bluuebunny/crossref_metadata_2025 huggingface dataset:
Amine oxidase, spermine, and hyperthermia induce cytotoxicity in P-glycoprotein overexpressing multidrug resistant Chinese hamster ovary cells
Authors: Lord-Fontaine Stephanie , Agostinelli Enzo , Przybytkowski Ewa , Averill-Bates Diana A
Published: April 2011

Multidrug resistance is a major obstacle for the successful use of chemotherapy. The multidrug resistance phenotype is often attributed to overexpression of P-glycoprotein, which is an energy-dependent drug efflux pump. We investigated a new strategy to overcome multidrug resistance, using purified bovine serum amine oxidase, which generates two major toxic products from the polyamine spermine. The cytotoxicity of the aldehyde(s) and H2O2, produced by the enzymatic oxidation of micromolar concentrations of spermine, was evaluated in multidrug resistant Chinese hamster ovary cells CHRC5 with overexpression of P-glycoprotein, using a clonogenic cell survival assay. We examined the ability of hyperthermia (42°C), and inhibition of cellular detoxification systems, to sensitize multidrug resistant cells to spermine oxidation products. Severe depletion of intracellular glutathione was achieved using L-buthionine sulfoximine and inhibition of glutathione S-transferase by ethacrynic acid. CHRC5 cells showed no resistance to the toxic oxidation products of spermine, relative to drug-sensitive AuxB1 cells. Exogenous catalase protected cells against cytotoxicity of H2O2, but spermine-derived aldehyde(s) still caused some cytotoxicity. Hyperthermia (42°C) enhanced cytotoxicity of spermine oxidation products. Cytotoxic responses in CHRC5 cells were compared to the drug-sensitive cells, to determine whether there are differential responses. CHRC5 cells were more sensitive to the cytotoxic effect of spermine oxidation products under more extreme conditions (higher temperature, higher spermine concentration, and longer exposure time). Glutathione depletion or glutathione S-transferase inhibition also led to enhanced cytotoxicity of spermine oxidation products in CHRC5 and AuxB1 cells. Our findings suggest that hyperthermia, combined with toxic oxidation products generated from spermine and amine oxidase, could be useful for eliminating drug-sensitive and multidrug resistant cells.Key words: amine oxidase, spermine, multidrug resistance, P-glycoprotein, hyperthermia.
Effects of ATP on regulation of galactosyltransferase-I activity responsible for synthesis of the linkage region between the core protein and glycosaminoglycan chains of proteoglycans
Authors: Higuchi Tsuyoshi , Tamura Shinri , Tanaka Kanji , Takagaki Keiichi , Saito Yoshiharu , Endo Masahiko
Published: April 2011

We report that ATP enhances the activity of galactosyltransferase-I, which synthesizes the linkage region between glycosaminoglycan chains and the core proteins of proteoglycans. The enzyme activity in cell-free fractions prepared from cultured human skin fibroblasts was measured by high-performance liquid chromatographic detection of galactosyl-xylosyl-(4-methylumbelliferone) produced from 4-methylumbelliferyl-β-D-xyloside used as an acceptor. ATP at 2 mM increased the enzyme activity by about 60% in the 110 × g supernatant of the cell homogenate, but not in the supernatant or precipitate fractions obtained by 100 000 × g centrifugation. When both fractions (the 100 000 × g supernatant and precipitate) were mixed, the additional ATP increased the enzyme activity. This increase was canceled by heat treatment or trypsin digestion of the 100 000 × g supernatant. In addition, the 100 000 × g precipitate, which was prepared from the 110 × g supernatant preincubated with ATP, exhibited increased activity, and this increase was abolished by alkaline phosphatase treatment. These results suggest that a protein kinase in the 100 000 × g supernatant activates galactosyltransferase-I activity.Key words: ATP, enzyme activator, galactosyltransferase-I, proteoglycan linkage region.
Identification of the S6 kinase activity stimulated in quiescent brine shrimp embryos upon entry to preemergence development as p70 ribosomal protein S6 kinase: Isolation ofArtemia franciscanap70S6kcDNA
Authors: Santiago Jorge , Sturgill Thomas W
Published: April 2011

We previously demonstrated that a protein kinase responsible for phosphorylating 40S ribosomal subunits is activated in quiescent Artemia franciscana embryos within 15 min of restoration of normal tonicity and incubation at 30°C. Here, we identify the activated S6 kinase as A. franciscana p70 ribosomal S6 kinase (p70S6k) subsequent to the isolation of an Artemia p70S6kcDNA. The protein conceptually translated from cDNA has 70% similarity and 64% identity to both Drosophila melanogaster and human p70S6k. Southern blot analysis is consistent with presence of a single p70S6kgene. Two transcripts of 5.4 and 2.7 kb were found. Abundance of both mRNAs increased dramatically around 4 h of preemergence development, and exhibited different steady-state level variation thereafter. Stimulated S6 kinase activity, partially purified by Superose 6 chromatography, correlated best with the slowest migrating, ~65 kDa, form detected by Western analysis using a specific polyclonal antibody made to a peptide from the predicted p70S6kNH2-terminus. Furthermore, the A. franciscana p70S6kwas immunoprecipitated with the same antibody, showing in parallel an S6 kinase activity similar to peak profiles. We conclude that the stimulated S6 kinase activity is that of an ortholog of human p70S6kthat may be involved in the regulation of protein synthesis during preemergence development in A. franciscana species.Key Words: p70 ribosomal S6 kinase cDNA, brine shrimp, development.
β1 integrin-extracellular matrix protein interaction modulates the migratory response to chemokine stimulation
Authors: Shenoy Priti S , Uniyal Shashi , Miura Kohei , McColl Christopher , Oravecz Tamas , Morris Vincent L , Chan Bosco M.C
Published: April 2011

It is well established that chemokines have a major role in the stimulation of cell movement on extracellular matrix (ECM) substrates. However, it is also clear that ECM substrates may influence the ability of cells to undergo migration. Using the migration chamber method, we assessed the migratory response of human embryonic kidney-293 (HEK) transfectant cells expressing the CC chemokine receptor 5 (CCR5) (HEK-CCR5) to stimulation by chemokines (macrophage inflamatory protein (MIP)-1α, MIP-1β, and regulated on activation normal-T cell expressed and secreted (RANTES)) on ECM substrates (collagen type I and fibronectin). Using filters coated with collagen (20 µg/mL), results showed that the chemokines differed in their ability to elicit cell movement according to the order MIP-1β > RANTES [Formula: see text] MIP-1α. In contrast, using filters coated with fibronectin (20 µg/mL), all three chemokines were similar in their ability to stimulate migration of HEK-CCR5 cells. In addition, the migratory response with respect to the concentrations of ECM substrates appeared biphasic; thus, chemokine-stimulated cell movement was inhibited at high ECM concentrations (100 µg/mL). To determine the involvement of β1 integrins, results showed that the migratory response to chemokine stimulation on collagen was largely inhibited by monoclonal antibody (mAb) to α2β1; however, complete inhibition required a combination of mAbs to α1β1 and α2β1. In comparison, migration on fibronectin was inhibited by mAb to α3β1 and α5β1. Our results suggest that the migratory response to CCR5 stimulation may vary quantitatively with both the CCR5 ligand (MIP-1α, MIP-1β, and RANTES), as well as the nature and concentration of the ECM substrate involved.Key words: chemokines, integrins, cell movement, extracellular matrix proteins, CCR5.
CBP, a transcriptional coactivator and acetyltransferase
Authors: McManus Kirk J , Hendzel Michael J
Published: April 2011

The CREB binding protein (CBP) was first identified as a protein that specifically binds to the active phosphorylated form of the cyclic-AMP response element binding protein (CREB). CBP was initially defined as a transcriptional coactivator that, as a result of its large size and multiple protein binding domain modules, may function as a molecular scaffold. More recently, an acetyltransferase activity, both of histones and nonhistones, has been found to be essential for transactivation. In this review, we will discuss the current understanding of the acetyltransferase specificity and activity of the CBP protein and how it may function to coactivate transcription. We will also examine the regulation of the CBP histone acetyltransferase activity in the cell cycle, by signal-transduction pathways and throughout development.Key words: CBP, acetyltransferase, chromatin, acetylation, p300.
Processing of the insulin-like growth factor-IImannose 6-phosphate receptor in isolated liver subcellular fractions
Authors: Tahiri Khadija , Cam Laurence , Desbuquois Bernard , Chauvet Geneviève
Published: April 2011

A truncated, soluble form of the insulin-like growth factor-IImannose 6-phosphate (IGF-IIM6P) receptor has been identified in serum and shown to be released from cultured tissues and cells, liver being the main contributor to serum receptor in adult rats. In the present study, the processing of the IGF-IIM6P receptor has been characterized in isolated liver subcellular fractions using ligand binding, affinity crosslinking, and Western immunoblotting techniques. The receptor in plasma membrane fractions differed from that in Golgi-endosomal fractions by: (i) a lower molecular size upon reducing polyacrylamide gel electrophoresis (245 vs. 255 kDa); (ii) a less tight membrane association as judged upon extractibility by NaCl; and (iii) the inability to recognize antibody anti-22C, directed against the cytoplasmic domain of the receptor. Incubation of cell fractions at 30°C led to a pH- and time-dependent release of the receptor into the medium. The pH optimum for release was 5.5 in the Golgi-endosomal fraction and 7.5 in plasma membrane fractions; at this pH, approximately 2% and 20%30% of total receptors were released per hour, respectively. Receptor release was inhibited in a dose-dependent manner by aprotinin, benzamidine, and leupeptin in the Golgi-endosomal fraction, and by 1,10 phenanthroline in plasma membrane fractions, although high concentrations were required for inhibition. The receptor released from Golgi-endosomes showed a 510 kDa reduction in size and a loss of ability to recognize antibody anti-22C, but that released from plasma membranes showed little or no changes in size. We conclude that soluble, carboxy-terminally truncated forms of the IGF-IIM6P receptor are generated from the intact receptor in isolated Golgi-endosomal and plasma membrane fractions. However, receptor processing in these fractions exhibits different properties, suggesting the involvement of different proteases.Key words: insulin-like growth factor-IImannose 6-phosphate (IGF-IIM6P) receptor, liver, plasma membrane, Golgi apparatus, endosomes.
Folding pathway for partially folded rabbit muscle creatine kinase
Authors: Park Yong-Doo , Ou Wen-Bin , Yu Tian-Wei , Zhou Hai-Meng
Published: April 2011

Rabbit muscle creatine kinase (CK) was modified by 5,5'-dithio-bis(2-nitrobenzoic acid) accompanied by 3 M guanidine hydrochloride denaturation to produce a partially folded state with modified thiol groups. The partially folded CK was in a monomeric state detected by size exclusion chromatography, native-polyacrylamide gel electrophoresis, circular dichroism, and intrinsic fluorescence studies. After dithiothreitol (DTT) treatment, about 70% CK activity was regained with a two-phase kinetic course. Rate constants calculated for regaining of activity and refolding were compared with those for CK modified with various treatments to show that refolding and recovery of activity were synchronized. To further characterize the partially folded CK state and its folding pathway, the molecular chaperone GroEL was used to evaluate whether it can bind with partly folded CK during refolding, and 1-anilinonaphthalene-8-sulfonate was used to detect the hydrophobic surface of the monomeric state of CK. The monomeric state of CK did not bind with GroEL, although it had a larger area of hydrophobic surface relative to the native state. These results may provide different evidence for the structural requirement of GroEL recognition to the substrate protein compared with previously reported results that GroEL bound with substrate proteins mainly through hydrophobic surface. The present study provides data for a monomeric intermediate trapped by the modification of the SH groups during the refolding of CK. Schemes are given for explaining both the partial folding CK pathway and the refolding pathway.Key words: creatine kinase; partially folded state; reactivation; refolding; GroEL; intermediate.
Keratin-mediated resistance to stress and apoptosis in simple epithelial cells in relation to health and disease
Authors: Marceau Normand , Loranger Anne , Gilbert Stéphane , Daigle Nathalie , Champetier Serge
Published: April 2011

Epithelial cells such as hepatocytes exhibit highly polarized properties as a result of the asymmetric distribution of subsets of receptors at unique portions of the surface membrane. While the proper targeting of these surface receptors and maintenance of the resulting polarity depend on microtubules (MTs), the Golgi sorting compartment, and different actin-filament networks, the contribution of keratin intermediate filaments (IFs) has been unclear. Recent data show that the latter cytoskeletal network plays a predominant role in providing resistance to various forms of stress and to apoptosis targeted to the surface membrane. In this context, we first summarize our knowledge of the domain- or assembly-related features of IF proteins and the dynamic properties of IF networks that may explain how the same keratin pair K8/K18 can exert multiple resistance-related functions in simple epithelial cells. We then examine the contribution of linker protein(s) that integrate interactions of keratin IFs with MTs and the actin-cytoskeleton network, polarity-dependent surface receptors and cytoplasmic organelles. We next address likely molecular mechanisms by which K8/K18 can selectively provide resistance to a mechanical or toxic stress, or to Fas-mediated apoptosis. Finally, these issues on keratin structurefunction are examined within a context of pathological anomalies emerging in tissue architecture as a result of natural or targeted mutations, or posttranslational modifications at specific amino acid residues. Clearly, the data accumulated in recent years provide new and significant insights on the role of K8/K18, particularly under conditions where polarized cells resist to stressful or apoptotic insults.Key words: keratins, desmosomes, Fas, Golgi, microtubules, actin, hepatocyte.
Checkpoints for vesicular traffic?
Authors: Fiset Annie , Faure Robert
Published: April 2011

During interphase the transport of material between different intracellular organelles requires accurate regulation of fusiogenic domains. Recent studies on hepatic endosomes indicated that compartmentalized Cdk2  cyclin E complexes act by braking fusion events. These Cdk2 complexes integrate tyrosine phosphorylation and dephosphory lation inputs, resulting in the control of the number of rounds of fusion at discrete domains. This leads to changes in the intracellular location of internalized receptors and ultimately their biological response.Key words: vesicular traffic, Cdk2, receptors tyrosine kinases.
Protein translocon at the Arabidopsis outer chloroplast membrane
Authors: Hiltbrunner A , Bauer J , Alvarez-Huerta M , Kessler F
Published: April 2011

Chloroplasts are organelles essential for the photoautotrophic growth of plants. Their biogenesis from undifferentiated proplastids is triggered by light and requires the import of hundreds of different precursor proteins from the cytoplasm. Cleavable N-terminal transit sequences target the precursors to the chloroplast where translocon complexes at the outer (Toc complex) and inner (Tic complex) envelope membranes enable their import. In pea, the Toc complex is trimeric consisting of two surface-exposed GTP-binding proteins (Toc159 and Toc34) involved in precursor recognition and Toc75 forming an aequeous protein-conducting channel. Completion of the Arabidopsis genome has revealed an unexpected complexity of predicted components of the Toc complex in this plant model organism: four genes encode homologs of Toc159, two encode homologs of Toc34, but only one encodes a likely functional homolog of Toc75. The availability of the genomic sequence data and powerful molecular genetic techniques in Arabidopsis set the stage to unravel the mechanisms of chloroplast protein import in unprecedented depth.Key words: Arabidopsis, genetics, chloroplast, protein import.
Synthesis and processing of bone sialoproteins during de novo bone formation in vitro
Authors: Zhu Xin-Li , Ganss Bernhard , Goldberg Harvey A , Sodek Jaro
Published: April 2011

Bone sialoprotein (BSP) and osteopontin (OPN) are sulphated and phosphorylated sialoglycoproteins that regulate the formation of hydroxyapatite crystals during de novo bone formation. To gain insights into the relationship between the synthesis and posttranslational modification of BSP and OPN and the mineralization of bone, pulsechase studies were conducted on cultures of newly forming bone nodules produced by fetal rat calvarial cells in vitro. Cultures were pulse labelled with35SO4, or with either32PO4or [γ-32P]ATP to study intracellular and extracellular phosphorylation, respectively, and chased in isotope-free medium for various times up to 24 h. The presence of radiolabelled BSP and OPN was determined in the cells, in culture medium, and in various tissue compartments obtained by dissociative extraction with 4 M GuHCl (G1), 0.5 M EDTA (E), and again with 4 M GuHCl (G2) and a bacterial collagenase digestion of the demineralized collagenous tissue residue. With each isotope employed, radiolabelled BSP and OPN were detected in the E extract within the 1-h chase period and increased in amount with time. Similarly,35SO4- and32PO4-labelled BSP increased in the G2 extract, but OPN was not detected. In the G1 extract the35SO4-labelled BSP decreased with chase time, whereas the32PO4-labelled BSP increased. No differences were evident in the profiles of BSP labelled with32PO4or [γ-32P]ATP. In the absence of β-glycerophosphate, which is required for optimal mineralization of the bone nodules,35SO4-labelled BSP was increased in the medium and G1 extract and decreased in the E extract and G2 extract after 3 h. In addition to differences in the tissue compartmentalization of BSP and OPN, these studies indicate that35SO4is lost from BSP during mineralization and that isoforms of BSP exist with a selective affinity for the organic and mineral phases. Moreover, the additional phosphorylation of BSP and OPN catalyzed by ectokinase activity does not appear to alter the distribution of these sialoproteins.Key words: biosynthesis, bone, bone sialoprotein, osteopontin, mineralization, posttranslational modification.
Alterations of intermediate filaments in various histopathological conditions
Authors: Cadrin Monique , Martinoli Maria-Grazia
Published: April 2011

Intermediate filament proteins belong to a multigene family and constitute an important cytoskeletal component of most vertebrate cells. Their pattern of expression is tissue specific and is highly controlled during embryonic development. Numerous pathologies are known to be associated with modifications of intermediate filament organisation, although their precise role has not yet been elucidated. The present review focuses on the most recent data concerning the possible causes of intermediate filaments disorganization in specific pathologic conditions affecting the epidermis, the liver, and the nervous system. We discuss the formation of abnormal intermediate filament networks that arise as a consequence of mutations that directly affect intermediate filament structure or are induced by multifactorial causes such as modifications of post-translational processes and changes in the levels of expression.Key words: intermediate filaments, phosphorylation, Mallory bodies, Lewy bodies, degenerative diseases.
Biliary secretion and actin–cytokeratin filament distribution in rat hepatocytes during phalloidin-induced cholestasis
Authors: Loranger Anne , Marceau Normand , Tuchweber Beatriz , Youseff Ibrahim
Published: April 2011

The relationship between bile secretion (bile flow, bile acids, phospholipids, and cholesterol) and distribution of actin microfilaments (MFs) and cytokeratin (CK) intermediate filaments (IFs) was examined in hepatocytes of rats injected with a single low dose of phalloidin. This treatment induced a transient cholestasis characterized by a rapid development period (0–90 min postinjection) and a slow recovery period (24 h and 5 days postinjection). No significant changes were observed in bile acid secretion during the 5-day period. The phospholipid output dropped to less than 25% at 90 min and was back to the normal value at 24 h postinjection. In a parallel way, the cholesterol secretion dropped to 30% but came back to only 60% of the control level. Nile Red staining demonstrated a concomitant accumulation of lipids both in the cytoplasm and at the surface membrane. Immunostaining of the actin MFs and CK IFs showed that, in contrast with controls where both cytoskeletal networks were preferentially and uniformly localized at the surface membrane (i.e., sinusoidal, basolateral, and canalicular regions), the toxin treatment led to a major targeting of actin to the pericanalicular region at 24 h and a massive accumulation of well-preserved CK IFs in the cytoplasm at 5 days. Interestingly, this accumulation of CK IFs was not linked to any significant variations in CK isoforms. Together, these data indicate that a selective binding of the toxin to sinusoidal membrane actin at the time of injection triggers a sequence of events that culminate in delayed accumulation of actin MFs at the canalicular pole and of CK IFs in the cytoplasm. Moreover, the reversible perturbation of the bile secretory activity implies a functional adaptation of the hepatocytes that parallels the phalloidin-induced reorganization of both cytoskeleton networks.Key words: phalloidin, actin, cytokeratins, liver, cholestasis.
Molecular cloning of chicken calcyclin (S100A6) and identification of putative isoforms
Authors: Allen Bruce G , Andrea Jacquelyn E , Sutherland Cindy , Schönekess Brett O , Walsh Michael P
Published: April 2011

A full-length cDNA encoding smooth muscle calcyclin (S100A6) was cloned from chicken gizzard, using reverse transcription - polymerase chain reaction techniques. The deduced amino acid sequence contains 92 residues with 12 substitutions and a 2 amino acid C-terminal extension when compared with human calcyclin. Calcyclin was purified from chicken gizzard by Ca2+-dependent hydrophobic chromatography, heat treatment, and anion-exchange chromatography. N-terminal sequencing of two CNBr peptides confirmed its identity as calcyclin. Two isoforms of calcyclin (A and B), which differ with respect to the presence or absence of a C-terminal lysine, were identified and the native protein was shown to exist as noncovalently associated homodimers (AA and BB) and heterodimers (AB). Incubation of purified calcyclin AA with an extract of chicken gizzard did not result in degradation of calcyclin A or appearance of calcyclin B, suggesting that calcyclin B is a bona fide isoform rather than a proteolytic fragment generated during purification. Western blotting of chicken tissues with anti-(gizzard calcyclin) indicated abundant expression of calcyclin in smooth muscle tissues, including esophagus, large intestine, and trachea, with lower levels in lung, heart, kidney, and brain, and none detectable in liver or skeletal muscle.Key words: Ca2+-binding proteins, calcyclin, smooth muscle, cDNA cloning, isoforms.
Effect of herbimycin A on hsp30 and hsp70 heat shock protein gene expression inXenopuscultured cells
Authors: Briant Douglas , Ohan Nicholas , Heikkila John J
Published: April 2011

We have examined the effect of herbimycin A, a benzoquinoid ansamycin antibiotic, on the pattern of gene expression in amphibians. Exposure of Xenopus laevis A6 kidney epithelial cells to 1 µg/mL herbimycin A induced the synthesis of the heat shock proteins hsp30 and hsp70 as well as 33- and 45-kDa proteins. Enhanced synthesis of a 34-kDa protein appears to be specific to herbimycin A because its synthesis did not increase after heat shock (35°C). In addition, the synthesis of hsp30 and hsp70 induced by herbimycin A was accompanied by an increase in their mRNAs. Herbimycin A induced a transient accumulation of hsp30 and hsp70 mRNA, which peaked between 4 and 6 h. Finally, concurrent treatment of cells with 0.5 µg/mL herbimycin A and a mild heat shock of 27°C yielded a synergistic accumulation of hsp30 and hsp70 mRNA. These studies demonstrate that herbimycin A can induce the expression of a set of stress proteins in amphibians and that concurrent treatment with herbimycin A and mild heat shock has a synergistic effect on the accumulation of hsp30 and hsp70 mRNA. Key words: heat shock, heat shock proteins, Xenopus, herbimycin A, mRNA.
High-resolution imaging at the cellular and subcellular levels in flattened whole mounts of early zebrafish embryos
Authors: Yager Thomas D , Ikegami Richard , Rivera-Bennetts Alma K , Zhao Cida , Brooker Deborah
Published: April 2011

We describe a rapid and sensitive method for high-resolution imaging at the cellular and subcellular levels in the whole-mount zebrafish embryo. The procedure involves fixing and staining the embryo, followed by deyolking and flattening it under a cover slip, to produce a planar mount that is 20 to 100 µm thick. Such a flattened whole mount allows imaging with a spatial resolution of ~500 nm in the x-y plane and does not require the use of embedding, sectioning, confocal microscopy, or computational deblurring procedures. We can resolve all individual nuclei and chromosome sets in the embryo, up to the late gastrula stage (10 000 cell stage). In addition, older embryos (through the segmentation stage) can also be examined, with the preservation of significant morphological detail. Because of its ability to resolve subcellular detail, the flattened whole-mount method can provide significant biological information beyond what can be obtained from conventional (three-dimensional) whole mounts. We have used the flattened whole-mount method to study subcellular events related to progression through the cell cycle or to apoptosis, in cells of the early zebrafish embryo. A specific DNA-binding dye (Hoechst 33258) or an antibody against a chromosomal protein (histone H1) was used to stain the nuclei of individual cells in the embryo. This allowed us to determine the spatial positions of all the individual cells, and also their stages in the cell cycle. A terminal transferase (TUNEL) assay was used to detect apoptotic cells. This combination of specific stains allowed us to study the behaviors of groups of cells in situ, within the developing zebrafish embryo.
Heat shock genes and the heat shock response in zebrafish embryos
Authors: Krone Patrick H , Lele Zsolt , Sass Jennifer B
Published: April 2011

Heat shock genes exhibit complex patterns of spatial and temporal regulation during embryonic development in a wide range of organisms. Our laboratory has initiated an analysis of heat shock protein gene expression in the zebrafish, a model system that is now utilized extensively for the examination of early embryonic development of vertebrates. We have cloned members of the zebrafish hsp47, hsp70,\i and hsp90 gene families and shown them to be closely related to their counterparts in higher vertebrates. Whole mount in situ hybridization and Northern blot analyses have revealed that these genes are regulated in distinct spatial, temporal, and stress-specific manners. Furthermore, the tissue-specific expression patterns of the hsp47 and hsp90 alpha genes correlate closely with the expression of genes encoding known chaperone targets of Hsp47 and Hsp90 in other systems. The data raise a number of interesting questions regarding the function and regulation of these heat shock genes in zebrafish embryos during normal development and following exposure to environmental stress.
Molecular cloning and expression of avian smooth muscle S100A11 (calgizzarin, S100C)
Authors: Schönekess Brett O , Walsh Michael P
Published: April 2011

S100A11 (calgizzarin or S100C), a member of the S100 family of Ca2+-binding proteins, was first identified in chicken gizzard smooth muscle and subsequently detected in several mammalian species and tissues. We now report the full-length coding sequence of avian smooth muscle S100A11. The cloned nucleotide sequence is 515 bases in length, which includes in-frame start and stop codons and encodes a protein of 101 amino acids. The chicken S100A11 sequence differs from human S100A11 at 25 positions (9 conserved) and is four residues shorter (overall identity 72.4%, similarity 81%). The protein contains two EF hands and conserved hydrophobic residues involved in dimer formation. Cloned avian S100A11 expressed in Escherichia coli and purified by Ca2+-dependent hydrophobic interaction chromatography and ion-exchange chromatography was recognized by polyclonal antibodies raised against tissue-purified protein and, like tissue-purified S100A11, bound45Ca2+in a gel overlay assay. Key words: S100A11, calgizzarin, Ca2+-binding protein, smooth muscle, avian.
Spatiotemporal changes in cytokeratin expression in the neonatal rat ovary
Authors: Pan Jie , Auersperg Nelly
Published: April 2011

Ovarian granulosa cells are derived embryologically from two keratin-positive epithelia of mesodermal origin, the ovarian rete and the ovarian surface epithelium. In the rat, presumptive granulosa cells still express keratin at birth but as they acquire functions related to oocyte support and steroidogenesis in the maturing ovary they lose this epithelial differentiation marker. Using double-label immunofluorescence microscopy, we examined the distribution of keratin-expressing granulosa cells in rat ovaries on days 1-10 postpartum in relation to (i) laminin and collagen type IV in follicular basement membranes, (ii) the zona pellucida, and (iii) 3β-hydroxysteroid dehydrogenase activity. Keratin was present in most (pre)granulosa cells on days 1-3. As the cells became multilayered in growing follicles, keratin was retained by granulosa cells adjacent to follicular basement membranes but disappeared from cells that were displaced towards follicular centers. From day 7 on, large follicles lacked keratin altogether. Laminin was a consistent component of follicular basement membranes at all ages, while collagen IV varied and diminished in parallel with keratin. 3β-Hydroxysteroid dehydrogenase was demonstrable in stromal interstitial cells from day 7 on. Zona pellucida first appeared in primary follicles adjacent to keratin-positive cells and subsequently became surounded with keratin-negative granulosa cells in growing follicles. The results suggest different roles for laminin and collagen IV in follicular basement membranes and support the hypothesis that keratin expression by granulosa cells depends on paracrine interactions with the ovarian stroma. In early growing follicles, these interactions may be interrupted by physical removal from the vicinity of the basement membranes as the granulosa cells become multilayered. In the more mature follicles, the loss of keratin from all granulosa cells suggests that the required stromal signals cease, perhaps as the perifollicular stroma differentiates into the theca.Key words: ovary, differentiation, keratin, basal membrane, development.
The presence of an unusual PKC isozyme profile in rat liver cells
Authors: Al-Mazidi Hayfa A , Kleine Leonard P , Franks Douglas J
Published: April 2011

We have previously shown that protein kinase C (PKC) is involved in the mitogenic response of T51B cells to epidermal growth factor. In fact, epidermal growth factor was an excellent mitogen, even after prolonged pretreatment of cells with TPA, suggesting that the PKC isoform implicated in proliferation is not down-regulated by 12-O-tetradecanoyl phorbol-13-acetate (TPA). We have now determined that the PKC isozymes -α, -βI, -δ, -ε, and -ζ are present in T51B cells. All five isoforms are associated with the plasma membrane and the cytoplasm and are either in or around the nucleus. PKC-βIhas a slightly different subcellular profile from that of the other isoforms in that it is clearly and strongly associated with the nuclear membrane. Also, a unique and novel pattern is obtained from immunoblots with anti-PKC-βI. PKC-βIis detected as a single band of 70 kDa in the cytosolic fraction and as a doublet of 65 and 77 kDa in the membrane fraction. PKC-α, -δ, and -ε were down-regulated by pretreatment of cells with TPA, while PKC-ζ was unaffected. Of particular interest was the fact that TPA did not down-regulate PKC-βI. In fact, the amount of this isoform associated with the plasma membrane increased. These findings indicate that it is probably PKC-βIthat is involved in the mitogenic response of T51B cells to epidermal growth factor. Since PKC-ζ is also not down-regulated by TPA, the possible involvement of this isoform needs to be resolved.Key words: protein kinase C, intracellular localization, cell proliferation, liver.
Spatial and temporal resolution of functional magnetic resonance imaging
Authors: Menon Ravi S , Gati Joseph S , Goodyear Bradley G , Luknowsky David C , Thomas Christopher G
Published: April 2011

Functional magnetic resonance imaging has become an invaluable tool for cognitive neuroscience, despite the fact that many of the physiological mechanisms giving rise to the effect are not well understood. We review the known biochemical and physiological basis of the technique and discuss how, within the noted limits, one might fully exploit the spatial and temporal resolution that is intrinsic to the very high magnetic fields that we use for human studies. This noninvasive brain mapping technique relies on the changes in blood oxygenation, blood volume, and blood flow, and we discuss some of the issues influencing the effects of these hemodynamic parameters on image intensity.Key words: fMRI, magnetic resonance imaging, BOLD, neuroimaging, functional imaging.
Solid-state 2H NMR study of methyl-d3-cobalamin
Authors: Garbutt Jennifer R , Goward Gillian R , Kirby Christopher W , Power William P
Published: April 2011

A solid-state 2H NMR study of methyl-d3-cobalamin has been performed as a function of temperature to provide information concerning the character and energetics of the motion performed by this unique bioorganometallic methyl group. Analysis of the 2H NMR line shape indicates that the methyl group undergoes rapid three-fold rotation, and that the Co-C-2H angle lies between 105.9 and 109.5°. Determination of the spin-lattice relaxation times T1 shows that the relaxation is anisotropic, consistent with a "jumping" motion of the methyl group rather than rotational diffusion. This also provides the activation energy to methyl jumps as 8.3 ± 1.3 kJ/mol. It is proposed that this energetic barrier may be a useful probe of changes in the electronic character of the Co-C bond that accompany the biological role of this molecule in such enzymes as methionine synthase.Key words: cobalamin, solid-state NMR, deuterium NMR, molecular dynamics.
The role of the N-acetylaspartate multiplet in the quantification of brain metabolites
Authors: Thompson Richard B , Allen Peter S
Published: April 2011

N-Acetylaspartate, whose methyl singlet is the primary magnetic resonance marker of neuronal dysfunction, also gives rise to a sequence-dependent, strongly coupled multiplet that overlaps the resonances of several other metabolites. Results are presented in this paper of a full numerical calculation of the response of the strongly coupled aspartate multiplet of N-acetylaspartate to a PRESS pulse sequence employing practical slice-selective pulses. These calculations, confirmed by experiments on phantoms, demonstrate the ability to predict the dependence of the response of strongly coupled spins on pulse design, as well as on interpulse evolutions, thereby facilitating a more rigorous comparison of the use of spectral fitting routines employed to extract metabolite concentrations on different instruments.Key words: magnetic resonance spectroscopy, N-acetylaspartate, NAA, brain, quantification, PRESS.
A Zn(II)-translocating P-type ATPase from Proteus mirabilis
Authors: Rensing Christopher , Mitra Bharati , Rosen Barry P
Published: April 2011

A mutant of Proteus mirabilis had been previously isolated as defective in swarming. The mutation had been found to be in a gene related to the Escherichia coli zntA gene, which encodes the ZntA Zn(II)-translocating P-type ATPase. In this study the P. mirabilis genewas expressed in an E. coli strain in which the zntA gene had been disrupted. The P. mirabilis gene complemented the sensitivity to salts of zinc and cadmium. Everted membrane vesicles from the zntA-disrupted strain lost ATP-driven 65Zn(II) uptake. Membranes from the complemented strain had restored 65Zn(II) transport. These results demonstrate that the P. mirabilis homologue of ZntA is a Zn(II)-translocating P-type ATPase.Key words: zinc resistance, P-type ATPase, Proteus mirabilis.
Complex interactions between skeletal muscle ryanodine receptor and dihydropyridine receptor proteins
Authors: Leong Peng , MacLennan David H
Published: April 2011

Evidence for functional interactions between the Ca2+ release channel in the skeletal muscle sarcoplasmic reticulum (the ryanodine receptor) and the L-type Ca2+ channel in the sarcolemma (the dihydropyridine receptor), leading to excitation-contraction coupling, is reviewed and experimental systems used to identify candidate sites of interaction are outlined.Key words: sarcoplasmic reticulum, excitation-contraction coupling.
Pre-steady state transport by erythrocyte band 3 protein: uphill countertransport induced by the impermeant inhibitor H2DIDS
Authors: Jennings Michael L , Whitlock Jonathan , Shinde Anjali
Published: April 2011

Pre-steady state Cl- efflux experiments have been performed to test directly the idea that the transport inhibitor H2DIDS (4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate) binds preferentially to the outward-facing state of the transporter. Cells were equilibrated with a medium consisting of 150 mM sodium phosphate, pH 6.2, N2 atmosphere, and 80-250 µM 36Cl-. Addition of H2DIDS (10-fold molar excess compared with band 3) induces a transient efflux of Cl-, as expected if H2DIDS binds more tightly to outward-facing than to inward-facing states. The size of the H2DIDS-induced efflux depends on the Cl- concentration and is about 700 000 ions per cell at the highest concentrations tested. The size of the transient efflux is larger than would be expected if the catalytic cycle for anion exchange involved one pair of exchanging anions per band 3 dimer. These results are completely consistent with a ping-pong mechanism of anion exchange in which the catalytic cycle consists of one pair of exchanging anions per subunit of the band 3 dimer.Key words: anion exchange, erythrocyte, stilbenedisulfonate, chloride.
Cell lineage in the developing neural tube
Authors: Kalyani Anjali J , Rao Mahendra S
Published: April 2011

Acquisition of cell type specific properties in the spinal cord is a process of sequential restriction in developmental potential. A multipotent stem cell of the nervous system, the neuroepithelial cell, generates central nervous system and peripheral nervous system derivatives via the generation of intermediate lineage restricted precursors that differ from each other and from neuroepithelial cells. Intermediate lineage restricted neuronal and glial precursors termed neuronal restricted precursors and glial restricted precursors, respectively, have been identified. Differentiation is influenced by extrinsic environmental signals that are stage and cell type specific. Analysis in multiple species illustrates similarities between chick, rat, mouse, and human cell differentiation. The utility of obtaining these precursor cell types for gene discovery, drug screening, and therapeutic applications is discussed.Key words: stem cells, oligodendrocytes, astrocytes, neurons, spinal cord.
Interactions between peripherin and neurofilaments in cultured cells: disruption of peripherin assembly by the NF-M and NF-H subunits
Authors: Beaulieu Jean-Martin , Robertson Janice , Julien Jean-Pierre
Published: April 2011

Neurofilaments are the principal intermediate filament type expressed by neurons. They are formed by the co-assembly of three subunits: NF-L, NF-M, and NF-H. Peripherin is another intermediate filament protein expressed mostly in neurons of the peripheral nervous system. In contrast to neurofilaments, peripherin can self-assemble to establish an intermediate filament network in cultured cells. The co-expression of neurofilaments and peripherin is found mainly during development and regeneration. We used SW13 cells devoid of endogenous cytoplasmic intermediate filaments to assess the exact assembly characteristics of peripherin with each neurofilament subunit. Our results demonstrate that peripherin can assemble with NF-L. In contrast, the co-expression of peripherin with the large neurofilament subunits interferes with peripherin assembly. These results confirm the existence of interactions between peripherin and neurofilaments in physiological conditions. Moreover, they suggest that perturbations in the stoichiometry of neurofilaments can have an impact on peripherin assembly in vivo.Key words: peripherin, neurofilament, SW13 cells, intermediate filament.
Advances in the osteoblast lineage
Authors: Aubin Jane E
Published: April 2011

Osteoblasts are the skeletal cells responsible for synthesis, deposition and mineralization of the extracellular matrix of bone. By mechanisms that are only beginning to be understood, stem and primitive osteoprogenitors and related mesenchymal precursors arise in the embryo and at least some appear to persist in the adult organism, where they contribute to replacement of osteoblasts in bone turnover and in fracture healing. In this review, we describe the morphological, molecular, and biochemical criteria by which osteoblasts are defined and cell culture approaches that have helped to clarify transitional stages in osteoblast differentiation. Current understanding of differential expression of osteoblast-associated genes during osteoprogenitor proliferation and differentiation to mature matrix synthesizing osteoblasts is summarized. Evidence is provided to support the hypothesis that the mature osteoblast phenotype is heterogeneous with subpopulations of osteoblasts expressing only subsets of the known osteoblast markers. Throughout this paper, outstanding uncertainties and areas for future investigation are also identified.Key words: skeletal development, differential gene expression, heterogeneity.
Odontoblast commitment and differentiation
Authors: Ruch Jean Victor
Published: April 2011

Histological and cytological organization confer specificity to the odontoblasts. These postmitotic, neural crest derived, polarized cells are aligned in a single layer at the periphery of the dental pulp and secrete the organic components of predentin-dentin. The developmental history of these cells demands a cascade of epigenetic signalling events comprising the acquisition of odontogenic potential by neural crest cells, their patterning in the developing jaws, the initiation of odontogenesis through interaction with the oral epithelium, commitment, and tooth-specific spatial distribution of competent preodontoblasts able to overtly differentiate. Recent experimental investigations are critically summarized, many open questions are stressed, and current hypotheses concerning the control of terminal odontoblast differentiation are outlined.Key words: odontoblast, neural crest, oral ectoderm, differentiation.
Genistein increases metallothionein expression in human intestinal cells, Caco-2
Authors: Kuo Shiu-Ming , Leavitt Penny S
Published: April 2011

Flavonoids found in common vegetables, fruits, and legumes have been shown to possess antioxidant property. This study is the first to demonstrate that one member of the flavonoid family, genistein, can induce the expression of metallothionein (a metal-binding protein with antioxidant property). We found the effect of genistein to be time- and dose-dependent (10-100 µM). The effect can be observed at both protein and mRNA levels and was synergistic to that of 30 µM zinc. Genistein was shown previously to interact with the estrogen receptor and induce gene expression similar to estrogens at a lower affinity. We thus tested the hypothesis that the effect of genistein on metallothionein expression was mediated through the steroid hormone pathway. We found that various glucocorticoids do not affect metallothionein expression in Caco-2 cells. 17beta-estradiol at 10-100 µM (concentrations much higher than needed to activate the estrogen response element) induced metallothionein expression in Caco-2 cells. However, a synthetic estrogen, diethylstilbestrol, did not increase metallothionein level at 10 µM. 17beta-Estradiol also did not act synergistically with zinc. Thus, genistein may enhance metallothionein expression through an uncharacterized mechanism. Further studies are needed to delineate the molecular mechanism and to determine whether the expression of other genes is also affected by genistein.Key words: metallothionein gene expression, genistein, flavonoid, estrogen, intestine.
Molecular cloning of a centrin homolog fromMarsilea vestitaand evidence for its translational control during spermiogenesis
Authors: Hart Peter E , Wolniak Stephen M
Published: April 2011

Spermiogenesis in the water fern Marsilea vestita is a process that reaches completion 11 h after dry microspores are immersed in an aqueous medium at 20°C. Each microspore produces 32 spermatozoids and each spermatozoid has a coiled cell body and approximately 140 cilia. The spermatids make basal bodies de novo, from a structure known as a blepharoplast. From the onset of development, the spores contain a large quantity of protein and stored mRNA. We have found previously that centrin, a protein involved in the function of microtubule organizing centers and present in association with basal bodies in motile cells, is made in large quantity approximately 4 h after the microspores are placed into liquid medium. In this paper, we show that a centrin cDNA (MvCen1) we isolated from M. vestita closely resembles centrin cDNAs from other eukaryotic organisms. MvCen1, synthesized in Escherichia coli as a GST-fusion protein, reacted with anti-centrin monoclonal antibodies on immunoblots. Northern blot analysis demonstrates that centrin mRNA is present in the dry microspore at the time of imbibition, at levels that remain constant over 10 h of development and are unaffected by treatment of spores with alpha-amanitin. The centrin transcripts, stored in dry microspores, cannot be translated in vitro for at least 30 min after imbibition.Key words: Marsilea vestita, spermatozoid, spermiogenesis, centrin, MTOC.
Pax7is expressed in the capsules surrounding adult mouse neuromuscular spindles
Authors: Rodger J , Ziman M R , Papadimitriou J M , Kay P H
Published: April 2011

The multigene Pax family of transcription factors plays an important role in the development of the central nervous system as well as in organ morphogenesis. Expression of one of the members of the family, Pax7, has been described in embryonic muscle and in both embryonic and adult brain. We recently detected Pax7 transcripts in RNA isolated from adult mouse skeletal muscle and brain and here use in situ hybridisation to localise the expression within these tissues. Pax7 expression was observed in neural cells of the brain and in cells of neural crest origin in the inner and outer capsules of neuromuscular spindles. The results suggest that Pax7 may be implicated in the formation and maintenance of neuromuscular contacts within the muscle spindle throughout life.Key words: Pax7 expression, skeletal muscle, neuromuscular spindle, basal lamina, Schwann cells.