bluuebunny/crossref_metadata_2025 huggingface dataset:
Bcl‐2 protects from oxidative damage and apoptotic cell death without interfering with activation of NF‐κB by TNF
Authors: Albrecht Hugo , Tschopp Jürg , Jongeneel C.Victor
Published: July 2002

The recent demonstration of the anti‐oxidant properties of the Bcl‐2 gene product suggested that expression of Bcl‐2 may interfere with the nuclear migration of the NF‐κB transcription factor, which is thought to depend on the presence of reactive oxygen intermediates. In mouse L cells, overexpression of Bcl‐2 interfered with the activation of NF‐κB by H2O2. However, Bcl‐2 had no effect on the activation of NF‐κB by TNF, even though it protected cells from TNF‐induced apoptosis. The effects of exogenous pyrrolidine dithiocarbamate were very similar to those of Bcl‐2 overexpression. We conclude that the protective effects of anti‐oxidants against induced apoptotic cell death are unrelated to their ability to interfere with NF‐κB activation.
Rapid kinetics of G protein subunit association: A rate‐limiting conformational change?
Authors: Neubig Richard R. , Connolly Mark P. , Remmers Ann E.
Published: July 2002

G protein subunit association and dissociation are thought to play an important role in signal transduction. We measured αβγ heterocomplex formation using resonance energy transfer. Fluorescein‐labelled α(F‐α) emission was quenched ∼ 10% on mixing with eosin‐labelled βγ(E‐βγ). Unlabelled βγ did not quench F‐α fluorescence. Stopped‐flow kinetics showed a t ½ ranging from 2.5 s to 0.25 s for 50 nM to 1200 nM E‐βγ. The rate saturated at high E‐βγ concentrations consistent with a two‐step mechanism. We report the first rapid‐mix studies of G protein subunit association kinetics which suggest that α and βγ combine by a two‐step process with a maximal rate of 4.1 ± 0.4 s−1.
Nanomolar concentrations of nitric oxide reversibly inhibit synaptosomal respiration by competing with oxygen at cytochrome oxidase
Authors: Brown Guy C. , Cooper Chris.E.
Published: July 2002

Nitric oxide (NO) reversibly inhibited oxygen consumption of brain synaptosomes. Inhibition was reversible, occurred at the level of cytochrome oxidase, and was apparently competitive with oxygen, with half‐inhibition by 270 nM NO at oxygen concentrations around 145 μM and by 60 nM NO at around 30μM O2. Isolated cytochrome oxidase was inhibited by similar levels of NO. These levels of NO are within the measured physiological and pathological range for a number of tissues and conditions, suggesting that NO inhibition of cytochrome oxidase and the competion with oxygen may occur in vivo.
Immunolocalization of X‐arrestin in human cone photoreceptors
Authors: Sakuma H. , Inana G. , Murakami A. , Higashide T. , McLaren M.J.
Published: July 2002

X‐arrestin is a recently identified retina‐specific gene of unknown function. Affinity‐purified anti‐peptide antibody to human X‐arrestin was prepared, and used in Western blot analysis of human retinal proteins and for immunohistochemistry on human retinal sections. By Western blot analysis, the antibody specifically bound to an ≈47 kDa protein, and by indirect immunofluorescence specifically labeled cone photoreceptors with greatest intensity in their outer segments. In single and double label experiments, the localization of X‐arrestin immunoreactivity was compared with immunolabelling patterns obtained with antibodies to red/green cone opsin, rhodopsin, and S‐antigen. The results showed that X‐arrestin is expressed in red‐, green‐ and blue‐sensitive cones in the human retina.
Rhythm generation in organotypic medullary cultures of newborn rats
Authors: Baker R.E. , Ballantyne D. , Bingmann D. , Jones D. , Widman G.
Published: July 2002

Organotypic transverse medullary slices (obex level) from six‐day‐old rats, cultured for two to four weeks in chemically defined medium contained rhythmically discharging neurones which were activated by CO2 and H+. The mechanisms underlying this rhythmicity and the spread of excitation and synaptic transmission within this organotypic tissue were examined by modifying the composition of the external solution. Our findings showed that (1) Exposure to tetrodotoxin (0.2 μM) or to high magnesium (6 mM) and low calcium (0.2 mM) concentrations abolished periodic activity. (2) Neither the blockade of GABAergic potentials with bicuculline methiodide (200 μM) and/or hydroxysaclofen (200 μM) nor the blockade of glycinergic potentials with strychnine hydrochloride (100 μM) abolished rhythmicity. (3) While atropine sulphate (5 μM) was ineffective in modulating periodic discharges nicotine (100 μM) — like CO2 — shortened the intervals between the periodic events; hexamethonium (50–100 μM) reduced both periodic and aperiodic activity. (4) Exposure to the NMDA antagonist 2‐aminophosphonovaleric acid (50 μM) suppressed periodic events only transiently. In the presence of 2‐aminophosphonovaleric acid rhythmicity recovered. However, the AMPA‐antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (10–50 μM), abolished periodic activity reversibly within less than 5 min. When 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione and nicotine were administered simultaneously periodic events persisted for up to 10 min. These findings indicate that synaptic excitatory drive is a prerequisite for the generation of rhythmic discharges of medullary neurones in this preparation. This drive may activate voltage‐dependent channels or it may facilitate endogenous cellular mechanisms which initiate oscillations of intracellular calcium concentration. To test the latter possibility (5) calcium antagonists were added to the bath saline. The organic calcium antagonists verapamil and flunarizine (50–100 μM each) and the inorganic calcium antagonists cobalt (2 mM) and magnesium (6 mM) suppressed periodic activity and abolished or weakened the chemosensitivity towards CO2/acidosis. (6) Dantrolene (10 μM), an inhibitor of intracellular calcium release decreased the periodicity, while thapsigargin (2 μM) which blocks endoplasmic Ca2+‐ATPase, transiently accelerated the occurrence of periodic events. (7) Oscillations of intracellular free calcium concentrations in Fura‐2 AM‐loaded cells were weakened or abolished by cobalt (2 mM). The results of (5)–(7) indicate that transmembrane calcium fluxes as well as intracellular Ca2+‐release and ‐clearance mechanisms are a prerequisite for intracellular free calcium oscillations which may be important in the generation of rhythmic discharges in medullary neurones.
Dysthymia and depressive disorders: dopamine hypothesis
Authors: Gessa GL
Published: July 2002

This paper reviews the recent literature supporting the hypothesis that reduced neurotransmission in the mesolimbic dopamine (DA) system may sustain some of the symptoms of depressive conditions including dysthymia. Experimental evidences indicate that mesolimbic DA plays a crucial role in controlling incentive, motivation and reward. Additionally, in different models of depression, a reduced DA activity in the limbic system, reversed by chronic antidepressant treatment, is observed. Finally, different antidepressants, irrespective of their acute action on the uptake of norepinephrine or serotonin, have the common property when given chronically to potentiate behavioural responses to DA agonists. The DA hypothesis of depression offers an explanation for the antidepressive effect of drugs such as sulpiride and amisulpride given at low doses, that preferentially block DA autoreceptors and thereby increase DA output.
The efficacy of a dose-escalated application of transdermal nicotine plus sulpiride in Tourette’s syndrome
Authors: Dursun SM , Reveley MA
Published: July 2002

The addition of a dose-escalated regimen of transdermal nicotine patches (TNP) improved tics of a non-smoker patient with Tourette's syndrome, whose tics did not respond to sulpiride. This improvement, as assessed by the Yale Global Tic Severity Scale, lasted for up to 8 weeks, but not 16 weeks. An explanation for the efficacy of TNP is presented.
Mutagenesis of the proposed iron‐sulfur cluster binding ligands in Escherichia coli biotin synthase
Authors: Hewitson Kirsty S , Baldwin Jack E , Shaw Nicholas M , Roach Peter L
Published: July 2002

Biotin synthase (BioB) is a member of a family of enzymes that includes anaerobic ribonucleotide reductase and pyruvate formate lyase activating enzyme. These enzymes all use S‐adenosylmethionine during turnover and contain three highly conserved cysteine residues that may act as ligands to an iron‐sulfur cluster required for activity. Three mutant enzymes of BioB have been made, each with one cysteine residue (C53, 57, 60) mutated to alanine. All three mutant enzymes were inactive, but they still exhibited the characteristic UV‐visible spectrum of a [2Fe‐2S]2+ cluster similar to that of the wild‐type enzyme.
The Saccharomyces cerevisiae DNA damage checkpoint is required for efficient repair of double strand breaks by non‐homologous end joining
Authors: de la Torre-Ruiz Maria-Angeles , Lowndes Noel F.
Published: July 2002

In this work we report that the Saccharomyces cerevisiae RAD9, RAD24, RAD17, MEC1, MEC3 and RAD53 checkpoint genes are required for efficient non‐homologous end joining (NHEJ). RAD9 and RAD24 function additionally in this process. Defective NHEJ in rad9Δ–rad24Δ, but not yku80Δ cells, is only partially rescued by imposing G1 or G2/M delays. Thus, checkpoint functions other than transient cell cycle delays may be required for normal levels of NHEJ. Epistasis analysis also indicated that YKU80 and RAD9/RAD24 function in the same pathway for repair of lesions caused by MMS and γ‐irradiation. Unlike NHEJ, the checkpoint pathway is not required for efficient site‐specific integration of plasmid DNA into the yeast genome, which is RAD52‐dependent, but RAD51‐independent.
Pro‐atherogenic factors induce telomerase inactivation in endothelial cells through an Akt‐dependent mechanism
Authors: Breitschopf Kristin , Zeiher Andreas M , Dimmeler Stefanie
Published: July 2002

Advanced aging may contribute to impairment of angiogenesis and development of vascular diseases. Telomerase was shown to delay endothelial cell (EC) senescence. Therefore, we determined the regulation of telomerase activity in EC. Inhibition of phosphoinositol 3‐kinase (PI3K) suppressed telomerase activity, whereas inhibitors directed against ERK1/2 or protein kinase C had no effect. Dominant negative Akt significantly reduced telomerase activity. Moreover, pro‐atherogenic stimuli such as oxidized low density lipoprotein led to an inactivation of Akt and diminished telomerase activity. Thus, the PI3K/Akt pathway plays an important role in the regulation of telomerase activity. Pro‐atherosclerotic factors impair telomerase activity and thereby may promote EC aging.
Two KaiA‐binding domains of cyanobacterial circadian clock protein KaiC
Authors: Taniguchi Yasuhito , Yamaguchi Akihiro , Hijikata Atsushi , Iwasaki Hideo , Kamagata Kyoko , Ishiura Masahiro , Go Mitiko , Kondo Takao
Published: July 2002

kaiABC, a gene cluster, encodes KaiA, KaiB and KaiC proteins that are essential to circadian rhythms in the unicellular cyanobacterium Synechococcus sp. strain PCC 7942. Kai proteins can interact with each other in all possible combinations. This study identified two KaiA‐binding domains (CKABD1 and CKABD2) in KaiC at corresponding regions of its duplicated structure. Clock mutations on the two domains and kaiA altered the strength of CKABD–KaiA interactions assayed by the yeast two‐hybrid system. Thus, interaction between KaiA and KaiC through CKABD1 and CKABD2 is likely important for circadian timing in the cyanobacterium.
TFIIH: from transcription to clinic
Authors: Egly Jean-Marc
Published: July 2002

Once a large proportion of the genes responsible for genetic disorders are identified in the post‐genome era, the fundamental challenge is to establish a genotype/phenotype relationship. Our aim is to explain how mutations in a given gene affect its enzymatic function and, in consequence, disturb the life of the cell. Genome integrity is continuously threatened by the occurrence of DNA damage arising from cellular exposure to irradiation and genotoxic chemicals. This mutagenic or potentially lethal DNA damage induces various cellular responses including cell cycle arrest, transcription alteration and processing by DNA repair mechanisms, such as the nucleotide excision repair (NER) pathway. Disruption of NER in response to genotoxic injuries results in autosomal recessive hereditary diseases such as Xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD). One of the most immediate consequences of the induction of strand‐distorting lesions is the arrest of transcription in which TFIIH plays a role in addition to its role in DNA repair. The observations made by clinicians close to XP, TTD and CS patients, suggested that transcription defects responsible for brittle hair and nails for TTD, or developmental abnormalities for CS, resulted from TFIIH mutations. Here a story will be related which could be called ‘a multi‐faceted factor named TFIIH’. As biochemists, we have characterized each component of TFIIH, three of which are XPB and XPD helicases and cdk7, a cyclin‐dependent kinase. With the help of structural biologists, we have characterized most of the specific three‐dimensional structures of TFIIH subunits and obtained its electron microscopy image. Together these approaches help us to propose a number of structure–function relationships for TFIIH. Through transfection and microinjection assays, cell biology allows us to determine the role of TFIIH in transcription and NER. We are thus in a position to explain, at least in part, transcription initiation mechanisms and their coupling to DNA repair. We now know how the XPB helicase opens the promoter region for RNA synthesis and that one of the roles of XPD helicase is to anchor the cdk7 kinase to the core‐TFIIH. In XP and CS associated patients, we have demonstrated that some XPD mutations prevent an optimal phosphorylation of nuclear receptors by cdk7 with, as a consequence, a drop in the expression of genes sensitive to hormone action. We have thus shown that hormonal responses operate through TFIIH. Careful analysis of each TFIIH subunit also shows how the p44 Ring finger participates in certain promoter escape reactions. We are also able to localize the action of TFIIH in the sequence of events that lead to the elimination of DNA lesions. Thanks to the combination of these different approaches we are obtaining a much clearer picture of the TFIIH complex and its integration into the life of the cell.
Characterization of seven murine caspase family members
Authors: Van de Craen Marc , Vandenabeele Peter , Declercq Wim , Van den Brande Ilse , Van Loo Geert , Molemans Francis , Schotte Peter , Van Criekinge Wim , Beyaert Rudi , Fiers Walter
Published: July 2002

© 1997 Federation of European Biochemical Societies.
A functional bipartite nuclear localisation signal in the cytokine interleukin‐5
Authors: Jans David A. , Briggs Lyndall J. , Gustin Sonja E. , Jans Patricia , Ford Sally , Young Ian G.
Published: July 2002

Interleukin (IL)‐5 is central in regulating eosinophilia in allergic disease and parasitic infections. We have identified a bipartite nuclear localisation signal (NLS) within amino acids 95–111 of human IL‐5 (hIL‐5), also present in mouse IL‐5 (mIL‐5). hIL‐5 and mIL‐5 were labelled fluorescently, and nuclear uptake subsequent to membrane binding and internalisation by intact receptor expressing cells visualised and quantified using confocal laser scanning microscopy. hIL‐5 and mIL‐5 were shown to be transported to the nucleus in in vivo and in vitro nuclear protein import assays. The hIL‐5 NLS was able to target a heterologous protein to the nucleus both in vivo and in vitro. Mutations within the proximal arm of the NLS abrogated nuclear targeting activity, confirming its bipartite nature. The results imply a nuclear signalling role for IL‐5 additional to pathways linked to the membrane receptor system.
Mutagenesis of two N‐terminal Thr and five Ser residues in HslV, the proteolytic component of the ATP‐dependent HslVU protease
Authors: Yoo Soon Ji , Shim Yoon Kyung , Seong Ihn Sik , Seol Jae Hong , Kang Man-Sik , Chung Chin Ha
Published: July 2002

HslVU in E. coli is a new type of ATP‐dependent protease consisting of two heat shock proteins: the HslU ATPase and the HslV peptidase that has two repeated Thr residues at its N terminus, like certain β‐type subunit of the 20S proteasomes. To gain an insight into the catalytic mechanism of HslV, site‐directed mutagenesis was performed to replace each of the Thr residues with Ser or Val and to delete the first or both Thr. Also each of the five internal Ser residues in HslV were replaced with Ala. The results obtained by the mutational analysis revealed that the N‐terminal Thr acts as the active site nucleophile and that certain Ser residues, particularly Ser124 and Ser172, also contribute to the peptide hydrolysis by the HslVU protease. The mutational studies also revealed that both Thr, Ser103, and Ser172, but not Ser124, are involved in the interaction of HslV with HslU and hence in the activation of HslU ATPase as well as in the HslVU complex formation.
Cloning and analysis of a novel human putative DNA methyltransferase
Authors: Van den Wyngaert Ilse , Sprengel Jörg , Kass Stefan U. , Luyten Walter H.M.L.
Published: July 2002

DNA methylation is intricately involved in a variety of cellular processes, such as differentiation, cell cycle progression, X‐chromosome inactivation and genomic imprinting. However, little is known about how specific DNA methylation patterns are established and maintained. Previously one mammalian DNA methyltransferase has been described, but there has been considerable speculation about the presence of a second activity capable of methylation. Here we report the identification and characterization of a novel human putative DNA methyltransferase. Using a bioinformatics screen we have identified several expressed sequence tags which show high sequence similarity to the Schizosaccharomyces pombe gene pmt1+. The cDNA for PuMet (for putative DNA methyltransferase) was cloned and the predicted amino acid sequence deduced. The gene is ubiquitously expressed, albeit at low levels. Like several other DNA methyltransferases, the bacterially overexpressed protein is not active in methylation assays.
Functional coupling between human E‐type Ca2+ channels and μ opioid receptors expressed in Xenopus oocytes
Authors: Ottolia Michela , Platano Daniela , Qin Ning , Noceti Francesca , Birnbaumer Mariel , Toro Ligia , Birnbaumer Lutz , Stefani Enrico , Olcese Riccardo
Published: July 2002

Neuronal α1E Ca2+ channels were expressed in Xenopus laevis oocytes alone and in combination with the μ opioid receptor. Macroscopic currents were recorded under voltage clamp conditions. The stimulation of the morphine receptor by the synthetic [d‐Ala2,N‐Me‐Phe4,Gly‐ol5] enkephalin (DAMGO) produced a 20% reduction in the α1E ionic current. This effect was associated with a large change in the decay phase of the Ba2+ current. The effect of 1 μM DAMGO was fully antagonized by the universal μ opioid receptor antagonist naloxone and by the selective antagonist β‐funaltrexamine. The ionic current inhibition induced by DAMGO was partially recovered by preceding strong depolarizations. The injection of the catalytic subunit of pertussis toxin (A‐protomer) abolished the effect of DAMGO, suggesting the involvement of a GTP binding protein in the α1E modulation. The coexpression of the regulatory β2a Ca2+ channel subunit, together with the α1E subunit and the μ opioid receptor, prevented the reduction of the ionic current following the receptor stimulation with DAMGO, whereas the coexpression with the β3 subunit reduced by ∼50% the modulatory effect of DAMGO. The effect produced by the stimulation of the opioid receptor could be mimicked by coexpressing the α1E channel with the G‐protein βγ subunits.
Caspases disrupt mitochondrial membrane barrier function
Authors: Marzo Isabel , Susin Santos A , Petit Patrice X , Ravagnan Luigi , Brenner Catherine , Larochette Nathanael , Zamzami Naoufal , Kroemer Guido
Published: July 2002

Mitochondrial intermembrane proteins including cytochrome c are known to activate caspases. Accordingly, a disruption of the mitochondrial membrane barrier function with release of cytochrome into the cytosol has been shown to precede caspase activation in a number of different models of apoptosis. Here, we addressed the question of whether caspases themselves can affect mitochondrial membrane function. Recombinant caspases were added to purified mitochondria and were found to affect the permeability of both mitochondrial membranes. Thus, caspases cause a dissipation of the mitochondrial inner transmembrane potential. In addition, caspases cause intermembrane proteins including cytochrome c and AIF (apoptosis‐inducing factor) to be released through the outer mitochondrial membrane. These observations suggest that caspases and mitochondria can engage in a circular self‐amplification loop. An increase in mitochondrial membrane permeability would cause the release of caspase activators, and caspases, once activated, would in turn increase the mitochondrial membrane permeability. Such a self‐amplifying system could accelerate the apoptotic process and/or coordinate the apoptotic response between different mitochondria within the same cell.
Identification of a structural requirement for thyroid Na+/I− symporter (NIS) function from analysis of a mutation that causes human congenital hypothyroidism
Authors: Levy Orlie , Ginter Christopher S , De la Vieja Antonio , Levy Daniel , Carrasco Nancy
Published: July 2002

Patients with congenital lack of I− transport do not accumulate I− in their thyroids, often resulting in severe hypothyroidism. A single amino acid substitution in the thyroid Na+/I− symporter (NIS), proline replacing threonine at position 354 (T354P), was recently identified as the cause of this condition in two independent patients [1, 2]. Here we report that the lack of I− transport activity in T354P NIS generated by site‐directed mutagenesis, is not due to a structural change induced by proline, but rather to the absence of a hydroxyl group at the β‐carbon of the amino acid residue at position 354. Hence, this hydroxyl group is essential for NIS function.
The neurotransmitter role of diadenosine polyphosphates
Authors: Miras-Portugal M.Teresa , Gualix Javier , Pintor Jesús
Published: July 2002

Diadenosine polyphosphates present at the cytosol can be transported to secretory granules allowing their exocytotic release. Extracellularly, they can act through specific metabotropic or ionotropic receptors, or as analogues of P2X and P2Y nucleotide receptors. The specific ionotropic receptor P4 is present in synaptic terminals, and modulated by protein kinases (PK) A and C and protein phosphatases. Activation of PKA or PKC, directly or through membrane receptors, results in a decrease of affinity or in reduction of the Ca2+ transient respectively. Adenosine and ATP, both products of the extracellular destruction of diadenosine polyphosphates, acting through A1 or P2Y receptors respectively, are important physiological modulators at the P4 receptor.
Excitation delocalization in the bacteriochlorophyll c antenna of the green bacterium Chloroflexus aurantiacus as revealed by ultrafast pump‐probe spectroscopy
Authors: Savikhin Sergei , Buck Daniel R , Struve Walter S , Blankenship Robert E , Taisova Alexandra S , Novoderezhkin Vladimir I , Fetisova Zoya G
Published: July 2002

Room temperature absorption difference spectra were measured on the femtosecond through picosecond time scales for chlorosomes isolated from the green bacterium Chloroflexus aurantiacus. Anomalously high values of photoinduced absorption changes were revealed in the BChl c Qy transition band. Photoinduced absorption changes at the bleaching peak in the BChl c band were found to be 7–8 times greater than those at the bleaching peak in the BChl a band of the chlorosome. This appears to be the first direct experimental proof of excitation delocalization over many BChl c antenna molecules in the chlorosome.
Inhibition of Na,K‐ATPase activity by cGMP is isoform‐specific in brain endothelial cells
Authors: Pontiggia Luca , Winterhalter Kaspar , Gloor Sergio M
Published: July 2002

cGMP has been shown to either activate or inhibit Na,K‐ATPase activity. Using mouse brain endothelial cells which express both ouabain‐resistant α1 and ouabain‐sensitive α2 and α3 isoforms, we show that cGMP reduces total Na,K‐ATPase activity to about 58%. The inhibition is prevented by the protein kinase G (PKG)‐specific inhibitor KT5823, indicating that cGMP‐mediated activation of PKG leads to inhibition of the pump. A similar extent of inhibition is obtained with nitric oxide. cGMP‐induced inhibition acts mainly on α1 isoforms but hardly affects α2/α3 isoforms. These data suggest that inhibition of Na,K‐ATPase activity by cGMP occurs in an isoform‐selective manner in brain endothelial cells.
Cytokine‐inducible SH2 protein (CIS3) and JAK2 binding protein (JAB) abolish prolactin receptor‐mediated STAT5 signaling
Authors: Helman Daniel , Sandowski Yael , Cohen Yael , Matsumoto Akira , Yoshimura Akihiko , Merchav Shoshana , Gertler Arieh
Published: July 2002

The ability of five members of the cytokine‐inducible SH2 protein family (CIS1–4) and JAK2 binding (JAB) protein to affect prolactin receptor (PRLR)‐mediated activity was tested in human 293 embryonic kidney fibroblasts transiently transfected with rat PRLR, five concentrations of CIS/JAB Myc‐tagged cDNAs and a STAT5‐responsive reporter gene encoding luciferase. The protein expressions of CIS1, CIS2, CIS3 and JAB were comparable, whereas the level of CIS4 was slightly lower. PRLR‐mediated luciferase activity was abolished in a dose‐dependent manner in cells transfected with cDNA of CIS3 or JAB, even at concentrations below the level of protein detection by anti‐Myc antibody. In contrast, CIS1, CIS2 and CIS4 had little or no effect, despite similar levels of expression. CIS1 expression in postpartum mouse mammary glands was high and changed little in the course of 3 days. CIS2 and CIS3 expression was also high and increased further, whereas JAB expression was very low. These results hint that at least in mammary gland CIS3 is likely the main physiological negative regulator of the PRLR‐mediated JAK2/STAT5 pathway.
Dopamine D2 receptor dimers in human and rat brain
Authors: Zawarynski Paul , Tallerico Teresa , Seeman Philip , Lee Samuel P , O'Dowd Brian F , George Susan R
Published: July 2002

In order to determine whether dimers of dopamine D2 receptors can occur in mammalian brain, rat and human brain striatal membranes were photolabelled with two radioactive photoaffinity compounds selective for dopamine D2 receptors, [125I]azidophenethylspiperone and [125I]‐4‐azido‐5‐iodonemonapride. It was found that [125I]azidophenethylspiperone only labelled the D2 monomer, while [125I]‐4‐azido‐5‐iodonemonapride labelled both D2 monomers and dimers, despite the fact that very high concentrations (6 nM) of both radiocompounds were used. In addition, human cloned D2 receptors were probed with a D2‐specific antibody, revealing multiple bands indicating the existence of trimers, tetramers and pentamers of D2 receptors. The different D2‐binding patterns of the spiperone and benzamide congeners may explain the different densities of dopamine D2 receptors found with these two radioligands in human brain positron tomography in health and disease.
Specific sequence‐directed anti‐bilitranslocase antibodies as a tool to detect potentially bilirubin‐binding proteins in different tissues of the rat
Authors: Battiston Lucia , Macagno Annalisa , Passamonti Sabina , Micali Fulvio , Luigi Sottocasa Gian
Published: July 2002

The hypothesis that the uneven distribution of bilirubin in the organism, which occurs in hyperbilirubinemia, could reflect an uneven distribution of bilirubin‐binding proteins was tested by searching for peptides containing the bilirubin‐binding motif identified in bilitranslocase (Battiston et al., 1998). In the rat, positive proteins bands were found to be present only in the liver, gastric mucosa and central nervous system. The electrophoretic mobilities of the positive compounds in the liver and stomach were identical to that of purified bilitranslocase (38 kDa). In the brain, on the contrary, two peptides were found with molecular masses of 79 and 34 kDa, respectively. Their distribution pattern in the central nervous system was different for each of them.
Persistence of botulinum neurotoxin action in cultured spinal cord cells1,2
Authors: Keller James E , Neale Elaine A , Oyler George , Adler Michael
Published: July 2002

Primary dissociated fetal mouse spinal cord cultures were used to study the mechanisms underlying the differences in persistence of botulinum neurotoxin A (BoNT/A) and botulinum neurotoxin/E (BoNT/E) activities. Spinal cord cultures were exposed to BoNT/A (0.4 pM) for 2–3 days, which converted approximately half of the SNAP‐25 to an altered form lacking the final nine C‐terminal residues. The distribution of toxin‐damaged to control SNAP‐25 remained relatively unchanged for up to 80 days thereafter. Application of a high concentration of BoNT/E (250 pM) either 25 or 60 days following initial intoxication with BoNT/A converted both normal and BoNT/A‐truncated SNAP‐25 into a single population lacking the final 26 C‐terminal residues. Excess BoNT/E was removed by washout, and recovery of intact SNAP‐25 was monitored by Western blot analysis. The BoNT/E‐truncated species gradually diminished during the ensuing 18 days, accompanied by the reappearance of both normal and BoNT/A‐truncated SNAP‐25. Return of BoNT/A‐truncated SNAP‐25 was observed in spite of the absence of BoNT/A in the culture medium during all but the first 3 days of exposure. These results indicate that proteolytic activity associated with the BoNT/A light chain persists inside cells for >11 weeks, while recovery from BoNT/E is complete in <3 weeks. This longer duration of enzymatic activity appears to account for the persistence of serotype A action.
αCaMKII binding to the C‐terminal tail of NMDA receptor subunit NR2A and its modulation by autophosphorylation
Authors: Gardoni F. , Schrama L.H. , van Dalen J.J.W. , Gispen W.H. , Cattabeni F. , Di Luca M.
Published: July 2002

Ca2+/calmodulin‐dependent protein kinase II (CaMKII), a multifunctional, widely distributed enzyme, is enriched in post‐synaptic densities (PSDs). Here, we demonstrate that CaMKII binds to a discrete C‐terminal region of the NR2A subunit of NMDA receptors and promotes the phosphorylation of a Ser residue of this NMDA receptor subunit. Glutathione S‐transferase (GST)‐NR2A(1349‐1464) binds native CaMKII from solubilised hippocampal PSDs in ‘pull‐out’ and overlay experiments and this binding is competed by recombinant αCaMKII(1‐315). The longer GST‐NR2A(1244‐1464), although containing the CaMKII phosphosite Ser‐1289, binds the kinase with a lower efficacy. CaMKII association to NR2A(1349‐1464) is positively modulated by kinase autophosphorylation in the presence of Ca2+/calmodulin. These data provide direct evidence for a mechanism modulating the synaptic strength.
Long‐range electrostatic effects on peptide folding
Authors: Åqvist Johan
Published: July 2002

The reversible folding/unfolding of a short peptide in solution is studied by molecular dynamics simulations. The effects of long‐range electrostatic interactions are examined and found to be important both for the equilibrium between folded and unfolded states and the dynamics of the folding process. The neglect of long‐range electrostatics leads to an increased population of unfolded states and increased structural fluctuations. When such interactions are taken into account, the peptide unfolds and folds to the experimentally determined structure several times during a 25 ns trajectory, with approximately equal populations of folded and unfolded states in the neighborhood of its proposed melting temperature. The effect of using spherical boundary conditions rather than periodic ones does not appear to have any major effect on the folding dynamics.
An intronic polymorphic domain often associated with susceptibility to affective disorders has allele dependent differential enhancer activity in embryonic stem cells
Authors: Fiskerstrand C.E. , Lovejoy E.A. , Quinn J.P.
Published: July 2002

Variable number tandem repeats (VNTR) within non‐coding regions of a number of genes have been correlated with susceptibility to various disease states. In particular, a VNTR polymorphism of a 16 or 17 bp element within intron 2 of the human serotonin transporter gene has been correlated with a predisposition to affective disorders. We have demonstrated that this region will support differential levels of reporter gene expression in differentiating embryonic stem cells, this being dependent on the presence of 10 or 12 copies of the repeat. The VNTR domain can therefore act as a transcriptional regulator, a property which potentially contributes to disease susceptibility.
DNA methylation and chromosome instability in breast cancer cell lines
Authors: Vilain Armelle , Vogt Nicolas , Dutrillaux Bernard , Malfoy Bernard
Published: July 2002

We show that in a series of eight breast cancer cell lines, a direct relationship exists between the overall DNA demethylation and the percentage of rearranged chromosomes, except for cell lines with a highly rearranged genome which can be weakly demethylated. A real time fluorescent detection method was used to quantify by reverse transcription‐PCR the expression of the DNA methyltransferase 1 and of the newly discovered DNA demethylase. The overall DNA methylation status seems to result from a complex interplay between the expression of these two genes. Our results suggest that in these tumor cells, the overall DNA demethylation is implicated in one of the mechanisms at the origin of the genome instability and that besides the role of the DNA methyltransferase 1, that of the DNA demethylase may be essential in the control of DNA methylation.
Molecular cloning of a novel CC chemokine, interleukin‐11 receptor α‐locus chemokine (ILC), which is located on chromosome 9p13 and a potential homologue of a CC chemokine encoded by molluscum contagiosum virus
Authors: Ishikawa-Mochizuki Izumi , Kitaura Motoji , Baba Masataka , Nakayama Takashi , Izawa Dai , Imai Toshio , Yamada Hidekazu , Hieshima Kunio , Suzuki Ryuji , Nomiyama Hisayuki , Yoshie Osamu
Published: July 2002

Molluscum contagiosum virus (MCV) encodes a CC chemokine MC148R which is likely to have been acquired from the host. By a homology search employing MC148R as a probe, we have identified a novel CC chemokine whose gene exists next to the IL‐11 receptor α (IL‐11Rα) gene in both humans and mice. Thus, this chemokine maps to chromosome 9p13 in humans where IL‐11Rα has been assigned. We term this novel chemokine IL‐11Rα‐locus chemokine (ILC). ILC has the highest homology to MC148R among the known human CC chemokines. Furthermore, ILC is strongly and selectively expressed in the skin where infection of MCV also takes place. Thus, ILC is likely to be the original chemokine of MC148R.
Molecular cloning and biological activity of ecdysis‐triggering hormones in Drosophila melanogaster
Authors: Park Yoonseong , Zitnan Dusan , Gill Sarjeet S. , Adams Michael E.
Published: July 2002

Ecdysis‐triggering hormones (ETH) initiate a defined behavioral sequence leading to shedding of the insect cuticle. We have identified eth, a gene encoding peptides with ETH‐like structure and biological activity in Drosophila melanogaster. The open reading frame contains three putative peptides based on canonical endopeptidase cleavage and amidation sites. Two of the predicted peptides (DrmETH1 and DrmETH2) prepared by chemical synthesis induce premature eclosion upon injection into pharate adults. The promoter region of the gene contains a direct repeat ecdysteroid response element. Identification of eth in Drosophila provides opportunities for genetic manipulation of endocrine and behavioral events underlying a stereotypic behavior.
Maternal serum placental leucine aminopeptidase (P‐LAP)/oxytocinase and preterm delivery
Authors: Kozaki H , Itakura A , Okamura M , Ohno Y , Wakai K , Mizutani S
Published: July 2002

Objective: To test the applicability of maternal serum placental leucine aminopeptidase (P‐LAP) as a parameter for predicting preterm delivery. Patients and Methods: Maternal serum P‐LAP activity of 61 consecutive women with preterm labor at admission were assayed and examined to assess the relationship of levels to the incidence of preterm and term deliveries. Results: When P‐LAP activity of study patient serum was at or below the 10th percentile, we found a greater ratio of preterm delivery (P=0.0085) and an approximately 2.3‐fold increase risk. The sensitivity, specificity, positive predictive value and negative predictive value were 29, 97, 89 and 62%, respectively. Conclusion: Maternal serum P‐LAP activity decreases in cases with spontaneous preterm delivery and may be a clinically potential marker for some cases of preterm labor.
Transynaptic modulation by insulin‐like growth factor I of dendritic spines in Purkinje cells
Authors: Nieto‐bona M.P. , Garcia‐segura L.M. , Torres‐alemán I.
Published: July 2002

Purkinje cells synthesize insulin‐like growth factor I and express insulin‐like growth factor I receptors during their entire life. An additional source of insulin‐like growth factor I for these cells is provided by climbing fiber afferents originating in the inferior olive nucleus. Recently we found that insulin‐like growth factor I from the inferior olive is necessary for motor learning processes probably involving Purkinje cell synaptic plasticity. We now studied whether inferior olive insulin‐like growth factor I influences the synaptic structure of Purkinje cells, because changes in synaptic morphology are related to neuronal plasticity events. We injected an insulin‐like growth factor I antisense in the inferior olive of adult rats, a procedure which we previously found to elicit a significant and reversible decrease of insulin‐like growth factor I levels in the contralateral cerebellum. Ultrastructural analysis of the cerebellar cortex of these animals showed a significant reduction in the size of dendritic spines on Purkinje cells of antisense‐treated rats compared to controls. The decrease in spine size was linked to a diminished numerical density of dendritic spines on Purkinje cells, without affecting the numerical density of synapses in the molecular layer of the cerebellum. This reduction was not due to a change in the thickness of the molecular layer. Climbing or parallel fiber terminals were also unaffected.

Taken together with previous findings, these results support a role for insulin‐like growth factor I produced in the inferior olive in the maintenance of Purkinje cell synaptic plasticity.
Agony and ecstasy: a review of MDMA effects and toxicity
Authors: Burgess C. , O’Donohoe A. , Gill M.
Published: July 2002

Background

Background – Ecstasy is a recreational drug with an anecdotal reputation for safety. However, reports of adverse effects and fatalities have increased in the medical and popular press.

Methods

Method – Literature search and review.

Results

Results – Acute Ecstasy toxicity does not appear to be due to overdose and cannot be solely attributed to the nature of the usual ambient environment. Adverse effects include hyperthermia, seizures, cardiac arrhythmias, hepatotoxicity, hyponatraemia and many psychiatric disorders. Ecstasy causes serotonergic neurotoxicity in the brains of animals at doses close to those used by humans, but its long-term effect on the human brain is unknown.

Conclusions

Conclusion – Ecstasy toxicity should be considered in the differential diagnosis of a variety of medical and psychiatric conditions. Given its popularity, both the acute and the potential long-term effects are a cause for concern.
Prevalence of subthreshold forms of psychiatric disorders in persons making suicide attempts in Hungary
Authors: Balázs J. , Bitter I. , Lecrubier Y. , Csiszér N , Ostorharics G.
Published: July 2002

summary

Background– Suicide and suicide attempts have been associated to psychiatric illnesses; however, little is known about the role in suicide risk of those symptoms that do not meet the full criteria for a DSM-IV disorder. The aim of this study was to examine the prevalence of subthreshold psychiatric disorders among suicide attempters in Hungary.Methods– Using a modified structured interview (Mini International Neuropsychiatric Interview) determining 16 Axis I psychiatric diagnoses and their subthreshold forms defined by the DSM-IV and a semistructured interview collecting background information, the authors examined 140 consecutive suicide attempters, aged 18–65 years.Results– Eighty-three-point-six percent of the attempters had one or more current threshold diagnoses on Axis I and in addition more than three-quarters (78.6%) of the subjects had at least one subthreshold diagnosis. Six-point-four percent of the subjects (N = 9) had neither subthreshold nor threshold diagnoses at the time of their suicide attempts. Ten percent of the subjects (N = 14), not meeting the full criteria for any DSM-IV diagnoses, had at least one subthreshold diagnosis. In 68.6% of the subjects (N = 96), both subthreshold and threshold disorders were diagnosed at the time of their suicide attempts. The number of subthreshold and threshold diagnoses were positively and significantly related (χ2 = 5.12, df = 1,P < 0.05). Sixty-three-point-six percent of the individuals received two or more current threshold diagnoses on Axis I and 44.3% of the individuals (N = 62) had two or more subthreshold diagnoses at the time of their suicide attempts.Limitations– The subthreshold definitions in this study included only those forms of the disorders which required the same duration as the criteria DSM-IV disorder with fewer symptoms.Conclusions– Suicide attempts showed a very high prevalence of subthreshold disorders besides psychiatric disorders meeting the full criteria required according to the DSM-IV. Subthreshold forms of mental disorders need to be taken into account in suicide prevention.
Rheumatology Milestones 2.0: A Roadmap for Competency‐Based Medical Training of Rheumatology Fellows in the 21st Century
Authors: Liebowitz Jason E. , Torralba Karina D. , Kolfenbach Jason , Marston Bethany , Dua Anisha B. , O'Rourke Kenneth S. , McKown Kevin , Battistone Michael J. , Valeriano‐Marcet Joanne , Edgar Laura , McLean Sydney , Gouze Karen R. , Bolster Marcy B.
Published: November 2020

Objective

Since 2014, rheumatology fellows have been assessed not only based on their ability to provide patient care and possession of medical knowledge but also on their skill in serving as patient advocates, navigators of health systems, and members of a health care team. Such assessments have been carried out through the use of competency‐based milestones from the Accreditation Council of Graduate Medical Education (ACGME). However, a needs assessment has demonstrated interest in more context validity and subspecialty relevance since the development of the ACGME internal medicine (IM) subspecialty reporting milestones. The ACGME thus created a milestones working group, and the present study was undertaken to develop Rheumatology Milestones 2.0 as well as a supplemental guide to assist with implementation.

Methods

The working group, consisting of 7 rheumatology program directors, 2 division directors, a community practice rheumatologist, a rheumatology fellow in training, and a public member who is a rheumatology patient, was overseen by the ACGME vice president for milestones development and met through three 12‐hour, in‐person meetings to compose the rheumatology specialty milestones and supplemental guide within the ACGME Milestones 2.0 project.

Results

Informed by the needs assessment data and stakeholders, the working group revised and adapted the ACGME IM subspecialty reporting milestones to create a rheumatology‐specific set of milestones and a supplemental guide for their implementation.

Conclusion

The Rheumatology Milestones 2.0 provides a specialty‐specific, competency‐based evaluation tool that can be used by program directors, clinical competency committees, and others to assess the competencies of rheumatology fellows during training and help measure readiness for independent practice.
A Single‐Mode, Self‐Adapting, and Self‐Powered Mechanoreceptor Based on a Potentiometric–Triboelectric Hybridized Sensing Mechanism for Resolving Complex Stimuli
Authors: Wu Xiaodong , Zhu Juan , Evans James W. , Arias Ana C.
Published: November 2020

Human skin is equipped with slow adapting (SA) and fast adapting (FA) capabilities simultaneously. To mimic such functionalities, elaborately designed devices have been explored by integrating multiple sensing elements or adopting multimode sensing principles. However, the complicated fabrication, signal mismatch of different modules, complex operation, and high power‐consumption hinder their widespread applications. Here, a new type of single‐mode and self‐powered mechanoreceptor that can mimic both SA and FA via seamless fusion of complementary while compatible potentiometric and triboelectric sensing principles is reported. The resultant potentiometric–triboelectric hybridized mechanoreceptor exhibits distinctive features that are hard to achieve via currently existing methods, including single‐mode output (only voltage signal), greatly simplified operation (single‐measurement setup), ultralow power‐consumption (<1 nW), self‐adaptive response behavior, and good capability for resolving complex stimuli. Diverse mechanical characteristics, including magnitude, duration, frequency, applying and releasing speed, can be well interpreted with this single‐mode and self‐powered mechanoreceptor. Its promising application for monitoring object manipulations with a soft robotic gripper is explored. Furthermore, the versatility of the mechanoreceptor for resolving complex stimuli in diverse daily scenarios is demonstrated. This work presents a new design that will significantly simplify the fabrication/operation and meanwhile boost the functionality/energy‐efficiency of future electronic devices and smart systems.
Deterministic Assembly of Single Sub‐20 nm Functional Nanoparticles Using a Thermally Modified Template with a Scanning Nanoprobe
Authors: Meng Yan , Cheng Gang , Man Zaiqin , Xu Ya , Zhou Shuang , Bian Jie , Lu Zhenda , Zhang Weihua
Published: November 2020

A deterministic assembly technique for single sub‐20 nm functional nanoparticles is developed based on nanostructured templates fabricated by hot scanning nanoprobes. With this technique, single nanoparticles including quantum dots, polystyrene fluorescent nanobeads, and gold nanoparticles are successfully assembled into 2D arrays with high yields. Experimental and theoretical analyses show that the key for the high yields is the hot‐probe‐based template fabrication technique, which creates geometrical nanotraps and modifies their surface energy simultaneously. In addition to single nanoparticle patterning, further experiments demonstrate that this technique is also capable of building complex nanostructures, such as nanoparticle clusters with well‐defined shapes and heterogeneously integrated nanostructures consisting of quantum dots and silver nanowires. It opens the door to many important applications.