bluuebunny/crossref_metadata_2025 huggingface dataset:
Establishment of CHO cell lines expressing four N‐methyl‐D‐aspartate receptor subtypes and characterization of a novel antagonist PPDC
Authors: Uchino Shigeo , Watanabe Wakako , Nakamura Takeshi , Shuto Satoshi , Kazuta Yuji , Matsuda Akira , Nakajima-Iijima Sadayo , Kudo Yoshihisa , Kohsaka Shinichi , Mishina Masayoshi
Published: October 2002

To develop an assay system that allows the N‐methyl‐D‐aspartate (NMDA) receptor subtype‐selective antagonistic potency of drugs, we have established Chinese hamster ovary cell lines expressing the four NMDA receptor subtypes (GluRϵ1/ζ1–GluRϵ4/ζ1) heat‐indelibly. Using these clonal cells, we found that a novel antagonist, (1S,2R)‐1‐phenyl‐2[(S)‐1‐aminopropyl]‐N,N‐diethylcyclopropanecarboxamide, was less selective for the GluRϵ1/ζ1: the IC50 values for the GluRϵ1/ζ1–GluRϵ4/ζ1 were 41.7, 13.3, 12.6 and 11.5 μM, respectively, while two well‐known antagonists, DL‐2‐amino‐5‐phosphonovaleric acid and ifenprodil, showed the known potency and selectivity for each subtype. Thus, the established clonal cells are of use in characterizing the pharmacological properties of drugs that act on NMDA receptors.
The AP‐1 repressor, JDP2, is a bona fide substrate for the c‐Jun N‐terminal kinase
Authors: Katz Sigal , Heinrich Ronit , Aronheim Ami
Published: October 2002

The Jun dimerization protein 2 (JDP2) is a novel member of the basic leucine zipper family of transcription factors. JDP2 binds DNA as a homodimer and heterodimer with ATF2 and Jun proteins but not with c‐Fos proteins. JDP2 overexpression represses activating protein 1 transcription activity. Whereas JDP2 mRNA and protein levels are stable following different cell stimuli, JDP2 is rapidly phosphorylated upon UV irradiation, oxidative stress and low levels of translation inhibitor. The c‐Jun N‐terminal kinase phosphorylates JDP2 both in vitro and in vivo. JDP2 contains a putative consensus JNK docking‐site and a corresponding phosphoacceptor site. Substitution of threonine 148 to an alanine residue blocks JNK‐dependent JDP2 phosphorylation. Our data indicate that JDP2 is a bona fide substrate for the c‐Jun N‐terminal kinase. The precise role of JDP2 phosphorylation on its function is not yet known.
Signalling of GPI‐anchored CD157 via focal adhesion kinase in MCA102 fibroblasts
Authors: Liang Fubo , Chang Chan Fong
Published: October 2002

CD157, a glycosylphosphatidylinositol‐anchored protein, has previously been shown to mediate tyrosine phosphorylation of a 130 kDa protein (p130) in several cell lines. In this study, we have identified the p130 protein to be focal adhesion kinase (FAK or pp125FAK). FAK undergoes phosphorylation at Tyr‐397 and Tyr‐861 in intact MCA102 cells stably transfected with CD157 (MCA/CD157). MCA/CD157 cells, which displayed a rounded and compact cell morphology, exhibited a dispersed distribution, in contrast to a more closely associated and elongated spindle cell shape in the vector‐transfected cells. MCA/CD157 cells proliferated at a rate 20–25% slower than the control cells. Our results demonstrate, for the first time, that FAK is a downstream signalling molecule of CD157.
Dynamic flexibility in the Escherichia coli genome
Authors: Tsai Lu , Sun Zhirong
Published: October 2002

Empirical rules based on tetranucleotide parameters were presented to predict the structural parameters twist (Ω), roll (ρ), tilt (τ) and slide (Dy
). A statistical mechanical model was used to analyze the flexibility of the Escherichia coli genome. The replication terminus region displayed a low level of flexibility. A strong correlation can be seen between G+C content and flexibility. Average flexibilities in the coding regions were found to be significantly larger than those in non‐coding regions. The flexible characteristics in the 5′‐neighborhood of the coding regions and in three class sigma promoter sequences in the E. coli genome were also analyzed.
Amino acid residues before the hydrophobic region which are critical for membrane translocation of the N‐terminal domain of synaptotagmin II
Authors: Kida Yuichiro , Sakaguchi Masao , Fukuda Mitsunori , Mikoshiba Katsuhiko , Mihara Katsuyoshi
Published: October 2002

We examined the fine structure of the type I signal‐anchor sequence of synaptotagmin II, which has a 60‐residue N‐terminal domain followed by a hydrophobic region (H‐region), focusing on the hinge region between the N‐terminal and the H‐regions. It was found that the charged or highly polar residues support the translocation of the N‐terminal domain through the endoplasmic reticulum membrane at specific positions in the hinge. The residue requirement correlated with the turn propensity scale for transmembranes. It is suggested that a certain conformation, likely helical hairpin, in the hinge is critical for N‐terminal domain translocation.
A novel dicyclodextrinyl ditelluride compound with antioxidant activity
Authors: Ren Xiaojun , Xue Yan , Zhang Kun , Liu Junqiu , Luo Guimin , Zheng Jian , Mu Ying , Shen Jiacong
Published: October 2002

Reactive oxygen species (ROS) primarily arise from products of normal metabolic activities and are thought to be the etiology of many diseases. A novel dicyclodextrinyl ditelluride (2‐TeCD) compound was designed to be a functional mimic of the glutathione peroxidase that normally removes ROS. 2‐TeCD exhibited highly catalytic efficiency and good water solubility. Antioxidant activity was studied by using ferrous sulfate/ascorbate‐induced mitochondria damage model system. 2‐TeCD protected the mitochondria against oxidative damage in a dose‐dependent manner and exhibited also great antioxidant ability in comparison with 2‐phenyl‐1,2‐benziososelenazol‐3(2H)‐one. The mimic may result in better clinical therapies for the treatment of ROS‐mediated diseases.
Respiratory chain network in mitochondria of Candida parapsilosis: ADP/O appraisal of the multiple electron pathways
Authors: Milani Graziela , Jarmuszkiewicz Wiesl̵awa , Sluse-Goffart Claudine M. , Schreiber Angelica Z. , Vercesi Anibal E. , Sluse Francis E.
Published: October 2002

In this study we demonstrated that mitochondria of Candida parapsilosis contain a constitutive ubiquinol alternative oxidase (AOX) in addition to a classical respiratory chain (CRC) and a parallel respiratory chain (PAR) both terminating by two different cytochrome c oxidases. The C. parapsilosis AOX is characterized by a fungi‐type regulation by GMP (as a stimulator) and linoleic acid (as an inhibitor). Inhibitor screening of the respiratory network by the ADP/O ratio and state 3 respiration determinations showed that (i) oxygen can be reduced by the three terminal oxidases through four paths implying one bypass between CRC and PAR and (ii) the sum of CRC, AOX and PAR capacities is higher than the overall respiration (no additivity) and that their engagement could be progressive according to the redox state of ubiquinone, i.e. first cytochrome pathway, then AOX and finally PAR.
Manganese superoxide dismutase induction by iron is impaired in Friedreich ataxia cells
Authors: Jiralerspong Sarn , Ge Bing , Hudson Thomas J. , Pandolfo Massimo
Published: October 2002

Iron‐mediated oxidative stress has been implicated in the pathology of the neurodegenerative disease Friedreich ataxia (FRDA). Here, we show that normal upregulation of the stress defense protein manganese superoxide dismutase (MnSOD) fails to occur in FRDA fibroblasts exposed to iron. This impaired induction was observed at iron levels in which increased activation of the redox‐sensitive factor NF‐κB was absent. Furthermore, MnSOD induction could only be partially suppressed by antioxidants. We conclude that an NF‐κB‐independent pathway that may not require free radical signaling is responsible for the reduction of MnSOD induction. This impairment could constitute both a novel defense mechanism against iron‐mediated oxidative stress in cells with mitochondrial iron overload and conversely, an alternative source of free radicals that could contribute to the disease pathology.
Human monkeypox and smallpox viruses: genomic comparison
Authors: Shchelkunov Sergei N. , Totmenin Alexei V. , Babkin Igor V. , Safronov Pavel F. , Ryazankina Olga I. , Petrov Nikolai A. , Gutorov Valery V. , Uvarova Elena A. , Mikheev Maxim V. , Sisler Jerry R. , Esposito Joseph J. , Jahrling Peter B. , Moss Bernard , Sandakhchiev Lev S.
Published: October 2002

Monkeypox virus (MPV) causes a human disease which resembles smallpox but with a lower person‐to‐person transmission rate. To determine the genetic relationship between the orthopoxviruses causing these two diseases, we sequenced the 197‐kb genome of MPV isolated from a patient during a large human monkeypox outbreak in Zaire in 1996. The nucleotide sequence within the central region of the MPV genome, which encodes essential enzymes and structural proteins, was 96.3% identical with that of variola (smallpox) virus (VAR). In contrast, there were considerable differences between MPV and VAR in the regions encoding virulence and host‐range factors near the ends of the genome. Our data indicate that MPV is not the direct ancestor of VAR and is unlikely to naturally acquire all properties of VAR.
Choline containing metabolites during cell transfection: an insight into magnetic resonance spectroscopy detectable changes
Authors: Griffin J.L , Mann C.J , Scott J , Shoulders C.C , Nicholson J.K
Published: October 2002

Increases in choline containing metabolites have been associated with a number of disorders, including malignant cell growth. In this study, high resolution magic angle spinning 1H nuclear magnetic resonance spectroscopy was employed to monitor metabolite changes during cell transfection, and an increase in phosphocholine was detected. This increase appears to be correlated with cell membrane disruption associated with the insertion of plasmid DNA into cells, since the level of phosphocholine in mock transfected cells was comparable to that of control cells. These data suggest choline containing metabolite changes detected in vivo using magnetic resonance spectroscopy relate to cell membrane disruption.
A sequential two‐step proteolytic process in the carboxyl‐terminal truncation of precursor D1 protein in Synechocystis sp. PCC68031
Authors: Inagaki Noritoshi , Yamamoto Yumiko , Satoh Kimiyuki
Published: October 2002

The D1 protein of photosystem II is synthesized in precursor with a carboxyl‐terminal extension. Interestingly, there is quite a range in chain length of the extension, which roughly depends upon the class of organisms. In cyanobacteria, e.g. in Synechocystis sp. PCC6803, the extension consists of 16 amino acid residues, seven residues longer than its counterpart in higher plants. In this study, we examined the D1 processing in Synechocystis sp. PCC6803 by pulse‐chase experiments and detected a proteolytic intermediate of this process. This finding suggests that the elongated extension in this organism is excised with a sequential two‐step proteolysis, which differs markedly from the manner observed in higher plants.
Bcl‐2 phosphorylation is required for inhibition of oxidative stress‐induced lysosomal leak and ensuing apoptosis
Authors: Zhao Ming , Eaton John W. , Brunk Ulf T.
Published: October 2002

B‐cell leukemia/lymphoma 2 (Bcl‐2) blocks oxidant‐induced apoptosis at least partly by stabilizing lysosomes. Here we report that phosphorylation of Bcl‐2 may be required for these protective effects. J774 cells overexpressing wild‐type Bcl‐2 resist oxidant‐induced lysosomal leak as well as apoptosis, and this protection is amplified by pretreatment with phorbol 12‐myristate 13‐acetate (which promotes protein kinase C (PKC)‐dependent phosphorylation of Bcl‐2). In contrast, cells overexpressing the Bcl‐2 mutant S70A (which cannot be phosphorylated) are not protected in either circumstance. Transfection with Bcl‐2(S70E), a constitutively active Bcl‐2 mutant which does not require phosphorylation, is protective independent of PKC activation. In contrast, C2‐ceramide, a putative protein phosphatase 2A activator, abolishes the protective effects of wild‐type Bcl‐2 overexpression but does not diminish protection afforded by Bcl‐2(S70E). Additional results suggest that, perhaps as a consequence of lysosomal stabilization, Bcl‐2 may prevent activation of phospholipase A2, an event potentially important in the ultimate initiation of apoptosis.
Incorporation of oxyethylene units between hydrocarbon chain and pseudoglyceryl backbone in cationic lipid potentiates gene transfection efficiency in the presence of serum
Authors: Dileep P.V , Antony A , Bhattacharya Santanu
Published: October 2002

Four novel cationic lipids with different numbers of oxyethylene units at the linkage region between the pseudoglyceryl backbone and the hydrocarbon chains have been synthesized and used as mixtures with 1,2‐dioleoyl‐L‐α‐glycero‐3‐phosphatidyl ethanolamine (DOPE) for liposome‐mediated gene transfection. Incorporation of different numbers of oxyethylene (–CH2CH2O–) units between long hydrocarbon chain at the C‐1 and C‐2 positions of the pseudoglyceryl skeleton improved the transfection efficiency considerably compared to the one in which the chains were connected via simple ether links. A pronounced improvement in the gene transfer efficiency was observed with the unsymmetrical cationic lipid 3 in which the long hydrocarbon at the C‐1 position of the pseudoglyceryl segment is connected via two (–CH2CH2O–) units. Notably, the transfection ability of lipid 3 with DOPE in the presence of serum was significantly greater than LIPOFECTAMINE®. This suggests that introduction of oxyethylene units between long hydrocarbon chains at the C‐1 and C‐2 positions of the pseudoglyceryl skeleton provides a novel strategy to achieve efficient gene transfer, especially in conditions where the presence of serum is critical.
Mapping the receptor site for ergtoxin, a specific blocker of ERG channels
Authors: Pardo-López Liliana , Garcı́a-Valdés Jesús , Gurrola Georgina B. , Robertson Gail A. , Possani Lourival D.
Published: October 2002

We show here that ergtoxin (ErgTx) is a bona fide, specific blocker of the human ether‐a‐go‐go‐related gene (HERG) channels. It does not affect the function of either M‐eag or M‐elk channels. A chimeric construction containing a segment of the P‐region of M‐eag channel inserted into the HERG channel drastically diminished or completely abolished the inhibitory effect of ErgTx, whereas chimeras of the P‐region of HERG channel into M‐eag channels recovered the inhibitory effect. From the P‐region point mutants of HERG channel assays, only the mutant N598Q shows about 25% decrement of the ErgTx inhibitory effect. ErgTx recognizes the P‐region of HERG channels, blocking the channel function with a K
d in the order of 12 nM.
Uncoupling proteins outside the animal and plant kingdoms: functional and evolutionary aspects
Authors: Sluse Francis E. , Jarmuszkiewicz Wieslawa
Published: October 2002

The appearance of intracellular oxidative phosphorylation at the time of acquisition of mitochondria in Eukarya was very soon accompanied by the emergence of uncoupling protein, a carrier specialized in free fatty acid‐mediated H+ recycling that can modulate the tightness of coupling between mitochondrial respiration and ATP synthesis, thereby maintaining a balance between energy supply and demand in the cell and defending cells against damaging reactive oxygen species production when electron carriers of the respiratory chain become overreduced. The simultaneous occurrence of redox free energy‐dissipating oxidase, which has the same final effect, could be related to the functional interactions between both dissipative systems.
Inhibition of PI3K/p70 S6K and p38 MAPK cascades increases osteoblastic differentiation induced by BMP‐2
Authors: Viñals Francesc , López-Rovira Teresa , Rosa Jose Luis , Ventura Francesc
Published: October 2002

Bone morphogenetic proteins (BMPs) transdifferentiate C2C12 cells from the myogenic to the osteogenic lineage. In this work we examine the role of the phosphatidylinositol 3‐kinase/p70 S6 kinase (PI3K/p70 S6K) and p38 mitogen‐activated protein kinase (p38 MAPK) cascades in the osteogenic effects of BMP‐2. BMP‐2 stimulated both cascades transiently (maximal at 1 h and decreasing thereafter). In contrast, BMP‐2 had no effect on p42/p44 MAPK (Erks) stimulation. We also analyzed the effects of selective inhibitors of these pathways on the expression of osteogenic markers. Inhibitors of p38 MAPK (SB203580) or the PI3K/p70 S6K pathway (Ly294002 and rapamycin) not only fail to block the osteoblast phenotype induced by BMP‐2, measured as induction of Cbfa1 expression and transcriptional activity, but also potentiate the effect of BMP‐2 on late osteoblast markers, such as alkaline phosphatase activity and osteocalcin expression. These data suggest that, in contrast to their positive effect on myogenic differentiation, PI3K/p70 S6K and p38 MAPK cascades have a negative role in osteoblast differentiation.
Identification of a cDNA encoding a novel C18‐Δ9 polyunsaturated fatty acid‐specific elongating activity from the docosahexaenoic acid (DHA)‐producing microalga, Isochrysis galbana1
Authors: Qi Baoxiu , Beaudoin Frédéric , Fraser Tom , Stobart A.Keith , Napier Johnathan A , Lazarus Colin M
Published: October 2002

Isochrysis galbana, a marine prymnesiophyte microalga, is rich in long chain polyunsaturated fatty acids such as docosahexaenoic acid (C22:6n‐3, Δ4,7,10,13,16,19). We used a polymerase chain reaction‐based strategy to isolate a cDNA, designated IgASE1, encoding a polyunsaturated fatty acid‐elongating activity from I. galbana. The coding region of 263 amino acids predicts a protein of 30 kDa that shares only limited homology to animal and fungal proteins with elongating activity. Functional analysis of IgASE1, by expression in Saccharomyces cerevisiae, was used to determine its activity and substrate specificity. Transformed yeast cells specifically elongated the C18‐Δ9 polyunsaturated fatty acids, linoleic acid (C18:2n‐6, Δ9,12) and α‐linolenic acid (C18:3n‐3, Δ9,12,15), to eicosadienoic acid (C20:2n‐6, Δ11,14) and eicosatrienoic acid (C20:3n‐3, Δ11,14,17), respectively. To our knowledge this is the first time such an elongating activity has been functionally characterised. The results also suggest that a major route for eicosapentaenoic acid (C20:5n‐3, Δ5,8,11,14,17) and docosahexaenoic acid syntheses in I. galbana may involve a Δ8 desaturation pathway.
The absolute concentration of nigral neuromelanin, assayed by a new sensitive method, increases throughout the life and is dramatically decreased in Parkinson's disease
Authors: Zecca Luigi , Fariello Ruggero , Riederer Peter , Sulzer David , Gatti Alberto , Tampellini Davide
Published: October 2002

The concentration of neuromelanin (NM) in substantia nigra pars compacta (SNPC) has been measured in male and female normal subjects at different ages in the range 1–97 years old and in SNPC of parkinsonian patients. A very similar age trend of NM concentration was found in both sexes. In the first year of life NM was not detectable, between 10 and 20 years the NM levels were 0.3–0.8 μg/mg of SNPC, between 20 and 50 years were 0.8–2.3 μg/mg SNPC and between 50 and 90 were 2.3–3.7 μg/mg of SNPC. In parkinsonian subjects, the NM levels were 1.2–1.5 μg/mg of SNPC, which is less than 50% with respect to the age‐matched controls. These data demonstrate a continuous NM accumulation in SNPC neurons during aging, the presence of large amounts of NM in SNPC and severe depletion of NM in Parkinson's disease.
Prior induction of heme oxygenase‐1 with glutathione depletor ameliorates the renal ischemia and reperfusion injury in the rat
Authors: Horikawa Saburo , Yoneya Rika , Nagashima Yoji , Hagiwara Kiyokazu , Ozasa Hisashi
Published: October 2002

Heme oxygenase (HO)‐1 catalyzes the rate‐limiting step in heme degradation releasing iron, carbon monoxide, and biliverdin. Induction of HO‐1 occurs as an adaptive and protective response to oxidative stress. Ischemia and reperfusion (IR) injury seems to be mainly caused by the oxidative stress. In this study, we have examined whether prior induction of HO‐1 with buthionine sulfoximine (BSO), a glutathione (GSH) depletor, affects the subsequent renal IR injury. BSO (2 mmol/kg body weight) was administered intraperitoneally into rats, the levels of HO‐1 protein increased within 4 h after the injection. When BSO was administered into rats at 5 h prior to the renal 45 min of ischemia, the renal IR injury was assessed by determining the levels of blood urea nitrogen and serum creatinine, markers for renal injury, after 24 h of reperfusion. The renal injury was significantly improved as compared to the rats treated with IR alone. Administration of zinc‐protoporphyrin IX, an inhibitor of HO activity, reduced the efficacy of BSO pretreatment on the renal IR injury. Our findings suggest that the prior induction of HO‐1 ameliorates the subsequent renal IR injury.
How do 14‐3‐3 proteins work? – Gatekeeper phosphorylation and the molecular anvil hypothesis
Authors: Yaffe Michael B
Published: October 2002

14‐3‐3 proteins were the first signaling molecules to be identified as discrete phosphoserine/threonine binding modules. This family of proteins, which includes seven isotypes in human cells and up to 15 in plants, plays critical roles in cell signaling events that control progress through the cell cycle, transcriptional alterations in response to environmental cues, and programmed cell death. Despite over 30 years of research, distinct roles for most isotypes remain unknown. Though 14‐3‐3 proteins perform different functions for different ligands, general mechanisms of 14‐3‐3 action include changes in activity of bound ligands, altered association of bound ligands with other cellular components, and changes in intracellular localization of 14‐3‐3‐bound cargo. We present a speculative model where binding of 14‐3‐3 to multiple sites on some ligands results in global ligand conformational changes that mediate their biological effects. For these multi‐site ligands, one binding site is likely to function as a ‘gatekeeper’ whose phosphorylation is necessary for 14‐3‐3 binding but may not always be sufficient for full biological activity. If correct, then 14‐3‐3 may prove to be a bona fide phosphodependent signaling chaperone.
WW and SH3 domains, two different scaffolds to recognize proline‐rich ligands
Authors: Macias Maria J. , Wiesner Silke , Sudol Marius
Published: October 2002

WW domains are small protein modules composed of approximately 40 amino acids. These domains fold as a stable, triple stranded β‐sheet and recognize proline‐containing ligands. WW domains are found in many different signaling and structural proteins, often localized in the cytoplasm as well as in the cell nucleus. Based on analyses of seven structures of WW domains, we discuss their diverse binding preferences and sequence conservation patterns. While modeling WW domains for which structures have not been determined we uncovered a case of potential molecular and functional convergence between WW and SH3 domains. The binding surface of the modeled WW domain of Npw38 protein shows a remarkable similarity to the SH3 domain of Sem5 protein, confirming biochemical data on similar binding predilections of both domains.
Copper‐induced conformational change in a marsupial prion protein repeat peptide probed using FTIR spectroscopy
Authors: Gustiananda Marsia , Haris Parvez I. , Milburn Peter J. , Gready Jill E.
Published: October 2002

We report the first Fourier transform infrared analysis of prion protein (PrP) repeats and the first study of PrP repeats of marsupial origin. Large changes in the secondary structure and an increase in hydrogen bonding within the peptide groups were evident from a red shift of the amide I band by >7 cm−1 and an approximately five‐fold reduction in amide hydrogen–deuterium exchange for peptide interacting with Cu2+ ions. Changes in the tertiary structure upon copper binding were also evident from the appearance of a new band at 1564 cm−1, which arises from the ring vibration of histidine. The copper‐induced conformational change is pH dependent, and occurs at pH >7.
The sorLA cytoplasmic domain interacts with GGA1 and ‐2 and defines minimum requirements for GGA binding
Authors: Jacobsen Linda , Madsen Peder , Nielsen Morten S. , Geraerts Wijnand P.M. , Gliemann Jørgen , Smit August B. , Petersen Claus M.
Published: October 2002

We report that the Vps10p domain receptor sorLA binds the adaptor proteins GGA1 and ‐2, which take part in Golgi–endosome sorting. The GGAs bind with differential requirements via three critical residues in the C‐terminal segment of the sorLA cytoplasmic tail. Unlike in sortilin and the mannose 6‐phosphate receptors, the GGA‐binding segment in sorLA contains neither an acidic cluster nor a dileucine. Our results support the concept of sorLA as a potential sorting receptor and suggest that key residues in sorLA and sortilin conform to a new type of motif (Ψ–Ψ–X–X–∅) defining minimum requirements for GGA binding to cytoplasmic receptor domains.
Mutational analysis of the apical region of domain II of the HCV IRES
Authors: Kalliampakou Katerina I , Psaridi-Linardaki Loukia , Mavromara Penelope
Published: October 2002

The hepatitis C virus internal ribosome entry site (IRES) binds directly to the 40S ribosomal subunit via domains III/IV while domain II induces conformational changes on the ribosome which have been implicated in the decoding process. Here, we performed an extensive mutational study within the apical portion of domain II in order to address the functional role of this region on translation. Our results showed that the conservation of most nucleotides in this region was only partially related to the IRES function. Notwithstanding, however, selected single point mutations within the apical loop had a deleterious effect on IRES activity.
Inhibition of bacterial RNase P by aminoglycoside–arginine conjugates
Authors: Eubank Timothy D. , Biswas Roopa , Jovanovic Milan , Litovchick Alexander , Lapidot Aviva , Gopalan Venkat
Published: October 2002

The potential of RNAs and RNA–protein (RNP) complexes as drug targets is currently being explored in various investigations. For example, a hexa‐arginine derivative of neomycin (NeoR) and a tri‐arginine derivative of gentamicin (R3G) were recently shown to disrupt essential RNP interactions between the trans‐activator protein (Tat) and the Tat‐responsive RNA (trans‐activating region) in the human immunodeficiency virus (HIV) and also inhibit HIV replication in cell culture. Based on certain structural similarities, we postulated that NeoR and R3G might also be effective in disrupting RNP interactions and thereby inhibiting bacterial RNase P, an essential RNP complex involved in tRNA maturation. Our results indicate that indeed both NeoR and R3G inhibit RNase P activity from evolutionarily divergent pathogenic bacteria and do so more effectively than they inhibit partially purified human RNase P activity.
Synthesis, membrane insertion and assembly of the chloroplast‐encoded D1 protein into photosystem II
Authors: Zhang Lixin , Aro Eva-Mari
Published: October 2002

Rapid light‐dependent turnover of the chloroplast‐encoded D1 protein maintains photosystem II (PS II) functional over a wide range of light intensities. Following initiation of psbA mRNA translation, the elongating D1 is targeted, possibly by chloroplast signal recognition particle 54 (cpSRP54), to the thylakoid cpSecY translocation channel. Transmembrane domains of nascent D1 start interacting with other PS II core proteins already during the translocation process to ensure an efficient assembly of the multiprotein membrane complex. Here we review the progress recently made concerning the synthesis, targeting, membrane insertion and assembly to PS II of the chloroplast‐encoded D1 protein and discuss the possible convergence of targeting and translocation of chloroplast‐ and nuclear‐encoded thylakoid proteins.
Regulatory interactions in Arabidopsis thaliana acetohydroxyacid synthase
Authors: Lee Yu-Ting , Duggleby Ronald G
Published: October 2002

Acetohydroxyacid synthase (AHAS; EC 4.1.3.18) contains catalytic and regulatory subunits, the latter being required for sensitivity to feedback regulation by leucine, valine and isoleucine. The regulatory subunit of Arabidopsis thaliana AHAS possesses a sequence repeat and we have suggested previously that one repeat binds leucine while the second binds valine or isoleucine, with synergy between the two sites. We have mutated four residues in each repeat, based on a model of the regulatory subunit. The data confirm that there are separate leucine and valine/isoleucine sites, and suggest a complex pathway for regulatory signal transmission to the catalytic subunit.
Molecular simulations of the large conductance mechanosensitive (MscL) channel under mechanical loading
Authors: Bilston Lynne E , Mylvaganam Kausala
Published: October 2002

The MscL channel is a mechanosensitive channel which is gated by membrane stress or tension. Here, we describe a series of simulations which apply simulated mechanical stress to a molecular model of the MscL channel using two methods – direct force application to the transmembrane segments, and anisotropic pressure coupling. In the latter simulations, pressures less than that equivalent to a bilayer tension of 12 dyn/cm did not cause the channel to open, while pressures in excess of this value resulted in the channel opening. These results are in approximate agreement with experimental findings.
In vitro PKA phosphorylation‐mediated human PDE4A4 activation
Authors: Laliberté France , Liu Susana , Gorseth Elise , Bobechko Brian , Bartlett Adrienne , Lario Paula , Gresser Michael J , Huang Zheng
Published: October 2002

The PDE4 catalytic machinery comprises, in part, two divalent cations in a binuclear motif. Here we report that PDE4A4 expressed in Sf9 cells exhibits a biphasic Mg2+ dose–response (EC50 of ∼0.15 and >10 mM) in catalyzing cAMP hydrolysis. In vitro phosphorylation of PDE4A4 by the PKA‐catalytic subunit increases the enzyme's sensitivity to Mg2+, leading to 4‐fold increased cAMP hydrolysis without affecting its K
m. The phosphorylation also increases the potencies of (R)‐ and (S)‐rolipram without affecting CDP‐840 and SB‐207499. The results support that modulating the cofactor binding affinity of PDE4 represents a mechanism for regulating its activity.
A quantity control mechanism regulating levels of the HapE subunit of the Hap complex in Aspergillus nidulans: no accumulation of HapE in hapC deletion mutants
Authors: Kato M. , Tateyama Y. , Hayashi K. , Naruse F. , Oonishi R. , Tanoue S. , Tanaka A. , Kobayashi T. , Tsukagoshi N.
Published: October 2002

The Aspergillus nidulans CCAAT‐binding complex (Hap complex) consists of at least three subunits, HapB, HapC and HapE. To investigate the quantity control mechanisms of the subunits during assembly of the Hap complex, reconstitution studies with the recombinant subunits and extracts prepared from the respective hap subunit deletion mutants were carried out. Furthermore, Western blot analysis of the Hap subunits and Northern blot analysis of the hap genes with the respective deletion mutants were also performed. From all the results together, it was suggested that the number of the HapC molecule could adjust that of the HapE molecule by forming stable heterodimers prior to assembly of the Hap complex.
Exploring the subsite specificity of Schistosoma mansoni aspartyl hemoglobinase through comparative molecular modelling
Authors: Silva F.P. , Ribeiro F. , Katz N. , Giovanni-De-Simone S.
Published: October 2002

Blood flukes of the genus Schistosoma currently infect millions of people in tropical and subtropical countries. An enzyme playing a major role in hemoglobin (Hb) degradation by Schistosoma mansoni has been cloned and shown to be highly similar to the human cathepsin D aspartyl proteinase, although presenting a distinct substrate specificity from the latter. Investigating the structural features responsible for this difference has a major application in the design of selective anti‐schistosomal drugs. In order to achieve this goal a homology model for the S. mansoni aspartyl hemoglobinase was constructed and then used to simulate the complexes formed with two transition state analogues of Hb‐derived octapeptide substrates. Comparison with human cathepsin D showed that different pocket volumes and surface electrostatic potentials arise from substitutions in residues comprising the S4, S3, S2 and S3′ subsites. Since the primary specificity of the S. mansoni enzyme resembles that of HIV‐1 protease, we have discussed the applicability of current retroviral protease inhibitors as leads for the design of new anti‐schistosomal drugs.
Characterization of a bacterial tyrosine ammonia lyase, a biosynthetic enzyme for the photoactive yellow protein
Authors: Kyndt J.A. , Meyer T.E. , Cusanovich M.A. , Van Beeumen J.J.
Published: October 2002

During genome sequence analysis of Rhodobacter capsulatus, nearby open reading frames were found that encode a photoactive yellow protein (PYP) and a hypothetical biosynthetic enzyme for its chromophore, a tyrosine ammonia lyase (TAL). We isolated the TAL gene, overproduced the recombinant protein in Escherichia coli, and after purification analyzed the enzyme for its activity. The catalytic efficiency for tyrosine was shown to be approximately 150 times larger than for phenylalanine, suggesting that the enzyme could in fact be involved in biosynthesis of the PYP chromophore. To our knowledge it is the first time this type of enzyme has been found in bacteria.
pH‐Dependent channel activity of heterologously‐expressed main intrinsic protein (MIP) from rat lens
Authors: Drake K.Dawn , Schuette Diana , Chepelinsky Ana B , Jacob Tim J , Crabbe M.James C
Published: October 2002

Wild‐type rat lens main intrinsic protein (MIP) was heterologously expressed in the membrane of Spodoptera frugiperda (Sf21) cells using the baculovirus expression system and in mouse erythroid leukaemia cells (MEL C88). Both MEL and Sf21 cell lines expressing wild‐type MIP were investigated for the conductance of ions using a whole cell patch clamp technique. An increase in conductance was seen in both expression systems, particularly on lowering the pH to 6.3. In Sf21 cells, addition of antibodies to the NPA1 box resulted in a reduction of current flow. These results suggest that MIP has pH‐dependent ion channel activity, which involves the NPA1 box domain.
NF‐κB elements contribute to junB inducibility by lipopolysaccharide in the murine macrophage cell line RAW264.7
Authors: Frazier-Jessen Michelle R , Thompson Cynthia D , Brown Robert , Rawat Rashmi , Nordan Richard P , Feldman Gerald M
Published: October 2002

Macrophages respond to bacterial lipopolysaccharide (LPS) by activating latent cis‐acting factors that initiate transcription of immediate early genes. One such immediate early gene, junB, is induced by LPS in macrophages within 30 min. To identify elements that mediate the induction of junB by LPS, upstream and downstream sequences flanking the junB gene were examined by transient expression in the RAW264.7 murine macrophage cell line using a luciferase reporter gene vector containing the junB minimal promoter. A >10‐fold enhancement was associated with a 222 bp region downstream of the junB promoter in response to LPS. Transient reporter assays demonstrated that multiple nuclear factor (NF) κB sites are required for inducibility of junB by LPS in RAW264.7 cells. Electrophoretic mobility shift assays confirmed binding of LPS‐induced nuclear proteins included p50/p65 heterodimers at these NF‐κB sites.
Ribosomal RNA pseudouridines and pseudouridine synthases
Authors: Ofengand James
Published: October 2002

Pseudouridines are found in virtually all ribosomal RNAs but their function is unknown. There are four to eight times more pseudouridines in eukaryotes than in eubacteria. Mapping 19 Haloarcula marismortui pseudouridines on the three‐dimensional 50S subunit does not show clustering. In bacteria, specific enzymes choose the site of pseudouridine formation. In eukaryotes, and probably also in archaea, selection and modification is done by a guide RNA–protein complex. No unique specific role for ribosomal pseudouridines has been identified. We propose that pseudouridine's function is as a molecular glue to stabilize required RNA conformations that would otherwise be too flexible.
Malignant transformation by the eukaryotic translation initiation factor 3 subunit p48 (eIF3e)
Authors: Mayeur Greg L , Hershey John W.B
Published: October 2002

Several components of translation, e.g. eIF4E and PKR, are implicated in cancer. The e‐subunit (p48) of mammalian initiation factor 3 is encoded by the Int6 gene, a common site for integration of the mouse mammary tumor virus genome, leading to the production of a truncated eukaryotic initiation factor‐3e (eIF3e). Stable expression of a truncated eIF3e in NIH 3T3 cells causes malignant transformation by four criteria: foci formation; anchorage independent growth; accelerated growth; and lack of contact inhibition. Stable expression of full‐length eIF3e does not cause transformation. The truncated eIF3e also inhibits the onset of apoptosis caused by serum starvation.
Insertion and glycosylation of Pf3‐derived membrane proteins in microsomes
Authors: Ridder Anja , Thissen Laura , Killian Antoinette , de Kruijff Ben
Published: October 2002

To get insight into the insertion mechanism of small newly synthesized single‐spanning membrane proteins, Pf3 coat protein mutants were constructed with potential glycosylation sites in the N‐terminus. Some of these proteins, when synthesized in vitro in the presence of microsomes, became efficiently glycosylated, proving that they insert into the membrane and translocate their N‐terminus to the lumenal side. Such Pf3 constructs also insert efficiently into Escherichia coli vesicles and even in pure lipid vesicles, suggesting a common mechanism, which might be spontaneous. Glycosylation was sensitive to changes in the amino acid sequence of the N‐terminus, suggesting that it depends on the structure of the protein and/or its positioning with respect to the lipid–water interface.
Clusterin is an extracellular chaperone that specifically interacts with slowly aggregating proteins on their off‐folding pathway
Authors: Poon Stephen , Treweek Teresa M , Wilson Mark R , Easterbrook-Smith Simon B , Carver John A
Published: October 2002

Clusterin is an extracellular mammalian chaperone protein which inhibits stress‐induced precipitation of many different proteins. The conformational state(s) of proteins that interact with clusterin and the stage(s) along the folding and off‐folding (precipitation‐bound) pathways where this interaction occurs were previously unknown. We investigated this by examining the interactions of clusterin with different structural forms of α‐lactalbumin, γ‐crystallin and lysozyme. When assessed by ELISA and native gel electrophoresis, clusterin did not bind to various stable, intermediately folded states of α‐lactalbumin nor to the native form of this protein, but did bind to and inhibit the slow precipitation of reduced α‐lactalbumin. Reduction‐induced changes in the conformation of α‐lactalbumin, in the absence and presence of clusterin, were monitored by real‐time 1H NMR spectroscopy. In the absence of clusterin, an intermediately folded form of α‐lactalbumin, with some secondary structure but lacking tertiary structure, aggregated and precipitated. In the presence of clusterin, this form of α‐lactalbumin was stabilised in a non‐aggregated state, possibly via transient interactions with clusterin prior to complexation. Additional experiments demonstrated that clusterin potently inhibited the slow precipitation, but did not inhibit the rapid precipitation, of lysozyme and γ‐crystallin induced by different stresses. These results suggest that clusterin interacts with and stabilises slowly aggregating proteins but is unable to stabilise rapidly aggregating proteins. Collectively, our results suggest that during its chaperone action, clusterin preferentially recognises partly folded protein intermediates that are slowly aggregating whilst venturing along their irreversible off‐folding pathway towards a precipitated protein.
NMR studies of flexible peptides in cavities mimicking the synaptic cleft
Authors: Pavone Luca , Crescenzi Orlando , Tancredi Teodorico , Temussi Piero Andrea
Published: October 2002

The interaction of neuropeptides with post‐synaptic receptors is characterised by a high entropic barrier originating from the combination of nanomolar concentration with low conformer population. The influence of high viscosity environments on conformer distribution can help overcome this difficulty. In an attempt to simulate the physicochemical conditions of the synaptic cleft, 15N‐labelled enkephalin has been studied in polyacrylamide gels swollen by different aqueous solutions in the temperature range 273–293 K. Nuclear Overhauser enhancement spectra in the gel pores are consistent with a conformational selection or a slowing down of internal motions that can favour the interaction of the peptide with the receptor.
The interaction of the bisphosphorylated N‐terminal arm of cardiac troponin I‐A 31P‐NMR study
Authors: Schmidtmann Anja , Lohmann Karin , Jaquet Kornelia
Published: October 2002

Cardiac troponin I, the inhibitory subunit of the heterotrimeric cardiac troponin (cTn) complex is phosphorylated by protein kinase A at two serine residues located in its heart‐specific N‐terminal extension. This flexible arm interacts at different sites within cTn dependent on its phosphorylation degree. Bisphosphorylation is known to induce conformational changes within cTnI which finally lead to a reduction of the calcium affinity of cTnC. However, as we show here, the bisphosphorylated cTnI arm does not interact with cTnC, but with cTnT and/or cTnI.
The acidic regions of WASp and N‐WASP can synergize with CDC42Hs and Rac1 to induce filopodia and lamellipodia
Authors: Hüfner Katharina , Schell Barbara , Aepfelbacher Martin , Linder Stefan
Published: October 2002

The acidic (A) region of WASp family proteins is thought to represent a high‐affinity binding site for Arp2/3 complex without activating properties. Here we show that GST‐fused WASp‐A and N‐WASP‐A, but not a WASP‐A/W500S mutant, several truncated WASp‐A constructs or WAVE1‐A can pull down Arp2/3 complex from cell lysates. Significantly, WASp‐A and N‐WASP‐A synergistically trigger formation of filopodia or lamellipodia when coinjected with sub‐effective concentrations of V12CDC42Hs or V12Rac1, respectively, into macrophages. The ability of WASp family A region constructs to induce this effect is closely correlated with their ability to bind Arp2/3 complex in vitro. These results imply that (i) Arp2/3 complex is critically involved in filopodia and lamellipodia formation in macrophages and (ii) acidic regions of WASp and N‐WASP are not simply binding sites for Arp2/3 complex but can prime it for RhoGTPase‐triggered signals leading to actin nucleation.
Novel nuclear‐encoded proteins interacting with a plastid sigma factor, Sig1, in Arabidopsis thaliana1
Authors: Morikawa Kazuya , Shiina Takashi , Murakami Shinya , Toyoshima Yoshinori
Published: October 2002

Sigma factor binding proteins are involved in modifying the promoter preferences of the RNA polymerase in bacteria. We found the nuclear encoded protein (SibI) that is transported into chloroplasts and interacts specifically with the region 4 of Sig1 in Arabidopsis. SibI and its homologue, T3K9.5 are novel proteins, which are not homologous to any protein of known function. The expression of sibI was tissue specific, light dependent, and developmentally timed. We suggest the transcriptional regulation by sigma factor binding proteins to function in the plastids of higher plant.
Dual action of isoprenols from herbal medicines on both PPARγ and PPARα in 3T3‐L1 adipocytes and HepG2 hepatocytes
Authors: Takahashi Nobuyuki , Kawada Teruo , Goto Tsuyoshi , Yamamoto Takayuki , Taimatsu Aki , Matsui Naoko , Kimura Kazuhiro , Saito Masayuki , Hosokawa Masashi , Miyashita Kazuo , Fushiki Tohru
Published: October 2002

Several herbal medicines improve hyperlipidemia, diabetes and cardiovascular diseases. However, the molecular mechanism underlying this improvement has not yet been clarified. In this study, we found that several isoprenols, common components of herbal plants, activate human peroxisome proliferator‐activated receptors (PPARs) as determined using the novel GAL4 ligand‐binding domain chimera assay system with coactivator coexpression. Farnesol and geranylgeraniol that are typical isoprenols in herbs and fruits activated not only PPARγ but also PPARα as determined using the chimera assay system. These compounds also activated full‐length human PPARγ and PPARα in CV1 cells. Moreover, these isoprenols upregulated the expression of some lipid metabolic target genes of PPARγ and PPARα in 3T3‐L1 adipocytes and HepG2 hepatocytes, respectively. These results suggest that herbal medicines containing isoprenols with dual action on both PPARγ and PPARα can be of interest for the amelioration of lipid metabolic disorders associated with diabetes.
Involvement of phosphatidylinositol 3‐kinase in nuclear translocation of protein kinase C ζ induced by C2‐ceramide in rat hepatocytes
Authors: Calcerrada M.C , Miguel B.G , Martı́n L , Catalán R.E , Martı́nez A.M
Published: October 2002

In this study we report that protein kinase C ζ (PKC ζ), one of the atypical isoforms of the PKC family located predominantly in cytosol, is redistributed by C2‐ceramide treatment in isolated hepatocytes. PKC ζ increased in membrane and nuclear fractions after 30 min of treatment with C2‐ceramide in a dose‐ and time‐dependent manner. The action of C2‐ceramide was inhibited by wortmannin and LY 294002, indicating that C2‐ceramide‐induced PKC ζ increase in both nucleus and membrane fractions is mediated by phosphatidylinositol 3‐kinase (PI3‐kinase) activation. In addition, a significant translocation of PI3‐kinase to the nucleus was observed after C2‐ceramide treatment.
Identification of differentially expressed genes by cDNA‐AFLP technique during heat stress in cowpea nodules
Authors: Simões-Araújo Jean Luiz , Rodrigues Roberta L , de A. Gerhardt Liliane B , Mondego Jorge M.C , Alves-Ferreira Márcio , Rumjanek Norma Gouvêa , Margis-Pinheiro Márcia
Published: October 2002

Legume nodules formed by diazotrophic microorganisms are active sites for biological nitrogen fixation (BNF). In tropical regions, a significant part of N supply for soybean, peanut and bean crops is derived from BNF, which is nevertheless often limited by high temperature stress. In contrast, cowpea nodules are very resistant to high temperatures. To understand the molecular bases of thermotolerance during BNF under heat stress, we have used cDNA‐amplified fragment length polymorphism experiments to identify differentially expressed transcripts from cowpea nodules subjected to heat shock treatment. The expression profiles obtained showed approximately 600 bands, 55 up‐regulated and nine corresponding to genes repressed by heat stress. Twenty transcript‐derived fragments were isolated, cloned and sequenced. The Vigna unguiculata nodule and stress response transcripts present similarities to those that encode low molecular weight heat shock proteins, wound‐induced proteins, disease resistance protein, and xylan endohydrolase isoenzyme, as well as different housekeeping genes. The differential expression of 15 genes was confirmed by using Northern blot or reverse Northern hybridization experiments.