bluuebunny/crossref_metadata_2025 huggingface dataset:
Occurrence of thiamin pyrophosphate‐dependent 2‐oxoglutarate decarboxylase in mitochondria of Euglena gracilis
Authors: Shigeoka Shigeru , Onishi Toshio , Maeda Kozi , Nakano Yoshihisa , Kitaoka Shozaburo
Published: July 2002

2‐Oxoglutarate decarboxylase which catalyzes the conversion of 2‐oxoglutarate into succinate semialdehyde occurs in mitochondria of Euglena gracilis which lacks a 2‐oxoglutarate dehydrogenase complex. The enzyme reaction required thiamin pyrophosphate, MgCl2, 2‐mercaptoethanol and NADP+ for the maximum activity, and was not affected by pyruvate and oxalacetate. In the reaction, the enzyme consumed 2‐oxoglutarate, evolved CO2 and formed succinate semialdehyde in stoichiometric relationship. The maximum enzyme activity was found at pH 7.0 and 40° C, and K
m values for 2‐oxoglutarate and thiamin pyrophosphate were 0.33 and 0.056 mM, respectively. These results indicate that the thiamin pyrophosphate‐dependent Euglena decarboxylase belongs to a new type of decarboxylase to be designated as 2‐oxoglutarate decarboxylase. The probable role of the new decarboxylase in Euglena mitochondria is discussed with regard to the tricarboxylic acid cycle.
Amino acid substitutions in mitochondrial ATPase subunit 9 of saccharomyces cerevisiae leading to oligomycin or venturicidin resistance
Authors: Nagley Phillip , Hall Ruth M. , Ooi Beng Guat
Published: July 2002

A series of isonuclear oligomycin‐resistant mutants of Saccharomyces cerevisiae carrying mutations in the mitochondrial olil gene has been studied. DNA sequence analysis of this gene has been used to define the amino acid substitutions in subunit 9 of the mitochondrial ATPase complex. A domain of amino acids involved in oligomycin resistance can be recognized which encompasses residues in each of the two hydrophobic portions of the subunit 9 polypeptide that are thought to span the inner mitochondrial membrane. Certain amino acid substitutions also confer cross‐resistance to venturicidin: these residues define an inner domain for venturicidin resistance. The expression of venturicidin resistance resulting from one particular substitution is modulated by nuclear genetic factors.
Viable somatic hybrids are obtained by direct current electrofusion of chemically aggregated plant protoplasts
Authors: Chapel Marc , Montane Marie-Hélène , Ranty Benoit , Teissie Justin , Alibert Gilbert
Published: July 2002

Using low levels of polyethylene glycol as aggregating agent, protoplasts of Nicotiana glauca and N. langsdorffii were electropulsed (square wave direct current electric pulses) and on subsequent culture on a selective medium (without phytohormones), calli developed. The hybrid character of some of the calli was demonstrated by direct comparison of the isoelectrophoretic pattern of the ribulose‐1, 5‐bisphosphate carboxylase subunits with those of the parent plants. In optimum conditions, the electrofusion technique described is about 30‐fold more efficient for hybrid production than the conventional polyethylene glycol method when applied to the same plant systems.
Characterization of central melatonin receptors using 125I‐melatonin
Authors: Laudon Moshe , Zisapel Nava
Published: July 2002

The binding of 125I‐melatonin, a potent analog of melatonin, to rat brain synaptosomal preparations was investigated. 125I‐melatonin bound with high affinity (K
d = 38 nM) to a single class of sites (B
max = 81 protein). Kinetic studies indicated that binding was time‐dependent and reversible. Specific 125I‐melatonin binding was inhibited by melatonin, and was unaffected by other structurally related compounds including serotonin. Binding of 125I‐melatonin was greatly reduced if the synaptosomal preparations were pretreated by heat or trypsin but was unaffected by freeze‐thawing. These results suggest that 125I‐melatonin may serve as a valuable probe for studying melatonin receptors.
Type VI collagen is a major component of the human cornea
Authors: Zimmermann Dieter R. , Trüeb Beat , Winterhalter Kaspar H. , Witmer Rudolf , Fischer René W.
Published: July 2002

Collagen type VI is shown to be present in the human cornea. This finding is based on comparative peptide mapping relative to type VI collagen isolated from placenta and on immunoblotting using antibodies specific for human type VI collagen. Scanning of polyacrylamide gels indicates that type VI collagen comprises as much as one quarter of the dry weight of the cornea. Indirect immunofluorescence shows this collagen to be distributed throughout the corneal stroma. Thus, type VI collagen must be considered a major component of the extracellular matrix of the human cornea.
The conformation of apamin
Authors: Freeman C.M. , Catlow C.R.A. , Hemmings A.M. , Hider R.C.
Published: July 2002

Energy minimisation techniques are used as a tool to distinguish between different proposed models for the structure of the bee venom polypeptide apamin. The influence of electrostatic interactions on the resultant energies is noted. The model of Hider and Ragnarsson [(1980) FEBS Lett. 111, 189‐193] is found to be of consistently low energy.
Structural study of a mutant type I collagen from a patient with lethal osteogenesis imperfecta containing an intramolecular disulfide bond in the triple‐helical domain
Authors: Traub Wolfie , Steinmann Beat
Published: July 2002

We have built molecular models of collagen type I from a patient with lethal osteogenesis imperfecta incorporating one or two mutant α1(I)‐chains which contain a cysteine substituting a glycine near the C‐terminal end. In either case, the cysteines can only be accommodated with considerable distortion of the native collagen structure, which disrupts inter‐chain contacts. The disturbance of the triple helix is limited to a small local region. This suggests that the most important consequence of the mutation is delayed helix formation leading to overmodification and decreased collagen production, rather than the structural abnormality of the folded molecules, which are only marginally unstable.
Investigation of human plasma low density lipoprotein by three‐dimensional flourescence spectroscopy
Authors: Koller Ernst , Quehenberger Oswald , Jürgens Günther , Wolfbeis Otto S. , Esterbauer Hermann
Published: July 2002

Human plasma LDL exhibits a diffuse fluorescence (excitation 360 nm) in the 400–600 nm range. Application of three‐dimensional fluorescence spectroscopy shows the presence of 7 fluorophores in the lipid and 6 fluorophores in the protein domain. The 430 nm fluorescence in freshly prepared LDL and its apo‐B is most likely indicative for remnants of in vivo lipid peroxidation.
Pyruvate utilization by synaptosomes is independent of calcium
Authors: Kauppinen Risto A. , Nicholls David G.
Published: July 2002

The significance of Ca2+ is assessed for the activation of pyruvate oxidation by intact nerve terminals (synaptosomes). Titration of glucose‐depleted synaptosomes with pyruvate in the presence of either veratridine or uncoupler stimulates respiration in a Ca2+‐independent manner. Additionally, the ability of exogenous pyruvate to support the mitochondrial membrane potential in situ is independent of Ca2+. It is concluded that Ca2+ does not regulate pyruvate oxidation in intact synaptosomes.
Mechanism of glucagon stimulation of fructose‐1,6‐bisphosphatase in rat hepatocytes
Authors: Casteleijn Eric , van Rooij Henri C.J. , van Berkel Theo J.C. , Koster Johan F.
Published: July 2002

Fructose‐1,6‐bisphosphatase
Glucagon
Low‐Mr activator
(Rat hepatocyte)
DNA sequence preferences for the anti‐cancer drug mitoxanthrone and related anthraquinones revealed by DNase I footprinting
Authors: Fox K.R. , Waring M.J. , Brown J.R. , Neidle S.
Published: July 2002

The interaction has been studied of several anthraquinone‐based intercalating drugs, including the anti‐cancer agent mitoxantrone, with defined sites of DNA. A 160 base pair DNA sequence from tyrT was employed for footprinting with DNase I. The anthraquinones had aminoalkylamino substituents in various positions of the ring system. Inhibition of enzymatic cutting of the DNA was observed at various positions on the sequence, mostly around some of the pyrimidine‐3',5'‐purine sites. Enhancements to cutting were observed clustered around AT‐rich regions. The compounds showed differences in detailed footprinting behaviour, which have been related to differences in their mode of interaction with DNA as found in earlier computer modelling studies.
Expression, secretion and processing of hirudin in E. coli using the alkaline phosphatase signal sequence
Authors: Dodt Johannes , Schmitz Thomas , Schäfer Thomas , Bergmann Cornelia
Published: July 2002

A DNA fragment coding for the E. coli phoA signal peptide was synthesized and inserted into the expression vector pKK223‐3. A single HindIII restriction site is located just at the end of the signal sequence. A gene coding for the proteinase inhibitor hirudin, which has previously been synthesized, was inserted into this HindIII site. The hybrid protein was expressed under control of the tac‐promoter and secreted into the periplasm of E. coli. From the periplasmic fraction two processed proteins were isolated. One of these was identical with desulfatohirudin and also had similar biological properties.
The possible role of protein kinase C and phospholipids in the regulation of steroid production in rat Leydig cells
Authors: Themmen Axel P.N. , Hoogerbrugge Jos W. , Rommerts Focko F.G. , van der Molen Henk J.
Published: July 2002

We have studied the possible involvement of the activation of calcium‐dependent phospholipid‐activated protein kinase (PK‐C) in the stimulatory action of LHRH on Leydig cells, using 4β‐phorbol‐12‐myristate‐13‐acetate (PMA) and phospholipase C (PL‐C). LHRH agonist (LHRH‐A) and PL‐C had a large synergistic effect on LH‐stimulated steroid production, whereas PMA inhibited the effect of LH. However, PMA always caused an increase in steroid production stimulated by various doses of dibutyryl cAMP. LH and PMA stimulated the phosphorylation of 17 and 33 kDa proteins, whereas LHRH‐A and PL‐C had no effect. Of all effectors used, LH had the most pronounced effect on the synthesis of 14, 27 and 30 kDa proteins. The present results suggest that the mechanisms of action of LHRH‐A and PL‐C on steroid production in Leydig cells may be similar and different from PMA, and may involve stimulation of a specific type of PK‐C or hydrolysis of a specific pool of phospholipids.
Structure of Rhodopseudomonas sphaeroides R‐26 reaction center
Authors: Chang C.-H. , Tiede D. , Tang J. , Smith U. , Norris J. , Schiffer M.
Published: July 2002

The molecular replacement method has been succesfully used to provide a structure for the photosynthetic reaction center of Rhodopseudomonas sphaeroides at 3.7 Å resolution. Atomic coordinates derived from the R. viridis reaction center were used in the search structure. The crystallographic R‐factor is 0.39 for reflections between 8 and 3.7 Å. Validity of the resulting model is further suggested by the visualization of amino acid side chains not included in the R. viridis search structure, and by the arrangements of the reaction centers in the unit cell. In the initial calculations quinones or pigments were not included; nevertheless, in the resulting electron density map, electron density for both quinones qa and qb appears along with the bacteriochlorophylls and bacteriopheophytins. Kinetic analysis of the charge recombination shows that the secondary quinone is fully functional in the R. sphaeroides crystal.
Evidence that TraT interacts with OmpA of Escherichia coli
Authors: Riede Isolde , Eschbach Marie-Luise
Published: July 2002

The OmpA protein is one of the major outer membrane proteins of Escherichia coli. Among other functions the protein serves as a receptor for several phages and increases the efficiency of F‐mediated conjugation when present in recipient cells. TraT is an F‐factor‐coded outer membrane lipoprotein involved in surface exclusion, the mechanism by which E. coli strains carrying F‐factors become poor recipients in conjugation. To determine a possible interaction of TraT with OmpA, the influence of TraT on phage binding to cells was measured. Because TraT inhibits inactivation of OmpA‐specific phages it is suggested that TraT interacts directly with OmpA. Sequence homology of TraT with proteins 38, the phage proteins recognizing outer membrane proteins, supports this finding. A model of protein interactions is discussed.
Effect of chronic ethanol consumption on microsomal lipid peroxidation
Authors: Krikun Graciela , Cederbaum Arthur I.
Published: July 2002

Microsomes isolated from chronic ethanol‐fed rats displayed elevated rates of malondialdehyde production when compared to pair‐fed controls, but lower rates when compared to chow‐fed controls. These differences did not correlate with total content of cytochrome P‐450 or activity of NADPH‐cytochrome c reductase. Titration curves with the potent iron‐chelating agent desferrioxamine revealed that the content of iron was greater in microsomes from the chow‐fed and lowest in microsomes from the pair‐fed control. However, other variables must also exist since even when excess iron was added to the microsomes, the order of malondialdehyde production remained chow‐fed > chronic ethanol > pair‐fed control. The variabilities associated with the different controls and the role and content of transition metals such as iron probably contribute towards the divergent effects of ethanol on lipid peroxidation.
Inhibition of a calcium‐activated, non‐selective cation channel, in a rat insulinoma cell line, by adenine derivatives
Authors: Sturgess Nicholas C. , Nicholas Hales C. , Ashford Michael L.J.
Published: July 2002

The effects of adenosine and adenine nucleotides on a calcium‐activated non‐selective cation channel, present in the plasma membrane of an insulin‐secreting cell line CRI‐G1 were investigated. Single‐channel currents were recorded from inside‐out membrane patches and the adenine derivatives applied to the solution bathing the cytoplasmic aspect of the membrane surface. The activity of this channel is shown to be inhibited by all the derivatives tested. The potency sequence for inhibition was found to be AMP > ADP > ATP > adenosine.
Plasminogen activator inhibitor type‐1 : reactive center and amino‐terminal heterogeneity determined by protein and cDNA sequencing
Authors: Andreasen P.A. , Riccio A. , Welinder K.G. , Douglas R. , Sartorio R. , Nielsen L.S. , Oppenheimer C. , Blasi F. , Danø K.
Published: July 2002

Both the urokinase‐type and tissue‐type plasminogen activator can convert their 5̃4 kDa type‐1 inhibitor (PAI‐1) to an inactive form with a lower apparent molecular mass. We have determined the amino‐terminal amino acid sequences of human native and converted PAI‐1, and isolated PAI‐1 cDNA and determined the nucleotide sequence in regions corresponding to the amino‐terminus and the cleavage site. The data show that the conversion of the inhibitor consists of cleavage of an Arg‐Met bond 33 residues from the carboxy‐terminus, thus localizing the reactive center of the inhibitor to that position. In addition, a heterogeneity was found at the amino‐terminus, with a Ser‐Ala‐Val‐His‐His form and a two‐residue shorter form (Val‐His‐His‐) occurring in approximately equal quantities.
Tick‐borne encephalitis virus genome
Authors: Pletnev A.G. , Yamshchikov V.F. , Blinov V.M.
Published: July 2002

RNA of a flavivirus, tick‐borne encephalitis virus (TBEV; strain Sofjin), was subjected to reverse transcription and the DNA copy was transformed into double‐stranded DNA by the action of E. coli DNA‐polymerase I (Klenow fragment). This DNA was annealed with plasmid pBR322. The recombinant plasmids were cloned in E. coli K802. The nucleotide sequence of the inserts of the clones, coding for region structural proteins C, M, E and nonstructural protein NS1, was determined by the Maxam‐Gilbert method. The genes of structural proteins form a compact cluster. Homology has been studied of the TBEV sequences found with the structures of proteins and RNAs of other flaviviruses, yellow fever virus and West Nile virus, and a high degree of homology was found.
Influence of lipid peroxidation on β‐adrenoceptors
Authors: Kramer K. , Rademaker B. , Rozendal W.H.M. , Timmerman H. , Bast A.
Published: July 2002

The peroxidation of lipids in biological membranes is a destructive phenomenon that can be elicited in various ways. Surface receptor molecules that allow cells to respond to hormones are possibly inactivated during lipid peroxidation. Effects of lipid peroxidation on receptors have not been extensively examined thus far. This investigation shows that there is a decrease in β‐adrenoceptor density (measured as specific (—)‐[125I]iodocyanopindolol binding) during lipid peroxidation, in both lungs and erythrocytes of the rat. To this end, lung membranes (containing both β1‐ and β2‐adrenoceptors) and intact erythrocytes (containing a homogeneous β2‐adrenoceptor population) were pretreated with cumene hydroperoxide (lung membranes with 0.1 mM and erythrocytes with 1 mM) and Fe2+ (1 × 10−5 M) for 60 min which resulted in extensive lipid peroxidation measured as malondialdehyde formation. The ratio β1‐:β2‐adrenoceptor density in lung membranes after treatment with cumene hydroperoxide did not change and remained at 30%:70%. A single injection (i.p.) with the herbicide paraquat (50 , 24 h), which is known to cause lung damage via lipid peroxidation, resulted in similar alterations in receptor density to those caused by cumene hydroperoxide in the in vitro experiments.
Cytochrome P‐450‐dependent H2O2 production demonstrated in vivo
Authors: Premereur Noël , Van den Branden Christiane , Roels Frank
Published: July 2002

By administration of allylisopropylacetamide, an inhibitor of cytochrome P‐450, we demonstrated that cytochrome P‐450 is involved in the production of H2O2 during aminopyrine metabolism and phenobarbital induction in both the unanaesthetized guinea pig and rat. In the guinea pig we also found evidence for the existence of a basal cytochrome P‐450‐dependent H2O2 production, i.e. in the absence of exogenous substrate. Catalase participates in the decomposition of H2O2 produced in the endoplasmic reticulum where cytochrome P‐450 is localized.
A possible involvement of cyclic AMP in the expression of desensitization of the nicotinic acetylcholine receptor
Authors: Albuquerque E.X. , Deshpande S.S. , Aracava Y. , Alkondon M. , Daly J.W.
Published: July 2002

Forskolin, an activator of adenylate cyclase, and its analogs were studied on the nicotinic acetylcholine receptor‐ion channel complex (AChR) of rat and frog skeletal muscles. At nanomolar concentrations, forskolin caused desensitization of the AChR located at the junctional region of innervated and the extrajunctional region of chronically denervated rat soleus muscles. The desensitization of the AChR occurred without alteration of the conducting state (channel lifetime, conductance or bursting) as shown by single channel currents. Accordingly, forskolin decreased the peak amplitude of the repetitive evoked endplate currents in frog sartorius muscles. These findings taken together with the good correlation found between the effects of forskolin and its analogs on the desensitization of the nicotinic AChR and their ability to activate adenylate cyclase suggested a possible involvement of phosphorylation of AChR via cyclic AMP on the desensitization process.
Regulation of glycerol 3‐phosphate oxidation in mitochondria by changes in membrane microviscosity
Authors: Amler Evžen , Rauchová Hana , Svobodová Jaroslava , Drahota Zdeněk
Published: July 2002

The inhibition of glycerol 3‐phosphate oxidation by oleic acid correlates with changes in membrane microviscosity monitored by the steady‐state fluorescence anisotropy of DPH. The dynamic measurements indicate that the changes of both the limiting anisotropy and rotational relaxation time occur in a concentration range where the enzyme activity is strongly inhibited.
The calcitonin‐like sequence of the β CGRP gene
Authors: Alevizaki Maria , Shiraishi Akio , Rassool Feyruz V. , Ferner Gwendolynn J.M. , Maclntyre Iain , Legon Steve
Published: July 2002

We have identified a region within the β CGRP gene which has the potential to encode a novel calcitonin‐like peptide. The gene is located on the short arm of chromosome 11 (11p 12–14.2) and we suggest that it resulted from a local duplication of the α gene. We have been unable to detect the corresponding mRNA in a variety of tissues which express α‐calcitonin. It is not clear whether this sequence can be expressed in man.
Immunological cross‐reactivity of fungal and yeast plasma membrane H+‐ATPase
Authors: Vai Marina , Popolo Laura , Alberghina Lilia
Published: July 2002

The plasma membrane H+‐ATPases from fungi and yeasts have similar catalytic and molecular properties. A structural comparison has been performed using immunoblot analysis with polyclonal antibodies directed toward the 102 kDa polypeptide of the plasma membrane H+‐ATPase from Neurospora crassa. A strong cross‐reactivity is observed between the fungal H+‐ATPase and the enzyme from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Structural homologies are indicated also by the analysis of the cross‐reactive peptides originated by proteolytic digestion of Neurospora and S.cerevisiae purified enzymes. Neither enzyme from these two sources appears to be glycosylated by a highly sensitive concanavalin A affinity assay on blotted proteins. A glycoprotein of M
r 115000 and pI 4.8–5, which comigrates with a cell cycle‐modulated protein on 2D gel, is present in partially purified preparations of plasma membrane H+‐ATPase of S.cerevisiae and it is shown to be structurally unrelated to H+‐ATPase.
Regions of SV40 large T antigen necessary for oligomerization and complex formation with the cellular oncoprotein p53
Authors: Montenarh Mathias , Vesco Cesare , Kemmerling Gudrun , Müller Dorothee , Henning Roland
Published: July 2002

The simian virus 40 (SV40) T antigen is composed of 708 amino acids and forms monomers and various oligomers and, in small amounts, heterologous complexes with the cellular oncoprotein p53 (T‐p53). Using SV40 mutants coding for T antigen fragments which are either deleted in the N‐terminal half or truncated by various lengths at the C‐terminal end, we found that a region between amino acids 114 and 152 and a C‐terminal region up to amino acid 669 are essential for the formation of high Mr oligomers of T antigen. Furthermore, only the C‐terminal end up to amino acid 669 is essential for T‐p53 complex formation but not the N‐terminus up to amino acid 152.
Expression, secretion and folding of human growth hormone in Escherichia coli
Authors: Becker Gerald W. , Hsiung Hansen M.
Published: July 2002

An efficient secretion vector containing a gene coding for an E. coli signal peptide fused to human growth hormone (hGH) was cloned into E. coli. The recombinant fusion protein was expressed and correctly processed hGH was secreted into the periplasmic space at a yield of 10–15 μg hGH/A
600. Purification of hGH from the periplasmic fraction by anion exchange and size exclusion gave hGH of greater than 90% purity. Characterization by SDS‐PAGE, amino terminal analysis, trypsin mapping, and circular dichroism demonstrated that the fusion protein was correctly processed to authentic hGH and that the E. coli periplasm provided an appropriate environment for proper folding of hGH and disulfide bond formation.
Calcium induces cyclic GMP formation in Dictyostelium
Authors: Small N.V. , Europe-Firmer G.N. , Newell P.C.
Published: July 2002

(Dictyostelium)
cyclic GMP
Chemotaxis
Ca2+
Ca2+ activation of troponin C‐extracted vertebrate striated fast‐twitch muscle fibers
Authors: Babu Aravind , Pemrick Suzanne , Gulati Jagdish
Published: July 2002

Dual regulation
Myosin‐linked control
Troponin C
Calcium switch
Muscle contraction
Cross‐bridge mechanism
Weak bridge
Strong bridge
3‐Hydroxy‐3‐methylglutaryl‐CoA lyase deficiency studied using 2‐dimensional proton nuclear magnetic resonance spectroscopy
Authors: Iles Richard A. , Jago James R. , Williams Stephen R. , Chalmers Ronald A.
Published: July 2002

1H‐NMR
Metabolism
Inborn error
3‐Hydroxy‐3‐methylglutaryl‐CoA lyase
Enzyme deficiency
2‐dimensional NMR
Thiolation of preformed liposomes with iminothiolane
Authors: Lasch Jürgen , Niedermann Gabriele , Bogdanov Alexander A. , Torchilin Vladimir P.
Published: July 2002

Preformed phosphatidylethanolamine‐containing liposomes were thiolated with 2‐iminothiolane (Traut's reagent) and subsequently activated by mixed disulfide formation with 5,5′‐dithiobis(2‐nitrobenzoic acid). Up to 65% of amino groups of the outer liposomal lamella, corresponding to 230 SH‐groups per vesicle, were modified. Covalent attachment of thiolated α‐chymotrypsin to these thiol‐liposomes via S‐S bridges yielded a protein/lipid ratio of 3.6 × 10−4 mol enzyme/mol lipid.
Microsomal isoflavone 2′‐ and 3′‐hydroxylases from chickpea (Cicer arietinum L.) cell suspensions induced for pterocarpan phytoalexin formation
Authors: Hinderer W. , Flentje U. , Barz W.
Published: July 2002

Microsomal fractions derived from suspension‐cultured chickpea (Cicer arietinum L.) cells induced for phytoalexin biosynthesis catalyzed the monohydroxylation of 4′‐methoxyisoflavones (biochanin A and formononetin) in the 2′‐ and 3′‐positions. The reactions depended on NADPH and molecular oxygen. Post‐microsomal supernatants or microsomes from non‐induced cells are without detectable activity in the hydroxylase assay. 4′‐Hydroxyisoflavones (genistein and daidzein) were not hydroxylated to any significant extent. The occurrence of these hydroxylases proceeds concomitantly with the accumulation of two pterocarpan phytoalexins, medicarpin and maackiain, by induced cell cultures. The results are discussed with regard to the biosynthetic sequences in the conversion of isoflavones to pterocarpans.
Omega Conus geographus toxin: A peptide that blocks calcium channels
Authors: Feldman Daniel H. , Olivera Baldomero M. , Yoshikami Doju
Published: July 2002

We previously reported that omega Conus geographus toxin (ωCgTX), blocks evoked‐release of transmitter at synapses in frog and attenuates the Ca2+ component of the action potential of chick dorsal root ganglion neurons. We report here voltage‐clamp experiments on cultured chick dorsal root ganglion neurons which demonstrate that ωCgTX produces a persistent block of voltage‐gated Ca2+ currents. Thus, we conclude that ωCgTX inhibits synaptic transmission by blocking Ca2+ channels in the presynaptic nerve terminal. The toxin had no effect on K+ currents; however, in some but not all neurons, ωCgTX reduced Na+ currents by 10–25%. These findings suggest that ωCgTX should be useful as a probe to examine synaptic Ca2+ channels.
ATP‐sensitive K+ channels in human isolated pancreatic B‐cells
Authors: Ashcroft Frances M. , Kakei Masafumi , Kelly Ronan P. , Sutton Robert
Published: July 2002

Glucose‐stimulated insulin release from rodent pancreatic B‐cells is thought to be initiated by the closing of ATP‐sensitive K+ channels in the plasma membrane as a consequence of glucose metabolism. We have identified an ATP‐sensitive K+ channel in membrane patches excised from human B‐cells which is similar to that found in rodent B‐cells in conductance, kinetics, ATP sensitivity and its inhibition by sulphonylureas. In man, the ATP‐sensitive K+ channel may also have a central role in glucose‐stimulated insulin secretion and may be (linked to) the receptor for the hypoglycemic sulphonylureas.
Evidence for two H2O2‐binding sites in ferric cytochrome c oxidase Indication to the O‐cycle?
Authors: Vygodina Tatyana , Konstantinov Alexander A.
Published: July 2002

H2O2 addition to the oxidized cytochrome c oxidase reconstituted in liposomes brings about a red shift of the Soret band of the enzyme and an increased absorption in the visible region with two distinct peaks at ∼570 and 605 nm. Throughout pH range 6–8.5, the spectral changes at 570 nm and in the Soret band titrate with very similar pH‐independent K
d values of 2–3,μM. At the same time, K
d of the peroxide complex measured at 605 nm increases markedly with increased H+ activity reaching the value of 18 ± 2 μM at pH 6.0. This finding may indicate the presence of two different H2O2‐binding sites in the enzyme with different affinity for the ligand at acid pH. The Soret and 570 nm band effects are suggested to report H2O2 coordination to heme iron of a
3, whereas the maximum at 605 nm could arise from H2O2 binding to Cu
a3 followed by the enzyme transition into the ‘pulsed’ (or ‘420/605’) conformation. Possible implication of the two H2O2‐binding sites for the cytochrome oxidase redox and proton‐pumping mechanisms are discussed.
Prothoracicotropic hormone has an insulin‐like tertiary structure
Authors: Jhoti H. , McLeod A.N. , Blundell T.L. , Ishizaki H. , Nagasawa H. , Suzuki A.
Published: July 2002

A three‐dimensional model of PTTH‐II has been constructed using interactive computer graphics and energy minimisation techniques, assuming homology with porcine insulin, the structure of which has been determined by X‐ray analysis. The model shows that PTTH‐II can assume an insulin‐like tertiary structure, which is compact with the exception of the sequence variable NH2‐terminal amino acids of the B chain. Most of the hydrophobic core residues including A2 Ile, A6 Cys, A11 Cys, A16 Leu, A20 Cys, B11 Leu, B15 Leu and B19 Cys are identical in PTTH‐II and insulins. The glycines at A1, B8 and B23 allow the chain to assume the characteristic tertiary interactions of the insulin fold and although polypeptide chains are shorter at the COOH‐termini of the A and B chains and extended at the NH2‐terminus of the B chain, the insulin‐like tertiary structure can still be assumed. It is unlikely that PTTH‐II forms either dimers or hexamers, characteristic of porcine and human insulin, and the model is consistent with the inability of PTTH‐II to bind anti‐insulin antibodies or insulin receptors. A hydrophobic surface region of PTTH‐II may be involved in intermolecular actions of physiological relevance. We discuss the implications of our model for evolution of this family of hormones and growth factors.
Protein kinase C and cAMP‐dependent protein kinase induce opposite effects on actin polymerizability
Authors: Ohta Yasutaka , Akiyama Tetsu , Nishida Eisuke , Sakai Hikoichi
Published: July 2002

Protein kinase C phosphorylated muscle and non‐muscle monomeric actin more efficiently than filamentous actin in vitro. By sedimentation assay, the ratio of phosphorylated to unphosphorylated actin was much higher in sedimentable actin than in the non‐sedimentable form, suggesting that phosphorylated actin was more readily incorporated into F‐actin than unphosphorylated actin. In contrast, actin phosphorylated by cAMP‐dependent protein kinase was found to have weaker polymerizability than the unphosphorylated form. The phosphopeptide mapping pattern of actin phosphorylated by protein kinase C was different from that of actin phosphorylated by cAMP‐dependent protein kinase. Thus, both the protein kinases phosphorylate actin differently and induce opposite effects on actin polymerizability.
The mechanism of neuronal resistance and adaptation to hypoxia
Authors: Schurr Avital , Rigor Benjamin M.
Published: July 2002

In this work we provide a theoretical explanation for the observations that: (i) young animals are more resistant to hypoxia than adult ones and (ii) repeated exposure to a hypoxic insult increases the tolerance of young animals and isolated brain tissue to that insult. Considered here is the role of taurine, a putative Ca2+ transport modulator, in attenuating Ca2+ influx and overload in brain tissue upon hypoxia. It is proposed that the higher resistance of young animals to hypoxia stems from their higher brain content of taurine as compared with adults. The increased resistance to lack of oxygen upon re‐exposure to hypoxia may occur as a result of protein and coenzyme A (CoA) breakdown which leads to the accumulation of products like cystine, cysteine, cysteamine and other sulfur‐containing compounds. Upon reoxygenation, these compounds are oxidized to form taurine, which in turn attenuates neuronal Ca2+ accumulation. The sulfur‐containing compounds are considered to be natural scavengers of oxygen‐derived free radicals which are formed upon reoxygenation and have been implicated as a major component in the process leading to ischemic/hypoxic brain damage. Repeated hypoxic insults bring about the formation of higher levels of taurine and hence the observed adaptation to oxygen lack. The hypothesis presented here is supported by experimental observations in our laboratory and those of others.
Chromogranin B (secretogranin I), a putative precursor of two novel pituitary peptides through processing at paired basic residues
Authors: Benjannet S. , Leduc R. , Adrouche N. , Falgueyret J.P. , Marcinkiewicz M. , Seidah N.G. , Mbikay M. , Lazure C. , Chretien M.
Published: July 2002

During the course of reversed‐phase high‐pressure liquid chromatography (RP‐HPLC) purification of the 7B2 peptide originally isolated in our laboratory from human pituitary gland extracts, two novel peptides were identified and purified to homogeneity. The complete amino acid sequence of the first one was established in 1985 and recently found to be entirely homologous to positions 420–493 of the just published chromogranin B sequence. This peptide, denoted GAWK, could originate from chromogranin B following specific cleavage at the basic amino acids flanking both termini of GAWK. Moreover, another peptide isolated in our laboratory from the same source and denoted CCB has been discovered and its sequence is also part of the same chromogranin B molecule. Here again, this peptide, occupying positions 597–653 and located at the COOH‐terminal region of chromogranin B, could derive from specific processing at basic amino acids, Arg‐Lys‐Lys, present at positions 594–596. In a manner reminiscent of the relationship between pancreastatin and chromogranin A, it is proposed that both GAWK and CCB are produced from chromogranin B after specific processing at basic amino acids. These data are thus in favor of a putative role of chromogranins as precursors to potentially bioactive peptides.
A monoclonal antibody against a synthetic peptide reveals common structures among spectrins and α‐actinin
Authors: Närvänen Outi , Närvänen Ale , Wasenius Veli-Matti , Partanen Paul , Virtanen Ismo
Published: July 2002

A monoclonal antibody (Mab) against a synthetic peptide, SEDYGKDL, corresponding to one conserved sequence in the chicken α‐fodrin repeats reacts in immunoblotting with avian α‐spectrin and α‐fodrin, both mammalian spectrins and with mammalian α‐fodrin. This Mab also reacts with α‐actinin in both chicken and human cells. Our results confirm the previously detected structural homology between spectrins and α‐actinin and implicate their common evolutionary origin.
Cloning of human muscle phosphofructokinase cDNA
Authors: Nakajima Hiromu , Noguchi Tamio , Yamasaki Tomoyuki , Kono Norio , Tanaka Takehiko , Tarui Seiichiro
Published: July 2002

Three overlapping cDNA clones for human muscle phosphofructokinase (HMPFK) covering the complete coding sequence were isolated. The sequence included a poly(A) tail, a 399 bp 3′‐untranslated region, a 2337 bp coding region for 779 amino acid residues and a part of the 5′‐untranslated region. Homologies between HMPFK and rabbit muscle phosphofructokinase (RMPFK) were 96% of the amino acids and 89% of the nucleotides in the coding region. Like RMPFK, HMPFK also possessed the internal homology between C‐ and N‐halves in its primary structure. Cloning of HMPFK cDNA will help to identify the molecular defect in patients with glycogenosis type VII (HMPFK deficiency).
An extremely acidic amino‐terminal presequence of the precursor for the human mitochondrial hinge protein
Authors: Ohta Shigeo , Goto Kimihiko , Arai Hajime , Kagawa Yasuo
Published: July 2002

Mitochondrial hinge protein is a subunit of ubiquinol‐cytochrome‐c reductase in the respiratory chain and ‘hinges’ cytochrome c with cytochrome c
1. The protein is encoded in the nuclear genome, synthesized in the cytosol and then imported into the mitochondria. The cDNA of the human hinge protein has been cloned and its nucleotide sequence was determined. The deduced primary structure of the amino‐terminal presequence consists of 13 amino acid residues, of which 4 amino acids are acidic and only one is basic. Since the presequences of most other precursors are rich in basic amino acids, this sequence is unique for targeting mitochondria. Expression of the gene was repressed in the presence of a phorbol ester in human promyelocyticleukemia cells (HL‐60), and this repression was greater than that of the ADP/ATP translocator. These findings suggest that the hinge protein, the expression of which is well regulated, is imported into mitochondria via a specific pathway.
In vivo enzymology: 13C NMR measurement of a kinetic isotope effect for methanol oxidation in Methylosinus trichosporium OB3b
Authors: Mason Ralph P. , Sanders Jeremy K.M. , Cornish Alex
Published: July 2002

The competitive oxidation of 13CH3OH and 13CD3OH has been observed using in vivo 13C NMR spectroscopy. Simultaneous 1H and 2H decoupling gave isotopically shifted 13C singlets for the two methanol isotopomers. The measured enzymic isotope effect, kH/kD is approx. 1.8, indicating that CH bond cleavage is not rate‐determining.
The kinetics of calcium binding to fura‐2 and indo‐1
Authors: Jackson A.P. , Timmerman M.P. , Bagshaw C.R. , Ashley C.C.
Published: July 2002

The kinetics of Ca2+ dissociation from fura‐2 and indo‐1 were measured using a stopped‐flow spectrofluorimeter. The dissociation rate constants were 84 s−1 and 130 s−1, respectively, in 0.1 M KCl at 20°C. The rate constants were insensitive to pH over the range 7.0 to 8.0. The second order association rate constants were estimated indirectly to be in the region of 5 × 108 M−1·s−1 and thus approach the diffusion‐controlled limit. The results demonstrate that these new generation indicators are well‐suited to measure rapid changes in concentration of intracellular Ca2+.
Expression of ricin A chain in Escherichia coli
Authors: O'Hare Mary , Roberts Lynne M. , Thorpe Philip E. , Watson Graham J. , Prior Bernadette , Lord J.Michael
Published: July 2002

DNA encoding ricin A chain was derived from preproricin cDNA and ligated into the expression vector pDS5/3. Transcription is controlled from the coliphage promoter PN25 fused with the lac operator of E.coli. When induced, E.coli 71.18 cells transformed with the recombinant plasmid express ricin A chain which is soluble and has full biological activity.
Direct photoaffinity labeling of the putative sulfonylurea receptor in rat β‐cell tumor membranes by [3H]glibenclamide
Authors: Kramer Werner , Oekonomopulos Raymond , Pünter Jürgen , Summ Hans-Dieter
Published: July 2002

The oral antidiabetic sulfonylurea [3H]glibenclamide specifically binds to plasma membranes from a rat β‐cell tumor indicating a receptor for sulfonylureas in these membranes. Irradiation of [3H]glibenclamide at 254 or 300 nm in the presence of albumin resulted in covalent labeling of the albumin molecule. Direct photoaffinity labeling of β‐cell membranes with [3H]glibenclamide resulted in the covalent modification of two membrane polypeptides with apparent molecular masses 140 and 33 kDa. The extent of labeling of the 140 kDa polypeptide was specifically decreased by sulfonylureas. This suggests that a membrane polypeptide of 140 kDa is a component of the sulfonylurea receptor in the β‐cell membrane.
cDNA sequence of the β2‐subunit of human liver alcohol dehydrogenase
Authors: Ehrig Torsten , von Wartburg Jean-Pierre , Wermuth Bendicht
Published: July 2002

A cDNA library of mRNA from a human liver expressing the β2‐subunit of alcohol dehydrogenase was constructed in λgt11. One clone coding for 352 of a total of 374 amino acid residues of the β2‐subunit was isolated. The sequence differed from that of the β1‐subunit at one nucleotide position resulting in an Arg/His exchange at position 47 of the peptide chain, in agreement with data from protein sequence analysis [(1984) FEBS Lett. 173, 360–366].
Species‐specific differences in the toxicity of puromycin towards cultured human and Chinese hamster cells
Authors: Dudani Anil K. , Gupta Radhey S. , Gupta Rajni
Published: July 2002

The toxicity of the protein synthesis inhibitor puromycin towards a number of human and Chinese hamster cell lines has been examined. In comparison to cells of human origin, Chinese hamster cells exhibited about 25‐fold higher resistance towards puromycin. These differences appeared to be species related as all the cell lines from any one species showed similar sensitivity towards puromycin. The incorporation of [3H]leucine in the hamster cell lines was accordingly found to be more resistant to the inhibitory effects of puromycin as compared to human cells. Studies on the cellular uptake of [3H]puromycin showed that in comparison to human cells, the drug uptake/binding in the hamster cell lines was greatly reduced. However, protein synthesis in the extracts of hamster and human cells showed no significant differences in sensitivity towards puromycin. These results show that the observed species related differences in cellular toxicity to puromycin are due to differences in the cellular uptake/binding of the drug.
Ca2+ regulation of thyroid NADPH‐dependent H2O2 generation
Authors: Dupuy C. , Dème D. , Kaniewski J. , Pommier J. , Virion A.
Published: July 2002

A thyroid particulate fraction contains an NADPH‐dependent H2O2‐generating enzyme which requires Ca2+ for activity. A Chaps solubilized extract of the thyroid particulate fraction partially purified by DEAE chromatography did not show a dependence on Ca2+ for activity. Preincubation of the particulate fraction with Ca2+ yielded a preparation insensitive to Ca2+. The non‐particulate fraction obtained after incubation of the particles in the presence of Ca2+ was able to inhibit, in the presence of EGTA, the Ca2+‐desensitized particulate fraction and the enzyme isolated on DEAE. It is concluded that the reversible Ca2+ activation of the NADPH‐dependent H2O2 generation was modulated in porcine thyroid tissue by (a) calcium‐releasable inhibitor protein(s).
Is translation inhibited by noncognate ternary complexes?
Authors: Bilgin Neşe , Ehrenberg Måns , Kurland Charles
Published: July 2002

We studied the influence of an error‐prone isoacceptor (tRNALeu
4), as well as an intermediate (tRNALeu
2) and a weak (tRNAVal) competitor of tRNAPhe on the poly(Phe) synthesis rate. Even at very high excess concentrations of these noncognate ternary complexes there was no significant effect on the translation rate. Our result argues against the assertion that in vivo translation is slowed down by noncognate tRNA and favours the hypothesis that the incorrect ternary complex concentrations are too low to saturate the ribosomes in vivo.
Differential effects of maitotoxin on ATP secretion and on phosphoinositide breakdown in rat pheochromocytoma cells
Authors: Gusovsky Fabian , Daly John W. , Yasumoto Takeshi , Rojas Eduardo
Published: July 2002

Maitotoxin (MTX) induced exocytotic secretion of ATP from PC12 rat pheochromocytoma cells. The threshold for stimulation of secretion was at concentrations of about 2 ng/ml of MTX. Maximal release occurred at 40 ng/ml. MTX‐induced ATP release required the presence of calcium in the extracellular medium and could be inhibited by nifedipine, a specific blocker of voltage‐dependent calcium channels. In addition to the effects on ATP secretion from PC12 cells, MTX stimulated the breakdown of phosphoinositides, as measured by the accumulation of [3H]inositol phosphates. Maximal stimulation of phosphoinositide breakdown was reached at only 0.5–1.0 ng/ml MTX. MTX at concentrations required to evoke ATP release ( >2 ng/ml) had lesser or no effect on phosphoinositide breakdown. Although stimulation of phosphoinositide breakdown by MTX was dependent on extracellular calcium, it was insensitive to the calcium channel blockers nifedipine, D‐600 and cobalt ions. The different concentration range required to elicit these responses and the varying sensitivity to calcium channel blockers indicate that MTX‐evoked secretion and MTX‐stimulated phosphoinositide breakdown are independent phenomena in PC12 cells.
Right atrial dilatation increases inositol‐(1,4,5)trisphosphate accumulation Implications for the control of atrial natriuretic peptide release
Authors: von Harsdorf R. , Lang R. , Fullerton M. , Smith A.I. , Woodcock E.A.
Published: July 2002

Stretching the right atrium of isolated perfused [3H]inositol‐labelled rat hearts was shown to stimulate the phosphatidyl‐inositol turnover pathway as demonstrated by the accumulation of [3H]inositol‐(1,4,5)trisphosphate and its degradation products. Stimulation was detectable after 1 min with larger increases observed after 10 or 20 min. These findings demonstrate that the myocardium can respond to dilatation by an activation of the phosphatidylinositol turnover pathway. Such a mechanism has implications for the release of atrial natriuretic peptide following right atrial distention.
Native cytokines do not bind to uromodulin (Tamm‐Horsfall glycoprotein)
Authors: Moonen Peter , Gaffner Raymond , Wingfield Paul
Published: July 2002

Uromodulin bound with high affinity to human tumour necrosis factor (TNF) coated on microtitre plates. This interaction was not competitively inhibited by native TNF in solution. No interaction was observed between immobilized uromodulin and TNF in the liquid phase unless conditions were chosen which denatured the latter protein. Recombinant interleukin‐1α adsorbed on microtitre plates also interacted with uromodulin. However, gel filtration experiments demonstrated no interaction between the proteins in the liquid phase. These and additional results indicate that uromodulin interacts with denatured cytokines, but not with native, soluble cytokines.
α3β3 complex of thermophilic ATP synthase Catalysis without the γ‐subunit
Authors: Kagawa Yasuo , Ohta Shigeo , Otawara-Hamamoto Yoko
Published: July 2002

A complex of the α‐ and β‐subunits of thermophilic ATP synthase showed about 25% of the ATPase activity of the αβγ complex. The α3β3 hexamer structure was analyzed by sedimentation (11.2 S) and gel filtration (310 kDa). Dilution of the αβ complex caused dissociation of the complex and rapid loss of ATPase activity which was restored by addition of the γ‐subunit. A previous method using urea for isolating the subunits resulted in an αβ complex with lower activity than that prepared by over‐expression of the genes. The αβ‐ATP complex was formed from the αβ complex, ADP and Pi in the presence of dimethyl sulfoxide.
Proton‐motive‐force‐driven sucrose uptake in sugar beet plasma membrane vesicles
Authors: Lemoine Rémi , Delrot Serge
Published: July 2002

Highly purified plasma membranes were prepared by aqueous two‐phase partioning from sugar beet (Beta vulgaris L.) leaf microsomes. Uptake of sucrose was studied with non‐energized vesicles and with vesicles artificially energized by a pH gradient (ΔpH) or a pH gradient plus an electrical gradient (ΔpH+Δψ). In non‐energized vesicles, sucrose was taken up via a diffusion‐like system until attainment of concentration equilibrium. Energized vesicles rapidly accumulated sucrose with accumulation ratios of 6 and 12 (vs the external concentration) for vesicles energized by ΔpH and ΔpH + Δψ, respectively. Energized uptake exhibited saturation kinetics, and was sensitive to carbonyl cyanide m‐chlorophenylhydrazone and N‐ethylmaleimide.