bluuebunny/crossref_metadata_2025 huggingface dataset:
Bacterioferritins and ferritins are distantly related in evolution Conservation of ferroxidase‐centre residues
Authors: Andrews S.C. , Smith J.M.A. , Yewdall S.J. , Guest J.R. , Harrison P.M.
Published: July 2002

Iron‐storage proteins can be divided into two classes; the bacterioferritins and ferritins. In spite of many apparent structural and functional analogies, no significant amino acid sequence similarity has been detected previously. This report now reveals a distant evolutionary relationship between bacterioferritins and ferritins derived by ‘Profile Analysis’. Optimum alignment of bacterioferritin and ferritin sequences suggests that key residues of the ferroxidase centres of ferritins are conserved in bacterioferritins.
Distribution of glutathione peroxidases glutathione reductase in rat brain mitochondria
Authors: Panfili Enrico , Sandri Gabriella , Ernster Lars
Published: July 2002

The distribution of glutathione reductase (GR), glutathione peroxidase (GPx) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) in isolated rat brain mitochondria was investigated. using a fractionation procedure for the separation of inner and outer membranes, contact sites between the two membranes and a soluble fraction mainly originating from the mitochondrial matrix. The data indicate that GR and GPx are concentrated in the soluble fraction, with a minor portion of the two enzymes being associated with the contact sites. PHGPx is localized largely in the inner membrane. The possible functional significance of these findings is discussed.
Gulose as a constituent of a glycoprotein
Authors: Mengele Reiner , Sumper Manfred
Published: July 2002

The aldohexose gulose was identified as a constituent of a hydroxyproline‐rich glycopeptide derived from the glycoprotein SSG 185. This glycoprotein is part of the extracellular matrix of the green alga Volvox carteri. The gulose residue occupies a terminal position in the corresponding saccharide.
Enzymatic activity of adenylate cyclase toxin from Bordetella pertussis is not required for hemolysis
Authors: Ehrmann Ingrid E. , Weiss Alison A. , Goodwin M.S. , Gray Mary C. , Barry Eileen , Hewlett Erik L.
Published: July 2002

Adenylate cyclase (AC) toxin from Bardetella pertussis enters cells to cause supraphysiologic increases in cAMP, AC toxin is also hemolytic. Substitution of Lys‐58 with a methionine residue by site‐directed mutagenesis of the structural gene for AC toxin, cyaA, and introduction of this mutation onto the B. pertussis chromosome results in an organism that synthesizes an enzyme‐deficient AC toxin molecule. This mutant toxin molecule exhibits 1000‐fold reduction in enzymatic activity relative to wild‐type and has no toxin activity in J774 cells. The enzyme‐deficient toxin molecule is not, however, impaired in its ability to lyse sheep red blood cells. In order to ascertain the importance of these two separate activities of AC toxin in vivo the enzyme‐deficient organisms were used to infect infant mice. The hemolytic, enzyme‐deficient mutant organisms are reduced in virulence relative to wild‐type organisms after intranasal challenge indicating that, although the enzymatic activity of AC toxin does not contribute to hemolysis, it is this property of the toxin which is important for virulence of B. pertussis.
A TIM barrel protein without enzymatic activity? Crystal‐structure of narbonin at 1.8 Å resolution
Authors: Hennig Michael , Schlesier Bernhard , Dauter Zbigniew , Pfeffer Sabine , Betzel Christian , Höhne Wolfgang E. , Wilson Keith S.
Published: July 2002

The major protein component in seeds is storage protein. These have no known enzymatic activity and act to provide amino acids as a source of metabolites in the developing seedling. We report here the first three dimensional crystal structure of a seed storage globulin at high resolution. The molecule of the 2S globulin, narbonin, from Vicia narbonensis L. consists of an eight‐stranded parallel α/gb barrel structure similar to that observed in triose phosphate isomerase (TIM). Narbonin is the first protein with this topology possessing no known enzymatic activity. Because of the lack of sequence information most of the primary structure was determined directly from the electron density.
Identification of a novel transthyretin variant (Val30→Leu) associated with familial amyloidotic polyneuropathy
Authors: Nakazato Masamitsu , Ikeda Shu-ichi , Shiomi Kazutaka , Matsukura Shigeru , Yoshida Kunihiro , Shimizu Hideaki , Atsumi Tetsushi , Kangawa Kenji , Matsuo Hisayuki
Published: July 2002

A novel variant transthyretin which contains a leucine‐for‐valine substitution at position 30 was isolated and identified in the serum of a patient with familial amyloidotic polyneuropathy (FAP). The amino acid substitution was proven to result from a guanine‐to‐cytosine change at the first base of codon 30 located in exon 2 in the mutated transthyretin gene by restriction fragment length analysis on the amplified transthyretin gene using Cfr13 I. The study indicates that the point mutation of the transthyretin gene is a cause of the disorder.
Activation of protein kinase C by the 14‐3‐3 proteins homologous with Exol protein that stimulates calcium‐dependent exocytosis
Authors: Isobe Toshiaki , Hiyane Yuriko , Ichimura Tohru , Okuyama Tsuneo , Takahashi Nobuhiro , Nakajo Shigeo , Nakaya Kazuyasu
Published: July 2002

The 14‐3‐3 proteins are a family of acidic proteins found mainly in the brain and are suggested to have a role in monoamine synthesis based on their ability to activate tyrosine and tryptophan hydroxylases in the presence of type II Ca2+/calmodulin‐dependent protein kinase. Recently, however, it has been demonstrated that a member of the 14‐3‐3 family, termed Exol, stimulates Ca2+‐dependent exocytosis in permeabilized adrenal chromaffin cells, suggesting that this protein family may influence the protein kinase C‐mediated control of Ca2+‐dependent exocytosis. Here we show that the 14‐3‐3 proteins activate protein kinase C at about 2‐fold more than the known level of the activated protein kinase, i.e. the activity of protein kinase C in the presence of Ca2+ and phospholipids. This raises the possibility that the cellular activity of protein kinase C is regulated by diverse members of the 14‐3‐3 family and that the reported ability of Exol to reactivate Ca2+‐dependent exocytosis is based on its stimulatory effect on protein kinase C activity. The 14‐3‐3 family, therefore, appears to be a multifunctional regulator of cell signalling processes mediated by two types of Ca2+‐dependent protein kinase, protein kinase C and type II calmodulin‐dependent protein kinase.
The compact state of reduced bovine pancreatic trypsin inhibitor is not the compact molten globule
Authors: Gussakovsky Eugene E. , Hans Elisha
Published: July 2002

Reduced bovine pancreatic trypsin inhibitor (BPTI) has been shown to be in a compact state [(1988) Biochemistry 27, 8889–8893]. This leads to the proposal that this compact state may be a compact molten globule folding intermediate. Optical rotatory dispersion in the visible region failed to show the presence of pronounced secondary structures in the reduced UPTI and no binding or 8‐anilino‐1‐naphthalenesulphonic acid to reduced BPTI could be detected. Yet, no Cooperative thermal transition was detected by tyrosine fluorescence. These experiments show that reduced BPTI is not in the compact molten globule state.
The amino acid sequence of a type I copper protein with an unusual serine‐ and hydroxyproline‐rich C‐terminal domain isolated from cucumber peelings
Authors: Mann Karlheinz , Schäfer Wolfram , Thoenes Ulrich , Messerschmidt Albrecht , Mehrabian Zara , Nalbandyan Robert
Published: July 2002

We have determined the amino acid sequence of a small copper protein isolated from cucumber peelings. This cupredoxin contains 137 amino acids including a pyroglutamate as the first residue. The N‐terminal 110 amino acid‐long domain shows 30–37% identity to 2 other cupredoxins, stellacyanin and cucumber basic blue protein. A unique feature of this protein is a 27 amino acid‐long, C‐terminal domain rich in 4‐hydroxyproline and serine and resembling certain plant cell wall proteins. The prolines in this domain are hydroxylated to a different extent depending on the surrounding sequence.
TGMV replication protein AL1 preferentially binds to single‐stranded DNA from the common region
Authors: Thömmes P. , Osman T.A.M. , Hayes R.J. , Buck K.W.
Published: July 2002

The AL1 protein of tomato golden mosaic virus (TGMV) is encoded by the viral DNA and has been shown to be essential for viral DNA replication. We have over‐expressed the AL1 open reading frame in E. coli and purified the protein from bacterial extracts to near homogeneity. Using various different techniques we have studied the interaction of the AL1 protein with DNA. The AL1 protein is able to bind to DNA containing the common region of the viral genome, which can be demonstrated by photochemical cross‐linking. Binding is 4‐fold stronger to single‐stranded than to double‐stranded DNA. Antibodies against the AL1 protein can be used to precipitate the protein‐DNA complex. The binding to single‐ and double‐stranded DNA is specifically to the common region since a DNA fragment unrelated to TGMV is not shifted in a gel retardation assay.
Modulation of the skeletal muscle sodium channel α‐subunit by the β1 ‐subunit
Authors: Wallner Martin , Weigl Lukas , Meera Pratap , Lotan Ilana
Published: July 2002

Co‐expression of cloned sodium channel β1 ‐subunit with the rat skeletal muscle‐subunit (αμI) accelerated the macroscopic current decay, enhanced the current amplitude, shifted the steady state inactivation curve to more negative potentials and decreased the time required for complete recovery from inactivation. Sodium channels expressed from skeletal muscle mRNA showed a similar behaviour to that observed from , indicating that β1 restores ‘physiological’ behaviour. Northern blot analysis revealed that the Na+ channel β1‐subunit is present in high abundance (about 0.1%) in rat heart, brain and skeletal muscle, and the hybridization with untranslated region of the ‘brain’ β1 cDNA to skeletal muscle and heart mRNA indicated that the diffferent Na + channel α‐subunits in brain, skeletal muscle and heart may share a common β1 ‐subunit.
Expression of TIMP3 mRNA is elevated in retinas affected by simplex retinitis pigmentosa
Authors: Jones S.E. , Jomary C. , Neal M.J.
Published: July 2002

To explore the molecular and cellular mechanisms associated with photoreceptor death in retinitis pigmentosa (RP), we have investigated altered transcriptional activity in RP retinas by a differential cDNA screening approach. We identified a clone (K222) showing over‐expression in simplex RP retinas compared with controls. K222 encodes a partial cDNA of the human tissue inhibitor of metalloproteinases‐3 (TIMP3) gene, a member of a family of genes implicated in extracellular matrix (ECM) remodelling. Increased expression of TIMP3 in degenerating RP retinas may reflect restructuring of the ECM architecture, and disruption of photoreceptor—matrix interactions could contribute to activation of apoptotic cell death processes.
Multiple palmitoylation of synaptotagmin and the t‐SNARE SNAP‐25
Authors: Veit Michael , Söllner Thomas H. , Rothman James E.
Published: July 2002

Synaptotagmin, a likely calcium sensor for synaptic transmission, and SNAP‐25, a t‐SNARE of the presynaptic plasma membrane, are key proteins for the docking and fusion of synaptic and other vesicles. We report that both synaptotagmin and SNAP‐25 are palmitoylated with their fatty acids attached in a labile thioester‐type bond. A SNAP‐25 mutant with deleted palmitoylation sites is found exclusively in the cytosol after cell fractionation, whereas the palmitoylated form of SNAP‐25 is membrane‐bound, establishing that SNAP‐25 is membrane‐anchored via covalently linked palmitate.
Novel amplification of non‐photochemical chlorophyll fluorescence quenching following viral infection in Chlorella
Authors: Seaton George G.R. , Hurry Vaughan M. , Rohozinski Jan
Published: July 2002

In higher plants non‐photochemical dissipation of excess light, trapped by the pigment pool of photosystem II, prevents photodamage to the photosynthetic apparatus. We report here that an algal virus infecting Chlorella strain Pbi induces non‐photochemical quenching of photosystem II fluorescence, indicating enhanced loss of absorbed light energy from photosystem II. This phenomenon occurs soon after the establishment of the virus infection cycle and is observed at low irradiance (20 μmol quanta m−2 s−1). At low light, infection associated non‐photochemical quenching is not linked to extensive conversion of violaxanthin to antheraxanthin and zeaxanthin. However, such conversion occurs rapidly (2–10 min) in infected cells under conditions of high irradiance (100–300 μmol quanta m−2 s−1). Under similar conditions uninfected Chlorella cells do not display significant changes in non‐photochemical quenching.
Effect of glutathione, glutathione sulphonate and S‐hexylglutathione on the conformational stability of class pi glutathione S‐transferase
Authors: Erhardt Julija , Dirr Heini
Published: July 2002

The glutathione S‐transferases (GST) are a supergene family of phase II detoxification enzymes which catalyse the S‐conjugation between glutathione and an electrophilic substrate. The active site can be divided into two adjacent functional regions, a highly specific G‐site for binding the physiological substrate glutathione and a nonspecific H‐site for binding nonpolar electrophilic substrates. Equilibrium and kinetic unfolding experiments employing tryptophan fluorescence and enzyme activity measurements were performed to study the effect of ligand binding to the G‐site on the unfolding and stability of the porcine class pi glutathione S‐transferase against urea. The presence of glutathione caused a shift in the equilibrium‐unfolding curves towards lower urea concentrations and enhanced the first‐order rate constant for unfolding suggesting a destabilisation of the pGSTP1‐1 structure against urea. The presence of either glutathione sulphonate or S‐hexylglutathione, however, produced the opposite effect in that their binding to the G‐site appeared to exert a stabilising effect against urea. The binding of these glutathione analogues also reduced significantly the degree of cooperativity of unfolding indicating a possible change in the protein's unfolding pathway.
Differential effects of interleukin‐1β and interleukin‐2 on glia and hippocampal neurons in culture
Authors: Araujo Dalia M. , Cotman Carl W.
Published: July 2002

The present study was undertaken to assess the effects of interleukin‐1β (IL‐1β) and interleukin‐2 (IL‐2) on glial and neuronal cells in culture. The presence of IL‐1β‐like and IL‐2‐like immunoreactivity was detected in media collected from both astroglial and microglial cultures, indicating that both lymphokines can be released from either cell type. However, the levels measured in microglial media were significantly higher than in the astroglial media. Moreover, the content of IL‐1β‐like immunoreactive material in the media was approximately five‐ to 10‐fold greater than that of IL‐2, although exposure of both microglial and astroglial cultures to IL‐1β significantly enhanced this measure. A possible role for this glial‐derived IL‐1β as an astroglial growth factor was substantiated by experiments showing that the lymphokine increased the incorporation of [3H]thymidine into astroglial, but not microglial cultures. In contrast, IL‐2 did not significantly alter glial proliferation. In hippocampal neuronal cultures, these lymphokines affected neuronal survival differently. Thus, only the highest concentration (500 ng/ml) of IL‐1β tested decreased the long‐term (three day), but not the short‐term (one day), survival of these neurons, whereas neuronal survival was compromised by IL‐2 even after short‐term (one day) exposure. In addition, in the long‐term (three‐day‐old) neuronal cultures exposed to IL‐2, extensive cellular swelling, vacuolations and neurite retractions were noted, even in cultures exposed to relatively low concentrations (< 10 ng/ml) of the lymphokine. These effects were not apparent with IL‐1β or the other lymphokines tested, including IL‐3, IL‐4 and IL‐8. The results suggests that the glial‐derived lymphokines IL‐1β and IL‐2 may have different functions in the CNS. Whereas IL‐1β may have an important role in the developing brain as a maintenance and growth‐promoting factor, IL‐2 may function as an inhibitory factor, and may be of significance only in instances during which it accumulates in sufficiently high concentrations in the vicinity of neurons.
A novel C‐terminal kinesin subfamily may be involved in chromosomal movement in Caenorhabditis elegans1
Authors: Ali M.Yusuf , Siddiqui Z.K. , Malik A.B. , Siddiqui Shahid S.
Published: July 2002

C‐terminal kinesin motor proteins, such as the Drosophila NCD and yeast KAR3, are involved in chromosomal segregation. Previously we have described two orthologs of NCD in Caenorhabditis elegans, KLP‐3 and KLP‐17, which also participate in chromosome movement. Here we report cDNA cloning of klp‐15 and klp‐16, and the expression pattern of the genes encoding C‐terminal motor kinesins including klp‐15 and klp‐16. Interestingly KLP‐15 and KLP‐16 form a unique class of C‐terminal kinesins, distinct from the previously known C‐terminal motors in other organisms. Using in situ hybridization and RNA interference assay, we show that although all of these motors mediate chromosome segregation, they do so in a combination of unique and overlapping manners, suggesting a complex hierarchy of kinesin motor function in metazoans.
Absorption and metabolism of genistin in the isolated rat small intestine
Authors: Andlauer Wilfried , Kolb Jochen , Fürst Peter
Published: July 2002

Uptake and intestinal metabolism of physiologically active genistin were studied in an ex vivo intestinal perfusion model; luminally applied concentrations were 5.9, 12.0, and 23.8 μmol/l. The intestinal absorption of genistin was 14.9% (±2.3, n=9), irrespective of the amounts applied. The majority of the absorbed genistin appeared as genistein glucuronide (11.6%), also recovered as the main metabolite on the luminal side (19.5%). Minor amounts of genistin (1.3%) and genistein (1.9%) were found on the vascular side, whereas 15.4% of applied genistin was luminally cleaved to yield genistein. Sulfate derivatives of genistein or genistin were not observed.
Phosphorylation of CPI‐17, an inhibitory phosphoprotein of smooth muscle myosin phosphatase, by Rho‐kinase
Authors: Koyama Mutsumi , Ito Masaaki , Feng Jianhua , Seko Tetsuya , Shiraki Katsuya , Takase Koujiro , Hartshorne David J. , Nakano Takeshi
Published: July 2002

Phosphorylation of CPI‐17 by Rho‐associated kinase (Rho‐kinase) and its effect on myosin phosphatase (MP) activity were investigated. CPI‐17 was phosphorylated by Rho‐kinase to 0.92 mol of P/mol of CPI‐17 in vitro. The inhibitory phosphorylation site was Thr38 (as reported previously) and was identified using a point mutant of CPI‐17 and a phosphorylation state‐specific antibody. Phosphorylation by Rho‐kinase dramatically increased the inhibitory effect of CPI‐17 on MP activity. Thus, CPI‐17 as a substrate of Rho‐kinase could be involved in the Ca2+ sensitization of smooth muscle contraction as a downstream effector of Rho‐kinase.
Water and potassium dynamics inside the KcsA K+ channel
Authors: Guidoni Leonardo , Torre Vincent , Carloni Paolo
Published: July 2002

Molecular dynamics simulations and electrostatic modeling are used to investigate structural and dynamical properties of the potassium ions and of water molecules inside the KcsA channel immersed in a membrane‐mimetic environment. Two potassium ions, initially located in the selectivity filter binding sites, maintain their position during 2 ns of dynamics. A third potassium ion is very mobile in the water‐filled cavity. The protein appears engineered so as to polarize water molecules inside the channel cavity. The resulting water induced dipole and the positively charged potassium ion within the cavity are the key ingredients for stabilizing the two K+ ions in the binding sites. These two ions experience single file movements upon removal of the potassium in the cavity, confirming the role of the latter in ion transport through the channel.
IFNα sensitizes ME‐180 human cervical cancer cells to TNFα‐induced apoptosis by inhibiting cytoprotective NF‐κB activation
Authors: Suk Kyoungho , Kim Yun-Hee , Chang Inik , Kim Ja Young , Choi Yun-Hee , Lee Kye Young , Lee Myung-Shik
Published: July 2002

Tumor necrosis factor α (TNFα) induces apoptosis of a variety of tumor cell types. The anti‐tumor effect of TNFα is often augmented by interferon (IFN) γ. We hypothesized that IFNα, which shares many biological activities with IFNγ, might also synergize with TNFα for the induction of tumor cell death. We tested our hypothesis using ME‐180 human cervical cancer cells exposed to either IFNα or TNFα alone or both. We analyzed the death of ME‐180 cells by biochemical and cytological means, and investigated the molecular mechanism underlying cytotoxic synergism between the two cytokines. We found that (i) IFNα/TNFα synergistically induced apoptosis of ME‐180 cells, which was accompanied by activation of caspases‐3 and ‐8; (ii) IFNα induced signal transducer and activator of transcription (STAT) 1 phosphorylation, and transfection of phosphorylation‐defective STAT1 dominant‐negative mutant inhibited IFNα/TNFα‐induced apoptosis; (iii) inhibition of nuclear factor κB (NF‐κB) by proteasome inhibitor MG‐132 sensitized ME‐180 cells to TNFα alone; (iv) IFNα treatment attenuated TNFα‐induced NF‐κB reporter activity, while it did not inhibit DNA binding of NF‐κB. Taken collectively, our results indicate that IFNα sensitizes ME‐180 cells to TNFα‐induced apoptosis by inhibiting TNFα‐mediated cytoprotective NF‐κB activation, and this sensitizing effect of IFNα is mediated through a STAT1‐dependent pathway.
Backbone dynamics of the channel‐forming antibiotic zervamicin IIB studied by 15N NMR relaxation
Authors: Korzhnev Dmitry M , Bocharov Eduard V , Zhuravlyova Anastasya V , Orekhov Vladislav Yu , Ovchinnikova Tatyana V , Billeter Martin , Arseniev Alexander S
Published: July 2002

The backbone dynamics of the channel‐forming peptide antibiotic zervamicin IIB (Zrv‐IIB) in methanol were studied by 15N nuclear magnetic resonance relaxation measurements at 11.7, 14.1 and 18.8 T magnetic fields. The anisotropic overall rotation of the peptide was characterized based on 15N relaxation data and by hydrodynamic calculations. ‘Model‐free’ analysis of the relaxation data showed that the peptide is fairly rigid on a sub‐nanosecond time‐scale. The residues from the polar side of Zrv‐IIB helix are involved in micro–millisecond time‐scale conformational exchange. The conformational exchange observed might indicate intramolecular processes or specific intermolecular interactions of potential relevance to Zrv‐IIB ion channel formation.
Mutations converting cyclodextrin glycosyltransferase from a transglycosylase into a starch hydrolase
Authors: Leemhuis Hans , Dijkstra Bauke W. , Dijkhuizen Lubbert
Published: July 2002

Cyclodextrin glycosyltransferase (CGTase) efficiently catalyzes transglycosylation of oligo‐maltodextrins, although the enzyme also has a low hydrolytic activity. Its +2 substrate binding subsite, which contains the conserved Phe184 and Phe260 residues, has been shown to be important for this transglycosylation activity [Nakamura et al. (1994) Biochemistry 33, 9929–9936]. Here we show that the amino acid side chain at position 260 also controls the hydrolytic activity of CGTase. Three Phe260 mutants of Thermoanaerobacterium thermosulfurigenes CGTase were obtained with a higher hydrolytic activity than ever observed before for a CGTase. These Phe260 mutations even changed CGTase from a transglycosylase into a starch hydrolase.
The intracellular cytoplasmic domain of the Alzheimer's disease amyloid precursor protein interacts with phosphotyrosine‐binding domain proteins in the yeast two‐hybrid system
Authors: M. McLoughlin Declan , C.J. Miller Christopher
Published: July 2002

We have used the yeast two‐hybrid system to screen for proteins that interact with the carboxy‐terminal domain of APP. Six different clones were isolated and sequence analyses revealed that they encoded domains of a previously described neuronal protein Fe65, a homologue of Fe65 and a homologue of protein X11. All of these proteins contain one or more phosphotyrosine binding (PTB) domains. PTB domain proteins bind to the sequence Asn‐Pro‐X‐Tyr when the Tyr is phosphorylated and are believed to function in signal transduction. APP contains such a motif. These results are consistent with a role for APP in signal transduction mechanisms.
Complete amino acid sequence of the A chain of mistletoe lectin I
Authors: Soler Montserrat Huguet , Stoeva Stanka , Schwamborn Cornelia , Wilhelm Sabine , Stiefel Thomas , Voelter Wolfgang
Published: July 2002

The complete amino acid sequence of the A chain of mistletoe lectin I was determined via Edman degradation sequencing of the N‐terminus and tryptic and endoproteinase Asp‐N overlapping fragments, amino acid analysis and MALDIMS. The data obtained show a great homology with the chains of ribosome‐inactivating proteins such as ricin and abrin with 111 (abrin‐a) and 103 (ricin‐D) amino acid residues conserved, respectively. The knowledge of the primary structure of MLA will have a fundamental impact on elucidating the biological function of medically applied mistletoe lectins on a molecular basis.
The E. coli SRP: preferences of a targeting factor
Authors: De Gier Jan-Willem L , Valent Quido A , Von Heijne Gunnar , Luirink Joen
Published: July 2002

Research on the targeting of proteins to the cytoplasmic membrane of E. coli has mainly focused on the so‐called ‘general secretory pathway’ (GSP) which involves the Sec‐proteins. Recently, evidence has been obtained for an alternative targeting pathway in E. coli which involves the signal recognition particle (SRP). The constituents of this SRP pathway in E. coli are homologous to those of the well‐characterized eukaryotic SRP pathway, which is the main targeting pathway for both proteins translocated across and inserted into the endoplasmic reticulum membrane. However, until recently, no clear function could be assigned to the SRP in E. coli. New studies point to an important role of the E. coli SRP in the assembly of inner membrane proteins.
Interaction of Fas(Apo‐1/CD95) with proteins implicated in the ubiquitination pathway
Authors: Becker Karin , Schneider Pascal , Hofmann Kay , Mattmann Chantal , Tschopp Jürg
Published: July 2002

Fas(Apo‐1/CD95), a receptor belonging to the tumor necrosis factor receptor family, induces apoptosis when triggered by Fas ligand. Upon its activation, the cytoplasmic domain of Fas binds several proteins which transmit the death signal. We used the yeast two‐hybrid screen to isolate Fas‐associated proteins. Here we report that the ubiquitin‐conjugating enzyme UBC9 binds to Fas at the interface between the death domain and the membrane‐proximal region of Fas. This interaction is also seen in vivo. UBC9 transiently expressed in HeLa cells bound to the co‐expressed cytoplasmic segment of Fas. FAF1, a Fas‐associated protein that potentiates apoptosis (Chu et al. (1996) Proc. Natl. Acad. Sci. USA 92, 11894–11898), was found to contain sequences similar to ubiquitin. These results suggest that proteins related to the ubiquitination pathway may modulate the Fas signaling pathway.
Evidence for a role of C‐terminal amino acid residues in skeletal muscle Ca2+ release channel (ryanodine receptor) function
Authors: Gao Ling , Tripathy Ashutosh , Lu Xiangyang , Meissner Gerhard
Published: July 2002

The effects of deleting 1, 3 and 15 amino acid residues from the highly conserved C‐terminus of the tetrameric skeletal muscle ryanodine receptor (RyR) complex were determined. Immunoblot analysis indicated similar expression levels in HEK293 cells for full‐length and mutant proteins. Full‐length and RyR lacking the last amino acid showed [3H]ryanodine binding and single channel activities typical of native receptors. Deletion of 3 amino acids resulted in decreased activities, whereas deletion of 15 amino acids yielded an inactive RyR. These results suggest that the most 15 C‐terminal amino acids are important for the expression of a functional RyR complex.
Co‐expression of the proprotein‐processing endoprotease furin and its substrate transforming growth factor β1 and the differentiation of rat hepatocytes
Authors: Hoshino Hideki , Konda Yoshitaka , Takeuchi Toshiyuki
Published: July 2002

Furin, a member of the yeast Kex2 endoprotease family, converts a number of proproteins to their active forms. The liver produces a number of proproteins having a furin‐cleavable site; thus, furin may be involved in growth and differentiation both in the partially hepatectomized liver and in primary cultured hepatocytes. Furin mRNA levels are elevated in tissues regenerated from partially hepatectomized rat liver. In primary culture of rat hepatocytes, furin expression increases gradually with time, and its expression is greatly enhanced by transforming growth factor β1, whose processing from the precursor requires cleavage by furin. Thus, we suggest that the regeneration and differentiation of hepatocytes is dependent upon the co‐elevation of furin and transforming growth factor β1 mRNAs.
Two non‐proline cis peptide bonds may be important for factor XIII function
Authors: Weiss Manfred S , Metzner Hubert J , Hilgenfeld Rolf
Published: July 2002

The structure of recombinant human cellular factor XIII zymogen was solved in its monoclinic crystal form and refined to an R‐factor of 18.3% (R
free=23.6%) for all data between 40.0 and 2.1 Å resolution. Two non‐proline cis peptide bonds were detected. One is between Arg310 and Tyr311 close to the active site cysteine residue (Cys314) and the other is between Gln425 and Phe426 at the dimerization interface. The structure and the role of these cis peptides are discussed in the light of their possible importance for factor XIII function.
N‐Linked glycosylation of Drosophila rhodopsin occurs exclusively in the amino‐terminal domain and functions in rhodopsin maturation
Authors: Katanosaka Kimiaki , Tokunaga Fumio , Kawamura Satoru , Ozaki Koichi
Published: July 2002

Immature Drosophila rhodopsin is N‐glycosylated, but undergoes complete deglycosylation during the process of protein maturation. In order to elucidate the site of glycosylation and its role in rhodopsin synthesis, we investigated the in vitro and in vivo synthesis of rhodopsin whose putative N‐glycosylation sites (Asn‐20 and Asn‐196) were replaced by isoleucine. The results demonstrated that immature rhodopsin binds a single oligosaccharide chain exclusively at Asn‐20 in the N‐terminal extracellular domain. Furthermore, the results gave the first evidence directly indicating that deletion of the oligosaccharide chain markedly impedes rhodopsin maturation.
Molecular dynamics study of the LS3 voltage‐gated ion channel
Authors: Zhong Qingfeng , Moore Preston B , Newns Dennis M , Klein Michael L
Published: July 2002

Molecular dynamics calculations have been carried out on a model of the LS3 synthetic ion channel in a membrane mimetic environment. In the absence of an external electrostatic field, the LS3 channel, which consists of a bundle of six α‐helices with sequence Ac‐(LSSLLSL)3‐CONH2, exhibits large structural fluctuations. However, in the presence of the field, the bundle adopts a well defined coiled‐coil structure with an inner pore of water. The observed structural changes induced by the applied field are consistent with the proposed gating mechanism of the ion channel.
Inhibition of ascorbate peroxidase under oxidative stress in tobacco having bacterial catalase in chloroplasts
Authors: Shikanai Toshiharu , Takeda Toru , Yamauchi Haruko , Sano Satoshi , Tomizawa Ken-Ichi , Yokota Akiho , Shigeoka Shigeru
Published: July 2002

To analyze the potential of the active oxygen‐scavenging system of chloroplasts, we introduced Escherichia coli catalase into tobacco chloroplasts. Photosynthesis of transgenic plants was tolerant to high irradiance under drought conditions, while the wild plants suffered severe damage in photosynthesis under the same conditions. Irrespective of responses to the stress, ascorbate peroxidase was completely inactivated both in the transgenic and wild‐type plants. These findings are contrary to the established idea that the ascorbate peroxidase‐mediated antioxidative system protects chloroplasts from oxidative stress.
Glycan engineering of proteins with whole living yeast cells expressing rat liver α2,3‐sialytransferase in the porous cell wall
Authors: Sievi Eeva , Helin Jari , Heikinheimo Riikka , Makarow Marja
Published: July 2002

The N‐glycans of recombinant proteins produced via the secretory pathway of cultured mammalian cells are often undersialylated, and insect cells lack sialytransferases. Undersialylated glycoproteins are rapidly cleared from the circulation, compromising the effect of pharmaceuticals. We show that incubation with living Saccharomyces cerevisiae cells expressing the catalytic ectodomain of rat liver α2,3‐sialyltransferase (ST3Ne) in the porous cell wall resulted in sialylation of glycoproteins. The K
m values of the yeast enzyme for several substrates were similar to those of recombinant ST3Ne from insect cells and of authentic ST3N. The yeast strain provides an inexpensive self‐perpetuating source of ST3N activity for glycan engineering of recombinant proteins.
Characterisation of the conformational and quaternary structure‐dependent heparin‐binding region of bovine seminal plasma protein PDC‐109
Authors: Calvete Juan J. , Campanero-Rhodes Ma.Asunción , Raida Manfred , Sanz Libia
Published: July 2002

PDC‐109, the major heparin‐binding protein of bull seminal plasma, binds to sperm choline lipids at ejaculation and modulates capacitation mediated by heparin. Affinity chromatography on heparin‐Sepharose showed that polydisperse, but not monomeric, PDC‐109 displayed heparin‐binding capability. We sought to characterise the surface topology of the quaternary structure‐dependent heparin‐binding region of PDC‐109 by comparing the arginine‐ and lysine‐selective chemical modification patterns of the free and the heparin‐bound protein. A combination of reversed‐phase peptide mapping of endoproteinase Lys‐C‐digested PDC‐109 derivatives and mass spectrometry was employed to identify modified and heparin‐protected residues. PDC‐109 contains two tandemly arranged fibronectin type II domains (a, Cys24‐Cys61; b, Cys69‐Cys109). The results show that six basic residues (Lys34, Arg57, Lys59, Arg64, Lys68, and Arg104) were shielded from reaction with acetic anhydride and 1,2‐cyclohexanedione in heparin‐bound PDC‐109 oligomers. In the 1H‐NMR solution structures of single fibronectin type II domains, residues topologically equivalent to PDC‐109 Arg57 (Arg104) and Lys59 lay around β‐strand D on the same face of the domain. In full‐length PDC‐109, Arg64 and Lys68 are both located in the intervening polypeptide between domains a and b. Our data suggest possible quaternary structure arrangements of PDC‐109 molecules to form a heparin‐binding oligomer.
Protein kinase D activation by deletion of its cysteine‐rich motifs
Authors: Iglesias Teresa , Rozengurt Enrique
Published: July 2002

Protein kinase D is a serine/threonine kinase that binds phorbol esters in a phospholipid‐dependent manner via a tandemly repeated cysteine‐rich, zinc finger‐like motif (the cysteine‐rich domain). Here, we examined whether the cysteine‐rich domain plays an additional role in the control of the catalytic kinase activity independently of the binding of allosteric effectors. We found that deletion of cys1, cys2 or the entire cysteine‐rich domain increases the basal activity of protein kinase D leading to a constitutively active form of this enzyme. Our results demonstrate, for the first time, that the cysteine‐rich domain of Protein kinase D plays a negative role in the regulation of protein kinase D kinase activity.
Structure based development of novel specific inhibitors for cathepsin L and cathepsin S in vitro and in vivo
Authors: Katunuma N. , Murata E. , Kakegawa H. , Matsui A. , Tsuzuki H. , Tsuge H. , Turk D. , Turk V. , Fukushima M. , Tada Y. , Asao T.
Published: July 2002

Specific inhibitors for cathepsin L and cathepsin S have been developed with the help of computer‐graphic modeling based on the stereo‐structure. The common fragment, N‐(L‐trans‐carbamoyloxyrane‐2‐carbonyl)‐phenylalanine‐dimethylamide, is required for specific inhibition of cathepsin L. Seven novel inhibitors of the cathepsin L inhibitor Katunuma (CLIK) specifically inhibited cathepsin L at a concentration of 10−7 M in vitro, while almost no inhibition of cathepsins B, C, S and K was observed. Four of the CLIKs are stable, and showed highly selective inhibition for hepatic cathepsin L in vivo. One of the CLIK inhibitors contains an aldehyde group, and specifically inhibits cathepsin S at 10−7 M in vitro.
Pyrococcus furiosus glyceraldehyde 3‐phosphate oxidoreductase has comparable W6+/5+ and W5+/4+ reduction potentials and unusual [4Fe‐4S] EPR properties
Authors: Hagedoorn Peter L , Freije J.Robert , Hagen Wilfred R
Published: July 2002

Pyrococcus furiosus glyceraldehyde 3‐phosphate oxidoreductase has been characterized using EPR‐monitored redox titrations. Two different W signals were found. W1
5+ is an intermediate species in the catalytic cycle, with the midpoint potentials E
m(W6+/5+)=−507 mV and E
m(W5+/4+)=−491 mV. W2
5+ represents an inactivated species with E
m(W6+/5+)=−329 mV. The cubane cluster exhibits both S=3/2 and S=1/2 signals with the same midpoint potential: E
m([4Fe‐4S]2+/1+)=−335 mV. The S=1/2 EPR signal is unusual with all g values below 2.0. The titration results combined with catalytic voltammetry data are consistent with electron transfer from glyceraldehyde 3‐phosphate first to the tungsten center, then to the cubane cluster and finally to the ferredoxin.
Cysteinyl‐tRNA formation: the last puzzle of aminoacyl‐tRNA synthesis
Authors: Li Tong , Graham David E. , Stathopoulos Constantinos , Haney Paul J. , Kim Hyun-soo , Vothknecht Ute , Kitabatake Makoto , Hong Kwang-won , Eggertsson Gudmundur , Curnow Alan W. , Lin Winston , Celic Ivana , Whitman William , Söll Dieter
Published: July 2002

With the exception of the methanogenic archaea Methanococcus jannaschii and Methanobacterium thermoautotrophicum ΔH, all organisms surveyed contain orthologs of Escherichia coli cysteinyl‐tRNA synthetase (CysRS). The characterization of CysRS‐encoding (cysS) genes and the demonstration of their ability to complement an E. coli cysS
ts mutant reveal that Methanococcus maripaludis and Methanosarcina barkeri, two other methanogenic archaea, possess canonical CysRS proteins. A molecular phylogeny inferred from 40 CysRS sequences indicates that the CysRS of M. maripaludis and Methanosarcina spp. are specific relatives of the CysRS of Pyrococcus spp. and Chlamydia, respectively. This result suggests that the CysRS gene was acquired by lateral gene transfer in at least one euryarchaeotic lineage.
Uptake of [3H]bilirubin in freshly isolated rat hepatocytes: role of free bilirubin concentration
Authors: Mediavilla Maria Gabriela , Pascolo Lorella , Rodriguez Joaquin V. , Guibert Edgardo E. , Ostrow J.Donald , Tiribelli Claudio
Published: July 2002

Hepatocytic transport of physiological concentrations of unconjugated bilirubin (UCB) has not been determined in isolated liver cells. Initial uptake of highly purified [3H]UCB was measured in rat hepatocytes in the presence of human serum albumin at various free, unbound UCB concentrations, [UCB]. At [UCB]=42 nM (below aqueous solubility of 70 nM), uptake was strictly temperature dependent; this was much less evident at [UCB]=166 nM (supersaturated). At low, physiological UCB concentrations, specific UCB uptake showed saturative kinetics with an apparent K
m of 41 nM, indicating carrier‐mediated transport. With aqueous supersaturation, UCB entered hepatocytes mainly by passive diffusion.
The role of p38 mitogen‐activated protein kinase in IL‐6 and IL‐8 production from the TNF‐α‐ or IL‐1β‐stimulated rheumatoid synovial fibroblasts
Authors: Suzuki Masaki , Tetsuka Toshifumi , Yoshida Shinichi , Watanabe Nobuyuki , Kobayashi Masaaki , Matsui Nobuo , Okamoto Takashi
Published: July 2002

We examined the role of p38 mitogen‐activated protein (MAP) kinase in the tumor necrosis factor α (TNF‐α)‐ or interleukin‐1β (IL‐1β)‐induced production of interleukin‐6 (IL‐6) and interleukin‐8 (IL‐8) in fresh rheumatoid synovial fibroblast (RSF) cultures concomitantly with the induction of p38 MAP kinase activity. Pretreatment of RSF with a specific p38 MAP kinase inhibitor, SB203580, blocked the induction of IL‐6 and IL‐8 without affecting nuclear translocation of nuclear factor κB (NF‐κB) or IL‐6 and IL‐8 mRNA levels. These findings suggest that p38 MAP kinase inhibitor may have synergistic, rather than additive, effect for the treatment of rheumatoid arthritis.
Gender differences in defined contribution pension decisions
Authors: Bajtelsmit Vickie L. , Bernasek Alexandra , Jianakoplos Nancy A.
Published: July 2002

This paper considers gender differences in allocation of household wealth to defined contribution pensions. Using data from the 1989 Survey of Consumer Finances, we estimate the coefficient of relative risk aversion based on the allocation of wealth into defined contribution pensions. Unlike previous studies, we consider the problem in the context of the household’s overall portfolio. We find that women exhibit greater relative risk aversion in their allocation of wealth into defined contribution pension assets. © 1999 Elsevier Science Inc. All rights reserved.
WOMEN'S HEALTH CARE: A NEW PARADIGM FOR THE 21ST CENTURY
Authors: Klima Carrie S.
Published: July 2002

Oppression based on gender exists in all aspects of women's lives and transcends contemporary cultures, economic systems, and even health care services. Radical feminism provides an alternative philosophic framework of health care that is based on a women‐centered viewpoint, with the experiences of women as its unifying philosophy. Midwifery is a means to apply this new philosophic approach to the health care of women. A partnership between midwifery and feminist philosophy will allow women's voices to be heard, while guiding research in women's health care in new directions, and illuminating new approaches to current health problems. The new millennium provides an opportunity to explore an alternative framework and philosophy that will change the current paradigm of women's health care.
Experimental and numerical study of stratified viscous oil–water flow
Authors: Bochio Gustavo , Cely Marlon M. H. , Teixeira Arthur F. A. , Rodriguez Oscar M. H.
Published: February 2021

Abstract

Stratified two‐phase flows of oil and water are important to the energy industry, and models capable of predicting this type of flow are primordial. Many studies focus on fluids with low viscosity, but a high viscosity oil in the mixture significantly changes its behavior. We gathered experimental data of pressure drop, volumetric fractions, and flow‐pattern data of a stratified liquid–liquid flow with high viscosity ratio. In addition, a wire‐mesh sensor provided tomographic views of the flow. The data were compared with computational fluid dynamics (CFD) models using OpenFOAM and a one‐dimensional model. CFD simulations used an interface capturing method, and turbulence damping was introduced to avoid high eddy viscosity at the interface region. Reynolds Average Navier–Stokes and large eddy simulations were used to account for turbulence, and they showed significant differences. The comparisons showed good overall results for pressure drop, volumetric fractions, and phase distributions between CFD and experiments.
Untersuchungen zur tatsächlichen Temperaturbeanspruchung von Massivbrücken
Authors: Vill Markus , Vospernig Michael , Reiterer Michael , Eichinger‐Vill Eva M. , Kari Hannes
Published: February 2021

Temperatureinwirkungen sind für die wirtschaftliche Dimensionierung von Lager‐ und Fahrbahnübergangskonstruktionen sehr relevant. Zusätzlich führen größere Bemessungseinwirkungen, die zufolge Temperatur entstehen, auch bei integralen Brücken zu einer Erhöhung der Zwangsbeanspruchung. Die Entwicklung möglichst realistischer Temperaturansätze ist daher von großer technischer und wirtschaftlicher Bedeutung. Im Rahmen des Projekts „Modifiziertes Temperaturlastmodell für Eisenbahnbrücken (MTE 2.0)“, das von den Österreichischen Bundesbahnen initiiert wurde, konnten an sechs Massivbrücken Langzeittemperatur‐ und Verformungsmessungen durchgeführt werden. Zusätzlich erfolgten numerische instationäre Simulationen und Vergleichsrechnungen zur Untersuchung der thermischen Beanspruchungen, des Temperaturgradienten sowie zeitabhängiger Effekte (Kriechen, Schwinden). Schließlich wurden Temperaturdaten der Österreichischen Zentralanstalt für Meteorologie und Geodynamik (ZAMG) probabilistisch analysiert, um die Korrelation zwischen maximaler Außenlufttemperatur und konstantem Temperaturanteil für Brücken zu optimieren. Anhand dieser Langzeitmessungen, Simulationen und Analysen konnte die Basis für ein optimiertes Temperaturlastmodell für Massivbrücken geschaffen werden, das vor allem bei Tragwerken mit geringer Schlankheit und massigen Querschnitten von großer Bedeutung ist und wirtschaftliche Vorteile im Entwurf bringt, ohne die Zuverlässigkeit der Konstruktion negativ zu beeinflussen.
Freeze Control of Nanoparticle Aggregation and Exploration as Surface Enhanced Raman Scattering (SERS) Platform
Authors: Fukunaga Yu , Miyagawa Akihisa , Harada Makoto , Okada Tetsuo
Published: February 2021

We propose freezing as an efficient method for controlling silver nanoparticle (AgNP) aggregation. Solutes are expelled from the ice crystals and concentrated in a freeze concentrated solution (FCS) upon freezing. AgNPs are also concentrated in the FCS and aggregate depending on their concentration therein. Because the FCS volume is a function of temperature as well as the cryoprotectant type and concentration, the aggregation of AgNPs can be controlled using these experimental parameters. The freeze‐induced AgNP aggregation allows simple preparations of efficient platforms for surface enhanced Raman scattering (SERS) measurements. The SERS sensitivity can be optimized by the AgNP concentration in the FCS.
Delineation of groundwater quality locations suitable for target end‐use purposes through deep neural network models
Authors: Lee Sanghoon , Kaown Dugin , Koh Eun‐Hee , Ko Kyung‐Seok , Lee Kang‐Kun
Published: February 2021

Groundwater is the main source of water for beverages, and its quality varies depending on extraction location; this is particularly the case in regions with complex geology, topography, and multiple forms of land use. Thus, it is important to determine a suitable groundwater extraction location based on intended water use and the related water quality standards. In this study, deep neural network (DNN) models and GIS data relating to groundwater quality were applied to estimate potential maps of Gangwon Province in South Korea, where groundwater is frequently extracted for drinking purposes. These maps specify areas where the groundwater quality is conducive for being used as mineral water and water for brewing coffee (hereafter referred as “coffee water”). Sensitivity analysis identified how inputs were sensitive to model estimation and showed that land‐use variables were the most sensitive. The importance of each variable quantified how good or bad its region is for the desired groundwater. The overall features of importance were similar between mineral water and coffee water. However, with differences in hydrogeological units, carbonate rock was a variable of high positive importance for mineral water; metamorphic rock was its equivalent for coffee water. Our results offer a potential map of desired groundwater quality in the absence of a detailed understanding of the underlying hydrochemical processes governing groundwater quality. Additionally, the development of such a potential mapping model can help to determine the appropriate development area of groundwater for their respective purposes.