bluuebunny/crossref_metadata_2025 huggingface dataset:
Thrombin and IgE antigen induce formation of inositol phosphates by mouse E‐mast cells
Authors: Baranes D. , Liu F.T. , Razin E.
Published: July 2002

Stimulation of murine chondroitin sulfate E containing mast cells (E‐MC) in vitro either by thrombin or immunologically resulted in a rapid formation of inositol phosphates (IPs). Increase in all of the three IPs (IP1 IP2 and IP3) could be detected 20 s after stimulation. The depletion of Ca2+ from the medium resulted in more than 80% reduction in β‐hexosaminidase release from either thrombin or IgE antigen stimulated cells. However, both thrombin and IgE antigen increased the formation of IP3 under these conditions independent of the presence of extracellular Ca2+.
Structural relationship between link proteins and proteoglycan monomers
Authors: Périn J.-P. , Bonnet F. , Jollés P.
Published: July 2002

Structural homologies between link proteins and proteoglycan monomers are demonstrated. A possible redundancy in the proteoglycan monomers structure is discussed and the link proteins domains homologous to other proteins are specified.
Identification of a 110‐kDa glycoprotein involved in cell‐substratum adhesion
Authors: Neumeier R. , Hanski C. , Reutter W.
Published: July 2002

Plasma membrane glycoproteins are involved in cell‐matrix interactions. For identification of such glycoproteins a recently developed cell system was used. Two cell populations differing in their adhesion properties were selected from Morris hepatoma 7777. One population was able to grow as a monolayer, while the other proliferated in suspension. From both cell lines spontaneous revertants were selected. By using antibodies raised against plasma membranes of the hepatoma and of rat liver for sequential immunoprecipitation a glycoprotein of M
r, 110000 was identified. This glycoprotein was only expressed in adherent hepatoma cells as well as in normal rat liver, but was absent in non‐adherent hepatoma cells and in the in vivo growing Morris hepatoma 7777. This suggests that the glycoprotein is involved in cell‐substratum adhesion of hepatocytes and adherent hepatoma cells.
Mutations of Chlamydomonas reinhardtii affecting the cytochrome bf complex
Authors: Bendali D.S. , Sanguansermsri M. , Girard-Bascou J. , Bennoun P.
Published: July 2002

Cytochrome bf complex
Cytochrome b‐559
Iron‐sulfur center
Photosynthetic mutant
(Chlamydomonas reinhardtii)
Cholesterol uptake of isolated rat hepatocytes is accelerated by several kinds of phosphatidylcholine
Authors: Nagamura Yoichi , Sasaki Kunio , Ito Madoka , Saito Kuniaki , Ogitsu Naomichi , Ishiguro Isao
Published: July 2002

Cholesterol uptake
(Isolated rat hepatocyte)
Phosphatidylcholine
Unsaturated fatty acid
Dioleoylphosphatidylcholine
Dipalmitoylphosphatidylcholine
Homology between bacterial DNA and bovine mitochondrial DNA encoding cytochrome c oxidase subunit III
Authors: Fink Pamela S. , Whitford Tracy , Leffak Michael , Prochaska Lawrence J.
Published: July 2002

A segment of mitochondrial DNA encoding the bovine cytochrome c oxidase subunit III gene was isolated and inserted into an Escherichia coli plasmid vector. A 556 base pair fragment of the insert DNA representing about 70% of the 3′‐end of the subunit III gene was used to search for homology with bacterial DNA from strains that contain heme aa
3‐type cytochrome c oxidases. Bacillus subtilis, Thermus thermophilus, and PS3 DNAs all showed strong hybridization to the probe, whereas Paracoccus denitrificans and Rhodopseudomonas sphaeroides DNAs showed only weak hybridization to the probe, even under low stringency conditions.
Enhanced metabolism of phosphatidylinositol in Candida tropicalis in association with filamentous growth caused by ethanol
Authors: Uejima Yasuhide , Koga Teiichiro , Kamihara Teijiro
Published: July 2002

Candida tropicalis Pk 233 grows in filamentous form in ethanol‐supplemented medium, and myo‐inositol prevents the ethanol ettect [(1979) Bioch!em. Biophys. Res. Commun. 91, 351–355]. Cells, which were grown with ethanol to the log phase, exhibited an increased rate of phosphatidylinositol turnover as judged by pulse‐chase experiments with 32Pi. Such enhanced metabolism of phosphatidylinositol was not observed in fully developed filamentous cells at the stationary phase or in yeast‐like cells which were grown without added ethanol or with ethanol plus myo‐inositol. The metabolism of other phospholipids was not affected by ethanol.
Luteinizing hormone‐releasing hormone (LHRH)‐induced arachidonic acid release in rat granulosa cells Role of calcium and protein kinase C
Authors: Minegishi T. , Wang J. , Leung P.C.K.
Published: July 2002

In rat granulosa cells prelabeled with [3H]arachidonic acid, addition of LHRH, A23187 or 12‐0‐tetradecanoyl phorbol‐13‐acetate (TPA) enhanced the release of [3H]arachidonic acid into the culture medium. The effect of A23187 was significant as early as 5 min and the lowest effective dose was 5 × 10−8/M. On the other hand, TPA was effective only at dosages greater than 10−6M. These results suggest that the stimulatory effect of LHRH on arachidonic acid release is coupled more tightly to a Ca2+‐dependent rather than a protein kinase C‐mediated pathway.
The effects of protein synthesis inhibition, and of mutations rna1.1 and rna82.1, on the synthesis of small RNAs in yeast
Authors: Piper Peter W. , Lockheart Alan , Bellatin Jaime
Published: July 2002

Strains of Saccharomyces cerevisiae have been constructed that possess temperature‐sensitive defects in tRNA precursor (pre‐tRNA) splicing and which also lack the processing endonuclease that acts at the 3′‐terminus of 5 S rRNA and 35 S rRNA precursors (pre‐rRNAs). The unspliced pre‐tRNAs accumulated by such strains at the nonpermissive temperature are identical in structure to those accumulated by pre‐tRNA splicing‐defective strains with a functional pre‐5 S RNA processing enzyme. The pre‐RNA processing activity is therefore not obligatorily involved in maturation of several yeast tRNAs. However, gels of the pulse‐labelled RNAs of RNA82+ and rna82.1 strains provide evidence that this enzyme acts upon a few small unstable transcripts that are not 5 S RNA forms. The most prominent of these transcripts on gels was, in wild‐type strains, an RNA 145 ± 2 nucleotides in length.
α‐Melanocyte‐stimulating hormone secretion from permeabilized intermediate lobe cells of rat pituitary gland The role of guanine nucleotides
Authors: Yamamoto Toshifumi , Furuki Yoshihiro , Kebabian John W. , Spatz Maria
Published: July 2002

The non‐hydrolyzable GTP analogue, guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS) and cyclic AMP potentiated the Ca2+‐evoked secretion of α‐melanocyte‐stimulating hormone (α‐MSH) from permeabilized neurointermediate lobe (IL) cells of rat pituitary gland. The enhancement by Mg‐GTPγS (100 μM) and cyclic AMP (1 μM) depended on the intracellular CA2+ concentration (EC50 = 4.8 ± 1.8 and 4.6 ± 1.7 μM; mean ± SE, with and without Mg‐GTPγS and cyclic AMP, respectively). A similar effect was observed with guanine nucleotide triphosphate (GTP and GppNHp). Mg was absolutely required for this event. Neither Mg‐GTPγS nor cyclic AMP alone was effective in potentiating α‐MSH secretion. GDPβS blocked the Mg‐GTPγS (100 μM) and cyclic AMP augmented secretion of α‐MSH. Neither neomycin (which affects the process of inositol 1,4,5‐triphosphate‐mediated Ca2+ mobilization) or colchicine (which influences microtubule assembly) had an effect on the cyclic AMP and Mg‐GTPγS potentiation of α‐MSH secretion. These data suggest that the GTP‐binding protein may be involved in the regulation of α‐MSH secretion after Ca2+ entry into the cells, since the intracellular environment is controlled in the permeabilized cells.
Production of the constant domain of murine T‐cell receptor β‐chain in Escherichia coli
Authors: Kuwana Yoshihisa , Itoh Seiga , Nagase Fumihiko , Nakashima Izumi , Kurosawa Yoshikazu
Published: July 2002

T‐cell antigen receptor is a heterodimer of disulfide‐linked α‐ and β‐chains. Although the essential features of T‐cell receptor seem to be rather similar to those of immunoglobulin, the amount of T‐cell receptor expressed on the surface of a T‐cell is not large enough to be analyzed physico‐chemically. In this study, the DNA fragment encoding 120 amino acids from the 116th to the 235th of the murine, β‐chain which corresponds to the presumed constant domain was inserted into an expression vector in E. coli. A large amount of this 18 kDa protein was observed to be synthesized in E. coli, and might be a good source for the three dimensional analysis of the T‐cell receptor molecule.
Identification of the proteins in direct contact with duck globin mRNA
Authors: Lockard Raymond E.
Published: July 2002

Proteins in direct contact with translationally active and repressed duck globin mRNA were determined by irradiating blood or lysates with ultraviolet light. Cross‐linked proteins from polyribosomes and free mRNP particles were 14C‐labeled by reductive methylation and identified on SDS‐polyacrylamide gels upon autoradiography. Results indicate that ten cross‐linked proteins are common to both polysomal and free mRNP, however, a 44 kDa protein appears to be specific for repressed mRNP particles. Furthermore, the notable lack of cross‐linked proteins in the 20–30 kDa range in free mRNP supports the view that the characteristic low molecular mass ‘prosomal’ proteins, previously found associated with translationally repressed duck globin free mRNP [(1984) EMBO J. 3, 29–34], do not interact directly with the mRNA molecule.
Quantitative determination of virus‐membrane fusion events Fusion of influenza virions with plasma membranes and membranes of endocytic vesicles in living cultured cells
Authors: Nussbaum Ofer , Loyter Abraham
Published: July 2002

Incubation of fluorescently labeled influenza virus particles with living cultured cells such as lymphoma S‐49 cells or hepatoma tissue culture cells resulted in a relatively high degree of fluorescence dequenching. Increase in the degree of fluorescence (35–40% fluorescence dequenching) was observed following incubation at pH 5.0 as well as at pH 7.4. On the other hand, incubation of fluorescently labeled influenza virions with erythrocyte ghosts resulted in fluorescence dequenching only upon incubation at pH 5.0. Only a low degree of fluorescence dequenching was observed upon incubation with inactivated unfusogenic influenza or with hemagglutinino‐influenza virions. The results of the present work clearly suggest that the fluorescence dequenching observed at pH 5.0 resulted from fusion with the cells' plasma membranes, while that at pH 7.4 was with the membranes of endocytic vacuoles following endocytosis of the virus particles. Our results show that only the fluorescence dequenching observed at pH 7.4 − but not that obtained at pH 5.0 − was inhibited by lysosomotropic agents such as methylamine and ammonium chloride, or inhibitors of endocytosis such as EDTA and NaN3.
Family of Na+, K+‐ATPase genes Intra‐individual tissue‐specific restriction fragment length polymorphism
Authors: Sverdlov E.D. , Broude N.E. , Sverdlov V.E. , Monastyrskaya G.S. , Grishin A.V. , Petrukhin K.E. , Akopyanz N.S. , Modyanov N.N. , Ovchinnikov Yu.A.
Published: July 2002

Intra‐individual tissue‐specific restriction fragment length polymorphism (RFLP) has been demonstrated in DNA isolated from different mammalian tissues using cDNAs of α‐ and β‐subunits of Na+, K+‐ATPase as hybridization probes. We propose that the RFLPs could result from gene rearrangements in the gene loci for the α‐ and β‐subunits of Na+,K+‐ATPase. The changes in restriction patterns have been shown to occur during embryonic development and tumor formation. In addition, the tissue specificity of the expression of different genes of the family of Na+,K+‐ATPase genes and their low expression in tumor cells have been demonstrated.
Modulation of retinal transducin and phosphodiesterase activities by synthetic peptides of the phosphodiesterase γ‐subunit
Authors: Morrison Daniel F. , Rider Maureen A. , Takemoto Dolores J.
Published: July 2002

Synthetic peptides corresponding to various regions of the light‐activated guanosine 3′,5′‐cyclic monophosphate phosphodiesterase (PDE) γ‐subunit (PDEγ) from bovine retinal rod outer segments were synthesized and tested for their ability to inhibit PDE activity, and GTPase activity of transducin. One of these peptides, corresponding to PDEγ residues 31–45, inhibited PDE activity and GTPase activity in a dose‐dependent manner. The GTPase activity was inhibited by PDEγ‐3 non‐competitively. This region of the PDEγ subunit may be involved in the direct interaction of transducin and PDEαβ with PDEγ.
Metabolic stability of the fucose in rat transferrin
Authors: Regoeczi Erwin , Chindemi Paul A.
Published: July 2002

The metabolic behaviour of the chitobiose core fucose that is a natural constituent of a large proportion of rat transferrin molecules was studied in rats comparatively to that of the polypeptide portion of the glyco‐protein by using appropriate labels ([3H]fucose and 125I) and affinity chromatographic techniques (lentil‐Sepharose). No evidence was obtained to suggest that this residue is cleaved from the glycan in significant amounts before removal of the entire glycoprotein for catabolism. Similarly, [14C]fucose linked to GlcNAc residues in the antennae of human asialotransferrin was being eliminated in pigeons at the same rate as the polypeptide itself. It is concluded that in spite of transferrin's exposure to the cellular milieu, the fate of its fucose is distinctly different from that of the same in plasma membrane glycoproteins.
Two major tyrosine protein kinases of resting human T lymphocytes are down‐regulated following mitotic stimulation
Authors: Hall Belinda S. , Hoffbrand A.Victor , Wickremasinghe R.Gitendra
Published: July 2002

Human lymphocyte tyrosine protein kinases (TPKs) have been analyzed by gel‐filtration chromatography. The major TPK species with activity towards an exogenous tyrosine‐containing peptide had molecular masses of 70–100 kDa (TPK I) and 35–40 kDa (TPK II). TPKs I and II were distinct from the well‐characterized autophosphorylating lymphoid cell TPK, pp56lck [(1983) J. Biol. Chem. 258, 10738–107421]. Both TPK I and TPK II were down‐regulated following mitogenic stimulation of lymphocytes with phytohae‐magglutinin. By contrast, pp56lck remained clearly detectable in stimulated lymphocytes. We suggest that TPKs I and II may play a role in the regulation of the lymphocyte cell cycle.
A spectral hole‐burning study of long‐wavelength chlorophyll a forms in greening leaves at 5 K
Authors: Mauring Koit , Renge Indrek , Avarmaa Rein
Published: July 2002

Persistent holes have been burnt in the spectra of post‐etiolated maize leaves in the spectral interval 700–745 nm at 5 K. Narrow (< 1 cm−1) holes detectable both in fluorescence and excitation spectra correspond to the zero‐phonon purely electronic S1◀S0 transition of chlorophyll a rather than to the S1←S0 vibronic or S
1
←S0 higher singlet transitions. The spectral composition of the inhomogeneously broadened absorption and fluorescence contours of long‐wavelength chlorophyll a forms in plants is discussed in terms of the contributions of phonon‐wings, vibronic satellites, purely electronic S1◀S0 transitions and its higher excitonic components.
Role of nucleoside components and internucleotide phosphate groups of oligodeoxyribonucleotide template in its binding to human DNA polymerase α
Authors: Lavrik O.I. , Levina A.S. , Nevinsky G.A. , Podust V.N.
Published: July 2002

Affinity labelling of human placenta DNA polymerase α (EC 2.7.7.7) with the reactive oligodeoxyribonucleotide d(pT)2pC[Pt2+(NH3)2OH](pT)7 was used for quantitative analysis of enzyme interaction with oligodeoxyribonucleotides as templates. Dissociation constants and Gibb's energy values for different oligothymidylates d(pT)
n
T where n = 1–14 have been evaluated by competitive experiments of these ligands with Pt2+ reagent. The data obtained prove the formation of one Me2+‐dependent electrostatic contact and a hydrogen bond between the enzyme and one phosphate of these templates. One may suppose that the hydrophobic interaction of any other monomeric link of oligodeoxyribonucleotides with the enzyme template site takes place.
Cell‐free synthesis of alkaline lipase, a glyoxysomal membrane protein, from castor bean endosperm
Authors: Maeshima Masayoshi , Takeuchi Atsuko , Asahi Tadashi
Published: July 2002

Polyadenylated RNA from castor bean endosperm was translated in a wheat germ cell‐free protein synthesis system, and alkaline lipase, an integral glyoxysomal membrane protein, was immunoprecipitated. The apparent molecular mass of the lipase synthesized in vitro was slightly higher than that of the mature enzyme (62 kDa). When mRNA derived from free and membrane‐bound polysomes in the endosperms was translated in vitro, the lipase was predominantly recovered from the products of the free polysomal mRNA, suggesting that the membrane protein is post‐translationally inserted into the membrane. The amino‐terminal 8 amino acid residues of the mature lipase were sequenced.
Pertussis toxin distinguishes between muscarinic receptor‐mediated inhibition of adenylate cyclase and stimulation of phosphoinositide hydrolysis in Flow 9000 cells
Authors: Lo William W.Y. , Hughes John
Published: July 2002

Pretreatment of human embryonic pituitary tumour cells (Flow 9000) with pertussis toxin significantly reduced carbachol‐mediated inhibition of isoprenaline and prostaglandin E2 stimulation of cyclic AMP formation. This is in accord with an action on the inhibitory Gi‐protein by pertussis toxin. In contrast, pertussis toxin‐pretreatment had no effect on either muscarinic agonist or GTP[S] (a non‐hydrolysable GTP analogue) stimulation of [3H]inositol phosphate production in intact and permeabilized [3H]inositol‐prelabelled Flow 9000 cells, respectively. These results suggest that muscarinic receptors are linked to the inhibition of adenylate cyclase and the stimulation of phosphoinositidase C via two different G‐proteins in Flow 9000 cells.
Aspirin induces alterations in low‐density lipoprotein and decreases its catabolism by cultured human fibroblasts
Authors: Mazière C. , Goldstein S. , Moreau M. , Mazière J.C. , Polonovski J.
Published: July 2002

Aspirin interacts in vitro with human low‐density lipoprotein (LDL), which results in a decrease in free amino groups of apolipoprotein B and an increase of electrophoretic mobility of the particle. The aspirin‐treated LDL was less efficiently recognized than native LDL by the apo B/E receptor of fibroblasts. These results suggest that aspirin in long‐term treatment could influence the LDL‐receptor pathway. However, aspirin‐treated LDL did not bind to the scavenger receptor of macrophages.
Activation of sodium‐proton exchange is not a prerequisite for Ca2+ mobilization and aggregation in human platelets
Authors: Hunyady László , Sarkadi Balázs , Cragoe Edward J. , Spät András , Gárdos George
Published: July 2002

Recently it has been suggested [(1987) Nature 325, 456–458; (1987) FEBS Lett. 212, 123–126] that the activation of Na+/H+ exchange is a prerequisite for platelet aggregation and the development of the Ca2+ signal. As direct evidence for the role of the Na+/H+‐exchange pathway the inhibition of the Ca2+ signal by EIPA, a specific inhibitor of Na+/H+ exchange, was offered. Here we demonstrate that low concentrations of EIPA (below 1 μM) completely block Na+/H+ exchange while EIPA inhibits aggregation or Ca2+ mobilization only in concentrations 100‐times greater than 1 μM. Moreover, another amiloride analogue, CBDMB, developed to act predominantly on Na+/Ca2+ exchange, does not affect Na+/H+ exchange in platelets but blocks aggregation and Ca2+ mobilization. We conclude that while Na+/H+ exchange has a fundamental role in platelet functions it is not prerequisite for the development of Ca2+ signal and aggregation.
The subunits of specific odor‐binding glycoproteins from rat olfactory epithelium
Authors: Fesenko E.E. , Novoselov V.I. , Bystrova M.F.
Published: July 2002

The specific odor‐binding glycoproteins have been isolated from rat olfactory epithelium. They consist of two subunits, gp88 and gp55. Subunit gp88 is capable of odorant binding.
Demonstration by immunoblotting of heterogeneity in the autoantibody response directed against fat cells in Graves' disease
Authors: Weetman A.P. , Nutman T.B. , Baker J.R. , Burman K.D.
Published: July 2002

Guinea pig fat cell membranes (FCM) have been widely used in preference to thyroid membranes as a source of TSH receptors to investigate TSH receptor antibodies in Graves' disease, because FCM are ostensibly free of other thyroid antigens. However, by FCM immunoblotting we have found: (i) 8 of 10 normal sera bound to determinants at 38 and 190 kDa; (ii) 17 other determinants were recognised by 60% of Graves' or Hashimoto sera and by 20% of normal sera; (iii) three determinants at 65–90 kDa were recognised by 5 of 13 Graves' but by none of the normal or Hashimoto sera; and (iv) none of the determinants recognised appeared to be related to the TSH receptor.
Conformational responses of an arachidonate‐ and U46619‐binding haemoprotein in relation to platelet activation
Authors: Krishnan Lissy K. , Jamaluddin Moideen P.
Published: July 2002

Antibodies were raised, in rabbits, against an arachidonate‐ and U46619‐binding protein purified from calf platelets. Spectral measurements and immunodiffusion experiments were employed to follow conformational responses of the protein in relation to platelet activation. Upon treatment with the platelet agonists, arachidonate and PGH2, as well as the common haem ligands, imidazole and CN−, the purified protein had its Soret band red‐shifted, with hypochromicity, but the protein saturated with the agonists, not with the haem ligands, showed altered antigenic properties in immunodiffusion experiments. In an analogous manner activation of gel‐filtered calf platelets with high concentrations of ADP and A23187, as well as by cold, had Soret bands of extracts of sonicated platelets red‐shifted, with hypochromicity; concomitantly, antigenically different conformations of the protein appeared in Triton X‐100 extracts of the activated platelets. A protein immunologically related to the platelet protein was detected in Triton X‐100 extracts of calf neutrophils. It is suggested that conformational changes of the protein induced by arachidonate or prostaglandin endoperoxides or H2O2 formed in different compartments during platelet activation by different stimuli may be a biochemical mechanism of stimulus‐response coupling and that similar mechanisms might operate in other cell types.
Higher order folding of chromatin is induced in different ways by monovalent and by bivalent cations
Authors: Makarov V.L. , Smirnov I. , Dimitrov S.I.
Published: July 2002

The condensation of the 10 nm chromatin filament in the 30 nm fiber by monovalent cations, polyamines and bivalent cations was studied with light scattering at 90° and flow linear dichroism methods. It was found that monovalent cation‐ and polyamine‐induced folding was a two‐step process: a precondensation, when a rotation of nucleosomes takes place only, and a condensation step without changes in nucleosome orientation. Divalent cations affected the structure of chromatin in one step only ‐ condensation of the chromatin filament being accompanied by nucleosome reorientation.
Inhibition of taurocholate efflux from rat hepatic canalicular membrane vesicles by glutathione disulfide
Authors: Griffiths James C. , Sies Helmut , Meier Peter J. , Akerboom Theodorus P.M.
Published: July 2002

In right‐side out rat hepatic canalicular membrane vesicles glutathione disulfide (GSSG) inhibited the efflux of taurocholate approx. 70% in the presence or approx. 55% in the absence of a valinomycin‐mediated K+ diffusion potential; maximal inhibition occurred at 5 mM GSSG. The inhibition by GSSG was abolished by dithioerythritol. Neither dithioerythritol alone nor GSH inhibited taurocholate efflux. S‐(2,4‐Dinitrophenyl) glutathione and N‐ethylmaleimide showed intermediate inhibitory effects.
Demonstration of an epidermal growth factor‐dependent 58 kDa phosphoprotein secreted by rat kidney fibroblasts
Authors: Binas Bert , Grosse Richard
Published: July 2002

Epidermal growth factor and 12‐O‐tetradecanoylphorbol‐13‐acetate increased the amount of 32Pi found as phosphoserine in a major, hitherto not described 58 kDa phosphoprotein (pp58) secreted by normal rat kidney fibroblasts. Platelet‐derived growth factor, insulin, nerve growth factor and fibroblast growth factor did not affect pp58 while transforming growth factor β decreased the accumulation of radioactivity into pp58. Cycloheximide, actinomycin D and ammonium chloride suppressed the labelling of pp58.
Molecular cloning of Clostridium difficile toxin A gene fragment in λ gtll
Authors: Muldrow Lycurgus L. , Ibeanu Gordon C. , Lee Norveta I. , Bose Nripendra K. , Johnson Joe
Published: July 2002

Toxin A of Clostridium difficile has been purified and monospecific antiserum produced. A reliable procedure for isolation and restriction of C. difficile chromosomal DNA was developed which allowed for the construction of a genomic library in λ gtll. Approx. 35000 plaques were screened using anti‐toxin A which resulted in the identification of one stable positive clone, λ cd 19. Verification of the immunological identity of the isolated toxin A gene fragment in λ cd 19 was determined by affinity purifying toxin A antibodies specific for λ cd 19 gene product, and using these selected antibodies to probe a Western blot of purified toxin A. The insert in λ cd 19 was demonstrated to be a 0.3 kb fragment by restriction digestion, and by hybridization of the clone to a chromosomal digest of C. difficile. The peptide coded for by the toxin A gene fragment in λ cd 19 was not cytotoxic for 3T3 mammalian tissue culture cells.
The molecular basis of the inherited deficiency of androsterone UDP‐glucuronyltransferase in Wistar rats
Authors: Corser Robert B. , Coughtrie Michael W.H. , Jackson Michael R. , Burchell Brian
Published: July 2002

A major UDP‐glucurnoyltransferase isoenzyme in rat liver (51 kDa), corresponding to androsterone glucuronidating activity, has been identified by immunoblot analysis. This isoenzyme is absent from Wistar rats exhibiting the low androsterone (LA) UDP‐glucuronyltransferase activity phenotype. Northern blot analysis of total RNA from normal and androsterone glucuronidation deficient Wistar rats demonstrated that the mRNA encoding this protein was not synthesised. Differences in restriction fragment length observed on Southern blotting of genomic DNA from LA Wistar rats indicate that this inherited deficiency is the result of a deletion in the androsterone UDP‐glucuronyltransferase gene.
An asymmetric low‐spin EPR signal of cytochrome b6 in the cytochrome b6f‐complex from spinach chloroplasts
Authors: Nitschke Wolfgang , Hauska Günter
Published: July 2002

Cytochrome b
6 from the chloroplast cytochrome b
6
f‐complex shows an asymmetric g
z = 3.85 low‐spin EPR signal in addition to a signal at g
z = 3.6–3.7, similar to cytochrome b in mitochondrial or bacterial cytochrome bc
1‐complexes. However, the signal is more unstable and probably represents the highly strained conformation of only part of the low‐potential heme in cytochrome b
6.
Opposite effects of adenosine on two types of cAMP‐induced gene expression in Dictyostelium indicate the involvement of at least two different intracellular pathways for the transduction of cAMP signals
Authors: Spek Wouter , van Drunen Kees , van Eijk Ronald , Schaap Pauline
Published: July 2002

Adenosine promotes the cAMP‐induced increase of mRNAs, probed with the cDNAs D11 and D14, which are preferentially expressed in prestalk cells, while it inhibits cAMP‐induced prespore gene expression. Half‐maximal inhibition of prespore gene expression occurs at about 300 μM, while prestalk stimulation by adenosine occurs at about 100‐fold lower concentrations and requires the presence of cAMP. These results indicate that adenosine interferes with the transduction of cAMP to gene expression and suggest the involvement of two different adenosine target sites. Our data furthermore indicate that the transduction of extracellular cAMP to prespore gene or prestalk gene expression occurs via divergent pathways.
Diguanosine 5′ ,5‴‐P1 ,P4‐tetraphosphate and other purine nucleotides inhibit endoribonuclease VI from Artemia
Authors: Grau Pilar P. , Heredia Claudio F.
Published: July 2002

The activity of the endoribonuclease VI from Artemia is sensitive to several purine nucleotides. The enzyme is non‐competitively inhibited by diguanosine tetraphosphate (K
i = 75 μM), a nucleotide abundant in Artemia encysted gastrulae and located in the same particulate fraction as the gastrular ribonuclease. Diguanosine triphosphate and diadenosine tetraphosphate are less efficient inhibitors (K
i⋍200 μM). The ribonuclease is non‐competitively inhibited by 5′‐AMP (K
i = 10 μM) and 5′‐GMP (K
i = 50 μM) but is insensitive to the corresponding 5′‐phosphates of cytosine and uridine. Other purine mononucleotides inhibit the enzyme activity less efficiently. The modulation of the enzyme activity by these nucleotides is discussed in relation with the changes in ribonuclease activity during early development of Artemia.
Structure‐function relationship in Escherichia coli translational initiation factors Characterization of IF1 by high‐resolution 1H‐NMR spectroscopy
Authors: Paci Maurizio , Pon Cynthia L. , Gualerzi Claudio O.
Published: July 2002

Escherichia coli translational initiation factor IF1 was studied by 1H‐NMR spectroscopy at 400 MHz. IF1 displays a very well resolved spectrum in both aromatic and aliphatic regions. Other spectral characteristics include relatively narrow resonance lines and lack of relevant cross‐relaxation phenomena. The resonances of the aromatic residues, in particular of the two His and two Tyr, were assigned by selective chemical modifications and spectroscopic techniques to individual residues in the protein sequence. The relative mobility of various residues of IF1 has been evaluated on the basis of the spin‐lattice relaxation times which are rather short and homogeneous. Overall the factor appears to have a complex secondary and tertiary structure and to be a flexible protein whose residues have a high degree of internal mobility.
Activation of cyclic nucleotide phosphodiesterase by a monosaccharide precursor of Escherichia coli lipid A
Authors: Walters John D. , Jirsa Richard C.
Published: July 2002

The E. coli lipid A precursor lipid X (N2
,O3
‐diacylglucosamine 1‐phosphate) activates calmodulin‐dependent cyclic nucleotide phosphodiesterase in a noncooperative, calcium‐independent manner by increasing its V
max and decreasing its K
m for substrate. The glycolipid produces half‐maximal activation at 11 μg/ml and does not further enhance activation by calcium‐calmodulin. Lipid X activation of phosphodiesterase requires the presence of the O
3‐linked hydroxymyristoyl residue. These findings suggest that lipid X could produce some of its biological effects by modulating intracellular cAMP levels.
Membrane surface potential of Spiroplasma floricola
Authors: Schummer Ulrich , Schiefer Hans Gerd
Published: July 2002

Anionic charges, cytochemically identified as lipid phosphate groups, cover the outer membrane surface of Spiroplasma floricola. They induce a negative membrane surface potential which affects the distribution of ions, including protons. Accordingly, the pH at the interface differs from the bulk pH. By using the fluorescent lipoid pH indicator 4‐heptadecyl‐7‐ hydroxycoumarin, the pH at the membrane surface was determined. From the difference of the bulk and the interfacial pH the membrane surface potential of S. floricola was calculated to be ϕ = − 118 mV.
Conformational analysis of 16 S ribosomal RNA from Escherichia coli by scanning transmission electron microscopy
Authors: Mandiyan Valsan , Hainfeld James F. , Wall Joseph S. , Boublik Miloslav
Published: July 2002

Digitized images of molecules of 16 S rRNA from Escherichia coli, obtained by scanning transmission electron microscopy (STEM), provide quantitative structural information that is lacking in conventional electron micrographs. We have determined the morphology, total molecular mass, mass distribution within individual rRNA molecules and apparent radii of gyration. From the linear density (M/L) we have assessed the number of strands in the structural backbone of rRNA and studied the pattern of branching and folding related to the secondary and tertiary structure of rRNAs under various buffer conditions. Even in reconstitution buffer 16 S RNA did not show any resemblance to the native 30 S subunit.
The importance of threonine‐301 from cytochromes P‐450 (laurate (ω‐1)‐hydroxylase and testosterone 16α‐hydroxylase) in substrate binding as demonstrated by site‐directed mutagenesis
Authors: Imai Yoshio , Nakamura Masahiko
Published: July 2002

Threonine‐301 from rabbit liver cytochromes P‐450 (laurate (ω‐1)‐hydroxylase and testosterone 16α‐hydroxylase) has been replaced by histidine via site‐directed mutagenesis. In the oxidized state the mutant P‐450s exhibited typical low‐spin type absorption spectra of P‐450 and their reduced CO complexes showed a Soret peak at 450 nm. However, no spectral change was induced on addition of substrates for their wild‐type counterparts. The mutant P‐450s were also completely devoid of the hydroxylase activity. These findings suggest that threonine‐301, which is highly conserved in P‐450s and located at the distal heme surface, plays an important role in substrate binding.
Equivalent locations of sucrose synthase genes in chromosomes 7D of wheat, 7Ag of Agropyron elongatum, and 7H of barley
Authors: Maraña Carmen , García-Olmedo Francisco , Carbonero Pilar
Published: July 2002

Wheat/Agropyron 7D/7Ag recombinant chromosomes were analysed by Southern blotting, using cDNA radioactive probes corresponding to the Ss1 and Ss2 sucrose synthase genes of wheat. The genes were located in the central segments of the short arms of both chromosomes 7D and 7Ag. A similar analysis of wheat/barley addition lines demonstrated that DNA sequences that cross‐hybridized with the wheat probes were located in chromosome 7H of barley.
Redox transformations of quinone antitumor drugs in liver microsomes
Authors: Rumyantseva G.V. , Weiner L.M.
Published: July 2002

The hydroxyl radical has been spin trapped in microsomal and purified NADPH‐cytochrome P‐450 reductase systems in the presence of adriamycin, daunomycin and mitomycin C. The presence of a lag period in quinone‐stimulated spin‐adduct formation is associated with oxygen removal upon its reduction to H2O2. The hydroxy radical generation has been stimulated by the Fe‐EDTA complex and completely inhibited by catalase. The mechanism of redox transformations of anthracyclines in a microsomal system has been proposed The single electron reduced quinone‐containing anticancer antibiotics play the following roles: (i) they reduce oxygen to H2O2 and (ii) they reduce the ferric ions necessary for H2O2 decomposition with hydroxyl radical formation.
Association of a myristoylated protein with a biological membrane and its increased phosphorylation by protein kinase C
Authors: Utsumi Toshihiko , Yoshinaga Koji , Koga Daizo , Ide Akio , Nobori Koichi , Okimasu Eiji , Terada Shigeo , Utsumi Kozo
Published: July 2002

A hydrophilic enzyme, lysozyme, was myristoylated in vitro by the N‐hydroxysuccinimide ester of myristic acid, and the monomyristoylated lysozyme was isolated by CM‐cellulose cation‐exchange column chromatography. The monomyristoylated lysozyme associated with phospholipid vesicles, whereas the association of native lysozyme was negligible. The membrane‐associated monomyristoylated lysozyme was phosphorylated with partially purified rat brain Ca2+‐ and phospholipid‐dependent protein kinase (protein kinase C) in the presence of Ca2+, phosphatidylserine and phorbolmyristate acetate. Thus, the myristoylated lysozyme became a substrate of protein kinase C through its hydrophobic association with the membrane. The present results suggest that the myristoylation of cytoplasmic proteins may have an important role in signal transduction.
Mutagenesis directed by phosphotriester analogues of oligonucleotides: A way to site‐specific mutagenesis in vivo
Authors: Petrenko V.A. , Kipriyanov S.M. , Boldyrev A.N. , Pozdnyakov P.I.
Published: July 2002

A new approach to induce directed mutations in genes of study through simple cotransfection of E. coli cells by the mixture of primer and template was developed. This method is based on the use of synthetic phosphotriester analogues of oligonucleotides as site‐specific mutagenic primers. The achieved yield of mutant clones was 2–3%.
Effect of monensin on intracellular transport and posttranslational processing of 11 S globulin precursors in developing pumpkin cotyledons
Authors: Hayashi Makoto , Akazawa Takashi , Nishimura Mikio , Hara-Nishimura Ikuko
Published: July 2002

We examined the effect of monensin on the biosynthesis of 11 S globulin, a major storage protein in pumpkin cotyledons. In the developing cotyledons, proglobulin, a precursor of 11 S globulin, is synthesized on the endoplasmic reticulum (ER) and transported to vacuoles, where it is converted to the mature 11 S globulin molecule by the action of a thiol protease [(1985) Plant Physiol. 77, 747 752; (1987) Plant Physiol. 85,440–445]. The developing pumpkin cotyledons pretreated with the carboxylic ionophore monensin (10−6 M) were pulse‐chase labeled with [35S]methionine, and subsequently, vacuoles were isolated from the tissues. Although labeled proglobulin was detected in the isolated vacuoles, the conversion of proglobulin to 11 S globulin was strongly inhibited. However, monensin was shown not to inhibit the in vitro processing of proglobulin by vacuolar protease, whose optimum pH was ∼ 5.0. It appears, therefore, that monensin inhibits the posttranslational proteolytic cleavage of proglobulin and not its intracellular transport from the ER to the vacuoles. This inhibition may be due to an elevation of vacuolar pH by monensin.
Heterogeneity in an isolated membrane protein Has the ‘authentic cytochrome oxidase’ been identified?
Authors: Hartzell Charles R. , Beinert Helmut , Babcock Gerald T. , Chan Sunney I. , Palmert Graham , Scott Robert A.
Published: July 2002

The criteria of homogeneity or native state of a protein are prone to become ambiguous when applied to membrane proteins, such as cytochrome‐c oxidase, which are purified by extraction with detergents. Properties of the purified material depend on the detergent used and on details of the purification protocol followed with any single batch of a preparation. We present arguments to show that the evidence presently available in published form does not justify the designation [(1987) J. Biol. Chem. 262 3160–3164] of one type of preparation as being closer to the native state than others.
A potential role for U1 RNA genes in gene duplication and conversion events
Authors: Latchman David S.
Published: July 2002

A gene encoding U1 snRNA has been identified in Caenorhabditis elegans by homology to the human U1 gene. The gene lies at the boundary of a duplication event also involving the small heat shock protein genes. The possible role of the U1 sequence in mediating the duplication event is discussed.
A monoclonal anti‐thromboxane B2 antibody
Authors: Reinke Margot , Brune Kay
Published: July 2002

A monoclonal antibody against thromboxane B2 which may be used in standard fluid phase radioimmunoassays with a detection limit of around 40 pg and a binding affinity of 1.98 × 109 M−1 is described. Limited crossreactivity could be observed only with structurally closely related compounds such as 2,3‐dinor‐thromboxane B2 (8.9%), thromboxane B1 (15.7%) and thromboxane B3 (39.7%). Detectable crossreactivity with 11‐dehydro‐thromboxane B2 ω‐carboxy‐thromboxane B2‐hydroxy‐thromboxane B2, prostaglandins of the D‐, E‐ and F‐type as well as metabolites of prostacyclin was lacking. The monoclonal anti‐thromboxane B2 antibody proved well suited for measuring the thromboxane B2 content in tissue culture supernatants as well as in human serum.
Fast electron transfer from low‐ to high‐potential cytochrome b6 in isolated cytochrome b6f complex
Authors: Nitschke Wolfgang , Hauska Günter , Crofts Antony R.
Published: July 2002

The two hemes of cytochrome b
6 in the cytochrome b
6
f complex isolated from chloroplasts exhibit slight spectral differences in the α‐band at room temperature, sufficient to deconvolute redox kinetics of cytochrome b
6 into the contributions of the two components. The deconvoluted changes observed can be explained by fast electron transfer from low‐ to high‐potential cytochrome b
6.
Detection and microsequencing of juvenile hormone‐binding proteins of an insect by the use of an iodinated juvenile hormone analog
Authors: Kulcsár Peter , Prestwich Glenn D.
Published: July 2002

An [125I] iodinated juvenile hormone (JH) analog can be used as a sensitive and highly selective probe for the visualization of high‐affinity, (JH)‐specific binding proteins from insect hemolymph samples. The proteins can be detected in their native form using a two‐dimensional (isoelectric focusing then native gradipore gel) separation of the crude protein mixture containing the 125I‐labeled iodinated JH analog. The proteins can be transferred to activated glass fiber paper by electroblotting, and the location of the bound γ‐emitter can be found by exposure of the dried gel or the electroblot to X‐ray film. The radiolabeled protein spot can be excised from the Coomassie‐stained glass fiber paper and subjected directly to gas‐phase N‐terminal amino acid sequencing. This non‐destructive, non‐denaturing technique may have wide applicability in identifying and sequencing ligand‐specific binding proteins in complex mixtures.
Spectroscopic characterisation of the reaction centre of photosystem II from higher plants
Authors: Newell W.R. , van Amerongen H. , van Grondelle R. , Aalberts J.W. , Drake A.F. , Udvarhelyi P. , Barber J.
Published: July 2002

Two different photosystem II particles isolated from pea, a core complex and the D1/D2/Cyt b‐559 reaction centre (RC) complex, have been characterised by absorption, linear dichroism (LD) and circular dichroism (CD) spectroscopy. Only one carotenoid contributes to the LD of the reaction centre, in agreement with the biochemical analysis. This carotenoid is oriented parallel to the long axis of the reaction centre. The chlorophyll QY contribution to the LD is oriented perpendicular to the long axis of the reaction centre. The LD of the reaction centre carotenoid is reversed in the core complex. In addition, the contribution of a second carotenoid species can be observed. In the core, the two pools of carotenoid have a rather different orientation with respect to the membrane plane: one parallel, the other perpendicular. In addition, in the core the sign of the LD in the chlorophyll QY region is reversed and red‐shifted as compared to the reaction centre. These observations suggest that the reaction centre is oriented with its long axis perpendicular to the long axis of the PS II core and to the membrane plane. The circular dichroism CD of the reaction centre has intense peaks of opposite sign at 444 nm (negative) and 435 nm (positive), which we attribute to exciton coupling between Chl a molecules in the reaction centre. The RC has no CD in the range 460–650 nm; thus there is no exciton coupling between the carotenoid and the other pigments. The lack of CD in this region is consistent with the biochemical analysis of only one carotenoid per reaction centre. The larger core complex exhibits much weaker CD (per Chl). The CD in the QY absorption band indicates exciton coupling between chlorophyll molecules. The sign of the pair of peaks in this region is reversed in the core with respect to the reaction centre.
Linear gramicidin activates neutrophil functions and the activation is blocked by chemotactic peptide receptor antagonist
Authors: Jacob Jose
Published: July 2002

The activation of functional responses in rabbit peritoneal neutrophils by gramicidin and the chemotactic peptide, N‐formyl‐methionyl‐leucyl‐phenylalanine methyl ester, was studied. Gramicidin activated superoxide generation, lysosomal enzyme release and a decrease in fluorescence of chlortetracycline‐loaded cells, as for the chemotactic peptide. The maximum intensities of the responses by gramicidin were lower than that by chemotactic peptide. Responses by both these peptides could be inhibited by t‐butyloxycarbonyl‐methionyl‐leucyl‐phenylalanine, a chemotactic peptide receptor antagonist. Gramicidin gave responses at low doses comparable to that of the chemotactic peptide.
Differential potency and trans‐activation of normal and mutant T24 human H‐ras1 gene promoters
Authors: Spandidos Demetrios A. , Pintzas Alex
Published: July 2002

We have employed a short‐term transfection assay system in which we monitored the transient expression of the chloramphenicol acetyltransferase (CAT) gene linked to the promoter region of the normal and mutant T24 H‐ras1 gene or the human ε‐globin gene in Chinese hamster lung (CHL) cells or cells derived from them which carry and express one or the other of the polyoma virus early genes. Our findings can be summarized as follows: (i) The mutant T24 H‐ras1 promoter region behaves as a stronger promoter than the H‐ras1 gene in all these types of cells as well as in rat 208F fibroblast cells. (ii) In CHL cells expressing the polyoma large T antigen the normal and mutant T24 Ha‐ras1 promoters are not trans‐activated in these cells and only a 2.5‐fold activation of the ε‐globin promoter is observed. (iii) In cells expressing the polyoma middle T antigen both the normal and mutant H‐ras1 are trans‐activated whereas transcription from the ε‐globin promoter is not affected when compared to the normal CHL cells. (iv) In cells expressing the polyoma small T antigen the normal and mutant H‐ras1 as well as the ε‐globin promoters are trans‐activated. We suggest from these data that a tissue‐specific element exists in the promoter region of the H‐ras1 gene and that the polyoma middle and small T antigens trigger the expression of proteins that trans‐activate these promoters.
The effect of troponin C removal on the Ca2+‐sensitive binding of Mg2+AMPPNP to myofibrils
Authors: Johnson Robert E.
Published: July 2002

It was previously shown that when rabbit skeletal myofibrils are titrated with Mg2+ AMPPNP under conditions that result in the dissociation of cross‐bridges from the thin filaments (i.e. 50% ethylene glycol, O°C), Ca2+‐sensitive, biphasic binding is observed. These titrations have been repeated using myofibrils from which the troponin C has been selectively removed. The disappearance of both Ca2+ sensitivity and biphasic binding is taken as evidence that the Ca2+ sensitivity is due to Ca2+ binding to troponin C and the biphasic binding of Mg2+AMPPNP observed in intact myofibrils is not due to packing constraints or steric hindrance.
A monoclonal antibody to C1q which appears to interact with C½C½s2‐binding site
Authors: Hsiung Li-min , Dodds Alister W. , Mason Donald W. , Reid Kenneth B.M.
Published: July 2002

A monoclonal antibody (SB‐4) to human C1q was prepared. The equilibrium constant of the antibody for C1q was found to be greater than 1010 M−1. It has been shown that the antibody binds to the A‐B chain dimer, probably via the B chain of C1q. Pepsin digestion of C1q at pH 4.5, which fragments the globular regions but leaves the collagenous region intact, allowed the demonstration that the antigenic site is located in the collagenous region of the molecule. The effect of the antibody on haemolytic activity has shown that it is capable of inhibiting the formation of EAC1 cells from EAC1q cells plus C1r and C1s but is incapable of inhibiting the C1 activity of preformed EAC1 cells. This indicates that the binding of the antibody to the collagenous portion of the B chain of C1q probably prevents interaction between C1q and the C1r2‐C1s2 complex.