bluuebunny/crossref_metadata_2025 huggingface dataset:
Mode of action of bottromycin A2: Effect of bottromycin A2 on polysomes
Authors: Otaka Tadahiko , Kaji Akira
Published: July 2002

When bottromycin A2 was added to an in vitro protein synthesis system carried out by naturally occurring polysomes, it inhibited protein synthesis effectively. Examination of the 3 steps of peptide chain elongation revealed that the binding of aminoacyl‐tRNA to the polyribosomes was inhibited by bottromycin A2. In contrast, we concluded that the peptide bond formation and the translocation steps in this system were not inhibited by bottromycin A2 on the basis of the following observation: (1) the break‐down of polysomes, which is dependent of EFG, puromycin and RR (ribosome releasing) factor, was insensitive to bottromycin A2; (2) the puromycin dependent release of polypeptide from polysomes, with or without EFG, was not inhibited by bottromycin A2. Thus bottromycin specifically interferes with proper functioning of the A sites of polysomes. This is consistent with results obtained using the model system with synthetic polynucleotides.
Intermediate states between P* and Pf in bacterial reaction centers, as detected by the fluorescence kinetics
Authors: Sebban Pierre , Barbet Jean Claude
Published: July 2002

The fluorescence lifetimes of the reaction centers isolated from the wild‐typeRhodopseudomonas sphaeroidespurple bacterium and those from the R26 mutant strain, lacking the carotenoid, were measured at low redox potential. In addition to the prompt fluorescence occurring directly from P* and the long delayed emission related to the radical pair state Pf, two other components are present. We suggest that they may come from intermediate states between P* and Pf, or reflect the stabilization of Pf itself.
The primary structure of a 1,4‐β‐glucan cellobiohydrolase from the fungus Trichoderma reesei QM 9414
Authors: Fägerstam Lars G. , Göran Pettersson L. , Åke Engström J.
Published: July 2002

The sequence of the approx. 490 amino acid residues of the main 1,4‐β‐glucan cellobiohydrolase (CBH I) (EC 3.2.1.91) from culture filtrates of the fungus Trichoderma reesei QM 9414 has been established by automatic liquid phase Edman degradation. Peptides obtained by chemical and enzymatic cleavage of the reduced and S‐carboxymethylated protein were isolated by a combination of gel filtration and high‐performance liquid chromatography. The amino‐terminus of the single polypeptide chain is blocked by a pyroglutamyl residue. Most of the neutral carbohydrate present in the glycoprotein is bound within a short region near the carboxyl‐terminus. Three attachment sites of glucosamine residues have also been established.
Reconstitution of light‐harvesting chlorophyll‐protein complexes with Photosystem II complexes in soybean phosphatidylcholine liposomes
Authors: Murphy Denis J. , Crowther David , Woodrow Ian E.
Published: July 2002

Isolated Photosystem II (PS II) complexes and light‐harvesting chlorophyll‐protein complexes have been reconstituted, both separately and together, into soybean phosphatidylcholine liposomes. PS II activity, as measured by dichlorophenolindophenol reduction, increased by 70% following reconstitution of PS II complexes. Proteoliposomes containing both PS II and light‐harvesting chlorophyll‐protein complexes also exhibited enhanced quantum efficiencies at sub‐saturating light intensities. This indicates that the light‐harvesting complexes are interacting with the PS II complexes in proteoliposomes, to increase effectively their antenna size.
A gene fusion that localises the penicillin‐binding domain of penicillin‐binding protein 3 of Escherichia coli
Authors: Hedge Philip J. , Spratt Brian G.
Published: July 2002

A gene fusion that links the COOH‐terminal 349 amino acids of penicillin‐binding protein 3 (60 kDa) of E.coli to the NH2‐terminus of β‐galactosidase has been constructed. The fusion protein (38.5 kDa) retains the ability to bind benzylpenicillin with high affinity, establishing that the penicillin‐binding domain (and presumably the penicillin‐sensitive transpeptidase activity) of this high molecular mass penicillin‐binding protein is located on a COOH‐terminal functional domain.
Thiyl free radical production with hematoporphyrin derivative, cysteine and light: a spin‐trapping study
Authors: Buettner Garry R.
Published: July 2002

Hematoporphyrin derivativeCysteineThiyl free radicalSinglet oxygenHydroxyl radicalSpin‐trapping
Evidence for a chlorophylla/b—protein complex associated with Photosystem II
Authors: Remy R. , Ambard-Bretteville F.
Published: July 2002

A new chlorophylla/b—protein complex designated as LHCPx was separated by SDS—PAGE. This complex differs from the light‐harvesting chla/b—protein of PS II, by its chla/bratio, its fluorescence properties and its apoprotein composition. Evidence is provided that this complex corresponds to a part of PS I antenna.
The structure of the voltage‐sensitive sodium channel
Authors: Greenblatt R.E. , Blatt Y. , Montai M.
Published: July 2002

A variety of computer‐aided analyses was applied to the recently derived amino acid sequence of the Electrophorus electricus sodium channel protein in order to extract structural information such as hydrophobicity, periodicity, and secondary structure predictors. We propose a schematic model for the arrangement and folding of the polypeptide chain within the bilayer. The model consists of 4 homologous regions, each containing 8 membrane‐spanning (probably α‐helical) structures. Several of these structures are amphipathic with a repeat of 3.5 residues, 4 of which (one from each homologous region) are postulated to form a negatively charged channel lining. Gating currents are proposed to arise from voltage‐dependent separation of multiple ion pairs buried within the hydrophobic, intramembranous protein interior.
pH‐sensitive liposomes mediate cytoplasmic delivery of encapsulated macromolecules
Authors: Straubinger Robert M. , Düzgünes Nejat , Papahadjopoulos Demetrios
Published: July 2002

Negatively charged liposomes are endocytosed by the coated vesicle system and accumulate in acidic intracellular vesicles. Liposomes that become unstable at acidic pH improve cytoplasmic delivery of membraneimpermeant macromolecules such as calcein (CAL) and FITC dextran (18 or 40 kDa). Oleic acid (OA): phosphatidylethanolamine (PE) (3:7 mole ratio) liposomes become permeable to CAL at pH < 7.0. Control liposomes of phosphatidylserine : PE or OA : phosphatidylcholine are stable at pH 4–8. OA:PE liposomes promote cytoplasmic delivery of encapsulated CAL to CV‐1 cells, as evidenced by the emergence of diffuse, cytoplasmic CAL fluorescence. Delivery requires metabolic energy and is partially inhibited by chloroquine or monensin, which raise the pH of intracellular vesicles.
Gene expression in the polycistronic operons of Escherichia coli heat‐labile toxin and cholera toxin: a new model of translational control
Authors: Yamamoto Tatsuo , Suyama Akira , Mori Nozomu , Yokota Takeshi , Wada Akiyoshi
Published: July 2002

A new model is proposed based on the suggestion that stable local secondary structures of mRNA interfere with ribosome movement on mRNA and consequently reduce the translation rate. This model accounts for a different level of translation for each cistron in the polycistronic mRNA of Escherichia coli heat‐labile toxin (LT) and cholera toxin. We also conclude that the mRNA secondary structures have been conserved during the evolution of the toxin genes for its functional importance.
Light changes the membrane potential and ion balances of retinal rod disks
Authors: Hughes Simon M. , Brand Martin D.
Published: July 2002

Light stimulation of rod cells in vertebrate eyes may cause Ca2+release from the intracellular disks. Radiolabelled tracers show that light causes a small hyperpolarization of intact disk stacks and redistribution of the ions Ca2+and Cl−.
Complete amino acid sequence of the B875 light‐harvesting protein of Rhodopseudomonas sphaeroides strain 2.4.1
Authors: Theiler Rolf , Suter Franz , Pennoyer Jeffrey D. , Zuber Herbert , Niederman Robert A.
Published: July 2002

The complete amino acid sequence was determined for the α‐ and β‐chains of the B875 light‐harvesting protein purified from photosynthetic membranes of Rhodopseudomonas sphaeroides 2.4.1. The sequence of the B875‐α‐polypeptide was identical to that reported for the R26.1 carotenoidless mutant [(1985) Biochim. Biophys. Acta 806, 185‐186] and contained 58 amino acid residues with a blocked methionine and a glutamic acid at the N‐ and C‐termini, respectively. The B875‐β‐polypeptide contained 48 amino acid residues with alanine and phenylalanine as respective N‐ and C‐termini; although otherwise identical, the leucine at position 29 in the wild‐type strain was replaced by proline in the mutant. This radical amino acid substitution occurred within the central hydrophobic domain of the β‐polypeptide chain and is thought to result in a weakening of the structure of the α/β heterodimer since it was not possible to isolate the intact pigment‐protein complex from the R26.1 mutant strain.
Separation of phosphorylated from non‐phosphorylated LHCP polypeptides by two‐dimensional electrophoresis
Authors: Rémy R. , Ambard-Bretteville F. , Dubertret G.
Published: July 2002

‘In vitro’ phosphorylated thylakoid polypeptides were studied by means of different electrophoretic techniques. A highly resolving two‐dimensional electrophoresis method, recently developed in the laboratory using CHAPS and SDS as detergent for electrofocusing, allows the separation of each of the LHCP apoproteins into several molecular species. Those having more acidic isoelectric points correspond to the phosphorylated forms.
Modulation of intracellular Ca2+ in the parathyroid cell
Authors: Epstein Paul A. , Prentki Mark , Attie Maurice F.
Published: July 2002

Stimuli which enhance secretion from parathyroid cells such as low extracellular Ca2+ or Mg2+ are associated with a decrease in the cytosolic Ca2+ concentration as measured by quin2. Current evidence suggests that increased production of inositol 1,4,5‐triphosphate (IP3) releases Ca2+ from cellular stores thus increasing cytosolic Ca2+. We used saponin‐permeabilized dispersed bovine parathyroid cells to study the effect of IP3 on intracellular Ca2+. IP3 released Ca2+ from these cells in a dose‐dependent manner; half‐maximal response occurred with 0.3 μM IP3 and maximal response with 1.2 μM IP3. Permeabilized cells incubated in the presence of the mitochondrial inhibitor antimycin A released a similar amount of Ca2+ suggesting that IP3 releases Ca2+ from a non‐mitochondrial pool. These results suggest that IP3 regulates cytosolic Ca2+ in this system and may function as a second messenger controlling hormone secretion.
Evidence for resistance of the microenvironment of the primary plastoquinone acceptor (QA−·Fe2+) to mild trypsinization in PS II particles
Authors: Renger G. , Rutherford A.W. , Völker M.
Published: July 2002

Photosystem IISemiquinone‐iron complexTrypsinHerbicidePhotosythesis
Evidence for the electrochromic identification of the change of charges in the four oxidation steps of the photoinduced water cleavage in photosynthesis
Authors: Saygin Ö. , Witt H.T.
Published: July 2002

After dark adaptation of oxygen‐evolving photosystem (PS) II complexes, oscillatory absorption changes (stable >0.5 s) with a period of 4 were previously detected at 514 nm, probably due to electrochromic band shifts of carotenoids [(1984) FEBS Lett. 176, 83‐87]. To support this interpretation, we looked for corresponding phenomena in the 650–714 nm range. Here, oscillations were also observed with a difference spectrum giving evidence for an electrochromic band shift of the chlorophyllaabsorption band. The oscillation pattern 0:0:+:+ corresponds to those at 514 nm, supporting the former conclusion that the states S2and S3have a surplus charge compared to S0and S1. This is possible if the electron release pattern 1:1:1:1 is accompanied by an intrinsic proton release pattern 1:0:1:2 for the transition S0→ S1→ S2→ S3→ S0.
Metabolism of glucose via a modified Entner‐Doudoroff pathway in the thermoacidophilic archaebacterium Thermoplasma acidophilum
Authors: Budgen Nigel , Danson Michael J.
Published: July 2002

It has been found that the thermoacidophilic archaebacterium, Thermoplasma acidophilum, can metabolise glucose via a modified Entner‐Doudoroff pathway involving non‐phosphorylated intermediates. Pyruvate and glyceraldehyde are the first products, the glyceraldehyde then being further metabolised to a second molecule of pyruvate via 2‐phosphoglycerate. Intermediates of this pathway have been identified by enzymic analysis or by thin‐layer chromatography and the individual enzymes involved have been assayed and their kinetic parameters determined. Comparisons are made with the pathways of glucose metabolism in other archaebacteria. p]Archaebacteria
(Thermoplasma)
Entner‐Doudoroff pathway
Glucose metabolism
Primary structure of the α‐subunit of bovine adenylate cyclase‐inhibiting G‐protein deduced from the cDNA sequence
Authors: Nukada Toshihide , Tanabe Tsutomu , Takahashi Hideo , Noda Masaharu , Haga Kazuko , Haga Tatsuya , Ichiyama Arata , Kangawa Kenji , Hiranaga Masuyo , Matsuo Hisayuki , Numa Shosaku
Published: July 2002

The primary structure of the α‐subunit of the adenylate cyclase‐inhibiting G‐protein (gi) has been deduced from the nucleotide sequence of cloned DNA complementary to the bovine cerebral mRNA encoding the polypeptide. A much higher degree of amino acid sequence homology is observed between the α‐subunits of Gi and transducin (68%) than between those of Gi and the adenylate cyclase‐stimulating G‐protein (Gs) (43%) or between those of transucin and Gs (42%).
Homo‐phytochelatins are heavy metal‐binding peptides of homo‐glutathione containing Fabales
Authors: Grill E. , Gekeler W. , Winnacker E.-L. , Zenk H.H.
Published: July 2002

Exposure of several species of the order Fabales to Cd2+ results in the formation of metal chelating peptides of the general structure (γ‐Glu‐Cys)
n
‐β‐Ala (n = 2–7). They are assumed to be formed from homo‐glutathione and are termed homo‐phytochelatins, as they are homologous to the recently discovered phytochelatins. These peptides are induced by a number of metals such as Cd2+, Zn2+, Hg2+, Pb2+, AsO4
− and others. They are assumed to detoxify poisonous heavy metals and to be involved in metal homeostasis.
Oxygen exchange between the Fe(IV)O heme and bulk water for the A2 isozyme of horseradish peroxidase
Authors: Hashimoto Shinji , Nakajima Ryo , Yamazaki Isao , Tatsuno Yoshitaka , Kitagawa Teizo
Published: July 2002

Resonance Raman spectra were observed for compound II of horseradish peroxidase A2, and the Fe(IV)O stretching Raman line was identified at 775 cm−1. This Raman line shifted to 741 cm−1 upon a change of solvent from H2
16O to H2
18O, indicating occurrence of the oxygen exchange between the Fe(IV)O heme and bulk water. The oxygen exchange took place only at the acidic side of the heme‐linked ionization with pK
a = 6.9.
Randomization of the EF particles in thylakoid membranes of synechocystis 6714 upon transition from state I to state II
Authors: Olive J. , M'Bina I. , Vernotte C. , Astier C. , Wollman F.A.
Published: July 2002

In the cyanobacteriumSynechocystis6714 we show that changes in light energy distribution are controlled by the redox state of the PQ pool and we report on the deep ultrastructural modifications caused by these changes. We conclude that State I‐State II transitions correspond to an increased energy transfer between PS II and PS I through a randomization of the EF particles of the thylakoid membranes. This reorganization could involve a change in the interaction between PS II centers and the phycobilisomes.
Mössbauer spectroscopy of haemoglobins
Authors: Oshtrakh M.I. , Semionkin V.A.
Published: July 2002

Human normal, adult and foetal oxyhaemoglobins and oxyhaemoglobins from leukaemic patients were studied, by Mössbauer spectroscopy. The estimations of quadrupole splitting ΔEQand isomer shift δ allow one to distinguish between adult, foetal and leukaemic oxyhaemoglobins. The differences in the Fe2+electronic structure and active site molecular structure of foetal and leukaemic oxyhaemoglobins were analyzed.
The absence of a spectroscopically resolved intermediate state P+B−in bacterial photosynthesis
Authors: Breton J. , Martin J.-L. , Petrich J. , Migus A. , Antonetti A.
Published: July 2002

Reaction centers from the photosynthetic bacteriumRhodopseudomonas sphaeroideshave been excited either in the bacteriopheophytin band at 760 nm or in the accessory bacteriochlorophyll (B) band around 800 nm with laser pulses of 150 fs duration. Upon monitoring in the absorption band of the primary donor (P) at 860 nm, ultrafast energy transfer is observed which leads to the excited state of P in less than 100 fs. A transient bleaching recovering in 400 ± 100 fs is specifically detected upon excitation and observation in the 800 nm absorption band of B. However, upon direct excitation of P in the near infrared and using either normal or borohydride‐treated reaction centers, we have found no spectral or kinetic evidence indicating the presence of a transient intermediate state such as P+B−.
Evidence for two calcium transport systems in the photosynthetic bacteriumChromatium vinosum
Authors: Peach Cindy R. , Cobb Andrea D. , Smith James A. , Knaff David B.
Published: July 2002

Light‐dependent Ca2+efflux via the Ca2+/H+antiport in the photosynthetic purple sulfur bacteriumChromatium vinosumwas inhibited by three phenothiazines: chlorpromazine; trifluoperazine and phenothiazine. The inhibitors had no effect on Ca2+uptake byC. vinosumin the dark nor any effect on the light‐dependent efflux of either Na+or Tl+catalyzed, respectively, by theC. vinosumNa+/H+or K+/H+antiports. Ruthenium red and LaCl3, neither of which inhibited light‐dependent Ca2+efflux inC. vinosum, markedly inhibited Ca2+uptake in the dark byC. vinosumcells. Ruthenium red had no effect on the uptake of either Na+or the K+analog T1+byC. vinosumcells in the dark. These results have been interpreted in terms of two separate Ca2+transport systems inC. vinosum: (i) a phenothiazine‐sensitive and ruthenium red, La3+‐insensitive Ca2+/H+antiport responsible for Ca2+efflux in the light; and (ii) a ruthenium red and La3+‐sensitive but phenothiazine‐insensitive Ca2+uptake system.
ATP‐induced photochemical quenching of variable chlorophyll fluorescence
Authors: Schreiber U. , Rienits K.G.
Published: July 2002

ATP induces rapid quenching of fluorescence yield in light‐activated class D chloroplasts, distinguishable from the slower quenching caused by protein phosphorylation. The mechanism of the rapid quenching is shown to be photochemical, by application of the saturation pulse method with a modulated measuring system [(1986) Photosynth. Res. 10, 51‐62]. The effect is most pronounced at relatively low light intensities (optimum 2) without addition of electron acceptors. The properties of the ATP‐induced quenching with respect to ionophores, uncouplers and ATPase inhibitors suggest involvement of the reversible, light‐activated thylakoid ATPase. As a working hypothesis, it is proposed that part of PS II reaction centers, which are stroma‐exposed and in close proximity to CF0‐CF1, are modified by ATPase activity. Transformation of cytochromeb‐559 high‐potential form to low‐potential form by membrane acidification is discussed as a possible mechanism.
Phosphorylation of thylakoid proteins and synthetic peptide analogs
Authors: Bennett John , Shaw Elizabeth K. , Bakr Soliman
Published: July 2002

Spinach thylakoids contain at least 8 proteins (8.3–58 kDa) whose phosphorylation is strongly inhibited by the plastoquinone antagonist 2,5‐dibromo‐3‐methyl‐6‐isopropyl‐p‐benzoquinone (DBMIB). Reduction of DBMIB with ascorbate completely or partially relieves inhibition for all proteins other than the 27 kDa light‐harvesting complex (LHC) II and a synthetic dodecapeptide (MRKSATTKKAVC). The peptide, an analog of the phosphorylation site of pea 27 kDa LHC II, is phosphorylated with the same site specificity, kinetics, redox control and sensitivity to DBMIB/ascorbate as the protein itself. The data indicate that synthetic peptides can be used to study the number, substrate specificity and redox regulation of thylakoid protein kinases.
Isolation of the intact photosystem I reaction center core containing P700 and iron‐sulfur center FX
Authors: Golbeck John H. , Parrett Kevin G. , Mehari Tetemke , Jones Karen L. , Brand Jerry J.
Published: July 2002

The photosystem I reaction center core containing P700 and iron‐sulfur center FX has been isolated from a Synechococcus photosystem I particle with 6.8 M urea at pH 10.0 followed by sucrose density ultracentrifugation. The reaction center core has retained > 90% of FX and 100% of P700 (determined by optical spectroscopy) but is totally devoid of iron‐sulfur centers FA and FB (determined by optical and ESR spectroscopy). SDS‐PAGE indicates the retention of the 57 kDa reaction center polypeptide(s) but the total absence of the 16.4 and 8.1 kDa polypeptides. The loss of FA and FB is further reflected in the decline of acid‐labile sulfide from 11.8 ± 0.4 S2− /P700 in the control particle to 4.6 ± 0.3 S2− /P700 in the reaction center core. This preparation represents the first isolation of an intact reaction center core incorporating the components P700 and FX but totally lacking FA and FB.
Effects of long‐chain N‐acylethanolamines on lipid peroxidation in cardiac mitochondria
Authors: Parinandi N.L. , Schmid H.H.O.
Published: July 2002

A long‐chain N‐acylethanolamine (N‐oleoyl‐2‐aminoethanol) is shown to inhibit the production of thiobarbituric acid‐reactive substances in rat heart mitochondria treated with Fe2+ or Fe3+/ADP. The inhibition is concentration‐dependent in the range 50–150 μM of the agent and can be nearly complete depending on the type and amount of the free radical‐generating system. Structural analogues of N‐acylethanolamine are inhibitory as well, but neither oleic acid nor ethanolamine has measurable effects. N‐Oleoyl‐2‐aminoethanol affects peroxidation of linoleic acid micelles only minimally and has no effect on deoxyribose peroxidation.
Reconstitution of plastoquinone in the D1/D2/cytochrome b‐559 photosystem II reaction centre complex
Authors: Gounaris Kleoniki , Chapman David J. , Barber James
Published: July 2002

Reconstitution of plastoquinone in the photosystem II D1/D2/cytochrome b‐559 reaction centre complex, in the presence of the detergent Triton X‐100, is reported. Illumination of the reconstituted system results in the reduction of cytochrome b‐559, the process being partly herbicide‐sensitive. In addition, the reconstitution of plastoquinone results in the ability of the isolated reaction centre to catalyse the photoreduction of 2,6‐dichlorophenolindophenol in the presence of the exogenous electron donor diphenylcarbazide.
Phorbol 12‐myristate 13‐acetate modulates the cAMP‐induced light‐scattering response of a Dictyostelium discoïdeum cell population
Authors: Thiery R. , Klein R. , Tatischeff I.
Published: July 2002

The effect of phorbol 12‐myristate 13‐acetate upon the light‐scattering response to cAMP of a D. discoïdeum cell suspension was investigated. It was found that the first spike of the cAMP‐mediated light‐scattering change (peaking at about 15–20 s after stimulation) was inhibited by the phorbol ester. This effect was concentration dependent with an half‐maximum value for the inhibition of 4 nM. The inhibition was found to be maximal after a 10–20 min incubation time. The phorbol ester was shown to affect the dose‐response relationship between the cAMP concentration and the relative amplitude of the light‐scattering change, more by decreasing the number of cAMP receptors than by decreasing their apparent affinity for cAMP.
Expression of ricin B chain in Escherichia coli
Authors: Hussain Khalid , Bowler Christopher , Roberts Lynne M. , Lord J.Michael
Published: July 2002

DNA encoding ricin B chain was fused to that encoding the E. coli OmpA signal peptide using the expression secretion vector pIN‐111‐ompA. When induced, E. coli cells transformed with the recombinant plasmid express ricin B chain. The recombinant product accumulates in the periplasmic space in a soluble, biologically active form.
Detection of immunoreactive endothelin in plasma of hemodialysis patients
Authors: Totsune Kazuhito , Mouri Toraichi , Takahashi Kazuhiro , Ohneda Minoru , Sone Masahiko , Saito Takao , Yoshinaga Kaoru
Published: July 2002

Two types of radioimmunoassay (RIA) methods for measuring endothelin (ET) in human plasma were developed. One was an extraction procedure using a Sep‐Pak C18 cartridge, the other being a direct method. By the extraction method, plasma ET levels were lower than the detectable limit (7 pg/ml) in normal subjects and elevated in hemodialysis patients. The absolute values obtained via the direct method were 20‐times higher than those from extraction. Gel‐filtration experiments revealed that this discrepancy was mainly due to immunoreactive (IR‐) endothelin‐like substances of high molecular mass near 11.6 kDa (large IR‐ET). Extraction of the peptide by the C18 cartridge could eliminate interference by large IR‐ET and is important in the accurate measurement of ET concentrations in plasma.
In vivo measurement of spectroscopic and photochemical properties of intact leaves using the ‘mirage effect’
Authors: Havaux Michel , Lorrain Lucie , Leblanc Roger M.
Published: July 2002

This paper describes a new, highly sensitive, method for in vivo studies of photosynthesis based on the ‘mirage effect’ in which thermal energy dissipation from intact leaves, illuminated with intensity‐modulated light, is sensed through the periodic deflection of a laser beam propagating along the leaf surface. The photothermal deflection technique allows one to rapidly estimate the gross efficiency of photochemical energy storage by comparing the heat emission signal with and without an additional strong, photosynthetically saturating, non‐modulated light. In pea leaves, the maximal storage efficiency at low light intensities was shown to approach 55%. The general utility of this simple photothermal method is illustrated by examining the variation of the deflection signal under different conditions. The spectral resolution of this new method is shown to be much higher than that of the photoacoustic method.
Immunological detection of arrestin, a phototransduction regulatory protein, in the cytosol of nucleated erythrocytes
Authors: Mirshahi Massoud , Borgese Franck , Razaghi Ahmad , Scheuring Uwe , Garcia-Romeu Federico , Faure Jean-Pierre , Motais René
Published: July 2002

Cytosolic extracts of trout and turkey erythrocytes were tested for their immunoreactivity with polyclonal and monoclonal antibodies to retinal arrestin (S‐antigen), a cytosolic protein of photoreceptor cells involved in the desensitization of rhodopsin. After adsorption or immunoaffinity chromatography of the extracts, these antibodies specifically recognized a protein having a molecular weight similar to that of retinal arrestin. Because the G‐protein‐mediated transduction systems, such as visual and β‐adrenergic systems, display a high degree of structural and functional homology, the presence of arrestin‐like proteins in non‐photosensitive cells suggests that these proteins are involved in the transduction of chemical signals, with a possible role in receptor desensitization.
Purification of Shiga toxin by α‐d‐galactose‐(1 → 4)‐β‐d‐galactose‐(1 → 4)‐ β‐d‐glucose‐(1 →) receptor ligand‐based chromatography
Authors: Ryd Marie , Alfredsson Hans , Blomberg Lennart , Andersson Åsa , A. Lindberg Alf
Published: July 2002

A simple and rapid method for Shiga toxin purification based on specific binding to the Galα1 → 4Galβ1 → 4Glc globotrioside trisaccharide covalently linked to polyacryl/polyvinyl (Fractogel) has been developed. A cell‐free sonicate‐filtrate of Shigella dysenteriae type 1, strain 114Sd was passed over the globotrioside‐Fractogel column, and bound toxin eluted with 6 M guanidine‐HCl. A yield of 36 mg pure toxin/1 sonicate‐filtrate was otained, i.e. a one step 1224‐fold purification. The recovery of biologically active toxin was 87%. The toxin was purified to homogeneity as shown by native PAGE and SDS‐PAGE.
The effect of quercetin on cell cycle progression and growth of human gastric cancer cells
Authors: Yoshida Mitsumori , Sakai Toshiyuki , Hosokawa Nobuko , Marui Nobuyuki , Matsumoto Katsuhiko , Fujioka Akihiro , Nishino Hoyoku , Aoike Akira
Published: July 2002

Quercetin, a flavonoid, is found in many plants, including edible fruits and vegetables. We examined the effects on cell growth of human malignant cells derived from the gastrointestinal tract and on cell cycle progression. Quercetin markedly inhibited the growth of human gastric cancer cells and the IC
50 value was 32–55 μM. DNA synthesis was suppressed to 14% of the control level by the treatment with 70 μM quercetin for 2 days. Furthermore, quercetin blocked cell progression from the G1 to the S phase.
The transmembrane topology of the α subunit from the ATPase in Escherichia coli analyzed by PhoA protein fusions
Authors: Bjørbæk Christian , Foërsom Vibeke , Michelsen Ole
Published: July 2002

The atpB gene encodes the α subunit of the H+‐ATPase of E. coli. The topology of this membrane protein has been analyzed by PhoA fusions. The results support an eight transmembrane segment model that is consistent with the hydropathic profile.
NADPH‐dependent flavoenzymes catalyze one electron reduction of metal ions and molecular oxygen and generate hydroxyl radicals
Authors: Shi Xianglin , Dalal N.S.
Published: July 2002

This study reports a new property of the important NAD(P)H‐dependent flavoenzymes, glutathione reductase, lipoyl dehydrogenase and ferredoxin‐NADP+ oxidoreductase, that can catalyze a one electron reduction of metal ions such as chromium(VI) and vanadium(V). During the enzymatic reduction process, molecular oxygen is reduced to H2O2, which reacts with the reduced metal complexes to generate hydroxyl radicals. Since the hydroxyl radicals have been suggested to play an important role in Cr(VI) toxicity, this study provides a basis for a recent observation that Cr(VI) mutagenesis is strongly oxygen dependent. These results also point to an enzymatic pathway for the metabolism of some metal ions and concomitant generation of hydroxyl radicals.
Arrestin from nucleated red blood cells binds to bovine rhodopsin in a light‐dependent manner
Authors: Scheuring Uwe , Franco Michel , Fievet Bruno , Guizouarn Hélène , Mirshahi Massoud , Faure Jean-Pierre , Motais René
Published: July 2002

Using a panel of monoclonal antibodies, it has previously been demonstrated that the cytosol of nucleated red cells (trout and turkey) contains a protein similar to arrestin, a soluble protein found so far only in the photosensitive cells and which, by binding to photoexcited rhodopsin, inhibits the phototransduction process. The role of this arrestin‐like protein in non‐photosensitive cells is questionable. In this report we present evidence that partially purified red blood cell arrestin (RBC arrestin) behaves functionally like bovine retinal arrestin: it binds to phosphorylated bovine rhodopsin only when this receptor has been photoactivated. Thus RBC arrestin and bovine retinal arrestin are closely related both structurally and functionally. By analogy with the function of retinal arrestin, it is proposed that RBC arrestin is involved in desensitization of membrane transport proteins and/or adrenergic receptors.
In vitro protein translocation into inverted membrane vesicles prepared from Vibrio alginolyticus is stimulated by the electrochemical potential of Na+ in the presence of Escherichia coli SecA
Authors: Tokuda Hajime , Kim Young Jae , Mizushima Shoji
Published: July 2002

A protein translocation system was reconstituted from inverted membrane vesicles prepared from Na+ pump‐possessing Vibrio alginolyticus and purified Escherichia coli SecA. The translocation required ATP and was stimulated by the functioning of the Na+ pump, suggesting that the electrochemical potential of Na+ but not that of H+, is important for protein translocation in Vibrio.
Phosphorylation of chicken brain‐type creatine kinase affects a physiologically important kinetic parameter and gives rise to protein microheterogeneity in vivo
Authors: Quest Andrew F.G. , Soldati Thierry , Hemmer Wolfram , Perriard Jean-Claude , Eppenberger Hans M. , Wallimann Theo
Published: July 2002

In addition to the two monomer subunits of chicken brain‐type creatine kinase (B‐CK, EC, 2.7.3.2), termed Bb (basic) and Ba (acidic), another subspecies called Bb∗ was identified by chromatofocussing in the presence of 8 M urea (Quest et al., [20]). The latter low abundance protein species, isolated from tissue extracts, comigrated on 2D‐gels with three minor species (Bbl‐3), initially identified in immunoprecipitated, [35S]methionine labeled in vitro translation products of cDNA coding for the basic monomer Bb. During in vitro translation experiments in the presence of [32P]‐γ ‐ATP, Bbl‐3 were labeled while phosphatase treatment eliminated these minor species. It is concluded that Bb* is identical to Bbl‐3 and represents phosphorylated derivatives of Bb. B‐CK dimer populations from different tissues were separated by ion‐exchange chromatography and the Km
values of the resulting fractions were determined under phospho‐creatine (CP)‐limiting conditions. In fractions containing only Bb and Bb∗ two kinetically different enzyme species were detected (Km
values for CP = 1.6 mM and 0.8 mM), while fractions containing B‐CK dimers composed of the major Ba and Bb monomers, but no Bb∗, were homogeneous in this respect (Km
for CP = 1.6 mM). Phosphorylation of Bb to yield Bb∗ is concluded to reduce the Km
of B‐CK dimers for CP by about 50%. This Km
shift is within the range of CP concentrations found in tissues expressing the B‐CK isoform and may therefore be of physiological relevance.
Purification and some characteristics of chicken liver L‐2‐hydroxyacid oxidase A
Authors: Dupuis Liliane , Caro Josiana De , Brachet Patrick , Puigserver Antoine
Published: July 2002

The isozyme A of L‐2‐hydroxyacid oxidase is a peroxisomal flavoenzyme that catalyzes the oxidation of short‐chain aliphatic L‐2‐hydroxyacids in many tissues of higher organisms. A new purification procedure allowed us to obtain a 1400‐fold purified enzyme from chicken liver. The N‐terminal amino acid of the polypeptide chain was found to be blocked as that of spinach glycolate oxidase, contrastingly with that of rat kidney isozyme B. Its amino acid composition was comparable to that of other known L‐2‐hydroxyacid oxidases. Despite different substrate specificity, some immunological identity was observed between chicken liver L‐2‐hydroxyacid isozyme A and rat kidney isozyme B.
Evidence for gene duplication forming similar binding folds for NAD(P)H and FAD in pyridine nucleotide‐dependent flavoenzymes
Authors: McKie James H. , Douglas Kenneth T.
Published: July 2002

For pyridine nucleotide‐dependent flavoezymes, binding both FAD and NAD(P)H on a single amino‐acids chain, we have found a high degree of internal sequence similarity for certain regions of FAD and NAD (P)H binding portions of the chain for any given protein. This was the case for a range of enzymes classes, including disulphide oxidoreductases (such as glutathione reductase, trypanothione reductase, lipoamide dehydrogenase, mercuric reductase), mono‐ and dioxygenases, nitrite reductaSE, alkyl hydroperoxidase and NADH dehydrogenase from E. coli. This provides strong support for gene duplication as the origin of at least part of the FAD and NAD(P)H recognising domains of such enzymes.
Biphasic kinetics of inositol 1,4,5‐trisphosphate accumulation, in gastrin‐stimulated parietal cells Effects of pertussis toxin and extracellular calcium
Authors: Roche Serge , Gusdinar Tutus , Bali Jean-Pierre , Magous Richard
Published: July 2002

The effect of Pertussis toxin (PTx) and extracellular Ca2+ were investigated on gastrin‐induced Ins(1,4,5)P3 mass level in isolated gastric parietal cells. Basal Ins(1,4,5)P3 content was 5.48±0.49 pmol/500 000 cells. Gastrin (10 nM) induced a rapid increase in Ins(1,4,5)P3 content which was maximal after 15 s and corresponded to 2–2.5‐fold basal level; this Ins(1,4,5)P3 content then decreased within 30 s. After a longer time of gastrin exposure (> 1 min), a sustained and unexpected increased in Ins(1,4,5)P3 accumulation was observed which was maximal at 7.5 min (corresponding to 2.3–2.8‐fold basal value) and slightly decreased thereafter. PTx treatment of cells (200 ng/ml) for 3 h or removal extracellular Ca2+ did not affect the rapid rise, but drastically reduced the sustained increase in Ins(1,4,5)P3 content (60–100% inhibition); this inhibition was not evident after 10 min of hormone stimulation. Furthermore, diltiazem, a Ca2+ channel blocker, led to a similar inhibition of the sustained increase. We concluded that: (i) gastrin induced rapid increase in Ins(1,4,5)P3 content via a mechanism insensitive to PTx and to extracellular Ca2+, and (ii) gastrin induced a sustained increase in Ins(1,4,5)P3 level via a mechanism sensitive to PTx and to extracellular Ca2+. Even though the rapid rise in Ins(1,4,5)P3 content may be involved in the intracellular Ca2+ mobilization occurring after the first seconds of hormone stimulation, the physiological role of the sustained Ins(1,4,5)P3 increased level remains to be elucidated.
Evidence for the importance of a positive charge and an α‐helical structure of the C‐terminus for biological activity of human IL‐6
Authors: Lütticken Claudia , Krüttgen Alex , Möller Carsten , Heinrich Peter C. , Rose-John Stefan
Published: July 2002

Thirteen point mutations of human interleukin‐6 at the C‐terminus were constructed at the cDNA‐level. Two degenerate oligonucleotide primers were used for PCR synthesis of two groups of point mutations at positions 182 and 184 The mutated cDNAs were in vitro transcribed and translated and subsequently assayed for biological activity in the B9 cell proliferation test. Our results confirm our former findings obtained with deletion mutants on the importance of the C‐terminus of IL‐6 for biological activity. In addition, we now present evidence for the importance of an α‐helix at the C‐terminus of IL‐6 and the presence of the positive charge at position 182 for biological activity.
Cyanobacterial thylakoid membrane proteins are reversibly phosphorylated under plastoquinone‐reducing conditions in vitro
Authors: Harrison Michael A. , Tsinoremas Nicholas F. , Allen John F.
Published: July 2002

Reversible, light‐dependent protein phosphorylation was observed in isolated thylakoid membranes of the cyanobacterium Synechococcus 6301. A polypeptide of 15 kDA in particular was phosphorylated under plastoquinone‐reducing conditions and was not phosphorylated under plastoquinone‐oxidising conditions. Phosphorylation and dephosphorylation reactions involving this and several other membrane polypeptides showed sensitivity to inhibitors of protein kinases and phosphatases. Changes in phosphorylation state correlated with changes in low temperature fluorescence emission characteristic or changes in excitation energy distribution between the photosystems. The 15 kDa phosphopolypeptide is likely to be involved directly in light state adaptations in cyanobacteria.
A novel brain‐specific 25 kDa protein (p25) is phosphorylated by a Ser/Thr‐Pro kinase (TPK II) from tau protein kinase fractions
Authors: Takahashi Miho , Tomizawa Kayoko , Ishiguro Koichi , Sato Kazuki , Omori Akira , Sato Showbu , Shiratsuchi Akiko , Uchida Tsuneko , Imahori Kazutomo
Published: July 2002

A novel brain‐specific 25 kDa protein (p25) was purified from a bovine brain extract. The protein was phosphorylated by Ser/Thr‐Pro kinase (TPK II) in tau protein kinase fractions at the Ser residues of Ser‐Pro sequences. Using immunoblot analysis, the protein was found only in brain extracts, and was most abundant in the brain regions such as cerebrum and hippocampus, but less abundant in cerebellum, medulla oblongata and olfactory bulb. The protein was detected in rat, bovine and human brain extracts, indicating that this protein specifically exists in mammalian brain tissues.
Collagen gene expression during chondrogenesis from chick periosteum‐derived cells
Authors: Nakata Ken , Nakahara Haruhiko , Kimura Tomoatsu , Kojima Akira , Iwasaki Motoki , Caplan Arnold I. , Ono Keiro
Published: July 2002

Chick periosteum‐derived cells, which do not enter the chondrogenic cell lineage during normal bone development and growth, exhibit chondrogenic potential in high cell density culture conditions. In such cultures, collagen gene expression was temporally analyzed at the mRNA level by a reverse transcription PCR (RT‐PCR) procedure, which showed that α1(II) and α1(IX) collagen mRNAs are coordinately increased, coincident with the onset of overt chondrogenesis, and subsequently decreased as chondrocytes exhibited hypertrophic characteristics. α1(X) collagen mRNA was detected well before the onset of chondrogenesis and markedly increased along with the hypertrophic change. For α2(I) collagen, both the bone/tendon form and the cartilage form of mRNA were detected throughout the culture period. This culture system provides an experimental vehicle capable of investigating the molecular events involved in the full range of chondrogenic differentiation starting from uncommitted periosteum‐derived mesenchymal stem cells.
Expression of 9 Salmonella typhimurium enzymes for cobinamide synthesis Identification of the 11‐methyl and 20‐methyl transferases of corrin biosynthesis
Authors: Roessner Charles A. , Warren Martin J. , Santander Patricio J. , Atshaves Barbara P. , Ozaki Shin-ichi , Stolowich Neal J. , Iida Katsumi , Scott A.Ian
Published: July 2002

Nine of the cbi genes from the 17.5 kb cob operon of Salmonella typhimurium previously shown by genetic studies to be involved in the biosynthesis of cobinamide from precorrin‐2, have been subcloned and expressed in Escherichia coli. Seven of the gene products were found in the soluble fraction of cell lysates and have been purified. The gene products corresponding to cbi E, F, H and L were shown by SAM binding and by homology with other SAM‐binding proteins to be candidates for the methyltransferases of vitamin B12 biosynthesis. The enzymatic functions of the gene products of cbiL and cbiF are associated with C‐methylation at C‐20 of precorrin‐2 and C‐11 of precorrin‐3.
Stereospecific assignments and χ1 rotamers for FKBP when bound to ascomycin from 3JHα,Hβ and 3JN,Hβ coupling constants
Authors: Xu Robert X. , Olejniczak Edward T. , Fesik Stephen W.
Published: July 2002

3
J
Hα,Hβ and 3
J
N,Hβ coupling constants were measured for isotopically labeled FKBP when bound to the immunosuppressant, ascomycin, using a 1H‐coupled 3D HCCH‐TOCSY and 15N‐coupled 3D HSQC‐TOCSY experiment, respectively. From an analysis of these two sets of coupling constants, stereospecific β‐proton assignments and χ1 rotamers for FKBP have been obtained. All of the χ1 rotamers were consistent with the χ1 angles measured in the X‐ray crystal structure of the FKBP/FK506 complex, suggesting that the structures of the two complexes are similar.
Changes in chromatin structure due to hypomethylation induced with 5‐azacytidine or DL‐ethionine
Authors: Fajkus Jiří , Vyskot Boris , Bezděk Milan
Published: July 2002

Changes in chromatin structure of the HRS60 family of repetitive sequences in tobacco DNA were studied after hypomethylation induced with 5‐azacytidine or DL‐ethionine. The TaqI site in the HRS60 units lies in nucleosomal core regions and its cleavage is enhanced in the hypomethylated chromatin. In contrast, the cleavage of the Sau3A1 site located in linker DNA does not depend on the level of methylation of DNA.
Interaction of human myeloperoxidase with nitrite
Authors: Cooper Chris E. , Odell Eddy
Published: July 2002

EPR (electron paramagnetic resonance) and optical spectroscopy show that human neutrophil myeloperoxidase is converted from ferric high‐spin to low‐spin by the addition of nitrite. The Soret peak shifts from 429 to 447 nm and new peaks appear in the visible region at 573 and 627 nm; the EPR g‐values change from 6.84, 5.02, 1.95 to 2.55, 2.31, 1.82. Small differences are seen in the EPR (but, not optical) spectra of myeloperoxidase isoenzyme I compared to isoenzymes II and III. The reaction with nitrite is detectable by EPR in intact neutrophils and is thus a possible in vivo monitor of NO/nitrite production by these cells.
Brefeldin A inhibits protein synthesis in cultured cells
Authors: Fishman Peter H. , Curran Patricia K.
Published: July 2002

The fungal metabolite brefeldin A (BFA) is known to disrupt the Golgi apparatus resulting in redistribution of Golgi proteins to the endoplasmic reticulum and inhibition of protein secretion. BFA was found to inhibit protein synthesis in rat glioma C6 cells by up to 70% between 0.1 and 1,μg/ml. Inhibition was both time‐dependent and reversible. BFA inhibited protein synthesis to varying degrees in a number of other cell lines but not in BFA‐resistant marsupial kidney cells. The same concentrations of BFA which inhibited protein synthesis, also blocked the inhibitory effects of Pseudomonas exotoxin and ricin on BFA‐sensitive cells. BFA, however, was unable to block the inhibition of protein synthesis by the toxins in the resistant marsupial kidney cells.
cDNA cloning and expression of cysteine synthase B localized in chloroplasts of Spinacia oleracea
Authors: Saito Kazuki , Tatsuguchi Kazuyo , Murakoshi Isamu , Hirano Hisashi
Published: July 2002

The cDNA clones for cysteine synthase B, which is localized in chloroplasts of Spinacia oleracea L., were isolated by screening a library with synthetic oligonucleotides encoding a partial peptide sequence of the purified protein. Nucleotide sequence analysis revealed an open reading frame encoding a polypeptide of 383 amino acids containing a putative transit peptide of 52 amino acids. A bacterial expression vector of the cDNA clone could genetically complement an Escherichia coli auxotroph lacking cysteine synthase and could produce the functionally active and immuno‐reactive cysteine synthase in E. coli. RNA blot hybridization suggested that the transcripts were primarily accumulated in leaves of spinach.
A novel peptide designed for sensitization of terbium (III) luminescence
Authors: Clark Ian D. , Hill Irene , Sikorska-Walker Marianna , MacManus John P. , Szabo Arthur G.
Published: July 2002

Several synthetic peptides, modelled from a Ca2+‐binding loop of the EF‐hand family of proteins, were prepared containing cysteine residues. The peptide, GDKNADGFICFEEL, was labelled covalently at the cysteine residue (loop position 9) with iodoacetamidosalicylic acid. This novel conjugate is a metal‐binding loop containing a salicylic acid side chain that could not only chelate Tb3+ in conjunction with the other chelating groups in the sequence, but could also sensitize Tb3+ luminescence. The loop had a high Tb3+ affinity, with stoichiometric binding observed under experimental conditions. The luminescence from the Tb3+‐peptide complex was more than 10‐fold greater than the luminescence reported from a related peptide which contained Trp as the Tb3+ donor at loop position 7. This peptide has significant potential for use in lanthanide‐based time‐resolved luminescence immunoassays.