bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of DABCO (1,4‐diazabicyclo[2,2,2]‐octane) on singlet oxygen monomol (1270 nm) and dimol (634 and 703 nm) emission
Authors: Lengfelder Edmund , Cadenas Enrique , Sies Helmut
Published: July 2002

Monomol (1270 nm) and dimol (634 and 703 nm) emissions of singlet oxygen arising from the H2O2—OCl− reaction are compared by using a germanium detector and a red‐sensitive photomultiplier, respectively. The detection limits in both cases are compared and the possibility of their application to biological systems in order to detect singlet molecular oxygen is discussed. 1,4‐Diazabicyclo[2,2,2]‐octane (DABCO) enhances both dimol and monomol emission of singlet oxygen from the above reaction; however, the tentative ratio ‘monomol/dimol emission’ is lowered in the presence of the tertiary amine.
Phosphoenolpyruvate shuttle ‐ transport of energy from mitochondria to cytosol
Authors: Drahota Zdeněk , Rauchová Hana , Miková Marie , Kaul Phool , Bass Arnošt
Published: July 2002

Brown‐adipose tissue mitochondria of hamster and rat contain phosphoenolpyruvate carboxykinase (EC 4.1.1.32). In the presence of ketoglutarate and malate, phosphoenolpyruvate is formed and exported forms mitochondria. Phosphoenolpyruvate formation is inhibited by 1,2,3‐benzenetricarboxylate. It is proposed that phosphoenolpyruvate carboxykinase together with pyruvate carboxylase and pyruvate kinase forms a phosphoenolpyruvate shuttle through which energy produced by the Krebs cycle in mitochondria may be exported to cytosol.
A helix dipole model for alamethicin and related transmembrane channels
Authors: Mathew M.K. , Balaram P.
Published: July 2002

A molecular model is proposed for the transmembrane channels formed by alamethicin and related polypeptides. The channels consist of an aggregate of rod‐like helical polypeptides with a central aqueous core of ordered water. The helix dipole moment is considered to be the major factor modulating channel size, selectivity and field‐dependent transitions.
Critical role of sulfhydryl group(s) in ATP‐dependent Ca2+ sequestration by the plasma membrane fraction from rat liver
Authors: Bellomo Giorgio , Mirabelli Francesca , Richelmi Plinio , Orrenius Sten
Published: July 2002

The ATP‐dependent sequestration of Ca2+ by the plasma membrane fraction from rat liver is stimulated by reduced glutathione and dithiothreitol and inhibited by diamide and t‐butyl hydroperoxide. The inhibitory effect on Ca2+ sequestration by the oxidizing agents is prevented in the presence of the thiols. Our results therefore suggest that free sulfhydryl group(s) may be critical for the activity of hepatic plasma membrane Ca2+ translocase, and that inhibition of this activity by the oxidation of such group(s) may contribute to the perturbation of Ca2+ homeostasis during oxidative stress.
Orientation of skeletal muscle actin in strong magnetic fields
Authors: Torbet J. , Dickens M.J.
Published: July 2002

Actin
Magnetic orientation
Birefringence
α‐Helix
Muscle contraction
Chaotic dynamics in yeast glycolysis under periodic substrate input flux
Authors: Markus Mario , Kuschmitz Dietrich , Hess Benno
Published: July 2002

The numerical analysis for a glycolytic model containing the enzymes phosphofructokinase and pyruvate kinase reveals different types of entrainment, as well as chaotic response under sinusoidal substrate input. Entrainment with response periods 1, 2, 3, 5 and 7‐times the input flux period and aperiodic behaviour is verified by measurements of NADH fluorescence in extracts of Saccharomyces cerevisiae in the theoretically predicted range. The stroboscopic transfer function obtained from the aperiodic signal admits period 3, implying chaos according to the Li‐Yorke theorem.
The bee venom melittin induces lysis of arthropod granular cells and inhibits activation of the prophenoloxidase‐activating system
Authors: Söderhäll Kenneth
Published: July 2002

The bee venom melittin induced degranulation and lysis of isolated granular cells from freshwater crayfish at a concentration of 3.5 μM. Melittin also efficiently blocked β1,3‐glucan activation of the prophenoloxidase‐activating system present in the blood cells of arthropods. Therefore, it is suggested that melittin in the bee venom may function as a toxin towards parasitic or predatory insects on bees and the bee community.
Localization of the amino acid substitution site in a fast migrating variant of human serum albumin
Authors: ladarola P. , Minchiotti L. , Galliano M.
Published: July 2002

Albumin Mi/Fg is an Italian genetic variant of human serum albumin arising from a Lys → Glu substitution which has been located in a CNBr fragment (CNBr VII) corresponding to the ‐COOH terminal portion of the molecule [(1984) J. Chromatogr. 298, 336‐344]. Tryptic peptides of CNBr VII from normal and Mi/Fg albumin have been purified by reverse‐phase high‐performance liquid chromatography (RP‐HPLC) and submitted to comparative structural studies. The amino acid sequence of the tryptic peptide of Mi/Fg variant that differs from the corresponding fragment of the normal serum albumin shows that the Lys → Glu substitution responsible for this variant is located at postion 573. This region of the albumin molecule is involved in the binding of long chain fatty acids.
Influence of cyclosporin A on the respiration of isolated rat kidney mitochondria
Authors: Jung Klaus , Pergande Monika
Published: July 2002

Cyclosporin A toxicity
Rat kidney
Mitochondrion respiration
Control of protein synthesis by amino acid supply
Authors: Arnstein Henry R.V. , Barwick Carl W. , Lange Jeremy D. , Thomas Huw D.J.
Published: July 2002

The enzyme asparaginase, which hydrolyses asparagine to aspartic acid, inhibited cell‐free protein synthesis by reticulocyte lysates. The inhibition was rapid and complete when sufficient enzyme was added but could be prevented or reversed by the addition of asparagine. The initial effect of asparaginase appears to be a block in polypeptide chain elongation due to asparagine deprivation, but there are some indications that prolonged incubation under these conditions may give rise to a secondary decrease in initiation of protein synthesis.
Deficient production of lysyi oxidase in cultures of malignantly transformed human cells
Authors: Kuivaniemi Helena , Korhonen Riitta-Maaria , Vaheri Antti , Kivirikko Kari I.
Published: July 2002

Lysyi oxidase activity in the culture medium of eight malignantly transformed human cell lines was very low compared with that in four control fibroblast lines, being 9–16% in five sarcoma cell lines and 7–11% in three other tumour cell lines. The low enzyme activity was probably due to deficient enzyme synthesis rather than impaired secretion into the cell medium, as low activity was also found in urea extracts of the cell pellets. Lysyi oxidase production thus appears to be closely regulated with deficient collagen gene expression in malignant transformation.
Two types of complementary DNAs of rat brain protein kinase C
Authors: Ono Yoshitaka , Kurokawa Tsutomu , Fujii Tomoko , Kawahara Kenji , Igarashi Koichi , Kikkawa Ushio , Ogita Kouji , Nishizuka Yasutomi
Published: July 2002

Two types of complementary DNA clones for rat brain protein kinase C were isolated. These clones encode 671 and 673 amino acid sequences, which differ from each other only in the carboxyl‐terminal regions of approx. 50 amino acid residues. This difference seems to result from alternative splicing. Elucidation of the sequences of these cDNA clones as well as some peptides from the purified rat brain enzyme suggests the existence of an additional species of protein kinase C in this tissue. It is attractive to imagine that the heterogeneity of protein kinase C may reflect diverse pathways of signal transduction into the cell.
Neuronal nicotinic acetylcholine receptor β‐subunit is coded for by the cDNA clone α4
Authors: Whiting Paul , Esch Fred , Shimasaki S. , Lindstrom Jon
Published: July 2002

Acetylcholine receptors (AChRs) with high affinity for nicotine but no affinity for α‐bungarotoxin, which have been purified from rat and chicken brains by immuno‐affinity chromatography, consist of two types of subunits, α and β [1,2]. The β‐subunits form the ACh binding sites [3]. Putative nicotinic AChR subunit cDNAs α3 and α4 have been identified by screening cDNA libraries prepared from rat PC12 cells and rat brain with cDNA probes encoding the mouse muscle AChR α‐subunit. Here we determine the amino‐terminal amino acid sequence of the rat brain AChR β‐subunit by protein microsequencing to be the same as amino acid residues 27–43 of the protein which could be coded by α4. Further, we present evidence consistent with a subunit stoichiometry of α3β2 for this neuronal nicotinic AChR.
Differences in the half‐lives of some mitochondrial rat liver enzymes may derive partially from hepatocyte heterogeneity
Authors: Vargas Jose Luis , Roche Enrique , Knecht Erwin , Grisolía Santiago
Published: July 2002

The different turnover rates of rat liver mitochondrial enzymes make autophagy unlikely to be the main mechanism for degradation of mitochondria. Although alternatives have been presented, hepatocyte heterogeneity has not been considered. Lighter hepatocytes isolated in a discontinuous Percoll gradient contain more glutamate dehydrogenase (GDH) (half‐life 1 day) and a more active autophagic system than heavier hepatocytes. The latter contain more carbamoyl phosphate synthase (CPS) and ornithine carbamoyl transferase (OTC) (half‐lives 8 days) but less lysosomal activity. As expected, isolated autophagic vacuoles contain, relative to the mitochondrial content, 3‐times less OTC and CPS than GDH, probably reflecting a faster lysosomal engulfment of mitochondria in the light hepatocytes (which contain more GDH). These data may explain some of the half‐life differences of the enzymes studied.
Immunoreactive forms of caldesmon in cultivated human vascular smooth muscle cells
Authors: Glukhova Marina A. , Kabakov Alexander E. , Ornatsky Olga I. , Vasilevskaya Tatjana D. , Koteliansky Victor E. , Smirnov Vladimir N.
Published: July 2002

150 kDa caldesmon was shown to be characteristic of vascular smooth muscle cells in normal tissue rather than in subculture. Subcultured smooth muscle cells from human aorta contained only the 70 kDa immunoreactive form of caldesmon. During the course of primary culture the amount of 150 kDa caldesmon as well as metavinculin decreased significantly whilst 70 kDa caldesmon became the predominant form, and by the onset of cell division the 150 kDa form was practically substituted by 70 kDa caldesmon. The data show that the predominance of 150 kDa caldesmon is characteristic of contractile smooth muscle cells, while in proliferating cells 70 kDa caldesmon is expressed.
Two desensitization processes of GABA receptor from rat brain
Authors: Cash Derek J. , Subbarao Katragadda
Published: July 2002

Two rapid phases of GABA receptor desensitization, which proceeded with a 10‐fold difference in rates, were detected in two types of experiment with membrane vesicle preparations from rat cerebral cortex. (i) The time course of GABA‐mediated 36Cl− influx progressed in two phases. (ii) The 36Cl− influx was decreased, by preincubation with GABA, in two phases. Measurements were made in the time range 10–1000 ms. The major loss of channel opening activity occurred in the faster phase, which was complete in 100 ms with saturating GABA concentrations. The remaining activity decreased in a slower phase in a few seconds with a 10‐fold slower rate. The faster phase of desensitization was more than 10‐fold faster than previously observed and the slower phase was slightly faster than previously reported measurements with GABA receptor. Both desensitization processes had a similar dependence on GABA concentration with a half response at ∼ 100 μM GABA.
The amino acid sequence of the α‐ and β‐chains of the two hemoglobins of the Antarctic fish Notothenia coriiceps neglecta
Authors: D'Avino Rossana , Caruso Carla , Schininà M.Eugenia , Rutigliano Bruno , Romano Mario , Camardella Laura , Bossa Francesco , Barra Donatella , di Prisco Guido
Published: July 2002

The blood of the Antarctic fish Notothenia coriiceps neglecta contains two hemoglobins, Hb 1 and Hb 2, which have a β‐chain in common. We have elucidated the primary structure of the β‐chain (146 residues) and of the α‐chains (142 residues) of the two hemoglobins. The two α‐chains differ from each other by 51 residues; in comparison with globin sequences of temperate fishes, the α‐chain of Hb 1 is more similar to that of bluefin tuna than to the α‐chain of Hb 2 of the same species.
Pulse radiolysis study on the reactivity of Trolox C phenoxyl radical with superoxide anion
Authors: Cadenas Enrique , Merényi Gabor , Lind Johan
Published: July 2002

The reaction between the phenoxyl radical of Trolox C, a water‐soluble vitamin E analogue, and superoxide anion radical was examined by using the pulse radiolysis technique. The results indicate that the Trolox C phenoxyl radical may undergo a rapid one‐electron transfer from superoxide radical [k=(4.5±0.5) × 108 M−1·s−1] to its reduced form. This finding indicates that superoxide radical might play a role in the repair of vitamin E phenoxyl radical.
Rapid relaxation of single frog skeletal muscle fibres following laser flash photolysis of the caged calcium chelator, diazo‐2
Authors: Mulligan I.P. , Ashley C.C.
Published: July 2002

Diazo‐2 is a calcium chelator based on BAPTA [(1989) J. Biol. Chem., in press], whose electron withdrawing diazoacetyl group may be rapidly (2000 s−1) converted photochemically to an electron donating carboxymethyl group by exposure to near ultraviolet light, producing an increase in its calcium affinity (K
d changes from 2.2μM to 0.073 μM) without steric modification of the metal binding site. Photolysis of a 2 mM solution of this compound with a brief flash of light from a frequency‐doubled ruby laser (347 nm) caused single skinned muscle fibres from the semitendinosus muscle of the frog Rana temporaria to relax with a mean half‐time of 60.4±5 ms (range 30–100 ms, n = 15) at 12°C, which is faster than the relaxation observed in intact muscles (half‐time 133 ms at 14°C [(1986) J. Mol. Biol. 188, 325–342]) and similar to the rate of the fast phase of tension decay in intact single fibres (20 s−1 at 1O°C [(1982) J. Physiol. 329, 1–20]).
Mastoparan inhibits phosphoinositide hydrolysis via pertussis toxin‐intensive G‐protein in human astrocytoma cells
Authors: Nakahata Norimichi , Abe Marilene Tamiko , Matsuoka Isao , Nakanishi Hironori
Published: July 2002

Mastoparan inhibited [3H]inositol phosphate accumulation induced by carbachol as well as cyclic AMP accumulation induced by isoproterenol in 1321N1 human astrocytoma cells. Mastoparan inhibited GTPγS‐induced, but not Ca2+‐induced, [3H]inositol phosphate accumulation in membrane preparations with an IC
50 of approximately 10 μM. The inhibitory effect of mastoparan on carbachol‐induced [3pH]inositol phosphate accumulation was resistant to pertussis toxin (IAP) treatment in intact cells. These results suggest that mastoparan inhibits phospholipase C in human astrocytoma cells via a GTP binding protein, which is not a substrate for IAP.
Mastoparan, a peptide toxin from wasp venom, stimulates glycogenolysis mediated by an increase of the cytosolic free Ca2+ concentration but not by an increase of cAMP in rat hepatocytes
Authors: Tohkin Masahiro , Yagami Tatsurou , Matsubara Takashi
Published: July 2002

A wasp venom, mastoparan, rapidly increased the cytosolic free Ca2+ concentration ([Ca2+]i) and activated phosphorylase in rat hepatocytes in a concentration‐dependent manner. Mastoparan could increase [Ca2+]i even in the absence of extracellular Ca2+, but a larger increase was observed in the presence of extracellular Ca2+. Thus, mastoparan mobilized Ca2+ from intracellular and extracellular Ca2+ stores. It also activated inositol triphosphate (IP3) accumulation, but did not stimulate cAMP production. From these results, we conclude that mastoparan activates rat hepatic glycogenolysis mediated by the accumulation of IP3, which causes an increase of [Ca2+]i but not that mediated by cAMP.
pH‐dependent release of catecholamines from tyrosine hydroxylase and the effect of phosphorylation of Ser‐40
Authors: Haavik J. , Martinez A. , Fiatmark T.
Published: July 2002

Bovine adrenal tyrosine hydroxylase (TH) is isolated in a partially inhibited state with the feed‐back inhibitors adrenaline and noradrenaline tightly coordinated to high‐spin (S = ) Fe(III) at the active site. In addition to the charge‐transfer interaction with iron, an additional charged group in the polypeptide chain, with an apparent pK
a of about 5.3 at 4°C, is involved in the binding of catecholamines. Protonation of this group increases the pseudo‐first order rate constant for the dissociation of the TH‐[3H]noradrenaline complex more than 100‐fold at 4°C. At pH 7.0 and 30°C, phosphorylation of Ser‐40 causes a 6‐fold increase in the rate constant for this dissociation.
SBD, a novel structural subunit of the Drosophila nicotinic acetylcholine receptor, shares its genomic localization with two α‐subunits
Authors: Sawruk Erich , Udri Conny , Betz Heinrich , Schmitt Bertram
Published: July 2002

Nicotinic acetylcholine receptors (nAChRs) display marked heterogeneity in both vertebrates and invertebrates. Here we describe the structure of a cDNA from Drosophila melanogaster which encodes a novel nAChR β‐type subunit (SBD or β2). The deduced amino acid sequence of SBD displays remarkable similarity to the Drosophila α‐subunits, ALS and SAD, while homology to the Drosophila β‐subunit ARD is less pronounced. The temporal expression of sbd transcripts during Drosophila development is similar to that of other nAChR subunit mRNAs, with high levels being found during late embryonic and late pupal stages. In embryos, sbd and als transcripts are localized in the central nervous system. The sbd gene maps cytogenetically in proximity to the als and sad genes at position 96A of chromosome 3R, suggesting the existence of a nAChR gene cluster in invertebrates.
Tumor necrosis factor bifunctionally regulates matrix metalloproteinases and tissue inhibitor of metalloproteinases (TIMP) production by human fibroblasts
Authors: Ito Akira , Sato Takashi , Iga Tatsuya , Mori Yo
Published: July 2002

The production of tissue inhibitor of metalloproteinases (TIMP) in human uterine cervical fibroblasts was increased by human recombinant tumor necrosis factor α (hrTNF) at a low concentration (0.005 ) but the elevated synthesis was suppressed in a dose‐dependent manner at higher concentrations (up to 50 ). In contrast, the production of collagenase (EC 3.4.24.7) and stromelysin was stimulated at all the corresponding concentrations. In contrast, human recombinant interleukin‐1α (hrIL‐1, 10 ) coordinately induced these enzymes and TIMP production. The reduction of the elevated TIMP production by TNF was not due to the inhibition of TIMP secretion. These results suggest that TNF modulates the extracellular matrix degradation in human fibroblasts bifunctionally by the suppression of TIMP production in addition to the acceleration of matrix metalloproteinases production. Furthermore, the fact that TNF and IL‐1 differently controlled the production of TIMP suggests that the signal pathway of TNF for TIMP production is different from that of IL‐1.
Sodium‐activated potassium current in mouse diaphragm
Authors: Re Lamberto , Moretti Vincenzo , Rossini Luigi , Giusti Piero
Published: July 2002

The mouse diaphragm muscle fiber was studied using the loose patch clamp technique. The voltage gated sodium currents were evoked by step pulses from a holding potential of about −70 mV. Following the activation of the sodium current, a very large and fast outward current was evoked. The sensitivity of this current to 4‐aminopyridine and tetraethylammonium indicates the potassium ion as the possible carrier for the channel. Furthermore, the sensitivity to tetrodotoxin and extracellular sodium demonstrated the sodium dependence of this current.
Lipid‐protein interactions in membranes
Authors: Marsh Derek
Published: July 2002

The interactions of lipids with integral and peripheral proteins can be studied in reconstituted and natural membranes using spin label electron spin resonance (ESR) spectroscopy. The ESR spectra reveal a reduction in mobility of the spin‐labelled lipid chains on binding of peripheral proteins to negatively‐charged lipid bilayers. A selectivity of interaction has been established for different lipid species, and in certain cases evidence is obtained for a partial penetration of the peripheral proteins into the membrane. The latter may be relevant to the import mechanism of apocytochrome c into mitochondria. Integral proteins induce a more direct motional restriction of the spin‐labelled lipid chains, allowing the stoichiometry and specificity of the interaction, and the lipid exchange rate at the protein interface to be determined from the ESR spectra. In this way, a population of very slowly exchanging cardiolipin associated with the mitochondrial ADP‐ATP carrier has been identified. The residues involved in the specificity for charged lipids of the myelin proteolipid protein have been localized to the deletion in the DM‐20 mutant, and the difference in lipid‐protein interactions with the β‐sheet and α‐helical conformations of the M‐13 coat protein, has been characterized.
Inhibition of HIV‐reverse transcriptase activity by some phloroglucinol derivatives
Authors: Nakane Hideo , Arisawa Munehisa , Fujita Akio , Koshimura Saburo , Ono Katsuhiko
Published: July 2002

Four phloroglucinol derivatives, named mallotophenone (5‐methylene‐bis‐2,6‐dihydroxy‐3‐methyl‐4‐methoxyacetophenone), mallotochromene (8‐acetyl‐5,7‐dihydroxy‐6‐(3‐acetyl‐2,4‐dihydroxy‐5‐methyl‐6‐methoxybenzyl)‐2,2‐dimethylchromene), mallotojaponin (3‐(3,3(dimethylallyl)S‐(3(acetyl‐2,4‐dihydroxy‐5‐methyl‐6‐methoxybenzyl)‐phloracetophenone) and mallotolerin (3‐(3‐methyl‐2‐hydroxybut‐3‐enyl)‐5‐(3‐acetyl‐2,4‐dihydroxy‐5‐methyl‐6‐methoxybenzyl)‐phloracetophenone), have been tested for their ability to inhibit the activity of human immunodeficiency virus (HIV)‐reverse transcriptase. Under the reaction conditions with (rA)n · (dT)12–18 as the template · primer, the enzyme activity was inhibited by approximately 70% in the presence of 10 μ/ml mallotochromene or mallotojaponin, whereas mallotophenone and mallotolerin were much less inhibitory to the enzyme. The enzyme activity was also inhibited, though to lesser extent, by these compounds under similar conditions with initiated MS‐2 phage RNA as the template · primer, The mode of inhibition was, as analyzed with mallotojaponin, competivite with respect to the template · primer, (rA)n · (dT)12–18, and non‐competitive with respect to the triphosphate substrate, dTTP, The K
i value of mallotojaponin for HIV‐reverse transcriptase was determined to be 6.1 μM.
The X‐ray structure of a tetrapeptide bound to the active site of human cyclophilin A
Authors: Kallen Joerg , Walkinshaw Malcolm D.
Published: July 2002

Human cyclophilin A (165 residues) has peptidyl‐prolyl cis‐trans isomerase activity. Here we report a high‐resolution three‐dimensional X‐ray structure of a substrate, ac‐Ala‐Ala‐Pro‐Ala‐amc (ac. acetyl: amc. amidomethylcoumarin) bound to the active‐site of cyclophilin. The structure consisting of a dimer of complexes and 135 water molecules was refined to a crystallographic R‐factor of 17.7% for all data in the range 8 Å‐2.3 Å.
The role of conserved aspartate and serine residues in ligand binding and in function of the 5‐HT1A receptor: A site‐directed mutation study
Authors: Ho Begonia Y. , Karschin Andreas , Branchek Theresa , Davidson Norman , Lester Henry A.
Published: July 2002

Wild‐type and mutant serotonin 1A receptors were transiently expressed in COS‐7 cells using the infection‐transfection variant of the vaccinia virus/ T7 polymerase vector system. The amino acid substitutions in the transmembrane regions, Asp82→Asn82, Asp116 →Asn116, and Ser198→Ala198 all resulted in a decrease in affinity for 5‐HT by 60–100‐fold, without affecting the affinity for the antagonist, pindolol. The binding of agonist to the additional mutant, Thr199→Ala199, was too weak to be measured, 5‐HT induced GTPase activities for all receptors studied. These findings indicate that the residues mutated play an important role in the binding of the agonist and less critical roles in the binding of the antagonist pindolol.
Identification and endothelin‐induced activation of multiple extracellular signal‐regulated kinases in aortic smooth muscle cells
Authors: Yoshimasa Takaaki , Nakao Kazuwa , Suga Shin-ichi , Kishimoto Ichiro , Kiso Yoshiaki , Imura Hiroo
Published: July 2002

In order to explore intracellular signaling pathways of the mitogenic action of endolhelin (ET), we examined the effect of ET on activities of extracellular signal‐regulated kinases (ERKS) in rat aortic smooth muscle cells (SMCs). Treatment of rat aortic SMCs with ET‐1 increased kinase activities toward myelin basic protein (MBP). Both 43‐ and 41‐kDa proteins were activated when kinase assays were done in MBP‐containing polyacrylamide gels after SDS‐PAGE. These proteins were identified as ERK1 and ERK2 with immunoprecipitation and immunoblotting using antipeptide antibodies, respectively. These results indicate that ERKs mediate signal transduction by ET.
Role of liver‐type glucose transporter (GLUT2) in transport across the basolateral membrane in rat jejunum
Authors: Miyamoto Ken-ichi , Takagi Toshimitsu , Fujii Takeru , Matsubara Tomoko , Hase Kyoko , Taketani Yutaka , Oka Tatsuzo , Minami Hisanori , Nakabou Yukihiro
Published: July 2002

To obtain information on the regulation of glucose transport across the basolateral membrane (BLM) of intestinal epithelial cells, we measured the number of [3H]cytochalasin B binding sites and the level of liver‐type glucose transporter (GLUT2) protein in the BLM in the jejunum of rats (i) with diabetes (ii) given a high‐carbohydrate diet or (iii) with experimental hyperglycemia (12 h infusion of a high‐glucose solution). A glucose uptake and the number of d‐glucose inhibitable [3H]cytochalasin B binding sites in BLM vesicles were significantly increased in all three conditions. Western blot analysis showed that the amount of GLUT2 protein in BLM vesicles was increased in rats with diabetes and those given a high‐carbohydrate diet, but not in those with experimental hyperglycemia. These results suggest that there is a mechanism for rapid regulation of glucose transport in the BLM that does not depend on change in the amount of GLUT2.
Compartmentation of inorganic phosphate in perfused rat liver
Authors: Thiaudiere Eric , Gallis Jean-Louis , Dufour Sylvie , Rousse Nicole , Canioni Paul
Published: July 2002

Compartmentation of inorganic phosphate was studied in intact perfused rat liver at 4°C by 31P NMR. It was shown that decreases in cytosolic pH or cytosolic Pi concentration induced the appearance of an additional Pi resonance at low field which was assigned to Pi from an alkaline compartment. Valinomycin (K+ ionophore) induced a further splitting of the lines whereas nigericin (K+/H+ antiport) or potassium cyanide (inhibitor of cytochrome oxidase) had opposite effects. As valinomycin acts mainly on the cytosolic/mitochondrial K+ gradient and KCN on the mitochondrial respiratory chain, it was deduced that the alkaline compartment as revealed by the second Pi resonance was probably mitochondria. Single pi lines observed on perchloric extracts of livers exhibiting two resonances during cold perfusion confirmed that the split peaks in the intact liver indeed arose from the same molecular species.
Synechocystis 6803 plastocyanin isolated from both the cyanobacterium and E. coli transformed cells are identical
Authors: Hervás Manuel , Navarro Francisco , Navarro José A. , Chávez Sebastián , Díaz Antonio , Florencio Francisco J. , De la Rosa Miguel A.
Published: July 2002

Native plastocyanin from Synechocystis 6803 has been isolated and purified to electrophoretic homogeneity. The corresponding gene (petE) has been cloned and expressed in E. coli, thus leading to a protein completely identical to plastocyanin purified from the cyanobacterial cells. The petE gene product is correctly processed in E. coli as deduced from the N‐terminal amino acid sequences. These results, along with the identical physicochemical and kinetic properties of the two protein preparations, confirm that expression of petE in E. coli is an adequate tool to address the study of Synechocystis plastocyanin by site‐directed mutagenesis.
cDNA cloning of rat proteasome subunit RC10‐II, assumed to be responsible for trypsin‐like catalytic activity
Authors: Nishimura Chihiro , Tamura Tomohiro , Akioka Hiroshi , Tokunaga Fuminori , Tanaka Keiji , Ichihara Akira
Published: July 2002

The nucleotide sequence of a cDNA that encodes anew subunit, named RC10‐II, of the 20S proteasome of rat embryonic brain has been determined. The polypeptide predicted from the open reading frame consists of 205 amino acid residues with a calculated molecular weight of 22,965 and isoelectric point of 6.15. Computer analysis showed that RC10‐II belongs to the β‐type subgroup of proteasomes, differing clearly from α‐type subunits of the proteasome gene family. The primary structure of RC10‐II was found to contain the partial amino acid sequences of several fragments of subunit 13, which has a cysteinyl residue critical for the trypsin‐like catalytic activity, as reported by L.R. Dick et al. [Biochemistry 31, 7347‐7355, 1992], suggesting that RC10‐II is a proteasomal subunit necessary for the expression of tryptic activity.
The cyanobacterium, Synechococcus sp. PCC7942, possesses two distinct genes encoding cation‐transporting P‐type ATPases
Authors: Kanamaru Kengo , Kashiwagi Sciji , Mizuno Takeshi
Published: July 2002

P‐type (or E1 E2‐type) ATPases comprise a large family of prokaryotic and eukaryotic proteins capable of transporting a variety of cations, and function in a wide variety of cellular processes. The present study was carried out to search for genes encoding P‐type ATPases in the phototrophie cyanobacterium, Synechococcus sp. PCC7942. We succeeded in cloning two genes each encoding P‐type ATPases from this bacterium. It was found that Synechococcus at least, two distinct P‐type ATPases; one belongs to the family of typical prokaryotic P‐type ATPases and the other markedly resembles eukaryotic P‐type ATPases. An insertion mutant lacking either of these two ATPase‐genes was constructed. The results showed that the growth of these mutants is hypersensitive to osmotic stress upon addition of NaCl or sorbitol to the medium.
Transcription of the halophage ΦH repressor gene is abolished by transcription from an inversely oriented lytic promoter
Authors: Stolt Pelle , Zillig Wolfram
Published: July 2002

The temperate phage ΦH of the extremely halophilic archaebacterium Halobacterium salinarium encodes a repressor, Rep, which in the immune state represses the production of an early lytic transcript, denoted T4. Rep acts at the transcriptional level by blocking the promoter for T4. The promoter for the rep gene itself is positioned back to back to the promoter for T4, in a manner analogous to that of the cI/cro genes in bacteriophage γ. Transcription of the rep gene does not occur when the phage is growing lytically. We show that this represson of rep transcription during lytic growth is due to the transcription per se from the stronger, oppositely oriented promoter for T4, without the need of a phage gene product.
Human progelatinase A can be activated by matrilysin
Authors: Crabbe Thomas , Smith Bryan , O'Connell James , Docherty Andrew
Published: July 2002

The activation of human progelatinase A by other matrix metalloproteinases was studied by following both the loss of its N‐terminal propeptide and the accompanying increase in the rate of hydrolysis of a synthetic substrate. Activated stromelysin 1 was unable to cause any activation of progelatinase A beyond that slowly occuring by autolysis, but an 8 h incubation with activated matrilysin was able to produce 64% of the activity generated by incubation with (4‐aminophenylmercuric)acetate (APMA). Wild‐type progelatinase A and a mutant proenzyme that cannot become active were both cleaved by matrilysin to a lower molecular weight species that had lost the propeptide. This shows that matrilysin activates progelatinase A by removing the propeptide in a process that does not require any autolytic cleavages.
Identification of a latent MAP kinase kinase kinase in PC12 cells as B‐raf
Authors: Traverse Sarah , Cohen Philip
Published: July 2002

A latent MAP kinase kinase kinase activity previously detected after chromatography of PC12 cell extracts on Mono Q [(1992) FEBS Lett. 314, 461–465] has been identified as B‐Raf by immunoblotting and immunoprecipitation experiments and by its specific activation with B‐Raf antibodies. B‐Raf is phosphorylated after stimulation of PC12 cells with nerve growth factor or epidermal growth factor, but this is not accompanied by activation of the Mono Q‐purified enzyme whether assayed in the absence or presence of GTP‐Ras.
Plasma membrane fatty acid‐binding protein (FABPpm) is exclusively located in the maternal facing membranes of the human placenta
Authors: Campbell Fiona M. , Dutta-Roy Asim K.
Published: July 2002

We reported earlier the presence of a 40 kDa plasma membrane fatty acid‐binding protein (FABPpm) in human placenta. This protein is thought to be involved in the sequestration of unesterified free fatty acids bound to albumin from the maternal plasma for delivery to the fetus. However, its location in human placental syncytiotrophoblasts is not known. These cells are bipolar; one side facing maternal circulation (microvillous membranes), and the other side facing fetal circulation (basal membranes). Therefore, it is important to resolve the location of this protein in trophoblast membranes in order to understand fatty acid transport and metabolism in human placenta. Isolated plasma membranes vesicles were prepared respectively from the maternal facing microvillous and fetal facing surface of the human full‐term placental syncytiotrophoblast. Using these membrane preparations, fatty acid binding activity, the polyacrylamide gel electrophoresis radiobinding assay for FABPpm, and Western blot analysis of FABPpm were carried out to determine the location of this protein in these membranes. Based on the above studies we conclude that the FABPpm is located exclusively in the microvillous membranes. Since FABPpm may be responsible for FFA uptake, its location in the microvillous membranes favours the unidirectional flow of maternal FFA to the fetus.
Protein import into the nucleus
Authors: Schlenstedt Gabriel
Published: July 2002

The transport of proteins from the cytoplasm into the nucleus is a multistep process. The nuclear localization sequence (NLS) of a transport substrate associates with the heterodimeric NLS‐receptor which binds to a subset of proteins of the nuclear pore complex (NPC). Translocation through the NPC is energy‐DApenDAnt and requires the small GTPase Ran. Proteins that interact with Ran in either the GDP‐bound or the GTP‐bound state coordinate transfer through the NPC. Lastly, the NLS‐receptor/substrate complex and Ran reach the nuclear siDA of the NPC where the complex disassembles.
Up‐regulation of cyclooxygenase‐2 mRNA in the rat spinal cord following peripheral inflammation
Authors: Beiche Flora , Scheuerer Stefan , Brune Kay , Geisslinger Gerd , Goppelt-Struebe Margarete
Published: July 2002

Prostaglandins (PG) have been described as mediators in spinal nociceptive processing after peripheral inflammation. Enzymes essential for PG biosynthesis, cyclooxygenase isozymes COX‐1 and COX‐2, have not yet been investigated in the spinal cord. In two studies on rats with adjuvant‐induced peripheral inflammation levels of mRNA expression of both COX isoforms were analyzed in the lumbar section of the spinal cord using reverse transcription‐polymerase chain reaction (RT‐PCR) technique. We could show that mRNA of both COX isoforms is expressed constitutively in the spinal cord with COX‐2 as the predominant isoform. Six hours after induction of peripheral inflammation, levels of COX‐2 mRNA expression were raised significantly in respect to untreated control rats and returned to baseline within 3 days after induction of inflammation. COX‐2 might therefore be regarded as the COX isozyme responsible for spinal PG release in nociceptive processing under a peripheral inflammatory stimulus.
Activins as candidate cholinergic differentiation factors in vivo
Authors: Fann Ming‐Ji , Patterson Paul H.
Published: July 2002

A number of cytokine families have been implicated in shaping neuronal survival, growth and gene expression. The neuropoietic and transforming growth factor‐β (TGF‐β) cytokines, in particular, have emerged as candidates for regulating the phenotype of sympathetic neurons. Culture studies have shown that neuropoietic cytokines (such as leukemia inhibitory factor, ciliary neurotrophic factor, oncostatin M, growth promoting activity) can induce the cholinergic enzyme, choline acetyltransferase (ChAT) and several neuropeptides, whereas certain members of the TGF‐β family (activin A, bone morphogenetic proteins‐2 and ‐6) induce partially overlapping but distinct sets of transmitter and neuropeptide genes in sympathetic neurons.

Since activins can induce ChAT in cultured neurons, we have investigated whether these cytokines are expressed by the appropriate cells and tissues to make them candidates for the cholinergic differentiation factor that is known to alter the phenotype of sympathetic neurons that innervate the sweat gland in the footpad in vivo. In‐situ hybridization with the anti‐sense probe for activin βB specifically labels the sweat glands but not other tissues in the footpads of developing rats. Ribonuclease protection assays indicate that βB as well as the other activin and inhibin subunit mRNAs are expressed by a number of tissues, including footpad, hairy skin and submaxillary gland. Homogenates of developing rat footpads, however, failed to induce the set of neuropeptide genes in cultured sympathetic neurons that is characteristic for activins, although neuropoietic cytokine activity was readily detectable in this assay. Thus, while activin βB mRNA is expressed in the sweat gland, this tissue does not contain detectable activin protein as assayed by its ability to regulate neuronal gene expression. Moreover, activin subunit mRNAs are expressed by targets of noradrenergic sympathetic neurons in vivo, indicating that activin expression is not limited to targets of cholinergic neurons.
DNA neosynthesis in Auerbach plexus ganglia isolated from the rat hypertrophic gut: An electrophoretic analysis
Authors: Robecchi Maria G. Giacobini , Borrione Paolo , Canavese Marco , Guena Stefano , Paraninfo Adriana , Poncino Alessandro , Silengo Lorenzo
Published: July 2002

We analysed using electrophoresis the total genomic DNA extracted from isolated Auerbach plexus ganglia of the hypertrophic duodenum upstream from a partial experimental stenosis. Results indicated the presence of two extra‐bands migrating below the high molecular weight DNA, suggesting that DNA amplification is the basic mechanism of the DNA neosynthesis previously observed in myenteric neurons.
Polyamines and ripening of photoreceptor outer segments in chicken embryos
Authors: Taibi G. , Schiavo M.R. , Nicotra C.
Published: July 2002

Polyamines and their related monoacetyl derivatives were studied in rod outer segment (ROS) and cone outer segment (COS) of photoreceptor cells from chick embryo retina during eye development (7th–18th days). Putrescine was found to be necessary, in the second phase of retinogenesis, to sustain both ROS and COS differentiation and, after acetylation, γ‐aminobutyric acid synthesis. On the other hand, spermidine and even more spermine intervene in the third phase of development when photoreceptors mature. Moreover, the presence of N1‐acetylspermidine already at the 7th day indicates that in the outer segment of photoreceptor cells too, as in the whole retina, putrescine synthesis comes about by two pathways. One pathway involves ornithine decarboxylase; the other, spermidine/spermine N1‐acetyltransferase and FAD‐dependent polyamine oxidase activities that convert spermidine to putrescine via N1‐acetylspermidine. These different biosynthetic pathways are probably also decisive in permitting γ‐aminobutyric acid synthesis, which is very important in the ripening process of neural retina.
Circuit reorganization in Ara‐C‐treated cerebellar cultures chronically exposed to picrotoxin
Authors: Seil F.J. , Drake‐Baumann R.
Published: July 2002

Organotypic cerebellar cultures derived from neonatal mice were exposed to the DNA synthesis inhibitor, cytosine arabinoside, or to cytosine arabinoside plus picrotoxin, an anti‐GABA agent that increased neuronal activity, for the first five days in vitro. The group treated with cytosine arabinoside alone was subsequently maintained in standard nutrient medium, while the group exposed to both cytosine arabinoside and picrotoxin was continuously maintained in medium with incorporated picrotoxin. Granule cells were destroyed and astrocytes were functionally compromised in both culture groups, and both groups exhibited Purkinje cell axon collateral sprouting, with projection of sprouted inhibitory terminals to unensheathed Purkinje cell somata and to Purkinje cell dendritic spines in equal numbers. Spontaneous cortical discharge rates were the same in both groups, and antidromic stimulation of Purkinje cell axons induced inhibition of cortical activity. These results differed from those of a previous study in which chronic exposure of otherwise untreated cerebellar cultures to anti‐GABA agents increased the complement of inhibitory terminals on glially ensheathed Purkinje cell somata and resulted in a reduction of spontaneous cortical discharge rates. These differences were attributed to the failure of picrotoxin (1) to alter the plastic changes consequent to exposure to cytosine arabinoside, in which Purkinje cells had excess inhibitory projections, and (2) to extend inhibitory synaptogenesis in a system in which inhibitory synapse development was already enhanced.
Permeability function related to cerebral microvessel enzymes during ageing in rats
Authors: Agrawal Anju , Shuklaj R. , Tripathi L.M. , Pandey V.C. , Srimal R.C.
Published: July 2002

Cerebral microvessels from rats were prepared and characterized by their enrichment of specific markers, namely alkaline phosphatase (AP) and τ‐glutamyl transpeptidase (τ‐GT). Further, it was observed that AP and τ‐GT registered marked increase in aged rats. On the contrary, lactate dehydrogenase (LDH) activity decreased with the increasing age. Monoamine oxidase A activity in the microvessels decreased with age whereas MAO‐B moved in the reverse direction. No noticeable change was seen in acetylcholinesterase activity with increasing age of rats.
Neuroendocrine and immunological functions in depressed patients: a follow-up study
Authors: Cervera-Enguix S , Rodríguez-Rosado A
Published: July 2002

The development, evaluation and use of biological markers with a diagnostic purpose in psychiatry is extremely important. However, with certain exceptions, truly sensitive and specific markers have not yet emerged. In order to investigate the relationship between the immune apparatus and the hypothalamic-pituitary activity on the one hand, and the psychopathological state of the patients on the other, we used a longitudinal design and assessed monocyte parameters (HLA-DR, CD 35, vimentin filaments, and phagocytosis index) and neuroendocrine tests (dexamethasone suppression test [DST] and thyrotropin-releasing hormone [TRH] stimulation test) at intake and at follow-up in 49 depressed patients. Immunological parameters were compared with neuroendocrine tests in both phases of the study. The combined use of both immunological and neuroendocrine tests did not add sensitivity to the immunological identification of depressed patients. The findings lead us to consider the role of the monocyte parameters as sensitive depressive state markers, while the combined use of both neuroendocrine and immunological tests in current clinical practice would be debatable.
Biochemical characterization and subcellular localization of the sterol C‐24(28) reductase, Erg4p, from the yeast Saccharomyces cerevisiae
Authors: Zweytick Dagmar , Hrastnik Claudia , Kohlwein Sepp D , Daum Günther
Published: July 2002

The yeast ERG4 gene encodes sterol C‐24(28) reductase which catalyzes the final step in the biosynthesis of ergosterol. Deletion of ERG4 resulted in a complete lack of ergosterol and accumulation of the precursor ergosta‐5,7,22,24(28)‐tetraen‐3β‐ol. An erg4 mutant strain exhibited pleiotropic defects such as hypersensitivity to divalent cations and a number of drugs such as cycloheximide, miconazole, 4‐nitroquinoline, fluconazole, and sodium dodecyl sulfate. Similar to erg6 mutants, erg4 mutants are sensitive to the Golgi‐destabilizing drug brefeldin A. Enzyme activity measurements with isolated subcellular fractions revealed that Erg4p is localized to the endoplasmic reticulum. This view was confirmed in vivo by fluorescence microscopy of a strain expressing a functional fusion of Erg4p to enhanced green fluorescent protein. We conclude that ergosterol biosynthesis is completed in the endoplasmic reticulum, and the final product is supplied from there to its membranous destinations.
Trichodion, a new inhibitor of inflammatory signal transduction pathways from a Trichosporiella species
Authors: Erkel Gerhard
Published: July 2002

In a search for new inhibitors of the IFN‐γ mediated signal transduction in HeLa S3 cells using secreted alkaline phosphatase (SEAP) as reporter gene, the novel pyran‐dione trichodion was isolated from fermentations of the imperfect fungus Trichosporiella sp. 20‐95. The compound inhibits the IFN‐γ mediated expression of the reporter gene with IC50 values of 21–42 μM (5–10 μg/ml). The NF‐κB and AP‐1 mediated expression of the reporter gene are inhibited with IC50 values of 42–84 μM (10–20 μg/ml) and 21 μM (5 μg/ml) respectively. Western blotting with COX‐2 and NOS II antibodies showed that the expression of both proinflammatory enzymes is almost completely inhibited at 21–42 μM (5–10 μg/ml) in LPS/IFN‐γ stimulated J774 mouse macrophages. Studies on the mode of action of the compound revealed that the inhibition of the NF‐κB dependent pathway is due to the stabilization of the IκB protein and the inhibition of the IFN‐γ dependent signaling is caused by an inhibition of the phosphorylation of the STAT1α transcription factor.
Activation of signal transduction pathways involving trkA, PLCγ‐1, PKC isoforms and ERK‐1/2 by tetanus toxin
Authors: Gil Carles , Chaı̈b-Oukadour Imane , Pelliccioni Patricia , Aguilera José
Published: July 2002

Previous reports have demonstrated that tetanus toxin (TeTx) induces activation and down‐regulation of protein kinase C (PKC). In the present work the differential activation of PKC isoforms and of signal transduction pathways, including nerve growth factor receptor trkA, phospholipase Cγ‐1 (PLCγ‐1), and extracellular regulated kinases 1 and 2 (ERK‐1/2) by TeTx in a synaptosome‐enriched P2 fraction from rat brain is reported. TeTx induces clear translocation from the soluble (cytosolic) compartment to the particulate (membranous) compartment of PKC‐β, ‐γ and ‐δ isoforms, whereas PKC‐ϵ showed a slight decrease of its soluble fraction immunoreactivity. On the contrary, the PKC‐ζ isoform shows no consistent response, whereas down‐regulation of total PKC‐α immunoreactivity is shown. Immunoprecipitation assays against phosphotyrosine show an increase of trkA and PLCγ‐1 phosphorylation. Moreover, trkA activation is corroborated using phospho‐specific antibodies against phosphorylated trkA. On the other hand, TeTx‐induced stimulation of mitogen‐activated protein (MAP) kinase activity is observed, this event also being detected by Western analysis using phospho‐specific antibodies against ERK‐1/2.