bluuebunny/crossref_metadata_2025 huggingface dataset:
Prediction of sequential antigenic regions in proteins
Authors: Welling Gjalt W. , Weijer Wicher J. , van der Zee Ruurd , Welling-Wester Sytske
Published: July 2002

Prediction of antigenic regions in a protein will be helpful for a rational approach to the synthesis of peptides which may elicit antibodies reactive with the intact protein. Earlier methods are based on the assumption that antigenic regions are primarily hydrophilic regions at the surface of the protein molecule. The method presented here is based on the amino acid composition of known antigenic regions in 20 proteins which is compared with that of 314 proteins [(1978) Atlas of Protein Sequence and Structure, vol. 5, suppl. 3, 363‐373]. Antigenicity values were derived from the differences between the two data sets. The method was applied to bovine ribonuclease, the B‐subunit of cholera toxin and herpes simplex virus type 1 glycoprotein D. There was a good correlation between the predicted regions and previously determined antigenic regions.
Metabolism of the neurotoxin in MPTP by human liver monoamine oxidase B
Authors: Fritz Richard R. , Abell Creed W. , Patel Nutan T. , Gessner Wieslaw , Brossi Arnold
Published: July 2002

The neurotoxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) was oxidized to dihydropyridine MPDP+ and pyridine MPP+ by preparations of monoamine oxidase B (MAO B), including pure human liver MAO B:monoclonal antibody complex. K
m,app values for MPTP and benzylamine, a preferred MAO B substrate, were 316 and 64 μM, respectively. 4‐Phenyl‐1,2,3,6‐tetrahydropyridine (PTP), the nor derivative of MPTP, was also a substrate (K
m,app = 221μM). MPDP+ MPTP, and MPP+, but not PTP, were found tobe irreversible inhibitors of MAO B. Our studies support the hypothesis that MPTP is oxidized in primate brain by MAO B to MPDP+ which is then converted to MPP+ a major metabolite found in the substantia nigra.
Essential structure for full enterotoxigenic activity of heat‐stable enterotoxin produced by enterotoxigenic Escherichia coli
Authors: Yoshimura Shoko , Ikemura Haruo , Watanabe Hiroyuki , Aimoto Saburo , Shimonishi Yasutsugu , Hara Saburo , Takeda Tae , Miwatani Toshio , Takeda Yoshifumi
Published: July 2002

Several analogues of heat‐stable enterotoxins (STh and STP) produced by enterotoxigenic Escherichia coli were synthesized. Peptides (STh[6–18] and STP[5–17]) consisting of 13 amino acid residues from the Cys residue near the N‐terminus to the Cys residue near the C‐terminus and linked by three disulfide bonds had the same biological and immunological properties as native STh and STP, respectively. The results indicated that the sequence with the 13 amino acid residues and three disulfide linkages is essential for full biological activity of ST.
Electrogenic proton exchange between cytochrome a3 active center and M‐aqueous phase
Authors: Konstantinov Alexander , Vygodina Tatiana , Andreev Igor M.
Published: July 2002

The rate of cyanide binding with the oxidized cytochrome‐c oxidase in proteoliposomes is controlled by ionization of a protein group with pK~6.7, the ligand reacting with the protonated enzyme only [(1983) Bioorg. Chem. (USSR) 9,216‐227]. As shown here, the kinetics of cyanide binding depends on the pH inside the proteoliposomes. The reaction rate is affected by the electrical potential difference across the proteoliposome membranes as if the a
3‐linked ionizable group exchanged H+ with the proteoliposome interior electrogenically. The data corroborate a hypothesis on the existence of a proton well communicating cytochrome oxidase O2‐reducing center with the M‐aqueous phase.
Glucose stimulates the biosynthesis of rat I and II insulin to an equal extent in isolated pancreatic islets
Authors: Rhodes Christopher J. , Lucas Christine A. , Halban Philippe A.
Published: July 2002

The effects of glucose on insulin biosynthesis were studied by measuring the incorporation of radiolabelled amino acids into proinsulin/insulin in isolated rat islets. The islets were pulse labelled for 15 min with [3H]leucine (present in rat insulin I and II) or [35S]methionine (unique to rat insulin II) and then incubated for a 165 min post‐label (chase) period during which the majority of labelled proinsulin was converted to insulin but under conditions whereby > 95% of radiolabelled proinsulin or insulin was retained in the islets. The newly synthesized, labelled, insulin was analyzed by high performance liquid chromatography. Rat I and II insulin biosynthesis was stimulated by 16.7 mM glucose to the same extent.
The glucose sensor in HIT cells is the glucose transporter
Authors: Ashcroft Stephen J.H. , Stubbs Mark
Published: July 2002

The nature of the rate‐limiting step for glucose utilization by the clonal insulin‐producing cell line HIT‐T15 has been investigated. In contrast to the situation in islets of Langerhans, we find that the HIT cell glucose metabolism is limited by the rate of entry of glucose into the cell. This is evidenced by the low rate of sugar transport and by the marked reduction in the rate of glucose utilization elicited by inhibitors of the glucose transporter. As judged by competition with glucose, the HIT cell glucose transporter also transports mannose, 2‐deoxyglucose and 3‐O‐methylglucose but not L‐glucose or N‐acetylglucosamine. The K
m for glucose of the glucose transporter, measured as the concentration of glucose required for a half‐maximal rate of glucose utilization, is 4.3 mM, similar to the concentration reported to give half‐maximal insulin release. Glucose‐stimulated insulin release from HIT cells is inhibited by phloretin or cytochalasin B but not by mannoheptulose. We conclude that the secretory responses of HIT cells are consistent with the substrate‐site hypothesis, but that, in contrast to normal B‐cells, the glucose sensor which confers concentration‐dependence and specificity to sugar‐stimulated insulin release, is the glucose transporter.
Counterion collapse and the effect of diamines on bacteriorhodopsin
Authors: Marinetti Tim
Published: July 2002

A recent report of electrical measurements on oriented bacteriorhodopsin in gels [(1986) FEBS Lett. 195, 164–168] concluded that low concentrations of diamines reversed the direction of the proton pump. Calculations are presented which show that in low diamine concentrations, charge displacements of the counterion atmosphere in the direction opposite to proton pumping are expected following H+ ejection. It is also shown that the effect will be sharply reduced by raising the diamine concentration or by adding excess salt, as was observed. Hence it is not necessary to conclude that diamines reverse the direction of the proton pump itself.
On the role of tryptophan as a superexchange mediator for quinone reduction in photosynthetic reaction centers
Authors: Plato M. , Michel-Beyerle M.E. , Bixon M. , Jortner J.
Published: July 2002

The role of tryptophan (W) in the quinone reduction in the reaction centers of R. viridis and Rb. sphaeroides is studied by a quantum chemical calculation of the electronic coupling matrix elements within the bacteriopheophytin‐W‐quinone subsystem, which is based on the X‐ray structural data. The results favor the superexchange mechanism via W over the direct coupling. Uncertainties in the available X‐ray coordinates and structural relaxation accompanying the formation of the P+H− state result in large changes in the relative contributions of the electronic couplings due to electron and hole superexchange. Aromatic amino acid residues may serve as essential functional components in electron transfer.
MAP 30: a new inhibitor of HIV‐1 infection and replication
Authors: Lee-Huang Sylvia , Huang Philip L. , Nara Peter L. , Chen Hao-Chia , Kung Hsiang-fu , Huang Peter , Huang Henry I. , Huang Paul L.
Published: July 2002

A new inhibitor of human immunodeficiency virus (HIV) has been isolated and purified to homogeneity from the seeds and fruits of the Momordica charantia. This compound, MAP 30 (Momordica Anti‐HIV Protein), is a basic protein of about 30 kDa. It exhibits dose‐dependent inhibition of cell‐free HIV‐1 infection and replication as measured by: (i) quantitative focal syncytium formation on CEM‐ss monolayers; (II) viral core protein p24 expression; and (iii) viral‐associated reverse transcriptase (RT) activity in HIV‐1 infected H9 cells. The doses required for 50% inhibition (ID50) in these assays were 0.83, 0.22 and 0.33 nM, respectively. No cytotoxic or cytostatic effects were found under the assay conditions. These data suggest that MAP 30 may be a useful therapeutic agent in the treatment of HIV‐1 infections. The sequence of the N‐terminal 44 amino acids of MAP 30 has been determined.
Modulation of the affinity of aspartic proteases by the mutated residues in active site models
Authors: Goldblum Amiram
Published: July 2002

The active sites of 3 types of aspartic proteases are modeled, based on crystallographic coordinates of endothiapepsin and of a model of HIV‐1 protease. The enthalpies of deprotonation from neutral to mono‐anion and to dianion are calculated with semiempirical minimal neglect of differential overlap, hydrogen bonding corrected (MNDO/H). This quantum mechanical study of models for the active sites of pepsins, human renin and retroviral aspartic proteases demonstrates that the replacements ofThr‐218 from pepsins by Ala in human renin and of both Ser‐35 and Thr‐218 by alanines in retroviral proteases increases the proton affinity and modulates the charge distribution of those active sites compared to the pepsins.
Prediction of prolyl residues in cis‐conformation in protein structures on the basis of the amino acid sequence
Authors: Frömmel Cornelius , Preissner Robert
Published: July 2002

In proteins most peptide bonds are in trans ‐conformation: the torsion angle ω = 180°. Only few show cis‐conformation in known protein structures (ω = 0°). Most of them are prolyl residues. About 6% of about 4000 prolyl residues are in cis‐conformation. Between trans‐ and cis‐prolyl residues significant differences are observed in the surrounding sequences. E.g. there are large amounts of aromatic residues N‐terminally in case of cis‐prolyl residues, but in the case of trans‐prolyl residues more aromatic amino acids occur C‐terminally. But in all cases there are only complex patterns which are indicative of cis‐ and trans‐conformation, respectively. Considering the neighbours (±6 residues) of prolyl residues and their physicochemical properties we find 6 different patterns which allow one to assign correctly about 75% of known cis‐structured prolyl residues, whereby no false positive one is predicted.
Amylin activates glycogen phosphorylase in the isolated soleus muscle of the rat
Authors: Young Andrew A. , Mott David M. , Stone Karen , Cooper Garth J.S.
Published: July 2002

The pancreatic β‐cell hormone amylin acts in isolated rat skeletal muscle to decrease insulin‐stimulated incorporation of glucose into glycogen. It also increases blood levels of lactate and glucose in fasted rats in vivo. However, it remained uncertain whether amylin exerts direct effects to stimulate muscle glycogenolysis. We now report that amylin caused a dose‐dependent increase in activity of muscle glycogen phosphorylase in isolated rat soleus muscle by stimulating phosphorylase a. Insulin inhibited amylin‐stimulated activation of phosphorylase. Effects of amylin to stimulate muscle glycogenolysis are consistent with observed effects of amylin in vivo and could be a major mechanism whereby amylin modulates carbohydrate metabolism.
A secY homologue is found in the plastid genome of Cryptomonas Φ
Authors: Douglas Susan E.
Published: July 2002

An open reading frame with significant similarity to the secY gene of Escherichia coli has been found within a ribosomal protein operon on the plastid genome of the chlorophyll c‐containing alga Cryptomonas Φ. The gene encodes a protein or 420 amino acids (molecular weight 46.906 daltons) and contains ten potential membrane‐spanning domains, as in the E. coli homologue. This report of a secY homologue in a plastid genome provides preliminary evidence that a prokaryotic‐like protein export system may be operating in plastids.
Endogenous Xenopus‐oocyte Ca‐channels are regulated by protein kinases A and C
Authors: Bourinet E. , Fournier F. , Nargeot J. , Charnet P.
Published: July 2002

Calcium entry into Xenopus oocyte occurs mainly through voltage‐dependent calcium channels. These channels were characterized as belonging to a particular type of calcium channel insensitive to dihydropyridines, ω‐conotoxin, and Agelenopsis aperta venom, but blocked by divalent cations (Co, Cd, Ni). Intracellular injection of cAMP, or bath application of phorbol ester, induced a marked increase in calcium current amplitude and a slowing of the inactivation time‐course. Despite their different pharmacology, endogenous calcium channels, like cardiac or neuronal calcium channels, could be thus regulated by protein kinases A and C.
Sequence similarity of a hornet (D. maculata) venom allergen phospholipase A1 with mammalian lipases
Authors: Soldatova Lyudmila , Kochoumian Loucia , King Te Piao
Published: July 2002

We have determined the sequence of a venom allergen phospholipase A1 from white‐faced hornet (Dolichovespula maculata) by cDNA and protein sequencings. This protein of 300 amino acid residues (Dol m I) has no sequence similarity with other known phospholipases. But it has sequence similarity with mammalian lipases; about 40% identity in overlaps of 123 residues. Tests suggest that hornet phospholipase has weak lipase activity. Hornet venom has 3 major allergens, and another hornet allergen antigen 5 (Dot m V) was previously found to have sequence similarity with a mammalian testis protein and a plant leaf protein.
Photoaffinity labelling of human leukotriene C4 synthase in THP‐1 cell membranes
Authors: Ali Ambereen , Zamboni Robert J. , Ford-Hutchinson Anthony W. , Nicholson Donald W.
Published: July 2002

Human leukotriene C4 synthase specific activity in the human monocytic leukemia cell line THP‐1 (0.302 ± 0.062 nmol LTC4 formed · min−1 mg−1) was 7.6‐fold higher than in U937 cells (0.040 ± 0.017 nmol LTC4 formed · min−1 · mg−1) and comparable to dimethylsulfoxide‐differentiated U937 cells (0.399 ± 0.084 nmol LTC4 formed · min−1 · mg−1). Using the photoaffinity probe, azido[125I]‐LTC4, a single polypeptide with a molecular mass of 18 kDa was specifically labelled in THP‐1 microsomal membranes. The rank order of potencies for competition of azido[125I]‐LTC4 photolabelling of the 18 kDa protein by glutathione, leukotrienes and their analogs was found to be LTC2 > (azido[127I]‐LTC4 ≈ LTC4) > (LTD4 ≈ LTE4) > (LTA4 ≈ LTB4) > S‐hexyl glutathione > glutathione, corresponded with the rank order of potencies for inhibition of LTC4 synthase activity but not inhibition of microsomal glutathione S‐transferase activity. The 18 kDa protein specifically labelled by azido[125I]‐LTC4 had high specificity for LTC4 and closely related leukotrienes and was separable from microsomal glutathione S‐transferase. We conclude that azido[125I]‐LTC4 specifically photolabels LTC4 synthase which is an 18 kDa polypeptide or contains an 18 kDa subunit.
Movable lobes and flexible loops in proteins Structural deformations that control biochemical activity
Authors: Kempner E.S.
Published: July 2002

Two classes of protein whose structure is modified by small ligands are reviewed. Proteins of one group contain two massive domains joined by a flexible link; in response to small molecules, the two lobes approach and enclose the ligand. In the other, a short segment of amino acids moves as a flexible loop over the ligand which often is trapped in a non‐aqueous environment. Biochemical reaction rates are altered dramatically by these movements.
Interaction of phosphorylated elongation factor EF‐2 with nucleotides and ribosomes
Authors: Dumont-Miscopein Agnès , Lavergne Jean-Pierre , Guillot Dominique , Sontag Bruno , Reboud Jean-Paul
Published: July 2002

The intrinsic fluorescence emission spectrum of elongation factor EF‐2 due to the 7 Trp residues was not modified after complete phosphorylation of the factor by the specific Ca2+/Calmodulin‐dependent kinase III. The effect of nucleotide binding on this fluorescence revealed differences between phosphorylated and unmodified EF‐2. Low concentrations of GTP had a smaller quenching effect on the fluorescence of phosphorylated EF‐2 than on the fluorescence of unmodified EF‐2, whereas GDP had exactly the same quenching effect on the fluorescence of both samples. These results suggest that phosphorylation of EF‐2 decreased its affinity for GTP but not for GDP. Ability of phosphorylated EF‐2 to form a ternary complex with ribosomes in the presence of a non‐hydrolysable GTP analog and its ability to protect ribosomes against ricin‐inactivation were both decreased to the same extent. The lower affinity of phosphorylated EF‐2 for GTP could be responsible for a weaker and/or incorrect interaction of the factor with the ribosome, in particular with the ricin‐site of the 28‐S rRNA assumed to be involved in translocation initiation.
Molecular analysis of the IL‐6 receptor in human multiple myeloma, an IL‐6‐related disease
Authors: Rodriguez Carmen , Theillet Charles , Portier Marielle , Bataille Régis , Klein Bernard
Published: July 2002

A PCR‐SSCP approach was used to search for mutations in IL‐6 receptor genes in 9 human plasma cell lines (HMCL) and in tumor plasma cells from 19 patients with fulminating multiple myeloma, an IL‐6‐related disease. Whereas no mutation was found in the cytokine receptor homologous (CRH) domain of IL‐6Rα, DNA and RNA polymorphisms in the gp130 CRH domain was detected in tumoral samples as well as in blood samples from healthy donors. Finally, mutations in the gp130 critical cytoplasmic domain were found in one HMCL and in tumor plasma cells of one patient. Only the mutated allele was expressed in the HMCL.
Crystal structure of momordin, a type I Ribosome Inactivating Protein from the seeds of Momordica charantia
Authors: Husain Jasmine , Tickle Ian J. , Wood Stephen P.
Published: July 2002

A type I ribosome‐inactivating protein, extracted and purified from M. charantia seeds, was crystallised by vapour diffusion with polyethylene glycol at pH 7.2. X‐ray data were collected to 2.1 Å resolution and the structure solved by molecular replacement using the A‐chain coordinates of ricin. The overall fold of the protein is similar to ricin but there are differences in secondary structure, on the surface and in the active site cleft. These differences are probably due in part to the evolution of the protein without a B‐chain partner. The most extensive reorganisation occurs at the C‐terminus whereas Tyr70 shows the greatest change in the active site cleft.
The common C‐terminal sequences of substance P and neurokinin A contact the same region of the NK‐1 receptor
Authors: Bremer Andrew A. , Leeman Susan E. , Boyd Norman D.
Published: July 2002

Although neurokinin A (NKA), a tachykinin peptide with sequence homology to substance P (SP), is a weak competitor of radiolabeled SP binding to the NK‐1 receptor (NK‐1R), more recent direct binding studies using radiolabeled NKA have demonstrated an unexpected high‐affinity interaction with this receptor. To document the site of interaction between NKA and the NK‐1R, we have used a photoreactive analogue of NKA containing p‐benzoyl‐L‐phenylalanine (Bpa) substituted in position 7 of the peptide. Peptide mapping studies of the receptor photolabeled by 125I‐iodohistidyl1‐Bpa7NKA have established that the site of photoinsertion is located within a segment of the receptor extending from residues 178 to 190 (VVCMIEWPEHPNR). We have previously shown that 125I‐BH‐Bpa8SP, a photoreactive analogue of SP, covalently attaches to M181 within this same receptor sequence. Importantly, both of these peptides (125I‐iodohistidyl1‐Bpa7NKA and 125I‐BH‐Bpa8SP) have the photoreactive amino acid in an equivalent position within the conserved tachykinin carboxyl‐terminal tail. In this report, we also show that site‐directed mutagenesis of M181 to A181 in the NK‐1R results in a complete loss of photolabeling of both peptides to this receptor site, indicating that the equivalent position of SP and NKA, when bound to the NK‐1R, contact the same residue.
Electrical activity regulates AChR gene expression via JNK, PKCζ and Sp1 in skeletal chick muscle
Authors: Altiok Nedret , Changeux Jean-Pierre
Published: July 2002

Electrical activity of myotubes represses nicotinic acetylcholine receptor (AChR) gene expression. This effect is mimicked by okadaic acid and blocked by tetrodotoxin (TTX) or staurosporine in cultured myocytes [Altiok et al., EMBO J. 16 (1997) 717–725]. In this study, we investigated the mechanism of this repression. We show that addition of exogenous phospholipase D (PLD) and C inhibits AChR expression in a manner which parallels that of okadaic acid. Furthermore, okadaic acid caused an increase of the threonine phosphorylation of protein kinase Cζ (PKCζ) and activator of transcription factor (ATF2) and a decrease of the phosphorylation of Sp1. All these effects were reversed by staurosporine, and TTX also abolished ATF2 phosphorylation. These data reveal a possible involvement of PLD, c‐jun N‐terminal kinase, PKCζ and Sp1 in the repression of AChR genes by electrical activity.
Aquaporin 3, a glycerol and water transporter, is regulated by p73 of the p53 family
Authors: Zheng Xiangjian , Chen Xinbin
Published: July 2002

p73, a member of the p53 family, has been shown to exhibit similar biochemical activities to that of p53. However, in contrast to p53, p73 is rarely mutated in human tumors and p73 mutant mice develop neurological, pheromonal, and inflammatory defects, but not spontaneous tumors. Furthermore, p73 mutant mice are deficient in the physiological control of cerebral spinal fluid. To determine what mediates these p73 activities, cDNA subtraction assay was performed to identify cellular genes that are regulated by p73. We found that aquaporin 3 (AQP3), a glycerol and water transporter, is regulated by p73. In addition, we identified a potential p53 response element in the promoter of the AQP3 gene, which is responsive to p73. This suggests that AQP3 may mediate the activity of p73 in maintaining cerebral spinal fluid dynamics.
A novel strategy for the expression of foreign genes from plant virus vectors
Authors: Toth Rachel L , Chapman Sean , Carr Fiona , Santa Cruz Simon
Published: July 2002

Potato virus X (PVX)‐based vector constructs were generated to investigate the use of an internal ribosome entry site (IRES) sequence to direct translation of a viral gene. The 148‐nucleotide IREScp sequence from a crucifer‐infecting strain of tobacco mosaic virus was used to direct expression of the PVX coat protein (CP). The IRES was inserted downstream of the gene encoding green fluorescent protein (GFP) and upstream of the PVX CP, in either sense or antisense orientation, such that CP expression depended on ribosome recruitment to the IRES. Stem–loop structures were inserted at either the 3′‐ or 5′‐end of the IRES sequence to investigate its mode of action. In vitro RNA transcripts were inoculated to Nicotiana benthamiana plants and protoplasts: levels of GFP and CP expression were analysed by enzyme‐linked immunosorbent assay and the rate of virus cell‐to‐cell movement was determined by confocal laser scanning microscope imaging of GFP expression. PVX CP was expressed, allowing cell‐to‐cell movement of virus, from constructs containing the IRES sequence in either orientation, and from the construct containing a stem–loop structure at the 5′‐end of the IRES sequence. No CP was expressed from a construct containing a stem–loop at the 3′‐end of the IRES sequence. Our results suggest that the IRES sequence is acting in vivo to direct expression of the 3′‐proximal open reading frame in a bicistronic mRNA thereby demonstrating the potential of employing IRES sequences for the expression of foreign proteins from plant virus‐based vectors.
Protein kinase B (PKB/Akt) – a key regulator of glucose transport?
Authors: Hajduch Eric , Litherland Gary J , Hundal Harinder S
Published: July 2002

The serine/threonine kinase protein kinase B (PKB/Akt) has been shown to play a crucial role in the control of diverse and important cellular functions such as cell survival and glycogen metabolism. There is also convincing evidence that PKB plays a role in the insulin‐mediated regulation of glucose transport. Furthermore, states of cellular insulin resistance have been shown to involve impaired PKB activation, and this usually coincides with a loss of glucose transport activation. However, evidence to the contrary is also available, and the role of PKB in the control of glucose transport remains controversial. Here we provide an overview of recent findings, discuss the potential importance of PKB in the regulation of glucose transport and metabolism, and comment on future directions.
Protein translocation machineries of peroxisomes
Authors: Holroyd Claudia , Erdmann Ralf
Published: July 2002

Targeting of peroxisomal matrix and membrane proteins is performed by distinct transport machineries and requires the concerted action of at least 23 peroxins. Cargo recognition takes place in the cytosol and the multiple binding sites for peroxisomal signal sequence receptors at the peroxisomal membrane reflect the existence of an import cascade where the cargo‐loaded receptors successively interact with different components of the import machinery. These interactions are likely to trigger conformational changes of the proteins within the import cascade which are required for the consecutive steps of peroxisomal protein import: docking, translocation, cargo release and receptor recycling.
Inhibition of nucleoside diphosphate kinase activity by in vitro phosphorylation by protein kinase CK2 Differential phosphorylation of NDP kinases in HeLa cells in culture
Authors: Biondi Ricardo M. , Engel Matthias , Sauane Moira , Welter Cornelius , Issinger Olaf-G. , Jiménez de Asúa Luis , Passeron Susana
Published: July 2002

Although a number of nucleoside diphosphate kinases (NDPKs) have been reported to act as inhibitors of metastasis or as a transcription factor in mammals, it is not known whether these functions are linked to their enzymatic activity or how this protein is regulated. In this report, we show that in vitro protein kinase CK2 catalyzed phosphorylation of human NDPK A inhibits its enzymatic activity by inhibiting the first step of its ping‐pong mechanism of catalysis: its autophosphorylation. Upon in vivo 32P labeling of HeLa cells, we observed that both human NDPKs, A and B, were autophosphorylated on histidine residues, however, only the B isoform appeared to be serine phosphorylated.
Binding of the G protein βγ subunit to multiple regions of G protein‐gated inward‐rectifying K+ channels
Authors: Huang Chou-Long , Jan Yuh Nung , Jan Lily Y
Published: July 2002

We have previously shown that direct binding of the βγ subunit of G protein (Gβγ) to both the N‐terminal domain and the C‐terminal domain of a cloned G protein‐gated inward‐rectifying K+ channel subunit, GIRK1, is important for channel activation. We have now further localized the Gβγ binding region in the N‐terminal domain of GIRK1 to amino acids 34–86 and the Gβγ binding region in the C‐terminal domain of GIRK1 to two separate fragments of amino acids 318–374 and amino acids 390–462. Of the four cloned mammalian GIRK subunits, GIRK1–4, GIRK1 and 4 form heteromeric K+ channels in the heart and similar channels in the brain include heteromultimers of GIRK1 and 2, and possibly other GIRK homomultimers and heteromultimers. We found that the N‐terminal and the C‐terminal domains of all four GIRKs bound Gβγ. The Gβγ binding activities for the C‐terminal domains of GIRK2–4 were lower than that for the C‐terminal domain of GIRK1. The higher Gβγ binding activity for the C‐terminal domain of GIRK1 is due to amino acids 390–462 which are unique to GIRK1. We also found that the N‐terminal and C‐terminal domains of GIRKs interacted with each other, and the N‐terminal domain of either GIRK1 or GIRK4 together with the C‐terminal domain of GIRK1 exhibited much enhanced binding of Gβγ. These results are consistent with the idea that the N‐ and C‐terminal domains of the cardiac G protein‐gated K+ channel subunits may interact with each other to form higher affinity binding site(s) for Gβγ.
Androgen regulation of ornithine decarboxylase in human prostatic cells identified using differential display
Authors: Betts Alan M. , Waite Ian , Neal David E. , Robson Craig N.
Published: July 2002

Androgens are essential for normal prostate physiology and have a permissive role in the development and progression of prostate cancer. Using the mRNA differential display technique, ornithine decarboxylase (ODC) was identified to be up‐regulated by androgens in human prostatic LNCaP cells. On Northern analysis, the induction of ODC expression by 10 nM androgen was rapid and continued up to 48 h exposure with a maximum 6.3‐fold up‐regulation. The anti‐androgen Casodex inhibited the androgen‐induced up‐regulation of ODC, whereas the protein synthesis inhibitor cycloheximide did not. Together these data suggest that regulation is mediated through the androgen receptor protein and does require secondary protein synthesis, respectively. The kinetics of induction of ODC were almost identical to those of prostate specific antigen. Taken together these data suggest that ODC is directly regulated by androgens in LNCaP cells.
What nitrates tyrosine? Is nitrotyrosine specific as a biomarker of peroxynitrite formation in vivo?
Authors: Halliwell Barry
Published: July 2002

Peroxynitrite (ONOO−) is a ‘reactive nitrogen species’ that can be formed (among other reactions) by combination of superoxide (O2
·−) and nitric oxide (NO·) radicals. It is being increasingly proposed as a contributor to tissue injury in several human diseases. The evidence presented for peroxynitrite participation usually includes the demonstration of increased nitrotyrosine levels in the injured tissue. Indeed, this is often the only evidence presented: the assumption is that formation of nitrotyrosine is a biomarker specifically diagnostic of ONOO− production. The present article examines this assumption and concludes that nitrotyrosine is a biomarker for ‘nitrating species’ rather than being specific for ONOO−.
Differential expression of genes coding for ABC transporters after treatment of Arabidopsis thaliana with xenobiotics
Authors: Tommasini Roberto , Vogt Esther , Schmid Jürg , Fromentau Myriam , Amrhein Nikolaus , Martinoia Enrico
Published: July 2002

ATP‐binding cassette (ABC) transporters are thought to be involved in many cellular detoxification mechanisms. Performing a BLAST search, we found four distinct expressed sequence tags (EST) of Arabidopsis thaliana highly similar to the human and fungal glutathione‐conjugate ABC transporters. We studied the expression of the corresponding genes in response to various xenobiotics in an effort to gain information on their function. The abundance of transcripts corresponding to one of the genes (EST1) was not affected by the various compounds tested, whereas the abundance of transcripts corresponding to the other three genes (EST2, EST3, EST4) was increased by 1‐chloro‐2,4‐dinitrobenzene, primisulfuron and IRL 1803. Treatment of Arabidopsis with either primisufuron or IRL 1803 resulted in a more than 40‐fold increase in EST2‐specific transcripts.
Identification of the yeast ACR1 gene product as a succinate‐fumarate transporter essential for growth on ethanol or acetate
Authors: Palmieri Luigi , Lasorsa Francesco M , De Palma Annalisa , Palmieri Ferdinando , Runswick Michael J , Walker John E
Published: July 2002

The protein encoded by the ACR1 gene in Saccharomyces cerevisiae belongs to a family of 35 related membrane proteins that are encoded in the fungal genome. Some of them are known to transport various substrates and products across the inner membranes of mitochondria, but the functions of 28 members of the family are unknown. The yeast ACR1 gene was introduced into Escherichia coli on an expression plasmid. The protein was over‐produced as inclusion bodies, which were purified and solubilised in the presence of sarkosyl. The solubilised protein was reconstituted into liposomes and shown to transport fumarate and succinate. Its physiological role in S. cerevisiae is probably to transport cytoplasmic succinate, derived from isocitrate by the action of isocitrate lyase in the cytosol, into the mitochondrial matrix in exchange for fumarate. This exchange activity and the subsequent conversion of fumarate to oxaloacetate in the cytosol would be essential for the growth of S. cerevisiae on ethanol or acetate as the sole carbon source.
Activated human neutrophils rapidly break down nitric oxide
Authors: McBride Alan G , Brown Guy C
Published: July 2002

Isolated human neutrophils produced no detectable (<10 nM) nitric oxide (NO) before or after activation with phorbol 12‐myristate 13‐acetate (PMA) or a chemotactic peptide, N‐formyl‐l‐methionyl‐l‐leucyl‐l‐phenylalanine. Physiological levels of NO (1 μM) added before or after neutrophil activation had no effect on their respiratory burst oxygen consumption. Neutrophils activated with PMA caused very rapid breakdown of exogenously added NO. NO breakdown rates recorded at 250 nM NO were 0.09±0.02 and 3.77±0.23 nmol NO/min/106 cells (n=3) before and after activation respectively and addition of copper‐zinc superoxide dismutase during activation significantly decreased this rate (1.06±0.09 nmol NO/min/106 cells (n=3)), suggesting that superoxide (O−
2) production was mainly responsible for the NO breakdown. These results suggest that activation of human neutrophils in vivo will dramatically decrease surrounding NO levels, potentially causing vasoconstriction, platelet aggregation and adhesion and peroxynitrite (ONOO−) formation.
In vitro interaction of the carboxy‐terminal domain of lamin A with actin
Authors: Sasseville A.Marie-Josée , Langelier Yves
Published: July 2002

The nuclear lamina formed by lamins is localized between the inner nuclear membrane and chromatin. Lamins are thought to be implicated in the higher order chromatin structure. Interactions of lamins with chromatin have been described but the molecular components directly involved in these interactions remain to be identified. Using a GST‐C‐terminal domain of lamin A fusion protein to probe cellular extracts for interacting proteins, we have found that this domain of lamin A binds to nuclear actin. This result suggests that an actin‐based molecular motor linked to the lamina could be involved in the movement of chromatin domains.
The thermosome: archetype of group II chaperonins
Authors: Klumpp Martin , Baumeister Wolfgang
Published: July 2002

The thermosome, the chaperonin of the archaea, and its homologue from the cytosol of eukaryotes, known as TRiC or CCT, form a distinct subfamily of the chaperonins that does not depend on a co‐chaperonin for protein folding activity. Recent structural data obtained by cryo‐ electron microscopy and X‐ray crystallography provide the first insights into a novel mechanism remarkably different from that of the bacterial GroEL‐GroES system.
Cytochrome P450 immobilisation as a route to bioremediation/biocatalysis
Authors: Lamb Stephen B. , Lamb David C. , Kelly Steven L. , Stuckey David C.
Published: July 2002

The diverse substrate specificity of the cytochrome P450 (P450; CYP) enzyme superfamily offers the opportunity to develop enzymatic systems for environmental detoxification and biotransformations of drugs, pesticides and fine chemicals. Here we report on the immobilisation of a fusion protein between plant cytochrome P450‐71B1 (CYP71B1) and its electron donor, plant NADPH cytochrome P450 reductase using an oil‐in‐water macro‐emulsion, termed polyaphron, which contains a proportion of internal organic phase (φ) greater than 0.74. Efficiency of P450 immobilisation was greater than 85%, and in this state enzymatic activity could be measured for more than 24 h at 15°C. Chlortoluron, a recalcitrant herbicide pollutant in the environment, was shown to be metabolised, with the major metabolite (N‐monodemethylated chlortoluron) being separated from the substrate due to partitioning into the aqueous phase. The turnovers exhibited superactivity compared with those obtained using free enzyme located in membranes prepared following heterologous expression in Saccharomyces cerevisiae and Escherichia coli. The potential to exploit the unprecedented catalytic diversity of the P450 superfamily in biocatalysis is discussed.
Expression of the Escherichia coli cyo operon in Paracoccus denitrificans results in a fully active quinol oxidase of unexpected heme composition
Authors: Schröter Thomas , Winterstein Christine , Ludwig Bernd , Richter Oliver-Matthias H
Published: July 2002

The cyo operon coding for the membrane‐bound bo
3‐type quinol oxidase of Escherichia coli has been expressed in a Paracoccus denitrificans strain deleted in its endogenous ba
3 quinol oxidase. Using the P. denitrificans qox promoter, the His tagged protein complex is synthesized to a level comparable to that in E. coli and the enzyme purified in a single step on a metal‐chelating column. Whereas the activity of the isolated complex matches that of the oxidase purified directly from E. coli, the heterologously expressed oxidase does not show the characteristic heme composition but now carries heme a in its binuclear site.
Possible stage‐specific function of NF‐κB during pre‐B cell differentiation
Authors: Guerrini Luisa , Molteni Angela , Blasi Francesco
Published: July 2002

Lipopolysaccharide (LPS)‐induced differentiation of the murine pre‐B cell line 70Z/3 is a model for pre‐B to B cell differentiation and has been used to show that the transcription factor NF‐κB is essential to induce the expression of the Igκ gene. We have investigated the mechanism involved in late stages of the process when all cells have reached a more mature B phenotype, i.e. beyond 48 up to 96 h of LPS treatment. NF‐κB binding activity was induced at early times by LPS treatment, but its DNA binding activity disappeared after 84 h of LPS treatment. Accumulation of IκBα protein in the nucleus correlated with the disappearance of NF‐κB activity at 72, 84 and 96 h, and treatment of nuclear extracts of 72–96 h LPS‐treated cells with Na‐deoxycholate restored NF‐κB binding activity. The data indicate that NF‐κB, while important to initiate the process of Igκ gene transcription in 70Z/3 pre‐B cells, is no longer required for its maintenance in differentiated 70Z/3 cells.
Distribution and functional diversification of the ras superfamily inSaccharomyces cerevisiae
Authors: Garcia-Ranea Juan Antonio , Valencia Alfonso
Published: July 2002

The recent availability of the fullSaccharomyces cerevisiaegenome sequence offers a first opportunity to analyze the composition, function and evolution of GTPases in the ras‐p21 superfamily. This superfamily in yeast is composed of 29 proteins divided into five families: ras with four sequences implicated in cell signalling; rho, six genes related to the cell shape machinery; ypt‐rab, ten proteins with different roles in intracellular trafficking; arf‐sar, seven proteins related to vesicular trafficking in secretory pathways; and ran, two proteins acting as components of the nuclear transport system. The superfamily covers a wide range of cellular functions from signalling to intracellular trafficking, while conserving the structural framework and a common mechanism of GTP hydrolysis.
Novel defensin subfamily from spinach (Spinacia oleracea)
Authors: Segura Ana , Moreno Manuel , Molina Antonio , Garcı́a-Olmedo Francisco
Published: July 2002

Antimicrobial peptides (So‐D1‐7) were isolated from a crude cell wall preparation from spinach leaves (Spinacia oleracea cv. Matador) and, judged from their amino acid sequences, six of them (So‐D2‐7) represented a novel structural subfamily of plant defensins (group IV). Group‐IV defensins were also functionally distinct from those of groups I–III. They were active at concentrations <20 μM against Gram‐positive (Clavibacter michiganensis) and Gram‐negative (Ralstonia solanacearum) bacterial pathogens, as well as against fungi, such as Fusarium culmorum, F. solani, Bipolaris maydis, and Colletotrichum lagenarium. Fungal inhibition occurred without hyphal branching. Group‐IV defensins were preferentially distributed in the epidermal cell layer of leaves and in the subepidermal region of stems.
Loss of imprinting of IGF2 and not H19 in breast cancer, adjacent normal tissue and derived fibroblast cultures
Authors: van Roozendaal C.E.P , Gillis A.J.M , Klijn J.G.M , van Ooijen B , Claassen C.J.C , Eggermont A.M.M , Henzen-Logmans S.C , Oosterhuis J.W , Foekens J.A , Looijenga L.H.J
Published: July 2002

Insulin‐like growth factors are involved in the paracrine growth regulation of human breast tumor cells. IGF2 is imprinted in most tissues, and shows expression of the paternal allele only. To investigate whether disruption of this monoallelic IGF2 expression is involved in breast cancer development, a series of primary tumors and adjacent, histologically normal, breast tissue samples, as well as matched primary in vitro fibroblast cultures were studied. Biallelic expression (partial) of IGF2 was found in the majority of in vivo samples, and corresponding fibroblast cultures, while monoallelic expression was found in a normal breast sample. In contrast, H19, a closely apposed, but reciprocally imprinted gene, assumed to be regulated by a common control element, showed retention of monoallelic H19 expression in all in vivo and in the majority of in vitro samples. These data indicate that IGF2, but not H19, is prone to loss of imprinting in breast cancer.
A RNA polymerase with transcriptional activity at 0°C from the Antarctic bacterium Pseudomonas syringae
Authors: Uma S. , Jadhav R.S. , Seshu Kumar G. , Shivaji S. , Ray M.K.
Published: July 2002

A DNA‐dependent RNA polymerase was purified from the Antarctic psychrotrophic bacterium Pseudomonas syringae. The RNA polymerase showed a typical eubacterial subunit composition with β, β′, α2 and σ subunits. The subunits cross‐reacted with antibodies raised against holoenzyme and the individual subunits of the RNA polymerase of Escherichia coli. However, the enzyme was considered unique, since unlike the RNA polymerase of mesophilic E. coli it exhibited significant and consistent transcriptional activity (10–15%) even at 0°C. But, similar to the enzyme from the mesophilic bacterium, the RNA polymerase from P. syringae exhibited optimum activity at 37°C. The study also demonstrates that the RNA polymerase of P. syringae could preferentially transcribe the cold‐inducible gene cspA of E. coli only at lower temperatures (0–22°C). The polymerase was also observed to be relatively more rifampicin‐resistant during transcription at lower temperature.
Prenatal stress and postnatal development of neonatal rats — sex‐dependent effects on emotional behavior and learning ability of neonatal rats
Authors: Nishio Hiroaki , Kasuga Shigeo , Ushijima Mitsuyasu , Harada Yasuo
Published: July 2002

Maternal sound stress (800 Hz; 77 dB, every other minute for 15 min/day, from day 10 to 18 of gestation), combined with forced swimming stress (15 min/day), was found to cause potentiation of sound‐induced loss of locomotor activity, referred to as emotional behavior, of male offspring, but not that of female offspring, at 4 weeks of age. Maternal stress also caused an increase in the total number of errors by male, but not female offspring in the water‐maze test at 6 weeks of age. These effects of stress on emotional behavior and learning behavior were abolished when dams were pretreated with buspirone (30 min before the stress, from day 8 to 18 of gestation). Thus, prenatal stress might have sex‐dependent effects on emotional behavior and learning ability of neonatal rats.
Retinoic acid as a chemotactic molecule in neuronal development
Authors: Maden M. , Keen G. , Jones G.E.
Published: July 2002

We describe the results of experiments on dissociated cultures of embryonic chick neural tissue which were designed to investigate further the role of all‐trans‐retinoic acid (tRA) on neurite outgrowth and, by inference, on the developing nervous systemin vivo. We show that tRA increases both the number of neurites and the length of neurites extending from these chick neural tube cells at nM concentrations. Secondly, using the newly designed Dunn chamber in which stable gradients of compounds can be generated, we show that neurites respond to a gradient of tRA by growing up the gradient. These observations indicate a role for RAin vivo, not only in the initial outgrowth of neurites, but also in their guidance to the appropriate targets.
Retroviral transfection of the lacZ gene from Liz‐9 packaging cells to glioma spheroids
Authors: Øyvind Per Enger , Visted Therese , Thorsen Frits , Bjerkvig Rolf , Lund‐Johansen Morten
Published: July 2002

Despite the development of numerous vectors for gene transfection to gliomas, patient survival length remains unaffected in clinical trials. For glioma gene therapy to be successful, the extent of gene transfer to the solid tumor tissue has to be high. In the present work we review some of the vector types and strategies so far utilized in experimental and clinical glioma gene therapy. Since gene transfer efficacy into solid glioma tissue is unknown for many vectors, we studied the gene transfer efficacy into multicellular spheroids derived from a human glioma cell line GaMg as well as into spheroids derived from human glioma biopsies (glioblastoma multiforme, GBM). A replication deficient retroviral vector from the Liz 9 packaging cell line was used for transfer of the bacterial β‐galactosidase lacZ gene into the target tissue. Gene transfer was obtained by adding medium containing virus from the producer cells to the target tissue. The experiments were also conducted with EGF (epidermal growth factor) added to the medium. The data show that the transfection rate ranged from 0–4.5% where the transfection efficacy was higher in spheroids after the addition of EGF. Most of the transfected cells were found at the surface, but transfected cells could also be observed in the center of the spheroids. We conclude that using this vector system, the transfection efficacy was low, even if the number of replicating cells was increased by adding EGF. The findings are consistent, and may partly explain, the lack of effect using this vector system during in vivo studies.