bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective dehydrogenation (oxidation) of 3,4‐dimethoxybenzyl alcohol by a non‐heme iron lignin‐peroxidase reaction mimic
Authors: Tung Hui-Chan , Sawyer Donald T.
Published: July 2002

In pyridine, bis(2,2′‐bipyridine)iron(II) (Fe(bpy)2+
2) activates hydrogen peroxide for the efficient and selective catalytic dehydrogenation (oxidation) of veratryl alcohol (model‐substrate monomer for lignin; 3,4‐(MeO)2PhCH2OH). Several other complexes (FeII(OPPh3)2+
4, FeII(O2bpy)2+
2, FeII(MeCN)2+
4, FeII(PA)2, FeIIICl3) are effective catalysts for the dehydrogenation of veratryl alcohol and benzyl alcohol, but their selectivity (relative reactivity with 3,4‐(MeO)2PhCH2OH vs. PhCH2OH) is less than the 6.1 ratio that is observed for the optimized FeII(bpy)2+
2/H2O2/pyridine(py) system. The reactivities have been determined for several other methoxybenzyl alcohols that are model substrates for lignin (e.g.,4‐MeOPhCH2OH and (MeO)3PhCH2OH).
Natural human tumor necrosis factor beta (lymphotoxin) Variable O‐glycosylation at Thr7, proteolytic processing, and allelic variation
Authors: Voigt Christopher G. , Maurer-Fogy Ingrid , Adolf Günther R.
Published: July 2002

Natural human tumor necrosis factor beta (TNF‐β) purified from supernatants of a human B‐lymphoblastoid cell line was found to be heterogeneous in molecular mass, with seven components resolved by gel electrophoresis. All components are N‐glycosylate at Asn62., N‐glycosylation does not contribute to heterogeneity. In addition, part of the molecules are O‐glycosylated at Thr7; O‐glycosylation is heterogeneous due to variable decoration with neuraminic acid. The four lower molecular mass forms are derived from the full‐length protein by trypsin‐like proteolytic cleavage in the N‐proximal region; these clipped molecules lack O‐linked carbohydrates. Two allelic variants differing in amino acid position 26 (threonine/asparagine) were identified.
Preferential binding of α‐actinin to actin bundles
Authors: Grazi Enrico , Cuneo Paola , Magri Ermes , Schwienbacher Christine
Published: July 2002

At 37°C, the α‐actinin‐F‐actin binding isotherm is anomalous. In 6.7% polyethylene glycol 6000, concomitantly with the formation of actin bundles, the binding isotherm becomes hyperbolic (K
diss. = 11.3 μM). α‐Actinin increases the rigidity of the networks formed by actin bundles in polyethylene glycol and by paracrystalline actin in 16 mM MgCl2 but not by F‐actin. It is proposed that in the cell α‐actinin functions are mostly carried on by interaction with actin bundles.
Crystallization and preliminary X‐ray investigation of the Escherichia coli molecular chaperone cpn60 (GroEL)
Authors: Spangfort Michael D. , Surin Brian P. , Oppentocht Jantina E. , Weibull Claes , Carlemalm Eric , Dixon Nicholas E. , Svensson L.Anders
Published: July 2002

The Escherichia coli molecular chaperone, cpn60 (GroEL), has been purified from an overproducing E. coli strain and crystallized. Of the two crystal forms that were obtained, one was found to be suitable for crystallographic and structural studies at low resolution. Preliminary X‐ray investigation of the crystals show unit cell dimensions: a = 143.3, b = 154.6 and c = 265 Å, with α = 82°, β = 95° and γ = 107°. The space group is P1 and the crystals diffract to a maximum of 7 Å when using CuKα X‐rays from a rotating anode. Both electron microscopy and non‐denaturing electrophoretic analysis of redissolved cpn60 crystals show that cpn60 crystallizes in the native oligomeric form. Comparison between the dimensions of oligomeric cpn60 and the crystallographic unit cell volume suggests that the unit cell contains two oligomeric cpn60 molecules. The V
M value for two cpn60 molecules per unit cell is 3.5 Å3/Da, corresponding to a water content of 65%. Electrophoretic analysis under denaturing conditions shows that the cpn60 in crystals is heterogeneous, and this probably explains the limited resolution of the diffraction data.
Down‐regulation by retinoic acid of the catalytic subunit of protein phosphatase type 2A during granulocytic differentiation of HL‐60 cells
Authors: Tawara Isao , Nishikawa Masakatsu , Monta Koichi , Kobayashi Kazuhiko , Toyoda Hideki , Omay Serdar B. , Shima Hiroshi , Nagao Minako , Kuno Takayoshi , Tanaka Chikako , Shirakawa Shigeru
Published: July 2002

Activity of protein phosphatase measured in the absence of divalent cations was decreased by 50% during all‐trans retinoic acid (ATRA)‐induced HL‐60 cell differentiation into the granulocytic phenotype. Treatment of HL‐60 cells with ATRA led to a dramatic decrease in the amount of protein phosphatase type 2A (PP2A) protein, whereas that of protein phosphatase type l (PP1) protein was relatively constant, as detected by immunoblotting with antibodies specific to PP1 and PP2A. The decreased phosphatase activity may be mainly due to a decrease in the expression of the PP2A protein. The mRNA level ofPP2Aβ was markedly decreased within 5 h after addition of ATRA, but there was only a slight increase in the mRNA level of PP2Aα. Selective down‐regulation of PP2Aβ mRNA clearly preceded the cell differentiation induced by ATRA treatment. Thus, PP2A is down‐regulated during ATRA‐induced differentiation of HL‐60 cells into granulocytes.
Trehalose‐6‐phosphate, a new regulator of yeast glycolysis that inhibits hexokinases
Authors: Blázquez Miguel A. , Lagunas Rosario , Gancedo Carlos , Gancedo Juana M.
Published: July 2002

Trehalose‐6‐phosphate (P) competitively inhibited the hexokinases from Saccharomyces cerevisiae. The strongest inhibition was observed upon hexokinase II, with a K
i, of 40 μM, while in the case of hexokinase I the K
i was 200 μM. Glucokinase was not inhibited by trehalose‐6‐P up to 5 mM. This inhibition appears to have physiological significance, since the intracellular levels of trehalose‐6‐P were about 0.2 mM. Hexokinases from other organisms were also inhibited, while glucokinases were unaffected. The hexokinase from the yeast, Yarrowia lipolytica, was particularly sensitive to the inhibition by trehalose‐6‐P: when assayed with 2 mM fructose an apparent K
i, of 5 μM was calculated. Two S. cerevisiae mutants with abnormal levels of trehalose‐6‐P exhibited defects in glucose metabolism. It is concluded that trehalose‐6‐P plays an important role in the regulation of the first steps of yeast glycolysis, mainly through the inhibition of hexokinase II.
Identification of putative internalization signals in prion proteins
Authors: Kazim A.Latif
Published: July 2002

Prion proteins were found to contain regions in their cytoplasmic domains that have significant structural homologies to molecular trafficking signals for internalization of membrane proteins. These regions may facilitate the endocytosis of prion proteins, which appears to be a first step in their conversion to the abnormal, amyloidogenic form.
Vitamin B6 deficiency causes activation of RNA polymerase and general enhancement of gene expression in rat liver
Authors: Oka Tatsuzo , Komori Naomi , Kuwahata Masashi , Sassa Toshihiro , Suzuki Isao , Okada Mitsuko , Natori Yasuo
Published: July 2002

The effect of vitamin B6 deficiency on the activity of RNA polymerase and expression of several mRNAs in rat liver was investigated. The activities of RNA polymerase I and II in the liver of vitamin B6‐deficient rats were found to be higher than the control rats by 30%. The expression of several mRNAs, including mRNAs for β‐actin and glyceraldehyde‐3‐phosphate dehydrogenase, and the content of poly(A)+ RNA were also increased in vitamin deficiency. These observations suggest that vitamin B6 influences gene expression in the liver, at least in part, by modulating the activity of RNA polymerase.
Inhibition of an archaeal protein phosphatase activity by okadaic acid, microcystin‐LR, or calyculin A
Authors: Oxenrider Keith A. , Rasche Madeline E. , Thorsteinsson Marc V. , Kennelly Peter J.
Published: July 2002

Soluble extracts of the methanogenic archaeon, Methanosarcina thermophila TM‐1, contained a divalent metal ion‐stimulated protein‐serine phosphatase activity. This activity was sensitive to micromolar concentrations of okadaic acid, microcystin‐LR, or calyculin A, three compounds thought to be highly specific inhibitors of the type 1/2A/2B genetic superfamily of eukaryotic protein‐serine/threonine phosphatases. The observation that each of these three chemically unrelated compounds inhibited this archaeal protein phosphatase activity suggests the existence of structural homology, and perhaps even common genetic ancestry, with the type 1/2A/2B superfamily of protein‐serine/threonine phosphatases found in eukaryotic organisms.
Prevention of the induced synthesis and secretion of group II phospholipase A2 by brefeldin A
Authors: Sánchez Rosa M. , Vervoordeldonk Margriet J.B.M. , Schalkwijk Casper G. , van den Bosch Henk
Published: July 2002

Brefeldin A (BFA) has previously been shown to block protein secretion and to cause dismantling of the Golgi cisternae in many cultured cell lines. BFA was found to prevent the induced synthesis and secretion of 14 kDa group II phospholipase A2 (PLA2) in rat mesangial cells. Furthermore, BFA inhibited total protein synthesis although PLA2 appeared to be more sensitive to the effect of this compound than total protein synthesis assessed by amino acid incorporation. BFA was unable to block protein synthesis or PLA2 activity in the cell completely but secretion of enzymatic activity and PLA2 protein into the cell culture media was totally inhibited.
Affinity purification of molecular chaperones of the yeast Hansenula polymorpha using immobilized denatured alcohol oxidase
Authors: Evers Melchior E. , Huhse Bettina , Titorenko Vladimir I. , Kunau Wolf H. , Hartl Franz-Ulrich , Harder Wim , Veenhuis Marten
Published: July 2002

We used peroxisomal alcohol oxidase (AO) for the affinity purification of molecular chaperones from yeasts. Methodical studies showed that up to 0.8 mg of purified bacterial GroEL was able to bind per ml of immobilized denatured AO column material. Using crude extracts of Hansenula polymorpha or Saccharomyces cerevisiae, several proteins were specifically eluted with Mg‐ATP which were recognized by antibodies against hsp60 or hsp70. One H. polymorpha 70 kDa protein was strongly induced during growth at elevated temperatures, whereas a second 70 kDa protein as well as a 60 kDa protein showed strong protein sequence homology to mitochondrial SSCI and hsp60, respectively, from S. cerevisiae.
Effect of 67 kDa calcimedin on caldesmon functioning
Authors: Bogatcheva Natalia V. , Panaiotov Michail P. , Vorotnikov Alexander V. , Gusev Nikolai B.
Published: July 2002

Interaction of smooth muscle caldesmon with calmodulin, troponin C, S‐100 protein and 67 kDa calcimedin was analyzed. Native gel electrophoresis and crosslinking revealed the complex formation between caldesmon and three EF‐hand Ca‐binding proteins, whereas calcimedin did not interact with caldesmon. In the presence of Ca2+, calcimedin binds to actin‐tropomyosin without affecting the interaction of caldesmon with this complex. Although calcimedin reversed the inhibitory action of caldesmon on the actomyosin ATPase activity at a lower concentration than three other Ca‐binding proteins, this effect only slightly depends on Ca2+ and was observed at the concentration of calcimedin comparable to that of actin. It is concluded that calcimedin itself cannot be responsible for Ca‐dependent regulation of caldesmon functioning, but actin bundling induced by calcimedin (or by other actin binding proteins) decreases the inhibitory action of caldesmon on the actomyosin ATPase activity.
Protein kinase A modulates an endogenous calcium channel, but not the calcium‐activated chloride channel, in Xenopus oocytes
Authors: Chen Yan , Pollock Jonathan D. , Wang Yuhuan , DePaoli-Roach Anna A. , Yu Lei
Published: July 2002

In Xenopus oocytes, Ca2+ influx through an endogenous voltage‐gated Ca2+ channel activates a transient outward Cl− current (I
CI(Ca)), which is potentiated by cAMP increase. The site of cAMP effect appears to be the Ca2+ channel instead of the Ca2+‐activated Cl− channel, because cAMP potentiates the Ba2+ current through the Ca2+ channel in a similar way to the I
Cl(Ca), and cAMP does not potentiate the Ca2+‐dependent CI− current in cells treated with Ca2+ ionophore. Using the catalytic subunit of protein kinase A (PKA) and PKA inhibitors, it was shown that PKA is both necessary and sufficient for the cAMP effect on I
CI(Ca). Furthermore, the cAMP/PKA‐mediated potentiation of I
cI(Ca) was inhibited by both type 1 and type 2A protein phosphatases.
Pig leukocyte cysteine proteinase inhibitor (PLCPI), a new member of the stefin family
Authors: Lenarc̆ic̆ Brigita , Ritonja Anka , Dolenc Iztok , Stoka Veronika , Berbic̆ Selma , Pungerc̆ar Joz̆e , Štrukelj Borut , Turk Vito
Published: July 2002

A new stefin type low‐M
r, cysteine proteinase inhibitor (PLCPI) was isolated from pig polymorphonuclear leukocytes as a contaminant of the cathelin sample. The inhibitor consists of 103 amino acids, and its M
r, was calculated to be 11,768. The inhibitor exhibits considerable sequence identity with inhibitors from the stefin family, particularly with human stefin A. The PLCPI is a fast acting inhibitor of papain and cathepsins L and S (k
ass ⩾ 1 × 106 M−1 · s−1) and forms very tight complexes with these enzymes (K
i, ⩽ 190 pM). The affinity for cathepsins B and H (K
i ⩾ 125 nM) was lower. These results also show that the inhibitory activity previously ascribed to cathelin was due to the presence of PLCPI.
Effects of ionizing irradiation on endothelial cell transglutaminase
Authors: Korner Gil , Deutsch Varda R. , Viodavsky Israel , Eldor Amiram
Published: July 2002

The activity of transglutaminase (TG) was measured in cultures of bovine aortic and capillary endothelial cells (EC) following exposure to γ‐irradiation. Resting confluent EC express significant TG activity which fluctuates in growing cells. This activity was increased by 2‐fold following non‐lethal irradiation. The increase in TG activity was dose dependent up to 20 Gy and reached a plateau at 24–36 h after irradiation. Immunohistochemical studies showed a prominent increase in cytoplasmic TG following irradiation. Western blot analysis of whole cell extracts showed no increase in total cellular TG. Kinetic studies demonstrated that the affinity of the enzyme to its substrate was not altered, but the V
max was increased. TG has previously been shown to be stored in an inactive form in EC membranes. This activity could be recovered in normal EC, but not in irradiated EC, by the addition of potassium thiocyanate and dithiothreitol or 0.8 M Nad. An inhibitor of TG was previously demonstrated in the 100,000 × g particulate fraction of EC. Following irradiation, a significant decrease in this inhibitory activity was demonstrated. These results imply that the post‐irradiation enhancement of TG activity may be caused by activation of a latent cellular enzyme. This elevated TG activity may cross‐link adjacent cytoplasmic and membrane proteins and may thus play an active role in the enhanced apoptosis observed following irradiation of EC.
Penetration of oligonucleotides into mouse organism through mucosa and skin
Authors: Vlassov V.V. , Karamyshev V.N. , Yakubov L.A.
Published: July 2002

Benzylamide 5'‐32P‐oligonucleotide derivatives were shown to penetrate into mice organism when administered by various routes: intranasally, per os, intravaginally and per rectum. In all cases, the compounds are rapidly accumulated in blood and guts. Analysis of the radioactive material from blood and pancreas revealed intact oligonucleotides. Although concentrations of oligonucleotides in tissues differ considerably by the various methods of administration, the efficiency of delivery is sufficient to consider all the routes as being of therapeutic value. Dose effect on the efficiency of oligonucleotide penetration into mice suggests the transport to be a saturable process. Application of an oligonucleotide lotion on mice ear helices results in reproducible accumulation of radioactivity in the animal tissues. Effectiveness of oligonucleotide delivery into mouse through skin can be improved by using electrophoretic procedure.
Transcription of Drosophila mobile element gypsy (mdg4) in heat‐shocked cells
Authors: Lyubomirskaya N.V. , Arkhipova I.R. , Ilyin Yu.V.
Published: July 2002

Drosophila melanogaster Schneider 2 line cultured cells were subjected to stable transformation by co‐transfection with two plasmids, one of which conferred G418 resistance and another which contained the Drosophila retrotransposon, gypsy (mdg4), under the control of the heat‐shock protein 70 promoter. Transcription of the introduced constructs, as well as of endogenous gypsy, was examined under the condition of heat shock. Active degradation of pre‐existing gypsy transcripts was observed. During recovery, gypsy transcription was restored, but its termination and/or 3'‐end processing became aberrant.
Urea‐generated free rotating water molecules are active in the protein unfolding process
Authors: Khurgin Yu. , Maksareva E.
Published: July 2002

The critical urea concentration (C3
∗) which destabilizes the structure of bovine serum albumin and chymotrypsinogen was determined by UV difference spectroscopy. The increase of the relative content of mobile rotating water molecules in aqueous urea was formerly shown by millimeter spectroscopy [1]. The rise of rotator content at a urea concentration C3⩾ C3
∗ when the bulk water is practically exhausted is suggested as a main driving force of protein unfolding.
Structure and sequence of an intronless gene for human casein kinase II‐α subunit
Authors: Devilat Ignacio , Carvallo Pilar
Published: July 2002

Using sixteen different primers based on the cDNA sequence of the human casein kinase II‐α subunit, different fragments of this gene were amplified by PCR from human genomic DNA. The sizes of these fragments were identical to amplified cDNA, which suggests the existence of an intronless genomic gene. The amplification was carried out on whole blood genomic DNA from three different individuals. The total sequence of the amplified casein kinase II‐α gene showed more than 99% homology to the cDNA. The gene contains a noninterrupted open reading frame, as expected for the homolog cDNA. Although the gene sequence is complete, four point mutations were found. Since there are no interruptions of the open reading frame, this intronless gene might be expressed.
The region Ser333‐Arg356 of the α‐chain of human C4b‐binding protein is involved in the binding of complement C4b
Authors: Hessing Martin , Kanters Deon , Takeya Hiroyuki , van't Veer Cornelis , Hackeng Tilman M. , Iwanaga Sadaaki , Bourna Bonno N.
Published: July 2002

Human C4b‐binding protein (C4BP) functions as a cofactor to factor I in the degradation of C4b and accelerates the decay rate of the C4b2a complex. In this study we describe a monoclonal antibody directed against the α‐chain of C4BP that inhibits the binding of C4b to C4BP. In order to identify the structural domain of the a‐chain of C4BP that interacts with C4b, tryptie fragments of C4BP were generated. Amino acid sequence analysis of the fragments revealed that the residues Ser333‐Arg356 of the α‐chain of C4BP contain the epitope of this antibody, and as a consequence, that this part of the α‐chain of C4BP is likely to be involved in the interaction with C4b.
Characterization of glucose transport system in Drosophila Kc cells
Authors: Wang May-yun , Wang Chung
Published: July 2002

We have characterized the glucose transport system in the Kc cells of Drosophila melanogaster. The transporter is stereospecific and can be inhibited by both cytochalasin B and phloretin, with k
i, values of 4 μM and 30 μM for cytochalasin B and phloretin, respectively. Moreover, phloridzin does not affect uptake of glucose by Kc cells. Thus, on the basis of inhibitor studies, it appears that this insect cell line contains glucose transporters similar to those of the mammalian cells. However, probes prepared from rat transporter cDNAs showed no cross‐hybridization with Drosophila RNA under moderately stringent conditions. Sugar selectivity has also been investigated by competition assay. The k
m values for d‐glucose, 2‐deoxy‐d‐glucose, d‐fructose and d‐galactose are 2 mM, 6 mM, 17 mM and 16 mM, respectively. The results indicate that this transporter prefers d‐glucose and that it does not discriminate between d‐galactose and d‐fructose.
Glutathione in whole blood: a novel determination using double quantum coherence transfer proton NMR spectroscopy
Authors: Mason Ralph P. , Cha Gloria H. , Gorrie George H. , Babcock Evelyn E. , Antich Peter P.
Published: July 2002

Double quantum selective coherence transfer proton NMR spectroscopy has been used to observe glutathione in whole blood. The efficient water suppression of this technique avoids the need to resuspend the cells in D2O, hence avoiding equilibrium and kinetic isotope effects. Using this method we estimate the concentration of glutathione in fresh whole rabbit blood at ~1.7 mM.
A 19F NMR study of the interaction of 3‐fluoro‐4‐demethoxydaunomycin with the hexanucleotide d(TCCGGA)2
Authors: Barr Kathryn E. , Russell Richard A. , Warrener Ronald N. , Collins J.Grant
Published: July 2002

19F NMR spectroscopy has been used to study the binding of the fluorinated anthracycline 3‐fluoro‐4‐demethoxydaunomycin with the hexanucleotide d(TCCGGA)2. In the spectrum of the 1:1 anthracycline‐d(TCCGGA)2 complex four resonances of approximately equal intensity were observed. This indicated that 4‐demethoxydaunomycin intercalated at all possible sites with similar affinity. This suggests that the specific high affinity binding sites that are observed in anthracycline—DNA footprinting experiments are strongly regulated by the local DNA conformation.
Effect of hyperthermia on the extracellular matrix I. Heat enhances hyaluronan and inhibits sulphated glycosaminoglycan synthesis
Authors: Wegrowski Yanusz
Published: July 2002

The synthesis of sulphated glycosaminoglycans (GAG) and hyaluronan (HA) was studied in 3 cell strains incubated at 37°C or 42°C. Cells were labelled with [3H]glucosamine and [35S]sulphate. No incorporation of [35S]sulphate was observed at 42°C. Cellulose acetate electrophoresis of GAGs synthetized at 42°C showed only one [3H]glucosamine‐labelled band completely digested with Streptomyces hyaluronidase. The quantification of [3H]glucosamine‐labelled GAGs before and after hyaluronidase treatment indicated an increased rate of HA synthesis at 42°C as compared to the rate of synthesis at 37°C. These results suggest that hyperthermia stimulates HA synthesis and inhibits sulphated GAG synthesis in several cell strains which in turn may contribute to the modification of extracellular environment in inflammation.
Synthesis and binding characteristics of two sulfhydryl‐reactive probes for vasopressin receptors
Authors: Pávó Imre , Kojro Elzbieta , Fahrenholz Falk
Published: July 2002

The present study describes the synthesis and receptor binding affinities of the sulfhydryl‐reactive vasopressin analogs deamino[Dab(N
8‐N‐maleoyl‐β‐alanin e)4]AVP (1a) and deamino[Lys(N
8‐N‐maleoyl‐β‐alanine)8VP (2a). The analogs were obtained by introducing the sulfhydryl‐reactive maleoyl‐β‐alanyl group at the δ‐amino group of Dab4 in deamino[Dab4]IAVP (1) and at the ε‐amino group of Lys8 in deamino[Lys8]VP (2), which were synthesized by the solid‐phase method. Furthermore, the analog modified at Dab4 was prepared as tritium labeled compound (1b) after catalytic iodine tritium exchange at Tyr2 in deamino[Dab4]AVP. The sulfhydryl‐reactive vasopressin analogs retained high binding affinity for the V2 vasopressin receptor in membranes derived from bovine kidney inner medulla. Apparent dissociation constants K
d of 45 nM (compound 1a) and 15 nM (compound 2a) were determined. Incubation of the ligand receptor complexes at pH 5.5 resulted in dissociation of the sulfhydryl‐reactive vasopressin analogs from the V2 receptor. No indications of a covalent reaction between analogs 1a, 2a and 1b and sulfhydryl groups in or close to the hormone binding site of the V2 receptor were found.
Oxidised low density lipoproteins induce iron release from activated myoglobin
Authors: Rice-Evans Catherine , Green Emma , Paganga George , Cooper Christopher , Wrigglesworth John
Published: July 2002

Recent reports have detected the presence of iron in human atherosclerotic lesions [Biochem. J. 286 (1992) 901–905]. This study provides evidence for a biochemical mechanism whereby iron is released from myoglobin by low density lipoprotein (LDL) which has become oxidised by the ferryl myoglobin species. The haem destabilisation and iron release are inhibited by monohydroxamate compounds and desferrioxamine through their ability to inhibit the propagation of LDL oxidation. Thus, iron may derive from the myoglobin released from ruptured cells in the oxidising environment of the atherosclerotic lesion.
Amplification of superoxide anion generation in phagocytic cells by HIV‐1 infection
Authors: Kimura Takuro , Kameoka Masanori , Ikuta Kazuyoshi
Published: July 2002

Amplification of superoxide (O−
2) generation by HIV‐1 infection was examined in two human myeloid‐monocytic cell lines. The level of O−
2 generation in HL‐60 after infection became significantly higher than that of the steady‐state. A similar phenomenon was also shown in U937, but only after acquisition of O−
2 generation ability by differentiation to macrophages. By means of the NADPH oxidase‐coupled response in infected cells, we reconstituted the O−
2‐generating machinery in cell‐free system. The results suggested that cytosolic factor(s) exerted by infection might be responsible for the amplification of O−
2 generation. Thus, HIV‐1 infection could elevate the level of oxidative stress in macrophages which might play an important role in disease progression.
Fertilization of Xenopus eggs imposes a complete translational arrest of mRNAs containing 3'UUAUUUAU elements
Authors: Marinx Olivier , Bertrand Sophie , Karsenti Eric , Huez Georges , Kruys Véronique
Published: July 2002

The early embryonic development of Xenopus is mainly governed by post‐transcriptional regulations until the mid‐blastula transition. In this report, we present evidence demonstrating that fertilization of Xenopus eggs triggers a complete translational arrest of mRNAs containing UA‐rich elements in their 3′‐untranslated region. This control is maintained at least until the mid‐blastula transition. Neither maturation nor pseudo‐fertilization of the egg is sufficient for triggering this control, suggesting that components originating from the male gamete are involved in the mechanism. Moreover, this control is exerted whether the mRNA is polyadenylated or not.
Susceptibility of rat retina acyl‐CoA: 1‐acyl‐sn‐glycero‐3‐phosphocholine O‐acyltransferase and CTP:phosphocholine cytidylyltransferase activity to lipid peroxidation and hydroperoxide treatment
Authors: Anfuso Carmelina D. , Lupo Gabriella , Sipione Simonetta , Alberghina Mario
Published: July 2002

Two enzyme activities involved in phospholipid metabolism in the rat retina were determined after in vivo and in vitro peroxidation according to several model systems. The in vivo models were based on: (i) intravenous administration of a sonicated emulsion of phospholipid and linoleate photooxidized mixture to normal rat for a period of one week; (ii) acute injection of Fe2+ solution (20 mM) or (iii) 0.5 mg of hydroperoxylinoleate into the vitreous body, and collection of retinal tissue 4 h or 4 days later, respectively. Oleoyl CoA:lysophosphatidylcholine acyltransferase activity was unchanged or exhibited significant inhibition. On the contrary, CTP:phosphocholine cytidylyltransferase activity was stimulated. By incubating in vitro the retina with: (i) Fe2+‐ascorbate; (ii) photooxidized phospholipid mixture (0.1–5 mM) or individual phospholipid classes; (iii) hydroperoxylinoleate (0.25–2 mM), with or without Fe2+, a significant inactivation of acyltransferase (six‐fold maximum loss of initial activity) and a slight stimulation of cytidylyltransferase were seen. Altogether, the results suggest that in situ oxygen radical generation by a variety of agents irreversibly perturbs enzymes and/or membrane structures in which the enzymes are inserted; these events may be a causal factor in retinal degeneration accompanying some ocular diseases.
Polymerization of actin from the thymosin β4 complex initiated by the addition of actin nuclei, nuclei stabilizing agents or myosin S1
Authors: Reichert Andreas , Heintz Daniela , Voelter Wolfgang , Mihelic Mirna , Faulstich Heinz
Published: July 2002

Thymosin β4 forms a 1:1 complex with actin and thereby prevents polymerization. Rapid formation of filaments from this complex was observed, however, when actin trimers were added. Polymerization can likewise be initiated by the addition of one equivalent of phalloidin or, less effectively, cytochalasin B. Since both toxins, which reportedly support nucleation, have similar effects as the covalently linked actin trimers, it appears that the formation of filaments from the actin—thymosin β4 complex depends on the availability of stable actin nuclei. Remarkably, rapid polymerization was also observed if small amounts of myosin S1 were added, suggesting that also myosin, a protein functionally connected with polymeric actin, can serve as a nucleation center. Considering the existence of thymosin β4 and related peptides in numerous mammalian tissues, our data suggest that spontaneous formation of microfilaments in non‐muscle cells may be regulated at the level of nucleation. Uncontrolled polymerization induced by the formation of phalloidin‐stabilized nuclei may explain the acute toxic effects of phalloidin in hepatocytes.
Autoregulation of the human WT1 gene promoter
Authors: Malik Karim T.A. , Poirier Veronique , Ivins Sarah M. , Brown Keith W.
Published: July 2002

The human Wilms tumour suppressor gene, WT1, encodes a zinc‐finger protein which can function as a transcriptional activator or suppressor. This study reports the analysis of the human WT1 gene promoter, and demonstrates that high levels of WT1 expression lead to autosuppression of the WT1 promoter. Deletion analyses of the promoter region implicate sequences 5′ and 3′ of the transcriptional start site as being crucial in WT1 autosuppression. Loss or alteration of this function of WT1 may be important in tumourigenesis.
Solution structure of human interleukin‐1 receptor antagonist protein
Authors: Stockman Brian J. , Scahill Terrence A. , Strakalaitis Nancy A. , Brunner David P. , Yem Anthony W. , Deibel Martin R.
Published: July 2002

Interleukin‐1 receptor antagonist protein (IRAP) is a naturally occurring inhibitor of the interleukin‐1 receptor. In contrast to IL‐1β IRAP binds to the IL‐1 receptor but does not elicit a physiological response. We have determined the solution structure of IRAP using NMR spectroscopy. While the overall topology of the two 153‐residue proteins is quite similar, functionally critical differences exist concerning the residues of the linear amino acid sequence that constitute structurally homologous regions in the two proteins. Structurally homologous residues important for IL‐1 receptor binding are conserved between IRAP and IL‐1β. By contrast, structurally homologous residues critical for receptor activation are not conserved between the two proteins.
A fatty‐acid‐binding protein from wheat kernels
Authors: Castagnaro Atilio , García-Olmedo Francisco
Published: July 2002

A protein of about 7 kDa (W‐FABP) has been isolated from mature wheat kernels by H2O extraction and gel filtration of the extract, followed by two steps of high‐performance liquid chromatography. The N‐terminal amino acid sequence has been determined up to the 28th residue and found to be identical (except for positions 4 and 5) to that deduced from a barley cDNA (EMBL X15257), which had been improperly classified as a non‐specific lipid transfer protein (LTP2). Similarly with LTPs, W‐FABP does bind fatty acids, but in contrast, it is not significantly homologous to LTPs, it is not recognized by LTP antibodies, it has a more acidic isoelectric point (pH < 6.8 vs. pH > 9.6), and it does not show antibiotic properties.
A connexin‐32 mutation associated with Charcot‐Marie‐Tooth disease does not affect channel formation in oocytes
Authors: Rabadan-Diehl Cristina , Dahl Gerhard , Werner Rudolf
Published: July 2002

Members of the connexin family differ most in their carboxy‐termini, both with respect to sequence and length. In order to assess the contribution of this region to channel function, a series of carboxy‐terminal deletion mutants were tested in the paired‐oocyte expression system. Connexin‐32 can be truncated by 64 amino acids without detectable loss of its known channel properties. Removal of additional amino acids results in a progressive loss of function over a stretch of 4 amino acids. In addition to this effect of length the charge of the carboxy‐terminus appears to be another determinant of channel function. One of the fully functional deletion mutants, carrying a stop codon after amino acid‐219, had been reported to be associated with Charcot‐Marie‐Tooth disease. The implications of this finding are discussed.
Cloning, sequencing and overexpression of the Desulfovibrio gigas ferredoxin gene in E. coli
Authors: Chen Baowei , Menon Nanda K. , Dervertarnian Lisa , Moura Jose J.G. , Przybyla Alan E.
Published: July 2002

We have cloned the gene encoding Desulfovibrio gigas ferredoxin using a photodigoxigenin‐labelled probe synthesized with the polymerase chain reaction. The DNA sequence of the gene predicts a polypeptide of 58 residues after removal of the initial formyl methionine (polypeptide M
r = 6,276). The ferredoxin gene was expressed in aerobically grown E. coli behind the lac promoter of pUC18 resulting in a high level of ferredoxin expression which comprises about 10% of the total cell protein. EPR analysis of recombinant ferredoxin revealed the presence of a [3Fe‐4S] cluster which is characteristic of native D. gigas ferredoxin II.
Human urokinase receptor expression is inhibited by amiloride and induced by tumor necrosis factor and phorbol ester in colon cancer cells
Authors: Wang Yao , Jones Caroline J. , Dang Jinjun , Liang Xiaoming , Olsen Jane E. , Doe William F.
Published: July 2002

The modulation of urokinase plasminogen activator receptor (uPAR) gene expression by tumor necrosis factor alpha (TNFα), phorbol ester (PMA) and amiloride was studied in three colon cancer cell lines. uPAR mRNA and protein were induced by TNFα and by PMA but were inhibited by amiloride at concentrations of 0.1 to 1 mM in the presence or absence of TNFα and PMA. Nuclear run‐on transcription assay indicated that the effects of amiloride and TNFα were mediated at least in part at the transcriptional level, whereas PMA may act in part via a post‐transcriptional mechanism. These results suggested that uPAR gene expression is modulated by multiple signal transduction pathways.
A cytoplasmic domain is required for the functional interaction of SRI and HtrI in archaeal signal transduction
Authors: Krah Monika , Marwan Wolfgang , Oesterhelt Dieter
Published: July 2002

Phototaxis in the archaeon Halobacterium salinarium is mediated by a stable complex of the photoreceptor sensory rhodopsin I and its transducer HtrI, which relays the light stimulus to the signalling pathway. Removal of the cytoplasmic signalling domain of HtrI eliminated the SRI‐specific motor response to light stimulation and led to the loss of the spectroscopically detectable physical interaction of SRI and HtrI. A similar phenotype was obtained by deleting part of a cytoplasmic loop located between the second transmembrane helix of HtrI and the signalling domain. These results indicate that the photochemical behavior of sensory rhodopsin I is not determined by interaction with the transmembrane helices of HtrI per se but functionally coupled to the signalling domain. It is proposed that light excitation of SRI results in a conformational change of the transducer which is conducted by the cytoplasmic loop, an extra module not found in the eubacterial transducer homologues, and activates the signalling domain.
Thermostability of the barnase—barstar complex
Authors: Makarov A.A. , Protasevich I.I. , Lobachov V.M. , Kirpichnikov M.P. , Yakovlev G.I. , Gilli R.M. , Briand C.M. , Hartley R.W.
Published: July 2002

Scanning microcalorimetry was used to study heat denaturation of barnase in complex with its intracellular inhibitor barstar. The heat denaturation of the barnase—barstar complex is well approximated by two two‐state transitions with the lower temperature transition corresponding to barstar denaturation and the higher temperature one to barnase denaturation. The temperature of barnase melting in its complex with barstar is 20°C higher than that of the free enzyme. The barstar melting temperature is almost the same in the complex or alone (71°C at pH 6.2 and 68°C at pH 8.0). It seems possible that when barstar unfolds it can remain bound to barnase, while the latter unfolds only on dissociation of the denatured barstar.
Characterization of energetically functional inverted membrane vesicles from Corynebacterium glutamicum
Authors: Schrempp S. , Bayan N. , Shechter E.
Published: July 2002

We show that inverted membrane vesicles from Corynebacteriwn glutamicum, a Gram‐positive bacterium, are able to generate and maintain an electrochemical gradient of protons in response to the addition of NADH. This result indicates that the respiratory chain is intact and that the vesicles are reasonably impermeable to protons. These membrane vesicles may be the starting point for in vitro translocation studies of proteins in Gram‐positive bacteria.
PMA‐induced down‐regulation of the receptor for α2‐macroglobulin in human U937 cells
Authors: Conese Massimo , Cavallaro Ugo , Sidenius Nicolai , Olson David , Soria Marco R. , Blasi Francesco
Published: July 2002

Transcription and expression of the urokinase (uPA) receptor (uPAR) are strongly stimulated by PMA. As for uPAR, the expression of α2‐MR is regulated by PMA in U937 cells. Ligand blotting experiments with the 39 kDa receptor‐associated protein RAP, a ligand for α2‐MR, indicated that α2‐MR levels first increased and then decreased after PMA treatment. FACscan as well as immunoblotting analysis with α2‐MR‐specific antibodies showed an identical trend: α2‐MR levels increased within the first day of treatment with PMA, decreased at later times, and totally disappeared by three days of treatment. The effect of PMA was not due to transcriptional down‐regulation, as the α2‐MR mRNA level did not decrease at later times. Sensitivity of U937 cells to uPA‐saporin, a toxin conjugate that reguires binding to WAR for killing activity, was also markedly decreased. These results suggest that uPAR‐mediated endocytosis depends on α2‐MR expression.
Inactivation of oncoprotein binding by a single Cys706‐to‐Tyr substitution in the retinoblastoma protein
Authors: Saijo Masafumi , Kato Mitsuo V. , Sasaki Masao S. , Ishizaki Kanji , Taya Yoichi
Published: July 2002

We previously found a new single amino acid substitution at codon 706 (Cys‐to‐Tyr) of the retinoblastoma (RB) gene in a sporadic retinoblastoma patient. The glutathione S‐transferase‐RB fused protein containing this mutation was here tested for binding to SV40 large T antigen and adenovirus E1A protein, and was shown to have lost its binding affinity. Thus, Tyr, as well as Phe, residues substituted for Cys706 were found to abolish the RB protein activity.
Differential influence of DNA supercoiling on in vivo strength of promoters varying in structure and organisation in E. coli
Authors: Jyothirmai G. , Mishra Rakesh K.
Published: July 2002

DNA supercoiling is known to influence promoter activity in vitro and in vivo in a promoter‐dependent manner in prokaryotes. In order to investigate how topology may influence promoter function, we have studied two kinds of promoter variants, (i) where only the spacer region is altered, and (ii) where the same promoter is tandemly repeated in either the same or opposite orientation. These promoters respond very differently to alterations in DNA supercoiling, suggesting that the overall structure of the promoter and its context contribute to the differential response to alterations in supercoiling in vivo.
The 3D structure of a cyclosporin analogue in water is nearly identical to the cyclophilin‐bound cyclosporin conformation
Authors: Wenger Roland M. , France Julien , Bovermann Günter , Walliser Louis , Widmer Armin , Widmer Hans
Published: July 2002

The conformation of [d‐MeSer3‐d‐Ser‐(O‐Gly8]CS, a water soluble cyclosporin derivative, has been determined in (D6)DMSO and in water using NMR. In these polar solvents the conformation is identical and very similar to the structure found in the cyclophilin‐cyclosporin complex. However, it differs significantly from its conformation in deuterated chloroform. This demonstrates unambiguously that the large structure change is induced primarily by the polar solvent rather than by complex formation with cyclophilin.
Ileal expression of gastrin and cholecystokinin
Authors: Friis-Hansen Lennart , Rehfeld Jens F.
Published: July 2002

Antibodies against the common active site of cholecystokinin (CCK) and gastrin stain three endocrine celltypes in the gut: G‐cells (that synthesize gastrin) I‐cells (that synthesize CCK), and TG‐cells (whose product is unknown). In order to examine whether TG‐cells either process progastrin or proCCK in a specific manner, or express a novel gastrin‐CCK related hormone, we studied the distal porcine ileum, which have far more TGthan G‐ and I‐cells. Ileal CCK and gastrin mRNA corresponded to those of the antroduodenal mucosa. Gel, ion‐exchange and reversed‐phase chromatography monitored with sequence‐specific immunoassays showed that the ileal mucosa on average contain 0.3 and 7.6 progastrin and proCCK, respectively; 1.1 and 13.5 glycine‐extended intermediates; and 1.1 and 24.8 of bioactive carboxyamidated gastrin and CCK, respectively. Gastrin was present only as non‐sulfated gastrin‐34, whereas CCK occurred in forms similar to those of the proximal intestine. Although ileal progastrin processing is tissue specific, the amounts of gastrin and CCK are too small to explain the TG‐cells. Moreover, since the ileal extracts were without traces of other peptides having the C‐terminus common to gastrin and CCK, the results suggest that TG‐cells produce a peptide, which is only weakly related to gastrin and CCK.
Role of type II phospholipase A2 in the inflammatory process of carrageenan‐induced pleurisy in rats
Authors: Kakutani Makoto , Murakami Makoto , Okamoto Hitoshi , Kudo Ichiro , Inoue Keizo
Published: July 2002

In order to investigate the role of type II phospholipase A2 (PLA2) in the inflammatory process, the effect of a monoclonal antibody specific to type II PLA2 on carrageenan‐induced pleurisy was studied in rats. Intravenous injection of the antibody (MB5.2), which inhibits the catalytic activity of type II PLA2, significantly reduced both the pleural exudate volume and the intrapleural leukoeyte count, while a control antibody did not have any appreciable effect. MB5.2 caused no change in the level of type II PLA2 in the pleural fluid. These results suggest that type II PLA2 generated at inflamed sites, at least in part, have a crucial role in the pathogenesis of acute inflammation.
Arguments against a close relationship between non‐phosphorylating and phosphorylating glyceraldehyde‐3‐phosphate dehydrogenases
Authors: Michels S. , Scagliarini S. , Seta F.Della , Carles C. , Riva M. , Trost P. , Branlant G.
Published: July 2002

Non‐phosphorylating NADP‐dependent glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (EC 1.2.1.9) from spinach leaves was purified to homogeneity using an improved purification procedure. Thus, a major contaminant with molecular mass and ion‐exchange properties similar to non‐phosphorylating GAPDH was eliminated. Using this pure non‐phosphorylating GAPDH, cofactor stereospecificity was determined by 1H NMR. Analysis of the NADPH formed from the hydride transfer from glyceraldehyde‐3‐phosphate to [4‐2H]NADP showed that the enzyme belongs to the A‐stereospecific dehydrogenase family. This stereospecificity is the same as that described for the aldehyde dehydrogenase (ALDH) superfamily and opposite to that of the phosphorylating GAPDH. Moreover, results from peptide sequencing analysis suggest a similarity in sequence between the non‐phosphorylating GAPDH and ALDHs. Thus, the results taken all together strongly suggest that non‐phosphorylating GAPDH belongs to the ALDH family and has no close relationship to the phosphorylating GAPDH class.
Localization of phospholipid‐binding sites of caldesmon
Authors: Bogatcheva Natalia V. , Huber Pia A.J. , Fraser Iain D.C. , Marston Steven B. , Gusev Nikolai B.
Published: July 2002

The interaction of phosphatidylserine with intact smooth muscle caldesmon and caldesmon fragments obtained by bacterial expression was investigated by means of light scattering. Among these fragments only those derived from the C‐terminal part of caldesmon (so‐called domain 4) were able to interact with phospholipids. Fragments 606C (residues 606–756), H7 (566–710) and H2 (626–710) form tight complexes with phosphatidylserine, whereas fragments H8 (658–737), H9 (669–737) and fragment H4 (566–624) interact with phospholipids less effectively. It is concluded that the phospholipid‐binding site is located in the sequence 626–710 of caldesmon. This sequence contains calmodulin‐binding sites and serine residues phosphorylated by protein kinase C and pro‐directed protein kinases. This could explain the effects of calmodulin and phosphorylation on the caldesmon‐phospholipid interaction described earlier.
An isoform of the human calcitonin receptor is expressed in TT cells and in medullary carcinoma of the thyroid
Authors: Frendo J.L. , Pichaud F. , Mourroux R.DeLage , Bouizar Z. , Segond N. , Moukhtar M.S. , Jullienne A.
Published: July 2002

We amplified, using the polymerase chain reaction and calcitonin receptor (CTR) specific primers, RNA extracted from medullary thyroid carcinoma (MTC) and the derived TT cell line. Both secrete large amounts of calcitonin. Electrophoresis of amplification products revealed, in both cases, an ethidium bromide‐stained band that hybridized to a CTR probe. Sequencing the band amplified from TT cells revealed an open reading frame identical to the sequence of H‐CTR but lacking 16 amino acids in the first intracellular loop. This demonstrates the existence of an mRNA coding for a subtype of H‐CTR which is expressed in TT cells and MTC.
The accessibility of peptides bound to the mouse MHC class II molecule IEd studied by fluorescence
Authors: de Kroon Anton I.P.M.
Published: July 2002

The accessibility of fluorescently labeled (antigenic) peptides bound to the detergent‐solubilized mouse MHC class II protein IE d has been studied by fluorescence techniques. Based on the efficiency of fluorescence quenching by the aqueous quenchers iodide and TEMPOL, different degrees of accessibility of the peptide‐attached fluorescein moiety are distinguished in the lEd‐bound state, which depend on the nature of the peptide and on the site of attachment. These different extents of sequestration from the aqueous phase are reflected in the fluorescence properties of the corresponding NBD‐labeled peptides bound to IE d. The results provide information on the topology of class II bound peptides.