bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular mechanisms for mammalian melanogenesis
Authors: Sugumaran Manickam
Published: October 2002

Melanogenesis is an important biochemical process for the production of skin pigments which protect many animals from the damage of solar radiation. The abnormalities in melanogenesis are associated with albinism, vitiligo, as well as malignant melanoma in humans. In the lower forms of animals viz., insects, the exoskeleton is hardened to protect their soft bodies by a process called sclerotization, which is often accompanied by melanization. Recent advances in the biochemistry of sclerotization and melanization reveal remarkable similarity between these two processes. The seven stages of sclerotization are: (a) enzymatic oxidation of N‐acyldopamine, (b) Michael‐1,4‐addition reactions of N‐acyldopamine quinone, (c) tautomerization of quinone to quinone methide, (d) Michael‐1,6‐addition of quinone methides, (e) tautomerization of N‐acyldopamine quinone methide to 1,2‐dehydro‐N‐acyldopamine, (f) enzymatic oxidation of 1,2‐dehydro‐N‐acyldopamine, and (g) the reactions of resultant quinonoid compounds. Amazingly, striking similarities in the reaction sequences are found in the melanization process starting from dopa. These comparisons predict a central role for quinone methides as reactive intermediates during melanization. Accordingly, recent studies provide increasing evidence in favor of this proposition.
Time‐dependent and indirect effect of inorganic phosphate on force production in rat gastrocnemius exercising muscle determined by 31P‐MRS
Authors: Giannesini Benoı̂t , Izquierdo Marguerite , Confort-Gouny Sylviane , Cozzone Patrick J , Bendahan David
Published: October 2002

The relationship of inorganic phosphate (Pi) and its diprotonated form (H2PO4
−) to isometric force (F) was analyzed non‐invasively using 31P‐magnetic resonance spectroscopy. Rat gastrocnemius muscles were electrically stimulated at six different frequencies in order to produce different levels of fatigue. A curvilinear relationship was demonstrated between force production and [Pi] and [H2PO4
−] accumulation. [Pi] and [H2PO4
−] were correlated with F at the end of the stimulation period but not when F was maximal at the early stage of the stimulation period. Interestingly, the respective [Pi] and [H2PO4
−] did not differ significantly between these two stages demonstrating that [Pi] and [H2PO4
−] cannot be considered as direct effectors of fatigue. This time‐dependent and indirect effect of [Pi] and [H2PO4
−] on force production might be mediated by calcium ions.
Properties of the detergent solubilised cytochrome c oxidase (cytochrome cbb3) purified from Pseudomonas stutzeri
Authors: Urbani Andrea , Gemeinhardt Sabine , Warne Antony , Saraste Matti
Published: October 2002

Cytochrome cbb
3 is a cytochrome c‐oxidising isoenzyme that belongs to the superfamily of respiratory haem/copper oxidases. We have developed a purification method yielding large amounts of pure cbb
3 complex from the soil bacterium Pseudomonas stutzeri. This cytochrome cbb
3 complex consists of three subunits (ccoNOP) in a 1:1:1 stoichiometry and contains two b‐type and three c‐type haems. The protein complex behaves as a monomer with an overall molecular weight of 114.0±8.9 kDa and a s
0
20,w value of 8.9±0.3 S as determined by analytical ultracentrifugation. Crystals diffracting to 5.0 Å resolution have been grown by the vapour diffusion sitting drop method to an average size of 0.1×0.1×0.3 mm. This is the first crystallisation report of a (cbb
3)‐type oxidase.
Striatin, a calmodulin‐dependent scaffolding protein, directly binds caveolin‐1
Authors: Gaillard Stéphane , Bartoli Marc , Castets Francis , Monneron Ariane
Published: October 2002

Caveolins are scaffolding proteins able to collect on caveolae a large number of signalling proteins bearing a caveolin‐binding motif. The proteins of the striatin family, striatin, SG2NA, and zinedin, are composed of several conserved, collinearly aligned, protein–protein association domains, among which a putative caveolin‐binding domain [Castets et al. (2000) J. Biol. Chem. 275, 19970–19977]. They are associated in part with membranes. These proteins are mainly expressed within neurons and thought to act both as scaffolds and as Ca2+‐dependent signalling proteins [Bartoli et al. (1999) J. Neurobiol. 40, 234–243]. Here, we show that (1) rat brain striatin, SG2NA and zinedin co‐immunoprecipitate with caveolin‐1; (2) all are pulled down by glutathione‐S‐transferase (GST)–caveolin‐1; (3) a fragment of recombinant striatin containing the putative caveolin‐binding domain binds GST–caveolin‐1. Hence, it is likely that the proteins of the striatin family are addressed to membrane microdomains by their binding to caveolin, in accordance with their putative role in membrane trafficking [Baillat et al. (2001) Mol. Biol. Cell 12, 663–673].
ABCA3 is a lamellar body membrane protein in human lung alveolar type II cells1
Authors: Yamano Gen , Funahashi Hisayuki , Kawanami Oichi , Zhao Li-Xia , Ban Nobuhiro , Uchida Yoshiyuki , Morohoshi Toshio , Ogawa Junichi , Shioda Seiji , Inagaki Nobuya
Published: October 2002

The ABCA3 gene, of the ABCA subclass of ATP‐binding cassette (ABC) transporters, is expressed exclusively in lung. We report here the cloning, molecular characterization, and distribution of human ABCA3 in the lung. Immunoblot analysis using the specific antibody reveals a 150‐kDa protein in the crude membrane fraction of human lung. Immunohistochemical analyses of alveoli show that ABCA3 is expressed only in the type II cells expressing surfactant protein A. At the ultrastructural level, ABCA3 immunoreactivity was detected mostly at the limiting membrane of the lamellar bodies. Since members of the ABCA transporter family are known to be involved in transmembrane transport of endogenous lipids, our findings suggest that ABCA3 plays an important role in the formation of pulmonary surfactant in type II cells.
Cleavage of a tail‐anchored protein by signal peptidase
Authors: Nilsson IngMarie , Johnson Arthur E , von Heijne Gunnar
Published: October 2002

Tail‐anchored proteins are post‐translationally targeted and inserted into the endoplasmic reticulum membrane. They do not use the co‐translational signal‐recognition particle (SRP)‐dependent pathway, but rather utilize an ill‐defined, ATP‐dependent mechanism. Here, we show that a tail‐anchored protein can be cleaved by signal peptidase and that the sequence requirements for efficient cleavage seem to be the same as for cleavage of co‐translationally targeted SRP‐dependent proteins.
TGF‐β1 and Smad4 overexpression induce a less invasive phenotype in highly invasive spindle carcinoma cells
Authors: Santibáñez Juan Francisco , Quintanilla Miguel , Martı́nez Jorge
Published: October 2002

We have examined the effect of transforming growth factor β1 (TGF‐β1) and overexpression of the Smad4 gene on the phenotype of Car C, a ras mutated highly malignant spindle carcinoma cell line. TGF‐β1‐treated Car C cells overexpressing Smad4 spread with a flattened morphology with membrane ruffles abundant in vinculin and show a reduction in their invasive abilities. TGF‐β1 treatment and overexpression of Smad4 also enhanced the production of PAI‐1 measured by the activation of the p3TP‐lux reporter gene containing a PAI‐1‐related promoter. This activation was abolished with a dominant‐negative Smad4 construct. These results lead us to conclude that both TGF‐β1 and Smad4 overexpression reduce the invasive potential of Car C cells, probably via the Smad pathway.
Deletion of nine carboxy‐terminal residues of the Rubisco small subunit decreases thermal stability but does not eliminate function
Authors: Esquı́vel Maria G. , Anwaruzzaman M. , Spreitzer Robert J.
Published: October 2002

A recent X‐ray crystal structure of ribulose‐1,5‐bisphosphate carboxylase/oxygenase from the green alga Chlamydomonas reinhardtii lacks 13 carboxy‐terminal residues of the small subunit. To determine the importance of this divergent region, a non‐sense mutation was created that removes nine residues. This engineered gene was transformed into a Chlamydomonas strain that lacks the small‐subunit gene family. The resulting holoenzyme has a normal CO2/O2 specificity but decreased carboxylation V
max. Whereas wild‐type enzyme retained most of its carboxylase activity after a 10‐min incubation at 55°C, the mutant enzyme was inactivated. Thus, although disordered or divergent, the carboxy terminus is required for maximal activity and stability.
The functional specificity of NeuroD is defined by a single amino acid residue (N11) in the basic domain
Authors: Wang Xukun , Korzh Vladimir , Gong Zhiyuan
Published: October 2002

In zebrafish, the basic helix‐loop‐helix (bHLH) gene neuroD specifies distinct neurons in the spinal cord. A preliminary experiment indicated that a related bHLH gene, ndr1a, normally expressed only in the olfactory organ in late embryos, also functions as neuroD to induce ectopic formation of spinal cord neurons in early embryos after introduction of its mRNA into early embryos. To define the functional specificity of these bHLH proteins, several mutant forms with selected point mutations in the basic domain were constructed and tested for inducing sensory neurons in the spinal cord. Our data indicate that the functional specificity of NeuroD to define sensory neurons is mainly due to a single residue (asparagine 11) in its basic domain.
Flavonoids differentially regulate IFNγ‐induced ICAM‐1 expression in human keratinocytes: molecular mechanisms of action
Authors: Bito Toshinori , Roy Sashwati , Sen Chandan K. , Shirakawa Toshiro , Gotoh Akinobu , Ueda Masato , Ichihashi Masamitsu , Packer Lester
Published: October 2002

The effect of plant flavonoids on intercellular adhesion molecule‐1 (ICAM‐1) expression in human keratinocyte was investigated. ICAM‐1 is known to mediate skin inflammation. Among the flavonoids tested, taxifolin was the most potent in inhibiting interferon γ (IFNγ)‐induced ICAM‐1 protein as well as mRNA expression in human keratinocytes. Much smaller dosages of taxifolin were required in primary keratinocytes compared to HaCaT (immortalized cell) to achieve similar levels of inhibition in the inducible ICAM‐1 expression. Regulation of inducible ICAM‐1 expression by taxifolin was at transcriptional level by inhibiting the activation of signal transducers and activators of transcription (STAT)1 and protein tyrosine phosphorylation of Janus kinase (JAK)1 suggesting that the JAK–STAT pathway may be the molecular site of action of taxifolin. Finally, taxifolin pre‐treatment also potently inhibited IFNγ‐induced ICAM‐1 expression in a reconstructed human skin equivalent suggesting therapeutic potential of taxifolin in skin pathological conditions related to increased cell adhesion and inflammation.
First tau repeat domain binding to growing and taxol‐stabilized microtubules, and serine 262 residue phosphorylation
Authors: Devred François , Douillard Soazig , Briand Claudette , Peyrot Vincent
Published: October 2002

Tau phosphorylation plays a crucial role in microtubule stabilization and in Alzheimer's disease. To characterize the molecular mechanisms of tau binding on microtubules, we synthesized the peptide R1 (QTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQI), reproducing the first tau microtubule binding motif. We thermodynamically characterized the molecular mechanism of tubulin assembly with R1 in vitro, and measured, for the first time, the binding parameters of R1 on both growing and taxol‐stabilized microtubules. In addition, we obtained similar binding parameters with R1 phosphorylated on Ser262. These data suggest that the consequences of Ser262 phosphorylation on tau binding to microtubules and on tubulin assembly are due to large intramolecular rearrangements of the tau protein.
Evidence for non‐isostructural replacement of Zn2+ with Cd2+ in the β‐domain of brain‐specific metallothionein‐3
Authors: Palumaa Peep , Njunkova Olga , Pokras Lesja , Eriste Elo , Jörnvall Hans , Sillard Rannar
Published: October 2002

Metallothionein‐3 (MT‐3) is a brain‐specific MT, which is downregulated in Alzheimer's disease. The N‐terminal region of CdMT‐3 is highly dynamic and has escaped structural characterization by nuclear magnetic resonance. We have used electrospray ionization mass spectrometry to probe conformational states of cadmium‐ and zinc‐substituted metalloforms of MT‐3 and can demonstrate that the N‐terminal β‐domain of MT‐3 filled with Cd2+ has a more open conformation than that filled with Zn2+. The results suggest that the larger Cd2+ ions cannot isostructurally replace zinc in the β‐domain of MT‐3 whereas in the case of MT‐1 and MT‐2 the replacement is isostructural. Specific metal binding properties of the β‐domain of MT‐3 may be essential for fulfilling the specific role of MT‐3 in the brain.
Protection against experimental malaria associated with AMA‐1 peptide analogue structures
Authors: Salazar Luz M , Alba Martha P , Torres Maria H , Pinto Martha , Cortes Ximena , Torres Libardo , Patarroyo Manuel E
Published: October 2002

One Plasmodium falciparum malaria antigen is an integral membrane protein called apical membrane antigen‐1. High activity binding peptides to human red blood cells have been identified in this protein. 4337 is a conserved, non‐immunogenic peptide with high activity red blood cell binding and its critical residues have already been identified. Peptide analogues (with amino acids having the same mass but different charge) were generated to change their immunogenic and protective characteristics. Three analogues having positive or negative immunological results were studied by nuclear magnetic resonance. The studied peptides all had an α‐helix fragment, but in different peptide regions and extensions, except for randomly structured 4337. We show that altering a few amino acids induced immunogenicity and protectivity against experimental malaria and changed their three‐dimensional structure, suggesting a better fit with immune system molecules and that modified peptides having better immunological properties can be included in the design of new malaria multi‐component subunit‐based vaccine.
Type I chaperonins: not all are created equal
Authors: Levy-Rimler Galit , Bell Rachel E , Ben-Tal Nir , Azem Abdussalam
Published: October 2002

Type I chaperonins play an essential role in the folding of newly translated and stress‐denatured proteins in eubacteria, mitochondria and chloroplasts. Since their discovery, the bacterial chaperonins have provided an excellent model system for investigating the mechanism by which chaperonins mediate protein folding. Due to the high conservation of the primary sequence among Type I chaperonins, it is generally accepted that organellar chaperonins function similar to the bacterial ones. However, recent studies indicate that the chloroplast and mitochondrial chaperonins possess unique structural and functional properties that distinguish them from their bacterial homologs. This review focuses on the unique properties of organellar chaperonins.
Chlamydomonas, a model system for studying the assembly and dynamics of photosynthetic complexes
Authors: Rochaix Jean-David
Published: October 2002

The green unicellular alga Chlamydomonas reinhardtii has emerged as a powerful model system for studying the biosynthesis of the photosynthetic apparatus and the acclimation of this system to changes in light conditions. The assembly of the photosynthetic complexes involves the coordinate interaction between the nuclear and chloroplast genetic systems. Many factors involved in specific chloroplast post‐transcriptional steps have been identified and characterized. Chlamydomonas is able to adapt to changes in light quality and in cellular ATP content by performing state transition, a process that leads to a redistribution of light excitation energy between photosystem II and photosystem I and that involves the redox state of the plastoquinone pool, the cytochrome b
6
f complex and one or several kinases specific for the light‐harvesting system. Genetic approaches have provided new insights into this process.
Transport of protein toxins into cells: pathways used by ricin, cholera toxin and Shiga toxin
Authors: Sandvig Kirsten , van Deurs Bo
Published: October 2002

Ricin, cholera, and Shiga toxin belong to a family of protein toxins that enter the cytosol to exert their action. Since all three toxins are routed from the cell surface through the Golgi apparatus and to the endoplasmic reticulum (ER) before translocation to the cytosol, the toxins are used to study different endocytic pathways as well as the retrograde transport to the Golgi and the ER. The toxins can also be used as vectors to carry other proteins into the cells. Studies with protein toxins reveal that there are more pathways along the plasma membrane to ER route than originally believed.
Iron transport and signaling in Escherichia coli
Authors: Braun Volkmar , Braun Michael
Published: October 2002

Bacteria solve the iron supply problem caused by the insolubility of Fe3+ by synthesizing iron‐complexing compounds, called siderophores, and by using iron sources of their hosts, such as heme and iron bound to transferrin and lactoferrin. Escherichia coli, as an example of Gram‐negative bacteria, forms sophisticated Fe3+–siderophore and heme transport systems across the outer membrane. The crystal structures of three outer membrane transport proteins now allow insights into energy‐coupled transport mechanisms. These involve large long‐range structural transitions in the transport proteins in response to substrate binding, including substrate gating. Energy is provided by the proton motive force of the cytoplasmic membrane through the activity of a protein complex that is inserted in the cytoplasmic membrane and that contacts the outer membrane transporters. Certain transport proteins also function in siderophore‐mediated signaling cascades that start at the cell surface and flow to the cytoplasm to initiate transcription of genes encoding proteins for transport and siderophore biosynthesis.
Genetic effects on an animal model of anxiety
Authors: Flint Jonathan
Published: October 2002

Genetic effects on behavioural measures thought to model anxiety have been reported on 15 mouse chromosomes. In general the individual effect from each locus is small, contributing to 10% or less of the total variation, but through use of crosses between inbred rodents the power to detect such effects is high: 39 loci have been reported at stringent levels of significance. Novel multivariate analyses of these data go some way to characterizing the genetic architecture of anxiety and also to validating the tests that are used for its measurement. However, we are still some way from finding the molecular variants that explain the heritability of the trait.
Involvement of Elk‐1 in L6E9 skeletal muscle differentiation
Authors: Khurana Ashwani , Dey Chinmoy S
Published: October 2002

In L6E9 skeletal muscle cells ternary complex factor (TCF) Elk‐1 expression increased with the onset of skeletal muscle differentiation, whereas its activation decreased as a function of differentiation. Its expression was predominantly restricted to cytoplasm and activated ones were predominantly restricted to the nucleus of the differentiated cells. Inhibition of ERK‐1/‐2 activities by PD098059 resulted into significant reduction in Elk‐1 expression and phosphorylation during differentiation. In contrast, inhibition of p38 mitogen‐activated protein kinase (MAPK) enhanced Elk‐1 expression and activation, thereby mediating inhibition of skeletal muscle differentiation. Overexpression of inactive mutant Elk‐1 enhanced differentiation. Data suggest that ERK‐1/‐2 and p38 MAPK activities modulate Elk‐1 expression and activation to regulate skeletal muscle differentiation.
Identification of a caspase 3‐independent role of pro‐apoptotic factor Bak in TNF‐α‐induced apoptosis
Authors: Suyama Eigo , Kawasaki Hiroaki , Taira Kazunari
Published: October 2002

By using our recently developed gene discovery system, we have identified Bak, a member of the Bcl‐2 family, as a pro‐apoptotic factor in the tumor necrosis factor (TNF)‐α‐induced apoptotic pathway in caspase 3‐deficient cells. Unlike Bcl‐2, Bak stimulates several apoptotic pathways, however the molecular mechanism(s) of its action remains unclear. For example, it is unclear whether Bak induces apoptosis in caspase 3‐deficient cells. In this study, we examined the effects of overexpression of Bak in MCF‐7 cells that lack caspase 3. We found that despite the absence of caspase 3 in MCF‐7 cells, they were more sensitive to the cell death effects of Bak as compared to caspase 3‐expressing HeLa S3 cells. The targeting of Bak function by ribozymes suggests that Bak is required for the TNF‐α‐induced apoptotic pathway in caspase 3‐deficient cells. This study demonstrates the caspase 3‐independent function of Bak in the TNF‐α‐induced apoptotic pathway.
Stepwise rotation of the γ‐subunit of EF0F1‐ATP synthase observed by intramolecular single‐molecule fluorescence resonance energy transfer1
Authors: Börsch Michael , Diez Manuel , Zimmermann Boris , Reuter Rolf , Gräber Peter
Published: October 2002

The EF0F1‐ATP synthase mutants bQ64C and γT106C were labelled selectively with the fluorophores tetramethylrhodamine (TMR) at the b‐subunit and with a cyanine (Cy5) at the γ‐subunit. After reconstitution into liposomes, these double‐labelled enzymes catalyzed ATP synthesis at a rate of 33 s−1. Fluorescence of TMR and Cy5 was measured with a confocal set‐up for single‐molecule detection. Photon bursts were detected, when liposomes containing one enzyme traversed the confocal volume. Three states with different fluorescence resonance energy transfer (FRET) efficiencies were observed. In the presence of ATP, repeating sequences of those three FRET‐states were identified, indicating stepwise rotation of the γ‐subunit of EF0F1.
Desolvation shell of hydrogen bonds in folded proteins, protein complexes and folding pathways
Authors: Fernández Ariel
Published: October 2002

A few backbone hydrogen bonds (HBs) in native protein folds are poorly protected from water attack: their desolvation shell contains an inordinately low number of hydrophobic residues. Thus, an approach by solvent‐structuring moieties of a binding partner should contribute significantly to enhance their stability. This effect represents an important factor in the site specificity inherent to protein binding, as inferred from a strong correlation between poorly desolvated HBs and binding sites. The desolvation shells were also examined in a dynamic context: except for a few singular under‐protected bonds, the size of desolvation shells is preserved along the folding trajectory.
Toxicity evolution of Vipera aspis aspis venom: identification and molecular modeling of a novel phospholipase A2 heterodimer neurotoxin1
Authors: Jan Virginie , Maroun R.C , Robbe-Vincent Annie , De Haro Luc , Choumet Valérie
Published: October 2002

We report the simultaneous presence of two phospholipase A2 (PLA2) neurotoxins in the venom of Vipera aspis aspis, the first such observation. One is monomeric and identical to ammodytoxin B of Vipera ammodytes ammodytes. Its presence may result from gene flux after interbreeding between V. aspis aspis and V. ammodytes ammodytes. The second, a novel heterodimer named vaspin, is very similar to vipoxin of Vipera ammodytes meridionalis and to PLA2‐I of Vipera aspis zinnikeri. It may result from expression of preexisting genes, the acidic subunit evolving from an ancestor common to ammodytin I2 from V. ammodytes ammodytes, which we also found in V. aspis aspis.
Silencing of Bruton's tyrosine kinase (Btk) using short interfering RNA duplexes (siRNA)
Authors: Heinonen Juhana E. , Smith C.I.Edvard , Nore Beston F.
Published: October 2002

Tec family tyrosine kinases, Bruton's tyrosine kinase (Btk), Itk, Bmx, Tec, and Txk, are multi‐domain proteins involved in hematopoietic signaling. Here, we demonstrate that human Btk protein can transiently be depleted using double‐stranded short RNA interference (siRNA) oligonucleotides. Imaging and Western blotting analysis demonstrate that Btk expression is down regulated in heterologous systems as well as in hematopoietic lineages, following transfection or microinjection of Btk siRNA duplexes. The induction of histamine release, a pro‐inflammatory mediator, in RBL‐2H3 mast cells was reduced by 20–25% upon Btk down regulation. Similar, results were obtained when the Btk activity was inhibited using the kinase blocker LFM‐A13. These results demonstrate a direct role of Btk for the efficient secretion of histamine in allergic responses.
A single nucleotide polymorphism in the alternative oxidase gene among rice varieties differing in low temperature tolerance
Authors: Abe Fumitaka , Saito Koji , Miura Kiyoyuki , Toriyama Kinya
Published: October 2002

Alternative oxidase (AOX) is encoded in a multigene family, and multiple isoforms have been observed in various plant species. We found for the first time an allelic variation in the same AOX locus. On SDS–gel blots of callus protein of rice (Oryza sativa L.), varieties without the QTL for low temperature tolerance showed a 32‐kDa AOX band, whereas those with the QTL showed a 34‐kDa band. The variation was attributed to the substitution of Lys71 for Asn71 caused by a single nucleotide polymorphism between alleles of OsAOX1a, and was tightly linked to the presence of the QTL.
Inactivation of cellular caspases by peptide‐derived tryptophan and tyrosine peroxides
Authors: Hampton Mark B , Morgan Philip E , Davies Michael J
Published: October 2002

Peroxides generated on peptides and proteins within cells, as a result of radical attack or reaction with singlet oxygen, are longer‐lived than H2O2due to their poor removal by protective enzymes. These peroxides readily oxidize cysteine residues and can inactivate thiol‐dependent enzymes. We show here that Trp‐ and Tyr‐derived peptide peroxides, generated by singlet oxygen, inhibit caspase activity in the lysates of apoptotic Jurkat cells.N‐Ac‐Trp‐OMe peroxide was the most effective inhibitor, and was 30‐fold more effective than H2O2under identical conditions. As such, protein peroxides could modulate the progression of apoptosis in cells in which they are generated.
Caspase activation in etoposide‐treated fibroblasts is correlated to ERK phosphorylation and both events are blocked by polyamine depletion
Authors: Stefanelli Claudio , Tantini Benedetta , Fattori Monia , Stanic’ Ivana , Pignatti Carla , Clo Carlo , Guarnieri Carlo , Caldarera Claudio M , Mackintosh Caroline A , Pegg Anthony E , Flamigni Flavio
Published: October 2002

Activation of the extracellular signal‐regulated kinases (ERKs) 1 and 2 is correlated to cell survival, but in some cases ERKs can act in signal transduction pathways leading to apoptosis. Treatment of mouse fibroblasts with 20 μM etoposide elicited a sustained phosphorylation of ERK 1/2, that increased until 24 h from the treatment in parallel with caspase activity. The inhibitor of ERK activation PD98059 abolished caspase activation, but caspase inhibition did not reduce ERK 1/2 phosphorylation, suggesting that ERK activation is placed upstream of caspases. Both ERK and caspase activation were blocked in cells depleted of polyamines by the ornithine decarboxylase inhibitor α‐difluoromethylornithine (DFMO). In etoposide‐treated cells, DFMO also abolished phosphorylation of c‐Jun NH2‐terminal kinases triggered by the drug. Polyamine replenishment with exogenous putrescine restored the ability of the cells to undergo caspase activation and ERK 1/2 phosphorylation in response to etoposide. Ornithine decarboxylase activity decreased after etoposide, indicating that DFMO exerts its effect by depleting cellular polyamines before induction of apoptosis. These results reveal a role for polyamines in the transduction of the death signal triggered by etoposide.
Selective elimination of glutamate activation and introduction of fluorescent proteins into a Caenorhabditis elegans chloride channel
Authors: Li Ping , Slimko Eric M , Lester Henry A
Published: October 2002

Glutamate‐gated chloride (GluCl) channels from invertebrates can be activated by ivermectin (IVM) to produce electrical silencing in mammalian neurons. To improve this GluCl/IVM strategy, we sought to mutate the Caenorhabditis elegans GluCl channels so that they become insensitive to glutamate but retain their sensitivity to IVM. Based on structure–function studies of nicotinic acetylcholine receptor superfamily members, we tested in oocytes 19 point mutants at 16 residues in the β‐subunit likely to be involved in the response to glutamate. Y182F reduces the glutamate response by greater than six‐fold, with little change to IVM responses, when coexpressed with wild‐type (WT) GluCl α. For GluCl αβ(Y182F), the EC50 and Hill coefficient for glutamate are similar to those of WT, indicating that the mutant decreases the efficacy of glutamate, but not the potency. Also, fluorescent proteins (enhanced green fluorescent protein, enhanced yellow fluorescent protein, enhanced cyan fluorescent protein; XFP) were inserted into the M3–M4 loop of the GluCl α, β and β(Y182F). We found no significant functional difference between these XFP‐tagged receptors and WT receptors. The modified GluCl channel, without glutamate sensitivity but with a fluorescent tag, may be more useful in GluCl silencing strategies.
The Tzs protein from Agrobacterium tumefaciens C58 produces zeatin riboside 5′‐phosphate from 4‐hydroxy‐3‐methyl‐2‐(E)‐butenyl diphosphate and AMP
Authors: Krall Lilian , Raschke Maja , Zenk Meinhart H. , Baron Christian
Published: October 2002

The plant pathogen Agrobacterium tumefaciens produces cytokinins upon induction of the virulence genes by secondary metabolites from wounded plants, and these hormones are believed to stimulate the infection process. To study the biosynthetic pathway, the tzs gene, encoding the Tzs (trans‐zeatin‐synthesizing) protein from A. tumefaciens, was cloned and the protein was overproduced and purified. Analysis of its reactivity with radioactively labeled substrate demonstrated conversion of 4‐hydroxy‐3‐methyl‐2‐(E)‐butenyl diphosphate, a product of the deoxyxylulose phosphate pathway, with AMP to zeatin riboside 5′‐phosphate. This suggests that A. tumefaciens uses an alternative pathway of cytokinin biosynthesis, which had previously been hypothesized to operate in plants.
Actin's propensity for dynamic filament patterning
Authors: Schoenenberger Cora-Ann , Bischler Nicolas , Fahrenkrog Birthe , Aebi Ueli
Published: October 2002

Actin, through its various forms of assembly, provides the basic framework for cell motility, cell shape and intracellular organization in all eukaryotic cells. Many other cellular processes, for example endocytosis and cytokinesis, are also associated with dynamic changes of the actin cytoskeleton. Important prerequisites for actin's functional diversity are its intrinsic ability to rapidly assemble and disassemble filaments and its spatially and temporally well‐controlled supramolecular organization. A large number of proteins that interact with actin, collectively referred to as actin‐binding proteins (ABPs), carefully orchestrate different scenarios. Since its isolation in 1994 [Machesky, L.M. et al. (1994) J. Cell Biol. 127, 107–115], the Arp2/3 complex containing the actin‐related proteins Arp2 and Arp3 has evolved to be one of the main players in the assembly and maintenance of many actin‐based structures in the cell (for review see [Borths, E.L. and Welch, M.D. (2002) Structure 10, 131–135; May, R.C. (2001) Cell Mol. Life Sci. 58, 1607–1626; Pollard, T.D. et al. (2000) Rev. Biophys. Biomol. Struct. 29, 545–576; Welch, M.D. (1999) Trends Cell Biol. 11, 423–427]). In particular, when it comes to the assembly of the intricate branched actin network at the leading edge of lamellipodia, the Arp2/3 complex seems to have received all the attention in recent years. In parallel, but not so much in the spotlight, several reports showed that actin on its own can assume different conformations [Bubb, M.R. et al. (2002) J. Biol. Chem. 277, 20999–21006; Schoenenberger, C.‐A. et al. (1999) Microsc. Res. Tech. 47, 38–50; Steinmetz, M.O. et al. (1998) J. Mol. Biol. 278, 793–811; Steinmetz, M.O. et al. (1997) J. Cell Biol. 138, 559–574; Millonig, R., Salvo, H. and Aebi, U. (1988) J. Cell Biol. 106, 785–796] through which it drives its supramolecular patterning, and which ultimately generate its functional diversity.
RecQ helicases: at the heart of genetic stability
Authors: Cobb Jennifer A , Bjergbaek Lotte , Gasser Susan M
Published: October 2002

The checkpoint‐mediated control of DNA replication is essential for maintaining the stability of the genome and preventing cancer in humans. The RecQ family of helicases has been shown to be important for the maintenance of genomic integrity in organisms ranging from bacteria to man. We propose that the RecQ homologue, Sgs1p, has an important function in the S‐phase checkpoint response of budding yeast, where it may be both a ‘sensor’ for damage during replication and a ‘resolvase’ for structures that arise at paused forks. RecQ helicases may serve a unique function that integrates checkpoint proteins with the recombination and replication fork machinery.
Direct identification of PTEN phosphorylation sites
Authors: Miller Susan J. , Lou David Y. , Seldin David C. , Lane William S. , Neel Benjamin G.
Published: October 2002

The PTEN tumor suppressor gene encodes a phosphatidylinositol 3′‐phosphatase that is inactivated in a high percentage of human tumors, particularly glioblastoma, melanoma, and prostate and endometrial carcinoma. Previous studies showed that PTEN is a seryl phosphoprotein and a substrate of protein kinase CK2 (CK2). However, the sites in PTEN that are phosphorylated in vivo have not been identified directly, nor has the effect of phosphorylation on PTEN catalytic activity been reported. We used mass spectrometric methods to identify Ser370 and Ser385 as in vivo phosphorylation sites of PTEN. These sites also are phosphorylated by CK2 in vitro, and phosphorylation inhibits PTEN activity towards its substrate, PIP3. We also identify a novel in vivo phosphorylation site, Thr366. Following transient over‐expression, a fraction of CK2 and PTEN co‐immunoprecipitate. Moreover, pharmacological inhibition of CK2 activity leads to decreased Akt activation in PTEN+/+ but not PTEN−/− fibroblasts. Our results contrast with previous assignments of PTEN phosphorylation sites based solely on mutagenesis approaches, suggest that CK2 is a physiologically relevant PTEN kinase, and raise the possibility that CK2‐mediated inhibition of PTEN plays a role in oncogenesis.
Nucleic acid aptamers in cancer medicine
Authors: Cerchia Laura , Hamm Jörg , Libri Domenico , Tavitian Bertrand , de Franciscis Vittorio
Published: October 2002

Many signalling proteins involved in diverse functions such as cell growth and differentiation can act as oncogenes and cause cellular transformation. These molecules represent attractive targets for cancer diagnosis or therapy and are therefore subject to intensive investigation. Aptamers are small nucleic acid molecules, isolated from combinatorial libraries by a procedure termed SELEX, that bind to a target molecule by providing a limited number of specific contact points embedded in a larger, defined three‐dimensional structure. In some cases aptamers have the potential to inhibit the biological function of the molecule resulting in useful reagents for target validation in a variety of disease models.
Cardiolipin: a proton trap for oxidative phosphorylation
Authors: Haines Thomas H , Dencher Norbert A
Published: October 2002

The role of specific lipid structures in biological membranes has been elusive. There are hundreds of them in nature. Why has nature made them? How do they aid in the functioning of membrane proteins? Genetics with its ‘knock out’ organisms declares that functions persist in the absence of any particular lipid. Nonetheless some lipids, such as cardiolipin (CL), are associated with particular functions in the cell. It may merely expand the variety of culture conditions (pH, temperature, etc.) under which the wild‐type organism survives. This article explores a unique role of CL as a proton trap within membranes that conduct oxidative phosphorylation and therefore the synthesis of ATP. CL's pK2 (above 8.0) provides a role for it as a headgroup proton trap for oxidative phosphorylation. It suggests why CL is found in membranes that pump protons. The high pK2 also indicates that the headgroup has but one negative charge in the neutral pH range. Data on the binding of CL to all of the oxidative phosphorylation proteins suggest that the CL may aggregate the oxidative phosphorylation proteins into a patch while it restricts pumped protons within its headgroup domain – supplying protons to the ATP synthase with minimal changes in the bulk phase pH.
P14ARF promotes accumulation of SUMO‐1 conjugated (H)Mdm2
Authors: Xirodimas Dimitris P , Chisholm June , Desterro Joana M.S , Lane David P , Hay Ronald T
Published: October 2002

p14ARF tumour suppressor stabilises and activates p53 by directly interacting with (H)Mdm2 [(human) murine double minute 2 homologue] and inhibiting its E3 ubiquitin ligase activity. Here we demonstrate that p14ARF promotes accumulation of (H)Mdm2 conjugated to the small ubiquitin‐like protein SUMO‐1. Mutational analysis demonstrated that the N‐terminus of Mdm2 is a target for p14ARF‐mediated SUMO conjugation. SUMO modification requires residues 2–14 in p14ARF that interact with (H)Mdm2 and residues 82–101 in exon 2 involved in nucleolar localisation of p14ARF. These data suggest a novel role for p14ARF as a regulator of activity of (H)Mdm2, which could be related to its tumour suppressing activities.
Dual regulation of heat‐stable enterotoxin‐mediated cGMP accumulation in T84 cells by receptor desensitization and increased phosphodiesterase activity
Authors: Bakre Manjiri M. , Visweswariah Sandhya S.
Published: October 2002

We report the regulation of cGMP accumulation induced by the heat‐stable enterotoxin, STh, in the T84 human colonic cell line. STh binding to its receptor, guanylyl cyclase C (GCC), leads to elevated intracellular levels of cGMP. Prolonged exposure of T84 cells to STh induced refractoriness to further cGMP accumulation, without significant receptor internalization, but with reduced STh‐induced cGMP synthesis by the receptor. Significantly, increased degradation of cGMP by a cGMP‐specific phosphodiesterase was observed in desensitized cells. This is the first report on the desensitization of GCC, as well as the role of the Type V phosphodiesterase in inducing cellular refractoriness.
A product of growth arrest‐specific gene 6 modulates scavenger receptor expression in human vascular smooth muscle cells
Authors: Murao Koji , Imachi Hitomi , Sayo Yoshitaka , Hosokawa Hitoshi , Sato Makoto , Ishida Toshihiko , Nakano Toru , Kodama Tatsuhiko , Sasaguri Yasuyuki , Takahara Jiro
Published: October 2002

Although Gas6 is identified as a growth factor for vascular smooth muscle cells (VSMCs), its roles in these cells have not been clearly elucidated. To examine the role of Gas6 in atherosclerosis, we examined the effects of Gas6 on scavenger receptor family expression in VSMCs. Scavenger receptor class A, one of the scavenger receptor family members, was upregulated in VSMCs by Gas6. Furthermore, the atherogenic lipoprotein, oxidized LDL, induced Gas6 production in these cells. These results indicate that Gas6 plays an important role in foam cell formation in human VSMCs.
Immunocytochemical localization of atrial natriuretic factor (ANF) in rat female reproductive tract: evidence for a potential hormonal regulation
Authors: Russinova A. , Mourdjeva M. , Valkova C. , Kyurkchiev S. , Kehayov I.
Published: October 2002

In the present studies atrial natriuretic factor (ANF) was characterized immunocytochemically in the reproductive tract of immature female rats, and changes of ANF levels in response to different hormonal conditions were demonstrated. Administration of pregnant mare serum gonadotropin (PMSG) to immature animals has shown to be a useful method to synchronize growth, differentiation and atresia of ovarian follicles. ANF immunoreactivity was investigated in rat uterus and oviduct during follicular growth and estrogenic dominance (48 h after PMSG treatment) and during follicular atresia and progesterone dominance (96 h after PMSG treatment). Our immunocytochemical results showed that in rat uterus ANF was localized in endometrial mucosal and glandular epithelium and smooth muscle cells of the myometrium. In the oviduct ANF immunoreactivity was observed in mucosal cells and muscle layers. Immunocytochemical staining patterns and Western blot analysis revealed that ANF levels in rat uterus and oviduct are modulated by the hormonal status. ANF immunoreactivity was elevated during estrogenic dominance (48 h after PMSG) in uterus and oviduct. However, during progesterone dominance (96 h after PMSG) elevation of ANF immunoreactivity was observed in the uterus only. These results raise the possibility that ANF expression in rat oviduct is positively controlled by estrogen and negatively by progesterone. ANF staining in uterus during progesterone phase provides evidence that both estrogen and progesterone regulate ANF levels in uterus. The observed staining patterns indicate that ANF may have intracellular functions as well as a role in priming the extracellular environment. Accordingly, the possibility that ANF might be an important regulatory molecule for autocrine / paracrine communication within the female reproductive tract should be considered.
Citalopram for treatment-resistant obsessive-compulsive disorder
Authors: Pallanti S. , Quercioli L. , Paiva R.S. , Koran L.M.
Published: October 2002

We investigated the comparative efficacy of citalopram vs. citalopram administered with clomipramine, in treatment-resistant obsessive-compulsive disorder (OCD).

Sixteen adult outpatients participated in a 90-day, randomized, open-label trial. Eligible patients were aged 18 to 45 years, had moderate to severe DSM-III-R OCD of ≥ one year's duration, a baseline Yale-Brown scale (Y-BOCS) score of ≥ 25 and no other active axis I diagnosis, and had failed adequate clomipramine and fluoxetine trials.

The citalopram-plus-clomipramine group (n = 9) experienced a significantly larger percent decrease in mean Y-BOCS score by day 90 than the citalopram alone group (n = 7). Only one citalopram patient decreased her score by ≥ 35%, and two by ≥ 25%. All nine citalopram-plus-clomipramine patients experienced decreases of 35%. Side effects were mild to moderate in both groups. We also treated with citalopram six OCD patients who had not tolerated fluoxetine alone and clomipramine alone; three achieved Y-BOCS score decreases of ≥ 35% at 90 days.

Since citalopram does not significantly affect clomipramine metabolism, the improvement in the combined drug group is unlikely to have resulted from increased plasma clomipramine levels. Double-blind controlled trials are needed of citalopram in OCD, and of combining citalopram with clomipramine in treatment-resistant OCD.
Defective institutions and their consequences: lesson from China, 1980–1993
Authors: Wang Shaoguang
Published: October 2002

Now nearly everyone accepts the fact that institutions matter. However, institutions matter in more than one way. While sound institutions may help to reduce uncertainty and create order in human interactions, flawed institutions may produce results that go contrary to their designers' wishes, no matter what these wishes are. Defects in institutional arrangements may provide “wrong” opportunities, place “wrong” constraints, create “wrong” incentives, reward “wrong” behaviors and above all, structure human interactions in “wrong” ways. This article investigates what makes institutions defective and why defective institutions may cause ineffectiveness. It first identifies the fundamental underpinnings of effective institutions and probes their importance in generating efficiency. It then tries to explain why, in the absence of some of these key elements, defective institutions cannot deliver what effective institutions can. The case of fiscal relations in China is used to highlight the key propositions of the article.
A qualitative study of chronic heart failure patients’ understanding of their symptoms and drug therapy
Authors: Rogers Angie , Addington‐Hall Julia M. , McCoy Anne S.M. , Edmonds Polly M. , Abery Alison J. , Coats Andrew J.S. , Simon J. , Gibbs R.
Published: October 2002

Objectives:

To explore patients’ understanding of their symptoms and the treatment of their heart failure.

Design:

Qualitative analysis of in‐depth interviews, using a constant comparative approach.

Subjects:

27 patients identified by Cardiology and Care of the Elderly physicians as having (a) symptomatic heart failure (New York Heart Association functional classes II, III and IV) and (b) a hospital admission for heart failure in the previous 20 months.

Results:

Patients were aged between 38–94 years (mean 69), 20 were in NYHA functional class III or IV. All had at least one concurrent illness. Analysis of the data identified four key areas: patients had little understanding of the purpose of their medications, were concerned about both the quantity and combination of drugs they were prescribed, had difficulties in differentiating between the side effects of drugs and symptoms of heart failure, and had little knowledge to help them interpret and/or treat changing symptoms.

Conclusion:

Providing patients with relevant information about their medications may help to reduce anxiety about the drugs they are taking. Acknowledging the symptoms associated with heart failure and the likely side effects of treatments might improve patients’ ability to interpret, treat or relieve symptoms.
Limited Sonography in Collaborative Midwifery Practice
Authors: Menihan Cydney Afriat
Published: October 2002

In 1996, the American College of Nurse Midwives published guidelines for midwives who wish to incorporate limited third trimester sonography into their practices. First, however, certain didactic and clinical content needs to be disseminated through basic and continuing midwifery education. To assist the busy practitioner, information is offered related to recommended methods of obtaining learning validation and continuing competency in limited sonography.

This article discusses various clinical situations in which the assessment and management of a patient's condition might be improved by having ultrasonographic information immediately available. In addition, selected data obtained by limited sonography that may necessitate physician collaboration are described through clinical case presentations.

The pitfalls of limited sonography are also discussed by use of case descriptions detailing some of the risks inherent in performing a limited ultrasonogram. The recommendation of performing a complete uterine survey with each limited ultrasonogram is emphasized.

Finally, some of the controversies surrounding routine sonography in pregnancy and sonograms for entertainment are addressed.