bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunohistochemical localisation of the serotonin 5‐HT2B receptor in mouse gut, cardiovascular system, and brain
Authors: Choi Doo-Sup , Maroteaux Luc
Published: April 2003

We recently reported the cloning of a new member of the serotonin 5‐HT2 family, the 5‐HT2B receptor. We now report the production and characterisation of a specific antiserum directed against the C‐terminal portion of the mouse 5‐HT2B receptor. After affinity purification, this polyclonal antibody recognises specifically the mouse 5‐HT2B receptor. Immunohistochemical analysis of cryosections from various adult mouse tissues reveals a major 5‐HT2B receptor expression in stomach, intestine and pulmonary smooth muscles as well as in myocardium. Furthermore, the antiserum recognises specific areas of the mouse brain, including cerebellar Purkinje cells and their projection areas.
Activation of platelet‐derived growth factor (PDGF) receptor dephosphorylation in intact Swiss 3T3 cells by elevators of intracellular Ca2+ and cAMP
Authors: Böhmer Frank-D. , Böhmer S.-Annette
Published: April 2003

We investigated the modulation of platelet‐derived growth factor (PDGF) receptor dephosphorylation in Swiss 3T3 cells using a novel assay permitting monitoring of receptor dephosphorylation in intact cells. PDGF treatment of the cells reduced the receptor dephospphorylation rate to 41%, the elevators of intracellular Ca2+, A23187 and thapsigargin increasing it to 227 and 138%, respectively. The cAMP elevators forskolin and isobutylmethylxanthine also accelerated PDGF receptor dephosphorylation. The involvement of Ca2+‐ and cAMP‐dependent protein kinases in the regulation of PDGF receptor dephosphorylation is suggested.
Inhibition of protein kinase C μ by various inhibitors. Inhibition from protein kinase c isoenzymes
Authors: Gschwendt Michael , Dieterich Sabine , Rennecke Joerg , Kittstein Walter , Mueller Hans-Joachim , Johannes Franz-Josef
Published: April 2003

Various inhibitors were tested for their potential to suppress the kinase activity of protein kinase C μ (PKCμ) in vitro and in vivo. Among the staurosporine‐derived, rather selective PKC inhibitors the indolocarbazole Gö 6976 previously shown to inhibit preferentially cPKC isotypes proved to be a potent inhibitor of PKCμ with an IC5 of 20 nM, whereas the bisindolymaleimide Gö 6983 was extremely ineffective in suppressing PKCμ kinase activity with a thousand‐fold higher ICm of 20 μM. Other strong inhibitors of PKCμ were the rather unspecific inhibitors staurosporine and K252a. Contrary to the poor inhibition of PKCμ by Gö 6983, this compound was found to suppress in vitro kinase activity of PKC isoenzymes from all three subgroups very effectively with IC50 values from 7 to 60 nM. Thus, Gö 6983 was able to differentiate between PKCμ and other PKC isoenzymes being useful for selective determination of PKCμ kinase activity in the presence of other PKC isoenzymes.
A novel leptin receptor isoform in rat
Authors: Wang May-Yun , Yan Ting Zhou , Newgard Christopher B. , Unger Roger H.
Published: April 2003

Five mouse and human leptin receptors (Ob‐R) have recently been identified, a long isoform (Ob‐Rb), preferentially expressed in hypothalamus, and 4 short isoforms, Ob‐Ra, Ob‐Rc, Ob‐Rd, and Ob‐Re. We have identified a new short isoform in the rat, r‐OB‐Rf, with 6 C‐terminal amino acids and a 3′ untranslated region without homology to other Ob‐R isoforms. Its higher expression in rat liver and spleen compared to brain, stomach, kidney, thymus, heart, lung and hypothalamus, contrasts with Ob‐Ra and Ob‐Rb homologues and raises possibilities of as yet unidentified roles for members of the growing Ob‐R gene family.
Tyrosine dephosphorylation of glycogen synthase kinase‐3 is involved in its extracellular signal‐dependent inactivation
Authors: Murai Hiroshi , Okazaki Michiko , Kikuchi Akira
Published: April 2003

We examined whether extracellular signals regulate glycogen synthase kinase‐3 (GSK‐3) activity through tyrosine dephosphorylation of GSK‐3. In resting Chinese hamster ovary cells overexpressing the human insulin receptor (CHO‐IR cells), GSK‐3 was tyrosine‐phosphorylated and active. Insulin and 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) induced inactivation and tyrosine dephosphorylation of GSK‐3. It is known that Ser‐9 of GSK‐3β is phosphorylated in response to insulin and that the phosphorylation of this amino acid residue causes inactivation of GSK‐3β. However, the ectopically expressed GSK‐3βΔ9, in which the N‐terminal nine amino acids of GSK‐3β were deleted, was still inactivated and tyrosine‐dephosphorylated in response to insulin. Protein phosphatase 2A treatment partially reversed insulin‐induced GSK‐3β inactivation, but did not change GSK‐βΔ9 inactivation. In CHO‐IR cells where protein kinase C was down‐regulated, TPA neither inactivated nor tyrosine‐dephosphorylated GSK‐3. However, insulin inactivated and tyrosine‐dephosphorylated GSK‐3, although to a lesser degree than in the control cells. These results suggest that in addition to serine phosphorylation, tyrosine dephosphorylation of GSK‐3 is also important for the regulation of GSK‐3 activity in response to extracellular signals and that insulin regulates GSK‐3 activity through both protein kinase C‐dependent as well as protein kinase C‐independent pathways.
A sensor protein involved in induction of nitrate assimilation in Azotobacter chroococcum
Authors: Muñoz-Centeno María C. , Peciña Ana , Cejudo Francisco J. , Paneque Antonio
Published: April 2003

Nitrogen‐fixing Azotobacter chroococcum cells, but not ammonium‐ or nitrate‐grown cells, exhibited two polypeptide components of 22 and 35 kDa, respectively, that we termed P22 and P35. Bidimensional polyacrylamide gel electrophoresis analysis of preparations from N2‐fixing cells that had been transferred to nitrate medium and then incubated for 2 h revealed that P22 had shifted to a more acidic part of the gel while P35 did not change its electrophoretic pattern. Using [32P]orthophosphoric acid it could be demonstrated that the shift in mobility of P22 was due to the phosphorylation of the polypeptide dependent on nitrate (nitrite). The A. chroococcum TR1 strain, which is unable to use nitrate as a nitrogen source and displays activities of nitrogenase, nitrate reductase and nitrite reductase, exhibited both polypeptides. In contrast, P22 and P35 were absent from A. chroococcum MCD1, a mutant strain that cannot assimilate nitrate and lacks the nitrate‐reducing enzymatic system. The results suggest that P22 could act as a sensor protein for nitrate in A. chroococcum.
Delineating functionally important regions and residues in the cathepsin B propeptide for inhibitory activity
Authors: Chen Yanmin , Plouffe Céline , Ménard Robert , Storer Andrew C.
Published: April 2003

Synthetic peptides derived from the proregion of rat cathepsin B were used to identify functionally important regions and residues for cathepsin B inhibition. Successive 5 amino acid deletions of a 56 amino acid propeptide from both the N‐ and C‐termini has allowed the identification of two regions important for inhibitory activity: the NTTWQ (residues 21p–25p) and CGTVL (42p–46p) regions. Alanine scanning of residues within these two regions indicates that Trp‐24p and Cys‐42p contribute strongly to inhibition, their replacement by Ala resulting in 160‐ and 140‐fold increases in K

i
, respectively.
Effect of purified, soluble urokinase receptor on the plasminogen‐prourokinase activation system
Authors: Behrendt Niels , Danø Keld
Published: April 2003

The extracellular proteolytic pathway mediated by the urokinase plasminogen activator (uPA) is a cascade system, initiated by activation of the zymogen, pro‐uPA. Pro‐uPA as well as uPA binds to the cellular uPA receptor (uPAR) which has a central function in cell‐dependent acceleration of the cascade system. This role of uPAR is generally assumed to be a positioning effect since uPAR‐expressing cells exclusively stimulate the activation of cell surface‐bound plasminogen (Ellis et al. (1993) Methods Enzymol. 223, 223–233). However, it was recently reported that a recombinant, soluble uPAR (suPAR) was capable of accelerating plasminogen activation in solution (Higazi et al. (1995) J. Biol. Chem. 270, 17375–17380). In this work we show that suPAR as such has no accelerative role. In contrast, the progress of the activation reactions in a soluble system with pro‐uPA and plasminogen was found to be attenuated by suPAR. This delay of the activation system was shown to include a partial inhibition of the plasmin‐mediated activation of pro‐uPA as well as of the uPA‐mediated activation of plasminogen.
Identification of nuclear localization signals within the zinc fingers of the WT1 tumor suppressor gene product
Authors: Bruening Wendy , Moffett Peter , Chia Shea , Heinrich Gunther , Pelletier Jerry
Published: April 2003

WT1 encodes a zinc finger protein with a key role in urogenital development that is inactivated in a subset of Wilms' tumors. This tumor suppressor gene product contains an amino‐terminal dimerization domain required for trans‐inhibition of wild‐type WT1 activity by mutants defective for DNA binding. In the course of characterizing truncation mutants of WT1, we noted that the WT1 zinc fingers contain two functionally independent targeting signals required for nuclear localization of the protein. These novel signals lie within zinc fingers I and within zinc fingers II and III. We demonstrate that nuclear targeting of the WT1 homodimerization domain functionally antagonizes activity of the wild‐type protein activity.
Translocation of diacylglycerol kinase α to the nuclear matrix of rat thymocytes and peripheral T‐lymphocytes
Authors: Wada Ikuo , Kai Masahiro , Imai Shin-ichi , Sakane Fumio , Kanoh Hideo
Published: April 2003

The cytosolic α‐diacylglycerol kinase (DGK) was translocated to and tightly associated with the nuclear matrix when rat thymocytes and peripheral T‐lymphocytes were stimulated with concanavalin A or anti‐T‐cell receptor antibody. This translocation occurred rather slowly and was completed in 3–4 h after cell stimulation. We also detected significant accumulation of nuclear phosphatidic acid interpreted as being formed by the translocated enzyme. The enzyme translocation is not directly linked to phosphoinositide turnover and protein phosphorylation, since phorbol myristate acetate and calcium ionophore did not affect the cellular DGKα and since we detected no covalent modification of the enzyme molecule. Although the mechanisms underlying the enzyme translocation remain unknown, our results indicate that DGKα participates in nuclear phospholipid metabolism occurring at the intermediate stage of lymphocyte activation.
Regulation of intracellular pH by phospholipase A2 and protein kinase C upon neutrophil adhesion to solid substrata
Authors: Galkina S.I. , Sud'ina G.F. , Margolis L.B.
Published: April 2003

Adhesion to solid substrata has been shown to increase intracellular pH (pH(i)) of fibroblasts and of other cells (FEBS Lett. (1988) 234, 449–450; Proc. Natl. Acad. Sci. USA (1989) 86, 4525–4529; J. Biol. Chem. (1990) 265, 1327–1332; Exp. Cell Res. (1992) 200, 211–214; FEBS Lett. (1995) 374,17–20). We have found that the inhibitors of PLA2, 4‐bromophenacyl bromide and manoalide, completely blocked the increase of pH(i) and spreading of neutrophils upon adhesion to solid substrata. Inhibition of phospholipase C with neomycin or removal of extracellular Ca2+ affects neither neutrophil spreading nor their pH(i). Inhibition of PKC with H‐7 or staurosporin increased pH(i). PMA, an activator of PKC, dramatically decreased pH(i) but did not impair the spreading of neutrophils. The effect of arachidonic acid, a product of PLA2 activity, on neutrophil pH(i) and spreading was similar to that of PMA. H‐7, an inhibitor of PKC, partially blocked the effect of arachidonic acid (AA) on pH(i). BW755C, an inhibitor of AA metabolism by cyclooxygenase or lipoxygenase, affected neither the pH(i) nor cell spreading. We propose that the increase of pH(i) upon neutrophil adhesion is mediated by PLA2 activity, while PKC decreased pH(i). AA produced by PLA2 activates PKC, thus forming a feedback regulation of pH(i).
Chemical modification of the essential arginine residues of pyruvate dehydrogenase prevents its phosphorylation by kinase
Authors: Nemerya N.S. , Zemskova M.A. , Nyukhalkina I.A. , Khailova L.S.
Published: April 2003

The mechanism of regulatory phosphorylation of the pyruvate dehydrogenase component (E1) of muscle pyruvate dehydrogenase complex was studied. Chemical modification of the arginine residues essential for substrate binding was shown to prevent incorporation of 32P from [γ‐32P]ATP into E1 catalyzed by kinase and to exclude completely the interaction of holo‐E1 with pyruvate. It is proposed that negatively charged phosphoseryl residues may compete with pyruvate for the active site arginine and thereby block the substrate binding.
Evidence for the presence of kinetin in DNA and cell extracts
Authors: Barciszewski Jan , Siboska Gunhild E. , Pedersen Bent O. , Clark Brian F.C. , Rattan Suresh I.S.
Published: April 2003

In contrast to the current view that kinetin (N
6‐furfuryladenine) is an unnatural and synthetic compound, we have detected it in commercially available DNA, in freshly extracted cellular DNA from human cells and in plant cell extracts by two independent methods. First, we discovered that N
6‐furfuryladenine has electrochemical properties that can be applied for monitoring this modified based by a HPLC/UV/EC method. Second, we have confirmed electrochemical assignments by mass‐spectrometric analysis. A pathway of kinetin formation is proposed in which the formation of furfural by oxidative damage of the deoxyribose moiety of DNA is followed by its reaction with adenine residues to form N
6‐furfuryladenine. Since this modification can lead to mutations, the odd DNA base has to be removed by repair enzymes.
Identification and expression of six family genes in mouse retina
Authors: Kawakami Kiyoshi , Ohto Hiromi , Takizawa Toshihiro , Saito Takuma
Published: April 2003

We identified five cDNA clones of the Six gene family which are expressed in retina. They are Six2, Six3α and Six3β (which are derived from alternative splicing forms), Six5, and AREC3/Six4. All of these Six family genes possess extensive sequence similarity among each other in the so‐homologous region (Six domain and homeodomain) but differ greatly in structure in some other regions. The amino acid sequence similarity of the so‐homologous region to the previously identified AREC3/Six4 is 70.1% for Six2, 57.3% for Six3α and Six3β, and 70.3% for Six5. The expression of these genes was observed in inner and outer nuclear layer, ganglion cell layer, and pigment epithelium of mouse retina by in situ hybridization. The so‐homologous region of each Six family protein has specific DNA binding activity. Six5 and Six2 bind to the same sequence as does AREC3/Six4, while Six3 does not. These observations suggest that some of the Six family genes can regulate the same target genes.
Defective ATP‐dependent mucin secretion by cystic fibrosis pancreatic epithelial cells
Authors: Montserrat C. , Merten M. , Figarella C.
Published: April 2003

The response of confluent monolayers of normal and cystic fibrosis (CF) pancreatic epithelial cells to stimulation by extracellular ATP and ATP analogues was investigated in terms of mucin secretion. Mucin secretion was measured as release of M1 antigens by a direct sandwich enzyme immunoassay. Extracellular ATP provoked rapid (≤15 min) and strong mucin secretion (+480 ± 35%) by the normal pancreatic cell lines but was not able to induce mucin secretion by the CF cell lines. The order of efficacy of nucleotide agonists with ATP > ADP > AMP > adenosine was that of typical P2‐purinergic receptors. ATP induced a rapid and transient intracellular [Ca2+] mobilization in both normal and CF pancreatic epithelial cells. This work demonstrated that CFTR seemed to mediate ATP‐dependent mucin secretion.
The C‐terminal of rat 4‐hydroxyphenylpyruvate dioxygenase is indispensable for enzyme activity
Authors: Lee Meng-Huee , Zhang Zhi-Hong , MacKinnon Colin H. , Baldwin Jack E. , Crouch Nicholas P.
Published: April 2003

We have cloned and overexpressed rat 4‐hydroxyphenylpyruvate dioxygenase (4HPPD) in Escherichia coli. The soluble, active recombinant enzyme was shown to contain both 4HPPD and α‐ketoisocaproate dioxygenase (αKICD) activity. However, upon truncation of the 14 amino acids at the C‐terminus by site‐directed mutagenesis, the resulting mutant enzyme (rat F antigen) exhibited complete loss of 4HPPD and αKICD activities. This finding suggests that the C‐terminal extension domain plays an essential role in the catalytic activity of the enzyme.
Unusual enzyme characteristics of aspartyl‐tRNA synthetase from hyperthermophilic archaeon Pyrococcus sp. KOD1
Authors: Fujiwara Shinsuke , Lee SungGa , Haruki Mitsuru , Kanaya Shigenori , Takagi Masahiro , Imanaka Tadayuki
Published: April 2003

The aspA gene, encoding the aspartyl‐tRNA synthetase (AspRS) from the hyperthermophilic archaeon Pyrococcus sp. KOD1, was expressed in Escherichia coli. The KOD1 AspRS, which was purified to homogeneity and was shown to be functional in dimeric form, aminoacylated tRNA from KOD1. The optimum temperature for this activity was 65°C, which was lower than that for the cell growth of KOD1 (85°C). However, it increased to 75°C by the addition of polyamine molecules, such as putrescine, spermine, and spermidine. Analysis of the thermal denaturations of the enzyme and of KOD1‐tRNA indicated that neither of them was denatured at temperatures below 70°C. These results suggest polyamine is one of the factors which are required to stabilize the AspRS‐tRNA complex in vivo. In order to determine whether the nucleotide triphosphate (NTP) is required for Asp‐tRNA synthesis, the aminoacylation was examined in the presence of each of the four NTPs. AspRS most effectively aminoacylated tRNA in the presence of ATP. However, we also found that the enzyme aminoacylated it even in the presence of GTP and UTP as well. Archaeon synthetase may have an interesting system to utilize other NTPs than ATP. The extreme conditions of early life may have given rise to these unique characteristics which then disappeared from developed organisms through evolution.
Two phosphatidylethanol classes separated by thin layer chromatography are produced by phospholipase D in rat brain hippocampal slices
Authors: Sarri Elisabet , Servitja Joan-Marc , Picatoste Fernando , Claro Enrique
Published: April 2003

Noradrenaline‐ and ionomycin‐stimulated as well as basal phospholipase D activity from rat hippocampus produced, in the presence of ethanol, two different classes of [32P]phosphatidylethanol (designated I and II), which were separated by thin layer chromatography. Endogenous labeling experiments using 3H‐fatty acids showed that two different classes of phosphatidylcholine, separated by two‐dimensional TLC, one enriched with high incorporation of [3H]arachidonic acid (B) and the other with [3H]myristic acid (A), were the most likely sources for the two classes of phosphatidylethanol. Experiments where individual 32P‐phospholipids extracted from [32P]Pi‐labeled hippocampal slices were incubated with cabbage phospholipase D, in the presence of ethanol, showed that each class of [32P]phosphatidylcholine, i.e. A and B, produced a different band of [32P]phosphatidylethanol, with the same mobility in TLC as phosphatidylethanol II and I, respectively.
Purification of histone deacetylase HD1‐A of germinating maize embryos
Authors: Brosch Gerald , Goralik-Schramel Maria , Loidl Peter
Published: April 2003

We have purified the soluble nuclear histone deacetylase HD1‐A of germinating maize embryos. By a combination of 6 chromatographic steps we achieved a 77 000‐fold purification of an enzymatically active protein. Gel filtration chromatography revealed a molecular weight of 45 kDa of the native enzyme and electrophoretic analysis of the purified enzyme by SDS‐PAGE resulted in a single band at a molecular weight of 48 kDa, indicating that the enzyme is a monomer protein. When fractions with enzyme activity of different stages of chromatographic purification were subjected to isoelectric focusing, enzyme activity focused at a pH of around 6.4 as measured in an activity gel assay; second dimension SDS‐PAGE again revealed a protein spot at a molecular weight of 48 kDa.
Valproic acid, a mood stabilizer and anticonvulsant, protects rat cerebral cortical neurons from spontaneous cell death: a role of histone deacetylase inhibition
Authors: Jeong Mi Ra , Hashimoto Ryota , Senatorov Vladimir V , Fujimaki Koichiro , Ren Ming , Lee Min Soo , Chuang De-Maw
Published: April 2003

We studied the neuroprotective effects of valproic acid (VPA), a primary mood stabilizer and anticonvulsant, in cultured rat cerebral cortical neurons (CCNs). CCNs underwent spontaneous cell death when their age increased in culture. As shown by mitochondrial activity and calcein‐AM assays, treatment of CCNs with VPA starting from day 9 in vitro markedly increased viability and prolonged the life span of the cultures. The neuroprotective action of VPA was time‐dependent and occurred at therapeutic levels with a maximal effect at about 0.5 mM. LiCl (1 mM) also protected CCNs from aging‐induced, spontaneous cell death but less effectively. VPA‐induced neuroprotection in aging CCN cultures was associated with a robust increase in histone H3 acetylation levels and the protective effect was mimicked by treatment with a histone deacetylase inhibitor, trichostatin A, but not by VPA analogs which are inactive in blocking histone deacetylase. Our results suggest a role of histone deacetylase inhibition in mediating the neuroprotective action of VPA.
The major peptidyl‐prolyl isomerase activity in thylakoid lumen of plant chloroplasts belongs to a novel cyclophilin TLP20
Authors: Edvardsson Anna , Eshaghi Said , Vener Alexander V , Andersson Bertil
Published: April 2003

Fractionation of proteins from the thylakoid lumen of spinach chloroplasts combined with peptidyl‐prolyl cis/trans isomerase (PPIase) measurements revealed a major isomerase activity that was ascribed to a novel enzyme TLP20 (hylakoid umen PIase of kDa). TLP20 was inhibited by cyclosporin A and mass spectrometric sequencing of tryptic peptides confirmed its classification as a cyclophilin. Genes encoding similar putative thylakoid cyclophilins with a unique insert of three amino acids NPV in their N‐termini were found in chromosome 5 of both Arabidopsis and rice. TLP20 is suggested to be the major PPIase and protein folding catalyst in the thylakoid lumen of plant chloroplasts.
An improved rhodopsin/EGFP fusion protein for use in the generation of transgenic Xenopus laevis
Authors: Jin Shengnan , McKee Timothy D , Oprian Daniel D
Published: April 2003

Previous studies by Papermaster and coworkers introduced the use of rhodopsin–green fluorescent protein (rho–GFP) fusion proteins in the construction of transgenic Xenopus laevis with retinal rod photoreceptor cell‐specific transgene expression [Moritz et al., J. Biol. Chem. 276 (2001) 28242–28251]. These pioneering studies have helped to develop the Xenopus system not only for use in the investigation of rhodopsin biosynthesis and targeting, but for studies of the phototransduction cascade as well. However, the rho–GFP fusion protein used in the earlier work had only 50% of the specific activity of wild‐type rhodopsin for activation of transducin and only 10% of the activity of wild‐type in rhodopsin kinase assays. While not a problem for the biosynthesis studies, this does present a problem for investigation of the phototransduction cascade. We report here an improved rhodopsin/EGFP fusion protein in which placement of the EGFP domain at the C‐terminus of rhodopsin results in wild‐type activity for activation of transducin, wild‐type ability to serve as a substrate for rhodopsin kinase, and wild‐type localization of the protein to the rod photoreceptor cell outer segment in transgenic X. laevis.
A comparison of the association of spermine with duplex and quadruplex DNA by NMR
Authors: Keniry Max A
Published: April 2003

The association of [1′,1″‐13C2]spermine ([1′,1″‐13C2]N,N′‐bis(3‐aminopropyl)‐1,4‐butanediamine) with duplex and quadruplex DNA has been studied by nuclear magnetic resonance spectroscopy. 1D NOESY experiments using two‐way selective cross‐polarization (ISI‐SCP‐NOESY) showed spermine intramolecular NOEs are either weakly positive or weakly negative when spermine is complexed to duplex B‐DNA and linear four‐stranded quadruplex DNA. In contrast, large negative intramolecular NOEs are observed when spermine is complexed to two distinct forms of folded quadruplex DNA suggesting greater immobilization of spermine on these folded DNA quadruplexes. No changes in the quadruplex stem structure are observed but there are minor changes to the loop structure of a two‐stranded folded quadruplex on binding spermine.
Proton translocation by transhydrogenase
Authors: Jackson J.Baz
Published: April 2003

Transhydrogenase, in animal mitochondria and bacteria, couples hydride transfer between NADH and NADP+ to proton translocation across a membrane. Within the protein, the redox reaction occurs at some distance from the proton translocation pathway and coupling is achieved through conformational changes. In an ‘open’ conformation of transhydrogenase, in which substrate nucleotides bind and product nucleotides dissociate, the dihydronicotinamide and nicotinamide rings are held apart to block hydride transfer; in an ‘occluded’ conformation, they are moved into apposition to permit the redox chemistry. In the two monomers of transhydrogenase, there is a reciprocating, out‐of‐phase alternation of these conformations during turnover.
Flagellar movement driven by proton translocation
Authors: Blair David F
Published: April 2003

The bacterial flagellar motor couples ion flow to rotary motion at high speed and with apparently fixed stoichiometry. The functional properties of the motor are quite well understood, but its molecular mechanism remains unknown. Recent studies of motor physiology, coupled with mutational and biochemical studies of the components, put significant constraints on the mechanism. Rotation is probably driven by conformational changes in membrane–protein complexes that form the stator. These conformational changes occur as protons move on and off a critical Asp residue in the stator protein MotB, and the resulting forces are applied to the rotor protein FliG.
Of JAKs, STATs, blind watchmakers, jeeps and trains
Authors: Kerr Ian M. , Costa-Pereira Ana P. , Lillemeier Björn F. , Strobl Birgit
Published: April 2003

Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling is essential but not sufficient for full responses to the interferons (IFNs), most cytokines and some growth factors. The IFN‐γ and interleukin‐6 (IL‐6) response pathways have been used as model systems to investigate both the signals involved and their organisation. Activated STAT1 diffuses freely in the cytoplasmic and nuclear compartments of the cell providing a ‘random walk’ element in the IFN‐γ response. Completely foreign chimeric receptors and, remarkably, in the absence of STAT3, the endogenous IL‐6 receptor can efficiently mediate an IFN‐γ‐like response. Accordingly all of the signals required for an IFN‐γ response can be generated through physiological levels of a foreign ligand. JAK/STAT signalling, therefore, appears ‘soft‐wired’, modular and highly flexible with substantial overlap between different response pathways. The data are consistent with a generic or ‘core’ set of signals from JAK/receptor complexes with ‘add‐on’ modulation through specific receptor motifs. The cellular background likely profoundly affects the nature of the response.
Basolateral Ca2+‐dependent K+‐channels play a key role in Cl—secretion induced by taurodeoxycholate from colon mucosa
Authors: Moschetta Antonio , Portincasa Piero , Debellis Lucantonio , Petruzzelli Michele , Montelli Roberta , Calamita Giuseppe , Gustavsson Pontus , Palasciano Giuseppe
Published: April 2003

Abstract The diarrhea associated with malabsorption of bile salts such as the secondary hydrophobic taurodeoxycholate (TDC) may be partly explained by the TDC‐induced increase in colon Cl− secretion. We, therefore, investigated the effects of TDC (0.5–8 mM) on electrical parameters and electrolyte transport of rat proximal colon mucosa mounted in Ussing chambers. Colonic secretion, measured as short circuit current (I SC), progressively increased on mucosal incubation with TDC ranging 0.5–2 mM; up to TDC 2 mM, a spontaneous recovery toward control values with no changes in epithelial resistance (Rt), and lactate dehydrogenase (LDH) release was observed. In contrast, for TDC > 2 mM, I SC increased further and the effect was progressive and associated with a significant decrease in the Rt and increased LDH release, implying a cytolytic effect. Mucosal preincubation with the Cl− channel inhibitor 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid (NPPB), fully prevented the precytolytic effect of TDC on I SC. Serosal preincubation with furosemide, a Na+/K+/2Cl− cotransporter inhibitor, significantly reduced TDC‐induced increase in I SC. Inhibition of the basolateral Ca2+‐dependent K+ channel—rSK4—with serosal clotrimazole or incubation with mucosal Ca2+‐free (EGTA) buffer completely prevented precytolytic TDC‐induced increase in I SC. In conclusion, Cl− secretion is activated in colon mucosa by TDC low concentrations; while at higher concentrations, a detergent cytotoxic effect intervenes. Activation of the Ca2+‐dependent basolateral K+ pathway, through TDC‐induced apical Ca2+ influx, provides the Na+/K+/2Cl− basolateral activation, thereby the driving force for the apical exit of Cl− ions. These findings further enhance the knowledge of the pathogenic mechanisms of diarrhea associated with bile salt malabsorption.
Inhibition of Na+, K+‐ATPase activity in rat striatum by guanidinoacetate
Authors: Zugno Alexandra I. , Stefanello Francieli M. , Streck Emilio L. , Calcagnotto Thiago , Wannmacher Clóvis M.D. , Wajner Moacir , Wyse Angela T.S.
Published: April 2003

The aim of this work was to investigate the effect of guanidinoacetate (GAA), the principal metabolite accumulating in guanidinoacetate methyltransferase (GAMT)‐deficiency, on Na+, K+‐ATPase, Mg2+‐ATPase and acetylcholinesterase (AChE) activities in striatum of young rats. We also studied the kinetics of the inhibition of Na+, K+‐ATPase activity caused by guanidinoacetate. Guanidinoacetate did not alter acetylcholinesterase and Mg2+‐ATPase activities, but significantly inhibited Na+, K+‐ATPase activity. The apparent Km and Vmax of Na+, K+‐ATPase for ATP as substrate were 0.20 mM and 0.82 nmol inorganic phosphate (Pi) released per min per mg of protein, respectively. Ki value was 7.18 mM, and the inhibition was of the uncompetitive type. The results also showed a competition between guanidinoacetate and argininic acid (AA), suggesting a common binding site for the guanidino compounds (GC) on the enzyme. It is proposed that Na+, K+‐ATPase inhibition by guanidinoacetate may be one of the mechanisms involved in the neuronal dysfunction observed in GAMT‐deficiency and in other diseases which accumulate guanidinoacetate.
Parents' perceptions of their 1–6‐year‐old children's pain
Authors: Kankkunen Päivi M , Vehviläinen‐Julkunen Katri M , Pietilä Anna‐Maija K , Halonen Pirjo M
Published: April 2003

Background of the study. Parents' perceptions of children's pain may have influence on how children's postoperative pain is alleviated at home after discharge from hospital.

Purpose of the study. To describe Parents' perceptions of 1–6‐year‐old children's pain.

Methods. Mothers (N=201) and fathers (N=114) whose child had undergone a day surgery in 10 Finnish central hospitals between October 2000 and September 2001 filled in a questionnaire including statements of pain perceptions, a Visual Analogue Scale to assess children's pain intensity and Parents' Postoperative Pain Measure (PPPM) to measure children's pain behaviours.

Results. Most of the parents suggested that adults have the responsibility to alleviate child's pain and that alleviation of child's postoperative pain prevents the child's fears during future visits in child welfare clinic. However, majority of parents described that postoperative pain decreases every day or that pain is always a part of surgery. Differences in Parents' perceptions were found by both Parents' and children's background variables. Parents' perceptions of children's pain were related to children's pain intensity and pain behaviours after surgery.

Conclusions. Parents' perceptions of children's pain were related to children's pain after surgery at home. Adequate information of children's pain should be provided to the parents before discharge to promote children's pain alleviation at home. Special attention should be paid on Parents' expectations of boys’ higher pain tolerance.
Treatment of chronic pain with millimetre wave therapy (MWT) in patients with diffuse connective tissue diseases: a pilot case series study
Authors: Usichenko Taras I , Herget Horst F
Published: April 2003

Background. Pain relief is reported to be the most common clinical application of electromagnetic millimetre waves.

Aim. To evaluate safety and pain relief effect of millimetre wave therapy (MWT) for treatment of chronic joint pain in a group of patients with diffuse connective tissue diseases.

Methods. Twelve patients with diffuse connective tissue diseases received MWT in addition to their analgesic medication with non‐steroidal anti‐inflammatory drugs. MWT procedure included the exposure of tender points around the painful joints to electromagnetic waves with frequency 54–78 GHz and power density of . The time of exposure was 35±5 min and the total number of sessions ranged from 5 to 10 (median 6). Intensity of pain, medication requirement, joint stiffness and subjective assessment of therapy success were measured before, during and immediately after the treatment, and after a 6‐months follow‐up.

Results. No adverse effects of MWT were noted. Pain intensity and required medication decreased significantly after the treatment (p<0.05) and remained at the same level throughout the follow‐up period. The joint stiffness decreased and the subjective assessment of the treatment success after 6 month did not change except in only one patient.

Conclusion. MWT applied to tender points around the affected joints was safe under the conditions of our study and after an appropriate full‐scale double‐blind clinical study, may be recommended as an effective adjunct therapy for chronic pain treatment in patients with diffuse connective tissue diseases.
Statistical design of reverse dye microarrays
Authors: Dobbin K. , Shih J. H. , Simon R.
Published: April 2003

Motivation: In cDNA microarray experiments all samples are labelled with either Cy3 dye or Cy5 dye. Certain genes exhibit dye bias—a tendency to bind more efficiently to one of the dyes. The common reference design avoids the problem of dye bias by running all arrays ‘forward’, so that the samples being compared are always labelled with the same dye. But comparison of samples labelled with different dyes is sometimes of interest. In these situations, it is necessary to run some arrays‘ reverse’—with the dye labelling reversed—in order to correct for the dye bias. The design of these experiments will impact one’s ability to identify genes that are differentially expressed in different tissues or conditions. We address the design issue of how many specimens are needed, how many forward and reverse labelled arrays to perform, and how to optimally assign Cy3 and Cy5 labels to the specimens.

Results: We consider three types of experiments for which some reverse labelling is needed: paired samples, samples from two predefined groups, and reference design data when comparison with the reference is of interest. We present simple probability models for the data, derive optimal estimators for relative gene expression, and compare the efficiency of the estimators for a range of designs. In each case, we present the optimal design and sample size formulas. We show that reverse labelling of individual arrays is generally not required.

Contact: dobbinke@mail.nih.gov.

Supplementary information: Supplementary material referenced in the text is available at http://linus.nci.nih.gov/~brb/TechReport.htm

* To whom correspondence should be addressed.
MARAN: normalizing micro-array data
Authors: Engelen Kristof , Coessens Bert , Marchal Kathleen , De Moor Bart
Published: April 2003

Summary: MARAN is a web-based application for normalizing microarray data. MARAN comprises a generic ANOVA model, an option for Loess fitting prior to ANOVA analysis, and a module for selecting genes with significantly changing expression.

Availability: http://www.esat.kuleuven.ac.be/maran/

Contact: kristof.engelen@esat.kuleuven.ac.be

* To whom correspondence should be addressed.
ALES: cell lineage analysis and mapping of developmental events
Authors: Braun V. , Azevedo R. B. R. , Gumbel M. , Agapow P.-M. , Leroi A. M. , Meinzer H.-P.
Published: April 2003

Motivation: Animals build their bodies by altering the fates of cells. The way in which they do so is reflected in the topology of cell lineages and the fates of terminal cells. Cell lineages should, therefore, contain information about the molecular events that determined them. Here we introduce new tools for visualizing, manipulating, and extracting the information contained in cell lineages. Our tools enable us to analyze very large cell lineages, where previously analyses have only been carried out on cell lineages no larger than a few dozen cells.

Results: Ales (A Lineage Evaluation System) allows the display, evaluation and comparison of cell lineages with the aim of identifying molecular and cellular events underlying development. Ales introduces a series of algorithms that locate putative developmental events. The distribution of these predicted events can then be compared to gene expression patterns or other cellular characteristics. In addition, artificial lineages can be generated, or existing lineages modified, according to a range of models, in order to test hypotheses about lineage evolution.

Availability: The program can run on any operating system with a compliant Java 2 environment. Ales is free for academic use and can be downloaded from http://mbi.dkfz-heidelberg.de/mbi/research/cellsim/ales.

Contact: CellO@dkfz-heidelberg.de

* To whom correspondence should be addressed.
Improvement of the GenTHREADER method for genomic fold recognition
Authors: McGuffin Liam J. , Jones David T.
Published: April 2003

Motivation: In order to enhance genome annotation, the fully automatic fold recognition method GenTHREADER has been improved and benchmarked. The previous version of GenTHREADER consisted of a simple neural network which was trained to combine sequence alignment score, length information and energy potentials derived from threading into a single score representing the relationship between two proteins, as designated by CATH. The improved version incorporates PSI-BLAST searches, which have been jumpstarted with structural alignment profiles from FSSP, and now also makes use of PSIPRED predicted secondary structure and bi-directional scoring in order to calculate the final alignment score. Pairwise potentials and solvation potentials are calculated from the given sequence alignment which are then used as inputs to a multi-layer, feed-forward neural network, along with the alignment score, alignment length and sequence length. The neural network has also been expanded to accommodate the secondary structure element alignment (SSEA) score as an extra input and it is now trained to learn the FSSP Z-score as a measurement of similarity between two proteins.

Results: The improvements made to GenTHREADER increase the number of remote homologues that can be detected with a low error rate, implying higher reliability of score, whilst also increasing the quality of the models produced. We find that up to five times as many true positives can be detected with low error rate per query. Total MaxSub score is doubled at low false positive rates using the improved method.

Availability: http://www.psipred.net

Contact: l.mcguffin@cs.ucl.ac.uk

* To whom correspondence should be addressed.
Towards optimal views of proteins
Authors: Sverud Oscar , MacCallum Robert M.
Published: April 2003

Motivation: Graphical representations of proteins in online databases generally give default views orthogonal to the PDB file coordinate system. These views are often uninformative in terms of protein structure and/or function. Here we discuss the development of a simple automatic algorithm to provide a ‘good’ view of a protein domain with respect to its structural features.

Results: We used dimension reduction with the preservation of topology (using Kohonen’s self organising map) to map 3D carbon alpha coordinates into 2D. The original protein structure was then rotated to the view which corresponded most closely to the 2D mapping. This procedure, which we call OVOP, was evaluated in a public blind trial on the web against random views and a ‘flattest’ view. The OVOP views were consistently rated ‘better’ than the other views by our volunteers.

Availability: The source code is available from the OVOP homepage: http://www.sbc.su.se/~oscar/ovop

Contact: maccallr@sbc.su.se

* To whom correspondence should be addressed.
Estimation of unsaturated hydraulic conductivity using soil suction measurements obtained by an insertion tensiometer
Authors: Singh D N , Kuriyan Sneha J
Published: April 2003

To estimate the unsaturated soil hydraulic conductivity of a silty soil, an insertion tensiometer has been used for measuring its suction corresponding to different water contents. These suction values have been used for developing the soil-water characteristic curve (SWCC). The obtained SWCC has been compared with the trends predicted by various fits available in the literature. Further, with the help of the obtained SWCC, the unsaturated soil hydraulic conductivity has been estimated. The study demonstrates the usefulness of insertion tensiometers for measuring soil suction and for estimating its hydraulic conductivity.Key words: silty soil, suction, insertion tensiometer, soil-water characteristic curve, unsaturated soil hydraulic conductivity.
INDUCTIVE LEARNING APPROACHES TO RAINFALL-RUNOFF MODELLING
Authors: DAWSON CHRISTIAN W. , BROWN MARTIN R. , WILBY ROBERT L.
Published: April 2003

Trying to model the rainfall-runoff process is a complex activity as it is influenced by a number of implicit and explicit factors — for example, precipitation distribution, evaporation, transpiration, abstraction, watershed topography, and soil types. However, this kind of forecasting is particularly important as it is used to predict serious flooding, estimate erosion and identify problems associated with low flow. Inductive learning approaches (e.g. decision trees and artificial neural networks) are particularly well suited to problems of this nature as they can often interpret underlying factors (such as seasonal variations) which cannot be modelled by other techniques. In addition, these approaches can easily be trained on the explicit factors (e.g. rainfall) and the inexplicit factors (e.g. abstraction) that affect river flow. Inductive learning approaches can also be extended to account for new factors that emerge over a period of time. This paper evaluates the application of decision trees and two artificial neural network models (the multilayer perceptron and the radial basis function network) to river flow forecasting in two flood prone UK catchments using real hydrometric data. Comparisons are made between the performance of these approaches and conventional flood forecasting systems.
A SEMI-PARAMETRIC HYBRID NEURAL MODEL FOR NONLINEAR BLIND SIGNAL SEPARATION
Authors: PENG HANCHUAN , CHI ZHERU , SIU WANCHI
Published: April 2003

Nonlinear blind signal separation is an important but rather difficult problem. Any general nonlinear independent component analysis algorithm for such a problem should specify which solution it tries to find. Several recent neural networks for separating the post nonlinear blind mixtures are limited to the diagonal nonlinearity, where there is no cross-channel nonlinearity. In this paper, a new semi-parametric hybrid neural network is proposed to separate the post nonlinearly mixed blind signals where cross-channel disturbance is included. This hybrid network consists of two cascading modules, which are a neural nonlinear module for approximating the post nonlinearity and a linear module for separating the predicted linear blind mixtures. The nonlinear module is a semi-parametric expansion made up of two sub-networks, one of which is a linear model and the other of which is a three-layer perceptron. These two sub-networks together produce a "weak" nonlinear operator and can approach relatively strong nonlinearity by tuning parameters. A batch learning algorithm based on the entropy maximization and the gradient descent method is deduced. This model is successfully applied to a blind signal separation problem with two sources. Our simulation results indicate that this hybrid model can effectively approach the cross-channel post nonlinearity and achieve a good visual quality as well as a high signal-to-noise ratio in some cases.
SIMULINK-BASED HW/SW CODESIGN OF EMBEDDED NEURO-FUZZY SYSTEMS
Authors: Reyneri L. M. , Chiaberge M. , Lavagno L. , Pino B. , Miranda E.
Published: April 2003

We propose a semi-automatic HW/SW codesign flow for low-power and low-cost Neuro-Fuzzy embedded systems. Applications range from fast prototyping of embedded systems to high-speed simulation of Simulink models and rapid design of Neuro-Fuzzy devices. The proposed codesign flow works with different technologies and architectures (namely, software, digital and analog). We have used The Mathworks' Simulink© environment for functional specification and for analysis of performance criteria such as timing (latency and throughput), power dissipation, size and cost. The proposed flow can exploit trade-offs between SW and HW as well as between digital and analog implementations, and it can generate, respectively, the C, VHDL and SKILL codes of the selected architectures.
COOPERATIVE COEVOLUTION OF NEURAL REPRESENTATIONS
Authors: BROWN A. D. , CARD H. C.
Published: April 2003

A genetic algorithm (GA) is used to search for a set of local feature detectors or hidden units. These are in turn employed as a representation of the input data for neural learning in the upper layer of a multilayer perceptron (MLP) which performs an image classification task. Three different methods of encoding hidden unit weights in the chromosome of the GA are presented, including one which coevolves all the feature detectors in a single chromosome, and two which promote the cooperation of feature detectors by encoding them in their own individual chromosomes. The fitness function measures the MLP classification accuracy together with the confidence of the networks.
ON THE CAPACITY OF MULTILAYER NEURAL NETWORKS TRAINED WITH BACKPROPAGATION
Authors: MIRANDA E. N.
Published: April 2003

The capacity of a layered neural network for learning hetero-associations is studied numerically as a function of the number M of hidden neurons. We find that there is a sharp change in the learning ability of the network as the number of hetero-associations increases. This fact allows us to define a maximum capacity C for a given architecture. It is found that C grows logarithmically with M.
TUNING DIVERSITY IN BAGGED ENSEMBLES
Authors: CARNEY JOHN G. , CUNNINGHAM PÁDRAIG
Published: April 2003

In this paper, we investigate how the level of diversity amongst individual neural networks in a bagged ensemble can significantly influence overall ensemble generalization performance. We propose a new technique that tunes this diversity so that ensemble generalization performance is optimized and evaluate its performance on benchmark regression data-sets.