bluuebunny/crossref_metadata_2025 huggingface dataset:
Association of a cGMP-binding activity with nuclei isolated from amoebae of Dictyostelium discoideum and Polysphondylium violaceum
Authors: Butler Jeffrey R. , Coukell M. Barrie
Published: December 2009

When vegetative and early slug stage amoebae of Dictyostelium discoideum or Polysphondylium violaceum were lysed by filter breakage in a nuclear isolation buffer not containing detergents, substantial levels of a cGMP-binding activity with slow-dissociation kinetics were detected. After fractionation by centrifugation, 50% or more of this binding activity was associated with isolated nuclei. In addition, with Polysphondylium cells, the fraction of stable, nuclear-associated binding activity appeared to increase during cell aggregation. These results support the idea that cGMP might function in the nucleus during early development.Key words: cGMP-binding activity, nuclear isolation, Dictyostelium discoideum, Polysphondylium violaceum.
Expression and molecular cloning of drought-induced genes in the wild tomato Lycopersicon chilense
Authors: Chen Ri-Dong , Tabaeizadeh Zohreh
Published: December 2009

Protein synthesis and translatable mRNA population changes induced during water stress were studied in leaves of a drought-resistant wild relative of tomato, Lycopersicon chilense, using one- and two-dimensional polyacrylamide gel electrophoresis. Under our experimental conditions, water deficit did not significantly affect total protein synthesis capacity. However, it induced biphasic synthesis of a new set of proteins. These newly synthesized proteins resumed to control levels upon rehydration of the plants. Certain drought-induced proteins also accumulated in leaves subjected to heat shock (39 °C) or exogenous abscisic acid (ABA, 1 mM) treatments. A cDNA library was constructed using poly(A)+ RNA from leaves of plants exposed to drought stress for 4 days. Differential screening of the library identified three groups of clones corresponding to drought- and ABA-induced mRNAs. Northern blot analysis showed that the genes of selected clones respond differently to the different environmental stresses. Our data clearly demonstrate that water stress alters gene expression in L. chilense plants resulting in the synthesis of new proteins, of which several respond to high temperature stress and others to an osmotic effect. These responses are in part modulated by ABA.Key words: abscisic acid, drought, gene expression, Lycopersicon chilense.
Properties of phosphorylated protein intermediates of the bacterial phosphoenolpyruvate:sugar phosphotransferase system
Authors: Anderson J. W. , Waygood E. B. , Saier Jr. M. H. , Reizer J.
Published: December 2009

The phosphohydrolysis properties of the following phosphoprotein intermediates of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) were investigated: enzyme I, HPr, and the IIAGlc domain of the glucose enzyme II of Bacillus subtilis; and IIAGlc (fast and slow forms) of Escherichia coli. The phosphohydrolysis properties were also studied for the site-directed mutant H68A of B. subtilis IIAGlc. Several conclusions were reached. (i) The phosphohydrolysis properties of the homologous phosphoprotein intermediates of B. subtilis and E. coli are similar. (ii) These properties deviate from those of isolated Nδ1- and Nε2-phosphohistidine indicating the participation of neighbouring residues at the active sites of these proteins. (iii) The rates of phosphohydrolysis of the H68A mutant of B. subtilis IIAGlc were reduced compared with the wild-type protein, suggesting that both His-83 and His-68 are present at the active site of wild-type IIAGlc. (iv) The removal of seven N-terminal residues of E. coli IIAGlc reduced the rates of phosphohydrolysis between pH 5 and 8.Key words: phosphoenolpyruvate:sugar phosphotransferase system, phosphoproteins, phosphohistidine, phosphorylation, sugar transport.
Identification of proteins potentially involved in proximal–distal pattern formation in the regenerating forelimb of the newt
Authors: Carlone Robert L. , Boulianne Robert P. , Link Andrew J.
Published: December 2009

We have used high resolution two-dimensional gel electrophoresis to identify and characterize proteins that may represent products of genes involved in establishing positional information along the proximal–distal axis of the regenerating forelimb of the newt Notophthalmus viridescens. At least 24 proteins have been found whose synthesis and (or) abundance is increased in proximal (midstylopodial) regenerates relative to midzeugopodial (distal) regenerates at either of two regeneration stages, the early dedifferentiation and moderate bud stages. Four of these same proteins show an axial asymmetry at both stages. Ten distal-specific proteins were also identified, although only one was common to both stages. More significantly, 6 of these 34 proteins (molecular masses of 73, 73, 51.5, 44.0, 19.5, and 16.5 kilodaltons and isoelectric points of 6.70, 6.74, 6.0, 6.05, 5.9, and 6.98, respectively) are regulated to proximal levels by treatment of distal regenerates with retinoic acid (RA) at both stages. An additional five are proximalized by RA at only one regeneration stage. Since the effect of RA is to proximalize positional information in blastema cells, these 11 proteins represent gene products that could be involved in a biochemical cascade leading to the establishment of positional information in the regenerating limb along this axis.Key words: positional information, retinoic acid, regeneration, blastema, pattern formation, newt.
Routes of electron transfer in beef heart cytochrome c oxidase: is there a unique pathway used by all reductants?
Authors: Crinson M. , Nicholls P.
Published: December 2009

Cytochrome c oxidase oxidizes several hydrogen donors, including TMPD (N,N,N′,N′-tetramethyl-p-phenylenediamine) and DMPT (2-amino-6,7-dimethyl-5,6,7,8-tetrahydropterine), in the absence of the physiological substrate cytochrome c. Maximal enzyme turnovers with TMPD and DMPT alone are rather less than with cytochrome c, but much greater than previously reported if extrapolated to high reductant levels and (or) to 100% reduction of cytochrome a in the steady state. The presence of cytochrome c is, therefore, not necessary for substantial intramolecular electron transfer to occur in the oxidase. A direct bimolecular reduction of cytochrome a by TMPD is sufficient to account for the turnover of the enzyme. CuA may not be an essential component of the TMPD oxidase pathway. DMPT oxidation seems to occur more rapidly than the DMPT – cytochrome a reduction rate and may therefore imply mediation of CuA. Both "resting" and "pulsed" oxidases contain rapid-turnover and slow-turnover species, as determined by aerobic steady-state reduction of cytochrome a by TMPD. Only the "rapid" fraction (≈70% of the total with resting and ≈85% of the total with pulsed) is involved in turnover. We conclude that electron transfer to the a3CuB binuclear centre can occur either from cytochrome a or CuA, depending upon the redox state of the binuclear centre. Under steady-state conditions, cytochrome a and CuA may not always be in rapid equilibrium. Rapid enzyme turnover by either natural or artificial substrates may require reduction of both and two pathways of electron transfer to the a3CuB centre.Key words: cytochrome c oxidase, cytochrome a, respiration, cyanide, stopped flow.
L-Histidinol in experimental cancer chemotherapy: improving the selectivity and efficacy of anticancer drugs, eliminating metastatic disease and reversing the multidrug-resistant phenotype
Authors: Warrington R. C.
Published: December 2009

Human cancer chemotherapy is limited by two major problems: the failure of commonly used anticancer drugs to act against tumor cells in a specific manner and the ability of malignant cells to resist killing by antineoplastic agents. Experimentally, both of these problems can be solved by using L-histidinol in combination with conventional anticancer drugs. A structural analogue of the essential amino acid L-histidine and an inhibitor of protein biosynthesis. L-histidinol improves the selectivity and the efficacy of a variety of cancer drugs in several transplantable murine tumors. Furthermore, L-histidinol circumvents the drug-resistant traits of a variety of cancer cells, including those showing multidrug resistance. This review will summarize these properties of L-histidinol, present new evidence on its ability to increase the vulnerability of both drug-sensitive and drug-resistant human leukemia cells to various anticancer drugs, and show that, in addition to inhibiting protein synthesis, L-histidinol acts as an intracellular histamine antagonist. The establishment of a connection between the latter mechanism and the capacity to modulate anticancer drug action has resulted in a clinical trial in the treatment of human cancer.Key words: L-histidinol, antineoplastic agents, transplantable tumors, metastasis, multidrug resistance.
Dolichol: a curriculum cognitionis
Authors: Hemming Frank W.
Published: December 2009

Dolichols were first described about 30 years ago when animal tissues were being examined for the presence of a putative precursor to the polyisoprenoid side chain of ubiquinone. These long-chain 2,3-dihydro-polycis-isoprenoid alcohols are found in all eukaryotic organisms. In many plant tissues they are accompanied by families of other polyisoprenoid alcohols that are usually similar molecules and possess an unsaturated α-isoprene residue. Analogy with the role of bactoprenyl phosphates in the synthesis of bacterial wall glycans led to the discovery that the mono- and di-phosphates of dolichols function as cofactors in protein N-glycosylation, involving the formation of glycosylated derivatives of dolichol as intermediates. Variation of the concentration of dolichyl phosphate was shown to be one way of controlling protein N-glycosylation. This can be achieved by modification of the relative activities of dolichol kinase and dolichol phosphate phosphatase. Modulation of the biosynthetic pathway, still not fully understood, of dolichyl phosphate may also be an important factor. Several disease conditions involve aberrations in these pathways.Key words: dolichols, polyisoprenoid alcohols, N-glycosylation, O-mannosylation.
Effect of alcohol on urinary and blood dolichols
Authors: Roine Risto , Nykänen Irma , Salaspuro Mikko
Published: December 2009

Alcohol appears to affect dolichol metabolism, as both serum and urinary dolichol concentrations were found to be significantly higher in alcoholics than in social drinkers. Furthermore, acute heavy drinking (5.5 g alcohol/kg body weight during 42 h) increased urinary dolichol excretion significantly, whereas moderate drinking (60 g/day for 10 days) had no effect. Increased urinary dolichol concentrations in alcoholics returned rapidly to normal with a half-life decay of 3 days, whereas increased serum dolichol concentrations did not change during a 7-day observation period. The mechanism behind alcohol-induced alterations in dolichol metabolism remains unclear, but based on our results, it seems likely that serum and urinary dolichols are regulated independently from each other.Key words: polyprenols, biological membranes, substance abuse, lysosomes.
The importance of N-linked glycoproteins and dolichyl phosphate synthesis for fusion of L6 myoblasts
Authors: Jamieson James C. , Wayne Steven , Belo Robert S. , Wright Jim A. , Spearman Maureen A.
Published: December 2009

Myoblasts fuse to form multinucleated myotubes, one of the early steps in the formation of multinucleated muscle fiber. The fusion reaction is accompanied by biochemical differentiation resulting in the expression of a variety of enzyme activities and macromolecules, particularly creatine phosphokinase. The fusing myoblast is thus an excellent system for use in studies on the molecular basis of cellular recognition. This report focuses on the role played by glycoproteins in this process. It was found that alteration of cell-surface glycoproteins, using oligosaccharide-processing inhibitors that interfered with the synthesis of the high-mannose type of N-linked oligosaccharide, resulted in the inhibition of both the fusion reaction and biochemical differentiation as determined by measurement of creatine phosphokinase. Ketoconazole, compactin, and lovastatin, which affect dolichol and cholesterol biosynthesis, were also potent fusion inhibitors. These observations, coupled with earlier studies on the characterization of fusion-defective myoblast cell lines defective in glycoprotein biosynthesis, point to the importance of surface glycoproteins in cellular recognition in L6 myoblasts.Key words: myoblast, dolichol, glycoprotein, fusion, ketoconazole, compactin, lovastatin.
The occurrence of long chain polyprenols in leaves of plants of Rosaceae family and their isolation by time-extended liquid chromatography
Authors: Świeżewska Ewa , Chojnacki T. , Jankowski W. J. , Singh K. , Olsson J.
Published: December 2009

The long chain polyprenols composed of 30 and more isoprene units from leaves of plants belonging to the genera Potentilla and Rosa have been described. They occur in the form of fatty acid esters. The composition of polyprenol mixture was species dependent and its content reached ca. 0.5% wet weight. Large scale preparation of individual polyprenols from a natural polyprenol mixture was performed using time-extended liquid chromatography on the hydrophobic gel Lipidex-5000.Key words: long chain polyprenols, Rosaceae.
Blood dolichol in lysosomal diseases
Authors: Jokelainen Kalle , Salmela Katja S. , Humaloja Kari , Roine Risto , Autio Seppo , Arvio Maria , Järvelä Irma , Nykänen Irma , Palo Jorma , Salaspuro Mikko
Published: December 2009

Highly elevated serum total dolichol (free dolichol + dolichyl ester) concentrations have recently been found in two lysosomal storage diseases, aspartylglucosaminuria (AGU) and mannosidosis. The present study demonstrates that the increase of serum dolichol in AGU patients is caused by an increase of serum free dolichol. In 15 patients the mean serum level of free dolichol (227 ± 16 ng/mL) was 1.9 times higher (p < 0.001) than that in healthy controls (120 ± 6 ng/mL), while the amounts of dolichol fatty acid esters were similar in the patients and controls (110 ± 9 vs. 118 ± 6 ng/mL). In contrast, 10 patients with neuronal ceroid-lipofuscinosis (NCL) (three with infantile, four with juvenile, and three with variant late infantile NCL) had significantly (p < 0.01) lower mean serum levels of both free (79 ± 5 ng/mL) and total (159 ± 6 ng/mL) dolichol than age-adjusted healthy controls (free, 100 ± 6 ng/mL; total, 206 ± 14 ng/mL). Decreased blood dolichol has not been reported earlier for any other disease. We conclude that the increased serum free dolichol in AGU reflects disturbed lysosomal function and that the decreased free and esterified dolichols in NCLs speak against their presumed primary lysosomal nature.Key words: serum dolichol, aspartylglucosaminuria, neuronal ceroid-lipofuscinosis, lysosomal function, lysosomal storage disease.
Dolichyl-pyrophosphoryloligosaccharide protein oligosaccharide transferase in neuronal ceroid-lipofuscinosis
Authors: Dessel Guido Van , Lagrou Albert , Hilderson Herwig , Dierick Wilfried
Published: December 2009

The kinetics of the oligosaccharide transfer from oligosaccharyl pyrophosphoryldolichol to endogeneous protein acceptors in human fibroblasts were studied. No alterations in the transferase activity and enzyme characteristics could be observed in fibroblasts from neuronal ceroid-lipofuscinosis (NCL) patients. Analysis of urinary dolichol of two NCL patients also did not reveal substantial differences with respect to controls.Key words: dolichol, fibroblast, neuronal ceroid-lipofuscinosis, oligosaccharide transfer, urine.
Monomer and excimer fluorescence of horse plasma gelsolin labelled with N-(1-pyrenyl)iodoacetamide
Authors: Silva Beatriz E. Ruiz , Koepf Edward K. , Burtnick Leslie D. , Turro Nicholas J.
Published: December 2009

Horse plasma gelsolin was labelled with the sulfhydryl-specific fluorescent reagent N-(1-pyrenyl)iodoacetamide. The level of incorporation of probe was 1.6 ± 0.3 mol pyrene/mol gelsolin. The circular dichroism spectrum of pyrenyl-gelsolin and its ability to interact with muscle actin were not different from that found for unmodified gelsolin. The emission from pyrenyl-gelsolin was dominated by a broad emission band centred near 483 nm, characteristic of the presence of pyrene excimers. Analysis of excitation spectra for the monomer and excimer-type fluorescence suggested that ground-state interactions may occur between adjacent pyrenes in the gelsolin structure. In the case either of excimer formation or of ground-state pyrene–pyrene interactions in doubly labelled gelsolin molecules, the modified Cys residues must be in close proximity in the folded protein structure. Thermal denaturation of gelsolin could be monitored by observing the decrease in excimer emission that accompanied heating and unfolding of the tertiary structure. While heat treatment alone did not eliminate excimer fluorescence, digestion of gelsolin with chymotrypsin completely abolished such emission. Also, pyrenyl-gelsolin prepared and studied in 6 M guanidine-HCl exhibited fluorescence characteristic of pyrene monomers exclusively.Key words: gelsolin, pyrene excimer fluorescence, fluorescence lifetime, thermal denaturation.
Secretion of SPARC by endothelial cells transformed by polyoma middle T oncogene inhibits the growth of normal endothelial cells in vitro
Authors: Sage E. Helene
Published: December 2009

Endothelioma cells expressing the polyoma virus middle T oncogene induced hemangiomas in mice by the recruitment of nonproliferating endothelial cells from host blood vessels (Williams et al. 1989). I now report that SPARC, a Ca2+-binding glycoprotein that perturbs cell–matrix interactions and inhibits the endothelial cell cycle, is produced by endothelioma cells and is in part responsible for the alterations in the morphology and growth that occur when nontransformed bovine aortic endothelial cells are cocultured with endothelioma cells. Normal endothelial cells cocultured with two different middle T-positive endothelial cell lines, termed End cells, exhibited changes in shape that were accompanied by the formation of cell clusters. Media conditioned by End cells repressed proliferation of normal endothelial cells, but enhanced that of an established line of murine capillary endothelium. Radiolabeling studies revealed no apparent differences in the profile of proteins secreted by aortic or capillary cells cultured in End cell conditioned media. Characterization of proteins produced by End cells led to the identification of type IV collagen, laminin, entactin, and SPARC as major secreted products. Although SPARC did not affect the morphology of End or capillary cells, it was associated with overt changes in the shape of aortic endothelial cells. Moreover, SPARC and a synthetic peptide from SPARC domain II inhibited the incorporation of [3H]thymidine by aortic cells, but had minimal to no effect on the capillary endothelial cell line. The inhibition of growth exhibited by aortic endothelial cells cultured in End cell conditioned media could be partially reversed by antibodies specific for SPARC and SPARC peptides. These studies indicate a potential role for SPARC in the generation of hemangiomas by End cells in vivo, a process that requires normal (host) endothelial cells to disengage from the extracellular matrix, withdraw from the cell cycle, migrate, and reassociate into the disorganized cellular networks that comprise cavernous and capillary hemangiomas.Key words: endothelial cells, hemangioma, cell proliferation, SPARC.
Increased preproenkephalin A gene expression in the rat heart after induction of a myocardial infarction
Authors: Paradis Pierre , Dumont Michel , Bélichard Pierre , Rouleau Jean-Lucien , Lemaire Simon , Brakier-Gingras Léa
Published: December 2009

The expression of preproenkephalin A (ppENK) gene was investigated in the rat heart, following the onset of myocardial infarction induced by ligation of the left anterior descending coronary artery. The relative abundance of ppENK mRNA and the level of enkephalins were measured by Northern blot analysis and radioimmunoassay, respectively, in the ventricles from control-unoperated, sham-operated, and operated rats. Three hours after the surgery, a comparison between rats with infarction and sham-operated rats revealed that the relative abundance of ppENK mRNA and the level of enkephalins were increased three- to four- and two- to three-fold, respectively, in the ventricles of rats with infarction. No difference was observed between rats with infarction and sham-operated rats 24 h after the surgery, or between rats with infarction compared at time intervals of 3 and 24 h following the surgery. The abundance of the ppENK mRNA in the polysomal fraction of the ventricular septum was also measured 3 h after the surgery and found to be threefold higher in rats with infarction as compared with sham-operated rats. These results indicate that the level of enkephalins rapidly increases in the ventricles of rats following myocardial infarction, and that this higher level may be ascribed to a stimulation of the local synthesis of enkephalins.Key words: preproenkephalin A, enkephalins, gene expression, myocardial infarction.
Sphingomyelin-metabolizing enzymes and protein kinase C activity in liver plasma membranes of rats fed with cholesterol-supplemented diet
Authors: Nikolova-Karakashian Mariana N. , Gavrilova Nadja J. , Petkova Diana H. , Setchenska Milka S.
Published: December 2009

The effect of cholesterol-supplemented diet on the activities of rat liver plasma membrane sphingomyelin-metabolizing enzymes and protein kinase C was studied. Protein kinase C, phosphatidylcholine:ceramide-phosphocholine transferase, and phosphatidylethanolamine:ceramide-phosphoethanolamine transferase activities were found to increase continuously and almost in parallel during the experimental period on cholesterol diet (days 10, 20, and 30). Linear regression analysis showed a positive correlation between these activities with correlation coefficients r = 0.959 for protein kinase C and phosphatidylcholine:ceramide-phosphocholine transferase, and r = 0.998 for protein kinase C and phosphatidylethanolamine:ceramide-phosphoethanolamine transferase. On the other hand, protein kinase C activation does not correspond to sphingomyelinase activity changes. These data suggest that protein kinase C activation observed in cholesterol-enriched plasma membranes is due to increased production of diacylglycerol and increased acylation of sphingosine to ceramide.Key words: protein kinase C, sphingomyelin-metabolizing enzymes, cholesterol, plasma membranes.
Dihydrolipoamide dehydrogenase from Haloferax volcanii: gene cloning, complete primary structure, and comparison to other dihydrolipoamide dehydrogenases
Authors: Vettakkorumakankav Nataraj N. , Stevenson Kenneth J.
Published: December 2009

We used the N-terminal amino acid sequence of dihydrolipoamide dehydrogenase from Haloferax volcanii, to design and synthesize two oligonucleotide probes that were used to identify and clone a 4.3 kilobase pair (kbp) fragment from MboI restriction endonuclease digestion of Hf. volcanii genomic DNA. The nucleotide sequence of a 1.5-kbp region of this clone was determined and this revealed an open reading frame that translated into a protein with good homology to dihydrolipoamide dehydrogenase from other sources. The first 48 amino acids were identical with the N-terminal sequence data obtained from the purified protein. The complete primary structure of the halophilic dihydrolipoamide dehydrogenase was analyzed in terms of its homologies to dihydrolipoamide dehydrogenases from other sources and its molecular adaptations to high intracellular ionic strength.Key words: Archaea, halophile, flavoenzyme, nucleotide sequence.
Slow troponin C is present in both muscle and nonmuscle cells
Authors: Berezowsky Craig , Bag Jnanankur
Published: December 2009

A common primer was used for synthesis of cDNAs from both chicken fast and slow troponin C (TnC) mRNAs. Synthesis of double-stranded cDNAs and their amplification by polymerase chain reaction gave specific products corresponding to these mRNAs. This method was used for determining the presence of TnC mRNAs in various tissues. Our results show that while the fast TnC mRNA is expressed only in the muscle cells, slow TnC mRNA is expressed in a number of nonmuscle cells. Not all nonmuscle cells, however, express slow TnC mRNA. Liver and brain tissues showed the presence of high levels of this mRNA, while it was absent in chicken smooth muscle and embryonic skin. The slow TnC mRNA was very stable in cardiac muscle cells. It degrades with a half-life of approximately 94 h. The same mRNA was less stable in skeletal muscle and liver cells. The half-lives were found to be only between 13 and 16 h in these cells. Our results suggest that slow TnC mRNA may function as the nonmuscle isoform of this contractile protein. Since slow TnC mRNA is the only TnC isoform present in cardiac muscle, liver and brain, it is possible that besides its role in regulating contraction of striated muscle slow TnC can also function in processes other than muscle contraction.Key words: troponin C mRNA, cardiac muscle, skeletal muscle, polymerase chain reaction, nonmuscle troponin C.
Amylase cis-acting sequences mediate the alleviation of glucose repression by cAMP in Drosophila
Authors: Magoulas Charalambos , Loverre-Chyurlia Ada , Hickey Donal A.
Published: December 2009

α-Amylase gene expression is highly repressed by dietary glucose in Drosophila melanogaster. This glucose effect can be alleviated by exogenous adenosine 3′:5′-cyclic monophosphate (cAMP). Here, we show that the relief of glucose repression by cAMP occurs at the level of amylase mRNA abundance. Furthermore, exogenous cAMP was shown to alleviate glucose repression of the transient expression of an amylase gene construct in transformed Amynull larvae. This construct contains only 109 base pairs of the promoter region; this is the minimal length of upstream sequence which is necessary for wild-type levels of amylase gene expression. These results indicate that cis-acting promoter elements located close to the transcriptional start site of the Drosophila amylase gene mediate both glucose repression and the cAMP-derepression effects.Key words: Drosophila, glucose repression, cAMP effect, amylase gene.
Presence of a novel nuclear lamina protein in Raji cells
Authors: Foisy Sylvain , Joly Erik , Sakr Françoise , Levac Pascale , Bibor-Hardy Viviane
Published: December 2009

In mammalian tissues, the nuclear lamina is composed of the major lamins A, B, and C, and minor lamins D/E. Although lamin B is present in all cell types, lamins A and C are absent from embryonic cells and most undifferentiated cells from hematopoietic lineage. We have investigated the nuclear lamina protein composition of the Raji cell line, lymphoblast-like cells established from a Burkitt lymphoma patient. Lamins A and C were confirmed absent by immunodetection and Northern blot analysis. Besides lamins B and D/E, a protein migrating around 71 kilodaltons was recognized by a serum directed against the nuclear lamina of BHK-21 fibroblasts. Cellular localization by sequential extraction established this 71-kilodalton protein as an exclusive component of the nuclear lamina fraction. These results indicate that the nuclear lamina has a more complex composition than previously thought to be the case for cells devoid of lamins A and C.Key words: hematopoietic cells, nuclear envelope, lamins, mRNA, nuclear lamina proteins.
Towards an understanding of microtubule function and cell organization: an overview
Authors: MacRae Thomas H.
Published: December 2009

Microtubules exhibit dynamic instability, converting abruptly between assembly and disassembly with continued growth dependent on the presence of a tubulin–GTP cap at the plus end of the organelle. Tubulin, the main structural protein of microtubules, is a heterodimer composed of related polypeptides termed α-tubulin and β-tubulin. Most eukaryotic cells possess several isoforms of the α- and β-tubulins, as well as γ-tubulin, an isoform restricted to the centrosome. The isoforms of tubulin arise either as the products of different genes or by posttranslational processes and their synthesis is subject to regulation. Tubulin isoforms coassemble with one another and isoform composition does not appear to determine whether a microtubule is able to carry out one particular activity or another. However, the posttranslational modification of polymerized tubulin may provide chemical signals which designate microtubules for a certain function. Microtubules interact with proteins called microtubule-associated proteins (MAPs) and they can be divided into two groups. The structural MAPs stimulate tubulin assembly, enhance microtubule stability, and influence the spatial distribution of microtubules within cells. The dynamic MAPs take advantage of microtubule polarity and organization to vectorially translocate cellular components. The interactions between microtubules and MAPs contribute to the structural–functional integration that characterizes eukaryotic cells.Key words: tubulin, microtubules, microtubule-associated proteins.
Nuclear and chromatin composition of mammalian gametes and early embryos
Authors: Clarke Hugh J.
Published: December 2009

Changes in nuclear structure and chromatin composition regulate gene activity in many cell types and could play a similar role during early mammalian embryogenesis. Oocytes of the mouse contain the three major lamin species present in somatic cells, although lamin A synthesized by oocytes has a higher molecular mass than the somatic species. Oocyte chromatin contains core histones similar to those of somatic cells, as well as elements that are immunologically related to protamines. In contrast, somatic-type histone H1 is not present. DNA topoisomerase II has not yet been identified in mammalian oocytes, but is abundant in frog oocytes. In contrast to oocytes, sperm do not contain a typical nuclear lamina. DNA topoisomerase II is detectable until late spermiogenesis. Although the DNA of sperm is associated mainly with protamines, some histone may be retained. There is also evidence that the arrangement of the DNA in the nucleus is nonrandom. These results demonstrate differences in nuclear and chromatin composition between oocytes and sperm. After fertilization, the nuclei of cleavage-stage blastomeres undergo programmed modifications. Lamin B is synthesized, whereas lamin A is not. In addition, a set of nuclear proteins is transiently synthesized in mice at the two-cell stage. Changes in embryonic chromatin composition also occur. The relative abundance of transcripts from different core histone genes differs between mouse oocytes and blastocysts. Furthermore, somatic histone H1 becomes detectable beginning at the mid-four-cell stage. As well, during early cleavage stages, expression of plasmid-borne genes becomes dependent on enhancers. Thus, developmentally regulated changes in nuclear and chromatin composition occur during early mammalian embryogenesis, and these may be important for the initiation and regulation of embryonic gene activity.Key words: chromatin, nucleus, embryogenesis, gametogenesis, mammals.
Body axis determination during early development in amphibians
Authors: Savard Pierre
Published: December 2009

The specification of the main axes of the body is a phenomenon based on cell communication and is among the early crucial events of embryonic development. Upon fertilization, the amphibian egg reorganizes its cytoplasmic content, leading to the establishment of the future dorsal–ventral axis of the body. Heterogeneous distribution of maternal components confers cellular regionalization after only a few mitoses. Development up to the 4000-cell stage proceeds almost entirely on maternal materials, and during this period there is remodeling of the chromatin to set up specific gene expression in various regions of the embryo. The zygote at this stage has already undertaken cellular interactions leading to mesoderm formation and regionalization. Dorsal mesodermal components then induce the formation of the Spemann's organizer, a structure directly involved in the specification of the anterior–posterior axis of the embryo (head to tail). Molecular analysis of these phenomena has allowed the identification of growth-factor-like and transcription-factor-like proteins that have characteristics typical of specification factors. We will review the recent advances on these molecules and will also discuss the putative role of retinoic acid as a posteriorizing agent.Key words: chromatin remodeling, cellular regionalization, homeobox, inducing factor, retinoic acid.
Towards understanding the control of the division cycle in animal cells
Authors: Masui Yoshio
Published: December 2009

The author reviewed the historical process by which classical knowledge of cell division accumulated, to give rise to the molecular biology of the cell cycle, and discussed the perspective of this field of research. The study of the control of cell division began at the turn of the century. It was hypothesized that cell division was a physiological regulation necessary for growing cells to maintain a proper nucleocytoplasmic ratio to survive, which was later substantiated by the finding that amoeba cells could be prevented from dividing by repeated excision of the cytoplasm. However, the observation in Tetrahymena that heat-shocked cells grow exceedingly, but fail to divide, suggested that the cell required the accumulation of a labile "division protein" to initiate division. Mechanisms that control the cell cycle were studied in oocytes by nuclear transplantation and cytoplasmic transfer, and in cultured mammalian cells, protozoa, and Physarum Plasmodia by cell fusion. These experiments demonstrated the existence of cytoplasmic factors that control the cell cycle. Maturation promoting factor (MPF) thus discovered in frog oocytes became known to be an ubiquitous cytoplasmic factor that causes the transition from interphase to metaphase in all organisms. The insight into the molecular control of cell growth and division was gained from yeast cell genetics. For biochemical analysis of the cell cycle control, the method to observe the cell cycle in vitro was developed using frog egg extracts. Thus, MPF was identified as a cdc2 – cyclin protein complex. Its activity was found to depend on synthesis and phosphorylation of these proteins. However, recently it was found that there were cell cycle phenomena that were difficult to explain in these terms. Various other cellular factors, including nucleocytoplasmic ratio and microtubule assembly, were also found to control MPF, as well as the cell cycle. It remained open to future investigation how these factors control MPF to alter the pattern of the cell cycle.Key words: cell cycle, cytostatic factor, maturation promoting factor, nucleocytoplasmic relation.
Regulation of mammalian ribonucleotide reductase by the tumor promoters and protein phosphatase inhibitors okadaic acid and calyculin A
Authors: Hurta Robert A. R. , Wright Jim A.
Published: December 2009

A rapid elevation of ribonucleotide reductase activity was observed with BALB c/3T3 fibroblasts treated with 10 nM okadaic acid, a nonphorbol ester tumor promoter and protein phosphatase inhibitor. Northern blot analysis of the two components of ribonucleotide reductase (R1 and R2) showed a marked elevation of R1 and R2 mRNA expression. Western blot analysis with R1 and R2 specific monoclonal antibodies indicated that the increase in ribonucleotide reductase activity was primarily due to the elevation of the R2 rather than the R1 protein during treatment with okadaic acid. The okadaic acid induced elevations in R1 and R2 message levels occurred without a detectable change in the proportion of cells in S phase and were blocked by treatment of cells with actinomycin D, indicating the importance of the reductase transcriptional process in responding to the action of okadaic acid. Furthermore, down-regulation of protein kinase C with 12-O-tetradecanoylphorbol-13-acetate pretreatment abrogated the okadaic acid mediated elevation of ribonucleotide reductase mRNAs, consistent with the involvement of this signal pathway in the regulation of ribonucleotide reductase and the effects of okadaic acid. Treatment of cells with 2.5 nM calyculin A, another non-phorbol ester tumor promoter and protein phosphatase inhibitor, resulted in a rapid elevation of both R1 and R2 mRNA levels within 10 min of treatment. This first demonstration that the non-phorbol ester tumor promoters and protein phosphatase inhibitors can cause rapid alterations in ribonucleotide reductase gene expression suggests that (i) ribonucleotide reductase, particularly the R2 component, plays a fundamental role in the critical early events involved in the process of tumor promotion, and (ii) illustrates a role for cellular protein phosphatases in the regulation of ribonucleotide reductase and, through this process, the regulation of DNA synthesis.Key words: ribonucleotide reductase, DNA synthesis, okadaic acid, calyculin A, tumor promoter, protein phosphatase.
The distribution of centrosomes in migrating endothelial cells during wound healing in situ
Authors: Rogers Kem A. , Sandig Martin , McKee Nancy H. , Kalnins Vitauts I.
Published: December 2009

We have examined the distribution of centrioles in rabbit thoracic aortic endothelial cells induced to migrate by wounding the endothelium in situ. Following denudation of the endothelium from a segment of the aorta with a balloon catheter, a wound edge was created from which endothelial cells began to migrate onto the denuded surface. In this in situ model of cell migration, the position of centrioles was determined in cells along the wound edge by immunofluorescence and antibodies which specifically label these cell organelles, and then they were classified in relation to the nucleus and the direction of cell migration as being oriented toward the wound, in the center, or away from wound. At time 0, as in normal unwounded adult rabbit aorta, no preferential orientation of centrioles was evident. Within 12 h after wounding, the centrioles in about 53% of endothelial cells near the wound edge were oriented toward the wound, while in less than 20% of the cells they were oriented away from wound. At 24 h, in cells up to 800 μm from the wound edge, centrioles in only about 10% of the endothelial cells were oriented away from wound, while in about 52% of cells they were found in the center and in 38% of the cells they remained oriented toward the wound. At 48 h, up to 2000 μm from the wound edge, the majority of endothelial cells had their centrioles in the center, possibly as a result of an increase in mitotic index as cells replicate to reestablish an intact endothelium. The results of this study demonstrate that, in endothelial cells starting to migrate on a natural substratum in situ in response to wounding, most centrioles reorient toward the wound edge. This observation is consistent with the hypothesis that the centrosome is involved in defining the direction of cell migration in endothelial cells.Key words: centriole, in situ, endothelium, wound healing, aorta.
Cytoskeletal organization following cannabinoid treatment in undifferentiated and differentiated PC12 cells
Authors: Tahir Stephen K. , Trogadis Judy E. , Stevens John K. , Zimmerman Arthur M.
Published: December 2009

Confocal microscopy in association with three-dimensional reconstruction was used to examine the changes in the microtubules and microfilaments following cannabinoid treatment of PC12 cells. Microtubules and microfilaments were disrupted in a dose-dependent manner following treatment with 10–30 μM Δ9-tetrahydrocannabinol (THC). A disruption of microtubules and microfilaments was observed following treatment with 30 μM cannabidiol and cannabinol. The amount of microtubules and microfilaments was reduced in a dose-dependent manner following treatment with 10 and 20 μM THC. Cannabidiol and cannabinol reduced the amount of microtubules and microfilaments; however, the reduction was less than that observed with THC treatment. Following the addition of nerve growth factor, differentiated PC12 cells were generally more sensitive to cannabinoid treatments than undifferentiated cells. The possible mechanisms that may account for the changes in microtubules and microfilaments following cannabinoid treatment are discussed.Key words: cannabinoids, confocal microscopy, cytoskeleton, pheochromocytoma, PC12 cells, volume reconstruction.
Binding of pp60v-src to membranes: evidence for multiple membrane interactions
Authors: Silverman Lauren , Sigal Catherine T. , Resh Marilyn D.
Published: December 2009

Membrane association of pp60v-src, the myristylated transforming protein of Rous sarcoma virus, has been shown to be a receptor-mediated process, which is inhibited by myristylated src peptides containing the N-terminal 11 amino acids of the v-src sequence (MGYsrc). By cross-linking radiolabeled MGYsrc peptide to fibroblast membranes, a 32-kilodalton membrane protein was identified as a candidate src receptor. To elucidate the potential role of p32 in binding pp60v-src, we studied the relationship between binding of MGYsrc peptide and pp60v-src polypeptide to cellular membranes. The subcellular membrane distribution of p32 was distinct from that of pp60v-src in transformed cells. Moreover, under certain defined in vitro conditions, it was possible to inhibit peptide cross-linking to p32 without significantly affecting pp60v-src membrane binding. However, when internal sequences were removed from pp60v-src, the binding characteristics of the src deletion polypeptide and MGYsrc peptide became identical. These data indicate that the presence of internal membrane binding domains influences the interaction of myristylated N-terminal src sequences with p32, and suggest that accessory binding factors might be involved in establishing stable contact between pp60v-src and the membrane phospholipid bilayer.Key words: myristylation, pp60v-src, membrane binding, cross-linking.
Mutagenesis of Ste18, a putative Gγ subunit in the Saccharomyces cerevisiae pheromone response pathway
Authors: Whiteway Malcolm , Dignard Daniel , Thomas David Y.
Published: December 2009

The yeast STE18 gene product has sequence and functional similarity to the γ subunits of G proteins. The cloned STE18 gene was subjected to a saturation mutagenesis using doped oligonucleotides. The populations of mutant genes were screened for two classes of STE18 mutations, those that allowed for increased mating of a strain containing a defective STE4 gene (compensators) and those that inhibited mating even in the presence of a functional STE18 gene (dominant negatives). Three amino acid substitutions that enhanced mating in a specific STE4 (Gβ) point mutant background were identified. These compensatory mutations were allele specific and had no detectable phenotype of their own; they may define residues that mediate an association between the Gβ and Gγ subunits or in the association of the Gβγ subunit with other components of the signalling pathway. Several dominant negative mutations were also identified, including two C terminal truncations. These mutant proteins were unable to function in signal transduction by themselves, but they prevented signal transduction mediated by pheromone, as well as the constitutive signalling which is present in cells defective in the GPAI (Gα) gene. These mutant proteins may sequester Gβ or some other component of the signalling machinery in a nonfunctional complex. Key wordsi yeast, G protein, STE18, mutagenesis, pheromone response.
cAMP-dependent protein kinase, but not the cGMP-dependent enzyme, rapidly phosphorylates Δ-CREB, and a synthetic Δ-CREB peptide
Authors: Colbran Janet L. , Roach Peter J. , Fiol Carol J. , Dixon Jack E. , Andrisani Ourania M. , Corbin Jackie D.
Published: December 2009

Phosphorylation of the cAMP response element binding protein (CREB) by the catalytic subunit of cAMP-dependent protein kinase (cAK) has been implicated in the cAMP-dependent stimulation of gene transcription. Δ-CREB, a spliced variant of CREB, and CREBtide (KRREILSRRPSYR), a synthetic peptide based on the phosphorylation sequence in Δ-CREB, were tested as substrates of cAK. Phosphorylation of Δ-CREB (0.17 μM) was stoichiometric within 30 s when using a concentration of cAK which approximated the intracellular level (0.2 μM). The rate of phosphorylation of Δ-CREB was comparable to the rates of the best physiological substrates of cAK tested. The rate of CREBtide phosphorylation was at least as great as that of Δ-CREB, indicating that the peptide retained the determinants of Δ-CREB which were responsible for substrate efficacy. The apparent Km of CREBtide phosphorylation by cAK was 3.9 μM, which is 10-fold lower than that of kemptide (Km = 39 μM), the synthetic peptide substrate most often employed for cAK measurement. The Vmax values were 12.4 μmol/(min∙mg) for CREBtide and 9.8 μmol/(min∙mg) for kemptide. The apparent Km of CREBtide phosphorylation by cGMP-dependent protein kinase (cGK) was 2.9 μM and the Vmax value was 3.2 μmol/(min∙mg). Both Δ-CREB and CREBtide were phosphorylated at a much slower rate by cGK as compared with cAK, implying that the high cAK/cGK specificity exhibited by Δ-CREB was retained by the peptide. Taken together, the results indicated that Δ-CREB and CREBtide are among the best substrates tested for cAK and suggested that phosphorylation of CREB by this enzyme could occur in intact cells. However, cGK does not appear to be an adequate catalyst for this reaction.Key words: cAMP-dependent protein kinase, cGMP-dependent protein kinase, CREB.
n–3 and n–6 polyunsaturated fatty acids have different effects on acyl-CoA:cholesterol acyltransferase in J774 macrophages
Authors: Davis Philip J.
Published: December 2009

The effects of incubating J774 mouse macrophages with different fatty acids on cholesterol esterification were investigated. In cells incubated with n–3 polyunsaturated fatty acids, the rate of cholesterol esterification was significantly reduced compared with cells incubated with n–6 polyunsaturated fatty acids or with oleic acid. This change in cholesterol esterification appears to be the result of reductions in the activity of acyl-CoA:cholesterol acyltransferase (ACAT) in the endoplasmic reticulum of the macrophages incubated with the n–3 polyunsaturated fatty acids. No differences in microsomal cholesterol were observed among cells incubated with different fatty acids. However, cellular cholesterol levels were lower in cells incubated with n–3 polyunsaturated fatty acids. In microsomes from cells incubated with n–3 polyunsaturated fatty acids, both the Km and the Vmax of ACAT were lower than in microsomes from cells incubated with n–6 fatty acids or oleic acid. These findings may explain some of the reduction in atherosclerotic lesions that are observed with dietary fish oils that contain high levels of n–3 polyunsaturated fatty acids.Key words: n–3 polyunsaturated fatty acids, acyl-CoA:cholesterol acyltransferase, macrophages, cholesterol esterification.