release of arachidonate and its metabolites is not affected by phospholipase C‐γ2 over‐expression. Over‐expression of phospholipase C‐γ2 is also associated with an enhancement of platelet‐derived growth factor‐induced change in intracellular Ca2+. These results demonstrate that stimulation of recombinant human phospholipase C‐γ2 induces a change in the intracellular Ca2+ concentration, a release of arachidonic acid and formation of prostaglandins in NIH 3T3 cells. In control cells platelet‐derived growth factor‐induced activation of arachidonic acid cascade is rate‐limited by the endogenous phospholipase C.
Saturation kinetics of coenzyme Q in NADH and succinate oxidation in beef heart mitochondria
Authors: Estornell E. , Fato R. , Castelluccio C. , Cavazzoni M. , Castelli G.Parenti , Lenaz G.
Published: July 2002

The saturation kinetics of NADH and succinate oxidation for Coenzyme Q (CoQ) has been re‐investigated in pentane‐extracted lyophilized beef heart mitochondria reconstituted with exogenous CoQ10. The apparent ‘K
m’ for CoQ10 was one order of magnitude lower in succinate cytochrome c reductase than in NADH cytochrome c reductase. The K
m value in NADH oxidation approaches the natural CoQ content of beef heart mitochondria, whereas that in succinate oxidation is close to the content of respiratory chain enzymes.
Do cytosolic factors prevent promiscuity at the membrane surface?
Authors: Lithgow Trevor , Høj Peter B. , Hoogenraad Nicholas J.
Published: July 2002

From the point of view of a preprotein, escaping from the cytosol into a specific organelle must seem an arduous, almost impossible task. How is it that preproteins resist the temptation to fold prematurely, to avoid the multiple membrane surfaces in the cell, and manage instead to enter only the translocation apparatus of a single organelle?
Down‐regulation of monocytic VLA‐4 leads to a decreased adhesion to VCAM‐1
Authors: Tiisala Sinikka , Hakkarainen Merja , Majuri Marja-Leena , Mattila Petri S. , Mattila Pirkko , Renkonen Risto
Published: July 2002

The α4β1 integrin VLA‐4 is expressed on practically all leukocytes, except on mature granulocytes. Here we show that in vitro treatment of monocytic cells with phorbol‐12‐myristate‐13‐acetate (PMA) leads to a selective decrease in the VLA‐4 α‐chain expression, both at the RNA and protein level. Meanwhile the expression of β1 and that of α5 another α‐chain associating with β1 was seen to increase. The decrease of α4 expression was restricted to monocytic cells, and was not observed on other VLA‐4‐positive cells tested (MOLT‐4 T cells and HOS sarcoma cells). The down‐regulation of the VLA‐4 α‐chain was followed by a decreased binding capacity of the cells to recombinant VCAM‐1. This data indicates that while previous findings show that the integrin‐dependent adhesion may rapidly be regulated by altering the avidity of the interacting molecules, their quantitative modulation also has a clear impact on adhesion.
Epidermal growth factor inhibits phosphoenolpyruvate carboxykinase gene expression in rat hepatocytes in primary culture
Authors: Fillat Cristina , Valera Alfons , Bosch Fatima
Published: July 2002

Epidermal growth factor (EGF) decreased the basal, and blocked the dibutyryl cyclic AMP (Bt2cAMP)‐induced, expression of P‐enolpyruvate carboxykinase (GTP) (PEPCK) and tyrosine aminotransferase (TAT) genes in both rat hepatocytes in primary culture and the FTO‐2B hepatoma cell line. Treatment of hepatocytes with EGF in combination with phorbol ester (TPA) resulted in an additive decrease of PEPCK mRNA levels. Overnight pretreatment of hepatocytes with TPA, which is known to downregulate protein kinase C, abolished the TPA and reduced the EGF‐mediated inhibition of PEPCK gene expression. These results suggested that EGF caused its effect, at least in part, through protein kinase C.
δ Opioid receptor mediates phospholipase C activation via Gi in Xenopus oocytes
Authors: Miyamae Takeaki , Fukushima Nobuyuki , Misu Yoshimi , Ueda Hiroshi
Published: July 2002

Cloned mouse δ‐subtype opioid receptor (DOR1) was expressed in Xenopus oocytes to study the signal transduction. Opioid δ‐agonists evoked a calcium‐dependent chloride current in oocytes injected with mRNA derived from DOR1, together with that from the α subunit of Gi1. The δ‐agonist‐induced current was blocked by naltrindol, a δ‐specific antagonist. The δ‐agonist evoked no or very weak currents in oocytes with the α subunit of Gq or Go. These findings indicate the functional coupling between the opioid δ‐receptor and phospholipase C through an activation of Gi.
Biochemical characterization of a 34 kda ribonucleoprotein (p34) purified from the spinach chloroplast fraction as an effective phosphate acceptor for casein kinase II
Authors: Kanekatsu Motoki , Munakata Hiroshi , Furuzono Kazuhisa , Ohtsuki Kenzo
Published: July 2002

A 34 kDa ribonucleoprotein (p34) was purified to homogeneity from a 1.0 M KCl extract of spinach chloroplasts and characterized as an effective phosphate acceptor for casein kinase II (CK‐II). The N‐terminal 21 residues (W‐V‐A‐Q‐T‐S‐E‐E‐E‐Q‐E‐G‐S‐T‐N‐A‐V‐L‐E‐G‐E) of p34 were 95% identical with the sequence reported for 28RNP (plastid mRNA 3' end processing factor in chloroplast). Moreover, the findings that DNAs as well as RNAs significantly stimulate the CK‐II catalyzed phosphorylation of p34 in vitro and induce its conformational change, suggest that the physiological activity of p34‐bound RNA or DNA in chloroplast post‐transcriptional regulation is controlled by specific p34 phosphorylation by CK‐II.
Efficient enzymatic synthesis of the sialyl‐LewisX tetrasaccharide
Authors: de Vries Theodora , van den Eijnden Dirk H. , Schultz Jody , O'Neill Roger
Published: July 2002

Sialyl‐Lewisx (NeuAcα2→3Galβ1→4[Fucαl→3]GlcNAc] has been identified as a ligand for E‐selectin, P‐selectin and recently also for L‐selectin. We have synthesized the sialyl‐Lewisx tetrasaccharide by total enzymatic synthesis from N‐acetyllactosamine using a placental α2→3‐sialyltransferase specific for type‐2 chain acceptors, followed by a cloned human α1→3‐fucosyltransferase (FucTV, the ‘plasma‐type’ enzyme). This procedure resulted in the tetrasaccharide in a 61% overall yield.
Rapid alteration of c‐myc and c‐jun expression in leukemic cells induced to differentiate by a butyric acid prodrug
Authors: Rabizadeh Ester , Shaklai Mati , Nudelman Abraham , Eisenbach Lea , Rephaeli Ada
Published: July 2002

The novel prodrug of butyric acid (BA), pivaloyloxymethyl butyrate, has been shown, in vitro, to induce differentiation and inhibit leukemic cell proliferation. The prodrug affects the cells in vitro at lower concentration and at least 100 times faster than does (BA). We have compared the ability of BA with that of its prodrug AN‐9 to modulate the expression of the early regulating genes, c‐myc and c‐jun, in HL‐60 cells. Exposure of HL‐60 cells to the prodrug resulted in a decrease of c‐myc and an increase of c‐jun expression. The prodrug elicited this effect at lower concentrations and at least 100 times faster than BA. Since changes in the expression of c‐myc and c‐jun occur minutes after exposure of the cells to the prodrug, these genes are likely to play a major role in the early stages of the differentiation pathway.
In vitro cleavage of HPV16 E6 and E7 RNA fragments by synthetic ribozymes and transcribed ribozymes from RNA‐trimming plasmids
Authors: He Yu-Kai , Lu Chang-De , Qi Guo-Rong
Published: July 2002

A RNA‐trimming plasmid pRG523 is constructed, in which three Rz genes, GR5(5'‐cis‐Rz gene), HR2G(trans‐Rz gene) and GR3(3'‐cis‐Rz gene), are arranged in the order from 5' to 3' downstream from the T7 promoter. In vitro transcription of this plasmid shows that the trans‐Rz can be trimmed to definite lengths by the cis‐Rz on both sides of the trans‐Rz. In vitro cleavage of HPV16 E6 and E7 RNA fragments of different lengths by synthetic Rz and that of E7 RNA with a length of 171 nt by synthetic Rz and transcribed Rzs with different lengths of flanking sequences is studied. The results show that the non‐base‐pairing flanking sequences on both Rz and target RNA can affect the cleavage reaction.
Identification of toxigenic Vibrio cholerae from the Argentine outbreak by PCR for ctx A1 and ctx A2‐B
Authors: Varela Pablo , Rivas Marta , Binsztein Norma , Cremona Maria Laura , Herrmann Patricio , Burrone Oscar , Ugalde Rodolfo A. , Frasch Alberto C.C.
Published: July 2002

A polymerase chain reaction (PCR) to detect a region of the A1 cholera toxin gene was applied to the identification of 43 Vibrio cholerae strains isolated from the recent outbreak in Argentina. A good correlation was observed between the GM1‐enzyme‐linked immunosorbent assay (GM1‐ELISA) to detect the B subunit of the enterotoxin and PCR. However, a V. cholerae non‐01 strain that was negative by the ELISA test, was positive by the PCR assay for the Al region. A second PCR test to detect the A2‐B coding region was developed to solve this case. We propose that routine detection of toxigenic V. cholerae by PCR should include analysis of A2‐B coding region or the whole cholera toxin operon.
Apolipoprotein B exhibits phospholipase A1 and phospholipase A2 activities
Authors: Reisfeld Nurit , Lichtenberg Dov , Dagan Arie , Yedgar Saul
Published: July 2002

Low density lipoproteins (LDL) as well as isolated apolipoprotein B (ApoB) have been shown to exhibit phospholipase A2 (PLA2) activity toward phospholipids containing an oxidized or short fatty acyl chain at position 2. Some of these studies employed the fluorescent analogue of phosphatidyl choline (PC), C6‐NBD‐PC, containing NBD‐caproic acid (C6‐NBD‐FA) at position 2 as a substrate, representative of short fatty acyl chains. The release of NBD‐caproic acid from position 2 is attributed to PLA2‐catalysed hydrolysis. However, this fatty acid can be released also by other enzymatic pathways. In the present study we examined, and ruled out, other enzymatic pathways which may be responsible for the hydrolysis of fatty acids from position 2 of phospholipids. On the other hand, we found that LDL as well as isolated ApoB hydrolyse C6‐NBD‐FA from both carbon 1 and carbon 2 of these phospholipids, thus exhibiting independent and simultaneous activities of phospholipase A1 and phospholipase A2.
Detection of a plant protein analogous to the yeast spliceosomal protein, PRP8
Authors: Kulesza Helen , Simpson Gordon G. , Waugh Robbie , Beggs Jean D. , Brown John W.S.
Published: July 2002

We have investigated whether a splieeosomal protein analogous to the yeast protein, PRP8, was present in higher plants. A protein with a molecular weight > 200 kDa was detected in Western blots of tobacco (Nicotiana tabacumL.) nuclear extracts with affinity‐purified antibodies, raised against four different β‐galactosidase‐PRP8 fusion proteins. The > 200 kDa protein was also immunoprecipitated by antibodies against the snRNA‐specific trimethylguanosine cap structure and was, therefore, snRNP‐associated. The presence of this protein in plants, in addition to yeast,Drosophilaand humans, and the conservation of large size and epitopes highlights the importance of PRP8 in pre‐mRNA splicing.
The mechanism of Ba2+ ‐induced exocytosis from single chromaffin cells
Authors: von Rüden Ludolf , García Antonio G. , López Manuela G.
Published: July 2002

Dialysis of Ba2+ into voltage‐clamped single bovine chromaffin cells produced a concentration‐dependent increase in cell capacitance, reflecting an enhanced rate of exocytotic events. Between 0.1 and 1 mM, Ba2+ linearly increased both the rate and the total amount of exocytosis. In undamped cells also, extracellular Ba2+ induced the release of catecholamines, as assayed with a carbon‐fibre electrode in the amperometric mode. Additionally, extracellular application of Ba2+ increased the apparent internal Ca2+ concentration ([Ca2+]app) in fura‐2‐loaded chromaffin cells. These observations were made both in the presence and absence of external Ca2+ (Ca2+
0), as well as after depletion of the intracellular Ca2+ stores with ionomycin. Under current‐clamp conditions, Ba2+ induced pronounced depolarization of the cells. These results are compatible with the following conclusions: by blocking K+ channels, Ba2+ causes depolarization of chromaffin cells. This results in opening of voltage‐gated Ca2+ channels and Ba2+ entry into the cytosol. Ba2+ then directly triggers exocytotic events, although it induces exocytosis only at concentrations more than a 100‐fold higher than Ca2+. Various effects contribute to the generally observed greater secretory responses with Ba2+ as compared with Ca2+; these are the depolarizing effects of extracellular Ba2+, its greater entry through non‐inactivating Ca2+ channels and its poor intracellular buffering largely arising from its weak affinity for plasmalemmal Ca2+ extrusion mechanisms. In some cases, Ba2+ additionally induces release of Ca2+ from internal stores, as evidenced by its effect on fura‐2 fluorescence at different wavelengths.
Specific increase of genetic expression of parvalbumin in fast skeletal muscles of mdx mice
Authors: Gailly Ph. , Hermans E. , Octave J.N. , Gillis J.M.
Published: July 2002

Parvalbumin mRNA was assayed by Northern blot analysis in muscles from normal and dystropic (mdx) mice. Its content was found to be specifically higher in mdx fast muscles than in control preparations. This suggests an increased expression of the protein in dystrophin‐lacking fast fibres. A possible role in calcium homeostasis is discussed.
Receptor‐binding domain of human α2‐macroglobulin Expression, folding and biochemical characterization of a high‐affinity recombinant derivative
Authors: Holtet Thor Las , Nielsen Kåre Lehmann , Etzerodt Michael , Moestrup Søren Kragh , Gliemann Jørgen , Sottrup-Jensen Lars , Thøgersen Hans Christian
Published: July 2002

A recombinant version of the receptor binding domain (RBDv) of human α2‐macroglobulin (α2M) has been expressed in E. coli and refolded using a novel iterative procedure. RBDv (Val1299‐Ala1451) is extended by 15 residues at the N‐terminal side of the Lys1313‐Glu papain cleavage site in human α2M. RBDv contains the intra‐chain bridge Cys1329‐Cys1444 and is soluble and monomeric. Competition experiments with 125I‐labelled methylamine‐treated α2M reveal that RBDv binds to the placental receptor for transformed α2M with a K
d of 8 nM, i.e. the binding affinity of RBDv is of the same order of magnitude as the intrinsic affinity for binding of one domain in transformed α2M to one receptor molecule.
Prediction of the structural similarity between spermidine/putrescine‐binding protein and maltose‐binding protein
Authors: Matsuo Yo , Nishikawa Ken
Published: July 2002

A structural similarity between spermidine/putrescine‐binding protein and maltose‐binding protein has been predicted by a sequence‐structure compatibility method. The sequence alignment obtained by this method revealed a consensus sequence motif located on the surface loop between the first α‐helix and the second β‐strand, and the further analysis identified a similar motif in iron‐binding protein. The conservation of this motif among certain bacterial periplasmic binding proteins suggests a common functional role for this region as well as an evolutionary relationship between them.
Expression of functional Raphanus sativus antifungal protein in yeast
Authors: Vilas Alves Ana L. , De Samblanx Genoveva W. , Terras Franky R.G. , Cammue Bruno P.A. , Broekaert Willem F.
Published: July 2002

Rs‐AFP2 is a 51 amino acid cysteine‐rich peptide isolated from radish (Raphanus sativus) seeds that exhibits potent inhibitory activity against filamentous fungi. A cDNA clone encoding the Rs‐AFP2 preprotein was modified by recombinant DNA methods to allow expression in the yeast Saccharomyces cerevisiae. This peptide was expressed in yeast as a fusion protein carrying at its N‐terminus the prepro‐sequences derived from the precursor of the yeast pheromone mating factor α1. These sequences allow secretion of the biologically active peptide in a correctly processed form. Deletion of the mating factor α1 pro‐peptide drastically reduced the expression level of the peptide.
Heterologous expression of cDNAs encoding barley (Hordeum vulgare) (1 → 3)‐β‐glucanase isoenzyme GV
Authors: Xu Peilin , Harvey Andrew J. , Fincher Geoffrey B.
Published: July 2002

Two cDNAs have been isolated from libraries generated from poly(A)+RNA of young barley roots and leaves, using a cDNA encoding barley (1 → 3)‐β‐glucanase isoenzyme GII as a probe. Nucleotide sequence analyses and ribonuclease protection assays show that the two cDNAs differ only in the length of their 3′‐untranslated regions; the corresponding mRNAs are likely to originate from a single gene by tissue‐specific processing at separate polyadenylation sites. When the coding region of the cDNA is expressed in E. coli, the resultant protein catalyses the hydrolysis of (1 → 3)‐β‐glucan with an action pattern characteristic of a (1 → 3)‐β‐glucan endohydrolase (EC 3.2.1.39). The enzyme has been designated isoenzyme GV of the barley (1 → 3)‐β‐glucanase family.
Degradation of protein kinase Cα and its free catalytic subunit, protein kinase M, in intact human neuroblastoma cells and under cell‐free conditions
Authors: Shea Thomas B. , Beermann Mary Lou , Griffin William R. , Leli Ubaldo
Published: July 2002

Proteolytic cleavage of protein kinase C (PKC) under cell‐free conditions generates a co‐factor independent, free catalytic subunit (PKM). However, the difficulty in visualizing PKM in intact cells has generated controversy regarding its physiological relevance. In the present study, treatment of SH‐SY‐5Y cells with 2‐O‐tetradecanoylphorbol 13‐acetate resulted in complete down‐regulation of PKC within 24 h without detection of PKM. By contrast, low levels of PKM were transiently detected following ionophore‐mediated calcium influx under conditions which induced no detectable PKC loss. PKM was not detected during rapid cell‐free degradation of partially purified SH‐SY‐5Y PKCα by purified human brain mM calpain. However, when the kinetics of PKC degradation were slowed by lowering levels of calpain, PKM was transiently detected. PKM was also only transiently observed following calpain‐mediated degradation of purified rat brain PKCα. Densitometric analyses indicated that, once formed, PKM was degraded approximately 10 times faster than PKC. These data provide an explanation as to why PKM is difficult to observe in situ, and indicate that PKM should not be considered as an ‘unregulated’ kinase, since its persistence is apparently strictly regulated by proteolysis.
Evidence for redox forms of the Aequorea green fluorescent protein
Authors: Inouye Satoshi , Tsuji Frederick I.
Published: July 2002

Highly purified recombinant Aequoreagreen fluorescent protein is able to undergo a reversible oxidation‐reduction reaction in the presence of molecular oxygen. In the oxidized form in near UV light, the protein is highly fluorescent, but when reduced with sodium dithionite, it becomes completely non‐fluorescent. On exposure to molecular oxygen the reduced, non‐fluorescent protein reverts to its original fluorescent state.
Intracellular single chain Fv antibody inhibits Ras activity in T‐cell antigen receptor stimulated Jurkat cells
Authors: Werge Thomas M. , Baldari Cosima T. , Telford John L.
Published: July 2002

A mammalian expression vector directing the synthesis of a cytoplasmic single chain Fv version of the Y13—259 anti‐Ras antibody was constructed and co‐transfected into the human lymphoid cell line Jurkat together with a reporter construct containing the bacterial gene for chloramphenicol acetyl transferase under the transcriptional control of several copies of the binding site for the transcription factor NF‐AT. The Ras specific antibody interferes with NF‐AT activation upon direct activation of the T‐cell antigen receptor, whereas activation by direct protein kinase C stimulation is less sensitive to the anti‐Ras antibody. Furthermore, the observed inhibition is dependent on the ratio of antibody to reporter plasmid utilized in the transfection experiments.
Cloning and expression of three isoforms of the human EP3 prostanoid receptor
Authors: Adam Mohamed , Boie Yves , Rushmore Thomas H. , Müller Gretchen , Bastien Lison , McKee Katherine T. , Metters Kathleen M. , Abramovitz Mark
Published: July 2002

Functional cDNA clones coding for three isoforms of the human prostaglandin E receptor EP3 subtype have been isolated from kidney and uterus cDNA libraries. The three isoforms, designated hEP3‐I, hEP3‐II and hEP3‐III, have open reading frames corresponding to 390, 388 and 365 amino acids, respectively. They differ only in the length and amino acid composition of their carboxy‐terminal regions, beginning at position 360. The human EP3 receptor has seven predicted transmembrane spanning domains and therefore belongs to the G‐protein‐coupled receptor family. The rank order of potency for prostaglandins and related analogs in competition for [3H]PGE2 specific binding to membranes prepared from transfected COS cells was comparable for all three isoforms, and as predicted for the EP3 receptor, with PGE2 = PGE1 > PGF2α = iloprost > PGD2 ⪢ U46619. In addition, the EP3‐selective agonist MB28767 was a potent competing ligand with an IC50 value of 0.3 nM, whereas the EP1‐selective antagonist AH6909 gave IC50 values of 2–7 μM and the EP2‐selective agonist butaprost was inactive. In summary, we have cloned three isoforms of the human EP, receptor having comparable ligand binding properties.
Alteration of the specificity of ecotin, an E. coli serine proteinase inhibitor, by site directed mutagenesis
Authors: Pál Gábor , Sprengel Gunther , Patthy András , Gráf László
Published: July 2002

The gene of ecotin, an E. coli proteinase inhibitor, was cloned, and by site‐directed mutagenesis the active site residue of the protein, Met84, was mutated to Lys, Arg and Leu. The recombinant wild‐type and mutant inhibitors were overexpressed in E. coli, purified to homogeneity and their inhibitory effects on trypsin, chymotrypsin and elastase were compared. Of these serine proteinases trypsin is the most strongly inhibited by wild type ecotin and its mutants. According to our results the character of residue 84 of ecotin significantly but not dramatically modifies the specificity of the inhibitor.
Effects of peroxynitrite‐induced protein modifications on tyrosine phosphorylation and degradation
Authors: Gow Andrew J. , Duran Daniel , Malcolm Stuart , Ischiropoulos Harry
Published: July 2002

The ability of protein tyrosine kinases to phosphorylate a synthetic peptide was inhibited 51% by peroxynitritemediated nitration of tyrosine. Exposure of endothelial cells to peroxynitrite decreased the intensity of tyrosine phosphorylated proteins and increased the intensity of nitrotyrosine‐containing proteins. Peroxynitrite‐modified BSA was degraded by human red blood cell lysates. However, human plasma in a concentration‐, time‐, and temperature‐dependent manner, removed the protein nitrotyrosine epitope. These results suggest that tyrosine nitration interferes with phosphorylation and targets proteins for degradation. Specific enzymatic process(es) for removing nitrotyrosine may be present in vivo.
Reverse transcriptase inhibitors suppress telomerase function and induce senescence‐like processes in cultured mouse fibroblasts
Authors: Yegorov Yegor E. , Chernov Dmitry N. , Akimov Sergei S. , Bolsheva Nadezda L. , Krayevsky Alexander A. , Zelenin Alexander V.
Published: July 2002

Spontaneous transformation of mouse embryonic fibroblasts in the presence of the reverse transcriptase inhibitors azidothymidine and carbovir led to the formation of telomerase‐free clones. After prolonged cultivation of fibroblasts in the presence of carbovir, resistant cells with a very high level of telomerase activity were obtained. Azidothymidine and carbovir, but not dideoxycytidine, induced senescence‐like processes in cultures of immortal mouse fibroblasts. After long‐term incubation, cell proliferation gradually decreased, their morphology becoming similar to that of the senescent ones. The process was reversible: after inhibitor removal, the cells, including the giant ones, entered mitoses. All these data suggest that reverse transcriptase inhibitors block telomerase function in mouse cells.
THE PROTEOGLYCAN DSD‐1‐PG OCCURS IN PERINEURONAL NETS AROUND PARVALBUMIN‐IMMUNOREACTIVE INTERNEURONS OF THE RAT CEREBRAL CORTEX
Authors: Wintergerst Eva Sabine , Faissner Andreas , Celio Marco R.
Published: July 2002

Proteoglycans involved in the shaping of the developing brain are often preserved in the adult brain in more restricted locations. We have studied the fate of DSD‐1‐PG, a chondroitin sulfate proteoglycan containing the hybrid epitope DSD‐1. DSD‐1‐PG exerts neurite outgrowth promoting activity and has been shown to occur in the developing brain during late brain development and into adulthood. In the adult rat brain, monoclonal and polyclonal antibodies against DSD‐1‐PG labelled only the circumference of a selected subpopulation of neurons. These nerve cells invariably expressed the calcium‐binding protein parvalbumin. The label occupied the extracellular space in close vicinity to the cell body, surrounding axon terminals and glial end feet, but was absent from synaptic clefts. DSD‐1‐PG is thus shown to be an additional representative of the growing list of substances found in perineuronal locations in the adult mammalian brain.
Observation of DNA–polymer condensate formation in real time at a molecular level
Authors: Martin A.L. , Davies M.C. , Rackstraw B.J. , Roberts C.J. , Stolnik S. , Tendler S.J.B. , Williams P.M.
Published: July 2002

Dynamic real time assembly of toroidal and rod‐like DNA condensates has been visualised using atomic force microscopy. Imaging has been conducted in an aqueous environment allowing the visualisation of hydrated, pegylated‐polymer DNA condensates undergoing dynamic structural movement and conformational change. A major hurdle in the field of gene delivery is cellular transfection and the subsequent transfer of condensed genetic material to the cell nucleus. An increased understanding of the process of DNA condensation will aid the development and optimisation of gene delivery vectors.
Cyclic AMP‐elevating agents induce the expression of MAP kinase phosphatase‐1 in PC12 cells
Authors: Burgun Claude , Esteve Laurent , Humblot Nathalie , Aunis Dominique , Zwiller Jean
Published: July 2002

Stimulation of pheochromocytoma PC12 cells by cAMP‐elevating agents caused the induction of the immediate early gene3CH134, which encodes MAP kinase phosphatase‐1 (MKP‐1). Forskolin was as potent as serum in stimulatingMKP‐1gene expression, whereas dibutyryl‐cAMP and neuropeptide PACAP were less effective. Induction of theMKP‐1gene was accompanied by neo‐synthesis of MKP‐1 protein. MAP kinase activation was not involved in the cAMP‐inducedMKP‐1gene expression. The MAP kinase inactivation, that would result from MKP‐1 induction in response to increased intracellular cAMP level, contributes to explain how hormones or neurotransmitters signaling through cAMP influence cell growth and differentiation.
Biophysical phenotypes of SCN5A mutations causing long QT and Brugada syndromes
Authors: Baroudi Ghayath , Chahine Mohamed
Published: July 2002

Long QT and Brugada syndromes are two hereditary cardiac diseases. Brugada syndrome has so far been associated with only one gene, SCN5A, which encodes the cardiac sodium channel. However, in long QT syndrome (LQTS) at least six genes, including the SCN5A, are implicated. The substitution (D1790G) causes LQTS and the insertion (D1795) induces both LQTS and Brugada syndromes in carrier patients. hH1/insD1795 and hH1/D1790G mutant channels were expressed in the tsA201 human cell line and characterized using the patch clamp technique in whole‐cell configuration. Our data revealed a persistent inward sodium current of about 6% at −30 mV for both D1790G and insD1795, and a reduction of 62% of channel expression for the insD1795. Moreover, a shift of steady‐state inactivation curve in both mutants was also observed. Our findings uphold the idea that LQT3 is related to a persistent sodium current whereas reduction in the expression level of cardiac sodium channels is one of the biophysical characteristics of Brugada syndrome.
Redox‐regulated chaperone function and conformational changes of Escherichia coli Hsp33
Authors: Raman B , Siva Kumar L.V , Ramakrishna T , Mohan Rao Ch
Published: July 2002

We have studied the chaperone activity and conformation of Escherichia coli heat shock protein (Hsp)33, whose activity is known to be switched on by oxidative conditions. While oxidized Hsp33 completely prevents the heat‐induced aggregation of ζ‐crystallin at 42°C at a ratio of 1:1 (w/w), the reduced form exhibits only a marginal effect on the aggregation. Far UV–circular dichroism (CD) spectra show that reduced Hsp33 contains a significant α‐helical component. Oxidation results in significant changes in the far UV–CD spectrum. Near UV–CD spectra show changes in tertiary structural packing upon oxidation. Polarity‐sensitive fluorescent probes report enhanced hydrophobic surfaces in the oxidized Hsp33. Our studies show that the oxidative activation of the chaperone function of Hsp33 involves observable conformational changes accompanying increased exposure of hydrophobic pockets.
SufC hydrolyzes ATP and interacts with SufB from Thermotoga maritima
Authors: Rangachari K. , Davis C.T. , Eccleston J.F. , Hirst E.M.A. , Saldanha J.W. , Strath M. , Wilson R.J.M.(Iain)
Published: July 2002

Genetic experiments in bacteria have shown the suf operon is involved in iron homeostasis and the oxidative stress response. The sufB and sufC genes that always occur together in bacteria are also found in plants, and even the malaria parasite, associated with the plastid organelle. Although the suf operon is believed to encode an iron‐dependent ABC‐transporter there is no direct evidence. By immunolocalization we show here that SufB and SufC are associated with the membrane of Escherichia coli. We also present kinetic studies with a recombinant version of SufC from Thermotoga maritima that shows it is an ATPase and that it interacts with SufB in vitro.