bluuebunny/crossref_metadata_2025 huggingface dataset:
Reduced mitogenic stimulation of human lymphocytes by extremely low frequency electromagnetic fields
Authors: Conti Pio , Gigante Giovanni Ettore , Cifone Maria Grazia , Alesse Edoardo , Ianni Gianfranco , Reale Marcella , Angeletti Pietro Ubaldo
Published: July 2002

Blastogenesis of human peripheral blood lymphocytes stimulated in vitro by non‐specific mitogens (PHA, ConA, PWM) upon exposure to extremely low frequency EMF has been studied. Different frequencies of square waveforms have been used. PHA‐stimulation resulted in strong inhibitions as measured by [3H]thymidine incorporation. A frequency window (3–50 Hz) within which ConA‐induced blastogenesis was significantly inhibited has been individuated. The mitogenic effect of PWM was significantly affected only at 3 Hz.
Binding of optically pure (−)‐[3H]nicotine to rat brain membranes
Authors: Abood Leo G. , Grassi Susan , Costanza Maria
Published: July 2002

With the recent availability of (−)‐[3H]nicotine of high specific activity, binding studies were performed on rat brain membranes in the presence of a variety of nicotine analogues and cholinergic drugs. Both a higher affinity (K
d = 2 × 10−10 M) and a lower affinity (2 × 10−9 M) site were observed; the stereoselectivity of both sites being similar. A good correlation was observed between IC
50‐values and psychotropic potency of a series of N′‐alkyl substituted nicotine analogues.
Inhibition of anion transport across the red cell membrane by dinitrophenylation of a specific lysine residue at the H2DIDS binding site of the band 3 protein
Authors: Rudloff V. , Lepke S. , Passow H.
Published: July 2002

The inhibition of anion transport by dinitrophenylation of the red cell membrane is brought about by the modification of a single lysine residue located on the 17‐kDa segment of the band 3 protein. This residue is identical with Lys a, which is also capable of reacting with one of the two isothiocyanate groups of 4,4'‐diisothiocyano dihydro‐stilbene‐2,2'‐disulfonate (H2DIDS). The rate of reaction between Lys a and 1‐fluoro‐2,4‐dinitrobenzene is reduced when a second lysine residue on the 35‐kDa segment of the band 3 protein becomes dinitrophenylated. This latter residue is not identical with Lys b which is known to be present on the 35‐kDa segment and involved in the cross‐linking of this segment with the 17‐kDa segment by H2DIDS.
A novel super‐secondary structure of proteins and the relation between the structure and the amino acid sequence
Authors: Efimov A.V.
Published: July 2002

A novel protein super‐secondary structure which is referred to as an αα‐corner is considered. The αα‐corner is formed by two consecutive α‐helices packed approximately crosswise and connected by two or more peptide units. It is shown that the amino acid sequences coding for the αα‐corners have a strictly definite order of hydrophobic, hydrophilic and glycine residues. A hypothesis is suggested that the αα‐corner can be an embryo of protein folding.
Effect of photosynthesis on dark mitochondrial respiration in green cells
Authors: Singh Prikhshayat , Naik M.S.
Published: July 2002

Green plant cells can generate ATP in both chloroplasts and mitochondria. Hence the effect of photosynthesis on dark mitochondrial respiration can be considered at a variety of levels. Turnover of ceitric acid cycle dehydrogenases, which is essential for supply of carbon skeletons for amino acid synthesis, seems to be largely unaffected during photosynthesis. The source of carbon for the anaplerotic function of the citric acid cycle in light is however, not known with certainty. NADH generated in these reactions is probably not oxidised via the mitochondrial electron transfer chain coupled to ATP synthesis. However, it may be oxidised by the alternative cyanide‐insensitive pathway, exported to the cytosol via the oxaloacetate‐malate dicarboxylate shuttle or directly utilised for cytosolic nitrate reduction. Oxidation of succinate via cytochrome oxidase may also be similarly inhibited in light. Whether increase in the cytosolic ATP/ADP ratio in light is responsible for the inhibition of mitochondrial electron transfer to O2 is not clearly established, because the ATP/ADP ratio is reported to be already quite high in the dark. Effective collaboration between photophosphorylation and oxidative phosphorylation in order to maintain the cytosolic energy charge at a present high level is discussed.
Domain structure of flagellin
Authors: Fedorov O.V. , Kostyukova A.S.
Published: July 2002

The chemotaxis of bacteria such as Salmonella and Escherichia coli involves smooth swimming punctuated by periods of tumbling. In smooth swimming the flagellar filaments are left‐handed, in tumbling they are right‐handed with a different wavelength. The filaments are constructed from a globular protein, flagellin, by a process of self‐assembly. The existing models assume that the flagellin molecule is bistable and longitudinal rows of subunits take one of the two possible conformations. Such a model explains the observed different morphology of the flagellum. We have studied Salmonella and E. coli flagellins in polymeric and monomeric forms by scanning microcalorimetry and circular dichroism. We have inferred that a flagellin molecule consists of several domains, two of which are structured at physiological temperatures and are in the monomeric form, while the others acquire a regular form only in the process of polymerization. This phenomenon may be the basis of a process during which the flagellin molecule, fitting into the flagellum, acquires a conformation analogous to that of the neighbouring molecule in the longitudinal row.
Effects of lithium on angiotensin‐stimulated phosphatidylinositol turnover and aldosterone production in adrenal glomerulosa cells: a possible causal relationship
Authors: Balla T. , Enyedi P. , Hunyady L. , Spät A.
Published: July 2002

Turnover of 32P‐labelled phosphatidylinositol (PI) was examined in isolated adrenal glomerulosa cells. Increased incorporation of [32P] phosphate into PI in response to angiotensin II was completely prevented by Li+. A simultaneous accumulation of 32P activity in phosphatidic acid (PA) was also observed. Angiotensin II increased the breakdown of PI despite the presence of Li+. These results suggest that Li is a suitable tool to interrupt the accelerated PI cycle in angiotensin‐stimulated cells. Aldosterone production of superfused cells was inhibited by Li+ when the cells were stimulated with angiotensin II. On the other hand, Li+ did not inhibit the aldosterone response of the cells to ACTH, a hormone which acts via cyclic AMP and does not enhance PI turnover in these cells. On the basis of these results, we assume that the inhibitory effect of Li+ on aldosterone production is related to its effect on PI turnover.
Cytosolic free Ca2+ in isolated rat hepatocytes as measured by quin2
Authors: Berthon Brigitte , Binet Adrien , Mauger Jean-Pierre , Claret Michel
Published: July 2002

Cytosolic [Ca2+] has been measured by using the Ca2+‐sensitive indicator quin2 in rat liver cells. Optimal loading and hydrolysis have been obtained by equilibrating the cells with 50 μM quin2 acetoxymethyl ester for 150 s. The increase in [Ca2+]i initiated by noradrenaline and vasopressin was reduced but not abolished by removing external Ca2+.
Calcium reconstitutes high rates of oxygen evolution in polypeptide depleted Photosystem II preparations
Authors: Ghanotakis Demetrios F. , Babcock Gerald T. , Yocum Charles F.
Published: July 2002

Exposure of highly resolved Photosystem II preparations to 2 M NaCl produces an 80% inhibition of oxygen‐evolution activity concomitant with extensive loss of two water‐soluble polypeptides (23 and 17 kDa). Addition of Ca2+ to salt‐washed PS II membranes causes an acceleration in the decay of Z⨥, the primary donor to P‐680+, and we show here that this acceleration is due to reconstitution of oxygen‐evolution activity by Ca2+. Other cations (Mg2+, Mn2+, Sr2+) are much less effective in restoring oxygen evolution. On the basis of these observations we propose that Ca2+, perhaps in concert with the 23 kDa polypeptide, is an essential cofactor for electron transfer from the ‘S’‐states to Z on the oxidizing side of PS II.
Glutathione biosynthesis in the isolated perfused rat lung: utilization of extracellular glutathione
Authors: Berggren Margareta , Dawson Janet , Moldéus Peter
Published: July 2002

The isolated perfused rat lung catalyzed the biosynthesis of GSH when the sulfur amino acids cysteine or N‐acetylcysteine, but not methionine, were supplied in the perfusion medium. The lung also had the capacity to utilize extracellular GSH for this purpose. Replenishment of intracellular GSH in perfused lungs from diethylmaleate‐treated rats was pronounced even at 25 μM GSH in the perfusion medium. The utilization of extracellular GSH is probably primarily through extracellular break‐down and resynthesis rather than direct uptake as indicated by the inhibitory effect of the γ‐glutamylcysteine synthetase inhibitor, buthionine sulfoximine and the γ‐glutamyl transferase inhibitor, anthglutin. The results indicate that the lung in addition to the kidney may utilize circulating plasma GSH.
Regulation of neuropeptide Y (NPY) binding by guanine nucleotides in the rat cerebral cortex
Authors: Undén Anders , Bartfai Tamas
Published: July 2002

In the presence of GTP, GDP, GMPP(NH)P, GMPP(CH2)P, GMPP(S)P but not in the presence of GMP, cGMP or ATP, the high affinity binding of neuropeptide Y (NPY) was reduced in a dose‐dependent manner. GTP (0.1 mM) diminished the maximal binding capacity for 125I‐labelled NPY by 40% without any change in the equilibrium dissociation constant of the receptor 125I‐labelled NPY complex.
Conformational heterogeneity in polypeptide cardiac stimulants from sea anemones
Authors: Gooley Paul R. , Blunt John W. , Norton Raymond S.
Published: July 2002

High‐resolution 1H NMR spectra at 300 MHz of the polypeptide cardiac stimulants anthopIeurin‐A and Anemonia sulcata toxin II reveal conformational heterogeneity in both molecules. The two conformations, manifest in a number of split 1H resonances, are in slow exchange over a wide range of pH and temperature. Heterogeneity affects a region of these molecules containing the structurally and functionally important Asp residues. By comparison with a homologous polypeptide Anemonia sulcata toxin I, which does not show this type of heterogeneity, it is suggested that the heterogeneity may originate in cis‐trans isomerism of the Gly‐40 to Pro‐41 peptide bond.
Isozymes of glycogen synthase
Authors: Kaslow Harvey R. , Lesikar David D.
Published: July 2002

Antisera to rat skeletal muscle glycogen synthase failed to recognize liver glycogen synthase by electroblot analysis. The antisera recognized the enzyme in skeletal muscle, heart, fat, kidney, and brain. The results support the hypothesis that there are at least two isozymes of glycogen synthase, and that most tissues contain a form similar or identical to the skeletal muscle type. There is a virtual absence of the muscle‐type enzyme in adult rat liver.
Mitochondrial and cytoplasmic protein syntheses are not required for heat shock acquisition of ethanol and thermotolerance in yeast
Authors: Watson Kenneth , Dunlop Glen , Cavicchioli Rick
Published: July 2002

Heat shock acquisition of ethanol‐ and thermotolerance in Saccharomyces cerevisiae was not inhibited in cells incubated in the presence of cycloheximide or chloramphenicol. Respiratory‐deficient (ρ−) mutants also characteristically exhibited the heat shock response. It was concluded that mitochondrial and cytoplasmic protein syntheses are not required for heat shock acquisition of ethanol and thermotolerance in yeast.
Bacterial flagellar motors and osmoelectric molecular rotation by an axially transmembrane well and turnstile mechanism
Authors: Mitchell Peter
Published: July 2002

Bacterial ion‐driven flagellar motors are the smallest known rotatory mechanical devices, natural or artificial, their overall diameter being only about 25 nm or one millionth of an inch. They are unique in the fields of biology and engineering. This paper develops a possible osmoelectric or local electrokinetic mechanism of molecular rotatory motion in bilayer membranes, which may help to explain how bacterial flagellar motors work, and may incidentally encourage new developments in the bioenergetics and biomechanics of enzyme, osmoenzyme and porter action.
Activated polymorphonuclear leucocytes consume vitamin C
Authors: Hemilä Harri , Roberts Peter , Wikström Mårten
Published: July 2002

Polymorphonuclear leucocytes (PMN) are known to produce Superoxide and other oxygen derivatives upon activation as part of their microbicidal armory. Here we report that extracellular ascorbate is effectively oxidised by activated but not by resting human PMN in vitro. The oxidation of ascorbate is mainly caused by the Superoxide that is generated by the activated cells, as shown by its effective inhibition by Superoxide dismutase. However, myeloperoxidase, which may generate hypochlorite, also contributes to a significant extent. Ascorbate reduces Superoxide to peroxide, as indicated by measurements of the stoichiometry of ascorbate and oxygen consumption. These results support the notion that extracellular ascorbate may serve as an important physiological protecting agent against oxygen radical damage in inflammation.
Purification and partial amino acid sequence analysis of human erythrocyte acetylcholinesterase
Authors: Chhajlani Vijay , Derr Dwight , Earles Betty , Schmell Eli , August Thomas
Published: July 2002

A single step immunoaffinity purification procedure for human erythrocyte acetylcholinesterase is described which permitted the isolation of milligram quantities of enzyme from 10 U of erythrocytes, with 113 000‐fold purification and a yield of about 22%. In SDS‐PAGE analysis, the enzyme corresponds to a disulfide linked dimer of 140 kDa which is converted to a 70 kDa monomer upon disulfide reduction. The tryptic peptides generated from purified enzyme were separated by reverse‐phase HPLC. Five of these peptides were analysed to determine the amino acid sequences. The obtained sequences showed no homology to the already known amino acid sequences for human serum and brain butyryl‐cholinesterase and Torpedo californica acetylcholinesterase.
Purification and characterization of Ca2+/calmodulin‐dependent actin‐binding proteins from squid retina
Authors: Asai Haruo , Arai Takao , Fujii Toshihiro , Matsumoto Gen
Published: July 2002

Ca2+/calmodulin (CaM)‐dependent actin‐binding proteins (CABPs) of 92, 105, 120 and 135 kDa were purified from squid retina. These proteins were eluted from the CaM affinity column in a Ca2+‐dependent manner, and binding of the CABPs to F‐actin was regulated by Ca2+/CaM. Electron microscopic observations employing the low‐angle rotary shadowing technique showed the CABP molecules to have granular shapes similar to the granular proteins associated with actin filaments in squid rhabdomeral microvilli. We have previously reported that these actin filaments are fragmented upon exposure to light [(1988) J. Cell Biol. 106, 1151–1160]. Since the intracellular Ca2+ concentrations of the invertebrate retina are elevated during the light illumination, these results indicate that the CABPs are directly associated with the actin filament in the microvilli of the squid photoreceptors. We therefore suggest that the CABPs may regulate the light‐induced structural changes of the microvillar cytoskeleton.
Glycolysis in P‐glycoprotein‐overexpressing human tumor cell lines Effects of resistance‐modifying agents
Authors: Broxterman Henricus J. , Pinedo Herbert M. , Kuiper Catharina M. , Schuurhuis Gerrit J. , Lankelma Jan
Published: July 2002

We show that drugs, such as verapamil, which reverse multidrug resistance (MDR), in P‐glycoprotein‐overexpressing tumor cells, increased the rate of lactate production in four human MDR cell lines, but not in the parent, sensitive cell lines. The effect on glycolytic rate was maximal at a medium concentration of 2 μM verapamil. The glycolytic rate in sensitive (A2780) and MDR (2780AD) cells showed the same pH dependence, but the effect of verapamil was seen only in 2780AD cells at all pH values investigated (6.6, 7.4 and 8.2). A series of drugs such as nigericin, oligomycin, amiloride and monensin had similar effects in the two cells. Phorbol myristate acetate increased lactate formation in neither cell line. Verapamil induced an extra amount of ATP consumption in P‐glycoprotein‐expressing 2780AD cells of approx. 25 pmol/s per 106 cells, which was estimated to be about 10% of cellular energy turnover.
The primary structure of rat ribosomal protein L26
Authors: Paz Veronica , Olvera Joe , Chan Yuen-Ling , Wool Ira G.
Published: July 2002

The amino acid sequence of rat ribosomal protein L26 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed from the NH2‐terminal amino acid sequence of the protein. Rat L26 contains 145 amino acids and has a molecular mass of 17 266 Da. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 8–16 copies of the L26 gene. The mRNA for the protein is about 650 nucleotides in length. Protein L26 has a sequence of 9 residues that may be repeated in three places.
Na+/H+ exchange and Ca2+ influx
Authors: Siffert Winfried , Akkerman Jan Willem N.
Published: July 2002

Cell stimulation raises the cytosolic free Ca2+ concentration, [Ca2+]i, and induces activation of Na+/H+ exchange which raises the cytosolic pH, pHj. Recent studies have addressed the question whether Na+/H+ exchange plays a role in Ca2+ influx and, specifically, whether a rise in pHi alone suffices to open Ca2+ channels in the plasma membrane. Artificial cytosolic alkalinization can induce Ca2+ uptake across the plasma membrane of endothelial cells, lymphocytes and smooth muscle cells. Furthermore, inhibition of Na+/H+ exchange reduces aganist‐induced Ca2+ influx in endothelial cells and platelets which supports the concept that pHi may regulate the opening of Ca2+ channels in the plasma membrane. Although these findings argue in favour of a role of Na+/H+ exchange in Ca2+ influx, the onset of pHi and Ca2+ rises, measured with fluorescent indicators, suggests that the increase in [Ca2+]i distinctly precedes the increase in pHi. This challenges the concept that alkalinization per se is a sufficient signal for the opening of Ca2+ channels in the plasma membrane.
Protein S binding in relation to the subunit composition of human C4b‐binding protein
Authors: Hillarp Andreas , Hessing Martin , Dahlbäck Björn
Published: July 2002

The human regulatory complement component C4b‐binding protein (C4BP) circulates in plasma either as a free protein or in a bimolecular complex with the vitamin K‐dependent protein S. The major form of C4BP is composed of 7 identical, disulfide‐linked 70 kDa subunits (α‐chains), the arrangement of which gives the C4BP molecule a spider‐like appearance. Recently, we identified a unique 45 kDa subunit (β‐chain) in C4BP. We have now isolated a subpopulation of C4BP, which does notbind protein S. This C4BP species, which had a molecular weight slightly lower than that of the predominant form, was found to lack the β‐chain. Another lower molecular weight form of C4BP was also purified. It contained the β‐chain and was efficient in binding protein S. Its subunit composition was judged to comprise six α‐chains and one β‐chain. These results indicate C4BP in plasma to be heterogeneous at a molecular level vis‐a‐vis subunit composition and/or protein S binding ability and provide support for the concept that the β‐chain of C4BP contains the single protein S binding site.
Nuclear envelope assembly around sperm chromatin in cell‐free preparations from Drosophila embryos
Authors: Ulitzur Nirit , Gruenbaum Yosef
Published: July 2002

Chicken sperm chromatin initiated an assembly of interphase‐like nuclei in a cell‐free cytoplasmic preparation from 1–6 h old Drosophila melanogaster embryos. The formation of these interphase‐like nuclei from the condensed sperm chromatin happened in a series of distinct steps. Anti‐Drosophila lamin monoclonal antibody stained the assembled nuclei in a pattern indistinguishable from normal Drosophila nuclei. This assembly process required an ATP regenerating system and could be blocked by the addition of novobiocin into the cell‐free extract.
Secretion of islet amyloid polypeptide in response to glucose
Authors: Kanatsuka A. , Makino H. , Ohsawa H. , Tokuyama Y. , Yamaguchi T. , Yoshida S. , Adachi M.
Published: July 2002

The content of isolet amyloid polypeptide (IAPP) in isolated rat pancreatic islets was determined by a radioimmunoassay. Reverse‐phase high‐performance liquid chromatography analysis revealed that a main peak of IAPP immunoreactivity in the extracts from the islets corresponded to a synthetic rat IAPP. Secretion of IAPP from the cells is regulated by the extracellular glucose concentration. Thus, IAPP may be a novel regulator for glucose homeostasis and changes in the secretion perhaps relate to insular amyloid deposits and impaired glucose tolerance in type 2 diabetes mellitus.
A single point mutation confers tetrodotoxin and saxitoxin insensitivity on the sodium channel II
Authors: Noda Masaharu , Suzuki Harukazu , Numa Shosaku , Stühmer Walter
Published: July 2002

A single point mutation of the rat sodium channel II reduces its sensitivity to tetrodotoxin and saxitoxin by more than three orders of magnitude. The mutation replaces glutamic acid 387 with a glutamine and has only slight effects on the macroscopic current properties, as measured under voltage‐clamp in Xenopus oocytes injected with the corresponding cDNA‐derived mRNA.
Functional expression of the calcium release channel from skeletal muscle ryanodine receptor cDNA
Authors: Penner Reinhold , Neher Erwin , Takeshima Hiroshi , Nishimura Seiichiro , Numa Shosaku
Published: July 2002

Combined patch‐clamp and fura‐2 measurements were performed to study the calcium release properties of Chinese hamster ovary (CHO) cells transfected with the rabbit skeletal muscle ryanodine receptor cDNA carried by an expression vector. Both caffeine (1–50 mM) and ryanodine (100 μM) induced release of calcium from intracellular stores of transformed CHO cells but not from control (non‐transfected) CHO cells. The calcium responses to caffeine and ryanodine closely resembled those commonly observed in skeletal muscle. Repetitive applications of caffeine produced characteristic all‐or‐none rises in intracellular calcium. Inositol 1,4,5‐trisphosphate (IP3) neither activated the ryanodine receptor channel nor interfered with the caffeine‐elicited calcium release. These results indicate that functional calcium release channels are formed by expression of the ryanodine receptor cDNA.
Identification of the N‐tosyl‐L‐phenylalanyl chloromethylketone modification site in Thermus thermophilus elongation factor Tu
Authors: Peter Marcus E. , Brockmöller Jürgen , Jonák Jiří , Sprinzl Mathias
Published: July 2002

EF‐Tu from Thermus thermophilus was first labelled with N‐[14C]tosyl‐L‐phenylalaninechloromethylketone and then cleaved by the combined action of CNBr and trypsin. The resulting peptides were separated by reversed‐phase HPLC. Analysis of the isolated, labelled peptide led to the identification of a sequence which was identical to residues 76–88 in T. thermophilus EF‐Tu. The TPCK reactive site is at Cys‐82. Kinetic measurements f the incorporation of TPCK into native EF‐Tu and EF‐Tu nicked at position Arg‐59 were performed. The results provide evidence that the cleavage of the peptide bond between Arg‐59 and Gly‐60 does not lead to a dramatic conformational change of EF‐Tu at the aa‐tRNA binding site.
Proteolytic activity of the plum pox potyvirus NIa‐protein on excess of natural and artificial substrates in Escherichia coli
Authors: Garcia Juan Antonio , Riechmann José Luis , Martín María Teresa , Laín Sonia
Published: July 2002

The plum pox potyvirus (PPV) NIa protease expressed from a medium copy number plasmid was able to process an excess of substrate expressed from a high copy number plasmid, in a binary Escherichia coli expression system. The ΔB7 NIa protease mutant only partially processed the NIb‐CP junction but its efficiency was independent of the amount of substrate. The ΔB7 mutant essentially did not recognize an artificial cleavage site which was quite efficiently recognized by the wild‐type protease. No competitive inhibition of the proteolytic activity by the presence of excess of different protease mutants was observed.
Expression of mRNAs for collagens and other matrix components in dedifferentiating and redifferentiating human chondrocytes in culture
Authors: Elima Kati , Vuorio Eero
Published: July 2002

Cell cultures were initiated from epiphyseal cartilages, diaphyseal periosteum, and muscle of 16‐week human fetuses. Total RNAs isolated from these cultures were analyzed for the levels of mRNAs for major fibrillar collagens, two proteoglycan core proteins and osteonectin. In standard monolayer cultures the differentiated chondrocyte phenotype was replaced by a dedifferentiated one: the mRNA levels of cartilage‐specific type II collagen decreased upon subculturing, while those of types I and III collagen, and the core proteins increased. When the cells were transferred to grow in agarose, redifferentiation (reappearance of type II collagen mRNA) occurred. Fibroblasts grown from periosteum and muscle were found to contain mRNAs for types I and III collagen and proteoglycan cores. When these cells were transferred to agarose they acquired a shape indistinguishable from chondrocytes, but no type II collagen mRNA was observed.
Biochemical properties of the YPT‐related rab1B protein
Authors: Touchot N. , Zahraoui A. , Vielh E. , Tavitian A.
Published: July 2002

GTP binding protein; Superfamily, ras; Secretion
Enhancement of the ΔpH‐dependent dissipation of excitation energy in spinach chloroplasts by light‐activation: correlation with the synthesis of zeaxanthin
Authors: Rees D. , Young A. , Noctor G. , Britton G. , Horton P.
Published: July 2002

The extent of energy‐dependent quenching of chlorophyll fluorescence in broken spinach chloroplasts has been quantitatively related to the size of the thylakoid proton gradient as measured by the quenching of 9‐aminoacridine fluorescence by titration at constant irradiance with the uncoupler nigericin or by change in irradiance. It was found that chloroplasts prepared from leaves that had been pre‐illuminated with strong light for 30 min showed energy‐dependent quenching at a lower proton gradient than chloroplasts prepared from dark‐adapted leaves. Measurement of the carotenoid composition of the thylakoids showed that light treatment raised the ratio of zeaxanthin: violaxanthin. The possible dependence of energy‐dependent quenching on xanthophyll composition and the physiological implications of this light‐activation process to the regulation of photosynthetic electron transport are discussed.
Labaditin, a novel cyclic decapeptide from the latex of Jatropha multifida L. (Euphorbiaceae)
Authors: Kosasi S. , van der Sluis W.G. , Boelens R. , Hart L.A.'t , Labadie R.P.
Published: July 2002

Latex; Decapeptide, cyclic; Labaditin; NMR, 1H‐; Peptide isolation; (Jatropha multifida, Euphorbiaceae)
The structure of porin from Rhodobacter capsulatus at 0.6 nm resolution
Authors: Weiss M.S. , Wacker T. , Nestel U. , Woitzik D. , Weckesser J. , Kreutz W. , Weite W. , Schulz G.E.
Published: July 2002

Porin; Membrane protein structure; X‐ray structure; (Rhodobacter capsulatus)
The amino acid sequence of Erythrina corallodendron lectin and its homology with other legume lectins
Authors: Adar Rivka , Richardson Michael , Lis Halina , Sharon Nathan
Published: July 2002

Primary sequence; Sequence homology; Lectin, legume; Glycosylation site
High resolution 1H NMR study of the solution structure of the S4 segment of the sodium channel protein
Authors: Mulvey Denise , King Glenn F. , Cooke Robert M. , Doak David G. , Harvey Timothy S. , Campbell Iain D.
Published: July 2002

Na‐ channel; Peptide, S4‐; NMR; Peptide structure
Granulocyte/macrophage colony‐stimulating factor primes human neutrophils for increased diacylglycerol generation in response to chemoattractant
Authors: Tyagi Shiv Raj , Winton Elliot F. , Lambeth J.David
Published: July 2002

Diacylglycerol; Granulocyte/macrophage colony‐stimulating factor; Formyl‐methionyl‐leucyl‐phenylalanine; Human neutrophil; Respiratory burst
Quinine blocks the high conductance, calcium‐activated potassium channel in rat pancreatic β‐cells
Authors: Mancilla Edna , Rojas Eduarde
Published: July 2002

The [Ca2+]i‐activated K+‐channel, one of the 3 K+‐channels described in pancreatic β‐cells, is a high conductance, voltage‐dependent K+‐channel. Quinine, known to block [Ca2+]i‐activated K+‐channels in other cells, has been described to block the silent phase between the bursts of glucose‐evoked electrical activity in mouse pancreatic β‐cells, and to inhibit K+ efflux from rat pancreatic islets. We report here that quinine blocks the [Ca2+
i‐activated K+‐channel in rat pancreatic β‐cells, from the external side of the membrane. We also show that the blockade is characterized by fast flickering of the K+‐channel between the open and closed state. Mean open and closed times within bursts were found to be exponentially distributed, suggesting that the blockade by quinine involves obstruction on the K+ flow through the open to be exponentially distributed, suggesting that the blockade by quinine involves obstruction on the K+ flow through the open channel.
Isolation and amino acid sequence of a novel 6.8‐kDa mitochondrial proteolipid from beef heart
Authors: Terzi E. , Boyot P. , Van Dorsselaer A. , Luu B. , Trifilieff E.
Published: July 2002

We have isolated a 6.8 kDa proteolipid from an acidic chloroform/methanol extract of bovine cardiac muscle. The molecular mass of the polypeptide was measured by fast atom bombardment‐mass spectrometry (FAB‐MS) (m/z 6834.1). Its amino acid sequence was partly determined by direct sequencing and completed by characterization of cyanogen bromide and tryptic fragments (sequencing, FAB‐MS and amino acid analysis). The polypeptide consists of 60 amino acid residues. Polyclonal antibodies raised in rabbit allowed its localization by electroimmunoblotting in mitochondria.
Saturation transfer EPR measurements of the rotational motion of a strongly immobilized ouabain spin label on renal Na, K‐ATPase
Authors: Mahaney James E. , Girard Jaymee P. , Grisham Charles M.
Published: July 2002

The rotational motion of an ouabain spin label with sheep kidney Na,K‐ATPase has been measured by electron paramagnetic resonance (EPR) and saturation transfer EPR (ST‐EPR) measurements. Spin‐labelled ouabain binds with high affinity to the Na,K‐ATPase with concurrent inhibition of ATPase activity. Enzyme preparations retain 0.61 ± 0.1 mol of bound ouabain spin label per ATPase αβ dimer. The conventional EPR spectrum of the ouabain spin label bound to the ATPase consists almost entirely (> 99%) of a broad resonance which is characteristic of a strongly immobilized spin label. ST‐EPR measurements of the spin labelled ATPase preparations yield effective correlation times for the bound labels of 209 ± 11 μs at 0°C and 44 ± 4 μs at 20°C. These rotational correlation times most likely represent the motion of the protein itself rather than the independent motion of mobile spin probes relative to a slower moving protein. Additional ST‐EPR measurements with glutaraldehyde‐crosslinked preparations indicated that the observed rotational correlation times predominantly represented the motion of entire Na,K‐ATPase‐containing membrane fragments, rather than the motion of individual monomeric or dimeric polypeptides within the membrane fragment. The strong immobilization of the ouabain spin label will make it an effective paramagnetic probe of the extracellular surface of the Na,K‐ATPase for a variety of NMR and EPR investigations.
Phorbol 12‐myristate 13‐acetate (PMA) increases the expression of the nerve growth factor (NGF) gene in mouse L‐929 fibroblasts
Authors: Wion Didier , Grogan Donal Mac , Houlgatte Rémi , Brachet Philippe
Published: July 2002

The rise of the NGF mRNA pool which takes place following exposure of L‐929 fibroblasts to serum was prevented in the presence of 5 μM K‐252a, a compound which inhibits several species of protein kinase activities. To characterize further this phenomenon, L‐929 cells growing in a serum‐free medium were exposed to cyclic nucleotide analogs, to a divalent cation ionophore or to the phorbol ester PMA. Only this latter compound induced an enhancement of the NGF mRNA pool, suggesting an involvement of protein kinase C in the upregulation of the NGF transcripts. The effects of PMA or serum also require a synthesis of protein since the level of NGF transcripts remained stable in the presence of cycloheximide.
The ribonuclease from an extinct bovid ruminant
Authors: Stackhouse Joseph , Presnell Scott R. , McGeehan Gerard M. , Nambiar Krishnan P. , Benner Steven A.
Published: July 2002

The sequence of the ribonuclease from the ancestor of swamp buffalo, river buffalo, and ox, corresponding approximately to Pachyportax latidens, an extinct ruminant known from the fossil record, has been reconstructed using the rule of ‘maximum parsimony’. This protein and two sequences that may have been intermediates in the evolution of modern ribonuclease have been constructed in the laboratory by site‐directed mutagenesis, and their properties examined.
Effect of gangliosides on binding, internalization and cytotoxic activity of ricin
Authors: Tonevitsky Alexander G. , Zhukova Olgad S. , Mirimanova Natalia V. , Omelyanenko Vladimir G. , Timofeeva Natalia V. , Bergelson Lev D.
Published: July 2002

The only gangliosides in Burkitts' lymphoma EB‐3 cells is GM3. Treatment of Burkitts' lymphoma EB‐3 cells with ganglosides GM1 or GM3 results in their binding to and partial incorporation into the cell membrane. About 25% of cell‐associated ganglioside GM 1 can interact with the ricin. However, such an increase in the number of binding sites does not enhance but rather decreases the cytotoxic effect of ricin. A similar protective effect was observed when the cells were pretreated with ganglioside GM3. In contrast, the increase in ricin binding sites caused by pretreatment of the cells with neuraminidase was accompanied by increase in ricin cytotoxicity. These differences may be related to observed differences in the rate of ricin‐endocytosis by native and ganglioside‐treated cells.
A novel combination of prosthetic groups in a fungal laccase; PQQ and two copper atoms
Authors: Karhunen Eija , Niku-Paavola Marja-L. , Viikari Liisa , Haltia Tuomas , van der Meer Robert A. , Duine Johannis A.
Published: July 2002

Extracellular laccase (benzenediol:oxygen oxidoreductase EC 1.10.3.2) from the lignin‐degrading fungus, Phlebia radiata, was shown to contain a novel combination of electron carriers as its prosthetic groups. In addition to two copper atoms per enzyme molecule, one molecule of PQQ was included as a cofactor. The EPR spectrum exhibits features of type 1 and type 2 copper atoms. In the enzymatic reaction 4 molecules of lignin model compound, coniferyl alcohol, are oxidized per molecule of oxygen reduced to water. During the reaction coniferyl alcohol is transformed to dilignols.
Interaction between injected Ca2+ and intracellular Ca2+ stores in Xenopus oocytes
Authors: Dascal Nathan , Boton Rony
Published: July 2002

Upon two repetitive deep injections of Ca2+ into Xenopus oocyte (200–300 μn under the membrane), the amplitude of the transient Cl− current induced by the second injection is several‐fold higher than that of the first one. This ‘potentiation’ persists even at 60–90 min intervals between injections. However, in oocytes permeabilized to Ca2+ by the ionophore A23187 in a Ca2+‐free solution, the potentiation completely disappears after 30 min. It is proposed that the injected Ca2+ is largely taken up by the stores, whereas following the second injection, a higher proportion of Ca2+ reaches the membrane, since the stores are already loaded. In ionophore‐treated oocytes, the stores lose the accumulated Ca2+ over several minutes and are then ready to take up Ca2+ again, hindering its arrival at the membrane.
The cDNA and deduced amino acid sequence of the γ subunit of the L‐type calcium channel from rabbit skeletal muscle
Authors: Bosse E. , Regulla S. , Biel M. , Ruth P. , Meyer H.E. , Flockerzi V. , Hofmann F.
Published: July 2002

Complementary DNAs for the γ subunit of the calcium channel of rabbit skeletal muscle were isolated on the basis of peptide sequences derived from the purified protein. The deduced primary structure is without homology to other known protein sequences and is consistent with the γ subunit being an integral membrane protein.
Nonthermal 60 Hz sinusoidal magnetic‐field exposure enhances 45Ca2+ uptake in rat thymocytes: dependence on mitogen activation
Authors: Walleczek Jan , Liburdy Robert P.
Published: July 2002

The effect of a 60 Hz sinusoidal magnetic field of nonthermal intensity on Ca2+ metabolism in rat thymic lymphocytes (thymocytes) was assessed in resting cells and in cells activated with the mitogen Concanavalin A (Con A). A 60 min exposure at 37°C to an induced electric field of 1.0 produced an average 2.7‐fold increase in Con A‐dependent 45Ca2+ ‐uptake compared to non‐exposed, isothermal control cells. In contrast, 45Ca2+ uptake remained unaltered during exposure of resting thymocytes. It was also found that thymocytes with a diminished ability to mobilize Ca2+ in response to Con A were most sensitive to the 60 Hz magnetic field. Although the precise mechanism of field interaction is at present unknown, modulation of Ca2+ metabolism during cell activation may represent a common pathway for field coupling to cellular systems.
Primary structure and functional expression from cDN A of the cardiac ryanodine receptor/calcium release channel
Authors: Nakai Junichi , Imagawa Toshiaki , Hakamata Yasuhiro , Shigekawa Munekazu , Takeshima Hiroshi , Numa Shosaku
Published: July 2002

The sequence of 4968 (or 4976 with an insertion) amino acids composing the ryanodine receptor from rabbit cardiac sarcoplasmic reticulum has been deduced by cloning and sequencing the cDNA. This protein is homologous in amino acid sequence and shares characteristic structural features with the skeletal muscle ryanodine receptor. Xenopus oocytes injected with mRNA derived from the cardiac ryanodine receptor cDNA exhibit Ca2+‐dependent Cl− current in response to caffeine, which indicates the formation of functional calcium release channels. RNA blot hybridization analysis with a probe specific for the cardiac ryanodine receptor mRNA shows that the stomach and brain contain a hybridizable RNA species with a size similar to that of the cardiac mRNA. This result, in conjunction with cloning and analysis of partial cDNA sequences, suggests that the brain contains a cardiac type of ryanodine receptor mRNA.
Reaction of vanadium(V) with thiols generates vanadium(IV) and thiyl radicals
Authors: Shi Xianglin , Sun Xiaoya , Dalal N.S.
Published: July 2002

The in vivo toxicity of vanadium(V) has been found to correlate with the depletion of cellular glutathione and related non‐protein thiols. With a view to understanding the mechanism for this observation, we have investigated the oxidation of glutathione, cysteine N‐acetylcysteine and penicillamine by vanadium(V), using electron spin resonance (ESR) and ESR spin trapping methodology. The spin trap used was 5,5‐dimethyl‐1‐pyrroline 1‐oxide (DMPO). It is found that the oxidation of these thiols by vanadium(V) generates the corresponding thiyl radicals and vanadium‐ (IV) complexes. The results suggest that free radical reactions play a significant role in the depletion of cellular thiols by vanadium(V) and hence in vanadium(V) toxicity.
Reaction mechamism of oxidative rearrangement of flavanone in isoflavone biosynthesis
Authors: Hashim Muhammed Faisal , Hakamatsuka Takashi , Ebizuka Yutaka , Sankawa Ushio
Published: July 2002

Microsomes that were prepared from elicitor‐treated Pueraria lobata cell cultures catalyzed the conversion of liquiritigenin, a flavanone, into daidzein, an isoflavone. The reaction was resolved into two steps. 2, 7, 4'‐Trihydroxyisoflavanone was formed as a major product when liquiritigenin was incubated with carefully washed microsomes in the presence of NADPH. The structure of 2, 7, 4'‐trihydroxyisoflavanone was confirmed by mass and 1H NMR spectroscopies. The enzyme responsible for this rearrangement reaction is a cytochrome P‐450‐dependent monooxygenase. Upon treatment with a soluble enzyme fraction 2, 7, 4'‐trihydroxyisoflavone yielded daidzein quantitatively. The incorporation of 18O from 18O2 into the 2‐hydroxy group of 2, 7, 4'‐trihydroxyisoflavanone was demonstrated by the shift of molecular ion in its mass spectrum. Based on these observations a new reaction mechanism, hydroxylation associated with 1,2‐migration, is proposed for the oxidative rearrangement reaction catalyzed by the cytochrome P‐450 enzyme of Pueraria lobata.
A 15 kDa proteolipid found in mediatophore preparations from Torpedo electric organ presents high sequence homology with the bovine chromaffin granule protonophore
Authors: Birman Serge , Meunier François-Marie , Lesbats Bernard , Le Caer Jean-Pierre , Rossier Jean , Israël Maurice
Published: July 2002

Upon SDS PAGE of isolated mediatophore, an acetylcholine‐translocating protein, a doublet at 15 kDa was identified. Amino acid sequencing after CNBr cleavage gave a 17 residue‐long peptide completely homologous with a sequence of the proton‐translocating proteolipid from bovine chromaffin granules. A 51‐mer oligodeoxynucleotide corresponding to this sequence was used to screen a library of electric lobe cDNAs constructed in λZap II. A positive recombinant clone was isolated and found to encode the complete sequence of a 15.5 kDa protein highly homologous to the bovine chromaffin or yeast vacuolar ATPase proteolipid. In vitro translation of sense RNA transcripts of the clone indeed yielded a single 15 kDa proteolipid. Northern blot analysis showed that the 1.3 kb mRNA encoding this protein is significantly expressed in nervous tissues but not in electric organ or liver of Torpedo marmorata.
Ca2+‐dependent noradrenaline release from permeabilised PC 12 cells is blocked by botulinum neurotoxin A or its light chain
Authors: McInnes Colin , Dolly J.Oliver
Published: July 2002

Permeabilisation of PC12 cells with digitonin allowed a direct study of the intracellular action of botulinum neurotoxin A, one of a group of dichain proteins produced by Clostridium botulinum that causes the fatal neuroparalytic condition, botulism. Release of [3H]noradrenaline from these permeabilised cells could be evoked by Ca2+ and this was inhibited specifically by the neurotoxin in a dose‐dependent manner (half‐maximal dose ~2 nM under the conditions used). Inclusion of the reducing agent dithiothreitol (up to 10 mM) had no effect on the level of inhibition. Moreover, electrophoretic analysis showed that this treatment of the toxin in the native state caused negligible reduction of inter‐chain disulphide bonds. Toxininduced blockade of neurotransmitter release was incomplete and could not be overcome by increased Ca2+ concentration (100 μM). The observed toxin‐insensitivity of the release from intact PC12 cells must result from inefficient toxin uptake, relative to that in peripheral cholinergic neurones. Refolded light chain alone inhibited exocytosis to the same degree and with similar potency to that of the intact neurotoxin, an effect not altered by the heavy chain. This inhibitory activity of the light chain in PC12 cells accords with observations made in permeabilised chromaffin cells [(1989) J. Biol. Chem. 264, 10354‐10360; (1989) FEBS Lett. 255, 391‐394] but contrasts with invertebrate neurones, where intracellular injection of the same preparations of both chains were necessary for inhibition of quantal release of acetylcholine [(1988) Proc. Natl. Acad. Sci. USA 85, 4090‐4094]. These collective findings may signify an interesting difference in the release process in such diverse systems or denote a dissimilarity in the transport or processing of the toxin when applied into intact neurones or cells permeabilised by detergent or streptolysin.
Ubiquitinated protein conjugates are specifically enriched in the lysosomal system of fibroblasts
Authors: Laszlo Lajos , Doherty Fergus J. , Osborn Natasha U. , John Mayer R.
Published: July 2002

Ubiquitin‐protein conjugates are found by imniunogold electron microscopy to be enriched (12‐fold) in the lysosomal compartment of 3T3‐L1 fibroblasts. Treatment of fibroblasts with the cysteine protease inhibitor E‐64 leads to an expansion of the lysosomal compartment and as a result an increase in the cellular content of ubiquitin‐protein conjugates. There is no change in the specific enrichment of ubiquitin‐protein conjugates in the lysosomal compartment following E‐64 treatment. The results suggest that some ubiquitin‐protein conjugates may normally be degraded lysosomally following sequestration by microautophagy and imply that protein ubiquitination may be one of the signals for protein uptake into lysosomes.
Phosphorylation of a bacterial activator protein, OmpR, by a protein kinase, EnvZ, stimulates the transcription of the ompF and ompC genes in Escherichia coli
Authors: Aiba Hirofumi , Mizuno Takeshi
Published: July 2002

The OmpR of Escherichia coli is a positive regulator specific for the ompF and ompC genes, which encode outer membrane proteins OmpF and OmpC, respectively. The EnvZ protein is a protein kinase which specifically phosphorylates the OmpR protein. In this study, the results of in vitro transcription experiments revealed that the phosphorylation of the OmpR protein by the EnvZ protein stimulates the transcription of both the ompF and ompC genes.
Members of the α‐amylase inhibitors family from wheat endosperm are major allergens associated with baker's asthma
Authors: Gómez Luis , Martin Elena , Hernández Dolores , Sánchez-Monge Rosa , Barber Domingo , del Pozo Victoria , de Andres Belén , Armentia Alicia , Lahoz Carlos , Salced Gabriel , Palomino Filar
Published: July 2002

We have identified the major antigens or IgE binding components from wheat flour. Thirty‐five sera from patients with baker's asthma were used to analyze the reaction with wheat salt‐soluble proteins. We found a 15 kDa SDS‐PAGE band which reacted with all sera tested. Purified members of the α‐amylase inhibitor family, which are the main components of the 15 kDa band, were recognized by specific IgE when tested with a pool of reactive sera. Immunodetection after two‐dimensional electrophoretic fractionation of crude inhibitor preparations from wheat endosperms also detected several inhibitor subunits as major low‐molecular‐weight allergens.
Sphingomyelin is synthesized in the cis Golgi
Authors: Jeckel Dieter , Karrenbauer Achim , Birk Rolf , Richard Schmidt R. , Wieland Felix
Published: July 2002

We have employed in vitro a truncated ceramide analogue with 8 carbon atoms in the sphingosine and the fatty acyl residue, each, to investigate the activity of various membrane fractions to synthesize truncated sphingomyelin. This shortened ceramide readily diffuses through membranes and therefore can easily find access to the lumina of intact organelles. Sphingomyelin synthase activity resides in the Golgi apparatus, and after sucrose density gradient centrifugation of Golgi‐enriched fractions sphingomyelin synthesis follows a cis Golgi marker enzyme.