bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of protein synthesis initiation factor 2 from Xenopus laevis oocytes with GDP and GTP analogs
Authors: Carvallo Pilar , Allende Jorge E.
Published: July 2002

The structural specificity of the purified protein synthesis initiation factor 2 (eIF‐2) from X. laevis ovary towards analogs of GTP and GDP was studied. The relative affinity of the structural analogs was measured by their capacity to inhibit the formation of the [3H]GDP·eIF‐2 binary complex. The results obtained demonstrate that modifications in the ribose moiety are well tolerated by eIF‐2 which binds dGTP, 2′,3′‐dialdehyde GTP (oGTP) and 2′,3′‐dialdehyde GDP (oGDP) and even the dinucleotide cytidylyl(5′‐3′)guanosine 5′‐triphosphate (pppGpC). Substitution in the polyphosphate chain by phosphorothioate groups in the β and γ positions (GDPβS or GTPγS) does not abolish the affinity for the nucleotides and the presence of an imido group between the β and γ phosphates in guanyl‐5′‐yl imidodiphosphate (GppNHp) still permits a weaker but significant binding. Guanine 5′‐O‐(2‐fluorodiphosphate) (GDPβF) has an affinity considerably lower than GDPβS. Methylation of position 7 of the guanine (7‐m GDP), however, completely eliminates the interaction of GDP with eIF‐2. The analogs tested can be listed in the following order of descending affinities: GDP > GDPβS > oGDP⩾ GTPγS > GDPβF > pppGpC > GTP > GppNHp > oGTP ⪢ 7‐m GDP. Assays of the capacity of GTP analogs to form a ternary complex of the type met‐tRNAi·GTP·eIF‐2 or of GDP analogs to inhibit the formation of this complex reflect, in general, the same order of relative affinities except for pppGpC, which is weaker in its capacity to form a ternary complex than GppNHp or oGTP, although it has a higher affinity than these compounds in the formation of a binary complex.
Heterogeneity in cDNA clones encoding rice glutelin
Authors: Wang Co-Shine , Shastri Kamini , Wen Lisa , Huang Jenq-Kuen , Sonthayanon Burachai , Muthukrishnan S. , Reeck Gerald.R.
Published: July 2002

Clones encoding the rice storage protein glutelin were selected from a cDNA library of immature rice seeds. Sequence analysis and hybridization studies on these clones provide insight into the nature of heterogeneity in glutelin genes. Based particularly on major differences in the 3′‐noncoding regions, it appears that glutelin genes fall into two sub‐families.
Effect of lipid fluidity upon the activity and structure of the 39 kDa porin from Enterobacter cloacae 908S
Authors: Ghosh Robin , Aggeler Robert
Published: July 2002

The 39 kDa porin from Enterobacter cloacae 908S was isolated in a lipopolysaccharide‐free form using the non‐ionic detergent, octylpentaoxyethylene, and reconstituted into vesicles of dimyristoylphosphatidylcholine (DMPC) and dioleoylphosphatidylcholine (DOPC), respectively. Porin activity, measured by the rate of hydrolysis of the lipid‐impermeant β‐lactam cephazoline by entrapped lactamase, could be demonstrated for porin‐DMPC but not for porin‐DOPC vesicles, and for the former was significantly lower in the gel than in the liquid‐crystalline phase. The fluorescence changes are thought to arise from lipid phase‐induced structural/dynamic changes of the porin structure.
The effects of dietary fish oil on hepatic high density and low density lipoprotein receptor activities in the rat
Authors: Roach Paul D. , Kambouris Ambrosios M. , Trimble Rodney P. , Topping David L. , Nestel Paul J.
Published: July 2002

Rats were fed either a standard ration diet or that diet supplemented with 8% by wt of a marine fish oil or safflower oil. After 10 days, plasma triacylglycerols, total cholesterol, high density lipoprotein (HDL) cholesterol, hepatic cholesterol and fatty acid synthesis and hepatic low density lipoprotein (LDL) receptor activity were significantly depressed while HDL receptor activity was significantly increased in rats fed fish oil. Fish oil‐induced effects on cholesterol metabolism in the rat therefore include reciprocal changes in the activities of hepatic LDL and HDL receptors.
Fructose 2,6‐bisphosphate and glucose 1,6‐bisphosphate in rabbit erythroid cells during differentiation
Authors: Gallego Carme , Bartrons Ramon , Carreras José
Published: July 2002

Fructose 2,6‐bisphosphate concentration and 6‐phosphofructo‐2‐kinase activity markedly decrease during differentiation of rabbit erythroid cells, being higher in erythroblasts (654 ± 97 pmol/109 cells; 238 ± 81 Uμ/109 cells) than in reticulocytes (40 ± 15 pmol/109 cells; 11 ± 3 Uμ/109 cells) and much higher than in mature erythrocytes (10 ± 0.8 pmol/109 cells 2 ± 1 Uμ/109 cells). The enzymatic activities involved in glucose 1,6‐bisphosphate metabolism also decrease, but the levels of aldohexose 1,6‐bisphosphates remain essentially constant during differentiation of erythroid cells.
Cloning and localization of the repressor gene (c) of the Mu‐like transposable phage D108
Authors: Levin David B. , DuBow Michael S.
Published: July 2002

We have localized the D108 thermosensitive (cts) repressor gene to a region of DNA approx. 600 base pairs (bp) in length by sub‐cloning an RsaI restriction endonuclease fragment (bp 200 to bp 802 from the left‐end of the D108 genome). We determined that the gene product from this fragment appears to be the same size (19 kDa) as that expressed from clones containing larger fragments of D108 DNA. Results from in vitro gel electrophoresis band‐retardation and in vivo immunity assays show that the sub‐cloned repressor appears to be fully functional.
Oxidation of N‐alkyl and C‐alkylputrescines by diamine oxidases
Authors: Frydman Rosalía B. , Ruiz Oscar , Kreisel Miriam , Bachrach Uriel
Published: July 2002

N‐Methyl‐, N‐ethyl‐, N‐propyl‐ and N‐butylputrescine were assayed as substrates of diamine oxidases from pea seedling and pig kidney. With the exception of N‐methylputrescine they were found to be oxidized to the corresponding aminoaldehydes. 1‐Methyl‐, 2‐methyl‐, 1‐ethyl‐ and 1‐propylputrescine were oxidized by the oxidases at lower rates than the N‐alkylderivatives. 1,3‐Dimethylputrescine had negligible oxidation rates while 1,4‐dimethylputrescine (2,5‐diaminohexane) was not a substrate. The oxidation of putrescine by the kidney oxidase was inhibited by 1,4‐dimethylputrescine, while the pea oxidase was strongly inhibited by the former as well as by 2‐methylputrescine and 1,3‐dimethylputrescine. Serum amine oxidase did not oxidize the substituted putrescines although several of the latter inhibited spermidine oxidation by this oxidase.
12‐O‐Tetradecanoylphorbol‐13‐acetate induces the enhancer function of human T‐cell leukemia virus type I
Authors: Fujii Masahiro , Nakamura Masataka , Ohtani Kiyoshi , Sugamura Kazuo , Hinuma Yorio
Published: July 2002

Phorbol esters were employed in studies on the molecular mechanism of the induction of expression of human T‐cell leukemia virus type I (HTLV‐I) by a tumor promoter, 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). Experiments using the chloramphenicol acetyltransferase (CAT) system showed that CAT expression directed by the long terminal repeat (LTR) of HTLV‐I was induced by TPA, but not by 4α‐phorbol‐12,13‐didecanoate, which is not an activator of protein kinase C, and that like other known enhancers, irrespective of its position and orientation, a 230‐bp fragment in the U3 region of the HTLV‐I LTR confers susceptibility to induction by TPA.
Labeling of human erythrocyte membrane proteins by photoactivatable radioiodinated phosphatidylcholine and phosphatidylserine A search for the aminophospholipid translocase
Authors: Zachowski Alain , Fellmann Pierre , Hervé Paulette , Devaux Philippe F.
Published: July 2002

We have synthesized radioiodinated photoactivatable phosphatidylcholine (125‐N3‐PC) and phosphatidylserine (125I‐N3‐PS). After incubation with red blood cells in the dark, the labeled PC could be extracted but not the corresponding PS molecule, indicating that the latter was transported by the aminophospholipid translocase, but not the former. When irradiated immediately after incorporation, N3‐PS, but not N3‐PC, partially blocked subsequent translocation of spin‐labeled aminophospholipids. Analysis of probe distribution by SDSpolyacrylamide gel electrophoresis revealed that 125I‐N3‐PS labeled seven membrane bound components with molecular masses between 140 and 27 kDa: one (or several) of these components should correspond to the aminophospholipid translocase.
Is there a root effect in Xenopus hemoglobin?
Authors: Brunori Maurizio , Bellelli Andrea , Giardina Bruno , Condo Saverio , Perutz Max F.
Published: July 2002

The reaction of Xenopus hemoglobin with oxygen and carbon monoxide has been reinvestigated over the pH range 8.5–6.0, in the absence and presence of organic phosphates (2,3‐diphosphoglycerate or inositol hexakisphosphate), to establish if the tetramer can be stabilized in a T‐quaternary state by protons and polyphosphate; the equilibrium and kinetic data indicate that Xenopus hemoglobin does exhibit a Root effect. These new results are discussed with reference to those reported by Bridges et al. [(1985) Resp. Physiol. 61, 125‐136] on Xenopus blood and, more generally, to the molecular definition and the structural basis of the Root effect as an extreme form of the Bohr effect.
Macrophage colony‐stimulating factor purified from normal human urine Amino‐terminal sequence and amino acid composition
Authors: Sakai Nobuo , Umeda Toshihiko , Suzuki Hiroyasu , Ishimatsu Yoshiaki , Shikita Mikio
Published: July 2002

A macrophage colony‐stimulating factor (M‐CSF) was purified to homogeneity from a large amount of normal human urine. Microanalysis of the N‐terminal amino acid sequence up to residue 44 revealed only a single residue difference from that deduced by other workers from the nucleotide sequence of M‐CSF cDNA clones. The amino acid composition of the present preparation suggested that the M‐CSF which we purified possessed a structure fitting the sequence 1–190 of TPA30‐1 cell M‐CSF deduced by Wong et al. [(1987) Science 235, 1504–1508].
Iodination‐induced alterations in biochemical properties of human placental insulin receptor
Authors: Ganguly Sabyasachi
Published: July 2002

Insulin receptors from human placenta have been labeled by using an oxidative iodination procedure (iodogen‐mediated or chloramine‐T‐mediated), Bolton‐Hunter reagent or [3H]acetic anhydride. The oxidative iodination procedure reduces the affinity for 131I‐insulin and the receptor protein becomes fragmented into smaller pieces with an S
20,w value of 5–6. However, treatment with Bolton‐Hunter reagent or [3H]acetic anhydride does not alter the K
d of 131I‐insulin binding and the S
20,w value remains unchanged with respect to the native receptor. It is proposed that for labeling multisubunit sulfhydryl‐linked protein drastic oxidative iodination procedures should be avoided.
Hemoglobin Grange‐Blanche [β27(B9) Ala → Val], a new variant with normal expression and increased affinity for oxygen
Authors: Baklouti F. , Giraud Y. , Francina A. , Richard G. , Périer C. , Geyssant A. , Jaubert J. , Brizard C. , Delaunay J.
Published: July 2002

Hemoglobin Grange‐Blanche [β27(B9) Ala → Val] is a new variant found in a Portuguese family. The carriers present moderate erythrocytosis. Upon isoelectric focusing, Hb Grange‐Blanche was slightly more cathodic than Hb A. βGrange‐Blanche chain migrated like the Gγ chain when submitted to electrophoresis in the presence of urea‐Triton X‐100. The precentage of Hb Grange‐Blanche was about 50% in the heterozygous state. Oxygen affinity was increased (P
50 = 22 mmHg), but heme‐heme interaction was normal. An abnormal tryptic peptide (βT3) was isolated using HPLC. Its composition allowed us to deduce unambiguously the amino acid change. The latter is the third mutation found in position 27 of the β‐chain. Because of its normal expression and its elevated affinity for oxygen, Hb Grange‐Blanche contrasts with Hb Knossos [β27(B9) Ala → Ser], a β‐thalassemic variant with low affinity.
Inhibition of herpesvirus‐induced thymidine kinase and DNA polymerase by β‐hydroxynorvaline
Authors: Massare Michael J. , Blough Herbert A.
Published: July 2002

Treatment of HSV‐infected cells with 5–10 mM β‐hydroxynorvaline (Hnv), a threonine analog, specifically affects herpesvirus DNA replication: both the rate of and total DNA synthesis are reduced, the former ∼ 15‐fold by Hnv (6 h post‐infection) and the latter by 12‐fold (between 3 and 12 h post‐infection). The effect on DNA replication was due to inhibition of HSV‐1 thymidine kinase (TK) and DNA polymerase (DP) activities; the former is reduced by 75% and whereas DP returns to baseline levels (when compared to untreated and/or uninfected cells). Host cell TK and DP activities are unaffected. It is suggested that β‐hydroxynorvaline is incorporated into these enzyme(s), either close to or at the active site thus perturbing viral DNA synthesis. β‐Hydroxynorvaline should have unique utility as a targeted antiviral compound, acting on both membrane‐mediated phenomena (fusion, penetration and attachment) and DNA replication.
Variable base pairing in a helix of eubacterial 5 S ribosomal RNA points to the existence of a conformational switch
Authors: Van den Eynde Hilde , De Wachter Rupert
Published: July 2002

A survey of 160 published sequences of eubacterial 5 S rRNAs shows that there exists structural variability in one of the helices of the generally accepted secondary structure model. Four structural variants are found, which differ with respect to the position and the number of bulges present. Most eubacterial 5 S RNAs fit into at least two of these conformations. A reaction scheme connecting the four observed conformations by changes in the base pairing scheme is proposed. Each of the known 5 S RNA sequences fits into conformations interconvertible by the proposed reactions.
Calcium‐sensitive non‐muscle α‐actinin contains EF‐hand structures and highly conserved regions
Authors: Noegel Angelika , Witke Walter , Schleicher Michael
Published: July 2002

The F‐actin crosslinking molecule α‐actinin from the slime mould Dictyostelium discoideum carries two characteristics EF‐hand structures at the C‐terminus. The calcium‐binding loops contain all necessary liganding oxygens and most likely form the structural basis for the calcium sensitivity of strictly calcium‐regulated non‐muscle α‐actinins. Furthermore, the sequence exhibits at the N‐terminal site of the molecule a high degree of homology to chicken fibroblast α‐actinin. This stretch of amino acids appears to have remained essentially constant during evolution and might represent the actin‐binding site. The findings have led us to propose a model for the inhibitory action of Ca2+ on non‐muscle α‐actinins.
Alteration of the ATG start codon of the A* protein of bacteriophage ϕX174 into an ATT codon yields a viable phage indicating that A* protein is not essential for ϕX174 reproduction
Authors: Baas P.D. , Liewerink H. , van Teeffelen H.A.A.M. , van Mansfeld A.D.M. , van Boom J.H. , Jansz H.S.
Published: July 2002

Bacteriophage ϕX174 gene A encodes two proteins: the gene A protein and the smaller A* protein, which is synthesized from a translational start signal within the A gene in the same reading frame as the gene A protein. The gene A protein is involved in initiation, elongation and termination of rolling circle DNA replication. The role of the A* protein in the life cycle of ϕX174, however, is unknown. Using oligonucleotide‐directed mutagenesis a viable ϕXl74 mutant was constructed in which the ATG start codon of the A* protein was changed into an ATT codon. This mutant, ϕX‐4499T, does not synthesize A* protein. The burst size of ϕX‐4499T amounted to 50% of that of wild type ϕXI74. This indicates that A* protein, although advantageous for phage reproduction, is not essential during the life cycle of bacteriophage ϕX174.
Loss of inducible photorepair in a frog cell line hypersensitive to solar UV light
Authors: Chao Chuck C.-K. , Lin-Chao Sue
Published: July 2002

The induction of enzymatic photorepair (EPR) in ICR 2A frog cells and a derived mutant cell line DRP36 hypersensitive to solar UV was studied. Using clonogenic assays, when induced wild‐type cells demonstrated an 8‐fold increase of EPR the mutant cells displayed a near‐background level of inducible EPR. The constitutive EPR in mutant cells, however, was the same as in wild‐type cells. A mixed culture of ICR 2A and DRP36 cells showed an intermediate inducible EPR depending upon the cell ratio. Inducible EPR was also detected at the DNA level in wild‐type cells, but not in mutant cells.
Polymyxin B, a novel inhibitor of red cell Ca2+‐activated K+ channel
Authors: Varec̆ka L̆udovít , Peterajová Elena , Pogády Jozef
Published: July 2002

Polymyxin B (PXB), a cyclic peptide antibiotic, in concentrations 0.1–3.0 mg/ml (0.08–4.0 mmol/l), inhibited the K+ efflux induced by opening of the Ca2+‐activated K+ channel (the Gárdos effect) in intact human red blood cells. The inhibition was observed when the Gárdos effect was elicited by Ca2+ in the presence of vandate, or propranolol, in ATP‐depleted cells, and in A23187‐treated cells. The inhibition of the Gárdos effect is caused neither by the inhibition of the anion channel by PXB nor by the inhibition of Ca2+ entry. It can be ascribed to the inhibition of the Ca2+‐activated K+ channel. The mechanism of the inhibition remains to be elucidated.
A method to exchange alkali light chains on myosin subfragment 1
Authors: Crasnier M.
Published: July 2002

Exchange of bound alkali light chains on myosin by free alkali light chains is described. It was found that the yield of hybrid obtained was dependent on the incubation time in 4.7 M NH4Cl at pH 9.5. 60% recovery of S1 (A1) from S1(A2) was obtained using only a 2‐fold molar excess of A1 over S1(A2).
Crosslinking of trypsin digested acto‐heavy meromyosin as a probe of the affinity of the two myosin heads to actin
Authors: Pliszka Barbara
Published: July 2002

The interaction of the two heads of the myosin molecule with actin was studied by tryptic digestion of HMM in the presence of actin, followed by crosslinking the two nicked heavy chains with Nbs2 at the S2 region. In view of the protection by actin of the kDa junction against proteolysis, the percentage of the heads interacting with actin was estimated from the proportion of the 110 kDa to the 60 kDa digestion product. Under conditions such that about 50% of HMM heads were protected by actin (at an actin to HMM head molar ratio of 1:1 in the absence of nucleotide, or 3:1 in the presence of 5 mM ADP), the crosslinking of the digestion products yielded a 230 kDa (110+110 kDa), 125 kDa (60+60 kDa) and 175 kDa (60+110 kDa) species. Since the latter should be the only crosslinking product when only one head of HMM molecule is protected by actin, it is concluded that there is no preferential binding of one of the two HMM heads to actin in the presence of ADP or at equimolar actin to myosin heads ratio.
dam methylase from E. coli
Authors: Kriebardis Anastasios , Guschlbauer Wilhelm
Published: July 2002

The enzyme dam methylase which recognizes and methylates the adenine in the palindromic sequence GATC in DNA was isolated and the secondary structure was determined by CD spectroscopy and various predicting methods from the amino acid sequence. The interaction of dam methylase with S‐adenosylmethionine was studied by CD spectroscopy indicating a decrease of the percentage of α‐helix as the amount of S‐adenosylmethionine bound to the enzyme was increased.
Effects of organic solvents and orthophosphate on the ATPase activity of F1 ATPase
Authors: de Meis Leopoldo
Published: July 2002

The ATPase activity of soluble F1 ATPase of mitochondria is activated by Pi. The concentration of Pi required for half‐maximal activation decreases from a value higher than 50 mM to about 1 mM Pi when one of the organic solvents dimethyl sulfoxide (15 to 30%), methanol (7.5 to 15%) or ethylene glycol (10 to 30%) is added to the assay medium. This effect is observed in the presence of MgCl2 but not in the presence of CaCl2.
Adenosine and oxytocin reverse antagonism of cyclic AMP elevating agents to insulin activation of adipocyte pyruvate dehydrogenase
Authors: Wong Ellen H.A. , Smith Judith A. , Jarett Leonard
Published: July 2002

ACTH, isoprenaline, forskolin, and dibutyryl cyclic AMP prevented insulin from stimulating adipocyte pyruvate dehydrogenase in the presence of adenosine deaminase. Antagonism was reversed by N
6‐phenyliso‐propyladenosine as well as oxytocin. The stimulatory effects of insulin, adenosine and oxytocin on adipocyte pyruvate dehydrogenase appear to be through (a) mechanism(s) which is (are) similar or related.
NMR study of the interaction of P‐450 with 4‐methoxypyridine
Authors: Woldman Ya.Yu. , Weiner L.M. , Gulayeva L.F. , Lyakhovich V.V.
Published: July 2002

Longitudinal relaxation (T1) measurements for all lines (α‐CH, β‐CH, O‐CH3) in the 4‐methoxypyridine 1H‐NMR spectrum were used to study the interaction of 4‐methoxypyridine with purified microsomal cytochrome P‐450 from livers of phenobarbital‐treated rats. The paramagnetic contribution to the observed T1
−1 value was determined from its dependence on 4‐methoxypyridine concentration. In the P‐450‐4‐methoxypyridine complex the latter is oriented so that the nitrogen of pyridine is directed towards the Fe3+.
Sizing of single‐stranded regions in double‐stranded DNA by preparative benzoylated DEAE‐cellulose chromatography
Authors: Norris Murray D. , Stewart Bernard W.
Published: July 2002

The concentration of caffeine required to elute wholly single‐stranded DNA from benzoylated DEAE‐cellulose is proportional to the polynucleotide length. The use of benzoylated DEAE‐cellulose chromatography for isolating and sizing single‐stranded regions in double‐stranded DNA has been examined using a series of hybrid molecules. Restriction fragments of the replicating form of bacteriophage ϕX174 were hybridized to the intact ‘plus’ strand, thereby forming hybrids having single‐ and/or double‐stranded regions in the kilobase range. A series of such hybrid preparations were subject to caffeine concentration gradient elution from benzoylated DEAE‐cellulose. After logarithmic transformation, a linear relationship (R = 0.94) could be demonstrated between eluting caffeine concentration and single‐stranded length, irrespective of the length of associated double‐stranded regions or the location, within a given fragment, of unpaired nucleotides. Benzoylated DEAE‐cellulose chromatography may therefore be used to separate and characterize, on a preparative scale, double‐stranded DNA containing single‐stranded regions.
A resonance Raman study of the binding of ethanol and methanol to ferrihemoglobin
Authors: Muhoberac B.B. , Shelnutt J.A. , Ondrias M.R.
Published: July 2002

The interactions of ethanol and methanol with ferrihemoglobin were examined using resonance Raman spectroscopy. After binding either alcohol, the low‐frequency resonance Raman spectra of human ferrihemoglobin are almost identical to the unperturbed spectrum except for shifts in the 309 cm−1 band to higher frequency by as much as 8 cm−1. The ethanol‐induced shift is greater than that with methanol even though complex formation was less for ethanol than methanol. The spectral changes imply a site‐specific, similar binding of these alcohols to ferrihemoglobin which may involve steric interactions. Possible assignments of the 309 cm−1 band to structural features as well as potential mechanisms of the alcohol‐induced spectral changes are discussed.
A new approach to studies of cell‐antibody interactions using fluorescent lipid probes
Authors: Tonevitsky A.G. , Manevich E.M. , Zhukova O.S. , Arsen'eva E.L. , Bogacheva G.T. , Bergelson L.D.
Published: July 2002

The interaction of poly‐ and monoclonal antibodies against the L‐chain of human Ig with Burkitt lymphoma EB‐3 cells was studied using a fluorescent lipid probe, anthrylvinyl‐labelled sphingomyelin, incorporated into the cell plasma membrane. Binding of the antibodies to Ig receptors on the surface was shown to induce changes in the fluorescence polarization of the probe. The high sensitivity of the method allows one to detect less than 100 antibody molecules per cell. The possibility of using cells or liposomes carrying antigens and fluorescent lipids for the determination of antibodies in solution is discussed.
Na‐pump activity in rat kidney cortex cells and its relationship with the cell volume
Authors: Proverbio Fulgencio , Proverbio Teresa , Matteo Rosalía G. , Perrone Trina M. , Marín Reinaldo
Published: July 2002

The present work was undertaken to evaluate whether changes in cell water content of rat kidney cortex cells can modulate the transport activity of the ouabain‐insensitive Na pump as they modulate the ouabain‐insensitive Na+‐ATPase. It was found that there is a close relationship between the cell volume and activity of the Na pump, whereas Na,K‐pump activity is not affected by variations in cell volume. When the cell water content is low, Na‐pump activity (Na+ transport and Na+‐ATPase activity) is minimal. Increases in cell water content produce a concomitant increase in Na‐pump activity.
Stimulation of phosphoinositides breakdown by the heat stable E. coli enterotoxin in rat intestinal epithelial cells
Authors: Banik Napur , Ganguly Uma
Published: July 2002

Rat intestinal epithelial cells were labelled with [32P]Pi and extracted, and the phospholipids were analysed by thin‐layer chromatography. 32P‐incorporation in phosphatidylinositol (PI) and phosphatidylinositol 4‐phosphate (PIP) and phosphatidylinositol 4,5‐phosphate (PIP2) were measured in control and heat stable enterotoxin (ST)‐treated cells. ST was found to induce rapid degradation of PIP and PIP2. The degradation of inositol lipids was accompanied by an increase of water soluble inositol phosphate (IP1, IP2, IP3) compounds. There was a two‐fold increase of radioactivity in IP2 and IP3 but no significant change was observed in IP1. Phospholipase C activity was increased tenfold with substrate PIP2 in ST‐pretreated cells. The present study indicates that ST triggers another second messenger system by increasing the PIP2 hydrolysis with the enzyme phospholipase C.
Regulation by Ca2+ of membrane elasticity of bovine chromaffin granules
Authors: Miyamoto Shigeaki , Fujime Satoru
Published: July 2002

In a range of [Ca2+] similar to cytosolic transient, a drastic reduction from about 20 dyn/cm to almost zero was observed in the membrane elastic modulus of bovine chromaffin granules, isolated in a solution containing 0.3 M sucrose and 5 mM Hepes at pH 7.0, and measured by combination of osmotic swelling and dynamic light‐scattering (DLS) methods. This result suggests that the granule membrane becomes extremely flexible as a prelude to exocytosis.
A simple strategy to amplify specifically the HLA‐DQβ gene region with genomic DNA as template
Authors: Gu Xue Fan , Elion Jacques , Ouagued Malika , Clauser Eric , Assan Roger , Krishnamoorthy Rajagopal
Published: July 2002

The nature of codon 57 in the HLA‐DQβ gene was recently reported as a potential marker of genetic susceptibility to insulin‐dependent diabetes mellitus. When exploring the relevance of this marker by using genomic DNA amplification, we encountered difficulties resulting from the coamplification of the homologous DXβ region. A simple strategy is proposed to amplify the DQβ region exclusively. It involves the preliminary digestion of genomic DNA with a restriction enzyme which cleaves DXβ specifically, leaving intact the DQβ sequence. The amplified material is suitable for dot blot analysis and restriction enzyme digestion. This strategy is of general interest when homologous sequences impair the specificity of enzymatic DNA amplification.
Chromosomal localization of the gene coding for the β‐subunit of Na+,K+‐ATPase in the American mink (Mustela vison)
Authors: Khlebodarova T.M. , Karasik G.I. , Lapteva S.E. , Matveeva N.M. , Serov O.L. , Sverdlov E.D. , Broude N.E. , Modyanov N.N. , Monastyrskaya G.S.
Published: July 2002

The BATP gene coding for the β‐subunit of Na+,K+‐ATPase has been localized on chromosome 13 of the American mink (Mustela vison) using mink‐Chinese hamster somatic cell hybrids and pig cDNA clones as probes. The AATP gene for the α‐subunit of Na+,K+‐ATPase is on mink chromosome 2[(1987) FEBS Lett. 217, 42–44]. Consequently, the AATP and BATP genes for the Na+,K+‐ATPase occupy separate mink chromosomes.
Processing of preproglycinin expressed from cDNA‐encoding A1aB1b subunit in Saccharomyces cerevisiae
Authors: Utsumi Shigeru , Sato Toshiro , Kim Chan-Shick , Kito Makoto
Published: July 2002

The cDNA sequence encoding the glycinin A1aB1b subunit was placed under control of the repressible‐acid phosphatase promoter of the yeast Saccharomyces cerevisiae in an expression plasmid. Yeast transformants containing these plasmids synthesized A1aB1b protein. The signal sequence of the A1aB1b protein was cleaved at the right processing site and the A1aB1b protein was not glycosylated in yeast in the same manner as in soybean.
Protein NMR resonance assignment by isotropic mixing experiments on random fractionally deuterated samples
Authors: LeMaster David M.
Published: July 2002

The 108‐residue protein E. coli thioredoxin has been uniformly enriched to 50% with deuterium at all carbon‐bound hydrogen positions. Isotropic mixing (i.e. TOCSY) experiments have been conducted for both the deuterated and natural‐abundance samples. Using a 54 ms mixing time correlation peaks can be seen for all four protons on the benzenoid ring of tryptophan in both samples. The deuteration results in an average decrease in cross‐sectional area of a factor of 2–3 for the TOCSY cross‐peaks. The cross‐peak intensities for the deuterated sample systematically decrease as a function of the number of protons involved in the transfer process thus overcoming a common ambiguity in the TOCSY experiment.
Effect of amrinone on myocardial mitochondria function
Authors: Bobyleva-Guarriero Valentina , Mussini Cristina , Muscatello U. , Cappello C. , Mattioli G.
Published: July 2002

The effect of amrinone on cardiac mitochondria of guinea pig was studied. It was found that amrinone does not change the respiratory function of cardiac mitochondria in the presence of α‐ketoglutarate, whereas it inhibits glutamate oxidation. It was also found that amrinone strongly inhibits the activity of glutamic dehydrogenase of both crude extract from sonicated heart mitochondria and of purified preparation from bovine liver. This inhibition may explain the effect of amrinone on the oxidation of glutamate in mitochondria. These results are discussed in view of the contradictory effects of amrinone on cardiac and other tissues.
Two distinct types of ɛ‐binding site exist in chloroplast coupling factor (CF1)
Authors: Andralojc P.J. , Harris D.A.
Published: July 2002

Isolated chloroplast coupling factor (CF1), when depleted of its ɛ‐subunit, has a high ATPase activity which can be inhibited by binding ɛ to a single high‐affinity (K
d = 1.4 × 10−10 M) site. In CF1 reduced by dithiothreitol (DTT), however, ɛ at this binding site is no longer inhibitory. Instead, 3 equivalent, lower affinity (K
d = 6 × 10−8 M), inhibitory binding sites for ɛ are observed, whether or not the high‐affinity site contains a bound ɛ‐subunit. Binding of ɛ to the high‐affinity site protects CF1 against trypsin activation, while binding to the low‐affinity site does not, showing that occupation of each class of site has a different effect on CF1 structure. The effects of DTT can be interpreted in terms of a reduction in intersubunit cooperativity in CF1.
Stimulation and partial stabilization of human histidyl‐tRNA synthetase by hemoglobin
Authors: Biswas Tapas , Miller Frederick W. , Plotz Paul H.
Published: July 2002

Histidyl‐tRNA synthetase, an enzyme against which antibodies are directed in some patients with polymyositis, has been purified 5000‐fold from HeLa cells, but was extremely labile to dilution or on storage at −80°C. In order to facilitate study of the biochemical and immunological properties of the enzyme, a stabilizer was sought. Hemoglobin at 2 mg/ml was found to stimulate the enzyme and also partially preserved the activity of the enzyme stored at a low concentration (< 10μg/ml). Hematin, but not the globin protein, could substitute for hemoglobin in stimulating the enzyme.
Parallel measurement of oxoglutarate dehydrogenase activity and matrix free Ca2+ in fura‐2‐loaded heart mitochondria
Authors: Lukács Gergely L. , Kapus András , Fonyó Attila
Published: July 2002

The entrapment of the Ca2+ ‐sensitive fluorescence indicators fura‐2 or quin2 in the matrix space of isolated heart mitochondria renders possible the direct monitoring of the matrix free Ca2+ ([Ca2+]m) [(1987) Biochem. J. 248, 609–613]. In this paper the correlation between the [Ca2+]m and the in situ activity of oxoglutarate dehydrogenase (OGDH) in fura‐2‐loaded mitochondria is shown. At the initial value of [Ca2+]m, 64 nM, which corresponded to 0.36 nmol/mg mitochondrial Ca content, the OGDH activity was 12% of the maximal. Half‐maximal and maximal activation were attained at 0.8 and 1.6 μM [Ca2+]m, respectively. The results indicate that an increase of the mitochondrial Ca content in the physiological range enhances the OGDH activity by means of elevation of [Ca2+]m.
Effect of exogenously added acylphosphatases on inositol lipid metabolism in human platelets
Authors: Berti Andrea , Stefani Maurizio , Degl'Innocenti Donatella , Ruggiero Marco , Chiarugi Vincenzo , Ramponi Giampiero
Published: July 2002

In this paper we demonstrate that human platelets contain an acylphosphatase isoenzyme. We then investigated the of exogenously added human muscle and erythrocyte acylphosphatases on inositol lipid content in human platelets permeabilized with saponin. Alterations in the level of the polyphosphoinositides were observed: in particular, the levels of phosphatidylinositol 4,5‐bisphosphate, and of phosphatidylinositol 4‐monophosphate were decreased, whereas the level of phosphatidylinositol was increased. These results suggest that acylphosphatases promote polyphosphoinositide dephosphorylation, possibly through intracellular Ca2+ mobilization.
AsnI: A novel class II restriction endonuclease from Arthrobacter sp., strain N‐CM, recognizing 5′‐AT/TAAT‐3′
Authors: Rexer Bernhard U. , Jarsch Michael , Sagmeister Christine , Glück Barbara , Berger Gerhard , Kessler Christoph
Published: July 2002

A new class II restriction endonuclease, AsnI, with a novel sequence specificity was isolated from the Gram‐positive eubacterium Arthrobacter species, strain N‐CM. AsnI recognizes the unambiguously defined palindromic hexanucleotide
consisting of A‐ and T‐residues. The novel enzyme in the presence of Mg2+ cleaves specifically both strands as indicated by the arrows. The staggered cuts generate 5′‐protruding ends with single‐stranded 5′‐TA‐3′ dinucleotide extensions. The novel enzyme may be a useful tool for cloning experiments by complementation of the few enzymes such as PstI and PvuI cutting only once in the Ampr‐gene of plasmids pBR322 and pBR328.
Non‐enzymatic synthesis of hygrine from acetoacetic acid and from acetonedicarboxylic acid
Authors: Endo Tsuyoshi , Hamaguchi Nobuko , Hashimoto Takashi , Yamada Yasuyuki
Published: July 2002

Hygrine, an intermediate in biosynthesis of tropane alkaloids, was formed non‐enzymatically by the coupling of γ‐methylaminobutyraldehyde with either acetoacetic acid or acetonedicarboxylic acid. When these ketoacids were fed to a tropane alkaloid‐free culture of tobacco roots, hygrine formation was observed. A possible process of this reaction is discussed.
Proteins released from stimulated neutrophils contain very high levels of oxidized methionine
Authors: Beck-Speier I. , Leuschel L. , Luippold G. , Maier K.L.
Published: July 2002

In proteins released from quiescent human neutrophils during incubation, 21% of the methionine (Met) residues were found to be oxidized. However, the portion of oxidized Met in extracellular proteins increased to 66% after stimulating the cells with zymosan and to 75% after stimulation with phorbol myristate acetate (PMA). Generation of such high levels of oxidized Met in native proteins by activated neutrophils has, so far, not been observed. The presence of superoxide dismutase during incubation of PMA‐stimulated cells produced a negligible effect on methionine oxidation, while the presence of catalase resulted in a methionine suifoxide (Met(O)) content of only 28% in the released proteins. It is proposed that the conversion of Met to Met(O) in these proteins predominantly occurs by action of the myeloperoxidase/ H2O2/Cl− system in the extracellular space.
The turnover number for band 3‐mediated sulfate transport in phosphatidylcholine bilayers
Authors: Scheuring Uwe , Lindenthal Sabine , Grieshaber Gabriele , Haase Winfried , Schubert Dieter
Published: July 2002

The anion transport system of the human erythrocyte membrane was reconstituted in unilamellar phosphatidylcholine vesicles, and a vesicle subpopulation of a narrow size distribution was isolated from the sample by gel filtration. In this subpopulation, the turnover number of the transport protein (the band 3 protein) for sulfate transport was determined. It was found that, in the reconstituted system, the protein transports sulfate 5–10‐times faster than in the human erythrocyte membrane.
Effect of DNA supercoiling on in vitro transcription from the adenovirus early region 4
Authors: Handa Hiroshi , Watanabe Hajime , Suzuki Yoshiaki , Hirose Susumu
Published: July 2002

The effect of DNA supercoiling on in vitro transcription from the early region 4 (E4) promoter of adenovirus type 5 (Ad5) has been investigated by using a cell‐free transcription system that maintains supercoiled DNA templates. The supercoiled DNAs yield several‐fold higher levels of E4 transcripts and more faithfully reflect the regulation of in vivo transcription than the linear DNAs.
Conformational modification of muscle phosphofructokinase from Jaculus orientalis upon ligand binding
Authors: Tijane M. , El Hachimi Z. , Benjouad A. , Desmadril M. , Yon J.M.
Published: July 2002

Phosphofructokinase from Jaculus orientalis muscle is an allosteric enzyme regulated by substrates and nucleotide effectors. The conformational modifications upon ligand binding were probed by UV difference spectra and reactivities of thiol groups towards dithiobisnitrobenzoate and N‐ethylmaleimide. The binding of Fru‐6‐P induced significant perturbations in the environment of the aromatic residues and buried the most reactive on the three accessible cysteines per protomer. The same effect on thiol reactivity was observed upon binding of the activator AMP. Various perturbations of both difference spectra and thiol reactivity were detected in the presence of either MG‐ATP, an allosteric inhibitor, or MG‐ITP which is not an effector.
Evidence for the appearance of a reticulocyte population low in lipoxygenase mRNA during the recovery from a phenylhydrazine‐induced anemia in rabbits
Authors: Minich W.B. , Evdokimova V.M. , Oleinikov A.V. , Höhne M. , Thiele B.J. , Rapoport S.M.
Published: July 2002

It is shown that during recovery from a phenylhydrazine‐induced anemia in rabbits a selective decrease in lipoxygenase mRNA takes place with a corresponding shut‐off of the synthesis of the enzyme. It is suggested that a new population, ‘recovery’‐reticulocytes, makes its appearance in the peripheral blood. Their cells are more mature than the stress macroreticulocytes. A cell‐free system prepared from the recovery‐reticulocytes exhibits low endogenous synthesis of non‐globin polypeptides, even without nuclease treatment, but retains full capacity to be stimulated by exogenous mRNA.
Optimising selective deuteration of proteins for 2D 1H NMR detection and assignment studies Application to the Phe residues of Lactobacillus casei dihydrofolate reductase
Authors: Feeney J. , Birdsall B. , Akiboye J. , Tendler S.J.B. , Barbero J.Jiménez , Ostler G. , Arnold J.R.P. , Roberts G.C.K. , Kühn A. , Roth K.
Published: July 2002

A selectively deuterated dihydrofolate reductase from L. casei has been prepared containing partially deuterated aromatic amino acids. This provides simplified 2D NMR spectra and allows signals from all 8 Phe residues to be identified. The pattern of deuteration is such that (i) the only cross‐peaks detected in the aromatic region of the 2D COSY spectrum are those between the Phe 2′,6′ and 3′,5′ protons and (ii) chemical shift degeneracy in the aromatic region is removed thus allowing unambiguous assignment of cross‐peaks in 2D NOESY spectra required for specific assignment purposes.
Hysteretic interaction of NADH and Mg2+ with mammalian NADH:CoQ reductase from beef heart
Authors: Tushurashvili P.R. , Dekanosidze N.Z. , Inasaridze N.P. , Kekelidze T.N. , Tsartsidze M.A. , Lomsadze B.A.
Published: July 2002

Preincubation of submitochondrial particles (SMP) from beef heart in a reaction mixture containing low concentrations of Mg2+ induces a time lag in the NADH:oxidase activity. Preconditioning of the SMP by NADH, but not by NAD+, prevents the Mg2+‐related time lag. The data obtained show that there exists a tight binding site for Mg2+ regulating the rate of electron transfer from NADH to the natural acceptor. The ability of Mg2+ to form a catalytically inactive complex with the enzyme is regulated by NADH.
Regulation of enzymatic activity by kinase‐free phosphorylation
Authors: Vener Alexander V.
Published: July 2002

Inorganic pyrophosphatase activity is regulated by kinase‐free phosphorylation. Phosphorylation by ATP activates the enzyme and that by Pi eliminates the activating effect of ATP. Acyl phosphate formed in the reaction with ATP is a covalent intermediate of ATP hydrolysis in the regulatory site of the enzyme. Therefore, kinase‐free phosphorylation shares the properties of both regulatory and catalytic phosphorylations.
Mechanism of inhibition of eukaryotic translational initiation by the trinucleotide ApUpG
Authors: Wagner Thomas , Sigler Paul B. , Gross Martin
Published: July 2002

ApUpG, the oligoribonucleotide homologous to the initiation codon, as well as the tetranucleotides ApUpGpA and ApUpGpG block initiation of protein synthesis in the rabbit reticulocyte lysate. These oligonucleotides are recognized as translational initiation sites by the ribosomes, leading to a very large accumulation of complete, but inactive, 80 S initiation complexes, containing methionylated initiator tRNA and ApUpG in a 1:1 stoichiometry. ApUpG appears to inhibit by competing with endogenous globin mRNA for 80 S ribosomal couples, since the inhibition of protein synthesis by ApUpG can be largely relieved by increasing the globin mRNA. The 80 S · Met‐tRNAi
Met · ApUpG complexes are not formed in the absence of hemin, demonstrating that their formation requires the active recycling of eukaryotic initiation factor 2. In addition the trinucleotide correctly directs the Met‐tRNAi
Met into the ribosomal donor site, since the methionyl residue is puromycin‐reactive.
Antibodies against an inter‐domain segment of polypeptide chain inhibit active‐site coupling in the pyruvate dehydrogenase multienzyme complex
Authors: Radford Sheena E. , Perham Richard N. , Ullrich Stephen J. , Appella Ettore
Published: July 2002

A synthetic peptide, AAPAAAPAKQEAAAPAPAAKAEAPAAAPAAKA, proved to be an efficient and specific immunogen in rabbits. The amino acid sequence of the peptide is identical to that of the inter‐domain region (PEP3) linking the innermost of the three lipoyl domains to the dihydrolipoamide dehydrogenase‐binding domain in the dihydrolipoamide acetyltransferase chain of the pyruvate dehydrogenase complex of Escherichia coli. Fab fragments from anti‐PEP3 antibodies selectively inhibited active‐site coupling in the complex without affecting the individual activities of the three component enzymes, highlighting the role of the inter‐domain regions as flexible linkers in catalysis.
Production of plasmids giving high expression of recombinant DNA‐derived ovine growth hormone variants in Escherichia coli
Authors: Wallis O.Caryl , Wallis Michael
Published: July 2002

A method for the production of plasmids giving different levels of expression of ovine growth hormone (oGH) variants in E. coli is described. The cDNA sequence coding for mature oGH was inserted into the multiple cloning site of plasmid pUC8 and random deletions were then introduced 3′ to the initiation codon. Clones producing GH (with varying N‐terminal extensions) were identified by immunological screening. Levels of expression of GH‐related protein, measured by immunoassay or on SDS‐polyacrylamide gels, varied from over 20% to less than 0.05% of total cell protein. The coding sequence of plasmid pOGHe101, giving very high expression of variant oGH1, was determined.
Isolation of cDNA clones encoding the human Sm B/B′auto‐immune antigen and specifically reacting with human anti‐Sm auto‐immune sera
Authors: Sharpe N.G. , Williams D.G. , Howarth D.N. , Coles B. , Latchman D.S.
Published: July 2002

A cDNA clone for the human SmB and B′ auto‐immune antigens has been isolated by antibody screening of a cDNA expression library. The cDNA clone hybridises with two distinct mRNAs, one of which is expressed in a tissue‐specific manner. A fusion protein expressed from the cDNA clone was recognised by a number of sera from systemic lupus erythematosus (SLE) patients containing anti‐Sm antibodies but not by sera reactive with other auto‐immune antigens. The potential use of this clone in a diagnostic assay for SLE and in elucidating the processes regulating the expression of SmB and B′ is discussed.
Cloning of a DNA region of Actinoplanes teichomyceticus conferring teicoplanin resistance
Authors: Moroni M.C. , Granozzi C. , Lorenzetti R. , Parenti F. , Sosio M. , Denaro M.
Published: July 2002

Teicoplanin is a glycopeptide antibiotic, produced by Actinoplanes teichomyceticus, active against Gram positive bacteria and recently introduced into clinical practice. It blocks cell wall biosynthesis by inhibiting peptidoglycan polymerization. The mechanism(s) of resistance of the producer strains of this class of antibiotics have not yet been characterized. We have constructed a genomic bank of A. teichomyceticus in Streptomyces lividans. A clone from this bank, pTR168, was able to confer resistance to teicoplanin on its sensitive host. The restriction map of plasmid pTR168 and the hybridization pattern to A. teichomyceticus DNA were determined; we have also studied the mechanism of this resistance which seems correlated with a reduced binding of the antibiotic to the cell wall.
Fructose 2,6‐bisphosphate and glucose 1,6‐bisphosphate levels in erythrocytes with high and low 2,3‐bisphosphoglycerate content during postnatal development
Authors: Gallego Carme , Carreras José
Published: July 2002

In rabbit and sheep erythrocytes the concentrations of 2,3‐bisphosphoglycerate, fructose 2,6‐bisphosphate and glucose 1,6‐bisphosphate suffer important changes after birth, which differ in both species. The changes of fructose 2,6‐bisphosphate and glucose 1,6‐bisphosphate correlate with the changes in the levels of the enzymatic activities involved in their synthesis. The change of 2,3‐bisphosphoglycerate levels in rabbit but not in sheep erythrocytes could be explained by the changes of the phosphofructokinase/pyruvate kinase and 2,3‐bisphosphoglycerate synthase/2,3‐bisphosphoglycerate phosphatase activity ratios.
In vitro translation of halobacterial mRNA
Authors: Gropp R. , Oesterhelt D.
Published: July 2002

A system for in vitro translation of halobacterial mRNA is described. It is composed of 70S ribosomes, a nuclease‐treated S100 supernatant, yeast tRNA and halobacterial mRNA. Specific expression of proteins could be demonstrated by translation of messages from halophage ϑ1‐infected cells. However, under no conditions could a pure bacterio‐opsin message be observed to be active in translation. This points to a more complex translational property of bacterio‐opsin and possibly other membrane proteins in archaebacteria.