bluuebunny/crossref_metadata_2025 huggingface dataset:
Binding of inositol phosphates to arrestin
Authors: Palczewski Krzysztof , Pulvermüller Alexander , Buczylko Janina , Gutmann Caroline , Hofmann Klaus Peter
Published: October 2002

Arrestin binds to phosphorylated rhodopsin in its light‐activated form (metarhodopsin II), blocking thereby its interaction with the G‐protein, transducin. In this study, we show that highly phosphorylated forms of inositol compete against the arrestin‐rhodopsin interaction. Competition curves and direct binding assays with free arrestin consistently yield affinities in the micromolar range; for example, inositol 1,3,4,5‐tetrakisphosphate (InP4) and inositol hexakisphosphate (InP6bind to arrestin with dissociation constants of 12 μM and 5 μM, respectively. Only a small control amount of inositol phosphates is bound, when arrestin interacts with phosphorylated rhodopsin. This argues for a release of bound inositol phosphates by interaction with rhodopsin. Transducin, rhodopsin kinase, or cyclic GMP phosphodiesterase are not affected by inositol phosphates. These observations open a new way to purify arrestin and to inhibit its interaction with rhodopsin. Their physiological significance deserves further investigation.
A new distinct group of 2 S albumins from rapeseed
Authors: Monsalve Rafael I. , Lopez-Otin Carlos , Villalba Mayte , Rodríguez Rosalía
Published: October 2002

Two napins (nIa and nIb), isolated from Brassica napus (rapeseed) seeds, have been sequenced. The two proteins show the common structural pattern of the 2 S albumins, since they are composed of two disulfide‐linked chains of different size, yet they exhibit an atypical low molecular weight (12.5 kDa vs. 14.5 kDa of the major napins). High sequence similarity has been found between these 2 proteins, but only 54% similarity can be estimated from their comparison with the 14.5 kDa major napins. Thus, nIa and nIb are considered representatives of a new distinct group of rapeseed napins since all the previously known napins exhibit 95% sequence similarity. Unexpectedly, the similarity increases when compared with the 2 S proteins from other species.
Functional characterization of RK5, a voltage‐gated K+ channel cloned from the rat cardiovascular system
Authors: Blair T.Ann , Roberds Steven L. , Tamkun Michael M. , Hartshorne Robert P.
Published: October 2002

A voltage‐sensitive K+ channel previously cloned from rat heart designated RK5 (rat Kv4.2) (Roberds and Tamkun, 1991, Proc. Natl. Acad. Sci. USA 88, 1798–1802) was functionally characterized in the Xenopus oocyte expression system. RK5 is a homolog of the Drosophila Shal K+ channel, activates with a rise time of 2.8 ms, has a midpoint for activation of −1 mV and rapidly inactivates with time constants of 15 and 60 ms. RK5 is sensitive to 4‐AP, IC50= 5 mM, and is insensitive to TEA and dendrotoxins. The voltage dependence and kinetics of the RK5 induced currents suggest this channel contributes to the I
f10 current in heart.
Transcript hairpin structures are not required for RNA polymerase pausing in the gene encoding the E. coli RNase P RNA, M1 RNA
Authors: Ramamoorthy Ramesh , Schmidt Francis J.
Published: October 2002

Strong pauses at nucleotides +118 and +121 relative to the transcriptional start occur during in vitro transcription of the E. coli rnpB gene encoding the catalytic M1 RNA subunit of Ribonuclease P. These pauses are immediately downstream of 2 phylogenetically conserved stem‐loop structures in the RNA. In the present work, single‐base changes which disrupted Watson‐Crick base‐pairing in the hairpins were introduced into rnpB. Transcription studies in vitro with these modified templates revealed that none of the nucleotide changes predicted to increase or decrease the stability of the first hairpin significantly affected the pause half‐lives. A mutation which disrupted the second hairpin increased the pause half‐life 2‐fold. The data suggest that the upstream stem and loop structures in the transcript are not involved in the pausing event.
Inhibition of 2′‐5′ oligoadenylate synthetase by divalent metal ions
Authors: Hartmann Rune , Walko Gernot , Justesen Just
Published: October 2002

OAS1 is the small form and OAS2 is the medium form of the human interferon‐induced 2′‐5′ oligoadenylate synthetases. The p42 isoform of OAS1 and the p69 isoform of OAS2 have been expressed in insect cells and purified to give pure, highly active 2′‐5′ oligoadenylate synthetase. The catalysis of 2′‐5′ oligoadenylate synthesis is strictly dependent on double‐stranded RNA and magnesium ions. We have examined the effect of a series of divalent metal ions: copper, iron and zinc ions strongly inhibited the enzymatic activity, cobalt and nickel ions were partly inhibitory whereas calcium and manganese ions were without effect. However, manganese ions can replace magnesium ions as activator. The inhibitory effect of zinc ions was characterised in detail. The inhibitory constants of Zn2+ were estimated to be 0.10 mM for OAS1p42 and to 0.02 mM for OAS2p69. Cross‐linking experiments showed that zinc ions can control the oligomerisation by enhancing the formation of tetrameric forms of OAS1p42
GFP‐like chromoproteins as a source of far‐red fluorescent proteins
Authors: Gurskaya Nadya G. , Fradkov Arkady F. , Terskikh Alexey , Matz Mikhail V. , Labas Yulii A. , Martynov Vladimir I. , Yanushevich Yurii G. , Lukyanov Konstantin A. , Lukyanov Sergey A.
Published: October 2002

We have employed a new approach to generate novel fluorescent proteins (FPs) from red absorbing chromoproteins. An identical single amino acid substitution converted novel chromoproteins from the species Anthozoa (Heteractis crispa, Condylactis gigantea, and Goniopora tenuidens) into far‐red FPs (emission λ
max=615–640 nm). Moreover, coupled site‐directed and random mutagenesis of the chromoprotein from H. crispa resulted in a unique far‐red FP (HcRed) that exhibited bright emission at 645 nm. A clear red shift in fluorescence of HcRed, compared to drFP583 (by more than 60 nm), makes it an ideal additional color for multi‐color labeling. Importantly, HcRed is excitable by 600 nm dye laser, thus promoting new detection channels for multi‐color flow cytometry applications. In addition, we generated a dimeric mutant with similar maturation and spectral properties to tetrameric HcRed.
Cingulin interacts with F‐actin in vitro
Authors: D'Atri Fabio , Citi Sandra
Published: October 2002

Cingulin, a M
r 140–160 kDa protein of the cytoplasmic plaque of epithelial tight junctions (TJ), interacts in vitro with TJ proteins and myosin. Here we investigated cingulin interaction with actin, using His‐tagged, full‐length Xenopus laevis cingulin expressed in insect cells, and glutathione S‐transferase (GST) fusion proteins of fragments of cingulin expressed in bacteria. Purified full‐length cingulin co‐pelleted with F‐actin after high speed centrifugation, and promoted the sedimentation of F‐actin under low speed centrifugation, suggesting that cingulin is an actin‐cross‐linking protein. The actin interaction of GST fusion proteins containing fragments of Xenopus cingulin suggested that the F‐actin binding site is between residues 101 and 294.
Organic phosphates as a new class of soluble guanylate cyclase inhibitors
Authors: Suzuki Tsuneharu , Suematsu Makoto , Makino Ryu
Published: October 2002

This study aimed to examine effects of varied organic phosphates on activities of soluble guanylate cyclase (sGC). The enzyme was purified from bovine lung. Physiologically relevant concentrations of ATP, 2,3‐bisphosphoglyceric acid and inositol hexakisphosphate inhibited its enzyme activities under steady‐state conditions as well as those determined under stimulation with S‐nitroso‐N‐acetylpenicillamine, a nitric oxide donor, carbon monoxide or YC‐1. Lineweaver–Burk plot analyses revealed that these three organic phosphates act as competitive inhibitors. Other organic phosphates such as cardiolipin and sphingomyelin but not inorganic phosphates exhibited such inhibitory actions. These results suggest that organic phosphates serve as inhibitors for sGC‐dependent signaling events.
Cycloprodigiosin hydrocloride suppresses tumor necrosis factor (TNF) α‐induced transcriptional activation by NF‐κB
Authors: Kamata Keiko , Okamoto Shiho , Oka Shin-ichi , Kamata Hideaki , Yagisawa Hitoshi , Hirata Hajime
Published: October 2002

Cycloprodigiosin hydrochloride (cPrG·HCl) obtained from a marine bacterium Pseudoalteromonas denitrificans induces apoptotic cell death in various cancerous cell lines. cPrG·HCl alone caused a little cytotoxicity in HeLa cells, but it enhanced the apoptotic process progressively when co‐administered with tumor necrosis factor (TNF)α. Here we studied the effect of cPrG·HCl on TNFα‐induced activation of the transcription factor nuclear factor κB (NF‐κB). Luciferase gene reporter assays revealed that cPrG·HCl potently suppressed the TNFα‐ and the phorbol myristate acetate‐induced activation of NF‐κB. The suppression occurred in the presence of imidazole, indicating that it was not related to the intracellular acidification resulting from the intrinsic H+/Cl− symporter activity of cPrG·HCl. cPrG·HCl inhibited neither the TNFα‐induced phosphorylation and degradation of inhibitor of nuclear factor‐κB, nor the subsequent nuclear translocation and DNA binding of NF‐κB. cPrG·HCl also suppressed NF‐κB‐enhanced gene expression induced by Rac1, Cdc42, MEKK1, inhibitor of nuclear factor‐κα (IKKα), IKKβ, and a subunit of NF‐κB, p65. These results indicate that cPrG·HCl suppresses NF‐κB‐dependent gene expression through the inhibition of transcriptional activation.
Inter‐domain cross‐talk controls the NifA protein activity of Herbaspirillum seropedicae
Authors: Monteiro Rose A. , de Souza Emanuel M. , Wassem Roseli , Yates M.Geoffrey , Pedrosa Fabio O. , Chubatsu Leda S.
Published: October 2002

Herbaspirillum seropedicae is an endophytic diazotroph, which colonizes sugar cane, wheat, rice and maize. The activity of NifA, a transcriptional activator of nif genes in H. seropedicae, is controlled by ammonium ions through a mechanism involving its N‐terminal domain. Here we show that this domain interacts specifically in vitro with the N‐truncated NifA protein, as revealed by protection against proteolysis, and this interaction caused an inhibitory effect on both the ATPase and DNA‐binding activities of the N‐truncated NifA protein. We suggest that the N‐terminal domain inhibits NifA‐dependent transcriptional activation by an inter‐domain cross‐talk between the catalytic domain of the NifA protein and its regulatory N‐terminal domain in response to fixed nitrogen.
Multiple sites of interaction between the intracellular domains of an inwardly rectifying potassium channel, Kir6.2
Authors: Jones Phillippa A , Tucker Stephen J , Ashcroft Frances M
Published: October 2002

The amino‐terminal and carboxy‐terminal domains of inwardly rectifying potassium channel (Kir) subunits are both intracellular. A direct physical interaction between these two domains is involved in the response of Kir channels to regulatory factors such as G‐proteins, nucleotides and intracellular pH. We have previously mapped the region within the N‐terminal domain of Kir6.2 that interacts with the C‐terminus. In this study we use a similar in vitro protein–protein interaction assay to map the regions within the C‐terminus which interact with the N‐terminus. We find that multiple interaction domains exist within the C‐terminus: CID1 (amino acids (aa) 279–323), CID2 (aa 214–222) and CID3 (aa 170–204). These domains correlate with regions previously identified as making important contributions to Kir channel assembly and function. The highly conserved nature of the C‐terminus suggests that a similar association with the N‐terminus may be a feature common to all members of the Kir family of potassium channels, and that it may be involved in gating of Kir channels by intracellular ligands.
In silico screening for tumour‐specific expressed sequences in human genome
Authors: Baranova A.V. , Lobashev A.V. , Ivanov D.V. , Krukovskaya L.L. , Yankovsky N.K. , Kozlov A.P.
Published: October 2002

A computer‐based differential display tool named HsAnalyst has been developed and successfully used for the comparison of expression patterns in a set of tumours versus a set of normal tissues. A list of EST clusters highly represented in tumours and rarely observed in normal tissues has been developed as a resulting output file of the program. These differentially expressed EST clusters (genes) can be useful for developing new tumour markers and prognostic indicators for a wide set of human malignancies. Tumour‐specific protein‐coding genes may be considered a manifestation of tumour‐specific gene expression.
Cadmium inhibits epoxidation of diatoxanthin to diadinoxanthin in the xanthophyll cycle of the marine diatom Phaeodactylum tricornutum
Authors: Bertrand Martine , Schoefs Benoît , Siffel Pavel , Rohacek Karel , Molnar Istvan
Published: October 2002

Cd has pleiotropic effects on plant physiology and in particular on photosynthesis. It has not been established yet if Cd alters the functioning of the xanthophyll cycle. To answer this question, an exponentially growing culture of the marine diatom Phaeodactylum tricornutum was incubated with Cd (20 mg/l) for 24 h and irradiated with a light activating the xanthophyll cycle, which in diatoms, consists of the reversible deepoxidation of diadinoxanthin to diatoxanthin. The measurements show that the deepoxidation step is not influenced by Cd. In contrast, the Cd concentration used sharply inhibits the epoxidation of diatoxanthin to diadinoxanthin.
Involvement of fibrinogen specific binding in erythrocyte aggregation
Authors: Lominadze David , Dean William L
Published: October 2002

Increased fibrinogen concentration and erythrocyte aggregation are significant risk factors during various cardiovascular diseases and cerebrovascular disorders. Currently, fibrinogen‐induced erythrocyte aggregation is thought to be caused by a non‐specific binding mechanism. However, the published data on changes in erythrocyte aggregation during hypertension point to the possible existence of other mechanism(s). Therefore, we tested the hypothesis that specific binding of fibrinogen is involved in erythrocyte aggregation. It was found that Oregon Green 488‐labeled human fibrinogen specifically binds rat erythrocyte membranes with a K
d of 1.3 μM. Further experiments showed that the peptide Arg‐Gly‐Asp‐Ser blocked both fibrinogen‐induced aggregation of intact erythrocytes and specific binding of fibrinogen to the erythrocyte membranes. These results suggest that in addition to non‐specific binding, a specific binding mechanism is also involved in fibrinogen‐induced erythrocyte aggregation.
Enhanced activation of bound plasminogen on Staphylococcus aureus by staphylokinase
Authors: Mölkänen Tomi , Tyynelä Jaana , Helin Jari , Kalkkinen Nisse , Kuusela Pentti
Published: October 2002

Activation of plasminogen (plg) to plasmin by the staphylococcal activator, staphylokinase (SAK), is effectively regulated by the circulating inhibitor, α2‐antiplasmin (α2AP). Here it is demonstrated that intact Staphylococcus aureus cells and solubilized staphylococcal cell wall proteins not only protected SAK‐promoted plg activation against the inhibitory effect of α2AP but also enhanced the activation. The findings suggest that the surface‐associated plg activation by SAK may have an important physiological function in helping staphylococci in tissue dissemination. Amino acid sequencing of tryptic peptides originating from the 59‐, 56‐ and 43‐kDa proteins, isolated as putative plg‐binding proteins, identified them as staphylococcal inosine 5′‐monophosphate dehydrogenase, α‐enolase, and ribonucleotide reductase subunit 2, respectively.
Expression of hetN during heterocyst differentiation and its inhibition of hetR up‐regulation in the cyanobacterium Anabaena sp. PCC 7120
Authors: Li Bin , Huang Xu , Zhao Jindong
Published: October 2002

The hetN gene plays an important role in heterocyst differentiation and pattern formation. An immunoblotting study showed that the hetN gene in Anabaena sp. PCC 7120 was expressed in vegetative cells grown with combined nitrogen. After a switch to a medium without combined nitrogen, hetN expression first declined and was then followed by a rapid increase in its product, HetN, which was only present in mature heterocysts. HetN is located on both thylakoid membranes and plasma membranes as determined by immunoblotting using purified membranes. Overexpression of hetN completely prevented hetR up‐regulation under nitrogen‐deprivation conditions, suggesting that its role in pattern control may depend on its inhibition of hetR expression.
Molecular and structural analyses of a novel temperature stress‐induced lipocalin from wheat and Arabidopsis
Authors: Frenette Charron Jean-Benoit , Breton Ghislain , Badawi Mohamed , Sarhan Fathey
Published: October 2002

Two cDNAs corresponding to a novel lipocalin were identified from wheat and Arabidopsis. The two cDNAs designated Tatil for Triticum aestivum L. temperature‐induced lipocalin and Attil for Arabidopsis thaliana temperature‐induced lipocalin encode polypeptides of 190 and 186 amino acids respectively. Structure analyses indicated the presence of the three structurally conserved regions that characterize lipocalins. Sequence analyses revealed that this novel class of plant lipocalin shares homology with three evolutionarily related lipocalins: the mammalian apolipoprotein D (ApoD), the bacterial lipocalin and the insect Lazarillo. The comparison of the putative tertiary structures of both the human ApoD and the wheat TaTIL suggest that the two proteins differ in membrane attachment and ligand interaction. Northern analyses demonstrated that Tatil and Attil transcripts are upregulated during cold acclimation and heat‐shock treatment. The putative functions of this novel class of plant lipocalins during temperature stresses are discussed.
Opening of ligand‐gated cation channel families by calpain inhibitors1
Authors: Suzuki Makoto , Ohki Gaku , Mochizuki Toshio , Somlo Stefan , Ishibashi Kenichi , Imai Masashi
Published: October 2002

The class of Ca2+‐permeable cation channels is composed of large families with six transmembrane segments including transient receptor potential, vanilloid receptor (VR), polycystin, epithelial calcium channels and melastatin (MLS). However, most of them are functionally silent and unexpressed in mammalian cells. An investigation of associated proteins made us believe that the blockade of calpain opens the silent channels. Using 1 μM of blockers in whole cellular patch pipette fill we measured currents of Chinese hamster ovary cells transfected by VR‐like 1 and 2, polycystin‐2, or a MLS‐like new member (MLS3S). Significant conductance of every clone with a characteristic rectification by blockers was demonstrated. The permeability of Ca2+ to them is similar to that reported. Western blot suggested that blockers did not affect the assembly of the protein but enabled its cleavage. Therefore, investigation of these families with the blockers may boost our knowledge of electrophysiologic function.
Identification of catalytically important amino acids in human ceruloplasmin by site‐directed mutagenesis
Authors: Brown Mark A. , Stenberg Leisa M. , Mauk A.Grant
Published: October 2002

The involvement of amino acid residues previously proposed on the basis of structural data to have roles in the ferroxidase and diamine oxidase activities of human ceruloplasmin was investigated. Variants of human ceruloplasmin, in which residues proposed to be involved in electron transfer and/or iron‐binding had been altered by site‐directed mutagenesis, were expressed in HEK293 cells. E633A and E597A/H602A variants exhibited reduction in both activities by 50–60% compared to recombinant wild‐type ceruloplasmin. The variant E935A/H940A had reduced ferroxidase activity (50%) but unaltered diamine oxidase activity, whereas the variant E971A exhibited enhanced diamine oxidase activity. For the L329M variant, both activities were identical to those of wild‐type ceruloplasmin.
Progression of dystrophic features and activation of mitogen‐activated protein kinases and calcineurin by physical exercise, in hearts of mdx mice
Authors: Nakamura Akinori , Yoshida Kunihiro , Takeda Shin'ichi , Dohi Naoko , Ikeda Shu-ichi
Published: October 2002

We have previously demonstrated that calcineurin and p38 mitogen‐activated protein kinase (MAPK) are up‐regulated in the hearts of mdx mice. However, the degree of up‐regulation observed was variable, which may reflect variable levels of daily physical activities among the mice. To investigate whether or not exercise affects dystrophic features and activates intracellular signaling molecules in mdx hearts, we subjected mdx and C57BL/10 mice to treadmill exercise and examined intracellular signaling molecules in cardiac muscles, at the protein level. The heart to body weight ratio was significantly increased in exercised mdx mice. Histopathology in exercised mdx hearts showed extensive infiltration of inflammatory cells, together with increases in interstitial fibrosis and adipose tissues, all of which were not observed either in exercised C57BL/10 or non‐exercised mdx hearts. Phosphorylated p38 MAPK, phosphorylated extracellular signal‐regulated kinase 1/2 and calcineurin, but not phosphorylated c‐Jun N‐terminal kinase 1, were up‐regulated in exercised mdx hearts compared to exercised C57BL/10 or non‐exercised mdx hearts. These data suggest that physical exercise accelerates the dystrophic process through activation of intracellular signaling molecules in dystrophin‐deficient hearts.
The ATP‐binding cassette (ABC) transporter Bpt1p mediates vacuolar sequestration of glutathione conjugates in yeast
Authors: Klein Markus , Mamnun Yasmine M. , Eggmann Thomas , Schüller Christoph , Wolfger Hubert , Martinoia Enrico , Kuchler Karl
Published: October 2002

Vacuolar sequestration or cellular extrusion of glutathione‐conjugated xenobiotics and catabolites by ATP‐binding cassette (ABC) transporters is an important detoxification mechanism operating in many species. In this study, we show that the yeast ABC transporter Bpt1p, a paralogue of Ycf1p, acts as an ATP‐dependent vacuolar pump for glutathione conjugates. Bpt1p, which is inhibited by vanadate and glibenclamide, accounts for one third of the total vacuolar transport of glutathione conjugates. Furthermore, immunoblot analyses show that Bpt1p levels are strongly elevated in early stationary phase, consistent with a function of Bpt1p in vacuolar detoxification.
Group B streptococcus (GBS) modifies macrophage phosphatidylserine metabolism during induction of apoptosis
Authors: Buratta Sandra , Fettucciari Katia , Mambrini Raffaela , Fetriconi Ilaria , Marconi Pierfrancesco , Mozzi Rita
Published: October 2002

Group B streptococcus (GBS) induced macrophage apoptosis by which it could avoid host defence mechanisms. Macrophages, which constitutively express phosphatidylserine (PtdSer) on the outer leaflet of plasma membrane, increased PtdSer exposure during GBS‐induced apoptosis. Induction of apoptosis decreased PtdSer radioactivity of macrophages incubated with [3H]serine. The effect appeared not due to increasing conversion of PtdSer to phosphatidylethanolamine or phosphatidylcholine nor to the release of radioactive membrane vesicles. The radioactivity in lysoPtdSer was also reduced. These results confirm that induction of apoptosis involves a modification of PtdSer metabolism and point out the typical features of the GBS‐induced apoptosis with respect to other models of apoptosis.
Induced chirality upon binding of cis‐parinaric acid to bovine β‐lactoglobulin: spectroscopic characterization of the complex
Authors: Zsila Ferenc , Imre Tı́mea , Szabó Pál T. , Bikádi Zsolt , Simonyi Miklós
Published: October 2002

Binding of the polyunsaturated cis‐parinaric acid to bovine β‐lactoglobulin (BLG) was studied by circular dichroism (CD), electronic absorption spectroscopy and mass spectrometry methods. Upon protein binding, the UV absorption band of parinaric acid is red shifted by ca. 5 nm, showing hypochromism and reduced vibrational fine structure, suggesting that the ligand binds as a monomer in non‐planar geometry. In the CD spectra measured at pH 7.36 and 8.5 a strong, negative Cotton band appears centered at 310 nm (Δϵ=−25 M−1 cm−1) corresponding to the long‐wavelength absorption band of cis‐parinaric acid. The source of this induced optical activity is the helical distortion of the polyene chromophore caused by the chiral protein environment. From CD spectral data the value of the association constant was calculated to be 4.7×105 M−1 at pH 7.36. CD and mass spectrometry measurements showed that parinaric acid binds weakly to BLG in acidic solution, though small peaks at mass 18559 and 18645 can be obtained in the reconstructed electrospray mass spectrum; these correspond to the binding of parinaric acid in 1:1 stoichiometry to both monomer variants of BLG B and A. The hydrophobic interior cavity of BLG was assigned as the primary binding site of cis‐parinaric acid.
Identification of G protein‐coupled receptor genes from the human genome sequence
Authors: Takeda Shigeki , Kadowaki Shiro , Haga Tatsuya , Takaesu Hirotomo , Mitaku Shigeki
Published: October 2002

We have identified novel G protein‐coupled receptors (GPCRs) with no introns in the coding region from the human genome sequence: 322 olfactory receptors; 22 taste receptors; 128 registered GPCRs for endogenous ligands; 50 novel GPCR candidates homologous to registered GPCRs for endogenous ligands; and 59 novel GPCR candidates not homologous to registered GPCRs. The total number of GPCRs with and without introns in the human genome was estimated to be approximately 950, of which 500 are odorant or taste receptors and 450 are receptors for endogenous ligands.
The C‐terminal domains of TACE weaken the inhibitory action of N‐TIMP‐3
Authors: Lee Meng-Huee , Verma Vandana , Maskos Klaus , Becherer J.David , Knäuper Vera , Dodds Philippa , Amour Augustin , Murphy Gillian
Published: October 2002

Tumor necrosis factor‐α converting enzyme (TACE) is an ADAM ( isintegrin nd etalloproteinases) that comprises an active catalytic domain and several C‐terminal domains. We compare the binding affinity and association rate constants of the N‐terminal domain form of wild‐type tissue inhibitor of metalloproteinase (TIMP‐3; N‐TIMP‐3) and its mutants against full‐length recombinant TACE and the truncated form of its catalytic domain. We show that the C‐terminal domains of TACE substantially weaken the inhibitory action of N‐TIMP‐3. Further probing with hydroxamate inhibitors indicates that both forms of TACE have similar active site configurations. Our findings highlight the potential role of the C‐terminal domains of ADAM proteinases in influencing TIMP interactions.
Cdc48 can distinguish between native and non‐native proteins in the absence of cofactors
Authors: Thoms Sven
Published: October 2002

The ATPase Cdc48 is required for membrane fusion and protein degradation. Recently it has been suggested that Cdc48 in a complex with Ufd1 and Npl4 acts as an ubiquitin‐dependent chaperone. Here it is shown that recombinant Cdc48 alone can distinguish between the native and the non‐native conformation of model substrates. First, Cdc48 prevents luciferase from aggregating following a heat shock. Second, it inhibits the aggregation of rhodanese upon dilution. Third, Cdc48 binds specifically to heat‐denatured luciferase. These chaperone‐like functions seem to be independent of ATPase activity. Furthermore, Cdc48 can act as a co‐chaperone in the Hsc70–Hsp40 chaperone system. These results show that Cdc48 possesses chaperone‐like activities and can functionally interact with Hsc70. Cdc48's ability to recognise denatured proteins can also be a source of unspecific binding in biochemical interaction experiments.
Development of photo‐crosslinking reagents for protein kinase–substrate interactions
Authors: Parang Keykavous , Kohn Jeffrey A. , Saldanha S.Adrian , Cole Philip A.
Published: October 2002

The identification of relevant protein kinase–protein substrate partners remains a serious challenge on a genome‐wide scale. The design and synthesis of a photo‐activatable nucleotide reagent to crosslink protein kinases with their substrates is described in which an azido group is appended to the γ‐phosphoryl and purine moieties of ATP. In the absence of UV, compounds of this class were shown to act as competitive inhibitors versus ATP and non‐competitive inhibitors versus peptide substrate for the protein tyrosine kinase Csk, suggesting that they can form a ternary complex with kinase and protein substrate. In vitro experiments with protein kinases indicate the bifunctional reagent can induce covalent protein–protein crosslinking that is dependent on UV irradiation. That significant kinase–substrate crosslinking occurs is suggested by the fact that this crosslinking is competitively inhibited by ATP. The crosslinked adducts can be readily cleaved by phosphodiesterase which supports the model for crosslinking and provides a simple method to deconvolute the linked protein partners.
Complex N‐glycosylated form of nicastrin is stabilized and selectively bound to presenilin fragments
Authors: Tomita Taisuke , Katayama Ryohei , Takikawa Rie , Iwatsubo Takeshi
Published: October 2002

The transmembrane glycoprotein nicastrin is a component of presenilin (PS) protein complex that is involved in γ‐cleavage of βAPP and site‐3 cleavage of Notch. PS undergoes endoproteolysis, and the proteolytic fragments are incorporated into the high molecular weight protein complexes that are highly stabilized. Here we show that Endo H‐resistant, N‐glycosylated form of nicastrin (p150‐NCT) is highly stabilized and selectively bound to PS fragments. Moreover, loss‐of‐function mutations of nicastrin inhibited formation of fully glycosylated p150‐NCT as well as stabilization of nicastrin, suggesting that glycosylation and stabilization of nicastrin polypeptides are tightly correlated with its function.
Short‐time non‐enzymatic nitric oxide synthesis from L‐arginine and hydrogen peroxide induced by shock waves treatment
Authors: Gotte G. , Amelio E. , Russo S. , Marlinghaus E. , Musci G. , Suzuki H.
Published: October 2002

The evidence that nitric oxide (NO) production is possible by a non‐enzymatic pathway has already been shown under restrictive experimental conditions. Here we show that NO can non‐enzymatically be formed with short‐time kinetics (min), by ‘bombing’ with shock waves a solution containing 1 mM hydrogen peroxide and 10 mM L‐arginine. This procedure is widening its medical application with surprisingly positive effects in tissue regeneration and our finding could be one of the first steps for the understanding of the biochemical responsible for these therapeutical effects.
Identification of a dominant self‐ligand bound to three HLA B44 alleles and the preliminary crystallographic analysis of recombinant forms of each complex
Authors: Macdonald Whitney , Williams David S , Clements Craig S , Gorman Jeffery J , Kjer-Nielsen Lars , Brooks Andrew G , McCluskey James , Rossjohn Jamie , Purcell Anthony W
Published: October 2002

A naturally processed and presented ligand that is shared by human leukocyte antigen (HLA) B*4402, B*4403 and B*4405 molecules has been identified in peptides isolated from immunoaffinity purified HLA B44 complexes. This peptide derived from HLA DPα residues 46–54, an endogenous product of HLA DP expressed in the cell line Hmy2.C1R, is a prominent peptide in the mass spectra of species isolated as bound peptides from each allele when the three HLA B44 subtypes were introduced as transfected gene products. Recombinant truncated forms of HLA B*44051–276, HLA B*44031–276, HLA B*44021–276 and β2‐microglobulin have been prepared as inclusion bodies in Escherichia coli and refolded in the presence of the DPα46–54 peptide and purified by a combination of size exclusion and anion exchange chromatography. This material was determined to be correctly folded based on detection of a conformational epitope recognized by the W6/32 monoclonal antibody. Large, plate‐like crystals of the three complexes were produced using polyethylene glycol as the precipitant. All the crystals belong to the space group P212121 with unit cell dimensions of approximately a=51, b=82, c=110 Å. The crystals of three B44/DPα complexes diffracted to a resolution of 1.9 Å or better. For the first time, using this natural, high abundance ligand of the HLA B44 molecules we have successfully expressed and refolded the three HLA B44 molecules and produced crystals amenable to structural studies.
OSBPa, a predicted oxysterol binding protein of Dictyostelium, is required for regulated entry into culmination
Authors: Fukuzawa Masashi , Williams Jeffrey G.
Published: October 2002

The oxysterol binding proteins (OSBPs) are believed to control cholesterol homeostasis but their precise mechanism of action is not well understood. The Dictyostelium osbA gene encodes a predicted OSBP, OSBPa, which lacks the PH domain that in most other OSBPs directs targetting to the Golgi. OSBPa instead localises selectively to the cell periphery and also, in some cells, to the perinuclear region. OSBPa null strains form normal fruiting bodies but are defective in the regulation of the transition from slug migration to culmination. Thus a plasma membrane‐enriched OSBP family member is essential for correct regulation of the slug–fruiting body switch.
Inositol hexakisphosphate (InsP6) can weaken the Ca2+‐dependent membrane binding of C2AB domain of synaptotagmin I
Authors: Lu Ying-Jie , He Yuhong , Sui Sen-fang
Published: October 2002

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca2+ sensor for rapid exocytosis. In the present work, two fragments of the large cytoplasmic domain of synaptotagmin I, C2A and C2AB, were compared by combining surface plasmon resonance with circular dichroism and fluorescence techniques. C2AB and C2A had almost identical membrane binding constants, indicating that C2A is the predominate domain to bind to negatively charged phospholipids. After reacting with inositol hexakisphosphate (InsP6) a conformational change of C2AB was detected in the presence of liposome. The InsP6 binding notably weakened the Ca2+‐dependent C2AB‐membrane interaction, which suggests that InsP6 may act as a modulator of neurotransmitter release by altering the state of synaptotagmin–phospholipid interaction.
Proteasomal activity modulates TGF‐ß signaling in a gene‐specific manner
Authors: Zhang Fan , Mönkkönen Mia , Roth Stina , Laiho Marikki
Published: October 2002

Transforming growth factor‐β (TGF‐β) signaling relies on Smad‐signaling pathway controlled in part by the proteasome. Here we demonstrate that inhibition of the proteasome function in mink epithelial cells accumulates both positive and negative modulators of TGF‐β signaling, phospho‐Smad2 and SnoN. Inhibition of the proteasome led to abrogation of TGF‐β target gene regulation in a gene‐specific manner. While regulation of p15Ink4b and myc by TGF‐β are lost, PAI‐1 induction, previously shown to occur in a Smad3‐dependent manner, was not affected by treatment of the cells with the proteasomal inhibitor MG132. The results suggest that proteasomal activity is required for TGF‐β signaling in a gene‐specific manner.
Activity, tissue distribution and site‐directed mutagenesis of a human peptide methionine sulfoxide reductase of type B: hCBS1
Authors: Jung Stephan , Hansel Alfred , Kasperczyk Hubert , Hoshi Toshinori , Heinemann Stefan H.
Published: October 2002

Human CBS1 is a methionine sulfoxide reductase of type B (MSRB) as it specifically reduced Met‐R‐SO in peptides with dithiothreitol or the thioredoxin system as reductants. Mutation C169S in the active site completely abolished enzymatic activity, while mutation W110A only reduced activity and C105S had no effect. Like human MSRA, hCBS1 showed in vivo reducing activity coexpressed with the Drosophila ShC/B potassium channel in oocytes, by accelerating the overall inactivation time course. hCBS1‐encoding mRNA is most abundant in muscle tissues, especially in the heart and thereby shows an expression pattern different to the human MSRA.
Phosphorylation of the regulatory subunit of smooth muscle protein phosphatase 1M at Thr850 induces its dissociation from myosin
Authors: Velasco Guillermo , Armstrong Chris , Morrice Nick , Frame Sheelagh , Cohen Philip
Published: October 2002

Rho kinase is known to control smooth muscle contractility by phosphorylating the 110 kDa myosin‐targetting subunit (MYPT1) of the myosin‐associated form of protein phosphatase 1 (PP1M). Phosphorylation of MYPT1 at Thr695 has previously been reported to inhibit the catalytic activity of PP1. Here, we show that the phosphorylation of Thr850 by Rho kinase dissociates PP1M from myosin, providing a second mechanism by which myosin phosphatase activity is inhibited.
Human gastrin‐releasing peptide receptor mediates sustained CREB phosphorylation and transactivation in HuTu 80 duodenal cancer cells
Authors: Qu Xiangping , Xiao Dongmei , Weber H.Christian
Published: October 2002

The G protein‐coupled human gastrin‐releasing peptide receptor (hGRP‐R) is frequently found aberrantly expressed in human cancers of the colon, stomach, and lung, and its ligand‐specific activation has been implicated in cell proliferation and differentiation. Here, we demonstrated hGRP‐R activation stimulated sustained cyclic AMP response element binding protein (CREB) phosphorylation and transactivation in duodenal cancer cells through a protein kinase C and partially p38 mitogen‐activated protein kinase‐dependent pathway. In contrast, intracellular calcium, ERK1/2, protein kinase A, and PI3 kinase were not involved. This novel signaling mechanism might be of importance for regulation of CREB‐dependent gene expression in human cancer expressing functional hGRP‐R.
Dimerization, ubiquitylation and endocytosis go together in growth hormone receptor function
Authors: Strous Ger J , Gent Jürgen
Published: October 2002

Internalization of membrane proteins has been studied for more than three decades without solving all the underlying mechanisms. Our knowledge of the clathrin‐coated endocytosis is sufficient to understand the basic principles. However, more detailed insight is required to recognize why different proteins enter clathrin‐coated pits with different rates and affinities. In addition to clathrin coat components, several adapter systems and even more accessory proteins have been described to preselect membrane proteins before they can enter cells. Recent experimental data have identified the ubiquitin–proteasome system as a regulatory system both in endocytic and lysosomal membrane traffic. This system is well‐known for its basic regulatory function in protein degradation, and controls a magnitude of key events. In this review, we will discuss the complexity and implications of this mechanism for membrane trafficking with emphasis on the growth hormone receptor.
Quality assessment of multiple alignment programs
Authors: Lassmann Timo , Sonnhammer Erik L.L
Published: October 2002

A renewed interest in the multiple sequence alignment problem has given rise to several new algorithms. In contrast to traditional progressive methods, computationally expensive score optimization strategies are now predominantly employed. We systematically tested four methods (Poa, Dialign, T‐Coffee and ClustalW) for the speed and quality of their alignments. As test sequences we used structurally derived alignments from BAliBASE and synthetic alignments generated by Rose. The tests included alignments of variable numbers of domains embedded in random spacer sequences. Overall, Dialign was the most accurate in cases with low sequence identity, while T‐Coffee won in cases with high sequence identity. The fast Poa algorithm was almost as accurate, while ClustalW could compete only in strictly global cases with high sequence similarity.
A molecular view on pluripotent stem cells
Authors: Eiges Rachel , Benvenisty Nissim
Published: October 2002

Pluripotent stem cells are undifferentiated cells that are capable of differentiating to all three embryonic germ layers and their differentiated derivatives. They are transiently found during embryogenesis, in preimplantation embryos and fetal gonads, or as established cell lines. These unique cell types are distinguished by their wide developmental potential and by their ability to be propagated in culture indefinitely, without loosing their undifferentiated phenotype. This short review intends to give a general overview on the pluripotent nature of embryo‐derived stem cells with a focus on human embryonic stem cells.
A novel repeat in the melanoma‐associated chondroitin sulfate proteoglycan defines a new protein family
Authors: Staub Eike , Hinzmann Bernd , Rosenthal André
Published: October 2002

The human melanoma‐associated chondroitin sulfate proteoglycan (MCSP) and its rat ortholog NG2 are thought to play important roles in angiogenesis‐dependent processes like wound healing and tumor growth. Based on electron microscopy studies, the highly glycosylated ectodomain of NG2 has been subdivided into the globular N‐terminus, a flexible rod‐like central region and a C‐terminal portion in globular conformation. We identified a novel repeat named CSPG in the central ectodomain of NG2, MCSP and other proteins from fly, worm, human, sea urchin and a cyanobacterium which shows similarity to cadherin repeats. As earlier electron microscopy studies indicate, the folding of the tandem repeats compresses the length of the proposed repeat region by a factor of ∼10 compared to the fully extended peptide chain. We identified two conserved negatively charged residues which might govern the binding properties of CSPG repeats. The phyletic distribution of CSPG repeats suggests that horizontal gene transfer contributed to their evolutionary history.
Intracellular localization of Herpes simplex virus type 1 thymidine kinase fused to different fluorescent proteins depends on choice of fluorescent tag
Authors: Söling Ariane , Simm Andreas , Rainov Nikolai G
Published: October 2002

Gene therapy employing the suicide gene/prodrug activating system Herpes simplex virus type 1 thymidine kinase (HSV‐TK)/ganciclovir (GCV) is effective in killing malignant tumor cells. Labeling of the HSV‐TK enzyme with fluorescent proteins makes possible the non‐invasive imaging of transduction efficiency, enzyme localization and activity in cell culture and in animal models of human cancers. Here we report the expression of HSV‐TK tagged with different fluorescent proteins (EGFP, DSRed1, DsRed2, dsdrFP616) and show that intracellular localization of the fusion products depends on the nature of the fluorescent tag despite the presence of several nuclear targeting signals within the enzyme itself. Coexpression of red fluorescent HSV‐TK fusion proteins with TK‐EGFP or untagged HSV‐TK allowed these proteins to enter the nucleus by inhibiting formation of red fluorescent protein oligomers. As enzyme localization may influence HSV‐TK activity, this observation is of potential importance to gene therapy studies.
Characterization of the spermidine synthase‐related gene family in Arabidopsis thaliana
Authors: Hanzawa Yoshie , Imai Akihiro , Michael Anthony J , Komeda Yoshibumi , Takahashi Taku
Published: October 2002

The Arabidopsis genome contains four genes that encode proteins similar to both spermidine synthase and spermine synthase of other organisms. Our previous study revealed that one of these genes, designated ACAULIS5 (ACL5), encodes spermine synthase and that its null mutation results in a severe defect in the elongation of stem internodes. Here we report the characterization of the other three genes, designated SPDS1, SPDS2 and SPDS3. Our results showed that SPDS1 and SPDS2 possess spermidine synthase activity in yeast spermidine synthase‐deficient mutants, but the enzyme activity of SPDS3 remained to be determined. RNA gel blot analysis revealed that all of these genes are expressed in all plant organs but show different responses to exogenous plant hormones, suggesting that they are involved in different aspects of growth by modulating the contents of polyamines in plant cells.
YIL113w encodes a functional dual‐specificity protein phosphatase which specifically interacts with and inactivates the Slt2/Mpk1p MAP kinase in S. cerevisiae
Authors: Collister Michelle , Didmon Mark P , MacIsaac Fiona , Stark Michael J , MacDonald Neil Q , Keyse Stephen M
Published: October 2002

We show here that the YIL113w gene of Saccharomyces cerevisiae encodes a functional protein phosphatase. Yil113p shows no activity in vitro towards either phosphorylated casein or myelin basic protein. However, Yil113p dephosphorylates activated extracellular signal‐regulated kinase 2 MAP kinase indicating that it is a dual‐specificity MAP kinase phosphatase. In support of this we find that Yil113p specifically interacts with the stress‐activated Slt2/Mpk1p MAP kinase of S. cerevisiae. Furthermore, expression of Yil113p causes the dephosphorylation of Slt2/Mpk1p in vivo, while expression of an inactive mutant of Yil113p causes the accumulation of phosphorylated Slt2/Mpk1p. We conclude that the physiological target of YIL113p is Slt2/Mpk1p.
Effect of Bacillus anthracis lethal toxin on human peripheral blood mononuclear cells
Authors: Popov Serguei G , Villasmil Rafael , Bernardi Jessica , Grene Edith , Cardwell Jennifer , Popova Taissia , Wu Aiguo , Alibek Darya , Bailey Charles , Alibek Ken
Published: October 2002

Lethal toxin (LeTx) plays a central role in anthrax pathogenesis, however a cytotoxicity of LeTx has been difficult to demonstrate in vitro. No cytolytic effect has been reported for human cells, in contrast to murine cell lines, indicating that cell lysis can not be considered as a marker of LeTx activity. We have recently shown that murine macrophage‐like RAW 264.7 cells treated with LeTx or infected with anthrax spores underwent changes typical of apoptotic death. Here we demonstrate that cells from human peripheral blood display a proapoptotic behavior similar to murine cells. TUNEL assay detected a nucleosomal degradation typical of apoptosis in peripheral blood mononuclear cells (PBMC) treated with LeTx. Membrane staining with apoptotic dyes was detected in macrophages derived from monocytes in presence of LeTx. The toxin inhibited production of proinflammatory cytokines in PBMC stimulated with a preparation of Bacillus anthracis cell wall. Infection of PBMC with anthrax spores led to the appearance of a large population of cells stained positively for apoptosis, with a reduced capacity to eliminate spores and vegetative bacteria. The aminopeptidase inhibitor, bestatin, capable of protecting cells from LeTx, restored a bactericidal activity of infected cells. These findings may be explained by LeTx expression within phagocytes and support an important role of LeTx as an early intracellular virulence factor contributing to bacterial dissemination and disease progression.
Human immune cells express ppMCH mRNA and functional MCHR1 receptor
Authors: Verlaet Myriam , Adamantidis Antoine , Coumans Bernard , Chanas Grazyna , Zorzi Willy , Heinen Ernst , Grisar Thierry , Lakaye Bernard
Published: October 2002

Melanin‐concentrating hormone (MCH) is highly expressed in the brain and modulates feeding behavior. It is also expressed in some peripheral tissues where its role remains unknown. We have investigated MCH function in human and mouse immune cells. RT‐PCR analysis revealed a low expression of prepro‐MCH and MCH receptor 1 (MCHR1) but not of MCHR2 transcript in tissular and peripheral blood immune cells. FACS and in vitro assay studies demonstrated that MCHR1 receptor expression on most cell types can trigger, in the presence of MCH, cAMP synthesis and calcium mobilization in peripheral blood mononuclear cells (PBMCs). Moreover, MCH treatment decreases the CD3‐stimulated PBMC proliferation in vitro. Accordingly, our data indicate for the first time that MCH and MCHR1 may exert immunomodulatory functions.