bluuebunny/crossref_metadata_2025 huggingface dataset:
N‐terminal heterogeneity of methylamine dehydrogenase from Thiobacillus versutus
Authors: Beeumen Jozef Van , Driessche Gonzalez Van , Huitema Fienke , Duine J.A. , Canters Gerard W.
Published: July 2002

The N‐terminal processing of MADH from the bacterium T. versutus and the N‐terminal heterogeneity of the isolated α subunit of the α2β2 protein complex was demonstrated by a combination of Edman sequence analysis of an electroblotted band, in situ digested with pyroglutamate aminopeptidase, and accurate mass determination of the homogenous subunit by the technique of electrospray ionisation mass spectrometry. From this study, it appears that the corresponding gene of the α subunit contains 395 amino acids and that it is preceded by a leader sequence of 31 residues.
Epitope mapping of a monoclonal antibody which binds HIV‐1 Gag and not the Gag‐derived proteins
Authors: Sarubbi Edoardo , Denaro Maurizio
Published: July 2002

Monoclonal antibody (MAb) 1G12 binds the uncleaved HIV‐1 Gag polypeptide (p55), but fails to recognize the final products of the proteolytic processing [Sarubbi, E. et al. (1991) FEBS Lett. 279, 265‐269]. In this report we show that binding of MAb 1G12 to a 110‐residue Gag fragment containing the p17–p24 cleavage site prevents proteolysis of this site by the HIV‐1 protease. Competition studies with synthetic peptides have been performed to map the binding site of MAb 1G12 on Gag. The antibody recognizes a sequential epitope that spans the HIV‐1 protease cleavage site; determinants located on both p17 and p24 are required for antibody binding. MAb 1G12 is also shown to lack any cross‐reactivity with other HIV‐1 protease cleavage sites.
Lymphotoxin gene expression by melanocytes and melanoma cell lines and persistence of unspliced mRNA
Authors: Melani Cecilia , Silvani Anna , Parmiani Giorgio , Colombo Mario P.
Published: July 2002

Human melanoma cell lines express many different cytokines [1], including lymphotoxin (LT), the production of which has been considered to be restricted to cells of the lymphocytic lineage in response to cell activation. LT expression by melanomas is constitutive and characterized by the presence of two mRNAs. In the present paper we report an analysis of the origin of the two LT‐specific transcripts in four human melanoma cell lines at different stages of progression and in four melanocytic cell lines. Reverse transcription‐polymerase chain reaction (RT‐PCR) performed with primers lying in the first and fourth exons and hybridization with intron probes showed a spliced and a full‐unspliced LT mRNA. This pattern was also displayed by one out of four melanocyte cell lines. Western blot analysis indicated that LT RNA is properly translated to a 23–25 kDa protein and immunocytochemistry showed its localization within the cytoplasm and on the cell membrane.
Differential modulation of pancreatic β‐cell bursting by intracellular pH in the presence and absence of a K‐ATP channel blocker
Authors: Barbosa Rui M. , Salgado António P. , Santos Rosa M. , Rosário Luís M.
Published: July 2002

The study of the influence of intracellular pH (pHi) changes on the mechanism underlying pancreatic β‐cell bursting has been hampered by concomitant effects on the activity of background ATP‐dependent K+ (K‐ATP) channels. β‐cells were made to burst in the absence of active K‐ATP channels by raising external Ca2+ in the presence of 11 mM glucose and tolbutamide. An alkalinizing pH; shift (exposure to 20 mM NH4C1) increased the burst active phase duration. Conversely, an acidifying shift (NH4Cl withdrawal) suppressed the electrical activity. This is the mirror image of the effects recorded in the absence of tolbutamide. Glibenclamide and quinine suppressed the alkalinization‐evoked hyperpolarization. This study emphasizes the differential sensitivity of different β‐cell ion channels to pHi and the prevalent role of K‐ATP channels as electrical transducers of cytoplasmic pH changes under regular physiological conditions.
Two distinct modalities of NMDA‐receptor inactivation induced by calcium influx in cultured rat hippocampal neurons
Authors: Schinder Alejandro F. , Montal Mauricio
Published: July 2002

Repetitive stimulation of glutamate (glu) receptors elicits increasingly smaller ionic currents in hippocampal neurons. To investigate mechanisms underlying this phenomenon, voltage clamp whole‐cell currents evoked by glu (100 μM) were recorded from hippocampal neurons in culture. These currents were primarily carried by N‐methyl‐d‐aspartate‐receptor (NMDA‐R) channels, as shown by the voltage‐dependent sensitivity to extracellular Mg2+ blockade, and inhibition by the specific antagonist MK‐801. In the presence of 2.2 mM extracellular Ca2+ ([Ca2+]e, repetitive glu applications (15 episodes of 4 s/min) elicited progressively smaller currents that stabilized at 45% of their initial peak value. Replacement of [Ca2+
e, by Ba2+ produced similar effects. This phenomenon, defined as interepisode inactivation, was exacerbated by elevating [Ca2+]e to 11 mM, attenuated by reducing [Ca2+
e to 0.22 mM, and further diminished by shortening the length of the glu pulse to 2 s. Current decay exhibited during individual stimuli, or intraepisode inactivation, was dependent on [Ca2+]e yet remained stable during repetitive stimulation. We conclude that interepisode and intraepisode inactivations of NMDA‐R currents are the expression of two distinct processes triggered by Ca2+. These modalities of inactivation may arise from Ca2+ binding either to the receptor or to closely associated regulatory proteins.
Carbohydrate structures of the cell adhesion molecule, contact site A, from Dictyostelium discoideum
Authors: Yoshida Motonobu , Matsui Taei , Fuse Goro , Ouchi Seiji
Published: July 2002

We determined the carbohydrate structures of contact site A from Dictyostelium discoideum. The carbohydrate moieties of contact site A were released by hydrazinolysis. Fractionation of the deacidified oligosaccharide mixture by Bio‐Gel P‐4 column chromatography revealed that it was composed of four major oligosaccharides. Their respective structures were determined by sequential exoglycosidase digestion. It is known that contact site A consists of two kinds of carbohydrates, I and II. Taking together the previous and the present results, it was deduced that carbohydrate I comprises N‐linked oligosaccharides and carbohydrate II O‐linked ones. Furthermore, the relative molar contents of GalNAc and GlcNAc in reducing terminal suggested that contact site A contains 67% of N‐linked and 33% of O‐linked oligosaccharides.
Stimulation of tyrosine phosphorylation without inositol lipid hydrolysis in human B lymphocytes on engaging CD72
Authors: Kamal Mohammed , Knox Kirstine , Finney Michael , Michell Robert H. , Holder Michelle J. , Gordon John
Published: July 2002

Occupancy of CD72 on resting tonsillar B cells by monoclonal antibodies (mAb) promotes entry into the G1 phase of the cell cycle with an accompanying increase in MHC Class II expression and provides a co‐stimulus to immobilized anti‐μ for driving DNA synthesis. We now report that engagement of CD72 by mAb stimulates tyrosine phosphorylation in B cells with a peak of activity seen at 5–10 min. Two major substrates of 29 and 57 kDa showed a basal level of phosphorylation which increased with time, while a 40 kDa protein and several other minor components were phosphorylated de novo on the addition of mAb to CD72. Inositol lipid hydrolysis was found to be unperturbed, although a shallow rise in the basal level of intracellular free Ca2+ was provoked on engaging CD72. Receptor cross‐linking was not a requirement for signaling human B cells through CD72: simple occupancy by univalent antibody was sufficient both to trigger the rise in basal [Ca2+]i and to promote DNA synthesis.
SH‐containing compounds as allosteric effectors of glyceraldehyde‐3‐phosphate dehydrogenase
Authors: Kuzminskaya Elena V. , Asryants Regina A. , Nagradova Natalia K.
Published: July 2002

The rate of hydrolysis of 3‐phosphoglyceroyl‐holoenzyme, a covalent intermediate of glyceraldehyde‐3‐phosphate dehydrogenase catalyzed reaction, is considerably decreased in the presence of micromolar concentrations of reduced glutathione, cysteine or dithiothreitol with K
i, values of 0.78 μM, 0.6 μM and 10 μM, respectively. The maximal effect is achieved at a molar ratio [effector]/[tetrameric enzyme] close to unity, which points to subunit cooperativity involved in the stabilization of the covalent intermediate against hydrolysis. The effect is specific for acylholoenzyme conformation and insignificant in the case of hydrolysis of acylated apoenzyme species. The ability of the effectors to stabilize the reaction intermediate against spontaneous hydrolysis, in which water replaces inorganic phosphate as the acyl group acceptor, may be a factor contributing to the specificity and effectiveness of the enzyme catalysis.
Ribosomal proteins, TL4 and TL5, from Thermus thermophilus form hybrid complexes with 5 S ribosomal RNA from different microorganisms
Authors: Gongadze G.M. , Tishchenko S.V. , Sedelnikova S.E. , Garber M.B.
Published: July 2002

Hybrid complexes of the ribosomal proteins, TL4 and TL5, from Thermus thermophilus with 5 S ribosomal RNA from Escherichia coli and Bacillus
stearothermophilus have been prepared. There was no competition between the two proteins for the binding sites. Stoichiometry of 5 S RNA binding for both proteins was 1:1 (protein/RNA). The TL4 protein competed with the E. coli ribosomal L5 protein, and the TL5 protein competed with theE. coli ribosomal proteins, L18 and L25, for binding with 5 S RNA.
Phosphatidic acid induces the respiratory burst of electropermeabilized human neutrophils by acting on a downstream step of protein kinase C
Authors: Mitsuyama Takashi , Takeshige Koichiro , Minakami Shigeki
Published: July 2002

Phosphatidic acid (PA) dose‐dependently induced Superoxide (O2
−) production of electropermeabilized human neutrophils but not of intact neutrophils, indicating that PA induces the activation of NADPH oxidase by acting on an intracellular target. The O2
− production by PA was not inhibited by protein kinase C (PKC) inhibitors, such as staurosporine and calphostin C, and an inhibitor of PA phosphohydrolase, propranolol. These observations suggest that the activation of the oxidase by PA is independent of the activity of PKC and may dominate the activation by diacylglycerol which is formed from PA via the action of PA phosphohydrolase. Furthermore, the production by PA, as well as that by phorbol myristate acetate, was inhibited by cyclic AMP and GDPßS. Therefore, PA seems to act at a site downstream of PKC.
Phosphatidylcholine enhances the activity of rat liver type II phosphatidylinositol‐kinase
Authors: Olsson Henric , Martínez-Arias Wilma , Jergil Bengt
Published: July 2002

A PtdIns 4‐kinase was purified extensively from rat liver exocytotic vesicles. The enzyme had a low K
m for ATP, was inhibited by adenosine, and had an apparent molecular mass of 54 kDa, indicating it to be a type II Ptdlns‐kinase. The activity of the purified enzyme was enhanced several‐fold by PtdCho, and to some extent by other phospholipids with basic polar head groups, and was inhibited by PtdSer. Kinetic analyses, presenting the substrate in mixed micelles of Triton X‐100, Ptdlns and PtdCho, showed that the effect of PtdCho was both to increase V
max and to decrease the apparent K
m for micellar PtdIns.
Isolation and partial characterization of cholesterol pronucleating hydrophobic glycoproteins associated to native biliary vesicles
Authors: Miquel Juan Francisco , Núñez Liliana , Rigotti Attilio , Amigo Ludwig , Brandan Enrique , Nervi Flavio
Published: July 2002

Cholesterol is transported both in unilamellar phosphatidylcholine vesicles and in bile salts‐mixed micelles in native bile. The vesicular carrier of biliary lipids apparently has a well defined protein profile with a potent cholesterol crystallization‐promoting activity. This study was conducted to identify and further characterize these vesicular proteins and to test the effect of isolated vesicular proteins on the cholesterol crystal formation in supersaturated model bile. The results confirmed that proteins are a constant component of highly purified biliary vesicles both in hepatic and gallbladder bile. Immunoglobulins (IgA, IgG and IgM) and albumin are associated to the purified hepatic biliary vesicles. Furthermore, four different hydrophobic glycoproteins with a molecular mass of 130, 114, 86, and 62–67 kDa were isolated. These glycoproteins showed no reactivity with anti‐human whole serum or anti‐immunoglobulin antibodies, suggesting that these proteins are biliary‐specific. Isolated 130, 114 and 62–67 kDa vesicular glycoproteins significantly decreased the cholesterol nucleation time in artificial model bile. We concluded that some, but not all, vesicular‐bound hydrophobic glycoproteins have cholesterol pronucleating activity and they may be involved in the pathogenesis of cholesterol gallstone disease.
A sialic acid analogue acting as a receptor determinant for binding but not for infection by influenza C virus
Authors: Brossmer Reinhard , Isecke Rainer , Herrler Georg
Published: July 2002

We describe a synthetic sialic acid analogue, 9‐thioacetamido‐N‐acetylneuraminic acid (9‐thioacetamido‐Neu5Ac), which is recognized by the receptor‐binding activity of influenza C virus, but is resistant to the receptor‐destroying enzyme (acetylesterase) of this virus. Following transfer of the analogue to the surface of receptor‐negative cells, influenza C virus is able to attach to these cells, but is unable to infect the cells. This result suggests that inactivation of virus receptors by the receptor‐destroying enzyme is essential for initiation of infection. Because of their unique properties such analogues promise to be powerful chemotherapeutic agents.
Interaction of avidin with spin‐labelled N‐biotinyl phosphatidylethanolamine in a lipid membrane
Authors: Swamy Musti J. , Marsh Derek
Published: July 2002

N‐Biotinyl phosphatidylethanolamine spin labelled at the C‐14 position of the sn‐2 chain has been incorporated at a level of 1 mol% in bilayers of dimyristoyl phosphatidylcholine, and the effects on the chain mobility of binding avidin to the biotin lipid headgroup have been studied by electron spin resonance spectroscopy. In the fluid phase, avidin causes a large and selective restriction in the chain motion of the biotin lipids to which it is attached, without perturbing appreciably the mobility of the bulk lipid chains. This specific type of lipid‐protein interaction is different in kind from that observed both with integral and peripheral membrane proteins and may be involved in transmembrane communication on ligand binding to lipid headgroups, as well as lateral communication (at high packing densities) between proteins with covalent lipid anchors.
Effects of sulfhydryl reagents on the Cys65 mutant of the transposon Tn10‐encoded metal‐tetracycline/H+ antiporter of Escherichia coli
Authors: Yamaguchi Akihito , Kimura Tomomi , Sawai Tetsuo
Published: July 2002

The Cys65 mutant of the Tn10‐encoded metal‐tetracycline/H+ antiporter is the only one which is inactivated by sulfhydryl reagents among the Cys mutants of the putative loop2–3 region [Yamaguchi, A. et al. (1992) J. Biol. Chem. 267, 19155–19162]. The tetracycline transport activity of the Cys65 mutant was completely abolished by N‐ethylmaleimide; however, methyl methanethiosulfonate only abolished 45% of the activity, even in the presence of an excess of the reagent. Since N‐ethylmaleimide did not further inactivate the methyl methanethiosulfonate‐treated antiporter, it is clear that the modified antiporter molecule with a small substituent, a thiomethyl group, had significant but lower activity than the unmodified antiporter. The binding of [14C]N‐ethylmaleimide to the Cys65 mutant was inhibited in the predsence of tetracycline. These findings indicate that position 65 is close to the site of the interaction with the substrate and the modification of the side chain at this position caused steric hindrance as to substrate translocation.
Targeted delivery of hygromycin B using reconstituted Sendai viral envelopes lacking hemagglutinin‐neuraminidase
Authors: Bagai Sangeeta , Sarkar Debi P.
Published: July 2002

Hygromycin B was encapsulated in reconstituted Sendai viral envelopes containing only the fusion (F) protein (F‐virosomes). Incubation of loaded F‐virosomes with cultured HepG2 cells resulted in fusion mediated delivery of hygromycin B to the cell cytoplasm, as was inferred from inhibition of DNA synthesis. Binding of the F‐virosomes to HepG2 cells was mediated by the interaction of terminal β‐galactose residues of fusion protein with asialoglycoprotein receptor on HepG2 cells, subsequently leading to fusion between the two membranes. The cytotoxic effect of hygromycin B enclosed in F‐virosomes was comparable with that of F,HN‐virosomes containing both hemagglutinin‐neuraminidase (HN) and F protein and F,HNred‐virosomes containing HN whose disulfide bonds were irreversibly reduced (HNred). Hygromycin B loaded fusogenic liposomes were prepared by coreconstituting the viral envelope containing only fusion protein with exogenous lipids. These fusogenic liposomes were found to be more active than F‐virosomes at the same fusion protein concentrations.
A factor protecting mammalian [75Se]SeCys‐tRNA is different from EF‐1α
Authors: Yamada Kenichiro , Mizutani Takaharu , Ejiri Shin-ichiro , Totsuka Tsuyoshi
Published: July 2002

In Escherichia coli, an elongation factor (EF‐Tu‐like) specific to SeCys‐tRNA, SELB, has been identified; however, a mammalian counterpart of SELB has not been reported to date. We searched for and found this factor in bovine liver extracts using the assay of [75Se]SeCys‐tRNA protecting activity against alkaline hydrolysis (SePF activity). We found SePF activity in the protein extracts of the precipitate (microsomal fraction) collected at 150,000 × g from bovine liver. The proteins were separated by Sephacryl S‐300 chromatography, and the SePF and EF‐1α activities were found in the same fraction, indicating that SePF and EF‐1α have the same molecular mass (approximately 50 kDa). We then chromatographed this active fraction using CM‐Sephadex C‐25 columns. The SePF activity was eluted after the peak of EF‐1α activity. This result indicated that this SePF activity was not dependent on EF‐1α. In addition to performing these two chromatographies, we investigated pure EF‐1α from Bombyx mori but could not detect any SePF activity in B. mori EF‐1α. Thus we showed that the SePF activity in bovine liver differs from that of EF‐1α in eukaryotes. Therefore the factor protecting [75Se]SeCys‐tRNA in bovine liver is not EF‐1α and must be a SELB‐like factor.
Ability of MBP or RBP signal peptides to influence folding and in vitro translocation of wild‐type and hybrid precursors
Authors: Rosemond M.Jane Cox , Strobel Sharon M. , Raya Paul H. , Bassford Philip J.
Published: July 2002

Maltose‐binding protein (MBP), whose export in E. coli is dependent upon the chaperone SecB, and ribose‐binding protein (RBP), whose export is SecB‐independent, have been used to generate hybrid secretory proteins. Here, in vitro techniques were used to analyze MBP, RBP, RBP‐MBP (RBP signal and MBP mature), and MBP‐RBP (MBP signal and RBP mature). In protease‐protection experiments, RBP folded considerably faster than MBP, RBP‐MBP, or MBP‐RBP. Only the folding properties of proteins containing the MBP mature moiety were influenced by SecB. In post‐translational translocation assays, MBP exhibited the highest translocation efficiency. The hybrids RBP‐MBP and MBP‐RBP showed intermediate levels, and RBP translocation was not detected in these assays. These experiments demonstrate the influence of the signal peptide in determining folding properties and translocation efficiency of precursor secretory proteins.
Aminoacyl‐tRNAs diversity before and unity after interaction with EF‐Tu:GTP
Authors: Barciszewski Jan , Sprinzl Mathias , Clark Brian F.C.
Published: July 2002

Evidence is presented for a new role for elongation factor EF‐Tu. This involves conformational restraint or conformational selection of any aminoacyl‐tRNA for channeling it to the ribosomal decoding site.
Substrate, substrate analogue, and inhibitor interactions with the ferrous active site of catechol 2,3‐dioxygenase monitored through XAS studies
Authors: Bertini Ivano , Briganti Fabrizio , Mangani Stefano , Nolting Hans F. , Scozzafava Andrea
Published: July 2002

The interactions of catechol (substrate), 2‐hydroxy‐pyridine‐N‐oxide (substrate analogue) and 2‐bromophenol (inhibitor) with the extradiol cleaving catechol‐2,3‐dioxygenase from Pseudomonas putida mt‐2 have been monitored through X‐ray absorption spectroscopy (XAS). The analysis of the data provides details about the mode of coordination of the substrate and of the inhibitors to the active site of the enzyme.
Intrinsic ligand binding properties of the human and bovine α,‐interferon receptors
Authors: Lim Jin-kyu , Xiong Jingwei , Carrasco Nancy , Langer Jerome A.
Published: July 2002

The Type I interferon receptor (IFN‐αR) interacts with all IFN‐αs, IFN‐β and IFN‐ω, and seems to be a multisubunit receptor. To investigate the role of a cloned receptor subunit (IFN‐αR1), we have examined the intrinsic ligand binding properties of the bovine and human IFN‐αR1 polypeptides expressed in Xenopus laevis oocytes. Albeit with different efficiencies, Xenopus oocytes expressing either the human or bovine IFN‐αR1 polypeptide exhibit significant binding and formation of crosslinked complexes with human IFN‐αA and IFN‐αB. Thus, the IFN‐αR1 polypeptide most likely plays a direct role in ligand binding.
Differentiation of HL‐60 cells is promoted by H‐toxin of Clostridium septicum
Authors: Morinaga Naoko , Kato Iwao , Noda Masatoshi
Published: July 2002

H‐toxin of Clostridium septicum potentiated dimethyl sulfoxide(DMSO)‐induced differentiation of human promyelocytic leukemia HL‐60 cells which were monitored by nuclear morphology and production of oxidative radicals. But, H‐toxin did not induce differentiation of HL‐60 cells in the absence of DMSO. These phenomena were not observed by staphylococcal leukocidin, a cytotoxin affecting to HL‐60 cells. In HL‐60 cells, ADP‐ribosylation of 118, 93, 75 and 58 kDa membrane proteins was observed, but the ADP‐ribosylation was not detected either in differentiated HL‐60 cells by DMSO or in normal polymorphonuclear leukocytes of human. When the membranes of HL‐60 cells were incubated with H‐toxin, ADP‐ribosylation of the membrane proteins was inhibited. Such suppressive effects on ADP‐ribosylation were not observed by DMSO or staphylococcal leukocidin. These data suggest that inhibition of the ADP‐ribosylation by H‐toxin may play an important role in potentiation of DMSO‐induced differentiation of HL‐60 cells.
Aspartic proteinases — Fourier transform IR studies of the aspartic carboxylic groups in the active site of pepsin
Authors: Iliadis Georgios , Zundel Georg , Brzezinski Bogumil
Published: July 2002

Fourier transform (FTIR) difference spectra of pepsin minus diazoacetylnorleucine methyl ester (DAN) or minus diazoacetyl‐l‐phenylalanine methyl ester (DAP) modified pepsin, respectively, demonstrated that Asp‐215 is not deprotonated in pepsin. The FTIR difference spectrum of pepsin minus 1,2‐epoxyparanitrophenoxypropane (EPNP) modified pepsin demonstrates that Asp‐32 is present in pepsin as CO−
2 anion. The position of the v(C – O) vibration demonstrates that no (O … H … O)− hydrogen bond between Asp‐215 and Asp‐32 is formed. Furthermore, no H3O+ is present in the active center. Studies of the complex of pepsin with the inhibitor pepstatin prove that the inhibitor removes the water from the active site and Asp‐32 becomes protonated.
GDP/GTP exchange reaction‐stimulating activity of Rabphilin‐3A for Rab3A small GTP‐binding protein
Authors: Fujita Yasuyuki , Sasaki Takuya , Araki Keishi , Takahashi Kazuo , Imazumi Katsunori , Kato Masaki , Matsuura Yoshiharu , Takai Yoshimi
Published: July 2002

Rabphilin‐3A is a putative target protein for Rab3A, a small GTP‐binding protein particularly implicated in neurotransmitter release. Rabphilin‐3A interacts more preferentially with GTP‐Rab3A than with GDP‐Rab3A. Moreover, Rabphilin‐3A shows a weak activity to stimulate the GTPase activity of Rab3A and a strong activity to inhibit the Rab3A GTPase‐activating protein (GAP)‐stimulated GTPase activity of Rab3A. Here, we show that Rabphilin‐3A has another activity to stimulate the GDP/GTP exchange reaction of Rab3A. Rabphifin‐3A may keep Rab3A continuously in the GTP‐bound form by converting again GDP‐Rab3A, which may be converted from GTP‐Rab3A by Rab3A GAP, to GTP‐Rab3A, until the function of Rab3A is accomplished.
Calmodulin is intrinsically LESS effective than troponin C in activating skeletal muscle contraction
Authors: Brandt Philip W. , George Samuel E. , Schachat Fred
Published: July 2002

Calmodulin (CaM) and troponin C (TnC) are evolutionarily and structurally homologous, yet they are not functionally interchangeable. In particular, CaM cannot effectively substitute for TnC as an activator of skeletal muscle contraction. To determine if this is a consequence of CaM's weak association with troponin T and I or the result of a more fundamental mechanistic defect, we have used CaM and a CaM[TnC] chimera, CaM[3,4 TnC], that stably associates with the thin filament. Replacement of TnC with CaM or CaM[3,4 TnC] reveals that CaM‐like molecules reduce the Ca2+‐sensitivity and cooperativity of activation, as well as the maximal Ca2+‐activated tension. These observations indicate that CaM‐like molecules are unable to continuously maintain the activated state of the thin filament.
CCK‐JMV‐180 acts as an antagonist of the CCKA receptor in the human IMR‐32 neuroblastoma cell line
Authors: Schaeffer P. , Prabonnaud V. , Roux M. , Gully D. , Herbert J.M.
Published: July 2002

[25I]Cholecystokinin‐8‐S (CCK‐8‐S) bound to a single class of saturable binding sites on the human neuroblastoma cell line IMR‐32 (K
D = 4 ± 1.5 nM, B
max = 10,500 ± 3,500 sites/cell (n = 6)). These binding sites were of the CCKA type, as demonstrated by the differential inhibition of the binding of [125I]CCK‐8‐S and CCK‐8‐S‐induced 45Ca2+ efflux by the specific CCKA antagonist SR 27897 and the specific CCKB antagonist PD 134,308. CCK‐JMV‐180, an analogue of CCK‐8‐S which has been shown to activate 45Ca2+ efflux in rat cells in a manner similar to CCK‐8‐S, acted as a potent antagonist of CCK‐8‐S‐induced 45Ca2+ efflux (IC50 = 50 nM) and inhibited [125I]CCK‐8‐S binding to IMR‐32 cells (IC50 = 1.7 nM). These results show that, unlike its CCK‐like effect in various animal systems, CCK‐JMC‐180 acts as an antagonist of CCKA receptors in the human neuroblastoma cell line IMR‐32.
Heterologous expression of an active rat regulatory protein of glucokinase
Authors: Detheux Michel , Van Schaftingen Emile
Published: July 2002

The cDNA presumed to encode the rat liver regulatory protein of glucokinase has been expressed in Escherichia coli and a partially soluble protein has been obtained. This recombinant protein was partially purified and found to have the same apparent molecular mass as the regulatory protein purified from rat liver. Like the latter, it inhibited rat liver glucokinase competitively with respect to glucose and its effect was sensitive to fructose 6‐phosphate and fructose 1‐phosphate.
In vivo and in vitro effects of mutagenesis of active site tyrosine residues of mercuric reductase
Authors: Rennex Denise , Pickett Mark , Bradley Mark
Published: July 2002

X‐ray crystal structure analysis of mercuric reductase suggested that the binding site for Hg2+ consisted of two tyrosine residues, Tyr264 and Tyr605, as well as two cysteine residues, Cys207 and Cys628. We have previously shown that mutagenesis of Tyr605 to Phe lowered the k
cat of the enzyme 6‐fold, whereas the same mutation of Tyr264 resulted in a reduction of 160‐fold [(1993) Biochemistry 32, 7475–7478]. Tyr605 occupies the same position in mercuric reductase as the active site His residue in the related enzyme glutathione reductase. The mutation of Tyr605 of mercuric reductase to a His residue produced a 24‐fold decrease in k
cat and a 15‐fold decrease in k
cat and a 15‐fold decrease in K
m. The in vivo resistance to Hg2+ of E. coli strains carrying wild type or mutant mer A genes correlated with the in vitro measurements of k
cat/K
m for mercuric reductase activity.
Regulation of low density lipoprotein receptor activity in Chinese hamster ovary cells transfected with the c‐fms gene
Authors: Yamamoto Koji , Shimano Hitoshi , Inaba Toshimori , Gotoda Takanari , Harada Kenji , Shimada Masako , Watanabe Yoshiro , Osuga Jun-ichi , Kawamura Masako , Yazaki Yoshio , Yamada Nobuhiro
Published: July 2002

Chinese hamster ovary (CHO) cells were transfected with the human c‐fms gene, which encodes the receptor for macrophage colony‐stimulating factor, to examine the effects on the low density lipoprotein (LDL) receptor activity. Degradation of [125I]LDL was significantly reduced in the transfected CHO cells as compared to non‐transfected cells when incubated in lipoprotein‐deficient serum. Quantitative analysis of LDL receptor mRNA using a competitive PCR method demonstrated that LDL receptor suppression occurred at transcription. These findings suggest that expression of the c‐fms gene is involved in the regulation of LDL receptors on macrophages.
Autoantigen Ku protein is involved in DNA binding proteins which recognize the U5 repressive element of human T‐cell leukemia virus type I long terminal repeat
Authors: Okumura Koichi , Takagi Shin , Sakaguchi Gaku , Naito Kazumi , Minoura-Tada Naoko , Kobayashi Hironao , Mimori Tsuneyo , Hinuma Yorio , Igarashi Hisanaga
Published: July 2002

We have identified and analyzed a 27‐nucleotide sequence (U5 repressive element, designated as U5RE) at the U5 region of the human T‐cell leukemia virus type I (HTLV‐I) long terminal repeat (LTR) which is required for HTLV‐I basal transcriptional repression. The basal promoter strength of constructs that contained deletions in the U5 region of the LTR was analyzed by chloramphenicol acetyltransferase (CAT) assays following transfection of HeLa cells or Jurkat T‐cells in the presence or absence of viral transactivator tax protein. We consistently observed a 2‐to 5‐fold increase in basal promoter activity when sequences between +277 to +306 were deleted. In vivo competition experiments suggested that the U5 DNA fragment from +269 to +295 contains a functional repressive element (USRE). Using gel mobility shift assays, we have purified a highly enriched fraction that could specifically bind U5RE. This DNA affinity column fraction contained three major detectable proteins on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis with silver staining: 110‐, 80‐ and 70‐kDa proteins. The 110‐kDa protein appeared to be a novel DNA‐binding protein whose characteristics are still obscure, while the 70‐ and 80‐kDa proteins were shown to be related to the human autoantigen Ku, the Ku (p70/p80) complex, as demonstrated by amino acid sequencing and immunological analyses. As Ku is known to be involved in transcriptional regulation, the specific interaction of Ku with U5RE raises intriguing possibilities for its function in HTLV‐I basal transcriptional repression.
Cloning of the Na+‐translocating NADH‐quinone reductase gene from the marine bacterium Vibrio alginolyticus and the expression of the β‐subunit in Escherichia coli
Authors: Hayashi Maki , Hirai Kouji , Unemoto Tsutomu
Published: July 2002

The Na+‐translocating NADH‐quinone reductase purified from the marine bacterium Vibrio alginolyticus is composed of three subunits, α, β and γ. From the N‐terminal amino acid sequences of each subunit and its polypeptide fragment obtained by partial digestion with V8 protease, oligonucleotides corresponding to forward and reverse primers for each gene (NQR A, B and C) encoding the α, β and γ subunit, respectively, were synthesized. Using these primers, a part of each gene was amplified from the chromosomal DNA of V. alginolyticus by a PCR method, and the PCR products were used for the cloning of the NQR gene in λ phage. Among the subclones selected by probe C, the expression of the β‐subunit as a gene product was detected in Escherichia coli membranes by activity staining and Western blotting.
Formation of sulphmyoglobin during expression of horse heart myoglobin in Escherichia coli
Authors: Lloyd Emma , Mauk A.Grant
Published: July 2002

Expression of recombinant horse heart myoglobin in Escherichia coli has been found to result in the production of both native and variable amounts (~ 16–17% total) of two sulphmyoglobin isomers. The recombinant sulphmyoglobin produced consists primarily of the A and B isomers as identified by 1H NMR spectroscopy with no evidence for production of the C isomer. Conversion of recombinant sulphmyoglobin to the native protein can be achieved by reconstitution with protohaem IX. The possible relationship of this observation to recombinant expression of other heme proteins is discussed.
Simvastatin‐sodium delays cell death of anoxic cardiomyocytes by inhibition of the Na+/Ca2+ exchanger
Authors: Bastiaanse E.M.Lars , Atsma Douwe E. , Kuijpers Marinette M.C. , Van Der Laarse Arnoud
Published: July 2002

When incubated under anoxic conditions, cultured neonatal cardiomyocytes undergo cell necrosis. Simvastatin‐sodium, the bioactive metabolite of simvastatin (a potent serum cholesterol‐lowering drug), delayed the anoxia‐induced myocyte necrosis in a dose‐dependent manner. This beneficial effect of simvastatin‐sodium could not be attributed to its cholesterol‐lowering properties. We found that simvastatin‐sodium, at concentrations of 20 and 50 μM, attenuated the rise in intracellular Ca2+ concentration ([Ca2+]i) measured with Fura‐2 in anoxic cardiomyocytes. In a test of sarcolemmal Na+/Ca2+ exchange activity, simvastatin‐sodium attenuated the rise of[Ca2+]i upon incubation in sodium‐free buffer, which normally causes a reversal of Na+/Ca2+ exchange and cellular calcium overload. The inhibitory action of simvastatin‐sodium on the sarcolemmal Na+/Ca2+ exchanger could well explain the cardioprotective effect of the drug on myocytes subjected to anoxia.
d‐Ribose stabilizes precursor and mature ribose‐binding proteins of Escherichia coli
Authors: Lee Heeyong , Kim Hyoungman
Published: July 2002

Heat‐ and guanidine hydrochloride‐induced unfolding and refolding of precursor as well as mature ribose‐binding proteins of Escherichia coli were studied in the presence of d‐ribose using intrinsic tyrosine fluorescence and circular dichroism. The precursor and mature proteins have shown virtually identical unfolding‐folding behavior. It was observed that d‐ribose refolds partially unfolded precursor and mature ribose binding proteins into native structure and decreases the unfolding rate of the these proteins. The conformational stabilities of these proteins were found to increase with increasing d‐ribose concentration.
Diurnal expression of NGF1‐A mRNA in retinal degeneration slow (rds) mutant mouse retina
Authors: Agarwal Neeraj
Published: July 2002

The retinal degeneration slow (rds) mutant mouse is a model for studying the retinal dystrophy for human disease, retinitis pigmentosa (RP). To continue our effort towards a possible mechanism of photoreceptor cell death in retinal dystrophies, we have studied the impact of the rds mutation on diurnal expression of a ‘zinc‐finger’ DNA‐binding protein, NGF1‐A mRNA in the isolated retinas ofrds mutant mice compared to those of BALB/c mice. Background levels of NGF1‐A mRNA were maintained during the subjective light period. Higher levels of NGF1‐A mRNA were observed immediately after the light offset and peaked two hours into the light offset for both the BALB/c and the rds mutant retinas and remained higher for several hours in the dark. If the animals were left continuously in light during the subjective dark period, NGF1‐A mRNA levels were not induced and remained lower. On the other hand NGF1‐A mRNA levels were transiently induced during the transition of the dark‐to‐light phase. These data suggest that NGF1‐A mRNA is differentially regulated by light and dark stimuli in the retina and an absence of rod outer segments in the rds mutant retina does not alter the normal diurnal cycle of NGF1‐A mRNA expression.
Changes of the expression of protein substrates of Ca2+/calmodulin‐dependent protein kinase II in neonate and adult rats
Authors: Sugiura Hiroko , Yamauchi Takashi
Published: July 2002

We studied the expression of whole protein substrates of Ca2+/calmodulin‐dependent protein kinase II (CaM kinase II) in the forebrain of neonate and adult rats. Protein substrates were determined by phosphorylation of the soluble and particulate fractions by CaM kinase II with [γ‐33P]ATP. Phosphorylated proteins were analyzed by SDS‐PAGE and two‐dimensional gel electrophoresis. More than 50 endogenous proteins were found to be phosphorylated by CaM kinase II in both soluble and particulate fractions. The expression of about 15 protein substrates increased in the particulate fraction from neonate to adult rats, and that of several proteins also changed in the soluble fraction. These findings suggest that the expression of protein substrates was regulated during development as well as that of CaM kinase II itself.
Transcription factor GATA‐1‐multiprotein complexes and chicken erythroid development
Authors: Penner C.Gail , Davie James R.
Published: July 2002

The chicken erythrocyte transcription factor, GATA‐1, is associated with several non‐DNA binding proteins. We show that GATA‐1 multiprotein complexes exist in primitive and definitive erythrocytes. These complexes bind to GATA motifs of the p‐globin promoter and histone H5 enhancer with high affinity, and to the chicken β‐globin promoter specialized TATA element and enhancer GATA with low affinity. The low affinity β‐globin TATA element would allow basal transcription factors to displace the GATA‐1 multiprotein complex. Further, our results suggest that p‐globin promoter's low affinity Sp1 binding site and reduced levels of Sp1 in definitive cells prevent its expression in these cells.
Purification and characterization of a ubenimex (Bestatin)‐sensitive aminopeptidase B‐like enzyme from K562 human chronic myeloid leukemia cells
Authors: Yamada Masatoshi , Sukenaga Yoshikazu , Fujii Hideji , Abe Fuminori , Takeuchi Tomio
Published: July 2002

A ubenimex‐sensitive aminopeptidase B‐like enzyme was purified from the non‐membrane‐bound fraction of K562 cells by a series of chromatographic procedures and slab‐gel electrophoresis. The apparent molecular mass of the enzyme was estimated to be 73 kDa by SDS‐PAGE. The aminopeptidase activity was activated by chloride ions and inhibited by Zn2+, Cu2+, Cd2+, and p‐chloromercuribenzoic acid. Ubenimex was a potent inhibitor of this aminopeptidase in the nanomolar range. The sequence of the N‐terminus of the protein was not determined. Partial amino acid sequencing revealed that the N‐terminus of this aminopeptidase B‐like enzyme was blocked by acylation. The partial sequences of the two fragments produced by CNBr cleavage and an acylamino acid‐releasing reaction showed this enzyme to be a new aminopeptidase.
Biochemical, structural, and biological properties of human thioredoxin active site peptides
Authors: Oblong John E. , Berggren Margareta , Powis Garth
Published: July 2002

The human redox protein thioredoxin is an autocrine growth factor for some cancer cells. Redox activity is essential for this function but other required structural features of thioredoxin are not known. Two 8‐mer peptides (I and II) and one 14‐mer peptide (III) were designed based on the amino acid sequence of the redox active site of thioredoxin. Peptide I and peptide III contained the wild‐type sequence of thioredoxin while peptide II contained serine residues in place of the catalytically active cysteines. Circular dichroism spectroscopy indicated that all three peptides were comprised mainly of random coil, with peptide III containing slightly more ordered secondary structure. Peptides I and III were substrates for thioredoxin reductase with K
M values of 890 and 265 μM, respectively. The redox inactive peptide II could not compete with thioredoxin for reduction by thioredoxin reductase in a coupled insulin reduction assay. However, peptide II was a competitive inhibitor for the reduction of 5,5'‐dithiobis‐(2‐nitrobenzoic acid) by thioredoxin reductase. All three peptides gave only background levels of stimulation of the proliferation of Swiss 3T3 murine fibroblasts when compared to the stimulation caused by thioredoxin. These results suggest that while the ability of thioredoxin to stimulate cellular proliferation is redox‐dependent, more information than that contained in the redox active site domain alone defined by 14 amino acids is required.
Import of a mitochondrial presequence into P. denitrificans
Authors: Roise David , Maduke Merritt
Published: July 2002

According to the endosymbiont hypothesis, mitochondria are descended from ancient aerobic bacteria that were engulfed by protoeukaryotic cells. Experiments described here show that a synthetic peptide corresponding to a yeast mitochondrial targeting sequence can be imported into Paracoccus
denitrificans, a soil bacterium thought to be closely related to the protomitochondrion. The import is very similar to that observed with isolated yeast mitochondria. The results suggest that the protomitochondrion may have been inherently able to translocate mitochondrial presequences. This ability may partly explain the development of the protein import process during the evolution of the mitochondrion.
Mitochondrial ATP hydrolysis and ATP depletion in thymocytes and Ehrlich ascites carcinoma cells
Authors: Chernyak B.V. , Dedov V.N. , Gabai V.L.
Published: July 2002

When Ehrlich ascites carcinoma (EAC) cells and thymocytes were treated with uncoupler or rotenone in glucose‐free medium, rapid ATP depletion was observed in both types of the cells. Oligomycin slowed down ATP loss in thymocytes, but not in EAC cells. Thus, mitochondrial ATP hydrolysis appears to be significant in deenergized thymocytes in contrast to EAC cells, in which other ATP consuming reactions were prevailing. Complete deenergization of mitochondria by uncoupler or rotenone in these cells resulted in inactivation of mitochondrial ATPase by 65–75%. The effect was observed after complete and rapid (20–30 s) disruption of the cells with detergent, Lubrol WX. ATPase was blocked by the specific inhibitor protein (IF1) as it was shown by the studies on reactivation of this enzyme. When respiration is blocked but ATP content is supported by glycolysis, mitochondrial ATPase is not suppressed by IF1, and maintains the energization of mitochondria. It is concluded that under complete de‐energization of mitochondria IF1, significantly inhibits mitochondrial ATP hydrolysis and may slow down ATP loss in thymocytes and EAC cells.
Site‐directed mutagenesis of two conserved charged amino acids in the N‐terminal region of α subunit of E. coli‐F0F1
Authors: Hase Barbara , Werner-Grüne Sabine , Deckers-Hebestreit Gabriele , Strotmann Heinrich
Published: July 2002

Two conserved charged amino acids of the N‐terminal ‘crown’ region of the α subunit of E. coli‐F1, α‐D36 and α‐R40 were exchanged for chemically related (α‐D36→K, α‐R40→K) of unrelated amino acids (α‐D36→K, α‐R40→G), respectively, by employing oligonucleotide‐directed mutagenesis. ATP formation and ATP hydrolyzing activity of isolated plasma membrane vesicles was strongly inhibited in mutant HS2 (α‐D36→K), but only slightly affected in the other mutants. The inhibition is not due to a lower content of F0F1 in HS2. In this mutant the extent of the proton gradient generated by ATP hydrolysis was more than 80% inhibited; in all other transformants much smaller effects were observed. The proton gradient established by NADH oxidation was 33% decreased in HS2, but was decreased to a lesser extent in all other mutants. After blockage of F0 by DCCD treatment, the same NADH‐induced proton gradient was obtained in all transformants including HS2. This and the fact that the activity of NADH oxidation was unchanged indicate increased proton leakiness of F0F1 carrying the α‐D36αK mutation. In F1 α‐D36 is located in a domain contacting the β subunit in the vicinity of the arginine β‐R52. The effect of α‐D36→K replacement on catalysis and coupling thus may be due to an electrostatic repulsive effect in the crown region which alters the α and β subunit interaction.
Highly efficient production of GFP and its derivatives in insect cells for visual in vitro applications
Authors: Oker-Blom Christian , Orellana Adelina , Keinanen Kari
Published: July 2002

We have generated recombinant baculoviruses for expression of the green fluorescent protein (GFP), a bright GFP mutant (S65T), and a GFP‐streptavidin fusion protein in Sf9 and High Five insect cell lines. At 3–4 days post infection, about 30% of the total protein contents was represented by the recombinant protein products, giving the infected insect cells a bright green color which was clearly visible by eye in daylight. The isolated GFP‐streptavidin fusion protein, which possessed fluorescence properties identical to those of the native GFP, was capable of binding biotin as shown by using biotinylated beads as well as biotinylated antibody complexes decorating surface expressed GluR‐6 glutamate receptor in live and fixed insect cells. The exceptionally high expression levels of GFP and GFP (S65T) and the GFP‐streptavidin fusion protein in recombinant baculovirus infected insects should facilitate production of GFP derivatives for in vitro applications.
Cloning and sequencing of winged bean (Psophocarpus tetragonolobus) basic agglutinin (WBA I): presence of second glycosylation site and its implications in quaternary structure
Authors: Sharma Vivek , Srinivas V.R. , Surolia Avadhesha
Published: July 2002

We report cloning of the DNA encoding winged bean basic agglutinin (WBA I). Using oligonucleotide primers corresponding to N‐ and C‐termini of the mature lectin, the complete coding sequence for WBA I could be amplified from genomic DNA. DNA sequence determination by the chain termination method revealed the absence of any intervening sequences in the gene. The DNA deduced amino acid sequence of WBA I displayed some differences with its primary structure established previously by chemical means. Comparison of the sequence of WBA I with that of other legume lectins highlighted several interesting features, including the existence of the largest specificity determining loop which might account for its oligosaccharide‐binding specificity and the presence of an additional N‐glycosylation site. These data also throw some light on the relationship between the primary structure of the protein and its probable mode of dimerization.