bluuebunny/crossref_metadata_2025 huggingface dataset:
Progesterone and cell–cell adhesion interact to regulate rat granulosa cell apoptosis
Authors: Peluso John J. , Pappalardo Anna
Published: December 2009

Ovarian follicles are composed of both small and large granulosa cells, but only the large granulosa cells undergo apoptosis within 24 h of culture in serum-free medium. The present study was designed to assess the relationship between cell–cell contact, progesterone treatment, and granulosa cell apoptosis. For this study, individual large granulosa cells were isolated from immature rat ovaries after sequential incubation with EGTA and EGTA–sucrose solutions. Granulosa cells were then cultured for 24 h in RPMI-1640 (control) supplemented with progesterone and (or) the progesterone antagonist RU 486. The cells were then fixed and assessed for apoptosis by either electron microscopy or in situ end labeling of DNA fragments. After 24 h of culture, the proportion of apoptotic granulosa cells was twofold lower for aggregated cells compared with single granulosa cells (p < 0.05). Aggregated granulosa cells were observed to be connected by gap junctions. Compared with controls, progesterone reduced and RU 486 increased the percentage of single and aggregated apoptotic granulosa cells present after culture. In the presence of RU 486, progesterone reduced the percentage of apoptotic single granulosa cells from 84 ± 4% (RU 486 alone) to 66 ± 8%. In granulosa cell aggregates, progesterone reduced the incidence of apoptosis from 86 ± 3% to 44 ± 7% (p < 0.05). Progesterone in the presence of RU 486 was more effective in inhibiting apoptosis of aggregated granulosa cells than in single granulosa cells (p < 0.05). Taken together, these data indicate that (i) progesterone acts through the progesterone receptor to inhibit granulosa cell apoptosis and (ii) cell–cell adhesion enhances progesterone's anti-apoptotic actions.Key words: rat, ovary, granulosa cell, apoptosis.
In situ end labeling of fragmented DNA in induced ovarian atresia
Authors: D'herde Katharina , Pestel Guido De , Roels Frank
Published: December 2009

Apoptosis is studied in a model of induced follicular atresia in the ovary of Japanese quail (Coturnix coturnix japonica) by in situ end labeling of DNA fragments in granulosa cells using two different techniques (incorporation of labeled nucleotides by DNA polymerase I or terminal deoxynucleotidyl transferase). The most remarkable observation related to apoptosis in this model is the predominant cytoplasmic localization of labeled DNA fragments, while DNA fragmentation appears to be absent from compacted chromatin masses of apoptotic nuclei and apoptotic nuclear fragments. Unstained apoptotic bodies are present adjacent to stained ones, so that their detection rate on hematoxylin + eosin stained sections is better than on the in situ end-labeled sections. This suggests that DNA fragmentation is a late event or not obligatory in apoptotic granulosa cell death. In contrast to similar studies on atretic granulosa in mammalian models, the process of apoptosis is asynchronous in the granulosal epithelium, with a majority of nuclei with normal chromatin configuration remaining negative for DNA fragmentation. Finally it is shown that the techniques used are not specific for apoptosis, as DNA fragmentation in necrotic granulosa cells is detected as well.Key words: programmed cell death, apoptosis, in situ DNA end labeling, endonuclease, necrosis.
Separate pools of endonuclease activity are responsible for internucleosomal and high molecular mass DNA fragmentation during apoptosis
Authors: Pandey Siyaram , Walker P. Roy , Sikorska Marianna
Published: December 2009

The oligonucleosomal pattern of DNA fragmentation is the best-characterized biochemical marker of apoptosis and believed to be generated by a, as yet unidentified, Ca2+,Mg2+-dependent endonuclease. All apoptotic cells fragment their genome. However, not every cell type undergoing apoptosis is capable of internucleosomal DNA cleavage. We have analyzed the endonuclease activities and patterns of DNA fragmentation in four established cell lines undergoing apoptosis following serum deprivation, i.e., rat 5123tc hepatoma and PC12 pheochromocytoma, as well as human MCF7 breast and DU145 prostatic carcinoma cells. Whereas apoptotic 5123tc and PC12 cells degraded their DNA into oligonucleosomes, the MCF7 and DU145 cells generated only >50 kilobase pairs (kbp) DNA fragments. However, when isolated nuclei from all four cell lines were incubated with both Ca2+ and Mg2+ ions, their DNA was cleaved into internucleosomal fragments. Following washing with a low ionic strength buffer, the nuclei could only degrade DNA to >50-kbp fragments. DNA ladders were produced again in these washed nuclei after reconstitution with the nuclear wash, which contained an endonucleolytic activity of approximately 97 kilodaltons. These experiments showed that cells maintain separate pools of endonucleolytic activities responsible for the high and low molecular mass DNA fragmentation, and depending on the cell type, one or both enzymatic pools become activated during apoptosis.Key words: chromatin, nucleus, PC12, MCF7, DU145.
Kinetics of apoptosis and secondary necrosis in cultured rat thymocytes and S.49 mouse lymphoma and CEM human leukemia cells
Authors: Cejna M. , Fritsch G. , Printz D. , Schulte-Hermann R. , Bursch W.
Published: December 2009

Cell culture systems are widely used to study metabolic changes during apoptosis. In cell culture, unlike in vivo, apoptotic cells are not phagocytosed and eventually lyse (secondary necrosis). This is of practical importance because metabolic changes seen in cultures may be due to the transition from apoptosis to necrosis, rather than to the induction of apoptosis itself. In the present study, we followed the kinetics of the occurrence of several indicators of cell death in rat thymocytes and mouse lymphoma (S.49), and human leukemia (CEM) cell cultures after dexamethasone treatment (10−6 M). The presence of apoptosis and secondary necrosis was demonstrated by electron microscopy. Nuclear condensation and fragmentation, which are considered to reflect early stages of apoptosis, were visualized with Hoechst fluorescent dye H 33258 for quantitative determination by light microscopy. In S.49 and CEM cultures their incidence increased after glucocorticoid treatment, but remained at relatively low levels not exceeding 6–9% until 36 h (S.49) or 3–4% until 92 h (CEM). The trypan blue positive cells, however, increased steadily to about 60%. Furthermore, flow cytometry (single parameter DNA analysis after propidium iodide staining) revealed the occurrence of cells with reduced DNA fluorescence. Morphological and biochemical (internucleosomal DNA cleavage) analysis of FACS-sorted cells showed that early after dexamethasone the majority of them were apoptotic. In S.49 and CEM cell cultures no clear-cut time lag between increase in cells with reduced DNA fluorescence, chromatin condensation/fragmentation, and the uptake of trypan blue could be detected. Rat thymocytes, however, exhibited a time lag between the increase in cell number with reduced DNA fluorescence and trypan blue index, but only until 8 h after treatment. Thereafter, again no clear-cut time lag between these indicators of cell death could be observed. This suggests that, at least in thymocyte cultures, secondary necrosis ensues apoptosis and both can be discriminated for a short period of time. Within this period of time we could detect increased levels of transglutaminase mRNA levels. On the other hand, clusterin mRNA levels did not increase after dexamethasone treatment. Taken together, these findings suggest that rat thymocytes, S.49, and CEM cells enter apoptosis asynchronously during the entire period of investigation and then rapidly progress into secondary necrosis.Key words: cell culture, apoptosis, secondary necrosis, electron microscopy, flow cytometry, Hoechst 33258, transglutaminase, clusterin.
High-pressure freeze fixation reveals novel features during ontogenesis of the vegetative cell inLedebouriapollen: an ultrastructural and cytochemical study
Authors: Hess Michael W.
Published: December 2009

The ultrastructure of the vegetative cell in the pollen of Ledebouria socialis Roth (Hyacinthaceae) was investigated from microspore mitosis to anthesis. As a result of the good preservation quality achieved with high-pressure freeze fixation and freeze substitution, novel structural features were observed. Extensive endomembrane compartments emerging at the onset of lipid and starch mobilization, were identified as protein bodies by using video-enhanced contrast light microscopy. Thus, proteins, apart from starch and lipids, represent a third class of important intermediary storage substances in developing pollen. The close spatial relationship between protein bodies, endoplasmic reticulum (ER), and storage lipids suggest that protein bodies and ER contribute to lipid digestion. Immediately prior to anthesis the protein bodies become transformed into unspecialized vacuoles as a result of the gradual dissolution of their contents; the formation of the protein bodies remains still to be elucidated. The ER proliferates extensively during pollen ontogenesis, thereby changing its ultrastructure and spatial organization. Microfilaments were detected during all developmental stages, in particular microtubule-associated single microfilaments. The microfilaments are likely to be composed of actin as shown by immunogold labeling.Key words: angiosperm pollen, freeze substitution, protein bodies, microfilaments, Hyacinthaceae.
Characterization, chromosomal mapping, and expression of different polyubiquitin genes in tissues from control and heat-shocked maize seedlings
Authors: Liu Ling , Maillet Daniel S. , Frappier J. Roger H. , Walden David B. , Atkinson Burr G.
Published: December 2009

Polyubiquitin transcripts accumulate in plant and animal cells following a heat shock. Most species have a few to several polyubiquitin genes; within a species, the genes may differ in nucleotide (nt) sequence and (or) the number of 228-nt repeats encoding the ubiquitin monomer. This study examines three maize (inbred Oh43) polyubiquitin genes. Two of the genes, MubG9 and MubG5, possess five repeats; the third, MubG1 possesses seven repeats. Sequence analyses of the genomic clones, MubG9 and MubG1 and a cDNA clone, MubG5, reveal that they differ primarily from each other in their nt sequences 5′ and 3′ to their open reading frames. MubG1 contains a 1004-base pair (bp) intron in its 5′ untranslated region. Using gene-specific probes, we show that the amount of polyribosome-associated mRNA transcripts from MubG9 isolated from 2- and 5-day old plumules and radicles is unchanged by heat shock. While the amount of transcript from MubG1 and MubG5 on the polyribosomes in plumules and radicles of 2-day-old seedlings is also unchanged by heat shock, the levels of these transcripts are elevated considerably in similar tissues from heat-shocked 5-day-old seedlings. Similar or identical gene-specific probes were employed to map the genes using the recombinant inbred method. MubG9 maps to chromosome 4L position 186; MubG1 maps to 5L104 and MubG5 to 4L188. The opportunity to use gene-specific probes extends the evidence for distinct modulation (time and tissue) of polyubiquitin gene expression in maize and provides the basis for locus assignment within the genome.Key words: ubiquitin, maize, heat shock, heat-shock proteins, gene expression, chromosome map.
Prior treatment with heat shock attenuates the stress response in isolated working rat hearts
Authors: Dakin Todd A. , Currie R. William
Published: December 2009

We examined the expression of the mRNAs encoding for the inducible heat shock protein (HSP) 71 and the constitutively synthesized HSP73 in control and 24-h post-heat-shocked (post-HS) hearts during isolated working heart perfusion. Paired control and 24-h post-HS rat hearts were perfused in the working heart mode for 1, 2, 3, or 4 h. Aortic and coronary flow rates and heart rates were not different between the control and 24-h post-HS hearts during the perfusion periods. After perfusion, total RN A was extracted and separated by gel electrophoresis. RNA was blotted to membranes, subsequently probed with 32P-labelled cDNA probes for HSP71 and HSP73 transcripts, and autoradiographed. Control hearts showed a sharp increase in transcripts for HSP71 and a more moderate increase in transcripts for HSP73 accumulation during perfusion. However, the increase in HSP71 and HSP73 transcripts in the HS hearts was markedly less than that in the control hearts. This suppression in gene expression in the HS hearts seems to suggest a negative control mechanism regulating transcription of mRNA encoding HSP71 and HSP73.Key words: mRNA, heart, Northern analysis, working heart perfusion, heat shock.
Glycosaminoglycans from two human malignant mesothelioma cell lines: determination, distribution, and effect of platelet-derived growth factor on their synthesis
Authors: Tzanakakis George N. , Karamanos Nikos K. , Klominek Julius , Hjerpe Anders
Published: December 2009

The synthesis and distribution of glycosaminoglycans (GAGs) were studied in two human malignant mesothelioma cell lines: one with fibroblast-like morphology and the other with epithelial differentiation. Analyses using highly sensitive high-pressure liquid chromatography techniques and agarose gel electrophoresis showed that these cells produce not only hyaluronan (HA) but also galactosaminoglycans (GalAGs, chondroitin sulfate and (or) dermatan sulfate) and heparan sulfate (HS). In both cell lines most of the HA (87–90%) and GalAGs (57–66%) are secreted into the extracellular matrix. Although HS is mainly bound to the cell surface in fibroblast-differentiated cells (75%), in epithelial type cells only 40% occurs in the cell-associated fraction. The amounts of secreted GAGs are 6- to 8-fold higher in epithelial than in fibroblast-like mesothelioma cultures. In cells with the fibroblast phenotype, the β-homodimer of platelet-derived growth factor (PDGF) in a concentration of 1.5 ng/mL stimulates HA and GalAG synthesis 5-fold and that of HS 10-fold, whereas higher concentrations suppress this stimulatory effect. The stimulatory effect, observed at low concentrations of this growth factor, was completely blocked by the addition of antibodies against this factor. In epithelially differentiated cells, the production of all GAGs was suppressed after addition of this factor, even at low concentrations. We therefore suggest that mesothelioma cells can produce GAGs, the synthesis of which is dependent on the presence and concentration of PDGF β-homodimer. The differences between the two cell lines regarding the effect of this growth factor on GAG synthesis indicates that the regulation of this synthesis is complex, other factors also being important.Key words: mesothelioma, differentiation, glycosaminoglycans, synthesis, growth factors.
Calnexin: a molecular chaperone with a taste for carbohydrate
Authors: Williams David B.
Published: December 2009

Calnexin is an integral membrane protein of the endoplasmic reticulum (ER) that binds transiently to a wide array of newly synthesized membrane and secretory proteins. It also exhibits prolonged binding to misfolded or incompletely folded proteins. Recent studies have demonstrated that calnexin functions as a molecular chaperone to facilitate the folding and assembly of proteins in the ER. It is also a component of the quality control system that prevents proteins from progressing along the secretory pathway until they have acquired proper tertiary or quaternary structure. Most proteins that are translocated into the ER are glycosylated at Asn residues, and calnexin's interactions are almost exclusively restricted to proteins that possess this posttranslational modification. The preference for glycoproteins resides in calnexin's ability to function as a lectin with specificity for the GlC1Man9GlcNAc2oligosaccharide, an early intermediate in the processing of Asn-linked oligosaccharides. Calnexin also has the capacity to bind to polypeptide segments of unfolded glycoproteins. Available evidence suggests that calnexin utilizes its lectin property during initial capture of a newly synthesized glycoprotein and that subsequent association (and chaperone function) is mediated through polypeptide interactions. Unlike other molecular chaperones that are soluble proteins, calnexin is an intrinsic component of the ER membrane. Its unique ability to capture unfolded glycoproteins through their large oligosaccharide moieties may have evolved as a means to overcome accessibility problems imposed by being constrained within a lipid bilayer.Key words: protein folding, molecular chaperones, calnexin, quality control, endoplasmic reticulum.
Physiological role of GlpB of anaerobic glycerol-3-phosphate dehydrogenase ofEscherichia coli
Authors: Varga Monica E. R. , Weiner Joel H.
Published: December 2009

Anaerobic sn-glycerol-3-phosphate dehydrogenase of Escherichia coli is encoded by an operon of three genes, glpACB. The promoter distal gene, glpB, encodes a 44-kilodalton polypeptide that is not part of the purified soluble dehydrogenase. By recombinant plasmid complementation, in a strain harboring a chromosomal deletion of glpACB, we found that all three genes were essential for anaerobic growth on glycerol-3-phosphate (G3P). By isolation of inner membrane preparations we confirmed the cytoplasmic membrane localization of GlpB. GlpB displayed an electron paramagnetic resonance spectrum that suggested the presence of iron–sulfur center(s) within GlpB. We used this spectrum to show that the center(s) were reduced by the artificial reductant dithionite and by the physiological substrate G3P but not by lactate or formate. The center(s) were oxidized by fumarate. These data indicated that GlpB mediates electron transfer from the soluble GlpAC dimer to the terminal electron acceptor fumarate via the membrane-bound menaquinone pool.Key words: glycerol-3-phosphate dehydrogenase, anaerobic electron transport, membrane proteins, ferredoxin, Escherichia coli.
Platelet glutathione transport: characteristics and evidence for regulation by intraplatelet thiol status
Authors: Sexton Daniel J. , Mutus Bulent
Published: December 2009

The present study demonstrates the carrier-mediated uptake of intact glutathione (GSH) by human platelets. Platelet GSH uptake was characterized as being Na+independent and saturable. The KM, apparentand Vmax, apparentfor GSH uptake in platelet plasma membrane vesicles were 28.0 ± 8.4 μM and 263.5 ± 28.5 pmol/min per mg protein, respectively. The transport was inhibited by GSH analogs and enhanced by KCl-induced membrane depolarization. GSH transport may be regulated by the intracellular thiol status, since the depletion of intraplatelet GSH with 100 μM 1-chloro-2,4-dinitrobenzene (CDNB) increased GSH uptake by ~40%. The KM, apparentand Vmax, apparentfor GSH uptake in intact platelets changed from 99.5 ± 15 μM and 42 ± 7.5 pmol/min per 109platelets, respectively, to 33.7 ± 6.7 μM and 21.5 ± 6.9 pmol/min per 109platelets, respectively, on reducing intraplatelet GSH with 100 μM CDNB.Key words: glutathione, platelets, transport.
Electron donation to photosystem II by diphenylcarbazide is inhibited both by the endogenous manganese complex and by exogenous manganese ions
Authors: Rashid Abdur , Popovic Radovan
Published: December 2009

Diphenylcarbazide (DPC) is an efficient electron donor to the inactive oxygen-evolving complex of photosystem II (PSII). We investigated the role of manganese on the rate of electron donation from DPC to PSII in both Mn-depleted (Tris washed) and Mn-retaining (NaCl washed) PSII preparations. The rate of electron donation from DPC to PSII was significantly higher in Mn-depleted than in Mn-retaining preparations, indicating a negative role of native Mn complex on DPC electron donation. The apparent Km values for DPC were found to be 0.11 and 0.17 mM for Mn-depleted and Mn-retaining PSII preparations, respectively. This difference in the Km values also indicates an antagonistic effect of endogenous Mn cluster on electron donation from DPC, which was markedly inhibited by exogenous Mn2+. However, the magnitude of inhibition was greater in Mn-depleted than in Mn-retaining PSII preparations. This indicates a higher accessibility of DPC to PSII in the absence of native Mn complex. Our results suggest (i) that Mn, either endogenous or added, acts as an accessibility barrier for DPC to donate electrons to PSII and (ii) that the native Mn complex not only functions as an accumulator of oxidizing equivalents but may also protect PSII from exogenous reductants.Key words: photosystem II, extrinsic polypeptides, Mn complex, electron transport, diphenylcarbazide.
Biological specificity and measurable physical properties of cell surface receptors and their possible role in signal transduction through the cytoskeleton
Authors: Forgacs G.
Published: December 2009

It is proposed that the binding specificities of cell adhesion molecules are manifested in their measurable physical properties. A method specifically designed to measure the interfacial tension of cell aggregates is described. With the introduction of a statistical mechanical model, the measured values of tensions for aggregates consisting of genetically engineered cells with controlled adhesive properties are used to obtain information on the strength of individual receptor–ligand bonds. The strength of binding must depend on the receptor and its ligand and reflects the amino acid sequence of the binding proteins. Many of the cell surface receptors, being transmembrane proteins, are attached to the various macromolecular networks of the cytoskeleton; therefore, it is suggested that their ligation and ensuing conformational change may substantially affect the mechanical state of the cytoskeletal assemblies. Since these assemblies are believed to actively participate in intracellular signaling by transmitting signals from the cell membrane into the nucleus, the cell adhesion molecules may influence signaling in a predictable way through their measurable physical characteristics. In particular, varying bond strength at the cell surface may lead to differential gene regulation.Key words: cell adhesion, surface tension, signaling, network, filament.
Influence of surface charges on cell adhesion: difference between static and dynamic conditions
Authors: Sabri Siham , Pierres Anne , Benoliel Anne-Marie , Bongrand Pierre
Published: December 2009

We tested the hypothesis that nonspecific repulsion, as a result of electrostatic forces and (or) steric stabilization effects, impaired adhesion more efficiently under dynamic than under static conditions. Cells from the human monocytic line THP1 were plated on a glass surface. Spherical particles bearing monoclonal antibodies specific for antigens expressed by THP1 cells (CD11b, CD18, CD35, CD64) were then added and adhesion was quantified. The effect of neuraminidase treatment of THP1 cells was also studied. Adhesion was then measured in a flow chamber under low shear flow (wall shear rate was 11 or 22 s−1), allowing a quantitative determination of cell adhesion frequency. The following conclusions were obtained: (i) under static conditions, neuraminidase treatment had little effect on adhesion (only CD18-mediated interaction was significantly increased at 4 °C after enzyme treatment); (ii) under dynamic conditions, neuraminidase treatment significantly increased binding; (iii) surprisingly, there was no clear relationship between the length of adhesion molecules involved in the interaction and binding efficiency; and (iv) such parameters as cell shape and topographical distribution of adhesion molecules may strongly influence adhesion under flow. It is concluded that a dynamic reorganization of the pericellular matrix following intercellular contact may play an important role in regulating adhesion.Key words: cell adhesion, laminar flow, steric stabilization, electrostatic repulsion, monocytes.
Changes in surface topography in endothelial monolayers with time at confluence: influence on subcellular shear stress distribution due to flow
Authors: Barbee Kenneth A.
Published: December 2009

It is well known that the morphology of endothelial cells in culture changes dramatically as they go from subconfluence to confluence. After reaching confluence, however, the morphology continues to change but much more subtly as cell density increases and they become more uniform in size and shape. Measurements of surface topography of confluent cells by atomic force microscopy (AFM) showed that cell heights became more uniform and that the root-mean-square amplitude of surface undulations decreased by 7% compared with monolayers that had just reached confluence. Computational fluid dynamics simulations of flow over the endothelial surface geometries measured by AFM showed that the change in topography with time after confluence altered the shear stress distribution, resulting in an increase in the stress concentrations experienced by the cells despite the reduced amplitude of the surface undulation. These data suggest that the starting point for in vitro experiments may influence the measured responses to shear stress, particularly transient responses that occur before structural adaptation takes place. In addition, changes in surface topography may reflect changes in cell tension, cytoskeletal structure, and adhesion to the substratum, all of which are associated with the regulation of growth in anchorage-dependent cells.Key words: cell mechanics, mechanotransduction, cell structure, cell culture.
Insulin does not disrupt actin microfilaments, microtubules, and in vitro aortic endothelial wound repair
Authors: Eshraghi Shervin , Gotlieb Avrum I.
Published: December 2009

In the face of small denuding injuries, the endothelium undergoes a process of rapid repair involving actin microfilaments, microtubules, and centrosomes to reestablish an intact monolayer. Failure to maintain an intact endothelial monolayer is an important factor in the pathogenesis of the atherosclerotic plaque. It was hypothesized that increased susceptibility to atherosclerosis in diabetes mellitus may be, in part, due to delayed reendothelialization following endothelial injury. To test this, the effects of high insulin concentrations on the reendothelialization of small wounds were examined using an in vitro porcine aortic endothelial cell wound model. Elevated concentrations of insulin did not disrupt the confluent endothelial monolayer or alter endothelial cell shape. Insulin also did not induce detectable alterations in the distribution of microtubules and microfilaments in the confluent monolayer. High insulin did not reduce the extent of reendothelialization of a linear wound made in the confluent monolayer. Centrosomal reorientation was similar to that of control wounded cultures as was the reorganization of the microfilaments and microtubules. The data suggest that the atherogenic effects of hyperinsulinemia are not due to disruption of endothelial repair.Key words: actin, microtubules, endothelial repair, insulin, diabetes.
Mechano-reception in osteoblast-like cells
Authors: Jones David , Leivseth Gunnar , Tenbosch Jochen
Published: December 2009

Response to mechanical stimulation is a basic biological phenomenon. Nearly all cells process mechanical input and respond to it by inducing and modulating biochemical pathways. In organisms with tissues, if the average mechanical load is increased, some tissues can increase their performance and often increase their bulk by cell division. A reduction in mechanical loading decreases performance, catabolic activity gains, and the tissue degenerates. The process of anabolism and catabolism regulated by mechanical loading is a second-to-second, minute-to-minute, and hour-to-hour process that works together with local and systemic hormones to ensure that the tissue can meet the demands of the mechanical environment. On the other hand, a mechanical load that is too high can cause tissue and matrix failure and damage to the cells, which can result in inflammation. In this paper, we review the possible biophysical and cell biological mechanisms that might be responsible for transducing physiological and hyperphysiological mechanical loading into the biological response of skeletal cells. We speculate on what the mechanism of mechano-transduction in bone might be compared with that of other cells and on how information produced by mechanical loading might be passed on to other cells to achieve a coordinated tissue response.Key words: mechano-transduction, osteoblasts, bone, calcium, phospholipase C.
Regulation of cell volume and intracellular pH in hyposmotically swollen rat osteosarcoma cells
Authors: Lo C. , Ferrier J. , Tenenbaum H. C. , McCulloch C. A. G.
Published: December 2009

The maintenance of cell volume involves transduction of a volume-sensing signal into effectors of volume-regulatory transporters. After exposure to anisotonic conditions, cells undergo compensatory volume changes that are mediated by active transport and passive movement of ions and solutes. Intracellular pH (pHi) homeostasis may be compromised during these processes. We have studied pHiand some of the signal transduction mechanisms involved in the regulatory volume decrease (RVD) that occurs after exposure to hypoosmolar conditions in rat osteosarcoma cells, ROS 17/2.8. Cells were loaded with BCECF; pHiand cell volume were estimated by dual excitation ratio fluorimetry. Swelling of cells in 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES) buffered hypotonic medium induced a rapid cell swelling followed by an incomplete RVD of ~30% in suspended (i.e., round) cells and ~60% in attached (i.e., spread) cells that was independent of subpassage number. RVD was inhibited by ouabain, valinomycin, and high external [K+], all of which should reduce the cell membrane electrochemical gradient for K+. Inhibition of RVD was induced also by decreasing intracellular [Ca2+] with B APTA–AM and by depletion of Cl−, indicating the role of calcium-regulated K+and Cl−efflux during RVD. Depolymerization of actin filaments by cytochalasin D prolonged the RVD three-fold and nonspecific activation of GTP-binding proteins up-regulated RVD. In attached cells the hypoosmolar-induced swelling caused a large reduction in pHi(~0.7 units), which was sustained as long as cells were in hypoosmotic medium. The reduction of pHiinduced by cell swelling was inhibited by Na+-free extracellular medium, ouabain, the tyrosine kinase inhibitor genistein, and to a lesser extent by Cl−-free medium. However, amiloride failed to inhibit the hypoosmolar-induced reduction of pHi. Collectively these data indicate that RVD of ROS 17/2.8 cells in HEPES-buffered medium is dependent on conductive efflux of K+and Cl−that is regulated by cell shape, actin, and GTP-binding proteins. The sustained inhibition of pHihomeostasis induced by cell swelling may reflect the existence of cell volume sensing mechanisms that operate through tyrosine kinases to regulate pHi.Key words: cell volume, pH, osteoblast, G proteins, actin.
A milestone in ribosomal crystallography: the construction of preliminary electron density maps at intermediate resolution
Authors: Schlünzen F. , Hansen H. A. S. , Thygesen J. , Bennett W. S. , Volkmann N. , Harms J. , Bartels H. , Krumbholz S. , Levin I. , Zaytzev-Bashan A. , Geva M. , Weinstein S. , Agmon I. , Sharon R. , Dribin A. , Maltz E. , Peretz M. , Weinrich V. , Franceschi F. , Böddeker N. , Morlang S. , Berkovitch-Yellin Z. , Yonath A. , Sagi I.
Published: December 2009

Preliminary electron density maps of the large and the small ribosomal particles from halophilic and thermophilic sources, phased by the isomorphous replacement method, have been constructed at intermediate resolution. These maps contain features comparable in size with what is expected for the corresponding particles, and their packing arrangements are in accord with the schemes obtained by ab-initio procedures as well as with the motifs observed in thin sections of the crystals by electron microscopy. To phase higher resolution data, procedures are being developed for derivatization by specific labeling of the ribosomal particles at selected locations with rather small and dense clusters. Potential binding sites are being inserted either by site directed mutagenesis or by chemical modifications to facilitate cluster binding on the surface of the halophilic large and the thermophilic small ribosomal particles, which yield the crystals diffracting to highest resolution (2.9 and 7.3 Å (1 Å = 0.1 nm), respectively). For this purpose, the surface of these ribosomal particles is being characterized and procedures are being developed for quantitative detachment of selected ribosomal proteins and for their incorporation into core particles. The genes of these proteins are being cloned, sequenced, mutated to introduce reactive side groups, mainly cysteines, and overexpressed. In parallel, two in situ small and stable complexes were isolated from the halophilic ribosome. Procedures for their crystal production in large quantities are currently being developed. Models, reconstructed at low resolution from crystalline arrays of ribosomes and their large subunits, are being used for initial low-resolution phasing of the X-ray amplitudes. The interpretation of these models stimulated the design and the crystallization of complexes mimicking defined functional states of a higher quality than those obtained for isolated ribosomes. These models also inspired modelling experiments according to results of functional studies, performed elsewhere, focusing on the progression of nascent proteins.Key words: ribosomes, crystallography, undecagold cluster, heteropolyanions.
A model of the translational apparatus based on a three-dimensional reconstruction of theEscherichia coliribosome
Authors: Frank Joachim , Zhu Jun , Radermacher Michael , Verschoor Adriana , Lata Ramani K. , Penczek Pawel , Grassucci Robert , Agrawal Rajendra K. , Srivastava Suman , Li Yanhong
Published: December 2009

The morphology of the Escherichia coli ribosome, i.e., its shape at moderate to low (20–40 Å (1 Å = 0.1 nm)) resolution, provides important constraints in modeling both the folding of ribosomal RNA and the translational process. A new reconstruction, obtained by low-dose cryoelectron microscopy and image processing of single ribosomes, contains clues to the way in which the ribosome interacts with the key functional ligands: the mRNA and the A- and P-site tRNAs. It also suggests possible pathways of the nascent polypeptide chain. From an interpretation of these clues in the light of existing knowledge, a plausible model for the locations and interactions of key components of protein synthesis is suggested.Key words: three-dimensional reconstruction, cryoelectron microscopy, messenger RNA, A- and P-site RNAs, exit tunnels.
Genomic evolution drives the evolution of the translation system
Authors: Andersson S. G. E. , Kurland C. G.
Published: December 2009

Our thesis is that the characteristics of the translational machinery and its organization are selected in part by evolutionary pressure on genomic traits have nothing to do with translation per se. These genomic traits include size, composition, and architecture. To illustrate this point, we draw parallels between the structures of different genomes that have adapted to intracellular niches independently of each other. Our starting point is the general observation that the evolutionary history of organellar and parasitic bacteria have favored bantam genomes. Furthermore, we suggest that the constraints of the reductive mode of genomic evolution account for the divergence of the genetic code in mitochondria and the genetic organization of the translational system observed in parasitic bacteria. In particular, we associate codon reassignments in animal mitochondria with greatly simplified tRNA populations. Likewise, we relate the organization of translational genes in the obligate intracellular parasite Rickettsia prowazekii to the processes supporting the reductive mode of genomic evolution. Such findings provide strong support for the hypothesis that genomes of organelles and of parasitic bacteria have arisen from the much larger genomes of ancestral bacteria that have been reduced by intrachromosomal recombination and deletion events. A consequence of the reductive mode of genomic evolution is that the resulting translation systems may deviate markedly from conventional systems.Key words: translation, evolution, genome, reassignment, rearrangement.
Structure and function of 5S rRNA in the ribosome
Authors: Bogdanov Alexey A. , Dontsova Olga A. , Dokudovskaya Svetlana S. , Lavrik Inna N.
Published: December 2009

5S rRNA is a small RNA molecule that is a component of a ribosome from almost all living organisms. In this review, we discuss the biogenesis of 5S rRNA and its properties as an independent structural domain of a ribosome as well as the current concepts concerning the higher order structure of 5S rRNA in free state and in its complexes with ribosomal proteins and its folding in the ribosome. Special attention is paid to recent experimental approaches that have been useful in 5S rRNA studies. Our own data on topography of 5S rRNA in the ribosomes are discussed in detail. The hypothesis describing the possible functional role of 5S rRNA for ribosome functioning is discussed.Key words: 5S rRNA, ribosomes, 23S rRNA, site-directed chemical cross-linking, RNA folding.
Antibiotic inhibition of the movement of tRNA substrates through a peptidyl transferase cavity
Authors: Porse Bo T. , Rodriguez-Fonseca Cristina , Leviev Ilia , Garrett Roger A.
Published: December 2009

The present review attempts to deal with movement of tRNA substrates through the peptidyl transferase centre on the large ribosomal subunit and to explain how this movement is interrupted by antibiotics. It builds on the concept of hybrid tRNA states forming on ribosomes and on the observed movement of the 5′ end of P-site-bound tRNA relative to the ribosome that occurs on peptide bond formation. The 3′ ends of the tRNAs enter, and move through, a catalytic cavity where antibiotics are considered to act by at least three primary mechanisms: (i) they interfere with the entry of the aminoacyl moiety into the catalytic cavity before peptide bond formation; (ii) they inhibit movement of the nascent peptide along the peptide channel, a process that may generally involve destabilization of the peptidyl tRNA, and (iii) they prevent movement of the newly deacylated tRNA between the P/P and hybrid P/E sites on peptide bond formation.Key words: peptidyl transferase cavity, transient tRNA states, antibiotics, inhibitory mechanism, subunit–subunit interactions.
The pseudouridine residues of ribosomal RNA
Authors: Ofengand James , Bakin Andrei , Wrzesinski Jan , Nurse Kelvin , Lane Byron G.
Published: December 2009

Pseudouridine (Ψ), the most common single modified nucleoside in ribosomal RNA, has been positioned in the small subunit (SSU) and large subunit (LSU) RNAs of a number of representative species. Most of the information has been obtained by application of a rapid primed reverse transcriptase sequencing technique. The locations of these Ψ residues have been compared. Many sites for Ψ are the same among species, but others are distinct. In general, the percentage Ψ in multicellular eukaryotes is greater than in prokaryotes. In LSU RNA, the Ψ residues are strongly clustered in three domains, all of which are near or connected to the peptidyl transferase center. There is no apparent clustering of Ψ in SSU RNA. The Ψ sites in LSU RNA overlap those for the methylated nucleosides, but this is not the case in SSU RNA. There are 265 Ψ sites known to nucleotide resolution, of which 246 are in defined secondary structures, and 112 of these are in nonidentical structural contexts. All 246 Ψ sites can be classified into five structural types. Two Escherichia coli Ψ synthases have been cloned and characterized, one for Ψ516 in SSU RNA and one for Ψ746 in LSU RNA. The Ψ746 synthase recognizes free RNA, but the Ψ516 enzyme requires an intermediate RNP particle. Possible functional roles for Ψ in the ribosome are discussed.Key words: pseudouridine, sequencing, modified nucleosides, ribosomal RNA.
Variety of nonsense suppressor phenotypes associated with mutational changes at conserved sites in Escherichia coli ribosomal RNA
Authors: Murgola Emanuel J. , Pagel Frances T. , Hijazi Kathryn A. , Arkov Alexey L. , Xu Wenbing , Zhao Song Q.
Published: December 2009

To screen for ribosomal RNA mutants defective in peptide chain termination, we have been looking for rRNA mutants that exhibit different patterns of suppression of nonsense mutations and that do not suppress missense mutations at the same positions in the same reporter gene. The rRNA mutations were induced by segment-directed randomly mutagenic PCR treatment of a cloned rrnB operon, followed by subcloning of the mutagenesis products and transformation of strains containing different nonsense mutations in the Escherichia coli trpA gene. To date, we have repeatedly obtained only two small sets of mutations, one in the 3′ domain of 16S rRNA, at five nucleotides out of the 610 mutagenized (two in helix 34 and three in helix 44), and the other in 23S rRNA at only four neighboring nucleotide positions (in a highly conserved hexanucleotide loop) within me 1.4 kb mutagenized segment. There is variety, however, in the suppression patterns of the mutants, ranging from suppression of UAG or UGA, through suppression of UAG and UGA, but not UAA, to suppression of all three termination codons. The two helices in 16S rRNA have previously been associated both physically and functionally with the decoding center of the ribosome. The 23S region is part of the binding site for the large subunit protein L11 and the antibiotic thiostrepton, both of which have been shown to affect peptide chain termination. Finally, we have demonstrated that the 23S mutant A1093, which suppresses trpA UGA mutations very efficiently, is lethal at temperatures above 36 °C (when highly expressed). This lethality is overcome by secondary 23S rRNA mutations in domain V. Our results suggest that specific regions of 16S and 23S rRNA are involved in peptide chain termination, that the lethality of A1093 is caused by high-level UGA suppression, and that intramolecular interaction between domains II and V of 23S rRNA may play a role in peptide chain termination at the UGA stop codon.Key words: 16S and 23S rRNAs, PCR mutagenesis, nonsense suppression, peptide chain termination, intramolecular interaction.
Structure and evolution of mammalian ribosomal proteins
Authors: Wool Ira G. , Chan Yuen-Ling , Glück Anton
Published: December 2009

Mammalian (rat) ribosomes have 80 proteins; the sequence of amino acids in 75 have been determined. What has been learned of the structure of the rat ribosomal proteins is reviewed with particular attention to their evolution and to amino acid sequence motifs. The latter include: clusters of basic or acidic residues; sequence repeats or shared sequences; zinc finger domains; bZIP elements; and nuclear localization signals. The occurrence and the possible significance of phosphorylated residues and of ubiquitin extensions is noted. The characteristics of the mRNAs that encode the proteins are summarized. The relationship of the rat ribosomal proteins to the proteins in ribosomes from humans, yeast, archaebacteria, and Escherichia coli is collated.Key words: ribosomes, ribosomal proteins, amino acid sequence, evolution.
Proteins P1, P2, and P0, components of the eukaryotic ribosome stalk. New structural and functional aspects
Authors: Remacha Miguel , Jimenez-Diaz Antonio , Santos Cruz , Briones Elisa , Zambrano Reina , Gabriel M. A. Rodriguez , Guarinos E. , Ballesta Juan P. G.
Published: December 2009

The eukaryoic ribosomal stalk is thought to consist of the phosphoproteins P1 and P2, which form a complex with protein P0. This complex interacts at the GTPase domain in the large subunit rRNA, overlapping the binding site of the protein L11-like eukaryotic counterpart (Saccharomyces cerevisiae protein L15 and mammalian protein LI2). An unusual pool of the dephosphorylated forms of proteins P1 and P2 is detected in eukaryotic cytoplasm, and an exchange between the proteins in the pool and on the ribosome takes place during translation. Quadruply disrupted yeast strains, carrying four inactive acidic protein genes and, therefore, containing ribosomes totally depleted of acidic proteins, are viable but grow with a doubling time threefold higher than wild-type cells. The in vitro translation systems derived from these stains are active but the two-dimensional gel electrophoresis pattern of proteins expressed in vivo and in vitro is partially different. These results indicate that the P1 and P2 proteins are not essential for ribosome activity but are able to affect the translation of some specific mRNAs. Protein P0 is analogous to bacterial ribosomal protein L10 but carries an additional carboxyl domain showing a high sequence homology to the acidic proteins P1 and P2, including the terminal peptide DDDMGFGLFD. Successive deletions of the P0 carboxyl domain show that removal of the last 21 amino acids from the P0 carboxyl domain only slightly affects the ribosome activity in a wild-type genetic background; however, the same deletion is lethal in a quadruple disruptant deprived of acidic P1/P2 proteins. Additional deletions affect the interaction of P0 with the P1 and P2 proteins and with the rRNA. The experimental data available support the implication of the eukaryotic stalk components in some regulatory process that modulates the ribosomal activity.Key words: ribosomal stalk, acidic proteins, phosphorylation, GTPase domain, translation regulation.
Relocation of the unusualVAR1 gene from the mitochondrion to the nucleus
Authors: Sanchirico Marie , Tzellas Andrew , Mason Thomas L. , Fox Thomas D. , Conrad-Webb Heather , Perlman Philip S.
Published: December 2009

The Varl protein (Var1p) is an essential, stoichiometric component of the yeast mitochondrial small ribosomal subunit, and it is the only major protein product of the mitochondrial genetic system that is not part of an energy transducing complex of the inner membrane. Interestingly, no mutations have been reported that affect the function of Var1p, presumably because loss of a functional mitochondrial translation system leads to an instability of mtDNA. To study the structure, function and synthesis of Varlp, we have engineered yeast strains for the expression of this protein from a nuclear gene, VAR1U, in which 39 nonstandard mitochondrial codons were converted to the universal code. Immunoblot analysis using an epitope-tagged form of Var1Up showed that the nuclear-encoded protein was expressed and imported into the mitochondria. VAR1Uwas tested for its ability to complement a mutation in mtDNA, PZ206, which disrupts 3′-end processing of the VAR1 mRNA, causing greatly reduced synthesis of Var1p and a respiratory-deficient phenotype. Respiratory growth was restored in PZ206 mutants by transformation with a centromere plasmid carrying VAR1Uunder ADH1 promoter control, thus proving that VAR1 function can be relocated from the mitochondrion to the nucleus. Moreover, epitope-tagged Var1Up co-sedimented specifically with small ribosomal subunits in high salt sucrose gradients. The relocation of VAR1 from the mitochondrion to the nucleus provides an excellent system for the molecular genetic analysis of structure–function relationships in the unusual Var1 protein.Key words: Saccharomyces cerevisiae, VAR1 gene, mitochondria, ribosome assembly, gene relocation, RNA processing, nuclear–mitochondrial interaction.