bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein synthesis is involved in the modulation of the level of the phosphoenolpyruvate carboxykinase mRNA by changes in cell volume in isolated rat hepatocytes
Authors: Quillard Muriel , Husson Annie , Chedeville Arlette , Fairand Alain , Lavoinne Alain
Published: July 2002

The mechanism of action of hydration state was studied on phosphoenolpyruvate carboxykinase (PCK) gene expression in isolated rat hepatocytes. Hypoosmolarity decreased the level of the PCK mRNA after a lag period of about 60 min. The decreasing effect of hypoosmolarity was totally blocked by inhibitors of both protein synthesis and gene transcription. Moreover, hypoosmolarity specifically increased the synthesis of a 45 000 M
r protein, which decreased in the presence of inhibitors of transcription. A close relationship between the synthesis of the 45 000 M
r protein and the decrease in the PCK mRNA level was observed, suggesting that this protein might potentially be involved in the regulation of the level of the PCK mRNA by cell swelling.
Effect of heparin on phosphorylation site specificity of neuronal Cdc2‐like kinase
Authors: Qi Zhong , Zhu Xiaodong , Goedert Michel , Fujita Donald J , Wang Jerry H
Published: July 2002

Neuronal Cdc2‐like kinase (Nclk) can be stimulated by heparin in a substrate‐dependent manner. While phosphorylation of tau is markedly enhanced by heparin, phosphorylation of histone H1 by Nclk is essentially unaffected. A histone H1 peptide, HS(9–18): PKTPKKAKKL, and its substitution analogues were used to examine the basis of the differential heparin effect. In the presence of heparin, the phosphorylation site specificity of Nclk is altered and only proline immediately following the phosphorylation site is still an essential substrate determinant. This change in the site specificity may adequately account for the differential heparin effect on histone H1 and tau phosphorylation.
Presence and localization of oscillin in human spermatozoa in relation to the integrity of the sperm membrane
Authors: Montag M , Parrington J , Swann K , Lai F.A , van der Ven H
Published: July 2002

We investigated the presence and localization of oscillin in human spermatozoa in relation to the integrity of the sperm membrane, which was assessed by the hypo‐osmotic swelling (HOS) test. We found no gross differences in the presence of oscillin in semen samples from men who presented with 70%, 40%, 25% or 2% of membrane‐intact spermatozoa. By immunofluorescence, membrane‐intact (HOS‐positive) spermatozoa showed staining of a single band at the equatorial region, whereas over 80% of HOS‐negative spermatozoa consistently showed a diffuse distribution of oscillin over the sperm head. However, some individuals presented with up to 50% of HOS‐positive spermatozoa showing an aberrant localization of oscillin. We found a significant correlation rate (r=0.70, P<0.05) between the percentage of HOS‐positive spermatozoa with an equatorial oscillin localization and the fertilization rates achieved after intracytoplasmic sperm injection. These data suggest that the localization of oscillin in human spermatozoa might have an impact on egg activation and fertilization rates.
DNA binding of polyomavirus large T‐antigen: kinetics of interactions with different types of binding sites
Authors: Bondeson Kåre , Rönn Ola , Magnusson Göran
Published: July 2002

Polyomavirus large T‐antigen binds to GRGGC sites in double‐stranded viral DNA, regulating transcription and replication. Using surface plasmon resonance to record interactions of large T‐antigen with different types of binding sites, we found that the configuration of recognition motifs influenced both the association and dissociation rates. Particularly, the complex formed at the origin of DNA replication was labile. A comparison of the interactions between large T‐antigen and binding sites with one, two and four GRGGC motifs in tandem showed a strong preference for dimer binding, without detectable co‐operativity between dimers. Sodium chloride stabilised the complexes, whereas the dissociation increased rapidly by increasing pH above 7.0.
Trapping of conformations of the Escherichia coli F1 ATPase by disulfide bond formation
Authors: Aggeler Robert , Grüber Gerhard , Capaldi Roderick A
Published: July 2002

A mutant of Escherichia coli F1F0‐ATPase, αS411C/βY331W/βE381C/γC87S, has been generated. CuCl2 treatment of this mutant led to cross‐linking between α and β subunits in yields of up to 90%. This cross‐linking across non‐catalytic site interfaces inhibited ATP hydrolysis activity. In the absence of cross‐linking, MgATP bound in catalytic sites of the mutant with three different affinities of 0.1 μM, 6 μM and 60 μM, respectively, values that are comparable to wild‐type. For MgADP, there was one tight site (0.34 μM) and two sites of lower affinity (each 27 μM), again comparable to wild‐type enzyme. After cross‐linking all three catalytic sites bound MgATP or MgADP with the same relatively low affinity (∼60 μM). Thus cross‐linking fixed all three catalytic sites in the same conformation. Trypsin cleavage experiments showed that cross‐linking fixed the ε subunit in the ATP+EDTA conformation.
The GXGXG motif in the pICln protein is not important for the nucleotide sensitivity of the pICln‐induced Cl− current in Xenopus oocytes
Authors: Voets Thomas , Buyse Gunnar , Droogmans Guy , Eggermont Jan , Nilius Bernd
Published: July 2002

It has been proposed that the pICln protein forms a nucleotide‐sensitive plasma membrane anion channel with a GXGXG motif being an essential component of the extracellular nucleotide‐binding site. To evaluate this hypothesis, we have performed voltage‐clamp experiments on Xenopus laevis oocytes injected with RNA encoding a rat mutant pICln in which the three glycines of the putative nucleotide‐binding site have been changed into alanines (G54A; G56A; G58A). The injected oocytes displayed outwardly rectifying anion currents, which were voltage‐dependently blocked by extracellular cAMP, but which were not affected by removal of extracellular Ca2+. Furthermore, the mutation did not affect the voltage‐dependent inactivation. We therefore conclude that there is no evidence in favour of an extracellular nucleotide‐binding site in pICln.
Effect of oxygen free radicals on myosin in muscle fibres
Authors: Könczöl Franciska , Lőrinczy Dénes , Belagyi Joseph
Published: July 2002

Experiments were performed on glycerol‐extracted muscle fibres prepared from psoas muscle of rabbit in the presence of hydroxyl free radical generating system. Short irradiation of spin‐labelled muscle fibres by UV light showed the interaction of probe molecules with oxygen free radicals. The intensity of the EPR signal from maleimide or isothiocyanate spin labels attached to the essential thiol groups decreased following irradiation. Oxygen free radicals affected the rate constant of the transition AM.ADP.Vi→AM.ADP in the ATP hydrolysis cycle. It was found that the essential –SH groups of myosin were involved in the oxidation of sulphydryls by Ce(IV). Ce(IV) complexed to nitrilotriacetic acid in the presence of spin trap produced long‐lived free radicals located partly on SH‐1 sulphydryls.
Evidence that protein kinase A activity is required for the basal and tax‐stimulated transcriptional activity of human T‐cell leukemia virus type‐I long terminal repeat
Authors: Turgeman Hava , Aboud Mordechai
Published: July 2002

The present study was undertaken to investigate the role of protein kinase A (PKA) in the control of human T‐cell leukemia virus type‐I (HTLV‐I) long terminal repeat (LTR) expression, since this issue is still controversial. For this purpose we employed two human T‐cell lines; the Jurkat cells in which long exposure to diBu‐cAMP severely down‐regulated the catalytic subunit of PKA (PKA‐C), and H‐9 cells in which such exposure markedly increased PKA‐C level. Transient transfection assays revealed that addition of diBu‐cAMP 1 h before or after transfection profoundly increased HTLV‐I LTR directed CAT expression and synergistically enhanced its stimulation by the viral transactivator tax gene product in both cell lines. However longer exposure to diBu‐cAMP before transfection reduced LTR‐CAT expression to below its basal level and completely abolished its stimulation by tax in Jurkat cells, and this diBu‐cAMP inhibitory effect could be abrogated by co‐transfection of a PKA‐C expressing vector. By contrast, in H‐9 cells, this long exposure to diBu‐cAMP continued enhancing LTR‐CAT expression and its tax‐mediated transactivation, and this stimulatory effect of diBu‐cAMP could be diminished by the PKA‐specific inhibitor N‐[2‐(p‐bromocinnamylamine)ethyl]‐5‐ isoquinolinsulfonamide (H‐89). Notably, in the absence of diBu‐cAMP treatment H‐89 reduced LTR‐CAT expression to below its basal level and prevented its stimulation by tax in both cell lines. Together these findings indicate not only that cAMP‐activated PKA stimulates HTLV‐I LTR expression and its transactivation by tax, but even in the absence of PKA activating signals the basal HTLV‐I LTR expression as well as its stimulation by tax are both dependent on a basal PKA activity.
An NMR‐based identification of peptide fragments mimicking the interactions of the cathepsin B propeptide
Authors: Yu Youlu , Vranken Wim , Goudreau Nathalie , de Miguel Elisa , Magny Marie-Claude , Mort John S , Dupras Robert , Storer Andrew C , Ni Feng
Published: July 2002

Selected fragments of the 62‐residue proregion (or residues 1p–62p) of the cysteine protease cathepsin B were synthesized and their interactions with cathepsin B studied by use of proton NMR spectroscopy. Peptide fragments 16p–51p and 26p–51p exhibited differential perturbations of their proton resonances in the presence of cathepsin B. These resonance perturbations were lost for the further truncated 36p–51p fragment, but remained in the 26p–43p and 28p–43p peptide fragments. Residues 23p–26p or TWQ25A in the N‐terminal 1p–29p fragment did not show cathepsin B‐induced resonance perturbations although the same residues had strongly perturbed proton resonances within the 16p–51p peptide. Both the 1p–29p and 36p–51p fragments lack a common set of hydrophobic residues 30p–35p or F30YNVDI35 from the proregion. The presence of residues F30YNVDI35 appears to confer a conformational preference in peptide fragments 16p–51p, 26p–51p, 28p–43p and 26p–43p, but the same residues induce the aggregation of peptides 16p–36p and 1p–36p. The peptide fragment 26p–43p binds to the active site, as indicated by its inhibition of the catalytic activity of cathepsin B. The cathepsin B prosegment can therefore be reduced into smaller, but functional subunits 28p–43p or 26p–43p that retain specific binding interactions with cathepsin B. These results also suggest that residues F30YNVDI35 may constitute an essential element for the selective inhibition of cathepsin B by the full‐length cathepsin B proregion.
Purification and properties of exopolyphosphatase isolated from Saccharomyces cerevisiae vacuoles
Authors: Andreeva Nadezhda A , Kulakovskaya Tatiana V , Kulaev Igor S
Published: July 2002

An exopolyphosphatase (polyPase) with a specific activity of 60 U/mg protein has been purified from the vacuolar sap of Saccharomyces cerevisiae. The molecular mass of the intact enzyme was found to be 245 kDa. It is highly specific towards high‐molecular polyphosphates (polyP). The activity with polyP9 is 24% of that with polyP208. The apparent K
m for polyP15 and polyP208 hydrolysis is 93 and 2.4 μM, respectively. The enzyme is slightly active with polyP3 and adenosine‐5′‐tetraphosphate, but does not hydrolyze pyrophosphate, ATP, GTP and p‐nitrophenylphosphate. It is stimulated by divalent metal cations. Co2+, the best activator, stimulates it 6‐fold. Antibodies that inhibit the cell envelope and cytosol polyPases of S. cerevisiae have no effect on the vacuolar polyPase. The vacuolar polyPase differs from other yeast polyPases in molecular mass, substrate specificity and effects of activators.
Mutational analysis of Glu272 in elongation factor 1A of E. coli
Authors: Mansilla F. , Knudsen C.R. , Clark B.F.C.
Published: July 2002

In our previous work (Mansilla et al. (1997) Protein Eng. 10, 927–934) we showed that Arg7 of Escherichia coli elongation factor Tu (EF1A) plays an essential role in aminoacyl‐tRNA (aa‐tRNA) binding. Substitution of Arg7 by Ala or Glu lost this activity. We proposed that Arg7 forms a salt bridge with the charged conserved amino acid Glu272 (Asp284 in Thermus aquaticus) thereby binding the N‐terminal region of the protein to domain 2 and thus completing the conformational rearrangement needed for binding aa‐tRNA. In this work we have mutated Glu272 to arginine, either alone (Glu272Arg), or in combination with one of the above mentioned mutations (Arg7Glu/Glu272Arg) in order to test this hypothesis. Our results show that, in confirmation of our thesis based on structural knowledge, the substitution of Glu272 (Asp284) decreases the ability of EF1A:GTP to bind aa‐tRNA.
CD95 (Fas/APO‐1) induces an increased phosphatidylserine synthesis that precedes its externalization during programmed cell death
Authors: Aussel Claude , Pelassy Claudette , Breittmayer Jean Philippe
Published: July 2002

CD95 (Fas, APO‐1)‐induced programmed cell death (apoptosis) in T cell lines is accompanied by a rapid flip‐flop of phosphatidylserine (PtdSer). Externalization of this phospholipid has been previously recognized as one of the early detectable events of cells undergoing apoptosis. We show here that CD95 induces a rapid (detectable at time <15 min), strong (2.5‐fold) but transitory neosynthesis of PtdSer in the Jurkat cell line that precedes its externalization. PtdSer decarboxylation, a mitochondrial specific process, was strongly inhibited by CD95 suggesting that changes in mitochondrial activity take place in the early events of Fas‐induced apoptosis and participate in the increased PtdSer synthesis observed. In cells undergoing apoptosis, newly synthesized PtdSer first exposed at the cell surface was in part shed with CD95‐induced plasma membrane vesicles, a process that likely explains the transitory effect observed.
Isolation of a cDNA for a novel 120‐kDa GTP‐binding protein expressed in motor neurons in the salmon brain
Authors: Kubokawa Kaoru , Miyashita Tomoyuki , Kubo Yoshihiro
Published: July 2002

We isolated a cDNA encoding a novel protein with GTP‐binding motifs from the salmon brain. The cloned 5.5‐kb cDNA encoded 971 amino acids, which showed limited homology with members of large GTP‐binding proteins, such as dynamin, MX protein and VPS1. By in vitro translation analysis, the size of the encoded protein was estimated to be 120 kDa. The recombinant protein including the putative GTP‐binding domain was shown to bind to GTP in vitro. mRNA was strongly expressed in the brain, ovary and skeletal muscle. In the brain, expression of the mRNA was observed specifically in motor neurons, in nucleus oculomotorius, in nucleus valvulae lateralis, in the medulla oblongata and in the spinal cord. To determine the cell biological significance of the encoded protein, we transfected CHO cells with wild type or a putative constitutive active mutant of the mG120 (‐kDa protein expressed in otor neurons) and examined morphological changes. However, no clear changes were observed. The functional role of this novel motor neuron‐specific large GTP‐binding protein remains to be elucidated.
Towards a mutant analysis of the tertiary structures of functional DNA‐binding motifs
Authors: Barker Andrew , Müller-Hill Benno
Published: July 2002

Transcription factors have specific regions, often α helices, with which they recognise DNA. These regions are more or less disordered off DNA. Some examples are listed here. However, a detailed mutant analysis of this phenomenon is missing. It could show to what extent DNA binding in vitro and in vivo is harmed when such a region is artificially made rigid by suitable substitutions and could reveal how much transcription factors have improved by having been selected to carry unstable recognition domains.
Identification and characterisation of a novel splice variant of synaptojanin1
Authors: Woscholski Rüdiger , Finan Peter M , Radley Elisabeth , Parker Peter J
Published: July 2002

Synaptojanin1, the major constitutively active PtdInsP3 5‐phosphatase activity in rat brain, is one of two closely related proteins both extensively spliced in their C‐terminal proline rich domain. We describe here the discovery of a novel splice variant of synaptojanin1 which misses the major N‐terminal part of the SAC1 domain. This ΔSAC‐synaptojanin1 is expressed in rat brain tissue as shown by Northern and Western analysis. However, the deletion of the SAC1 domain does not alter PtdInsP3 5‐phosphatase activity demonstrating that the SAC1 domain is not necessary for catalytic function.
Down‐regulation of negative acute‐phase response genes by hypotonic stress in HepG2 hepatoma cells
Authors: Claeyssens Sophie , Banine Fatima , Rouet Philippe , Lavoinne Alain , Salier Jean-Philippe
Published: July 2002

An increased hepatocellular hydration state (HS) that can be induced by hypotonic stress or a high glutamine uptake modulates the transcription of given genes in liver. This could be important in the acute phase (AP) of a systemic inflammation where both HS and glutamine uptake transiently increase in liver. In HepG2 hepatoma cells cultured in conditions of hypotonic stress or a high extracellular glutamine availability, a specifically decreased expression of two human mRNAs, namely those of α1‐microglobulin/bikunin precursor (AMBP) and α2‐HS‐glycoprotein, that are also down‐regulated in liver by AP, could be seen. A functional analysis of the AMBP promoter indicated that this hypotonic stress‐induced down‐regulation takes place at a transcriptional level. In these experiments, the mRNA level and transcription of the glyceraldehyde‐3‐phosphate dehydrogenase gene that are known to be unmodified in AP did not exhibit any change. Given that hypotonic stress also up‐regulates the transcription of a liver gene that is also up‐regulated in AP [Meisse et al. (1998) FEBS Lett. 422, 346–348], the AP‐associated increase in hepatocellular HS now appears to participate in the transcriptional control of both sets of genes that are up‐ or down‐regulated in AP.
Intracellular free calcium concentration in human taste bud cells increases in response to taste stimuli
Authors: Fujiyama Rie , Miyazaki Toshihiro , Miyamoto Takenori , Okada Yukio , Mizuno Akio , Inokuchi Tsugio , Sato Toshihide
Published: July 2002

We examined changes of intracellular free calcium concentration [Ca2+]i elicited by taste stimuli of sucrose, denatonium and NaCl in the taste buds of seven human fungiform papillae. In one taste bud we observed an increase in [Ca2+]i induced by only NaCl. In another bud an increase of [Ca2+]i in response to both NaCl and sucrose was found. The Ca2+ responses to NaCl and sucrose occurred in differential areas within the one taste bud. In the other five fungiform papillae [Ca2+]i was not changed by the taste stimuli. These results suggest that an increase of [Ca2+]i participates in taste transduction mechanisms for sucrose and NaCl, and that taste cells in one taste bud may respond to differential stimuli.
Paracrine regulation of talin mRNA expression by androgen in human prostate
Authors: Betts A.M , Collett G.P , Neal D.E , Robson C.N
Published: July 2002

Androgens are essential for normal prostate physiology and are intimately associated with the growth and progression of prostate cancer. However, few androgen regulated genes in the prostate have been identified. Using the mRNA differential display technique a 164‐bp cDNA fragment was identified as being androgen regulated in the human prostate. Nucleotide sequence analysis of this fragment revealed 84% homology with the gene encoding the cytoskeletal protein talin. Confirmation of the androgen regulation of this gene was carried out using Northern analysis. Primary prostatic stromal cells treated with conditioned medium (CM) from androgen‐treated primary prostatic epithelial cells showed an approximate 2‐fold reduction in talin mRNA levels compared with stromal cells treated with CM from epithelial cells not exposed to androgens. Expression of talin mRNA in human prostatic tissue was confirmed by in situ hybridisation. The highest levels of expression were present in the epithelial cells, with lower levels of expression in the stroma. Thus, androgen regulation of talin expression may play a role in normal and/or aberrant growth and development of the prostate.
The permeability transition pore induced under anaerobic conditions in mitochondria energized with ATP
Authors: Kuzminova Alevtina E , Zhuravlyova Anastasiya V , Vyssokikh Mikhail Yu , Zorova Ljubava D , Krasnikov Boris F , Zorov Dmitry B
Published: July 2002

The role of oxygen in the induction of mitochondrial permeability transitions was studied. Oxygen consumption, swelling, membrane potential and calcium transport were recorded simultaneously in isolated rat liver mitochondria. Oxygen depletion was accomplished by saturating the medium with N2 and allowing either mitochondrial respiration or glucose/glucose oxidase to consume the residual oxygen. Upon anaerobiosis, mitochondria were supplemented with 500 μM ATP to support succinate‐driven membrane potential. Under these conditions, 100 μM Ca2+ induced cyclosporin A‐sensitive permeability transitions. To eliminate the possible inhibition of permeability transition by high concentrations of adenine nucleotides, anaerobic mitochondria were also energized by the combination of 20 μM ADP and phosphoenolpyruvate/pyruvate kinase. These mitochondria also underwent Ca2+‐induced permeability transition. Under both of these conditions, namely the addition of ATP as a single or through actions of pyruvate kinase, the respiratory components were totally reduced. Thus, oxygen is not a necessary factor for mitochondria to undergo permeability transitions.
Structure of the human transcription factor TFIIF revealed by limited proteolysis with trypsin
Authors: Yong Cai , Mitsuyasu Hiromichi , Chun Zhang , Oshiro Satoru , Hamasaki Naotaka , Kitajima Shigetaka
Published: July 2002

In this study, the human general transcription factor IIF (TFIIF), a heteromeric complex of RAP74 and RAP30 subunits, was subjected to limited proteolysis with trypsin. The central region of RAP74 was demonstrated to be highly sensitive to trypsin while both the N‐ and C‐terminal regions contained trypsin‐resistant structures. In contrast, RAP30 digestion occurred after proteolysis of RAP74. The digestion pattern of RAP74 recruited into the preinitiation complex showed no marked difference from that of IIF, while RAP30 in the complex was protected from trypsin. These results indicate that RAP74 apparently contains three structural domains, the central one of which is externally surfaced and unstructured, but RAP30 is internally wrapped by RAP74. Furthermore, the accessibility of the central region of RAP74 is unaltered in the minimal preinitiation complex, while RAP30 is involved in promoter recognition through its DNA binding activity.
An engineered site for protein kinase C flanking the SV40 large T‐antigen NLS confers phorbol ester‐inducible nuclear import
Authors: Xiao Chong-Yun , Jans David A
Published: July 2002

Nuclear import of simian virus SV40 large tumour antigen (T‐ag) is enhanced by the protein kinase CK2 (CK2) site flanking the nuclear localisation sequence (NLS). We report here that replacement of this site with a consensus site for protein kinase C (PK‐C) can alter the regulation of T‐ag nuclear import and render it inducible by phorbol ester. Measurement of nuclear import kinetics using fluorescently labelled proteins and confocal laser scanning microscopy show that the introduced PK‐C site is functional in enhancing T‐ag nuclear import compared to a protein lacking the CK2 site. Treatment with the PK‐C activator phorbol 12‐myristate 13‐acetate (PMA) further increases the level of maximal nuclear accumulation and the initial nuclear import rate. This engineered PMA‐responsive NLS may have application in targeting of molecules of interest to the nucleus in response to agents stimulating PK‐C.
Protein kinase CK2 is altered in insulin‐resistant genetically obese (fa/fa) rats
Authors: Roher Nerea , Miró Francesc , José Marta , Trujillo Ramon , Plana Maria , Itarte Emilio
Published: July 2002

Hepatic insulin receptor levels in 6‐week‐old obese (fa/fa) rats were about 2‐fold lower than those from lean (Fa/−) rats, which agrees with their insulin‐resistant state. Nuclear protein kinase CK2 activity and protein content in livers from obese (fa/fa) rats were similar to those of lean (Fa/−) animals but the cytosolic levels were reduced to half, due to a decrease in the 39‐kDa catalytic subunit. Marked increases in activity, due to rises in the 44‐kDa and 39‐kDa catalytic subunits, were seen in the 16 000×g sediments (M1) from insulin‐resistant rats, with moderate changes in the 100 000×g sediments (M2). The increase in CK2 binding to M1 did not require increases in the molecular chaperone grp94, which was unaltered in insulin‐resistant rats.
(−)‐2C‐Methyl‐d‐erythrono‐1,4‐lactone is formed after application of the terpenoid precursor 1‐deoxy‐d‐xylulose
Authors: Fellermeier Monika A , Maier Ulrich H , Sagner Silvia , Bacher Adelbert , Zenk Meinhart H
Published: July 2002

Application of [1,2‐14C]1‐deoxy‐d‐xylulose, the committed precursor of terpenoids, thiamine and pyridoxol, to a variety of plant species resulted in the labelling of an unknown metabolite. The isolation and purification of this metabolite from Ipomoea purpurea plants fed with 1‐deoxy‐d‐xylulose (DX), followed by NMR analysis, resulted in the identification of its structure as (−)‐2C‐methyl‐d‐erythrono‐1,4‐lactone (MDEL). MDEL has been previously isolated as a stress metabolite of certain plants. A hypothetical biosynthetic scheme is given.
The effect of Lp3 enlargement on the folding and catalysis of hepatitis delta virus cis‐cleaving ribozyme
Authors: Wu Huey-Nan , Lee Jing-Yu , Ping Yueh-Hsin , Wang Chen-Nai
Published: July 2002

Oligonucleotides were inserted into the Lp3 region of a hepatitis delta virus (HDV) cis‐cleaving ribozyme and the effect of Lp3 enlargement on ribozyme folding was examined by assaying the activity of each mutant. The location of insertion and the sequence of the inserted oligonucleotide had distinct effects on ribozyme activity, and the HDV ribozyme was capable of adopting the active structure for cis‐cleavage when a pentanucleotide was inserted into the 3′ portion of Lp3. Furthermore, whether the insertion mutant cis‐cleaved or not, the structure of Lp3 was altered by the inserted oligonucleotide. These findings disclose that the 5′ rather than the 3′ portion of Lp3 is critical for ribozyme folding and catalysis.
Induction of a spectroscopically defined transition by guanidinium hydrochloride on a recombinant calcium binding protein from Entamoeba histolytica
Authors: Gopal B , Krishna Rao J.V , Thomas C.J , Bhattacharya A , Bhattacharya S , Murthy M.R.N , Surolia A
Published: July 2002

Sequence analysis and metal ion binding studies reported earlier have established that the calcium binding protein (CaBP) from the parasitic ameboid Entamoeba histolytica protein has four canonical EF hand motifs which bind calcium. Equilibrium denaturation studies on both the apo and the holo forms of this protein indicate the presence of stable transition intermediates at low denaturant concentrations as revealed by the binding of the non‐specific hydrophobic dye ANS. Fast reaction kinetics shows that the binding of the Gdn+ ions at or near the Ca2+ sites in the N‐terminal domain influences metal ion binding to the sites in the C‐terminal domain. Isothermal calorimetric titrations performed using low GdnHCl concentrations reveal the presence of two binding sites of low affinity, both being endothermic in nature. Thus the stabilization of CaBP observed at low GdnHCl concentration represents a native‐like intermediate, with the Gdn+ ions mimicking Ca2+ binding at the N‐terminal domain of this protein.
Probing the potential metal binding site in Escherichia coli 3‐deoxy‐d‐arabino‐heptulosonate 7‐phosphate synthase (phenylalanine‐sensitive)
Authors: Sundaram Appavu K , Howe David L , Sheflyan Galina Ya , Woodard Ronald W
Published: July 2002

The active site residues of the proposed metal binding site of DAH 7‐P synthase (phe) were probed by site‐directed mutagenesis of C61 to glycine and serine, H64 to glycine, and with the double mutant C61H/H64C. While C61S and C61H/H64C were inactive, both C61G and H64G were active. All mutants, regardless of enzymatic activity, bound one equivalent of Fe2+ per monomeric unit. Even though C61 and H64 were shown not to be metal ligands for the DAH 7‐P synthase (phe), they may provide some of the backbone interactions/secondary structural elements necessary to properly form the metal binding pocket.
Cloning of a cDNA encoding a new calcium‐binding protein from Dictyostelium discoideum and its developmental regulation1
Authors: Han Young-Hoon , Kang Sa-Ouk
Published: July 2002

By employing 2D‐PAGE, a protein differentially expressed during the development of Dictyostelium discoideum was discovered. The full cDNA of this protein was cloned using RT‐PCR. The deduced protein is composed of 166 amino acid residues containing four EF‐hand domains typical for calcium‐binding proteins and was named CBP3. This protein shows little amino acid sequence homology with the other calcium‐binding proteins from D. discoideum except EF‐hand domains. The CBP3 mRNA was absent in vegetative amebas and accumulated maximally at 6 h of the development on filters. The mRNA level decreased thereafter and disappeared after 12 h of the development, while the protein level peaked at 8 h of development and remained constant thereafter. The mobility of CBP3 on SDS gel was shifted by treatment with EGTA, confirming the Ca2+‐binding activity of the protein.
Proposition for the biochemical mechanism occurring in the sucrose isomerase active site
Authors: Veronese Thierry , Perlot Patrice
Published: July 2002

Sucrose conversion by sucrose isomerase from Protaminobacter rubrum, Serratia plymuthica and Erwinia rhapontici was investigated in the presence of different monosaccharides in the reaction mixture. These conditions led to inhibitory effects and to glucosyl transfer of the glucose moiety of sucrose to the exogen monosaccharide. Comparison of the structure of the different inhibitors and acceptors has allowed us to suppose the binding of the sucrose molecule into the active site, and thereafter, to propose the possible biochemical reactions leading to the formation of different products from sucrose. This study also underlines the close homologies between these enzymes.
Origins of globular structure in proteins
Authors: Tolstoguzov Vladimir
Published: July 2002

Thermodynamic incompatibility of polymers in a common solvent is possibly a driving force for formation and evolution of globular protein structures. Folding of polypeptide chains leads to a decrease in both excluded volume of molecules and chemical differences between surfaces of globular molecules with chemical information hidden in the hydrophobic interior. Folding of polypeptide chains results in ‘molecular or thermodynamic mimicry' of globular proteins and in at least more than 10‐fold higher phase separation threshold values of mixed protein solutions compared to those of classical polymers. Unusually high co‐solubility might be necessary for efficient biological functioning of proteins, e.g. enzymes, enzyme inhibitors, etc.
Intercellular traffic of human immunodeficiency virus type 1 transactivator protein defined by monoclonal antibodies
Authors: Demirhan Ilhan , Chandra Angelika , Hasselmayer Oliver , Chandra Prakash
Published: July 2002

Monoclonal antibodies (mAbs) directed against the amino‐terminal region (N‐terminal sequence 2–19) of transactivator protein (tat) of HIV‐1 have been shown to inhibit intercellular transactivation mediated by the extracellular tat protein. The intracellular transactivation was not significantly affected by anti‐tat mAbs. The specificity of anti‐tat mAbs in abolishing the transactivating potential of extracellular tat is documented by studies with mAbs to HIV‐1 reverse transcriptase, or to a human mammary cancer protein. None of these antibodies showed any inhibitory effect on intercellular transactivation. Specific interaction of anti‐tat IgG with tat protein expressed in Jurkat cells is further supported by experiments on immunoblotting. Extracellular tat is responsible for signals which induce a variety of biological responses in HIV‐infected cells, as well as in uninfected cells. The fact that anti‐tat mAbs can abolish the intercellular traffic of tat protein offers a unique strategy in the development of vaccines against AIDS.
Undercarboxylation of recombinant prothrombin revealed by analysis of γ‐carboxyglutamic acid using capillary electrophoresis and laser‐induced fluorescence
Authors: Vo Hung C , Britz-Mckibbin Philip , Chen David D.Y , MacGillivray Ross T.A
Published: July 2002

The γ‐carboxyglutamic acid (Gla) content of several variants of human prothrombin has been measured by using capillary electrophoresis and laser‐induced fluorescence (CE‐LIF). Both plasma‐derived prothrombin and recombinant prothrombin contain ten residues of Gla per molecule of protein. In contrast, a variant of human prothrombin (containing the second kringle domain of bovine prothrombin) was separated into two populations that differed in their Gla content. Direct measurement of the Gla content showed an association with the presence or absence of the calcium‐dependent conformational change that is required for prothombinase function. Thus, the CE‐LIF assay is useful in determining the carboxylation status of recombinant proteins.
Redistribution of protein kinase C isoforms in rat pancreatic acini during lactation and weaning
Authors: Rodrı́guez-Martı́n E. , Boyano-Adánez M.C. , Bodega G. , Martı́n M. , Hernández C. , Quin Y. , Vadillo M. , Arilla-Ferreiro E.
Published: July 2002

Freshly enzymatically isolated pancreatic acini from lactating and weaning Wistar rats were used to investigate the role of protein kinase C (PKC) isoforms during these physiologically relevant pancreatic secretory and growth processes. The combination of immunoblot and immunohistochemical analysis shows that the PKC isoforms α, δ, and ϵ are present in pancreatic acini from control, lactating and weaning rats. A vesicular distribution of PKC‐α, ‐δ, and ‐ϵ was detected by immunohistochemical analysis in the pancreatic acini from all the experimental groups. PKC‐δ showed the strongest PKC immunoreactivity (PKC‐IR). In this vesicular distribution, PKC‐IR was located at the apical region of the acinar cells. No differences were observed between control, lactating and weaning rats. However, the immunoblot analysis of pancreatic PKC isoforms during lactation and weaning showed a significant translocation of PKC‐δ from the cytosol to the membrane fraction when compared with control animals. Translocation of PKC isoforms (α, δ and ϵ) in response to 12‐O‐tetradecanoyl phorbol 13‐acetate (TPA) 1 μM (15 min, 37°C) was comparable in pancreatic acini from control, lactating and weaning rats. In the control group, a significant translocation of all the isoforms (α, δ and ϵ) from the cytosol to the membrane was observed. The PKC isoform most translocated by TPA was PKC‐δ. In contrast, no statistically significant increase in PKC‐δ translocation was detected in pancreatic acini isolated from lactating or weaning rats. These results suggest that the PKC isoforms are already translocated to the surface of the acinar cells from lactating or weaning rats. In addition, they suggest that isoform specific spatial PKC distribution and translocation occur in association with the growth response previously described in the rat exocrine pancreas during lactation and weaning.
Gender related differences in ATP‐dependent transport of dinitrophenyl‐glutathione conjugate across murine canalicular liver plasma membrane
Authors: Srivastava Sanjay K. , Hu Xun , Xia Hong , Pal Ajai , Guo Jianxia , Orchard John L. , Singh Shivendra V.
Published: July 2002

The present study reports gender related differences in ATP‐dependent transport of dinitrophenyl‐glutathione (GSH) conjugate (DNP‐SG), a model GSH xenobiotic conjugate, across murine canalicular liver plasma membrane (cLPM). ATP‐dependent transport of DNP‐SG across female A/J mouse cLPM was mediated by two components, a high‐affinity and a low‐affinity component, with corresponding K
m of 18 μM (V
max 0.02 nmol/min·mg) and 500 μM (V
max 0.23 nmol/min·mg), respectively. On the other hand, only one component for the ATP‐dependent transport of DNP‐SG was observed in male mouse cLPM (K
m 130 μM; V
max 0.18 nmol/min·mg). Moreover, the rate of ATP‐dependent transport of DNP‐SG was markedly higher in the cLPM fraction of male mouse compared with that of the female. Presence of two transport components in female mouse cLPM, but only one system in the cLPM fraction of male mouse, was confirmed by measuring DNP‐SG mediated stimulation of ATP hydrolysis (DNP‐SG ATPase activity). To the best of our knowledge, the present study is the first report on gender related differences in ATP‐dependent murine canalicular transport of GSH conjugates.
Hypothetical protein KIAA0079 is a mammalian homologue of yeast Sec24p
Authors: Tani Katsuko , Oyama Yuki , Hatsuzawa Kiyotaka , Tagaya Mitsuo
Published: July 2002

The Sec23p‐Sec24p complex is a component of COPII‐coated vesicles that mediate protein transport from the endoplasmic reticulum in yeast. The mammalian hypothetical protein KIAA0079 (KIAA0079p) exhibits sequence similarity to yeast Sec24p. KIAA0079p was co‐eluted with mammalian Sec23p on gel filtration. In vitro binding experiments revealed that the C‐terminal region of KIAA0079p binds to the N‐terminal region of mammalian Sec23p. Overexpression of KIAA0079p caused a defect in protein export from the endoplasmic reticulum. These results support the idea that KIAA0079p is a functional homologue of yeast Sec24p.
Differential potency of two crosslinking plant lectins to induce formation of haptenic‐sugar‐resistant aggregates of rat thymocytes by post‐binding signaling
Authors: Timoshenko A.V. , Gorudko I.V. , Cherenkevich S.N. , Gabius H.-J.
Published: July 2002

To evaluate the significance of post‐binding events for stable aggregate formation, the aggregation/dissociation of rat thymocytes initiated by two crosslinking plant lectins, namely concanavalin A (Con A) and Solanum tuberosum agglutinin (STA), were comparatively studied. Despite intimate cell contacts in the aggregates only Con A led to establishment of haptenic‐sugar‐resistant (HSR) complexes. The presence of inhibitor II of diacylglycerol kinase, a dual calmodulin antagonist/protein kinase C inhibitor (trifluoperazine), and a sulfhydryl group reagent (N‐ethylmaleimide) impaired this process. The obtained results indicate that the formation of HSR cellular contacts is not an automatic response to lectin‐dependent cell association. In contrast to STA, Con A binding elicits this reaction with involvement of diacylglycerol kinase, protein kinase C and/or calmodulin as well as thiol level perturbation, as inferred by the application of target‐selective inhibitors.
Identification, molecular cloning, and characterization of subunit 11 of the human 26S proteasome
Authors: Hoffman Laura , Gorbea Carlos , Rechsteiner Martin
Published: July 2002

We sequenced five peptides from subunit 11 (S11), a 43 kDa protein of the human 26S proteasome, and used this information to clone its cDNA. The S11 cDNA encodes a 376 amino acid protein with a pI of 5.6 and a molecular mass of 42.9 kDa. Translation of S11 RNA in the presence of [35S]methionine produces a radiolabeled protein that co‐migrates with S11 of the human 26S proteasome on SDS‐PAGE. Polyclonal antiserum made against recombinant S11 recognizes a protein of the same size in extracts of bacteria expressing S11 and in purified 26S proteasomes from human red blood cells or rabbit reticulocytes. The S11 sequence does not contain motifs that suggest a biological function. S11 is, however, the human homolog of Rpn9, a recently identified subunit of the yeast 26S proteasome.
A re‐investigation of the ribonuclease sensitivity of a DNA demethylation reaction in chicken embryo and G8 mouse myoblasts
Authors: Jost Jean-Pierre , Siegmann Michel , Thiry Stéphane , Jost Yan-Chim , Benjamin Don , Schwarz Steffen
Published: July 2002

Recently published results (Nucleic Acids Res. 26, 5573–5580, 1998) suggest that the ribonuclease sensitivity of the DNA demethylation reaction may be an experimental artifact due to the possible tight binding of the nucleases to the methylated DNA substrate. Using an improved protocol we show for two different systems that demethylation of hemimethylated DNA is indeed sensitive to micrococcal nuclease, requires RNA and is not an experimental artifact. The purified 5‐MeC‐DNA glycosylase from chicken embryos and G8 mouse myoblasts was first incubated for 5 min at 37°C with micrococcal nuclease in the presence of Ca2+ in the absence of the DNA substrate. Upon blocking the nuclease activity by the addition of 25 mM EGTA, the DNA demethylation reaction was initiated by adding the labeled hemimethylated DNA substrate to the reaction mixture. Under these conditions the DNA demethylation reaction was abolished. In parallel controls, where the purified 5‐MeC‐DNA glycosylase was pre‐incubated at 37°C with the nuclease, Ca2+ and EGTA or with the nuclease and EGTA, RNA was not degraded and no inhibition of the demethylation reaction was obtained. As has already been shown for chicken embryos, the loss of 5‐MeC‐DNA glycosylase activity from G8 myoblasts following nuclease treatment can also be restored by the addition of synthetic RNA complementary to the methylated strand of the substrate DNA. No reactivation of 5‐MeC‐DNA glycosylase is obtained by complementation with a random RNA sequence, the RNA sequence complementary to the non‐methylated strand or DNA, thus ruling out a non‐specific competition of the RNA for the binding of the nuclease to the labeled DNA substrate.
Dual specificity antibodies using a double‐stranded oligonucleotide bridge
Authors: Chaudri Zahida N. , Bartlet-Jones Michael , Panayotou George , Klonisch Thomas , Roitt Ivan M. , Lund Torben , Delves Peter J.
Published: July 2002

The covalent conjugation of oligonucleotides to antibody Fab’ fragments was optimized by using oligonucleotides modified with a hexaethylene linker arm bearing three amino groups. One oligonucleotide was coupled to antibody of one specificity and a complementary oligonucleotide to antibody of a second specificity. The antibodies were then allowed to hybridize by base pairing of the complementary nucleotide sequences and the generation of bispecific antibody was analyzed on SDS‐PAGE and confirmed using BIAcore analysis. The strategy of complementary oligonucleotide‐linked bispecific molecules is not limited to antibodies but is applicable to linking any two molecules of different characteristics.
Identification of the catalytic subunit of cAMP‐dependent protein kinase from the photosynthetic flagellate, Euglena gracilis Z1
Authors: Kiriyama Hiroyasu , Nanmori Takashi , Hari Kengo , Matsuoka Daisuke , Fukami Yasuo , Kikkawa Ushio , Yasuda Takeshi
Published: July 2002

A gene named epk2 that encodes the amino acid sequence of a protein kinase was identified from the photosynthetic flagellate, Euglena gracilis Z. Homology search and phylogenetic analysis revealed that the deduced amino acid sequence of epk2 is most similar to that of the catalytic subunit of cAMP‐dependent protein kinase (PKA). Northern blot analysis showed that Euglena cells express a 1.4‐kb transcript of this gene. When the EPK2 protein was coexpressed with the rat regulatory subunit of PKA in cultured mammalian cells, these two proteins were coimmunoprecipitated. The association of EPK2 and the rat regulatory subunit of PKA was not detected in the cell lysate incubated with cAMP. EPK2 immunoprecipitated from the transfected cells phosphorylated Kemptide, a synthetic peptide substrate for PKA, and the phosphorylation was inhibited by PKI, a PKA‐selective protein kinase inhibitor. These results indicate that EPK2 is a PKA homologue in the photosynthetic flagellate, and this is the first evidence for the occurrence of the PKA catalytic subunit in photosynthetic organisms.
Mutation of Lys‐75 affects calmodulin conformation
Authors: Medvedeva Marina V. , Polyakova Oxsana V. , Watterson D.Martin , Gusev Nikolai B.
Published: July 2002

Some properties of synthetic calmodulin and its five mutants with replacement of Lys‐75 were analyzed by means of electrophoresis, limited proteolysis and MALDI mass‐spectrometry. A double mutant of calmodulin containing insert KGK between residues 80 and 81 and replacement of Lys‐75 by Pro has a highly flexible central helix which is susceptible to trypsinolysis in the presence of Ca2+. Two mutants, K75P and K75E, having a distorted central helix demonstrate high resistance to trypsinolysis in the absence of Ca2+. Arg‐90 and Arg‐106 being the primary site of trypsinolysis of synthetic calmodulin are partially‐protected in K75P and K75E mutants. The central helix of K75A and K75V mutants is stabilized by hydrophobic interactions between residues located in positions 71, 72 and 75. In the presence of Ca2+, the central helix of K75V is resistant to trypsinolysis. Mutations K75A and K75V decrease the rate of trypsinolysis of the central helix with a simultaneous increase of the rate of trypsinolysis in the C‐terminal domain of calmodulin. It is concluded that the point mutation in the central helix has a long distance effect on the structure of calmodulin.
The HIV‐1 Vpr co‐activator induces a conformational change in TFIIB
Authors: Agostini Isabelle , Navarro Jean-Marc , Bouhamdan Mohamad , Willetts Karen , Rey Françoise , Spire Bruno , Vigne Robert , Pomerantz Roger , Sire Joséphine
Published: July 2002

Vpr is a HIV‐1 virion‐associated protein which plays a role in viral replication and in transcription and cell proliferation. We have previously reported that Vpr stimulates transcription of genes lacking a common DNA target sequence likely through its ability to interact with TFIIB. However, the molecular mechanism of the Vpr‐mediated transcription remains to be precisely defined. In this in vitro study, we show that the binding site of Vpr in TFIIB overlaps the domain of TFIIB which is engaged in the intramolecular bridge between the N‐ and C‐terminus of TFIIB, highly suggesting that binding of Vpr may induce a change in the conformation of TFIIB. Indeed, with a partial proteolysis assay using V8 protease, we demonstrate that Vpr has the ability to change the conformation of TFIIB. We investigated in this partial proteolysis assay a series of Vpr‐mutated proteins previously defined for their transactivation properties. Our data show a correlation between the ability of Vpr‐mutated proteins to stimulate transcription and their ability to induce a conformational change in TFIIB, indicating a functional relevance of the Vpr‐TFIIB interaction.
Characterization of an alternatively spliced isoform of rat vesicle associated membrane protein‐2 (VAMP‐2)
Authors: Mandic Robert , Lowe Anson W.
Published: July 2002

VAMPs are esicle ssociated embrane roteins that are essential for secretion. A spliced isoform of rat VAMP‐2, called VAMP‐2B, is characterized in this study. The VAMP‐2B transcript is the result of alternative RNA splicing in which an intron is retained. The predicted amino acid sequence of VAMP‐2B differs from VAMP‐2 at its carboxy‐terminal end. Because recent studies have shown that VAMP's carboxy‐terminal end influences the protein's sorting, the location of myc‐epitope tagged VAMP‐2B in PC12 cells was determined. Subcellular fractionation showed colocalization of myc‐VAMP‐2B and endogenous VAMP‐2. Thus alternative RNA splicing does not affect VAMP‐2 sorting in PC12 cells.