bluuebunny/crossref_metadata_2025 huggingface dataset:
An artificial crystalline DDT‐binding polypeptide
Authors: Moser Rudolf , Thomas Richard M. , Gutte Bernd
Published: July 2002

A hydrophobic 24‐residue polypeptide that could potentially form a four‐stranded antiparallel β‐pleated sheet and bind the insecticide DDT was designed and synthesized. The synthetic peptide aggregated in 1 M acetic acid but was monomeric in aqueous 50% ethanol. In the latter solvent the 24‐residue polypeptide and DDT formed a complex with an apparent dissociation constant of ≈2 × 10−5 M. The DDT binding of an analogue of this peptide possessing the same amino acid residues in a random sequence was more than 2 orders of magnitude and that of bovine serum albumin at least 3 orders of magnitude weaker. The designed polypeptide could be crystallized.
The nature of diene conjugation in human serum, bile and duodenal juice
Authors: Cawood P. , Wickens D.G. , Iversen S.A. , Braganza J.M. , Dormandy T.L.
Published: July 2002

Diene‐conjugated lipids have been located by HPLC in serum, bile and duodenal juice. Whether esterified or not the same predominant fatty acid is responsible for most of the diene conjugation in all of these biological fluids. Initial attempts to generate this fatty acid in pure lipid by classical lipid peroxidation in vitro were unsuccessful. Ultraviolet irradiation of free fatty acids in the presence of protein produced diene‐conjugated lipids similar to those found in vivo. The predominant diene‐conjugated fatty acid in vivo is an isomerised C18:2 compound.
Molecular cloning of a full‐length cDNA encoding the hemagglutinin‐neuraminidase glycoprotein of Sendai virus
Authors: Miura Naoyuki , Nakatani Yoshihiro , Ishiura Masahiro , Uchida Tsuyoshi , Okada Yoshio
Published: July 2002

We cloned a full‐length complementary DNA for the hemagglutinin‐neuraminidase (HN) mRNA of Sendai virus (HVJ) using a synthetic 27‐mer as a probe. Nucleotide sequence analysis showed that there is a long open reading frame on the mRNA that encodes a protein of 575 amino acids. The deduced amino acid sequence indicated that only one hydrophobic region sufficiently long to anchor the protein in the membrane and located near the N‐terminus (amino acids 35–60). It is suggested that HN protein is oriented with its N‐terminus inside the membrane.
Expression of functional (Na+ + K+)‐ATPase from cloned cDNAs
Authors: Noguchi Shunsuke , Mishina Masayoshi , Kawamura Masaru , Numa Shosaku
Published: July 2002

Functional (Na+ + K+)‐ATPase is formed in Xenopus oocytes injected with α‐ and β‐subunit‐specific mRNAs derived from cloned Torpedo californica cDNAs. Both the mRNAs are required for the expression of functional (Na+ + K+)‐ATPase.
Maximum activities of some key enzymes of glycolysis, glutaminolysis, Krebs cycle and fatty acid utilization in bovine pulmonary endothelial cells
Authors: Leighton Brendan , Curi Rui , Hussein Anwar , Newsholme Eric A.
Published: July 2002

Despite the importance of endothelial cells little is known about their metabolic fuel requirements. To provide some information in this area, the maximum catalytic activities of key enzymes of important metabolic pathways have been measured in bovine pulmonary endothelial cells. The results suggest that both glucose and glutamine are important fuels for these cells: in addition, the oxidation of fatty acids may also be of quantitative significance. The activity of glutaminase in these cells was about 20‐fold higher than that in lymphocyte, a cell which exhibits high rates of glutaminolysis. It is suggested that a high rate of glutamine metabolism by endothelial cells is important not only for energy provision but also for provision of nitrogen for biosynthetic purposes including production of local messengers.
Ca2 +‐mobilizing hormones elicit phosphatidylethanol accumulation via phospholipase D activation
Authors: Bocckino Stephen B. , Wilson Pamela B. , Exton John H.
Published: July 2002

Vasopressin, angiotensin II and epinephrine elicited the accumulation of phosphatidylethanol in rat hepatocytes exposed to ethanol and of phosphatidate in the absence of ethanol. When isolated liver plasma membranes were exposed to ethanol, GTPγS stimulated the production of phosphatidylethanol whereas phosphatidate was formed in the absence of ethanol. With increasing ethanol concentrations, phosphatidate formation declined whereas phosphatidylethanol production increased. These findings suggest that rat hepatocytes possess a hormone‐dependent phospholipase D activity that can also catalyse the formation of phosphatidylethanol.
Susceptibility of protein kinase C to oxidative inactivation: Loss of both phosphotransferase activity and phorbol diester binding
Authors: Gopalakrishna Rayudu , Anderson Wayne B.
Published: July 2002

Exposure of protein kinase C to low concentrations of either N‐chlorosuccinimide or H2O2 resulted in rapid and parallel loss of phosphotransferase activity and phorbol ester binding. This oxidative inactivation of protein kinase C also occurred in intact cells exposed to a low concentration of H2O2. With H2O2 treatment the rate of inactivation of protein kinase C in the cytosol of MCF‐7 cells was rather slower than that which occurred in the cytosol of PYS cells. However, in both cell types, the oxidative inactivation of membrane‐associated protein kinase C occurred rapidly in comparison to the enzyme in the cytosol. Prior treatment of cells with phorbol ester to induce membrane association (stabilization) of protein kinase C, followed by exposure to H2O2, resulted in increased inactivation of protein kinase C, suggesting that membrane association of protein kinase C increases its susceptibility to oxidative inactivation
Trehalose accumulates in Saccharomyces cerevisiae during exposure to agents that induce heat shock response
Authors: Attfield Paul V.
Published: July 2002

The storage disaccharide, trehalose, is accumulated in yeast during a temperature shift from 30 to 45°C. The response peaks at 90 min and is transient since levels of trehalose decline rapidly in cells returned to 30°C. Storage of trehalose is inhibited when cells are incubated in the presence of acridine orange or ethidium bromide prior to and during temperature shift, suggesting a requirement for de novo RNA synthesis. Accumulation of trehalose occurs when cells are exposed to either ethanol, copper sulphate or hydrogen peroxide at 30°C, indicating that the phenomenon may be a general response to physiological stress. Parallels are drawn between the trehalose accumulation response and the heat shock response in yeast.
Infection of tobacco protoplasts with in vitro transcribed tobacco mosaic virus RNA using an improved electroporation method
Authors: Watanabe Yuichiro , Meshi Tetsuo , Okada Yoshimi
Published: July 2002

We previously established an in vitro transcription system which can produce infectious tobacco mosaic virus (TMV) RNAs from cDNA templates [(1986) Proc. Natl. Acad. Sci. USA 83, 5043‐5047]. Here we report a useful system for the infection of tobacco protoplasts with a small amount of infectious RNAs, like in vitro transcripts from cDNA templates, involving an improved electroporation method. This system will facilitate the elucidation of the functions of the genomic RNA and its coding proteins using in vitro manipulated TMV‐RNAs.
Pseudo arylsulfatase A deficiency Biosynthesis of an abnormal arylsulfatase A
Authors: Ameen Mohammed , Chang Patricia L.
Published: July 2002

Pseudo arylsulfatase A deficiency, an asymptomatic condition, and metachromatic leukodystrophy, a severe neurodegenerative disease, are both associated with profound reductions of arylsulfatase A activity in man. We now report that with metabolic labelling, cultured pseudo deficient cells synthesized about 20% of the normal amount of arylsulfatase A at a reduced rate of apparent synthesis and increased rate of degradation. However, in the presence of ammonium chloride which stimulated secretion of lysosomal enzymes, these cells synthesized about 80% of the normal amount of enzyme protein. Hence, the defect in pseudo arylsulfatase A deficiency is associated with labile arylsulfatase A molecules which can be stabilized if they are diverted from intracellular storage.
Loss of transforming activity of plasmid DNA (pBR322) in E. coli caused by singlet molecular oxygen
Authors: Wefers Heribert , Schulte-Frohlinde Dietrich , Sies Helmut
Published: July 2002

Plasmid DNA pBR322 in aqueous solution was exposed to singlet molecular oxygen (1O2) generated by microwave discharge. DNA damage was detected as loss of transforming activity of pBR322 in E.coli (CMK) dependent on the time of exposure. DNA damage was effectively decreased by singlet‐oxygen quenchers such as sodium azide and methionine. Replacement of water in the incubation buffer by D2O led to an increase in DNA damage. 9,10‐Bis(2‐ethylene)anthracene disulfate was used as a chemical trap for 1O2 quantitation by HPLC analysis of the endoperoxide formed.
Estrogens as natural antioxidants of membrane phospholipid peroxidation
Authors: Sugioka Katsuaki , Shimosegawa Yasuko , Nakano Minoru
Published: July 2002

Estrogens, such as estrone, estradiol and estriol, were tested as possible antioxidants of lipid peroxidation induced by Fe3+‐ADP‐adriamycin or Fe3+‐ADP‐ascorbate. The estrogens with phenolic structure possessed substantial activities with respect to the inhibition of lipid peroxidation. Concentrations of estradiol and estriol required to achieve 50% inhibition of membrane phospholipid peroxidation were about 4‐ and 6‐times that of α‐tocopherol, respectively
Expression of the oxytocin gene in the large cells of the bovine corpus luteum
Authors: Fehr S. , Ivell R. , Koll R. , Schams D. , Fields M. , Richter D.
Published: July 2002

In the bovine ovary there is a delay of 4–6 days between the observed maximum of oxytocin mRNA and the peak in the luteal levels of oxytocin nonapeptide. This implies a maturation process involving components of the post‐translational processing pathway. In situ hybridization shows the oxytocin gene to be transcribed exclusively in the large cells of the corpus luteum at the beginning of the estrous cycle.
Stimulation of Ca2+ efflux from fura‐2‐loaded platelets activated by thrombin or phorbol myristate acetate
Authors: Pollock W.K. , Sage S.O. , Rink T.J.
Published: July 2002

We investigated the restoration of [Ca2+]i in fura‐2‐loaded human platelets following discharge of internal Ca2+ stores in the absence of external Ca2+. After stimulation by thrombin [Ca2+]i returned from a peak level of 0.6 μM to resting levels within 4 min. When ionomycin discharged the internal stores the recovery was slower with [Ca2+]i still elevated at around 0.5 μM after 5 min. Thrombin added shortly after ionomycin could accelerate the recovery of [Ca2+]i and restore resting levels within 5 min, an effect that was mimicked by phorbol‐12‐myristate‐13‐acetate (PMA). Since the continued presence of ionomycin precluded reuptake into the internal stores we conclude that thrombin and PMA stimulate Ca2+ efflux, perhaps via protein kinase C actions on a plasma membrane Ca2+ pump.
Trans splicing in vivo: joining of transcripts from the ‘divided’ gene for ribosomal protein S12 in the chloroplasts of tobacco
Authors: Zaita Norihiro , Torazawa Keita , Shinozaki Kazuo , Sugiura Masahiro
Published: July 2002

The ‘divided’ gene for chloroplast ribosomal protein S12 consists of 3 exons and the first exon (5'‐rps12) is located far from the second and third exons (3'‐rps12) in the tobacco chloroplast genome. Northern blot hybridization revealed that both 5'‐rps12 and 3'‐rps12 are transcribed in the chloroplasts. Reverse transcription analysis indicated that trans splicing between the 5'‐rps12 and 3'‐rps12 transcripts occurs in vivo.
Photoaffinity labelling of the phenylalkylamine receptor of the skeletal muscle transverse‐tubule calcium channel
Authors: Striessnig Jörg , Knaus Hans-Günther , Grabner Manfred , Moosburger Kurt , Seitz Werner , Lietz Helmut , Glossmann Hartmut
Published: July 2002

The tritiated arylazido phenylalkylamine (‐)‐5‐[(3‐azidophenethyl)[N‐methyl‐3H]methylamino]‐2‐(3,4,5‐tri‐methoxyphenyl)‐2‐isopropylvaleronitrile was synthesized and used to photoaffinity label the phenylalkylamine receptor of the membrane‐bound and purified calcium channel from guinea‐pig skeletal muscle trans‐verse‐tubule membranes. The photoaffinity ligand binds reversibly to partially purified membranes with a K
d of 2.0 ± 0.5 nM and a B
max of 17.0 ± 0.9 protein. Binding is stereospecifically regulated by all three classes of organic calcium channel drugs. A 155 kDa band was specifically photolabelled in transverse‐tubule particulate and purified calcium channel preparations after ultraviolet irradiation. Additional minor labelled polypeptides (92, 60 and 33 kDa) were only observed in membranes. The heterogeneous 155 kDa region of the purified channel was resolved into two distinct silver‐stained polypeptides after reduction (i.e. 155 and 135 kDa). Only the 155 kDa polypeptide carries the photoaffinity label and it is concluded that the 135 kDa polypeptide (which migrates as a 165 kDa band under alkylating conditions) is not a high‐affinity drug receptor carrying subunit of the skeletal muscle transverse‐tubule L‐type calcium channel.
Activation of phospholipase A2 by cannabinoids
Authors: Evans A.T. , Formukong E. , Evans F.J.
Published: July 2002

Cannabinoids Δ1‐tetrahydrocannabinol, cannabinol, cannabidiol and cannabigerol have been shown to affect directly the activity of phospholipase A2 in a cell‐free assay. The compounds produced a biphasic activation of the enzyme, with EC50 values in the range 6.0–20.0 × 10−6 M and IC50 values in the range 50.0–150.0 × 10−6M. These results correlated well with the relative potencies reported for the stimulation of prostaglandin release from human synovial cells in vitro, confirming that activation of phospholipase A2 is the predominant action of cannabinoids on arachidonate metabolism in tissue culture. However, since Δ1‐tetrahydrocannabinol is unique among these compounds in possessing cataleptic activity, it is unlikely that phospholipase A2 is the major receptor mediating the psychotropic effects of cannabis.
The measurement of free radical reactions in humans
Authors: Halliwell Barry , Grootveld Martin
Published: July 2002

The question as to whether free radical reactions are a major cause of tissue injury in human disease, or merely an accompaniment to such injury, is very difficult to answer because of lack of adequate experimental techniques. New techniques that are becoming available are discussed, with specific reference to their use in humans.
Identification of a new 84/82 kDa calmodulin‐binding protein, which also interacts with actin filaments, tubulin and spectrin, as synapsin I
Authors: Okabe Toshio , Sobue Kenji
Published: July 2002

A new 84/82 kDa calmodulin‐binding protein, which also interacts with actin filaments, tubulin and spectrin, was purified from the bovine synaptosomal membrane. The binding of calmodulin to this protein was Ca2‐dependent, and was inhibited by trifluoperazine, the association constant being calculated to be 2.2 × 106 M−1. Maximally, 1 mol of calmodulin bound to 1 mol of the purified protein. This protein was phosphorylated by both kinase II (Ca2+‐ and calmodulin‐dependent kinase) and cyclic AMP‐dependent kinase. In addition, antibody against this protein was demonstrated to have an immunological crossreactivity with synapsin I in the synaptosomal membrane.
Molecular cloning and characterization of a full‐length cDNA clone for human plasminogen
Authors: Forsgren Margaretha , Råden Benny , Israelsson Marianne , Larsson Kerstin , Hedén Lars-Olof
Published: July 2002

A human liver cDNA library enriched for full‐length clones was screened for plasminogen cDNA using a synthetic 24‐nucleotide probe derived from a reported partial cDNA sequence. 12 positive clones were identified and one of these was characterized in detail. The 2.7 kb insert contains the complete coding region. At 5 positions, it gives residues different from those reported in a previous amino acid sequence analysis of the protein. The present results show an extra lle at position 65, Gln instead of Glu at positions 53 and 342, Asn at position 88 instead of Asp, and Asp at position 453 rather than Asn. In the 3'‐non‐coding region an extension of 29 bases is found which does not contain any structure compatible with a known polyadenylation signal. Instead, the consensus signal AATAAA is placed at a distance of 46 bases upstream of the poly(A)‐tail.
Carbon flux through citric acid cycle pathways in perfused heart by 13C NMR spectroscopy
Authors: Malloy Craig R. , Sherry A.Dean , Jeffrey F.Mark H.
Published: July 2002

Mathematical models of the TCA cycle derived previously for 14C tracer studies have been extended to 13C NMR to measure the 13C fractional enrichment of [2‐13C]acetyl‐CoA entering the cycle and the relative activities of the oxidative versus anaplerotic pathways. The analysis is based upon the steady‐state enrichment of 13C into the glutamate carbons. Hearts perfused with [2‐13C]acetate show low but significant activity of the anaplerotic pathways. Activation of two different anaplerotic pathways is demonstrated by addition of unlabeled propionate or pyruvate to hearts perfused with [2‐13C]acetate. In each case, the amount of [2‐13C]acetate being oxidized and the relative carbon flux through anaplerotic versus oxidative pathways are evaluated.
Steady‐state rate of F1‐ATPase turnover during ATP hydrolysis by the single catalytic site
Authors: Milgrom Ya.M. , Murataliev M.B.
Published: July 2002

Under the conditions of ATP regeneration and molar excess of nucleotide‐depleted F1‐ATPase the enzyme catalyses steady‐state ATP hydrolysis by the single catalytic site. Values ofKm
= 10−8 M and Vm
= 0.05 s−1 for the single‐site catalysis have been determined. ADP release limits single‐site ATP hydrolysis under steady‐state conditions. The equilibrium constant for ATP hydrolysis at the F1‐ATPase catalytic site is ⩽ 0.7.
Adenylate ratios in the cytosol, chloroplasts and mitochondria of barley leaf protoplasts during photosynthesis at different carbon dioxide concentrations
Authors: Gardeström Per
Published: July 2002

Barley (Hordeum vulgare) protoplasts were incubated in darkness and in the light at saturating and limiting CO2 concentrations. The protoplasts were fractioned by a membrane filtration technique which allows quenching of the metabolism by acidification within about 0.1 s and the ATP/ADP ratios in the cytasol, chloroplasts and mitochondria were determined. It is concluded that the cytosolic ATP/ADP ratio is considerably higher during photosynthesis at limiting CO2 (which is the normal situation for a C3 plant in air) compared to photosynthesis at saturating CO2 or darkness.
Calmodulin antagonists increase free cytosolic calcium levels in plant protoplasts in vivo
Authors: Gilroy S. , Hughes W.A. , Trewavas A.J.
Published: July 2002

Inhibition by calmodulin antagonists has often been used to identify the involvement of calmodulin in physiological processes in plants. Therefore the role of calmodulin in the maintenance of cytosolic calcium levels was investigated using these drugs on carrot cell protoplasts loaded with the [Ca2+] indicator, quin 2. Resting [Ca2+] was 361 ±47 nM (n = 30). On incubation with the anticalmodulins, W7, TFP and tetracaine, calcium levels increased, over 15 min, to > 10−6 M (500 μM extracellular Ca2+) or 500 nM (1 mM extracellular EGTA). This rise in calcium level provides an alternative explanation for some of the effects of calmodulin antagonists on the direct involvement of calmodulin in plant processes.
Asparaginyl‐rhamnose: A novel type of protein‐carbohydrate linkage in a eubacterial surface‐layer glycoprotein
Authors: Messner Paul , Sleytr Uwe B.
Published: July 2002

The subunits of the crystalline surface layer of Bacillus stearothermophilus, strain NRS 2004/3a contain carbohydrates covalently linked to protein. Hydrolysis of a glycopeptide obtained by pronase digestion of the glycoprotein and analysis of the fragments revealed that rhamnose is N‐glycosidically linked to the amide nitrogen of an asparaginyl residue.
Identification of a tetrasialylated monofucosylated tetraantennary N‐linked carbohydrate chain in human platelet glycocalicin
Authors: Korrel Sophia A.M. , Clemetson Kenneth J. , van Halbeek Herman , Kamerling Johannis P. , Sixma Jan J. , Vliegenthart Johannes F.G.
Published: July 2002

Glycocalicin (140 kDa), the main constituent of the glycoprotein Ib α‐chain (150 kDa) of the human platelet membrane, contains 4 putative N‐glycosylation sites. For the structural analysis of the N‐glycosidic carbohydrate chains of glycocalicin, the glycoprotein has been subjected to the hydrazinolysis procedure. The acidic carbohydrate chains obtained were fractionated by ion‐exchange chromatography on DEAE‐Sephadex A‐25, subsequently analysed by sugar analysis, anion‐exchange chromatography on Mono Q HR 5/5 500 MHz 1H‐NMR spectroscopy. A novel tetrasialylated monofucosylated tetraantennary chain was identified in the glycoprotein. It could also be deduced that in all structures the α2→6‐linked NeuAc is attached exclusively at the Galβ1→4GlcNAcβ1→2Manα1→3 antenna, whereas the other antennae can be terminated with α2→3‐linked NeuAc. As minor constituents sialylated N‐linked carbohydrate chains with a terminal Fucα1→2Galβ1→. sequence were detected.
Sobemovirus genome appears to encode a serine protease related to cysteine proteases of picornaviruses
Authors: Gorbalenya Alexander E. , Koonin Eugene V. , Blinov Vladimir M. , Donchenko Alexei P.
Published: July 2002

A putative serine protease was identified among non‐structural proteins of southern bean mosaic virus (SBMV) by sequence comparison with cellular and viral proteases. The predicted SBMV proteased is played a significant similarity to cysteine proteases of picornaviruses, providing a possible evolutionary link between the two enzyme classes. It is suggested that SBMV follows the general expression strategy characteristic of other positive‐strand RNA viruses containing 5′‐terminal covalently linked proteins (VPg), i.e. generation of functional proteins by polyprotein processing.
Superoxide production from nonenzymatically glycated protein
Authors: Sakurai Tamiko , Tsuchiya Seishi
Published: July 2002

Nonenzymatically glycated human serum albumin and glycated poly‐lysine(Lys) in vitro brought about the reduction of nitroblue tetrazolium and ferricytochrome c at pH 9.06 and pH 7.8, respectively. This reduction was inhibited partially by superoxide dismutase (SOD). Glycated poly‐Lys caused the oxidation of NADH in the presence of LDH at pH 7.0 which was completely inhibited by SOD. Glycated material was found to function both as a reductant and an oxidant. The reactivity of glycated material is discussed and a possible mechanism by which superoxide is produced is proposed. Results may give a clue to diabetic complications.
The sexual inducer of Volvox carteri Primary structure deduced from cDNA sequence
Authors: Mages H.-W. , Tschochner H. , Sumper M.
Published: July 2002

The primary structure of the sexual inducer of Volvox carteri f. nagariensis has been deduced by cloning and sequence analysis of cDNA. The sexual inducer contains 208 amino acids including a signal sequence. A total of six potential N‐glycosylation sites are found within the polypeptide chain. At the genomic level, the sexual inducer protein is encoded in five exons.
Expression of the enkephalin precursor gene in rat Sertoli cells
Authors: Yoshikawa Kazuaki , Aizawa Takado
Published: July 2002

Searching for somatic cells expressing the preproenkephalin (A) gene in the testis, we have isolated Sertoli cells from the testes of 20‐day‐old rats. Cultured Sertoli cells contained a single species (about 1.5 kb) of preroenkephalin mRNA, and follicle‐stimulating hormone (FSH) transiently increased the mRNA abundance to a maximum (about 30 molecules per cell) at 12 h. Various compounds that activate the cyclic AMP system in Sertoli cells similarly increased the abundance of preproenkephalin mRNA. Moreover, FHS increased intracellular Met‐enkephalin immunoreactive peptides in Sertoli cells. Thusthe preproenkephalin gene expression in Sertoli is positvely regulated by FSH through the cyclic AMP system.
Chloride mediated inhibition of the phosphate and the sulfate transport by dipyridamole in human erythrocyte ghosts
Authors: Renner M. , Dietl M. , Schnell K.F.
Published: July 2002

The inhibition of the unidirectional phosphate and sulfate flux in human erythrocyte ghosts by dipyridamole has been investigated. The inhibition of the phosphate and the sulfate flux is mediated by chloride. At zero chloride, dipyridamole was found to be completely ineffective. At 10 mM chloride, dipyridamole acts as a noncompetitive inhibitor of the phosphate and the sulfate flux and elicits an up to 95% inhibition of the phosphate and the sulfate transport. The results of our studies provide strong evidence for a cooperative binding of chloride and of dipyridamole to the non‐protonated form of the band 3 membrane domain.
Growth inhibitory activity of atrial natriuretic factor in rat glomerular mesangial cells
Authors: Appel Richard G.
Published: July 2002

Atrial natriuretic factor (ANF) binding sites, or ANF‐induced cGMP accumulation, have been shown in numerous tissues that may not be intimately involved in volume homeostasis. In a series of experiments, we found that quiescent rat glomerular mesangial cells in culture could be reactivated to enter the cell cycle, and that ANF had potent inhibitory effects on proliferation of these cells. [3H]Thymidine incorporation increased by fivefold in reactivated compared to quiescent cells, and ANF in physiologic concentrations inhibited this incorporation by 48%. Thus, ANF appears to be a multifunctional peptide, consistent with a number of peptide growth factors.
Correlation between the distribution of the reversing factor and eukaryotic initiation factor 2 in heme‐deficient or double‐stranded RNA‐inhibited reticulocyte lysates
Authors: Matts Robert L. , Thomas N.Shaun B. , Hurst Robin , London Irving M.
Published: July 2002

The recycling of eukaryotic initiation factor eIF‐2 requires the exchange of GDP for GTP, in a reaction catalyzed by the reversing factor (RF). Recent studies have suggested that a 60 S ribosomal subunit‐bound eIF‐2 · GDP complex is an intermediate in protein chain initiation. We have monitored the distribution of RF in heme‐deficient and dsRNA‐inhibited lysates by immunoblot analysis of sucrose gradient fractions and have compared the distribution with that of eIF‐2(α‐32P). RF and eIF‐2(αP) were both found to be tightly associated with 60 S and 80 S ribosomes, as their distribution did not change in gradients containing up to 0.1 M K+. The association of eIF‐2(α‐32P) and RF with 60 S and 80 S ribosomes was enhanced in the presence of F−, indicating the presence of an endogenous ribosome‐associated phosphatase activity which is capable of dephosphorylating eIF‐2(αP) in the absence of F−. These observations are consistent with the hypothesis that under physiologic conditions, RF interacts with the 60 S‐bound eIF‐2 · GDP complex to promote the dissociation of GDP from eIF‐2 and the release of eIF‐2 from the 60 S subunit as a complex with RF.
Mechanical stimulation induces Ca2+i transients and membrane depolarization in cultured endothelial cells Effects on Ca2+i in co‐perfused smooth muscle cells
Authors: Goligorsky Michael S.
Published: July 2002

Cytosolic Ca2+ concentration and membrane potential were monitored in individual cultured enothelial cells mechanically stimulated with a micropipette attached to the stage of a microscope. Both dimpling and poking of endothelial cells resulted in Ca2+
i transients (from 63 ± 12 to 397 ± 52 nM, characterized by a refractory period of approx. 2 min) and cell depolarization. Ca2+
i transients of the reduced amplitude (201 ± 41 nM) were evoked by mechanical stimulation of endothelial cells incubated in a Ca2+‐free medium. Dimpling‐induced Ca2+
i transients were refractory to the pretreatments with pertussis toxin, colchicine, or cytochalasin B, and were not mimicked by an increase in the hydrodynamic pressure. In a co‐perfusion system (endothelium: smooth muscle), both the KCl‐induced depolarization and ionomycin‐induced increase in Ca2+
i in the endothelial cells resulted in the reduction of Ca2+
i in the smooth muscle cells. The data reported are consistent with the phenomenon of vascular relaxation in response to the increased blood flow. We hypothesize that the mechanical interaction of the formed elements with the microvascular endothelium can serve as a pacemaker for the sustained relaxation of vascular smooth muscle.
Human heart cytochrome c oxidase subunit VIII Purification and determination of the complete amino acid sequence
Authors: Van Kuilenburg André B.P. , Muijsers Anton O. , Demol Hans , Dekker Henk L. , Van Beeumen Jozef J.
Published: July 2002

Subunit VIII was purified from a preparation of the human heart cytochrome c oxidase and its complete amino acid sequence was determined. The sequence proved to be much more related to that of the bovine liver oxidase subunit VIII than to that found in bovine heart. Our finding of a ‘liver‐type’ subunit VIII in the human heart enzyme suggests that either there are no isoforms of human subunit VIII or the human oxidase does not show the type of tissue specificity that has been reported for the oxidase in other mammals.
Transforming growth factor β regulates the levels of different fibronectin isoforms in normal human cultured fibroblasts
Authors: Balza Enrica , Borsi Laura , Allemanni Giorgio , Zardi Luciano
Published: July 2002

Fibronectin (FN) polymorphism is caused by alternative splicing patterns in at least three regions of the primary transcript of a single gene. Using a monoclonal antibody (Mab) specific for an FN segment (ED‐A), that can be included or omitted from the molecule depending on the pattern of splicing, we have examined whether transforming growth factor β (TGF‐β) and dexamethasone, which are both known to increase the level of total FN, regulate the levels of different FN isoforms. We found that, while dexamethasone does not significantly change the ratio between the total FN and the ED‐A containing FN, TGF‐β preferentially increases the expression of the FN isoform containing the ED‐A sequence.
A novel dinitrophenylglutathione‐stimulated ATPase is present in human erythrocyte membranes
Authors: LaBelle Edward F. , Singh Shivendra V. , Ahmad Hassan , Wronski Leszek , Srivastava Satish K. , Awasthi Yogesh C.
Published: July 2002

Vesicles prepared from human erythrocyte membranes were found to catalyze ATP hydrolysis that was stimulated by dinitrophenylglutathione (Dnp‐SG). This activity was dependent on temperature and Me2+ and independent of ion pump ATPases present in erythrocyte membranes. The activity was a linear function of protein and time up to 60 min. The K
m values of ATPase for Dnp‐SG and ATP were found to be 49 μM and 1.67 mM, respectively. This suggests that in erythrocytes, the transport of Dnp‐SG requires direct enzymatic hydrolysis of ATP and both Dnp‐SG‐stimulated ATPase activity and the ATP‐dependent efflux of Dnp‐SG from erythrocytes represent different activities of the same protein.
Presence and characteristics of epidermal growth factor receptors in human fetal small intestine and colon
Authors: Pothier Pierre , Ménard Daniel
Published: July 2002

In the present study, we demonstrate for the first time the presence of important concentrations of EGF binding sites in isolated epithelial cells of both human fetal small intestine and colon as early as 12 weeks gestation. The pattern of EGF binding in the small intestine between 12 and 17 weeks show that binding was significantly higher (2.5‐fold) in younger fetuses than in older fetuses. Moreover, the fetal colon exhibited a much higher binding capacity (1.5–2.5 times) than corresponding intestinal cells for all age groups studied. Analysis of Scatchard representations reveal that the concentration of high‐ and low‐affinity binding sites in colonic epithelial cells are twice the values observed in corresponding intestinal cells. The present data raise interesting possibilities as to the role of this growth factor in human fetal gut development.
Oxygen radical induced alterations in polyclonal IgG
Authors: Griffiths H.R. , Lunec J. , Gee C.A. , Willson R.L.
Published: July 2002

In the sera and synovial fluid of patients with rheumatoid arthritis, part of the IgG fraction is found in an aggregated and fluorescent form. Oxygen‐free radicals have been implicated in this denaturation, although the precise radical species responsible is unknown. In this work, oxygen‐free radicals generated radiolytically were allowed to attack polyclonal IgG in solution. OH. radicals induced aggregation of the monomer and a new fluorescence appeared in the visible region (Ex 360 nm, Em 454 nm). The superoxide radical anion was found to be inert in both these respects, whilst peroxy radicals induced autofluorescence without concomitant aggregation. The results suggest that OH. and/or peroxy radical attack may be an in vivo mechanism for IgG denaturation.
Bleomycin resistance conferred by a drug‐binding protein
Authors: Gatignol Anne , Durand Henri , Tiraby Gérard
Published: July 2002

The protein coded by a bleomycin‐resistance gene (ble) cloned from producing actinomycetes was purified from a culture of a recombinant E.coli strain and its action on bleomycin was determined by in vitro assays. The protein binds reversibly in a one to one ratio to bleomycin which can no longer cleave DNA. The bleomycin resistance of cells harboring a ble gene could be accounted for by a sequestering effect of the bleomycin‐binding protein.
The extraction of high‐molecular‐mass DNA from hair shafts
Authors: Schreiber Arnd , Eberhard-Amtmann  , Storch Volker , Sauer Gerhard
Published: July 2002

A simple and efficient method is presented for the extraction of DNA from hair shafts. DNA preparations obtained by this approach can be made amenable to restriction enzyme digestion, thereby allowing further molecular biological analysis.
Photosynthetic reaction centre of Chloroflexus aurantiacus Primary structure of M‐subunit
Authors: Ovchinnikov Yu.A. , Abdulaev N.G. , Shmuckler B.E. , Zargarov A.A. , Kutuzov M.A. , Telezhinskaya I.N. , Levina N.B. , Zolotarev A.S.
Published: July 2002

The M‐subunit primary structure of the reaction centre (RC) from Chloroflexus aurantiacus composed of 306 amino acid residues has been determined by parallel analysis of the protein and corresponding DNA. The blocked N‐terminus as well as replacement of the essential histidine liganding Mg of an accessory bacteriochlorophyll in purple bacteria by leucine distinguishes the M‐subunit of Chloroflexus RC from that of purple bacteria.
The site of action of the A‐chain of mistletoe lectin I on eukaryotic ribosomes The RNA N‐glycosidase activity of the protein
Authors: Endo Yaeta , Tsurugi Kunio , Franz Hartmut
Published: July 2002

The site of action of the A‐chain of mistletoe lectin (ML‐A) from Viscum album on eukaryotic ribosomes was studied. Treatment of rat liver ribosomes with ML‐A, followed by treatment of the isolated rRNA with aniline, caused the release of a fragment with about 450 nucleotides from 28 S rRNA. Further analysis of nucleotide sequences of this fragment revealed that the aniline‐sensitive site of phosphodiester bond was between positions A‐4324 and G‐4325 in 28 S rRNA. These results indicate that ML‐A inactivates the ribosomes by cleaving a N‐glycosidic bond at A‐4324 of 28 S rRNA in the ribosomes as ricin A‐chain does.
Cystatin domains in alpha‐2‐HS‐glycoprotein and fetuin
Authors: Elzanowski Andrzej , Barker Winona C. , Hunt Lois T. , Seibel-Ross Elizabeth
Published: July 2002

We have found that chain A of alpha‐2‐HS‐glycoprotein contains two cystatin domains that show closest similarity to those of kininogen. Most likely, the two proteins diverged after the primary duplication of a single cystatin domain as the two cystatin domains of alpha‐2‐HS‐glycoprotein are more similar, especially in disulfide bonding, to the corresponding domains of kininogen than to each other. We also propose that the carboxyl‐terminal (non‐cystatin) parts of kininogen and alpha‐2‐HS‐glycoprotein contain homologous segments. We suggest that alpha‐2‐HS‐glycoprotein may act as an inhibitor of the cysteine proteinases responsible for bone resorption. We have also found that fetuin is closely related to alpha‐2‐HS‐glycoprotein.
Islet amyloid polypeptide: Identification and chromosomal localization of the human gene
Authors: Mosselman S. , Höppener J.W.M. , Zandberg J. , van Mansfeld A.D.M. , van Kessel A.H.M.Geurts , Lips C.J.M. , Jansz H.S.
Published: July 2002

Islet or insulinoma amyloid polypeptide (IAPP) is a 37 amino acid polypeptide isolated from pancreatic amyloid. Here, we describe the isolation and partial characterization of the human gene encoding IAPP. The DNA sequence predicts that IAPP is excised from a larger precursor protein and that its carboxy‐terminus is probably amidated. The predicted normally occurring IAPP is identical to the reported polypeptides isolated from pancreatic amyloid, except for the amidated carboxy‐terminus. IAPP specific polyadenylated RNAs of 1.6 kb and 2.1 kb are present in human insulinoma RNA. The human IAPP gene is located on chromosome 12.
Assignment of the human aminopeptidase N (peptidase E) gene to chromosome 15q13‐qter
Authors: Kruse Torben A. , Bolund Lars , Grzeschik Karl-Heinz , Ropers Hans Hilger , Olsen Jørgen , Sjöström Hans , Norén Ove
Published: July 2002

The gene for aminopeptidase N (EC 3.4.11.2) has been located on the human chromosome 15q13‐qter using HindIII‐cleaved DNA from a panel of hybrids between rodent and human cells. The Southern blots were probed by the 5′‐EcoRI fragment of the recently cloned human aminopeptidase N cDNA.
Activation enthalpies and pH dependence of phenol hydroxylase from Trichosporon cutaneum, in vitro and in situ
Authors: Mörtberg Monika , Neujahr Halina Y.
Published: July 2002

The effect of pH and temperature on phenol hydroxylase in vitro was compared to the corresponding effect on the enzyme in situ, in permeabilized cells. Activation enthalpies in situ were about 75–80% of those in vitro, in both cases decreasing with increasing pH (6.0–8.5). The order of addition of phenol and NADPH affected the K
m values for phenol at 25°C, but not at 10°C. The results support the idea that the enzyme in situ is in a more favourable position for catalysis than the purified enzyme and that slow conformational changes, triggered by binding of phenol, become rate limiting above 10°C.
Halothane, an inhalation anesthetic, activates protein kinase C and superoxide generation by neutrophils
Authors: Tsuchiya Masahiko , Okimasu Eiji , Ueda Wasa , Hirakawa Masahisa , Utsumi Kozo
Published: July 2002

The rate of superoxide generation of guinea pig intraperitoneal neutrophils by a chemotactic peptide or 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) was increased by 2‐bromo‐2‐chloro‐1,1,1,‐trifluoroethane (halothane), an inhalation anesthetic. This increase was inhibited by 1‐(5‐isoquinolinesulfonyl)methylpiperazine dihydrochloride (H‐7), a specific inhibitor of Ca2+‐ and phospholipid‐dependent protein kinase C (PKC). Halothane was found to significantly activate partially purified PKC. The activation required phosphatidylserine (PS) and Ca2+. Dioleoylglycerol‐ or TPA‐activated PKC activity was further increased by halothane. The cytoplasmic proteins of guinea pig neutrophils phosphorylated by halothane‐activated PKC were similar to those phosphorylated by PMA‐activated PKC. The phosphorylation of a 48 kDa protein, a phosphorylated protein required for NADPH oxidase activation, was also increased by halothane. These data suggest that the increase of superoxide production by halothane is correlated with its activation of PKC.
Plasminogen activator inhibitor 1 (PAI) is bound to vitronectin in plasma
Authors: Wiman Björn , Almquist Åsa , Sigurdardottir Olöf , Lindahl Tomas
Published: July 2002

Functionally active plasminogen activator inhibitor 1 (PAI) is bound to a discrete binding protein in plasma [(1988) Thromb. Haemost. 59, 392‐395]. The binding protein has now been partially purified using conventional chromatographic techniques. After addition of active PAI its complex with the binding protein was purified by chromatography on insolubilized monoclonal antibodies towards PAI. Dodecylsulphate (polyacrylamide gel electrophoresis revealed two main compounds with molecular masses of 50 and 75 kDa respectively. NH2‐terminal amino acid sequence analysis and immunoblotting analysis suggested that the two compounds were PAI (50 kDa) and vitronectin (75 kDa). We conclude that the PAI‐binding protein is identical to vitronectin.
A pulsed EPR study of redox‐dependent hyperfine interactions for the nickel centre of Desulfovibrio gigas hydrogenase
Authors: Chapman Alan , Cammack Richard , Hatchikian Claude E. , McCracken John , Peisach Jack
Published: July 2002

The nickel centre of hydrogenase from Desulfovibrio gigas was studied by electron spin echo envelope modulation (ESEEM) spectroscopy in the oxidized, unready (Ni‐A) and H2‐reduced active (Ni‐C) states, both in H2O and 2H2O solution. Fourier transforms of the 3‐pulse ESEEM, taken at 8.7 GHz, for Ni‐A and Ni‐C in H2O contained similar peaks with narrow linewidths at frequencies of 0.4, 1.2 and 1.6 MHz, and a broader peak centred at 4.5 MHz. At 11.6 GHz, the low frequency components showed small field‐dependent shifts, while the high frequency component was shifted to 5.1 MHz. These results are consistent with the presence of 14N, possibly from imidazole, coupled to the nickel centre. In 2H2O, Ni‐A was shown to be inaccessible for exchange with solvent deuterons. In contrast, Ni‐C was accessible to solvent exchange, with a deuterium population being in close proximity to the metal ion. Thus, the nickel environment of the active protein is different from that in the oxidized or unready state. On illumination of Ni‐C, although EPR changes are seen, 14N coupling remains, and for the 2H2O sample, deuterium coupling is also retained.
Bleomycin‐detectable iron in serum from leukaemic patients before and after chemotherapy Therapeutic implications for treatment with oxidant‐generating drugs
Authors: Halliwell B. , Aruoma O.I. , Mufti G. , Bomford A.
Published: July 2002

Patients with acute myeloid leukaemia show elevated plasma iron and, in 2/6 cases studied, low‐molecular‐mass iron complexes capable of stimulating radical reactions were present in the plasma. Shortly after the onset of chemotherapy, there is a sharp rise in transferrin saturation and all patients studied showed low‐molecular‐mass iron in their plasma. It is proposed that such iron could interact with oxidants generated by certain drugs (e.g. adriamycin or daunorubicin) to facilitate tissue damage, and that some of the side‐effects of chemotherapy might be ameliorated by careful co‐administration of small doses of desferrioxamine.
Inside‐out plant plasma membrane vesicles of high purity obtained by aqueous two‐phase partitioning
Authors: Larsson Christer , Widell Susanne , Sommarin Marianne
Published: July 2002

Highly purified plasma membranes obtained from leaves of sugar beet (Beta vulgaris L.) by aqueous two‐phase partitioning were separated into two fractions by further phase partition steps. The vesicles partitioning to the interface showed an ATP‐dependent H+‐uptake (measured using the pH probe acridine orange) and a negligible K+,Mg2+‐ATPase latency, while the vesicles partitioning in the upper phase showed only slow H+‐uptake and a high ATPase latency on addition of Triton X‐100. Based on these results the material at the interface is estimated to contain ∼90% sealed, inside‐out vesicles, and the material in the upper phase ∼90% sealed, right‐side‐out vesicles
An improved procedure for identifying and quantitating protein phosphatases in mammalian tissues
Authors: Cohen Philip , Klumpp Susanne , Schelling Donald L.
Published: July 2002

The type 2A protein phosphatases in mammalian tissue extracts are inhibited completely and specifically by 1–2 nMadaic acid. In contrast, type 1 protein phosphatases are hardly affected at these concentrations, complete inhibition requiring 1 μMadaic acid. These observations have been exploited to develop an improved procedure for the identification and quantitation of type 1, type 2A and type 2C protein phosphatases in tissue extracts.
Neutralization of lethal potency and inhibition of enzymatic activity of a phospholipase A2 neurotoxin, crotxin, by non‐precipitating antibodies (Fab)
Authors: Choumet Valérie , Jiang Ming-Shi , Radvanyi François , Ownby Charlotte , Bon Cassian
Published: July 2002

Rabbit antibodies were prepared against both purified catalytic (component‐B) and purified non‐catalytic (component‐A) subunits of crotoxin, the major phospholipase A2 neurotoxin from the South American rattlesnake. They cross‐react with crotoxin‐like toxins from the venom of several Crotalus species as well as with single‐chain phospholipase A2 neurotoxins from Crotalid and Viperid venoms (agkistrodontoxin and ammodytoxin A) but not from Elapid venoms (notexin). Immunological cross‐reactions of anti‐component‐A and anti‐component‐B sera with crotoxin and with its isolated components A and B showed that component‐A exposes determinants of low immunogenicity which are present on component‐B, whereas the major antigenic determinants of component‐B are not present on component‐A. Anti‐component‐B anti‐bodies, but not anti‐component‐A antibodies, neutralize the lethal potency of crotoxin and inhibit its enzymatic activity. Furthermore, non‐precipitating anti‐component‐B Fab fragments were as potent as antibodies, indicating that crotoxin neutralization results from the binding of the antibodies to the catalytic subunit, rather than the formation of an immuno‐precipitate.
Characterization of the locomotor depression produced by an A2‐selective adenosine agonist
Authors: Nikodijević Olga , Daly John W. , Jacobson Kenneth A.
Published: July 2002

Adenosine analogs, such as N6‐cyclohexyladenosine (CHA) that are selective for A1‐adenosine receptors, and analogs, such as 5'‐N‐ethylcarboxamidoadenosine (NECA) that are active at both a1 and A2 receptors, cause a profound depression of locomotor activity in mice via a central mechanism. The depression is effectively reversed by non‐selective adenosine antagonists such as theophylline. We report that 2‐[(2‐aminoethyl‐amino) carbonylethylphenylethylamino]‐5'‐N‐ethylcarboxamidoadenosine (APEC), an amine derivative of the A2 selective agonist, CGS21680, is a potent locomotor depressant in mice. The in vivo pharmacology is consistent with A2‐selectivity at a central site of action. Two parameters indicative of locomotor activity, horizontal activity and total distance travelled, were measured using a computerized activity monitor. From dose‐respon‐ se curves it was found that APEC (ED50 16 ) is more potent than CHA (ED50 60 ) and less potent than NECA (ED50 2 ). The locomotor depression by APEC was reversible by theophylline, but not by the A1‐selective antagonists 8‐cyclopentyltheophylline (CPT) and 8‐cyclo‐pentyl‐1,3‐dipropyl‐2‐thioxanthine, nor by the peripheral antagonists 8‐p‐sulfophenyltheophylline (8‐PST) and 1,3‐dipropyl‐8‐P‐sulfophenylxanthine. The locomotor activity depression elicited by NECA and CHA was reversed by A1‐selective antagonists. These results suggest that the effects of APEC are due to stimulation of A2 adenosine receptors in the brain.