bluuebunny/crossref_metadata_2025 huggingface dataset:
Dissection of functional domains in Bcl-2α by site-directed mutagenesis
Authors: Borner Christoph , Olivier Reynald , Martinou Isabelle , Mattmann Chantal , Tschopp Jurg , Martinou Jean-Claude
Published: December 2009

Bcl-2α is a mitochondrial or perinuclear-associated oncoprotein that prolongs the life span of a variety of cell types by interfering with programmed cell death. How Bcl-2 confers cell survival is unknown, although antioxidant and antiprotease functions have been proposed. In addition, protein structures of Bcl-2 that are crucial for its survival activity are still ill-defined. Bcl-2 can occur as Bcl-2α or Bcl-2β, two alternatively spliced forms which solely differ in their carboxyl termini. The finding that Bcl-2α is active and membrane bound, but Bcl-2β is inactive and cytosolic, indicates that the carboxyl terminus contributes to the survival activity of Bcl-2. This region contains two subdomains, a domain X with unknown function and a hydrophobic stretch reported to mediate membrane assocation of Bcl-2α. Recently Bcl-2-related proteins have been identified. These include Bax that heterodimerizes with Bcl-2 and, when overxpressed, counteracts Bcl-2. Bax contains two highly conserved regions of sequence homology with Bcl-2, referred to as Bcl-2 homology 1 and 2 (BH1 and BH2) domains. Site-directed mutagenesis studies have revealed that both domains are not only novel dimerization motifs for the interaction of Bax with Bcl-2 but also crucial for the survival activity of Bcl-2. Interestingly, the C-terminal end of BH2 encompasses the Bcl-2α/β splice site, as well as part of domain X in Bcl-2α. To better define the role of domain X and the hydrophobic C-terminal stretch of Bcl-2α for its survival activity, we created various deletion and truncation mutations in these regions by site-directed mutagenesis. We show here that membrane attachment and therefore the hydrophobic stretch is not required for the survival activity of Bcl-2, but part of domain X appears to be indispensable.Key words: apoptosis, Bcl-2, mutagenesis, cell survival, functional domains.
Modification of radiation-induced apoptosis in radiation- or hyperthermia-adapted human lymphocytes
Authors: Cregan S. P. , Boreham D. R. , Walker P. R. , Brown D. L. , Mitchel R. E. J.
Published: December 2009

We have investigated the influence of the cellular adaptive response to ionizing radiation on radiation-induced apoptosis in human cells. The adaptive response is believed to be a protective mechanism that confers resistance to the detrimental effects of ionizing radiation and that can be induced by different agents, including hyperthermia and radiation. We have used fluorescence analysis of DNA unwinding (FADU) to assay the induction of apoptosis in human peripheral blood lymphocytes by ionizing radiation. Using the FADU assay, we have observed the initial radiation-induced DNA damage, its subsequent disappearance due to enzymatic repair, and its time- and dose-dependent reappearance. We believe this reappearance of DNA damage to be indicative of the DNA fragmentation event associated with apoptosis. This interpretation has been supported at the individual cell level using an in situ terminal deoxynucleotidyl transferase (TDT) assay (Apoptag, Oncor Inc.), which detects the 3′-hydroxyl ends of fragmented DNA, and by fluorescence analysis of nuclear morphology in Hoechst 33258 stained cells. Pretreatment of cells with low-dose γ-radiation (0.1 Gy) or mild hyperthermia (40 °C for 30 min) altered the extent of radiation-induced (3 Gy) apoptosis. Both pretreatments sensitized lymphocytes to become apoptotic after the 3-Gy radiation exposure. This sensitization may represent an adaptive response mechanism that reduces the risk that genetically damaged cells will proliferate. The ability to modify the probability of radiation-induced apoptosis may lower the cancer risk from a radiation exposure.Key words: apoptosis, adaptive response, ionizing radiation, hyperthermia.
Expression of the mdr1 P-glycoprotein gene: a mechanism of escape from glucocorticoid-induced apoptosis
Authors: Gruol Donald J. , Bourgeois Suzanne
Published: December 2009

Glucocorticoid hormones cause apoptosis in the murine T-lymphoma cell line WEHI-7. Glucocorticoid receptors in these cells are cytoplasmic proteins that translocate to the nucleus upon binding hormone. Thus, regulation of cytoplasmic glucocorticoid concentrations controls the level of activated receptors and sensitivity to steroid-induced apoptosis. We found that expression of the mdr1 P-glycoprotein gene produces a reduced accumulation of dexamethasone in WEHI-7 cells. Concomitantly, there is a suppression of dexamethasone-induced changes in transcription and a decrease in steroid sensitivity. P-glycoproteins are known to cause an outward, ATP-dependent transport of a variety of unrelated hydrophobic drugs across the plasma membrane. Our results indicate that glucocorticoid transport by P-glycoproteins depends upon the presence of an hydroxyl group at position 11 of corticosteroids and is enhanced by hydroxyl groups at the positions 16, 17, and 21. The antiprogestin RU486, which contains a dimethyl aminophenyl substitution at the position 11, is not transported by the mdr 1 P-glycoprotein. We have found that RU486 is an inhibitor of P-glycoprotein function, indicating that steroid analogs could be useful chemosensitizers in patients undergoing chemotherapy.Key words: multidrug resistance, glucocorticoids, apoptosis, lymphomas, RU486.
Hormonal regulation of programmed cell death during amphibian metamorphosis
Authors: Tata Jamshed R.
Published: December 2009

Extensive programmed cell death (PCD) is initiated at the onset of amphibian metamorphosis, resulting in 100% of cells dying in some larval tissues, as during total regression of tail and gills. All cell death during metamorphosis is under the control of thyroid hormone (TH), which can initiate the process precociously in whole tadpoles or in individual tissues in culture. The hormone prolactin (PRL), given exogenously, prevents natural and TH-induced metamorphosis. We have exploited this dual hormonal regulation in premetamorphic Xenopus tails in organ culture to identify and characterize early genes that are TH-induced and considered important for initiating cell death. Among the earliest genes activated by TH are those encoding the two thyroid hormone receptors TRα and TRβ. This autoinduction of TR genes is considered important since, in blocking this process, PRL also inhibited the expression of other TH-inducible genes and prevented cell death. The expression of early genes other than TR genes, which are known to promote cell death or survival, is also considered to be important for the initiation of PCD during amphibian metamorphosis. We are, therefore, working on the identification, characterization, and expression of members of the Xenopus bcl-2-like gene family, as well as other genes, such as nur-77 and ICE, which may act as early genes during tadpole tail regression.Key words: cell death, thyroid hormones, Xenopus, metamorphosis, gene expression.
Metabolic events during programmed cell death in insect labial glands
Authors: Halaby Reginald , Zakeri Zahra , Lockshin Richard A.
Published: December 2009

The labial gland of Manduca sexta is a valuable system to study the mechanisms of programmed cell death since the death of the gland is nearly synchronous and, except for the anterior duct, involves all of the tissue. The gland degenerates in 5 days during pupation. Our previous work documents a drop in total protein synthesis as the gland degenerates. To evaluate potential causes of this altered protein synthesis, we monitored several parameters of metabolism in dying cells: levels of adenosine triphosphate to estimate the energy resources of the gland; reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to assess mitochondrial respiration; levels of acid phosphatase to assay lysosomal enzyme activity; and concentrations of cyclic nucleotides and inositol triphosphate to monitor signaling. While protein synthesis fell precipitously on day 0, total adenosine triphosphate and mitochondrial respiration were unchanged until the cells underwent massive collapse on day 3. Lysosomal acid phosphatase increased during early metamorphosis, and ultimately the bulk of the cytoplasm was destroyed in autophagic vacuoles. Changes in the concentrations of second messengers were modest and late. The relationships between the metabolism and the collapse of the labial gland are under investigation.Key words: programmed cell death, Manduca sexta, energetics, lysosomes, second messengers, protein synthesis.
Endonuclease activities, chromatin structure, and DNA degradation in apoptosis
Authors: Walker P. Roy , Sikorska Marianna
Published: December 2009

The degradation of DNA into a specific pattern of fragments is a characteristic feature of apoptosis. Initially, these fragments were visualized by conventional agarose gel electrophoresis as a DNA ladder consisting of multimers of approximately 200 base pairs. Subsequent analysis by pulsed-field gel electrophoresis showed that there were also fragments in the 50- to 300-kilobase range. These size classes of DNA are generated by endonuclease activity at internucleosomal, interloop, and interrosette sites in chromatin and are, therefore, specifically related to the structural organization of chromatin in the nucleus. We have recreated this pattern of degradation in isolated nuclei and established the specific cation requirements for each stage. A model of chromatin degradation in apoptosis is described that incorporates all of the known cation, enzymatic, and structural aspects of the process.Key words: apoptosis, endonuclease, chromatin, calcium, nucleus.
Identification of chromosomes implicated in suppression of apoptosis in somatic cell hybrids
Authors: Speevak Marsha D. , Chevrette Mario
Published: December 2009

In vitro exposure of tumorigenic cell lines to the chemotherapeutic agent PALA (N-(phosphonoacetyl)-L-aspartate) usually results in cell death (shown here to be apoptosis), followed by clonal growth of rare survivors. On the other hand, normal diploid cells respond to PALA by arresting in G1 and G2 of the cell cycle. It was previously suggested that growth control mechanisms might exist to prevent cells from entering S phase under toxic conditions and that genes involved in such mechanisms were mutated or deleted in tumor cells. Interestingly, the tumor suppressor gene p53, a putative G1 control gene, was shown to mediate PALA-induced growth arrest. However, growth arrest occurs in cells that lack wild-type p53, suggesting that other genes are involved as well. To identify these genes, we have generated whole cell hybrids between mouse melanoma and normal human fibroblast cells. At early passage, a whole cell hybrid (BHF12) responds to PALA with growth arrest, while at later passage, the same hybrid undergoes apoptosis. To determine which human chromosomes are required for the PALA-induced growth arrest phenotype, we isolated subclones of the hybrid and tested them for their PALA response. FISH (fluorescence in situ hybridization) and PCR (polymerase chain reaction) amplification have been used to identify the human chromosome content of BHF12 and its subclones. Several human chromosomes, in addition to chromosome 17 (the location of p53), are consistently associated with the growth arrest phenotype. These findings provide evidence that one or more genes in addition to p53 may be involved in the suppression of a drug-induced apoptosis pathway and may lie on one or more of these chromosomes.Key words: apoptosis, growth arrest, N-(phosphonoacetyl)-L-aspartate, somatic cell hybrids.
Effect of lipoproteins on cholesterol synthesis in rat Sertoli cells
Authors: Maboundou Jean-Claude , Fofana Mohamed , Fresnel Jacqueline , Bocquet Jean , Goff Dominique Le
Published: December 2009

Lipoprotein metabolism has been investigated in cultured rat Sertoli cells. Cells incubated with low-density lipoproteins (LDLs) or high-density lipoproteins (HDLs) showed a concentration-dependent decrease of sterol synthesis, indicating a net cholesterol delivery to the Sertoli cells. At 50 μg/mL, lipoproteins inhibited the incorporation of [14C]acetate into free cholesterol by 83% for the LDL and 47% for the HDL. Electron microscopic examinations of the Sertoli cells provide evidence of the internalization of gold-labelled HDL into coated pits and coated vesicles. Competitive studies between human LDL and rat HDL indicate that Sertoli cells take up cholesterol from LDL and HDL containing apolipoprotein (apo) E by common pathways. These results suggest that Sertoli cells possess apo B and E receptors for the uptake and degradation of LDL and HDL, although the basement membrane excludes the passage of LDL from blood capillaries to the Sertoli cells. At 50 μg/mL, apo-E-depleted HDL inhibited the incorporation of [14C]acetate into free cholesterol by 34%. Thus, this study shows that Sertoli cells are capable of taking up apo-E-depleted HDL cholesterol for cell metabolism.Key words: high-density lipoproteins, low-density lipoproteins, rat Sertoli cell.
Detection of dipeptidyl peptidase IV in glioma C6 and neuroblastoma SK-N-SH cell lines
Authors: Sedo Aleksi , Revoltella Roberto P.
Published: December 2009

The dipeptidyl peptidase IV (DPP-IV) activity of the rat glioma cell line C6 and the human neuroblastoma cell line SK-N-SH was investigated. DPP-IV fluorescent substrate was cleaved by both cell lines. The pH reaction optimum determined was typical for DPP-IV described in other cell models. The reaction was inhibited by specific inhibitors diprotins A and B. Enzyme activity was localized, both on the cell surface and intracellularly. Most of the DPP-IV activity was membrane bound. However, soluble intra-cellular activity was found in both cell lines. Secreted activity was not detected in either cell line. In the C6 line, but not in the SK-N-SH line, we demonstrated depression of the ratio of cell surface to total cell DPP-IV activity at higher cell densities, indicating possible enzyme redistribution during cell growth in culture. Identification of DPP-IV activity is the first step in our study of the role of DPP-IV in the neural system.Key words: dipeptidyl peptidase IV, glioma, neuroblastoma.
N-Ethylmaleimide-modified actin filaments do not bundle in the presence of α-actinin
Authors: Milzani Aldo , DalleDonne Isabella , Colombo Roberto
Published: December 2009

We show that the modification of actin subdomain 1 by N-ethylmaleimide (NEM), which binds Cys-374 close to the C-terminus of the molecule, inhibits the α-actinin-induced bundling of actin filaments. This effect is not merely related to the block of Cys-374, since N-(1-pyrenyl)iodoacetamide (pyrene-IA) is unable to prevent bundling. Considering that NEM (but not pyrene-IA) influences actin assembly, we suggest that the inhibition of the actin – α-actinin interaction is due to the chemical modification of actin Cys-374 which, by inducing a marked spatial reorganization of actin monomers, is able to modify both the intra- and inter-molecular interactions of this protein. Finally, NEM-modified actin filaments form bundles in the presence of polyethylene glycol 6000 since, in this case, the side by side association of actin filaments does not depend on the accessibility of binding sites nor on the formation of chemical bonds.Key words: chemically modified actin, N-ethylmaleimide, pyrene-IA, Cys-374, actin bundles, α-actinin.
HPr: a model protein
Authors: Anderson J. W.
Published: December 2009

Histidine-containing protein (HPr) is a central component of the bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS). This brief review covers recent structure–function studies on the active center of this protein: the role of the active center residues in phosphotransfer; the residues contributing to the phosphohydrolysis properties of HPr; and the contribution residues in HPr make to the pKaof the transiently phosphorylated active-site residue, His 15. As well, the potential for HPr to be used as a model protein for studying problems not directly associated with its function in the PTS is discussed.Key words: phosphoenolpyruvate: sugar phosphotransferase system, histidine-containing protein, active center, structure–function, model protein.
Normalization of pancreatic exocrine enzymes by islet transplantation in diabetic rats
Authors: Lee P. C. , Jordan M. , Pieper G. M. , Roza A. M.
Published: December 2009

In an effort to evaluate the effectiveness of islet transplantation in correcting exocrine dysfunction, young male Lewis rats were made diabetic by i.v. streptozotocin injection. Diabetes status was confirmed by decrease in insulin and increase in blood glucose and glycosylated hemoglobin levels. Pancreatic islets were isolated from age-matched control syngeneic rats by collagenase digestion followed by purification through a Ficoll gradient. Islets (~1200) were grafted to the liver by intraportal injection to animals at 8 weeks after diabetes was established. Transplanted rats were sacrificed 4 weeks after correction of hyperglycemia. Diabetes resulted in decrease in body weight. Transplantation reversed the body weight loss and led to a body weight gain. Diabetes resulted in a decrease in pancreatic amylase (1.4 ± 0.4 U/mg protein compared with a control value of 121.9 ± 3.2 U/mg protein) and a slight increase in lipase (87.3 ± 5.5 U/mg protein compared with a control value of 69 ± 4.7 U/mg protein). Transplantation completely normalized amylase (132.2 ± 25.0 U/mg protein) and lipase (56.3 ± 3.9 U/mg protein) in spite of an imperfect correction of blood insulin, glucose, and glycosylated haemoglobin levels in these rats. These data demonstrated that islet transplantation is very effective in correcting the exocrine enzyme changes resulting from diabetes. Evaluation of steady-state levels of amylase mRNA in these groups of animals by Northern blots showed a decrease in the amylase mRNA level in diabetes and a return to that of control in transplanted rats, indicating that the control of amylase expression is most likely at the pretranslational level.Key words: islet transplantation, exocrine enzymes, mRNA.
Characterization of amine oxidase activities in macrophages from human peripheral blood
Authors: Seiler Nikolaus , Moulinoux Jacques-Philippe , Havouis Rene , Toujas Louis
Published: December 2009

A sensitive assay for the determination of hydrogen peroxide formation and a method for the identification and characterization of amine oxidases in cells and other oxidase sources of limited accessibility are described. The enzymes are characterized by substrate and inhibitor patterns. The method was applied to the identification and characterization of polyamine oxidizing enzymes in macrophages from human peripheral blood. The major oxidase activity in these cells was found to be a tissue-type polyamine oxidase, but with distinct characteristics. Diamine oxidase and monoamine oxidase activities were also detected in homogenates of macrophages. Since the formation of toxic products by a releasable polyamine oxidase is supposed to be an integral part of a regulatory function of macrophages, we consider our work as a basis for the elucidation of this function.Key words: macrophages, amine oxidases, polyamine oxidase, diamine oxidase, monoamine oxidase, hydrogen peroxide, fluorimetry.
Detection of a glycosylated form of hen egg white lysozyme
Authors: Trudel Jean , Asselin Alain
Published: December 2009

By assaying lysozyme activity after denaturing polyacrylamide gel electrophoresis of commercial hen egg white lysozyme preparations, minor lysozymal activity was detected as an 18-kDa protein. After electrophoretic purification for microsequencing, the N-terminus sequence of the 18-kDa lysozyme was found to be identical with mature 14.4-kDa hen egg white lysozyme. The 18-kDa hen egg white lysozyme was judged to be glycosylated based on a 3.6-kDa decrease in molecular mass after N-glycosidase F treatment, binding to concanavalin A – Sepharose, and staining with periodate – Schiff's reagent. The minor form corresponded to about 0.3% of lyzozyme molecules.Key words: lysozyme, glycosylation, sequential PAGE, N-terminus microsequencing.
Cellular adhesiveness, contractility, and traction: stick, grip, and slip control
Authors: Opas Michal
Published: December 2009

Translocation of cells over solid substrata depends on generation of motive force, in crawling tissue cells, brought about by regulated contractility of intracellular actomyosin. Intracellular contractile machinery has a direct, structural connection to the cell surface. Hence, regulated adhesiveness of the cell surface provides a mechanism whereby a cell can fine tune the extent of tractional forces that are necessary for effective translocation. Cells are able to control adhesiveness of surfaces (stick), contractility (grip), and the extent of traction exerted on the substratum (slip). Here, I discuss several aspects of local (subcellular) regulation of adhesiveness and contractility and speculate on how cells, given a choice of the substratum, decide on how and where to apply traction.Key words: cell adhesion, focal contacts, cell motility, traction, cytomechanics.
Mechanoreception at the cellular level: the detection, interpretation, and diversity of responses to mechanical signals
Authors: Banes Albert J. , Tsuzaki Mari , Yamamoto Juro , Brigman Brian , Fischer Thomas , Brown Thomas , Miller Larry
Published: December 2009

Cells from diverse tissues detect mechanical load signals by similar mechanisms but respond differently. The diversity of responses reflects the genotype of the cell and the mechanical demands of the resident tissue. We hypothesize that cells maintain a basal equilibrium stress state that is a function of the number and quality of focal adhesions, the polymerization state of the cytoskeleton, and the amount of extrinsic, applied mechanical deformation. A load stimulus detected by a mechano-electrochemical sensory system, including mechanically sensitive ion channels, integrin–cytoskeleton machinery, and (or) a load-conformation sensitive receptor or nonreceptor tyrosine kinase, may activate G proteins, induce second messengers, and activate an RPTK or JAK/STAT kinase cascade to elicit a response. We propose the terms autobaric to describe a self-loading process, whereby a cell increases its stress state by contracting and applying a mechanical load to itself, and parabaric, whereby a cell applies a load to an adjacent cell by direct contact or through the matrix. We predict that the setpoint for maintaining this basal stress state is affected by continuity of incoming mechanical signals as deformations that activate signalling pathways. A displacement of the cytoskeletal machinery may result in a conformational change in a kinase that results in autophosphorylation and cascade initiation. pp60Src is such a kinase and is part of a mechanosensory protein complex linking integrins with the cytoskeleton. Cyclic mechanical load induces rapid Src phosphorylation. Regulation of the extent of kinase activation in the pathway(s) may be controlled by modulators such as G proteins, kinase phosphorylation and activation, and kinase inhibitors or phosphatases. Intervention at the point of ras–raf interaction may be particularly important as a restriction point.Key words: mechanoreception, cells, in vitro, load deformation.
Type I and type VIII adenylyl cyclases constitute a family whose activation is coupled to cellular deformation through the action of calcium–calmodulin
Authors: Watson Peter A. , Giger Kathryn E. , Kempinski Anna M.
Published: December 2009

In certain tissues and cells, increases in concentrations of the second messenger cAMP are seen in response to mechanical or deformational stimuli. Type I and type VIII adenylyl cyclases, representing members of a family of calcium–calmodulin-stimulated adenylyl cyclases, and type VII adenylyl cyclase were each stably expressed in human embryonal kidney (HEK) 293 cells. HEK 293 cells exogenously expressing either type I adenylyl cyclase or any one of three type VIII adenylyl cyclase splice variants respond to swelling with increases in cAMP, requiring the presence of calcium in the extracellular medium for such responsiveness. Type VII expressing HEK 293 cells failed to respond to swelling with increased cAMP but demonstrated potentiationof isoproterenol-stimulated activity. This is characteristic of the influence of protein kinase C on the activity of the type VII protein. The relative swelling responsiveness of HEK 293 cells expressing splice variants of the type VIII adenylyl cyclase is consistent with the relative EC50values for calcium–calmodulin stimulation of these splice variants. This is consistent with the involvement of calmodulin and the requirement for increases in intracellular calcium in mediating swelling-induced acceleration of type VIII adenylyl cyclase activity.Key words: mechanotransduction, cAMP, calcium, calmodulin, cytoskeleton.
Simian virus 40 transformation alters the actin cytoskeleton, expression of matrix metalloproteinases and inhibitors of metalloproteinases, and invasive behavior of normal and ataxia-telangiectasia human skin fibroblasts
Authors: Hansell Elizabeth J. , Frisch Steven M. , Tremble Patrice , Murnane John P. , Werb Zena
Published: December 2009

Alterations in the actin cytoskeleton of normal cells result in changes in cell shape and adhesiveness and induce expression of matrix-degrading matrix metalloproteinases. We examined the effect of simian virus 40 transformation of normal and ataxia-telangiectasia human skin fibroblasts, a process that produces actin reorganization, altered cell morphology, and altered cell behavior, on expression of genes of the matrix metalloproteinase and tissue inhibitor of metalloproteinases gene families. Simian virus 40 transformation induced collagenase-1 gene expression; in contrast, stromelysin-1, 72-kDa gelatinase (gelatinase A), tissue inhibitor of metalloproteinases-1, and tissue inhibitor of metalloproteinases-2 genes were repressed. Transformation also altered the response of the fibroblasts to 12-O-tetradecanoylphorbol-13-acetate. Collagenase mRNA was induced in 12-O-tetradecanoylphorbol-13-acetate treated transformed cells up to 50-fold more than in untreated transformed cells or in 12-O-tetradecanoylphorbol-13-acetate treated untransformed parent cells. In contrast, 12-O-tetradecanoylphorbol-13-acetate did not overcome the attenuated expression of stromelysin-1 in the simian virus 40 transformants. In addition, 92-kDa gelatinase (gelatinase B) was induced by 12-O-tetradecanoylphorbol-13-acetate only in the simian virus 40 transformants. The responses of gelatinase A and tissue inhibitor of metalloproteinases-1 to 12-O-tetradecanoylphorbol-13-acetate were unchanged. The pattern of altered proteinase expression after transformation was accompanied by a phenotypic alteration in cell invasion. The simian virus 40 transformants exhibited enhanced invasiveness through a basement-membrane-like matrix. These data demonstrate that enhanced invasiveness in simian virus 40 transformed cells is accompanied by changes in actin organization and expression of proteinases and inhibitors, as well as in the balance between proteinases and inhibitors in favor of proteinases.Key words: actin cytoskeleton, collagenase, metalloproteinase, tissue inhibitor of metalloproteinases, SV40 transformation, ataxia-telangiectasia.
Modeling chemoattractant-elicited relocalization of myosin filaments inDictyostelium
Authors: He Xiaoyi , Dembo Micah
Published: December 2009

Dictyostelium myosin is able to assemble into filaments that, when visualized under normal conditions, appear to be uniformly distributed throughout the cytoplasm. After stimulation by the chemoattractant cAMP, these filaments quickly diminish in the cellular medulla and accumulate in the cortex. A general hypothesis to explain the mechanism of this relocalization proposes that one or more of the chemical coefficients governing filament polymerization is precisely regulated by some sort of intracellular second messenger. To investigate this hypothesis we formulated a simple theoretical model of myosin polymerization and then used this model to analyze the resting state of the cell and various scenarios for initializing a transition to the activated state. In general, we found that the relocalization of filaments could be realized if a second messenger increased the elongation and (or) the nucleation coefficients for filament assembly in cortical ectoplasm and (or) if the messenger decreased these parameters in the cellular medulla. By comparing these limiting cases with experimental observations, we concluded that models in which redistribution of myosin is achieved by decreasing filament stability in the medulla are the most likely candidates.Key words: cytoskeleton, amoeboid motility, chemotaxis, Dictyostelium, myosin.
Properties of circulating leukocytes in spontaneously hypertensive rats
Authors: Shen K. , Sung K. -L. P. , Whittemore D. E. , DeLano F. A. , Zweifach B. W. , Schmid-Schönbein G. W.
Published: December 2009

The factors responsible for predisposition to progressive organ injury and vascular complications in arterial hypertension are uncertain. Recent evidence shows that leukocytes participate in cardiovascular conditions for which hypertension is a risk factor. Therefore, there is a need to define the properties of circulating leukocytes in hypertensives. There are about twice as many circulating leukocytes in spontaneous hypertensive rats (SHRs) compared with their normotensive controls, the Wistar–Kyoto rats (WKYs). The SHR neutrophils are viscoelastic and similar to neutrophils in WKYs but exhibit lower deformability in short-term elastic deformation. Mature SHRs have elevated levels of spontaneous pseudopod formation. Mild stimulation with N-formyl-Met-Leu-Phe or platelet-activating factor (10−8 M) results in a significantly enhanced level of neutrophil pseudopod formation in SHRs but not in WKYs. SHRs exhibit higher levels of spontaneous superoxide formation. Alkaline phosphatase content of individual circulating neutrophils in SHRs is on average lower while plasma levels of alkaline phosphatase in the same samples are elevated in the SHRs. Spontaneous degranulation of SHR neutrophils is also detectable with myeloperoxidase measurements. Such activity of circulating leukocytes poses a significant risk for vascular cytotoxicity in the hypertensive rats.Key words: neutrophil, degranulation, alkaline phosphatase, myeloperoxidase, nitro blue tetrazolium reduction, rheological properties, adhesion.
Endotoxin, tumor necrosis factor, and dexamethasone effects on human endothelial cell fibronectin dynamics: synthesis, matrix assembly, and receptor expression
Authors: Romer Lewis H. , Polin Richard A.
Published: December 2009

The three inflammatory modulators endotoxin, tumor necrosis factor (TNF) α, and dexamethasone (DEX) were studied for their effects on fibronectin (FN) dynamics in human umbilical vein endothelial cells. Cell culture supernatants were analyzed for new soluble pool FN synthesis. Endotoxin (LPS) (10 μg/mL) decreased the newly synthesized soluble pool of FN (p < 0.05). An increase in soluble FN was demonstrated with 1 and 10 ng/mL TNF α (p < 0.05). DEX decreased newly synthesized endothelial cell (EC) FN in the soluble pool at 4, 40, and 400 μg/mL (p < 0.05). Extracellular matrix FN content was examined using immunofluorescence. The thick FN mesh seen in control cells contrasted with a decreased FN matrix after treatment with each of the three study agents. Immunoprecipitation of the FN receptor α5β1integrin from [35S]methionine-labelled cell extracts demonstrated down regulation of receptor expression by both TNF α and DEX as compared with control samples. These data indicate that LPS, TNF α, and DEX may weaken EC–substratum adhesion by differential effects on FN synthesis and secretion, FN incorporation into the extracellular matrix, and down regulation of FN receptor expression.Key words: endothelium, fibronectin, extracellular matrix, integrin, cytokine.
Cytoskeletal mechanics of neurulation: insights obtained from computer simulations
Authors: Brodland G. Wayne , Clausi David A.
Published: December 2009

The morphogenetic movements associated with the process of neurulation have been the subject of much investigation during the last one hundred years. A plethora of experimental evidence has been generated regarding the forces that drive this seemingly simple process, and many theories about the mechanics of the process have been proposed. Recent computer simulations have proved useful for evaluating these theories from a mechanical perspective. In this work, computer simulations are used to investigate several theories about the forces that drive neurulation. A simplified version of a formulation previously presented by the authors provides the mathematical foundation for these simulations. The simulations confirm that forces generated by circumferential microfilament bundles (CMB's) in conjunction with notochord forces can produce the rolling motions characteristic of amphibian neurulation. They also support the notion that redundancies exist in the systems of forces available to drive neurulation shape changes. The shape changes that occur following a variety of surgical and teratogenic interventions are also simulated. These simulations corroborate the role of circumferential microfilament bundles as a primary force generator.Key words: neurulation, cytomechanics, computer simulations, finite element method.
Biomechanical feedback in morphogenesis, as exemplified by stretch responses of amphibian embryonic tissues
Authors: Beloussov L. V. , Luchinskaia N. N.
Published: December 2009

We explore the idea that morphogenetical processes may be self-regulated by the biomechanical feedback established between the active stress-generating devices and the passive stresses of stretching and (or) compression, these feedback directed towards hyperrestoration (restoration with overlapping) of the initial stress values. As an example, a stretch-induced behaviour of the pieces of ventral ectoderm of Xenopus laevis early gastrulae is considered. By stretching the explants in 1.3–1.7 times, we induced several active poststretching cell responses, including further autonomous elongation of an explant in the stretch direction and contraction in the perpendicular direction, as well as more complicated shape changes. At the cellular level, these responses were associated with the return of the stretched cells to isodiametrical shapes and with the production of extensive cell protrusions along the stretch direction. As shown by dissections, the stretch-induced tissue tensions were considerably diminished in the poststretching period. The results obtained are discussed within the framework of the hyperrestoration hypothesis.Key words: biomechanics, stretch responses, morphogenesis, amphibian embryonic tissue.
Global regulators of ribosome biosynthesis in yeast
Authors: Planta Rudi J. , Gonçalves Paula M. , Mager Willem H.
Published: December 2009

Three abundant ubiquitous DNA-binding protein factors appear to play a major role in the control of ribosome biosynthesis in yeast. Two of these factors mediate the regulation of transcription of ribosomal protein genes (rp-genes) in yeasts. Most yeast rp-genes are under transcriptional control of Rap1p (repressor–activator protein), while a small subset of rp-genes is activated through Abf1p (ARS binding factor). The third protein, designated Reb1p (rRNA enhancer binding protein), which binds strongly to two sites located upstream of the enhancer and the promoter of the rRNA operon, respectively, appears to play a crucial role in the efficient transcription of the chromosomal rDNA. All three proteins, however, have many target sites on the yeast genome, in particular, in the upstream regions of several Pol II transcribed genes, suggesting that they play a much more general role than solely in the regulation of ribosome biosynthesis. Furthermore, some evidence has been obtained suggesting that these factors influence the chromatin structure and create a nucleosome-free region surrounding their binding sites. Recent studies indicate that the proteins can functionally replace each other in various cases and that they act synergistically with adjacent additional DNA sequences. These data suggest that Abf1p, Rap1p, and Reb1p are primary DNA-binding proteins that serve to render adjacent cis-acting elements accessible to specific trans-acting factors.Key words: Abf1p, Rap1p, Reb1p, yeast, ribosome biosynthesis.
Pleiotropic effects of mutations at positions 13 and 914 inEscherichia coli16S ribosomal RNA
Authors: Brakier-Gingras Léa , Pinard Robert , Dragon François
Published: December 2009

Mutations at position 13 or 914 of Escherichia coli 16S ribosomal RNA exert pleiotropic effects on protein synthesis. They interfere with the binding of streptomycin, a translational miscoding drug, to the ribosomes. They increase translational fidelity, and this effect can be related to a perturbation of the higher order structure of the 530 stem–loop, a key region for tRNA selection. In contrast, the structure of the decoding center is not perturbed. The mutations also affect translational initiation, slowing down the formation of the 30S initiation complex. This effect can be related to a destabilization of the pseudoknot helix (17–19/916–918), at the convergence of the three major domains of 16S ribosomal RNA.Key words: ribosomal RNA, translational accuracy, translational initiation.
The elongating ribosome: structural and functional aspects
Authors: Nierhaus Knud H. , Beyer Dieter , Dabrowski Marylena , Schäfer Markus A. , Spahn Christian M. T. , Wadzack Jörg , Bittner Jörg-Uwe , Burkhardt Nils , Diedrich Gundo , Jünemann Ralf , Kamp Detlev , Voss Helga , Stuhrmann Heinrich B.
Published: December 2009

We determined the positions and arrangements of RNA ligands within the ribosome with a new neutron-scattering technique, the proton-spin contrast-variation. Two tRNAs were bound to the ribosome in the pre-translocational and the post-translocational state. The mass centre of gravity of both tRNAs resides at the subunit interface of the body of the 30S subunit. Both tRNAs are separated by an angle of 50–55°, and their mutual arrangement does not change during translocation. The mass centre of gravity moves by 13 ± 3Å(1Å = 0.1 nm) during translocation, corresponding well with the length of one codon. Using an RNase-digestion technique, the length of the mRNA sequence covered by the ribosome was determined to be 39 ± 3 nucleotides before and after translocation. The ribosome moves like a rigid frame along the mRNA during translocation. In contrast, both tRNAs seem to be located on a movable ribosomal domain, which carries the tRNAs before, during, and after translocation, leaving the microtopography of the tRNAs with the ribosome unaltered. This conclusion was derived from an analysis of the contract patterns of thioated tRNAs on the ribosome. The results have led to a new model of the elongation cycle, which reinterprets the features of the previous "allosteric three-sites model" in a surprisingly simple fashion. Finally, a mutational analysis has identified a single nucleotide of the 23S rRNA essential for the peptidyltransferase activity.Key words: tRNA–ribosomal contacts, neutron scattering, location of tRNAs in ribosomes, movement of ribosome on mRNA, peptidyltransferase.
Escherichia coliinitiator tRNA: structure–function relationships and interactions with the translational machinery
Authors: Mangroo Dev , Wu Xin-Qi , Rajbhandary Uttam L.
Published: December 2009

We showed previously that the sequence and (or) structural elements important for specifying the many distinctive properties of Escherichia coli initiator tRNA are clustered in the acceptor stem and in the anticodon stem and loop. This paper briefly describes this and reviews the results of some recently published studies on the mutant initiator tRNAs generated during this work. First, we have studied the effect of overproduction of methionyl-tRNA transformylase (MTF) and initiation factors IF2 and IF3 on activity of mutant initiator tRNAs mat are defective at specific steps in the initiation pathway. Overproduction of MTF rescued specifically the activity of mutant tRNAs defective in formylation but not mutants defective in binding to the P site. Overproduction of IF2 increased me activity of all mutant tRNAs having the CUA anticodon but not of mutant tRNA having me GAC anticodon. Overproduction of IF3 had no effect on the activity of any of me mutant tRNAs tested. Second, for functional studies of mutant initiator tRNA in vivo, we used a CAU→CUA anticodon sequence mutant mat can initiate protein synthesis from UAG instead of AUG. In contrast with me wild-type initiator tRNA, the mutant initiator tRNA has a 2-methylthio-N6-isopentenyl adenosine (ms2i6A) base modification next to the anticodon. Interestingly, this base modification is now important for activity of the mutant tRNA in initiation. In a miaA strain of E. coli deficient in biosynthesis of ms2i6A, the mutant initiator tRNA is much less active in initiation. The defect is specifically in binding to the ribosomal P site.Key words: initiator tRNA, initiation Factors, formylation, P site binding, base modification.
Histidine 229 in protein L2 is apparently essential for 50S peptidyl transferase activity
Authors: Cooperman Barry S. , Wooten Tammy , Traut Robert R. , Romero Daniel P.
Published: December 2009

It has recently been suggested that peptidyl transferase activity is primarily a property of ribosomal RNA and that ribosomal proteins may act only as scaffolding. On the other hand, evidence from both photoaffinity labeling studies and reconstitution studies suggest that protein L2 may be functionally important for peptidyl transferase. In the work reported here, we reconstitute 50S subunits in which the H229Q variant of L2 replaces L2, with all other ribosomal components remaining unchanged, and determine the catalytic and structural properties of the reconstituted subunits. We observe that mutation of the highly conserved His 229 to Gin results in a complete loss of peptidyl transferase activity in the reconstituted 50S subunit. This is strong evidence for the direct involvement of L2 in ribosomal peptidyl transferase activity. Control experiments show that, though lacking peptidyl transferase activity, 50S subunits reconstituted with H229Q-L2 appear to be identical with 50S subunits reconstituted with wild-type L2 with respect to protein composition and 70S formation in the presence of added 30S subunits. Furthermore, as shown by chemical footprinting analysis, H229Q-L2 appears to bind 23S RNA in the same manner as wild-type L2. Thus, the effect of H229 mutation appears to be confined to an effect on peptidyl transferase activity, providing the most direct evidence for protein involvement in this function to date.Key words: protein L2, site-specific mutagenesis, peptidyl transferase, reconstitution, histidine.
Fragmentation of the ribosome to investigate RNA–ligand interactions
Authors: Howard Barbara-Ann , Thom George , Jeffrey Ian , Colthurst Dave , Knowles David , Prescott Catherine
Published: December 2009

RNA molecules perform a variety of important and diverse functions and, therefore, an understanding of their structure and interaction with proteins and ligands is essential. Large RNA molecules (for example, the ribosomal RNAs) are complex and hence reports describing their fragmentation into functional subdomains has provided a means for their detailed analysis. We present here an in vivo approach to study RNA–ligand interactions. This is based on the concept that an RNA fragment could mimic a drug-binding site present on the intact molecule. Overexpression of the fragment would sequester the drug thereby permitting the continued functioning of the ribosome and, thus, ensuring cell viability. Accordingly, a fragment of 16S rRNA encompassing the spectinomycin-binding domain in helix 34 (nucleotides 1046–1065 and 1191–1211) was cloned and in vivo expression resulted in drug resistance. Furthermore, an RNA fragment lacking flanking sequences to helix 34 was also selected from among a pool of random rRNA fragments and shown to confer spectinomycin resistance. A similar in vitro approach is also described for the analysis of rRNA molecules that interact with the yeast elongation factor 3 (EF-3).Key words: rRNA, spectinomycin, EF-3.
Cotranslational folding of nascent proteins onEscherichia coliribosomes
Authors: Hardesty Boyd , Kudlicki Wieslaw , Odom O. W. , Zhang Tong , McCarthy Diane , Kramer Gisela
Published: December 2009

Evidence is presented for cotranslational folding of rhodanese or ricin during its synthesis on Escherichia coli ribosomes. During transcription–translation, full-length but enzymatically inactive polypeptides accumulated as peptidyl-tRNA on the ribosomes. These polypeptides were activated and released by subsequent incubation with the bacterial chaperones and with release factor (RF-2). Coumarin was incorporated cotranslationally at the N-terminus of the nascent protein from fluorophore-S-Ac-Met-tRNAf. Changes in fluorescence indicated that DnaJ bound to the nascent proteins and to a fluorescently labeled synthetic peptide corresponding to the N-terminal 17 amino acids of bovine rhodanese. This peptide also bound to 70S ribosomes or 50S subunits but not to 30S subunits. It inhibited activation and RF-2-dependent release of the full-length ribosome-bound rhodanese. A deletion mutant of rhodanese lacking the N-terminal 23 amino acids was not accumulated on the ribosome but was synthesized very efficiently. However, the protein that was formed was enzymatically inactive. DnaJ did not bind to this deletion mutant on ribosomes. We conclude that the chaperone-mediated reactions facilitate binding of the N-terminal sequence of nascent proteins to a specific site on 50S ribosomal subunits where it blocks release. The ribosome-bound protein undergoes chaperone-mediated reactions that are required for folding into an enzymatically active conformation.Key words: protein synthesis, ribosome, chaperone, protein folding, nascent peptide.
Constitutive expression of the E2F1 transcription factor in fibroblasts alters G0 and S phase transit following serum stimulation
Authors: Logan Thomas J. , Jordan Kelly L. , Hall David J.
Published: December 2009

The E2F1 transcription factor was constitutively expressed in NIH3T3 fibroblasts to determine its effect on the cell cycle. These E2F1 cell lines were not tightly synchronized in G0 phase of the cell cycle following serum starvation, as are normal fibroblasts. Instead, the cells are spread throughout G0 and G1 phase with a portion of the population initiating DNA synthesis. Upon serum stimulation, the remaining cells in G0/G1 begin to enter S phase immediately but with a reduced rate. Constitutive expression of E2F1 appears to primarily affect the G0 phase, since transit of proliferating E2F1 cell lines through G1 phase is the same as control cells. Consistent with a shortened G0 phase, the E2F1 cell lines have a significantly reduced cellular volume. Additionally, the first S phase after serum stimulation, but not subsequent S phases, is nearly doubled in the E2F1 cell lines compared with control cells. Cell lines expressing a deletion mutant of E2F1 (termed E2F1d87), known to significantly affect cell shape, have cell cycle and volume characteristics similar to the E2F1 expressing cells. However, all S phase durations are considerably lengthened and the cells demonstrate delayed growth after plating.Key words: cell cycle, E2F1 transcription factor, G0/G1 phase.