bluuebunny/crossref_metadata_2025 huggingface dataset:
A G‐protein of sarcoplasmic reticulum of skeletal muscle is activated by caffeine or inositol trisphosphate
Authors: Hasegawa Tohru , Kumagai Shuzo
Published: July 2002

Here we show the activation of G‐protein by inositol trisphosphate (IP3) or caffeine in sarcoplasmic reticulum (SR) of skeletal muscle and the consequent existence of a common mechanism of Ca2+ release from SR induced by caffeine and by IP3. (i) Indomethacin inhibits Ca2+ release induced by IP3 or caffeine. (ii) PGE1 does not induce Ca2+ release itself, but does stimulate Ca2+ release induced by IP3, or caffeine, from SR. (iii) Forskolin stimulates both types of Ca2+ release. The inhibitory effect of indomethacin on both forms of Ca2+ release, and the stimulatory effect of PGE1 and forskolin on either Ca2+ release suggest that there exists a common mechanism between IP3‐ and caffeine‐induced Ca2+ release. (iv) Caffeine or IP3 activates G‐protein via inhibition of a GTPase activity. (v) Indomethacin itself inactivates this G‐protein by stimulation of a GTPase activity and reverses the activation of G‐protein induced by IP3 or caffeine. (vi) PGE1 competes with the inhibitory effect of indomethacin on GTPase activity and PGE1 itself activates G‐protein through inhibition of GTPase activity. From these results, it could be suggested that caffeine or IP3 induces Ca2+ release from the SR via activation of G‐protein, which affects the Ca2+ channel and cAMP which seems to affect G‐protein via A‐kinase.
8‐Br‐cAMP inhibits the transient expression of firefly luciferase
Authors: Coker G.T. , Vinnedge L. , O'Malley K.L.
Published: July 2002

The genes for firefly luciferase and chloramphenicol acetyltransferase (CAT) were used as reporter genes to explore the activation of heterologous promoters by 8‐Br‐cAMP. Cells were transfected with a CAT gene/tyrosine hydroxylase promoter, which contains a cAMP response element. Extracts from cells treated with 8‐Br‐cAMP had 340% more enzyme activity than untreated cells. In contrast, treated cells transfected with a tyrosine hydroxylase/luciferase construct had 30% less activity than control cells. Simian virus and rous sarcoma virus promoters/luciferase constructs also had lower activities in cells treated with 8‐Br‐cAMP than untreated cells. The inhibition of luciferase enzyme activity by cAMP appears to be posttranscriptional since both luciferase and CAT RNA levels were similarly increased in cells treated with 8‐Br‐cAMP or 1‐methyl‐3‐isobutylmethylxanthine. The lower level of luciferase activity was not due to simple allosteric inhibition. We conclude that constructs using the firefly luciferase as a reporter gene are unsuitable for studying the effects of cAMP on the regulation of promoters.
Binding properties of T4 gene 32 protein fragments carrying partially cleaved terminal domains
Authors: Casas-Finet Jose R.
Published: July 2002

Analysis of fluorimetric equilibrium‐binding isotherms of a proteolytic fragment of bacteriophage T4 gene 32 protein (g32P) lacking residues 1–9 shows that this region contains the site responsible for the function of the NH2‐terminal ‘B’ domain (residues 1–21). The end codon of the frameshift mutant g32P‐PR201 has been identified as TAG at nucleotide position 852. The PR201 gene 32 product ends at Ser283 and carries a truncated COOH‐terminal ‘A’ domain (residues 253–301). Fluorimetric titrations of g32P‐PR201 with double‐stranded DNA show that the functional residues of the A domain are located within the region spanning residues 284–301.
δ°‐Thalassemia in cis of βKnossos‐globin gene Normal structure and normal transient expression of the δ‐globin gene
Authors: Ouazana R. , Bozon D. , Baklouti F. , Gonnet C. , Delaunay J. , Godet J.
Published: July 2002

We have previously described the first homozygous cases of Hb Knossos in an Algerian family. The Hb A2 was completely absent, ascertaining the presence of a δ°‐thalassemia determinant in cis of the βKnossoss gene. Here, we investigate the affected δ‐globin gene. The complete DNA sequence of the gene and its 5′ and 3′ flanking regions was determined. Only two nucleotide changes were recorded: a C → T substitution at −199 and an AT insertion at −448 upstream from the cap site. To examine the involvement of these changes in gene function, the δ‐gene was subcloned in an expression vector and introduced into COS cells. Analysis of RNA derived from these cells, using an S1 protection assay and dot‐blot hybridization, revealed qualitatively and quantitatively normal transcription. The loss of δ‐globin gene activity in vivo may be due to the alteration of a tissue‐specific control.
Regulation of DNA supercoiling in Escherichia coli: Genetic basis of a compensatory mutation in DNA gyrase
Authors: McEachern Fiona , Fisher L.Mark
Published: July 2002

Bacterial DNA supercoiling is controlled by balancing the supercoiling activity of DNA gyrase and the relaxing activity of DNA topoisomerase I. We have characterized the gyrB gene from a topA deletion mutant of Escherichia coli (DM800) that has a compensatory mutation in gyrB, lowering the activity of gyrase 10‐fold, and thereby redressing the intracellular level of supercoiling. The mutant gene differs from the wild type in carrying three rather than two direct tandem repeats of a 6 bp sequence encoding Ala‐Arg. We suggest this novel mutation affects domain spacing and was generated by an unequal crossing over event, possibly involving gyrase.
The noncompetitive blocker [3H]chlorpromazine labels segment M2 but not segment M 1 of the nicotinic acetylcholine receptor α‐subunit
Authors: Giraudat Jérôme , Galzi Jean-Luc , Revah Frédéric , Changeux Jean-Pierre , Haumont Pierre-Yves , Lederer Florence
Published: July 2002

The membrane bound acetylcholine receptor from Torpedo marmorata was photolabeled by the noncompetitive channel blocker [3H]chlorpromazine under equilibrium conditions in the presence of the agonist carbamoylcholine. The radioactivity incorporated into the AChR subunits was reduced by addition of phencyclidine, a specific ligand for the high‐affinity site for noncompetitive blockers. The α‐subunit was purified digested with trypsin and/or CNBr and the resulting fragments fractionated by HPLC. Sequence analysis resulted in the identification of Ser‐248 as a major residue labeled by [3H]chlorpromazine in a phencyclidine‐sensitive manner. This residue is located in the hydrophobic and putative transmembrane segment M2 of the α‐subunit, a region homologous to that containing the chlorpromazine‐labeled Ser‐262 in the δ‐chain [1] and the Ser‐254 and Leu‐257 in the β‐chain [2]. Extended sequence analysis of the hydrophobic segment M 1 further showed that no labeling occurred in this region.
Heterogeneous activation of protein kinase C during rat liver regeneration induced by carbon tetrachloride administration
Authors: Sasaki Yutaka , Hayashi Norio , Ito Toshifumi , Fusamoto Hideyuki , Sato Nobuhiro , Kamada Takenobu
Published: July 2002

During rat liver regeneration induced by carbon tetrachloride administration, the protein kinase C α subspecies was activated in a heterogeneous fashion, a higher number of hepatocytes expressing the protein kinase C α subspecies being detected in the pericentral zone than in the periportal zone. This zonal heterogeneity became maximal at 24 h after the treatment. The distribution of hepatocytes expressing the protein kinase C α subspecies was roughly coincident with that of hepatocytes exhibiting DNA synthesis. These results suggest that protein kinase C may play a crucial role in liver regeneration.
Non‐random spontaneous chain breakages occur in DNA methylated with dimethyl sulfate
Authors: Dölle Albert , Strätling Wolf H.
Published: July 2002

The method of in vivo footprinting uses partial methylation of the DNA in living cells with dimethyl sulfate (DMS), cleavage of the DNA chains at modified guanosines with piperidine, and mapping of the site of cleavage by the genomic sequencing technique of Church and Gilbert. Here we report on a spontaneous breakage reaction of DMS‐methylated DNA at guanosines as well as adenosines, which is highly non‐random with respect to the DNA sequence. In our in vivo genomic footprinting studies at the chicken lysozyme promoter this reaction gave rise to additional adenosine‐derived bands in the guanosine sequence ladder.
A model for the ability of drugs to induce enhanced D Nase I cleavage
Authors: Portugal José
Published: July 2002

A common property of sequence‐selective DNA‐binding drugs lies in their ability to induce an enhanced D Nase I cleavage in regions surrounding their binding sites. A hypothetical model to explain the enhancements induced by drug binding to the minor‐groove of DNA is presented. It involves the participation of three different single models: a mass action effect produced by the enzyme redistribution after drug binding; changes in the minor groove width size; and interactions between the enzyme and the drug so increasing the cleavage in places located close to the binding site. The model is tested by using statistical data analysis. The hypothetical model might explain the experimental results better than any of the single models alone but these models also appear to render significant results.
Inositol 1,3,4,5‐tetrakisphosphate and inositol 1,4,5‐trisphosphate act by different mechanisms when controlling C2+ in mouse lacrimal acinar cells
Authors: Changya L. , Gallacher D.V. , Irvine R.F. , Petersen O.H.
Published: July 2002

In internally perfused single lacrimal acinar cells the competitive inositol 1,4,5‐trisphosphate (Ins 1,4,5‐P3)‐antagonist heparin inhibits the ACh‐evoked K+ current response mediated by internal Ca2+ and also blocks both the Ins 1,4,5‐P3‐evoked transient as well as the sustained K+ current increase evoked by combined stimulation with internal Ins 1,4,5‐P3 and inositol 1,3,4,5‐tetrakisphosphate (Ins 1,3,4,5‐P4). When, during sustained stimulation with both Ins 1,4,5‐P3 and Ins 1,3,4,5‐P4, one of the inositol polyphosphates is removed, the K+ current declines; whereas removal of Ins 1,4,5‐P3 results in an immediate termination of the response, removal of Ins 1,3,4,5‐P4 only causes a very gradual and slow reduction in the current. Ins 1,3,4,5‐P4 is therefore not an acute controller of Ca2+ release from stores into the cytosol, but modulates the release of Ca2+ induced by Ins 1,4,5,P3 by an unknown mechanism, perhaps by linking Ins 1,4,5 P3‐sensitive and insensitive Ca2+ stores.
N‐Formyl‐methionyl‐leucyl‐phenylalanine induced accumulation of inositol phosphates indicates the presence of oligopeptide chemoattractant receptors on circulating human lymphocytes
Authors: Schubert Thomas , Müller Walter E.
Published: July 2002

Chemotaxis; Formylpeptide receptor; Inositol phosphate; Lithium ion; (Human lymphocyte)
Calcium‐dependent phospholipid binding proteins associated with the membranes of rabbit skeletal muscle
Authors: Melgunov Vladimir I. , Mamedova Nigyar A. , Akimova Elena I. , Adzhimolaev Tarikh A.
Published: July 2002

By using extraction in the presence of Ca2+ and Triton X‐100 and then in the presence of EGTA without detergent, a set of Ca2+‐dependent phospholipid binding proteins has been identified in the membranes of transverse tubules (T‐tubules) and sarcoplasmic reticulum (SR), isolated from rabbit skeletal muscles. Longitudinal SR, junctional SR and T‐tubule membranes yielded about 9, 14 and 3.3 μg of EGTA‐soluble proteins per 1 mg of membrane protein, respectively. In the presence of 1 mM CaCl2, 68 and 33 kDa proteins of T‐tubules and junctional SR as well as 30 kDa protein of T‐tubules were shown to bind to liposomes made of 1:1 mixtures of (i) phosphatidylcholine and (ii) phosphatidylserine, phosphatidic acid, or phosphatidyl ethanolamine. In the presence of EGTA, the above‐mentioned proteins were mostly found in the supernatants. Binding of the proteins with liposomes consisting of pure phosphatidylcholine was negligible.
Asp83, Glu113 and Glu134 are not specifically involved in Schiff base protonation or wavelength regulation in bovine rhodopsin
Authors: Janssen J.J.M. , De Caluwé G.L.J. , De Grip W.J.
Published: July 2002

Site‐specific mutagenesis was employed to investigate the proposed contribution of proton‐donating residues (Glu, Asp) in the membrane domains of bovine rhodopsin to protonation of the Schiff base‐linking protein and chromophore or to wavelength modulation of this visual pigment. Three point‐mutations were introduced to replace the highly conserved residues Asp83 by Asn (D83N), Glu113 by Gln (E113Q) or Glu134 by Asp (E134D), respectively. All 3 substitutions had only marginal effects on the spectral properties of the final pigment (⩽ 3 nm blue‐shift relative to native rhodopsin). Hence, none of these residues by itself is specifically involved in Schiff base protonation or wavelength modulation of bovine rhodopsin.
Identification of a putative amyloid A4‐generating enzyme as a prolyl endopeptidase
Authors: Ishiura Shoichi , Tsukahara Toshifumi , Tabira Takeshi , Shimizu Teruo , Arahata Kiichi , Sugita Hideo
Published: July 2002

The A4 amyloid peptide is deposited in Alzheimer's disease inside neurons as neurofibrillary tangles or extracellularly as vascular amyloid. The A4 peptide is cleaved off by an unidentified proteinase from a larger precursor protein (APP), which resembles a cell surface receptor. The proteinase, which cleaves off the membrane‐spanning domain of APP, may be important in amyloid formation. To evaluate this, a model peptide substrate, succinyl‐ isoleucyl‐alanine‐methylcoumarinamide, which is homologous to the C‐terminal portion of A4 peptide, was synthesized to screen the putative A4‐generating proteinase. On chromatographie purification, it was found that two proteinases are involved in the hydrolysis of the peptide, the major one being identified as a prolyl endopeptidase. This evidence may facilitate elucidation of the mechanism of amyloid deposition in Alzheimer's disease.
Suppression of nuclear ADP‐ribosyltransferase activity in Ehrlich ascites tumor cells by 5‐azacytidine
Authors: Hoshino Jiro , Nothacker Hans-Peter , Schalge Sigrid , Kröger Hans
Published: July 2002

Exposure of Ehrlich ascites tumor cells to 5‐azacytidine for 5 h resulted in a partial loss of ability of DNA to stimulate ADP‐ribosyltransferase activity, as assessed in a reconstituted in vitro enzyme system consisting of purified calf thymus enzyme, calf thymus whole histone and DNA isolated from the cells. The degree of suppression in vitro varied depending on the amount of histone and DNA added and it reached a maximum with a value of 83% and 62% of control for DNAs from cells exposed to 10 μM and 30 μM 5‐azacytidine, respectively, at a histone/DNA mass ratio of 0.4. In the absence of histone (conditions of auto‐ADP‐ribosylation of the enzyme), no suppression was detectable.
Degradation of 2‐phosphoglycerate by cytotoxin B of Clostridium difficile
Authors: Knoop Floyd , Martig Robert , Owens Michaela
Published: July 2002

Cytotoxin B of C. difficile was highly purified by selective ammonium sulfate precipitation, Biogel A5m chromatography, phenyl boronate hydrophobic interaction chromatography and ultracentrifugation. The final cytotoxic product had a specific activity of 7.8 × 108
protein and showed a single protein band with an estimated molecular weight of 163000 when subjected to SDS‐PAGE. Immunoelectrophoresis of the final product showed a single precipitin arc. The addition of cytotoxin B to imidazole‐HCl buffer (pH 7.4) containing MgSO4, KCl and the substrate 2‐phosphoglycerate resulted in the formation of phosphoenolpyruvate as demonstrated by spectrophotometric analysis. Phosphoglycerate conversion was absent when the cytotoxin was heat‐inactivated of reacted with specific antitoxin prior to assay.
The actin gelling activity of chicken gizzard α‐actinin at physiological temperature is triggered by water sequestration
Authors: Grazi Enrico , Trombetta Giorgio , Magri Ermes , Cuneo Paola
Published: July 2002

At 37°C, in the presence of 6% () polyethylene glycol 6000, 30 nM α‐actinin from chicken gizzard induces the gelation of 12 μM actin. Static measurement shows that the addition of 30 nM α‐actinin increases the rigidity of the system from 23.5 to 54 . According to the theory of osmoelastic coupling, also large additives, such as the proteins of the cell sap, are able to cause an osmotic stress equivalent to that caused by polyethylene glycol. We thus conclude that, in vivo, α‐actinin acts as an actin gelling protein.
The importance of Val‐157 hydrophobia interaction for papain inhibitory activity of an epoxysuccinyl amino acid derivative
Authors: Yamamoto Daisuke , Matsumoto Keita , Ohishi Hirofumi , Ishida Toshimasa , Inoue Masatoshi , Kitamura Kunihiro , Hanada Kazunori
Published: July 2002

Based on the crystal structure of the papain‐E‐64‐c complex, 3‐dimensional binding modes of a series of epoxysuccinyl amino acid derivatives to the papain active site have been constructed and the structure‐inhibitory activity relationship has been analyzed using the accessible surface area and nonbonded energy parameters. The result indicates the importance of the hydrophobic interaction between the amino acid side chain of the inhibitor and the papain Val‐157 residue for revealing the potent inhibitory activity.
A novel method for measuring membrane conductance changes by a voltage‐sensitive optical probe
Authors: Rosemberg Yosef , Korenstein Rafi
Published: July 2002

This study presents a method whose principles enable using a voltage‐sensitive optical probe, to quantitatively measure conductivity changes elicited in membrane vesicles and cells. The procedure is based on the fact that the amplitude of the transmembrane potential difference, established across a membrane by an external electric field, is decreased when membrane conductivity is increased upon incorporation of ionophores into the membrane. The method was applied to osmotically swollen thylakoid membranes whose membrane conductivity was changed by the addition of gramicidin or ionomycin. The electric field induced stimulated luminescence from photosystem I (electrophotoluminescence‐EPL) was used as a voltagesensitive optical probe. We calculated the induced conductance changes by using a calibrated EPL vs external electric field response curve and measuring the ionophore‐mediated attenuation of the EPL signal. The calculated ionophore‐unmodified conductance of the thylakoid membrane yields a value of 171 ± 56 . The value of the membrane conductance, modified by 10 nM gramicidin was found to be 190 ± 56 . The modified membrane conductance and the membrane conductance changes induced by 1 μM ionomycin in the presence of CaCl2 were found to be 186 ± 3 and 15 ± 3 , respectively.
Interaction of puromycin with acceptor site of human placenta 80 S ribosomes
Authors: Graifer D.M. , Fedorova O.S. , Karpova G.G.
Published: July 2002

The complex N‐AcPhe‐tRNAPhe · poly(U) · 80 S ribosome from human placenta was treated with puromycin taken in various concentrations. Based on the kinetic data of N‐acetylphenylalanyl‐puromycin formation, the association constant of puromycin with the acceptor site of the ribosome was estimated to be (3.96 ± 0.84) × 104 M−1 at 37°C.
Modulation of phosphorylation and dephosphorylation of keratin and other polypeptides by estradiol‐17β in rat vaginal epithelium
Authors: Gupta P.D. , Swarup G. , Reddy A.G.
Published: July 2002

Phosphorylation of keratin polypeptides was studied by incubating vaginal tissues (removed from estradiol primed and unprimed 30‐day‐old rats) with 32Pi. Analysis by SDS‐PAGE and autoradiography showed that on treatment with estradiol phosphorylation of 63 and 58 kDa keratin polypeptides increased 3‐ and 2‐fold respectively. Phosphorylation was maximal after 30 min of estradiol priming and decreased thereafter. Phosphorylation of some non‐keratin polypeptides (37, 34, 32 and 25 kDa) also showed time dependent variation. The results showed that estradiol can modulate phosphorylation‐dephosphorylation of keratins and other polypeptides in rat vaginal epithelial cells.
Comparative analysis of mitochondrial and amyloplast adenylate translocators
Authors: Pozueta-Romero Javier , Viale A.M. , Akazawa T.
Published: July 2002

Structurally intact and metabolically competent mitochondria isolated from liquid‐culture cells of sycamore (Acer pseudoplatanus L.) were shown to incorporate ADPglucose. Employing the double silicone oil layer filtering centrifugation method, we examined the kinetic properties of the uptake of various adenylates as well as the inhibitory effects exerted by carboxyatractyloside, atractyloside and bongkrekic acid, known specific inhibitors of the mitochondrial adenylate translocator. Immunoblot patterns of peptides derived from the partial proteolytic digestion of the mitochondrial and plastid adenylate translocators were shown to be essentially the same. We conclude that the molecular entities engaged in the adenylate transport system operating in two different organelles. mitochondria and amyloplasts, are very similar.
Amino acid sequences of nerve growth factors derived from cobra venoms
Authors: Inoue Seiji , Oda Toshio , Koyama Junichi , Ikeda Kiyoshi , Hayashi Kyozo
Published: July 2002

Amino acid sequences of nerve growth factors (NGFs), purified from the venoms of Indian cobra (Naja naja) and Thailand cobra (Naja naja siamensis) were determined. The sequence of N. naja NGF differed from that reported previously by Hogue‐Angeletti et. al. [(1976) Biochemistry 15, 26–34]. The sequence of N. naja siamensis NGF was identical to that of Formosan cobra Naja naja atra NGF, determined previously by Oda et. al. [(1989) Biochem, Int. 19, 909–917] and to that deduced from the nucleotide sequence of an NGF cDNA from the venom gland of N. naja siamensis, as reported by Selby et. al. [(1987) J. Neurosci. Res. 18, 293–298].
Biphasic expression of slow myosin light chains and slow tropomyosin isoforms during the development of the human quadriceps muscle
Authors: Laurent-Winter C. , Soussi-Yanicostas N. , Butler-Browne G.S.
Published: July 2002

Using a two‐dimensional electrophoresis technique coupled with sensitive silver staining, we have investigated the chronology of appearance of the myosin light chain and tropomyosin isoforms during early stages of human quadriceps development. Our results show that slow myosin light chains and the slow tropomyosin isoform are not detected at 6 weeks of gestation. These isoforms transiently appear between 12.5 weeks and 15 weeks of gestation and then disappear. The slow myosin light chains are re‐expressed at 31 weeks of gestation and the slow tropomyosin isoform later at 36 weeks of gestation, and normally remained expressed into the adulthood. Our study thus reveals a biphasic expression of the slow myosin light chains and the slow tropomyosin isoform in developing human quadriceps muscle.
Sodium dependent inositol transport in HL60 cells is not related to Na+/K+, ATPase activity
Authors: Baxter M.A. , Grafton G. , Bunce C.M. , Sheppard M.C. , Brown G.
Published: July 2002

In HL60 cells, inositol transport is sodium‐dependent but functionally independent of Na+/K+ ATPase activity. This observation has implications for the currently proposed theory for the development of diabetic complications.
Chemical modification of the sole histidine residue of smooth muscle caldesmon
Authors: Bonet-Kerrache Armelle A. , Walsh Michael P.
Published: July 2002

Caldesmon was stoichiometrically N‐carbethoxylated specifically at the only histidine residue (His‐610) with diethylpyrocarbonate. Carbethoxylation of a 1:1 molar complex of caldesmon and calmodulin in the presence of Ca2+ resulted in the stoichiometric N‐carbethoxylation of His‐610 of caldesmon and His‐107 of calmodulin. Carbethoxy‐caldesmon, like the unmodified protein, bound to immobilized calmodulin (in the presence of Ca2+) and to immobilized tropomyosin (at low ionic strength). The affinity of F‐actin for carbethoxy‐caldesmon (K
4 = 1.29 × 10−4M) was similar to that for unmodified caldesmon (K
4 = 0.88 × 10−4M), and the modified protein was as effective as control caldesmon in the inhibition of the actin‐activated MgATPase of skeletal muscle myosin. We conclude that the predicted basic amphiphilic α‐helical sequence (Arg‐593‐His‐610) does not represent the calmodulin‐binding site of caldesmon. Furthermore, His‐610 does not play a major role in the interaction of caldesmon with F‐actin or tropomyosin.
On the mechanism of rotenone‐insensitive reduction of quinones by mitochondrial NADH:ubiquinone reductase The high affinity binding of NAD+ and NADH to the reduced enzyme form
Authors: Čénas Narimantas K. , Bironaité Daiva A. , Kulys Juozas J.
Published: July 2002

NADH acts as an incomplete competitive inhibitor for 5,8‐dioxy‐1,4‐naphtoquinone during its rotenone‐insensitive reduction by mitochondrial NADH:ubiquinone reductase. NAD+ and ADP‐ribose act as incomplete mixed‐type inhibitors. K
i of NAD+ and NADH towards quinone are about one order less than towards ferricyanide. The bimolecular rate constant of the reduction of the enzyme by NADH in the quinone reductase reaction is about 2 times less than that of ferricyanide reductase reaction. These data indicate that the reduction site of 5,8‐dioxy‐1,4‐naphtoquinone is close to NAD+/NADH and ferricyanide binding site. It seems that during the steady‐state reduction of ferricyanide and 5,8‐dioxy‐1,4‐naphtoquinone these oxidizers react with NADH:ubiquinone reductase reduced to different extents.
Regulation of glutathione S‐transferase gene expression by phenobarbital in cultured adult rat hepatocytes
Authors: Vandenberghe Yves , Tee Lisa , Morel Fabrice , Rogiers Vera , Guillouzo André , Yeoh George
Published: July 2002

Previous studies, by using Northern blotting analyses, showed that phenobarbital (PB) affects the steady‐state mRNA levels of glutathione S‐transferase (GST) subunits , and 7 in both conventional cultures of adult rat hepatocytes and co‐cultures, with rat liver epithelial cells [Vandenberghe et al., 1989, FEBS Lett. 251, 59–64; Morel et al., 1989, FEBS Lett. 258, 99–102]. To determine whether PB acts at the transcriptional level, nuclear ‘run‐on’ experiments using cDNA probes hybridizing to GST subunits , and 7 mRNA were performed on purified nuclei isolated from control and PB treated hepatocytes seeded under conventional and co‐culture conditions. Data from this study demonstrate that the increase in steady‐state mRNA levels observed in both conventional culture and co‐culture after 4 days PB exposure results from an increased transcriptional activity or the GST genes. However, a substantial increase in steady‐state mRNA levels in the absence of a commensurate increase in transcriptional activity at 12 days of co‐culture, indicates that the barbiturate has also a stabilizing effect in vitro on the GST mRNAs.
Differences between embryonic and adult Torpedo acetylcholine receptor γ subunit
Authors: Souroujon Miriam C. , Carmon Sara , Safran Anat , Fuchs Sara
Published: July 2002

Antibodies to a synthetic peptide corresponding to residues 346–359 of the Torpedo acetylcholine receptor (AChR) γ subunit, were employed to compare the adult and embryonic receptor. This peptide contains a consensus phosphorylation site for cAMP‐dependent protein kinase (PKA). The anti‐peptide antibodies discriminated between adult and embryonic AChRs, and reacted preferentially with the adult γ form. These observed immunological differences did not seem to stem from different phosphorylation states. Our results suggest that the embryonic Torpedo AChR may have a γ‐like subunit that differs from the known adult form of this subunit, at least in the specific region that contains the phosphorylation site for PKA.
Cleavage reaction of a synthetic oligoribonucleotide corresponding to the autocleavage site of a precursor RNA from bacteriophage T4
Authors: Hosaka Hideo , Ogawa Takashi , Sakamoto Kensaku , Yokoyama Sigeyuki , Takaku Hiroshi
Published: July 2002

A fragment (GUUUCGUACAAAC) having a consensus sequence for the self‐cleavage domain in a precursor of an RNA molecule from T4‐infected Escherichia coli cells (p2Sp1; precursor of species 1) was chemically synthesized and found to be cleaved either between CA (139–140) or between UA (137–138) in the presence of monovalent cations and a non‐ionic detergent. The cleaved products had 5′‐hydroxyl and 3′‐phosphate groups, of which some were in the form 2′.3′‐cyclic phosphates.
Formation of enzyme—substrate disulfide linkage during catalysis by protein disulfide isomerase
Authors: Hu Chao-Hong , Tsou Chen-Lu
Published: July 2002

During the regeneration of native ribonuclease A (RNase) from the disulfide scrambled molecule by protein disulfide isomerase (PDI), the substrate forms a covalent intermediate with the enzyme through disulfide linkage(s). This has been shown by the appearance of a band at the molecular weight position expected in SDS‐PAGE at the same time as the increase in RNase activity. The new band decreased when the regeneration of RNase activity approached completion and disappeared by treatment of the reaction mixture with excess dithiothreitol.
Characterization of ursolic acid as a lipoxygenase and cyclooxygenase inhibitor using macrophages, platelets and differentiated HL60 leukemic cells
Authors: Najid A. , Simon A. , Cook J. , Chable-Rabinovitch H. , Delage C. , Chulia A.J. , Rigaud M.
Published: July 2002

A new property of ursolic acid, lipoxygenase and cyclooxygenase inhibition, has been described in an acetone‐extract of heather flowers (Calluna vulgaris) wllich could help explain the anti‐inflammatory characteristics of this plant. In mouse peritoneal macrophages, human platelets and differentiated HL60 leukemic cells, ursolic acid, at 1 μM, blocks arachidonate metabolism.
Inserting foreign peptides into the major coat protein of bacteriophage M13
Authors: Ilyichev A.A. , Minenkova O.O. , Kishchenko G.P. , Tat'kov S.I. , Karpishev N.N. , Eroshkin A.M. , Ofitzerov V.I. , Akimenko Z.A. , Petrenko V.A. , Sandakhchiev L.S.
Published: July 2002

Foreign DNA fragments were inserted into filamentous phage gene VIII to create hybrid B‐proteins with foreign sequences in the amino terminus. The hybrid proteins are incorporated into the virions which retain viability and infectivity. Virions with hybrid B‐proteins have the same contour length and the same number of B‐protein molecules as virions with natural B‐proteins. It was shown that for one of hybrid B‐proteins the position of the processing site had changed.
Analysis of the structure of photosystem I in cyanobacterial thylakoid membranes
Authors: Hefti Andreas , Ford Robert C. , Miller Mette , Cox Raymond P. , Engel Andreas
Published: July 2002

Using electron microscopy of negatively stained specimens, we have investigated the shape and degree or association or photosystem (PS) I complexes in cyanobacterial thylakoid membranes. When incubated at high concentrations of magnesium chloride (>0.15 M), the PSI complexes from small ordered arrays in the membrane composed of monomeric complexes in a PI square lattice of dimensions a=b=11 nm. Averaged projections of the complex resemble chose found for the purified PSI reaction centre arter reconstitution (Ford, R.C, Hefti, A. and Engel, A. (1990) EMBO J. 9, 3067‐3075). Some small differences in its shape are discussed, with particular reference to the differences in the polypeptide composition of the 2 preparations. We find that the complex remains in the monomeric form in the thylakoid membrane under all the conditions tested.
Group II phospholipase A2 induced by interleukin‐1β in cultured rat gingival fibroblasts
Authors: Shinohara Hiroyuki , Amabe Yukari , Komatsubara Takanori , Tojo Hiromasa , Okamoto Mitsuhiro , Wakano Yoichi , Ishida Hiroshi
Published: July 2002

Previously, we reported the presence of group II‐like phospholipase A2 activity in the soluble fraction of rat gingiva. In the present study, we found that treatment of rat gingival cells with human recombinant interleukin‐ 1β resulted in dose‐dependent stimulation of intracellular and extracellular phospholipase A2 activity. Antisera against group II phospholipase A2 totally blocked the interleukin‐1β‐induced phospholipase A2 activity, but antisera against group I phospholipase A2 did not. Moreover, immunoblot analysis showed that the induced phospholipase A2 was group II phospholipase A2. These findings suggest that the induced enzyme belongs to the group II phospholipase A2 family of proinflammatory enzymes.
Transcripts of the viroid central conserved region contain the local tertiary structural element found in full‐length viroid
Authors: Paul C.P. , Levine B.J. , Robertson H.D. , Branch A.D.
Published: July 2002

The viroid central conserved region (CCR) is highly conserved among different viroids and is thought to be involved in viroid replication. A novel tertiary structure occurs in the CCR of native circular potato spindle tuber RNAs. To permit more detailed studies of this structural element, a small RNA oligonucleotide containing the CCR of the viroid genome was synthesized. The tertiary structure of these CCR transcripts was examined by UV‐crosslinking of the RNA, followed by mapping of the crosslink using limited alkaline digestion and classical RNA secondary analysis. The CCR transcript was found to undergo UV‐crosslinking between the same two bases as in full‐length viroid, indicating that the tertiary structure is the same and that the CCR transcript will be useful for the affinity purification of host components.
Angiotensin II directly induces follicle rupture and oocyte maturation in the rabbit
Authors: Yoshimura Yasunori , Karube Masataka , Koyama Norihiro , Shiokawa Shigetatsu , Nanno Tomonori , Nakamura Yukio
Published: July 2002

To investigate the possible direct involvement of angiotensin II (Ang II) in ovulation and oocyte maturation, Ang II at 100 or 10 μg was administered at 2‐h intervals in the in‐vitro perfused rabbit ovaries. The addition of Ang II in the perfusate induced ovulation in vitro in the absence of gonadotropin, while ovulation did not occur in any contralateral control ovaries. However, the ovulatory efficiency in the Ang II‐treated ovaries was significantly lower than in hCG‐treated ovaries. Ang II significantly stimulated the meiotic maturation of ovulated ova and follicular oocytes. Concomitant addition of the specific receptor antagonist of Ang II, saralasin, 30 min before the onset of Ang II administration blocked Ang II‐induced ovulation in a complete manner. Although suralasin did not inhibit completely hCG‐induced ovulation and oocyte maturation, these results suggest that Ang II produced in the ovary may act locally in the process of ovulation.
Protein conformational changes and myelin solubilization by anion‐detergent solutions
Authors: Monreal Jaime , Carmona Pedro , Regueiro Pilar , Diaz Ricardo S.
Published: July 2002

The addition of sodium sulfate to a myelin suspension in sodium phosphate buffer at neutral pH, containing octyl glucoside detergent (OG), increases the membrane solubility more than 5‐fold by an unknown structural mechanism. FTIR spectroscopy has been applied to investigate anion effects on the conformational structure of myelin proteins. Sulfate and sulfate‐phosphate media, but not phosphate alone, induce a great conformational protein disorder. The addition of the detergent to the anion mixture solution prevents the myelin from protein denaturation. The conformational transitions have also been quantified through the amide I region. Explanations of these changes and their connections with myelin solubility are also included.
Analysis of differentially expressed genes in retinitis pigmentosa retinas Altered expression of clusterin mRNA
Authors: Jones S.E. , Meerabux J.M.A. , Yeats D.A. , Neal M.J.
Published: July 2002

The molecular and cellular processes underlying photoreceptor degeneration in retinitis pigmentosa (RP) are unknown. We have investigated gene expression in diseased retinas using differential hybridization screening of a retinal cDNA library with probes derived from normal and RP retinal RNA. Most differential clones detected corresponded to transcripts absent from the dystrophic state, including e.g. opsin. However, one clone was noticeably increased in RP in comparison with the control; partial sequencing showed it encoded clusterin. Increased expression of clusterin has been identified in several cases of tissues undergoing apoptosis (programmed cell death), and our finding suggests that the degenerative changes in advanced RP may represent another example or apoptosis, possibly with common causative mechanisms.
Lamin A gene expression is specifically suppressed in v‐src‐transformed cells
Authors: Ozaki Toshinori , Sakiyama Shigeru
Published: July 2002

By a differential screening procedure, we have obtained several cDNAs the expression of which was down‐regulated in v‐src‐transformed 3Y1 cells compared to parental 3Y1 cells. One of these cDNAs, termed N26, was extensively analyzed. Sequence analysis revealed that N26 cDNA was 3,095 nucleotides in length and contained an open reading frame of 665 amino acid residues. Based on an homology search at the nucleotide level, it was postulated that N26 gene encodes lamin A. The expression of the N26 gene was not suppressed in other types of transformed cells, such as v‐mos‐, v‐ras‐ and SV40‐transformed 3Y1 cells.
Detection of an enzyme bound γ‐glutamyl acyl ester of carbamyl phosphate synthetase of Escherichia coli
Authors: Lusty Carol J.
Published: July 2002

E. coli carbamyl phosphate synthetase binds 0.2–0.4 mol equivalents of glutamine in an acid resistant form. The bound material is quantitatively released as glutamate by weak base hydrolysis and as a mixture of 12% glutamate, 10% γ‐glutamylhydroxamate, and 70% pyrrollidonecarboxylic acid by hydrolysis with hydroxylamine. These results provide direct evidence for a γ‐glutamyl acyl ester on the enzyme. The absence of the acyl ester in a mutant carbamyl phosphate synthetase with a Cys269 → Ser substitution in the glutaminase subunit further suggests that the covalent intermediate is a thioester of Cys269. Under equilibrium conditions, the Cys269Ser mutant enzyme binds glutamine with a K
d of 7 ± 1 μM, indicating that Cys269 is essential for acyl ester formation but not for binding of glutamine.
On the activation—inactivation coupling in Shaker potassium channels
Authors: Lee Chyuan-Yih
Published: July 2002

The ‘ball‐and‐chain’ model suggests the existence of a negative site which may attract the positively charged inactivation ball to occlude the pore when the channel is in the open state. For Shaker K+ channels, we propose that the state‐dependent negative site be tryptophan‐435, which becomes negatively charged after receiving an electron from tyrosine‐445. The kinetic scheme for the channel's activation‐inactivation coupling as derived from the YW‐gated model resembles a successful ‘scheme 8’ proposed by Zagotta and Aldrich. Our model suggests that the final rapid voltage‐independent transition to the open state is due to the deprotonation of tyrosine‐445.
DNA binding of histone H1 is modulated by nucleotides
Authors: Nilsson Pia , Mannermaa Riitta-Maaria , Oikarinen Jouko , Grundström Thomas
Published: July 2002

Histone H1 acts as a general repressor of transcription in eukaryotes by organizing nucleosomes into inaccessible condensed forms of chromatin. The capability of H1 to bind to DNA with some sequence specificity is likely to be critical in the control of these processes. We show here that ATP and several other nucleotides, including non‐hydrolyzable derivatives, can inhibit DNA binding of H1. The results also show that ATP differentially affects binding of H1 to DNA in a fashion enhancing nucleotide sequence specificity of the binding. The study suggests a novel mechanism or modulation of H1 activity that has important implications for the role of H1 as a transcriptional regulator.
Light‐induced oxidation of iron atoms in a photosensitive nitrile hydratase
Authors: Honda Jun , Teratani Yoshitaka , Kobayashi Yoshio , Nagamune Teruyuki , Sasabe Hiroyuki , Hirata Akira , Ambe Fumitoshi , Endo Isao
Published: July 2002

The photoactivation process of a photosensitive nitrile hydratase (NHase) from Rhodococcus sp. N‐771 has been investigated by 57Fe Mössbauer spectroscopy and magnetic susceptibility measurements in order to clarify the behavior of iron atoms in the enzyme. Mössbauer spectra of inactive NHase gave two symmetric‐doublet components indicating the presence of two iron species, while that of the active NHase gave a single symmetric doublet indicating the presence of a single iron species. Magnetic susceptibility measurements of the inactive and active HNase both showed small effective magnetic moments. These results led us to conclude that one of the two iron atoms incorporated in the NHase is oxidized during photoactivation, namely from a low spin ferrous to a low spin ferric state. This is the first observation of an intramolecular photooxidation phenomena involving iron in a single protein molecule.
Phosphorescence and ODMR study of the binding interactions of acetylcholine receptor α‐subunit peptides with α‐cobratoxin
Authors: Tringali Arthur E. , Pearce Suman Frieda A. , Hawrot Edward , Brenner Henry C.
Published: July 2002

Optical detection of magnetic resonance (ODMR) and phosphorescence spectroscopy have been applied to synthetic peptides derived from the α‐subunit of the nicotinic acetylcholine receptor of Torpeda californica and their complexes with α‐cobratoxin (CBTX). TheCBTX Trp phosphorescence is strongly quenched by the proximal disulfide linkage, while the emission wavelengths and ODMR frequencies of the 18‐mer αl81–198 indicate a more hydrophobic Trp environment than in the 12‐mer α185–196. Binding to CBTX produces a subtle increase in the hydrophobicity of the Trp environment for the peptides, in qualitative agreement with a recently proposed binding model, in which a receptor Trp residue interacts strongly with a hydrophobic cleft of the toxin.
Interaction of VIP, PACAP and related peptides in normal and leukemic human monocytes and macrophages
Authors: Chedeville Annick , Mirossay Ladislav , Chastre Eric , Hurbain-Kosmath Ilse , Lopez Manuel , Gespach Christian
Published: July 2002

The activation of the cAMP signaling pathway by vasoactive intestinal peptide (VIP), pituitary adenylate cyclase‐activating peptide (PACAP) and related peptides was studied (i) in normal peripheral human monocytes and THP‐1 leukemic human monocytes, (ii) in their derived macrophage counterparts respectively obtained after spontaneous differentiation or retinoic acid (RA) treatment, and (iii) in human bronchoalveolar macrophages. In THP‐1 monocytes, PACAP increased basal adenylate cyclase activity 5.3‐fold, with an affinity 50‐times greater than that of VIP or helodermin (K
a = 3.2 × 10−11 M VIP), whereas in normal peripheral monocytes, PACAP and VIP exhibited similar affinities and only increased eAMP generation 2‐fold (EC50 = 10−9 M). Spontaneous and RA‐induced differentiation into normal and leukemic macrophages induced a progressive loss of cAMP production and regulation of superoxide anion production by VIP and related peptides. The neoplastic transformation in THP‐1 monocytes and the deficiencies in the cAMP cascade observed during the terminal differentiation of normal and leukemic human macrophages may relate to a differential genetic expression of the VIP/PACAP receptor subtypes, and alterations in the functional activity of the stimulatory and inhibitory Gs/Gi subunits of adenylate cyclase.
A synthetic peptide of the N‐terminus of ADP‐ribosylation factor (ARF) inhibits regulated exocytosis in adrenal chromaffin cells
Authors: Morgan Alan , Burgoyne Robert D.
Published: July 2002

We have investigated the role of ADP‐ribosylation factor (ARF) in regulated exocytosis in digitonin‐permeabilized adrenal ehromaffin cells by the use of a synthetic peptide, hARFl(2–17), based on the N‐terminus of the protein. hARFl(2–17) inhibited Ca2+‐dependent but not basal exocytosis, whereas equimolar levels of other synthetic peptides were ineffective. The inhibitory effect of hARFl(2–17) was dose‐dependent and half‐maximal at 12 μM. GTPγS‐induced secretion in the presence of non‐stimulatory CA2+ concentrations was also inhibited by hARFl(2–17). These results point to a hitherto unsuspected role for ARF in regulated exocytosis, and the potency of the hARFl(2–17) peptide suggests that ARF is essential for exocytosis in bovine adrenal chromaffin cells.
DIDS (4,4'‐diisothiocyanatostilbene‐2,2'‐disulfonic acid) inhibits an early step of protein translocation across the mammalian ER membrane
Authors: Jungnickel Berit , Rapoport Tom A.
Published: July 2002

Protein translocation across the endoplasmic reticulum (ER) membrane of yeast can be inhibited by agents believed to specifically affect the transport of ATP through the membrane (Mayinger P. and Meyer D.I. (1993) EMBO J. 12, 659‐666), suggesting the involvement of a translocation component in the lumen of the ER that binds ATP. We demonstrate that one of the inhibitors, 4.4'‐diisothiocyanatostilbene‐2.2'‐disulfonic acid (DIDS), also affects the translocation of proteins into mammalian microsomes. Translocation is blocked at the point of transfer of the nascent chain from the signal recognition particle (SRP) into the ER‐membrane. We also confirm that photoaffinity‐labelling of microsomes with 8‐azido‐ATP inhibits the same early step of protein translocation. Since this step is reported to not require ATP. these results raise the possibility that, in both cases, factor(s) other than ATP‐binding components of the translocation machinery are perturbed.
cDNA isolation and partial gene structure of the human α4(IV) collagen chain
Authors: Sugimoto Manabu , Oohashi Toshitaka , Yoshioka Hidekatsu , Matsuo Nobuhiko , Ninomiya Yoshifumi
Published: July 2002

A novel collagen IV chain, α4(IV), has recently been identified in basement membranes. We describe part of the primary structure of the human α4(IV) polypeptide for the first time, which has been determined by cloning and sequencing of cDNAs encoding 241 amino acid residues of the COL domain and 231 residues of the NC1 domain. We also characterized a genomic DNA fragment containing 4 exons coding for the entire NCl domain. Among five known α chains of collagen IV, the α4(IV) chain is distinct from the other four chains. However, it is more similar to the α2 (IV) chain than to the α1(IV), α3(IV) and α5(IV) chains in terms of amino acid sequence homology, domain structure of polypeptides and exon/intron structure of the genes, suggesting the presence of two phylogenetically distinct subclasses of collagen IV α chains; one composed of α2 and α4 chains and the other of α1, α3 and α5 chains.
Insight into the interactions between subunits I and II of the cytochrome c oxidase of the yeast Saccharomyces cerevisiae by means of extragenic complementation
Authors: Meunier Brigitte , Colson Anne-Marie
Published: July 2002

In yeast, revertants were selected from two respiratory deficient mutants carrying mutations in the catalytic subunits of cytochrome oxidase. From a mutant carrying a double mutation in the vicinity of the copper binding pocket in subunit II, two genetically independent revertants were obtained in which the same extragenic reversion mutation was observed, A147V, in the putative helix 4 of subunit I. A comparison with revertants derived from the second deficient mutant, carrying the deficiency mutation, S140L, in the loop 3–4 of subunit I, provides additional data in favour of an interaction between helix 4 of subunit I and subunit II.