bluuebunny/crossref_metadata_2025 huggingface dataset:
Fcε receptor mediated Ca2+ influx into mast cells is modulated by the concentration of cytosolic free Ca2+ ions
Authors: Dar Ora , Pecht Israel
Published: July 2002

The relationship between the Fcε receptor mediated stimulation of mast cells and the Ca2+ signal it induces were studied using thapsigargin (TG), a blocker of the endoplasmic reticulum Ca2+ pump. TG induced, in mucosal mast cells (RBL‐2H3 line), a dose‐dependent and an InsP3‐independent increase in [Ca2+]i (from resting levels of 83–150 nM to 600–680 nM), and a secretory response amounting to 30–50% of that observed upon FcεRI clustering. The TG induced rise of [Ca2+]i is most probably provided by both arrest of its uptake by the endoplasmic reticulum and influx from the medium. Thus, Ca2+ influx in mast cells may be modulated by the [Ca2+]i level.
Tyrosine kinase activity is involved in the protein kinase C induced expression ofinterleukin‐1β gene in monocytic cells
Authors: Palkama Tessa , Matikainen Sampsa , Hurme Mikko
Published: July 2002

The role of protein tyrosine kinases in the expression of interleukin‐1β (IL‐1β) gene in response to phorbol esters (PMA) in THP‐1 cell line was investigated. Genistein, a specific tyrosine kinase inhibitor, inhibited PMA induced IL‐1β protein and mRNA levels in THP‐1 cells. Genistein did not have a significant effect on PMA induced activity in transient transfection assays using reporter gene constructs containing the PMA responsive sequence of the IL‐ 1β gene linked to IL‐ 1β promoter, or five repeats of PMA responsive sites (AP‐1 sites) in front of a thymidine kinase promoter. This indicates that the tyrosine kinase activity required for the PMA induced IL‐1β expression is coupled downstream from or functions independent of the PMA induced AP‐1 activity.
Three‐dimensional structure of neurotoxin‐1 from Naja naja oxiana venom at 1.9 Å resolution
Authors: Nickitenko A.V. , Michailov A.M. , Betzel Ch. , Wilson K.S.
Published: July 2002

Neurotoxin‐1 from Naja naja oxiana venom (NTX‐1) has been crystallized by vapor diffusion in sitting drops. The crystals have cell dimensions of a = 25.2 Å,b = 75.6 Å, c = 35.9 Å, and are in space group P212121. Three‐dimensional data to 1.9 Å have been recorded by a Syntex P21 automatic diffractometer. The atomic structure of the toxin has been determined by molecular replacement using the α‐cobratoxin (α‐CTX) as the search model. The position of 534 non‐hydrogen protein atoms have been determined. The model contains 65 water molecules. Refinement has led to an R‐factor of 19.3% at 1.9 Å resolution. The secondary and tertiary structures of NTX‐1 have been analyzed and a comparison with structure of the α‐CTX has been made.
Ligand binding properties of human cellular retinoic acid binding protein II expressed inE. colias a glutathione‐S‐transferase fusion protein
Authors: Redfern Christopher P.F. , Wilson Katherine E.
Published: July 2002

To test the hypothesis that 9‐cis‐retinoic acid is a ligand for cellular retinoic acid binding protein II (CRABPII), human CRABPII was expressed as a glutathione‐S‐transferase fusion protein (GST‐CRABP II) and a single affinity purification step used to extract it from bacterial lysates. GST‐CRABP II boundall trans‐retinoic acid with high affinity (Kd14.2 ± 6.5 nM), but 9‐cis‐retinoic acid bound poorly. These studies suggest that 9‐cis‐retinoic acid is not a ligand for CRABP II. Their ease of purification makes GST‐CRABP fusion proteins useful tools for ligand binding studies with different retinoids.
A rice homeotic gene, OSH1, causes unusual phenotypes in transgenic tobacco
Authors: Kano-Murakami Yuriko , Yanai Tomoe , Tagiri Akemi , Matsuok Makoto
Published: July 2002

A rice gene, OSH1, which shares homology with animal homeobox genes, has been isolated. We have introduced the OSH1 cDNA into tobacco in order to examine its function. Expression of the OSH1 cDNA in tobacco induced morphological abnormalities in the leaves, petals and stems of the transformants suggesting that OSH1 functions as a morphological regulator. OSH1 cDNA expression was analyzed under the control of three different promoters. This work revealed that not only the level of OSH1 expression but also the site and timing of the expression affect the morphology of the plant.
Correlation between phospholipase A2 activity and intra‐Golgi protein transport reconstituted in a cell‐free system
Authors: Tagaya Mitsuo , Henomatsu Nobuhiro , Yoshimori Tamotsu , Yamamoto Akitsugu , Tashiro Yutaka , Fukui Toshio
Published: July 2002

A wide variety of phospholipase A2 inhibitors blocks intra‐Golgi protein transport reconstituted in a cell‐free system. Phospholipase A2 activity detectable under the protein transport assay conditions is actually inhibited by the inhibitors. There is a good correlation between the inhibition of protein transport and that of phospholipase A2 activity. Prolactin secretion from GH3 cells is also blocked by a membrane‐permeable phospholipase A2, inhibitor, suggesting the physiological relevance to inhibition of protein transport in vitro by phospholipase A2 inhibitors.
ADP‐ribosylation of Rho proteins inhibits sperm motility
Authors: Hinsch Klaus-Dieter , Habermann Barbara , Just Ingo , Hinsch Elvira , Pfisterer Susanne , Schill Wolf-Bernhard , Aktories Klaus
Published: July 2002

The highly homologous Rho proteins RhoA, RhoB and RhoC are low‐molecular‐mass GTP‐binding proteins. They are selectively ADP‐ribosylated by Clostridium botulinum ADP‐ribosyltransferase C3 (C3 exoenzyme). The biological function of the Rho proteins is still unclear; there is evidence that they are involved in the regulation of the filamental network of cells. Here we report that C3 exoenzyme‐like toxins ADP‐ribosylate small GTP‐binding proteins in bovine spermatozoa and inhibit sperm motility. These findings indicate that Rho proteins which reportedly regulate the microfilament system are basically involved in sperm motility.
Human interleukin‐13 activates the interleukin‐4‐dependent transcription factor NF‐IL4 sharing a DNA binding motif with an interferon‐γ‐induced nuclear binding factor
Authors: Köhler Ingrid , Alliger Peter , Minty Adrian , Caput Daniel , Ferrara Pascual , Höll-Neugebauer Bärbel , Rank Gerti , Rieber Ernst Peter
Published: July 2002

The effects of interleukin‐13 (IL‐13) and interleukin‐4 (IL‐4) on cellular functions were shown to be quite similar. We provide evidence that in monocytes as well as in T lymphocytes both IL‐4 and IL‐ 13 activate the same recently identified transcription factor NF‐IL4 which binds to the specific responsive element IL‐4RE. In addition, we show that a nuclear factor activated by interferon‐γ also interacts with the IL‐4RE. It differs from NF‐IL4 in the electrophoretic mobility of the complex with DNA, in its DNA‐binding specificity and in the proteins interacting with the DNA sequence. Sensitivity against various enzyme inhibitors suggests that components of the signal transduction pathway are shared by all three cytokines.
Induction of a tomato peroxidase gene in vascular tissue
Authors: Botella Miguel A. , Quesada Miguel A. , Medina María I. , Pliego Fernando , Valpuesta Victoriano
Published: July 2002

Expression of a tomato peroxidase gene that is constitutively expressed only in roots was induced in stems and leaves as a result of mechanical wounding. However, wound‐induction of TPX1 transcript accumulation in leaves was limited to the mid‐rib. No TPX1 transcript was detected in the lamina of the leaf after wounding. Peroxidase isozyme studies indicated the presence of a unique basic isoform in stems after wounding.
Nearest neighbor analysis of D1 and D2 subunits in the photosystem II reaction center using a bifunctional cross‐linker, hexamethylene diisocyanate
Authors: Tomo Tatsuya , Satoh Kimiyuki
Published: July 2002

A cross‐linked product between the D1 and D2 subunits was generated by treating isolated spinach PS II reaction center with a bifunctional cross‐linker, 1,6‐hexamethylene diisocyanate. To obtain information on the contact site(s) between D1 and D2 proteins, the cross‐linked product was cleaved by CNBr, the resultant fragments were separated using a reverse‐phase HPLC, and then the partial sequence of the cross‐linked polypeptide fragments were determined. By comparing the results with the deduced amino acid sequence of the D1 and D2 proteins from spinach, it is concluded that the C‐terminal domains of the D1 subunit (D308‐A334) and that of the D2 subunit (Y297‐L353) are in close proximity.
Molecular cloning, expression and chromosomal localisation of human AMP‐activated protein kinase
Authors: Beri Raj K. , Marley Anna E. , See Chee Gee , Sopwith William F. , Aguan Kripa , Carling David , Scott James , Carey Frank
Published: July 2002

A cDNA encoding rat liver AMP‐activated protein kinase (AMPK) was used to isolate human skeletal muscle AMPK cDNA clones. Human AMPK cDNA is more than 90% homologous to the rat sequence and predicts a protein of molecular mass 62.3 kDa, which closely agrees with the mass observed in Western blots of human tissues. AMPK antibodies were also shown to immunoprecipitate AMPK from human liver extracts. A cDNA probe was used to identify a 9.5kb transcript in several human tissues and to isolate human genomic clones. PCR mapping of rodent/human hybrid cell lines localised the human AMPK gene to chromosome 1, and fluorescent in situ hybridisation with a human genomic clone was used to sub‐localise the human AMPK gene to 1p31.
Cloning of the complete coding region for human protein phosphatase inhibitor 2 using the two hybrid system and expression of inhibitor 2 in E. coli
Authors: Helps Nicholas R. , Street Alasdair J. , Elledge Stephen J. , Cohen Patricia T.W.
Published: July 2002

The yeast two hybrid system has been employed to identify cDNAs encoding proteins which interact with the γ1, isoform of human protein phosphatase 1. Here we report the isolation of a cDNA encoding human protein phosphatase inhibitor 2. The deduced human sequence of 205 amino acids shows 92% identity to inhibitor 2 from rabbit. Human inhibitor 2 was expressed in E. coli and purified to homogeneity. The expressed human protein inhibited both native and bacterially expressed PP1, with the same K
i (1 nM) as inhibitor 2 purified from skeletal muscle. A gene or pseudogene for inhibitor 2 may be present near the major histocompatibility complex on chromosome 6.
A polycation causes migration of negatively charged phospholipids from the inner to outer leaflet of the liposomal membrane
Authors: Yaroslavov A.A. , Kul'kov V.E. , Polinsky A.S. , Baibakov B.A. , Kabanov V.A.
Published: July 2002

Aggregation of the negatively charged liposomes caused by the addition of the linear polycation, poly‐N‐ethyl‐4‐vinylpyridine bromide, was studied. At the point of maximal size and zero electrophoretic mobility of aggregates, the concentration of positive charges brought in by the adsorbed polycation was found to be equal to the total concentration of negative charges both on the outer and inner surface of the lipid bilayer. Since polycation saturation of the liposomal negative charges was found to occur without disruption of the membrane, it was concluded that the polycation induced migration of negatively charged phospholipid molecules from the inner to outer leaflet of the bilayer.
Homologues of the engrailed gene from five molluscan classes
Authors: Wray C.G. , Jacobs D.K. , Kostriken R. , Vogler A.P. , Baker R. , DeSalle R.
Published: July 2002

We used the polymerase chain reaction (PCR) to amplify, clone, and sequence 10 engrailed homeodomains from 8 species in the five major molluscan classes, including the serially organized chiton (Polyplacophora) lineage. The Drosophila melanogaster gene engrailed (en) is one of several genes involved in embyonic segment polarity determination. Studies of engrailed sequence and expression in molluscs are of interest due to questions regarding the evolution and homology of segmentation in these taxa. Nucleotide and deduced amino acid sequence comparisons reflect evolutionary conservation within helices of the en homeodomain and ancient divergences in the region 3′ to the homeodomain.
Activation of G protein‐coupled inward rectifier K+ channels in brain neurons requires association of G protein βγ subunits with cell membrane
Authors: Nakajima Yasuko , Nakajima Shigehiro , Kozasa Tohru
Published: July 2002

In cultured noradrenergic neurons from the rat locus coeruleus, application of recombinant G protein β
1
γ
2 subunits (30 nM) to the cytoplasmic side induced single channel activity similar to the somatostatin‐induced single channel activity of G protein‐coupled inward rectifier potassium channels (Kir (G)). In contrast, recombinant GTPγS‐activated, myristoylated α
i2 (100 nM) did not activate this brain Kir (G). Application of β
1
γ
2C68S (30 nM or 150 nM), in which the cysteine residue fourth from the carboxyl terminus of γ
2 was replaced by serine, failed to activate the brain Kir(G). This mutant lacks prenylation which is required for the association of βγ subunit with the cell membrane. Thus, our results suggest that the association of βγ subunit with the cell membrane is a prerequisite for activating Kir(G) channels.
Maternal mortality in a Kenyan pastoralist population
Authors: Mace R. , Sear R.
Published: July 2002

Objective: To measure maternal mortality among the Gabbra, a group of nomadic pastoralists living in a remote area of Kenya. Method: As part of a survey of 851 households, information on the number of sisters of respondents who died of pregnancy‐related causes was collected and the data were used to calculate maternal mortality statistics using the sisterhood (an indirect) method. Results: The maternal mortality ratio for this population was 599 deaths per 100 000 births (95% C.I. 424–775). The lifetime risk of dying around childbirth is 1 in 30, and the proportion of ever‐married sisters that died under 50 years of age who died from maternal causes is 0.48 (95% C.I. 0.38–0.58). Conclusion: The risk of dying of maternal causes is high in this population.
A sequence in the carboxy‐terminus of the α1C subunit important for targeting, conductance and open probability of L‐type Ca2+ channels
Authors: Kepplinger Klaus J.F , Kahr Heike , Förstner Günter , Sonnleitner Max , Schindler Hansgeorg , Schmidt Thomas , Groschner Klaus , Soldatov Nikolai M , Romanin Christoph
Published: July 2002

The role of the 80‐amino acid motif 1572–1651 in the C‐terminal tail of α1C Ca2+ channel subunits was studied by comparing properties of the conventional α1C,77 channel expressed in HEK‐tsA201 cells to three isoforms carrying alterations in this motif. Replacement of amino acids 1572–1651 in α1C,77 with 81 non‐identical residues leading to α1C,86 impaired membrane targeting and cluster formation of the channel. Similar to α1C,86, substitution of its 1572–1598 (α1C,77L) or 1595–1652 (α1C,77K) segments into the α1C,77 channel yielded single‐channel Ba2+ currents with increased inactivation, reduced open probability and unitary conductance, when compared to the α1C,77 channel. Thus, the C‐terminal sequence 1572–1651 of the α1C subunit is important for membrane targeting, permeation and open probability of L‐type Ca2+ channels.
Annexin XI may be involved in Ca2+‐ or GTP‐γS‐induced insulin secretion in the pancreatic β‐cell
Authors: Iino Satoshi , Sudo Toshiki , Niwa Tae , Fukasawa Tatsuya , Hidaka Hiroyoshi , Niki Ichiro
Published: July 2002

The aim of this study was to investigate possible involvement of annexin XI in the insulin secretory machinery. In fluorescence immunocytochemistry, annexin XI was found in the cytoplasm of pancreatic endocrine cells and a pancreatic β‐cell line, MIN6, in a granular pattern. MIN6 cells also possessed weak and diffused annexin XI immunoreactivity in the cytoplasm. Immunoelectron microscopy revealed annexin XI in the insulin granules. Insulin secretion from streptolysin‐O‐permeabilized MIN6 cells was inhibited by anti‐annexin XI antibody, when the release was stimulated by either Ca2+ or GTP‐γS, but not by a protein kinase C‐activating phorbol ester. Inhibition of insulin release by anti‐annexin XI antibody was reproduced in permeabilized rat islets. These findings suggest that annexin XI may be involved in the regulation of insulin secretion from the pancreatic β‐cells.
Evidence for the activation of PAR‐2 by the sperm protease, acrosin: expression of the receptor on oocytes
Authors: Smith Rosealee , Jenkins Alison , Lourbakos Afrodite , Thompson Philip , Ramakrishnan Vanitha , Tomlinson Jim , Deshpande Usha , Johnson David A. , Jones Roy , Mackie Eleanor J. , Pike Robert N.
Published: July 2002

Proteinase‐activated receptor‐2 (PAR‐2) is a member of a family of G‐protein‐coupled, seven‐transmembrane domain receptors that are activated by proteolytic cleavage. The receptor is expressed in a number of different tissues and potential physiological activators identified thus far include trypsin and mast cell tryptase. Acrosin, a trypsin‐like serine proteinase found in spermatozoa of all mammals, was found to cleave a model peptide fluorescent quenched substrate representing the cleavage site of PAR‐2. This substrate was cleaved with kinetics similar to those of the known PAR‐2 activators, trypsin and mast cell tryptase. Acrosin was also shown to induce significant intracellular calcium responses in Chinese hamster ovary cells stably expressing intact human PAR‐2, most probably due to activation of the receptor. Immunohistochemical studies using PAR‐2 specific antibodies indicated that the receptor is expressed by mouse oocytes, which suggests that acrosin may play additional role(s) in the fertilization process via the activation of PAR‐2 on oocytes.
Differential effects of annexins I, II, III, and V on cytosolic phospholipase A2 activity: specific interaction model
Authors: Kim Seung-Wook , Ko Jesang , Kim Jae Hong , Choi Eung Chil , Na Doe Sun
Published: July 2002

Annexins (ANXs) are a family of proteins with calcium‐dependent phospholipid binding properties. Although inhibition of phospholipase A2 (PLA2) by ANX‐I has been reported, the mechanism is still controversial. Previously we proposed a ‘specific interaction’ model for the mechanism of cytosolic PLA2 (cPLA2) inhibition by ANX‐I [Kim et al., FEBS Lett. 343 (1994) 251–255]. Here we have studied the cPLA2 inhibition mechanism using ANX‐I, N‐terminally deleted ANX‐I (ΔANX‐I), ANX‐II, ANX‐II2P112, ANX‐III, and ANX‐V. Under the conditions for the specific interaction model, ANX‐I, ΔANX‐I, and ANX‐II2P112 inhibited cPLA2, whereas inhibition by ANX‐II and ANX‐III was negligible. Inhibition by ANX‐V was much smaller than that by ANX‐I. The protein–protein interactions between cPLA2 and ANX‐I, ΔANX‐I, and ANX‐II2P112 were verified by immunoprecipitation. We can therefore conclude that inhibition of cPLA2 by specific interaction is not a general function of all ANXs, and is rather a specific function of ANX‐I. The results are consistent with the specific interaction model.
Location of a ligand recognition site of FMRFamide‐gated Na+ channels
Authors: Cottrell G.A. , Jeziorski M.C. , Green K.A.
Published: July 2002

The second FMRFamide‐gated Na+ channel (HtFaNaC), from Helisoma trivolvis, has been cloned. HtFaNaC has some different pharmacological properties to HaFaNaC, from Helix aspersa, which has enabled a rational approach to be made to start to identify the FMRFamide recognition site. Several chimeras were made by switching sections between the channels. The differences in sensitivity to FMRFamide, and amiloride, were assessed after expression in Xenopus oocytes. The data suggest that a recognition site for FMRFamide, and the potentiating action of amiloride, resides in a sequence of about 120 amino acids in the extracellular loop proximal to the first transmembrane segment.
Exogenously injected nuclear import factor p10/NTF2 inhibits signal‐mediated nuclear import and export of proteins in living cells
Authors: Tachibana Taro , Hieda Miki , Sekimoto Toshihiro , Yoneda Yoshihiro
Published: July 2002

p10/NTF2 is a cytosolic factor which is required for the translocation step in nuclear protein import in an in vitro assay with digitonin‐permeabilized cells. To study the functional roles of p10/NTF2 on protein transport between the nucleus and cytoplasm in living cells, recombinant p10/NTF2 was microinjected injo cultured mammalian cells. Cytoplasmically injected p10/NTF2 strongly inhibited the nuclear import of co‐injected NLS‐containing substrates in a dose‐dependent manner but had no effect on the diffusive import of small non‐nuclear proteins. Moreover, when injected into the cell nucleus, p10/NTF2 inhibited the nuclear export of NES‐containing substrates. The results suggest that the nuclear import factor p10/NTF2 may also be involved in the nuclear export of proteins and that the protein transport efficiency between the nucleus and cytoplasm may be regulated by the intracellular level of p10/NTF2.
Vertebrate evolution by interspecific hybridisation – are we polyploid?
Authors: Spring Jürg
Published: July 2002

For the growing fraction of human genes with identified functions there are often homologues known from invertebrates such as Drosophila. A survey of well established gene families from aldolases to zinc finger transcription factors reveals that usually a single invertebrate gene corresponds to up to four equally related vertebrate genes on different chromosomes. This pattern was before widely noticed for the Hox gene clusters but appears to be more general. Genome quadruplication by two rounds of hybridisation is discussed as a simple biological mechanism that could have provided the necessary raw material for the success of vertebrate evolution.
Imidazolone, a novel advanced glycation end product, is present at high levels in kidneys of rats with streptozotocin‐induced diabetes
Authors: Niwa Toshimitsu , Katsuzaki Tomoyuki , Ishizaki Yayoi , Hayase Fumitaka , Miyazaki Takashi , Uematsu Toshihiko , Tatemichi Noriyuki , Takei Yoshifumi
Published: July 2002

We produced a monoclonal antibody to imidazolones A and B, novel advanced glycation end products formed from the reaction of 3‐deoxyglucosone (3‐DG) with the guanidino group of arginine. Liquid chromatography/mass spectrometry demonstrated that the formation of imidazolone A by incubating 3‐DG with arginine is very rapid, reaching a maximum concentration within 24 h, but the formation of imidazolone B is very slow and low in quantity even after 2 weeks. Thus, at physiological conditions the formation of imidazolone A is dominant, while that of imidazolone B is negligible. Immunochemistry demonstrated that the imidazolone content in the kidneys of streptozotocin‐induced diabetic rats was significantly higher than in the control rats. Serum levels of 3‐DG in the diabetic rats were also significantly higher than in control rats. 3‐DG attacks the arginine residues of the tissue proteins, producing imidazolone at high levels in the kidneys affected by diabetic nephropathy.
Direct determination of the chemical composition of acetylcholinesterase phosphonylation products utilizing electrospray‐ionization mass spectrometry
Authors: Barak Ruth , Ordentlich Arie , Barak Dov , Fischer Meir , Benschop Hendrik P , De Jong Leo P.A , Segall Yoffi , Velan Baruch , Shafferman Avigdor
Published: July 2002

While non‐reactivability of cholinesterases from their phosphyl conjugates (aging) is attributed to an unimolecular process involving loss of alkyl group from the phosphyl moiety, no conclusive evidence is available that this is the only reaction path and involvement of other post‐inhibitory processes cannot be ruled out. To address this issue, molecular masses of the bacterially expressed recombinant human acetylcholinesterase and of its conjugates with a homologous series of alkyl methyl‐phosphonofluoridates, were measured by electrospray‐ionization mass spectrometry (ESI‐MS). The measured mass of the free enzyme was 64 700 Da (calculated 64 695 Da) and those of the methylphosphono‐HuAChE adducts, bearing isopropyl, isobutyl, 1,2‐dimethylpropyl and 1,2,2‐trimethylpropyl substituents, were 64 820, 64 840, 64 852 and 64 860 Da, respectively. These values reflect both the addition of the phosphonyl moiety and the gradual mass increase due to branching of the alkoxy substituent. The composition of these adducts change with time to yield a common product with molecular mass of 64 780 Da which is consistent with dealkylation of the phosphonyl moieties. Furthermore, in the case of 1,2‐dimethylpropyl methylphosphono‐HuAChE, the change in the molecular mass and the kinetics of non‐reactivability appear to occur in parallel indicating that dealkylation is indeed the predominant molecular transformation leading to ‘aging’ of phosphonyl‐AChE adducts.
A human gene encoding morphine modulating peptides related to NPFF and FMRFamide
Authors: Perry Stephen J. , Yi-Kung Huang Eagle , Cronk David , Bagust Jeffery , Sharma Ram , Walker Robert J. , Wilson Shelagh , Burke Julian F.
Published: July 2002

FMRFamide‐related peptides have been isolated from both invertebrates and vertebrates and exhibit a wide range of biological effects in rats. We show here that in humans 2 FMRFamide‐related peptides are encoded by a single gene expressed as a spliced mRNA. The larger predicted peptide (AGEGLNSQFWSLAAPQRFamide) differs from the peptide isolated from bovines (AGEGLSSPFWSLAAPQRFamide) by the substitutions of 2 amino acids. The shorter predicted peptide (NPSF, SQAFLFQPQRFamide) is 3 amino acids longer than the bovine 8 amino‐acid NPFF (FLFQPQRFamide) or the human NPFF peptide isolated from serum [5], suggesting that the encoded protein is subject to cleavage by a tripeptidyl peptidase or by a novel processing mechanism. On rat spinal cord, the larger peptide is indistinguishable in activity from the equivalent bovine peptide whereas the smaller extended peptide is inactive.
Proton pumping by cytochrome c oxidase is coupled to peroxidase half of its catalytic cycle
Authors: Vygodina T.V , Capitanio N , Papa S , Konstantinov A.A
Published: July 2002

The four‐electron reaction cycle of cytochrome oxidase is comprised of an eu‐oxidase phase in which the enzyme receives the first two electrons and reduces oxygen to bound peroxide and a peroxidase phase in which the peroxy state formed in the eu‐oxidase half of the cycle is reduced by the 3rd and 4th electrons to the ferryl‐oxo state and oxidized form, respectively. Here we show that the ferrocyanide‐peroxidase activity of cytochrome c oxidase incorporated in phospholipid vesicles is coupled to proton pumping. The H+/e
− ratio for the ferrocyanide‐peroxidase partial reaction is twice higher than for the overall ferrocyanide‐oxidase activity and is close to 2. These results show that proton pumping by COX is confined to the peroxidase part of the enzyme catalytic cycle (transfer of the 3rd and 4th electron) whereas the eu‐oxidase part (transfer of the first two electrons) may not be proton pumping.
In vitro membrane integration of leader peptidase depends on the Sec machinery and anionic phospholipids and can occur post‐translationally
Authors: van Klompenburg Wim , Ridder Anja N.J.A. , van Raalte Anne L.J. , Killian Antoinette J. , von Heijne Gunnar , de Kruijff Ben
Published: July 2002

A cell‐free system based on a lysate and membrane vesicles from Escherichia coli is used to study characteristics of the membrane integration reaction of the polytopic membrane protein leader peptidase (Lep). Integration into inverted inner membrane vesicles was detected by partial protection against externally added protease. Integration is most efficient when coupled to translation but can also occur post‐translationally and depends on the action of the proteinaceous Sec machinery and availability of anionic phospholipids. Lep is the first example of a membrane protein without cleavable signal sequence which requires anionic lipids for integration in vitro.
Muscle glycogen synthase translocates from the cell nucleus to the cytosol in response to glucose
Authors: Ferrer Juan C , Baqué Susanna , Guinovart Joan J
Published: July 2002

We have studied the intracellular localization of muscular glycogen synthase by fusing the green fluorescent protein (GFP) of the jelly‐fish Aequorea victoria to the N‐terminus of human muscle glycogen synthase (HMGS), and expressing the chimeric protein in C2C12, COS‐1 cells, and primary cultured rat hepatocytes. In contrast to what we have recently found for the hepatic glycogen synthase (Fernández‐Novell et al. (1997) Biochem. J. 321, 227–231), the GFP/HMGS fusion protein is localized to the nucleus of the cell in the absence of glucose, and in the presence of the sugar it is essentially found in the cytosol. Insulin is not required for the translocation of the enzyme.
Suramin enters and accumulates in low pH intracellular compartments of v‐sis‐transformed NIH 3T3 cells
Authors: Huang Shuan Shian , Koh Han A. , Huang Jung San
Published: July 2002

Using acridine orange as a reporter compound, we demonstrate that suramin enters and accumulates in low pH intracellular compartments (endosomes, lysosomes, and trans‐Golgi complex) of normal and v‐sis‐transformed NIH 3T3 cells. The concentration of suramin in these acidic compartments is estimated to be >150 μM, higher than the concentration known to completely inhibit interaction of the platelet‐derived growth factor (PDGF) receptor and v‐sis gene product. These results support the hypothesis that suramin reverses the transformed phenotype of v‐sis‐transformed cells by entering the cell via endocytosis and blocking interaction of the v‐sis gene product and PDGF receptor in intracellular organelles.
FNR is a direct oxygen sensor having a biphasic response curve
Authors: Jordan Peter A. , Thomson Andrew J. , Ralph Edward T. , Guest John R. , Green Jeffrey
Published: July 2002

FNR is a transcription regulator that controls the expression of target genes in response to anoxia. Anaerobiosis is accompanied by the acquisition of two [4Fe‐4S]2+ clusters per FNR dimer and the ability to bind DNA site‐specifically. Oxidation of the [4Fe‐4S]2+ form of FNR by O2 produced a non‐DNA‐binding, transcriptionally inactive form which also contains an iron‐sulfur cluster, recently identified by Mossbauer spectroscopy as a [2Fe‐2S] cluster (Khoroshilova et al., 1997, PNAS. 94, 6078). Complete conversion needed at least 2.5–3.0 molecules of O2 per [4Fe‐4S]2+ cluster. Using sub‐stoicheiometric amounts of air‐saturated buffer, stable equilibria were established in which the [4Fe‐4S]2+ and [2Fe‐2S]2+ forms co‐exist and no EPR detectable free ferric ions were released. In contrast, a 20‐fold molar excess K3Fe(CN)6 was required to oxidise the [4Fe‐4S]2+ cluster and in this case, ferric ions were released. FNR is therefore a sensitive O2 sensor.
Dictyostelium discoideum protein disulfide isomerase, an endoplasmic reticulum resident enzyme lacking a KDEL‐type retrieval signal
Authors: Monnat Jean , Hacker Ulrike , Geissler Heidrun , Rauchenberger Robert , Neuhaus Eva M. , Maniak Markus , Soldati Thierry
Published: July 2002

The primary activity of protein disulfide isomerase (PDI), a multifunctional resident of the endoplasmic reticulum (ER), is the isomerization of disulfide bridges during protein folding. We isolated a cDNA encoding Dictyostelium discoideum PDI (Dd‐PDI). Phylogenetic analyses and basic biochemical properties indicate that it belongs to a subfamily called P5, many members of which differ from the classical PDIs in many respects. They lack an intervening inactive thioredoxin module, a C‐terminal acidic domain involved in Ca2+ binding and a KDEL‐type retrieval signal. Despite the absence of this motif, the ER is the steady‐state location of Dd‐PDI, suggesting the existence of an alternative retention mechanism for P5‐related enzymes.
Cooperativity of phosphatidylinositol transfer protein and phospholipase D in secretory vesicle formation from the TGN – phosphoinositides as a common denominator?
Authors: Tüscher Oliver , Lorra Christoph , Bouma Barend , Wirtz Karel W.A , Huttner Wieland B
Published: July 2002

Phosphatidylinositol transfer protein (PITP) and phospholipase D (PLD) stimulate the formation of constitutive secretory vesicles (CSVs) and immature secretory granules (ISGs) from the trans‐Golgi network (TGN) in a cell‐free system. The stimulatory effects of PITP and PLD are additive. Stimulation by either PITP or PLD is blocked by geneticin, a member of the aminoglycoside antibiotics known to bind to phosphoinositides. Since the PLD we used is insensitive to geneticin, our results suggest that phosphoinositides promote secretory vesicle formation as downstream effectors of both PITP and PLD, possibly via the recruitment of proteins mediating membrane budding and fission.
Amyloid β‐protein (Aβ) associated with lipid molecules: immunoreactivity distinct from that of soluble Aβ
Authors: Yanagisawa Katsuhiko , McLaurin JoAnne , Michikawa Makoto , Chakrabartty Avijit , Ihara Yasuo
Published: July 2002

We previously identified a novel amyloid β‐protein (Aβ), that binds to GM1 ganglioside, in brains exhibiting the early pathological changes of AD. In this study, we raised monoclonal antibodies, using membrane fractions containing abundant GM1 ganglioside‐bound Aβ as antigens. Monoclonal antibody 4396, produced in this study, immunoprecipitates Aβ42 in the membrane fractions of brains with diffuse plaques, but does not react with soluble Aβ42 or GM1 ganglioside. Furthermore, this antibody recognizes the Aβ bound to lipid vesicles containing GM1 ganglioside, and unexpectedly, phosphatidylinositol. In contrast, a control anti‐Aβ monoclonal antibody does not recognize the Aβ bound to these lipid vesicles. These results indicate that Aβ associated with lipids has an immunoreactivity distinct from that of soluble A.
Characterization of protein kinase C‐mediated phosphorylation of the short cytoplasmic domain isoform of C‐CAM
Authors: Edlund Magnus , Wikström Kristina , Toomik Reet , Ek Pia , Öbrink Björn
Published: July 2002

C‐CAM is a ubiquitously expressed cell adhesion molecule belonging to the carcinoembryonic antigen family. Two co‐expressed isoforms, C‐CAM‐L and C‐CAM‐S, are known, having different cytoplasmic domains both of which can be phosphorylated in vivo. Here we have characterized the PKC‐mediated phosphorylation of the short cytoplasmic domain isoform, C‐CAM‐S. Phorbol myristyl acetate induced phosphorylation of C‐CAM‐S in transfected CHO cells. Using synthetic peptides and Edman degradation we identified Ser449 as the PKC‐phosphorylated amino acid residue. Binding experiments with modified peptides indicated that this phosphorylation decreases the ability of the cytoplasmic domain of C‐CAM‐S to bind calmodulin.
Expression profile and structural divergence of novel human annexin 311
Authors: Morgan Reginald O. , Fernandez Maria-Pilar
Published: July 2002

Systematic analysis of expressed sequence tags in dbEST yielded an expression profile of the ten known human annexins and led to the discovery of a novel subfamily expressed mainly in differentiating tissues. Full‐length cDNAs encoded a 338‐amino acid protein with less than 40% identity to other annexins, an atypical amino acid composition, and an insertion and deletion in internal repeat 3. The most striking feature was a complete ablation of all four type II calcium‐binding sites in the conserved tetrad core. Annexin 31 thus constitutes a unique, natural probe for investigating the role of membrane binding in annexin function.
Stimulation of NSF ATPase activity during t‐SNARE priming
Authors: Haynes Lee P. , Barnard Richard J.O. , Morgan Alan , Burgoyne Robert D.
Published: July 2002

N‐Ethylmaleimide‐sensitive factor (NSF) plays a key role in vesicular traffic by disassembling and priming SNARE proteins for their function in docking and fusion. We demonstrate that the ATPase activity of NSF is activated by α‐soluble NSF attachment protein (α‐SNAP) in a complex with syntaxin 1A. In addition, we show that a construct consisting of the H3 domain of syntaxin 1A (GST‐synt(195–263), which does not support NSF disassembly in the presence of MgATP gave a larger stimulation. NSF ATPase activation was specific and did not occur using mutant α‐SNAPs unable to bind GST‐synt or with mutated C‐termini. We suggest that activation of NSF ATPase activity in the SNARE complex may be essential to allow SNARE priming.
phd1+, a histone deacetylase gene of Schizosaccharomyces pombe, is required for the meiotic cell cycle and resistance to trichostatin A1
Authors: Kim Young Bae , Honda Akira , Yoshida Minoru , Horinouchi Sueharu
Published: July 2002

A gene named phd1
+ encoding a protein highly homologous to the yeast and human histone deacetylases, such as Saccharomyces cerevisiae Rpd3p and human HDAC1, was cloned from Schizosaccharomyces pombe. The immune complex isolated from S. pombe cells expressing Phd1 fused to the FLAG epitope showed histone deacetylase activity, which was inhibited by trichostatin A (TSA), a specific inhibitor of histone deacetylase. The null mutation of phd1
+ resulted in a marked decrease in the total cellular histone deacetylase activity and an increase in the sensitivity to TSA. Although the phd1 disruptant showed no obvious defect in the mitotic cell cycle or mating, both homothallic haploid and heterothallic diploid cells failed to form spores in the absence of phd1
+. These results indicate that phd1
+ encodes a histone deacetylase, which is involved in the meiotic cell cycle in S. pombe.
Synthesis and differentially regulated processing of proinsulin in developing chick pancreas, liver and neuroretina
Authors: Alarcón Cristina , Serna José , Pérez-Villamil Beatriz , de Pablo Flora
Published: July 2002

Regulated preproinsulin gene expression in non‐pancreatic tissues during development has been demonstrated in rodents, Xenopus and chicken. Little is known, however, about the synthesis and processing of the primary protein product, proinsulin, in comparison with these events in pancreas. Using specific antisera and immunocytochemistry, immunoblot and HPLC criteria, we characterize the differential processing of proinsulin in developing neuroretina, liver and pancreas. The chick embryo pancreas expresses the convertase PC2, and largely processes proinsulin to insulin. In contrast, little or no mature PC2 is present in embryonic liver and neuroretina and the (pro)insulin immunoactivity identified is predominantly proinsulin.
Isocitrate lyase of Ashbya gossypii – transcriptional regulation and peroxisomal localization
Authors: Maeting Ines , Schmidt Georg , Sahm Hermann , Revuelta José Luis , Stierhof York-Dieter , Stahmann K.-Peter
Published: July 2002

The isocitrate lyase‐encoding gene AgICL1 from the filamentous hemiascomycete Ashbya gossypii was isolated by heterologous complementation of a Saccharomyces cerevisiae icl1d mutant. The open reading frame of 1680 bp encoded a protein of 560 amino acids with a calculated molecular weight of 62 584. Disruption of the AgICL1 gene led to complete loss of AgIcl1p activity and inability to grow on oleic acid as sole carbon source. Compartmentation of AgIcl1p in peroxisomes was demonstrated both by Percoll density gradient centrifugation and by immunogold labeling of ultrathin sections using specific antibodies. This fitted with the peroxisomal targeting signal AKL predicted from the C‐terminal DNA sequence. Northern blot analysis with mycelium grown on different carbon sources as well as AgICL1 promoter replacement with the constitutive AgTEF promoter revealed a regulation at the transcriptional level. AgICL1 was subject to glucose repression, derepressed by glycerol, partially induced by the C2 compounds ethanol and acetate, and fully induced by soybean oil.
Affinity isolation of imidazoline binding proteins from rat brain using 5‐amino‐efaroxan as a ligand
Authors: Monks Lara K. , Cosgrove Karen E. , Dunne Mark J. , Ramsden Christopher A. , G. Morgan Noel , Chan Sue L.F.
Published: July 2002

We have employed an amino derivative of the imidazoline ligand, efaroxan, to isolate imidazoline binding proteins from solubilised extracts of rat brain, by affinity chromatography. A number of proteins were specifically retained on the affinity column and one of these was immunoreactive with an antiserum raised against the ion conducting pore component of the ATP‐sensitive potassium channel. Patch clamp experiments confirmed that, like its parent compound, amino‐efaroxan blocks ATP‐sensitive potassium channels in human pancreatic β‐cells and can stimulate the insulin secretion from these cells. The results reveal that a member of the ion conducting pore component family is strongly associated with imidazoline binding proteins in brain and in the endocrine pancreas.
Induction of oxidative DNA damage in anaerobes
Authors: Takeuchi Toru , Nakaya Yasuhiro , Kato Naoki , Watanabe Kunitomo , Morimoto Kanehisa
Published: July 2002

We compared oxidative DNA damage in strictly anaerobic Prevotella melaninogenica, aerotolerant anaerobic Bacteroides fragilis, and facultative anaerobic Salmonella typhimurium after exposure to O2 or H2O2. Using HPLC with electrochemical detection, we measured 8‐hydroxydeoxyguanosine (8OHdG) as a damage marker. O2 induced 8OHdG in P. melaninogenica but not in B. fragilis, which shows catalase activity, or in S. typhimurium. In P. melaninogenica, with catalase, O2 induced less 8OHdG; superoxide dismutase had no effect; with glucose and glucose oxidase, O2 induced more 8OHdG. H2O2 also markedly increased 8OHdG. O2 was suggested to induce 8OHdG through H2O2. O2 or H2O2 decreased survival only in P. melaninogenica. Highly sensitive to oxidative stress, P. melaninogenica could prove useful for investigating oxidative DNA damage.
A role for ADP ribosylation factor in the control of cargo uptake during COPI‐coated vesicle biogenesis
Authors: Malsam Jörg , Gommel Daniel , Wieland Felix T. , Nickel Walter
Published: July 2002

ARF‐mediated hydrolysis of GTP has been demonstrated to regulate coat disassembly of Golgi‐derived COPI transport vesicles (Tanigawa, G., Orci, L., Amherdt, M., Ravazzola, M., Helms, J.B. and Rothman, J.E. (1993) J. Cell Biol. 123, 1365–1371). In addition, a requirement for GTP hydrolysis at an early stage of COPI vesicle biogenesis has been established since cargo uptake is impaired in the presence of GTPγS (Nickel, W., Malsam, J., Gorgas, K., Ravazzola, M., Jenne, N., Helms, J.B. and Wieland, F.T. (1998) J. Cell Sci. 111, 3081–3090), a non‐hydrolyzable analogue of GTP. We now demonstrate that the GTPase involved in the regulation of cargo uptake is ARF, revealing a multi‐functional role of this GTPase in COPI‐mediated vesicular transport. The molecular mechanism of cargo uptake as well as the functional implications of these findings on the overall process of COPI vesicle biogenesis are discussed.
Inhibition of yeast inositol phosphorylceramide synthase by aureobasidin A measured by a fluorometric assay
Authors: Zhong Wenyan , Murphy Dennis J , Georgopapadakou Nafsika H
Published: July 2002

Inositol phosphorylceramide synthase (IPC synthase) is an essential and unique enzyme in fungal sphingolipid biosynthesis and is the target of the cyclic nonadepsipeptide antibiotic aureobasidin A. As a first step towards understanding the mechanism of aureobasidin A inhibition, we developed a fluorometric HPLC assay for IPC synthase using the Saccharomyces cerevisiae enzyme and the fluorescent substrate analog 6‐[N‐(7‐nitro‐2,1,3‐benzoxadiazol‐4‐yl)amino]‐hexanoyl ceramide (C6‐NBD‐cer). The kinetic parameters for C6‐NBD‐cer were comparable to those for the synthetic substrate N‐acetylsphinganine used previously. Aureobasidin A acted as a tight‐binding, non‐competitive inhibitor with respect to C6‐NBD‐cer and had a K
i of 0.55 nM.
Angiotensin in cardiac surgery: efficacy in patients on angiotensin converting enzyme inhibitors
Authors: Bennett Sean R. , McKeown June , Drew Phil , Griffin Steve
Published: July 2002

Background:

Patients presenting for cardiac surgery are often treated with angiotensin converting enzyme inhibitors (ACEIs), either for heart failure or hypertension. Control of systemic vascular resistance (SVR) during surgery can be difficult in such patients. Angiotensin II has been available as an unlicensed vasoconstrictor, but there is concern about renal damage and its use.

Aim:

This study compared a standard vasoconstrictor with angiotensin II and examined the effect on renal function after cardiac surgery.

Method:

Twenty consecutive, consenting patients scheduled for cardiac surgery that had been taking ACEIs for at least 6 months, were randomly assigned to receive either phenylephrine or angiotensin II for the control of SVR during and for 24 h after cardiac surgery. A pulmonary artery catheter was used to guide therapy. Creatinine clearance was measured before, 24 and 48 h after surgery.

Results:

Low SVR and blood pressure requiring intervention was seen in all patients, particularly during cardiopulmonary bypass. One patient in the control group failed to respond to P, but responded normally to angiotensin II. Neither drug caused renal impairment.

Conclusion:

Angiotensin II is a safe alternative to phenylephrine in patients on ACEIs and should be considered in patients who fail to respond to conventional vasoconstrictors.
HRD1 inhibits fatty acid oxidation and tumorigenesis by ubiquitinating CPT2 in triple‐negative breast cancer
Authors: Guo Xin , Wang Aman , Wang Wen , Wang Ya , Chen Huan , Liu Xiaolong , Xia Tian , Zhang Aijia , Chen Di , Qi Huan , Ling Ting , Piao Hai‐long , Wang Hong‐jiang
Published: November 2020

Dependence on glutamine and acceleration of fatty acid oxidation (FAO) are both metabolic characteristics of triple‐negative breast cancer (TNBC). With the rapid growth of tumors, accelerated glutamine catabolism depletes local glutamine, resulting in glutamine deficiency. Studies have shown that the use of alternative energy sources, such as fatty acids, enables tumor cells to continue to proliferate rapidly in a glutamine‐deficient microenvironment. However, the detailed mechanisms behind this metabolic change are still unclear. Herein, we identified HRD1 as a regulatory protein for FAO that specifically inhibits TNBC cell proliferation under glutamine‐deficient conditions. Furthermore, we observed that HRD1 expression is significantly downregulated under glutamine deprivation and HRD1 directly ubiquitinates and stabilizes CPT2 through K48‐linked ubiquitination. In addition, the inhibition of CPT2 expression dramatically suppresses TNBC cell proliferation mediated by HRD1 knockdown in vitro and in vivo. Finally, we found that the glutaminase inhibitor CB839 significantly inhibited TNBC cell tumor growth, but not in the HRD1 knock‐downed TNBC cells. These findings provide an invaluable insight into HRD1 as a regulator of lipid metabolism and have important implications for TNBC therapeutic targeting.
Stimuli‐Responsive Nanocomposite Hydrogels for Biomedical Applications
Authors: Lavrador Pedro , Esteves Marco R. , Gaspar Vítor M. , Mano João F.
Published: November 2020

The complex tissue‐specific physiology that is orchestrated from the nano‐ to the macroscale, in conjugation with the dynamic biophysical/biochemical stimuli underlying biological processes, has inspired the design of sophisticated hydrogels and nanoparticle systems exhibiting stimuli‐responsive features. Recently, hydrogels and nanoparticles have been combined in advanced nanocomposite hybrid platforms expanding their range of biomedical applications. The ease and flexibility of attaining modular nanocomposite hydrogel constructs by selecting different classes of nanomaterials/hydrogels, or tuning nanoparticle‐hydrogel physicochemical interactions widely expands the range of attainable properties to levels beyond those of traditional platforms. This review showcases the intrinsic ability of hybrid constructs to react to external or internal/physiological stimuli in the scope of developing sophisticated and intelligent systems with application‐oriented features. Moreover, nanoparticle‐hydrogel platforms are overviewed in the context of encoding stimuli‐responsive cascades that recapitulate signaling interplays present in native biosystems. Collectively, recent breakthroughs in the design of stimuli‐responsive nanocomposite hydrogels improve their potential for operating as advanced systems in different biomedical applications that benefit from tailored single or multi‐responsiveness.