bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of apoptosis in breast cancer cells in response to vitamin D and antiestrogens
Authors: Welsh JoEllen
Published: December 2009

1,25-Dihydroxycholecalciferol D3 (1,25(OH)2D3), the active metabolite of vitamin D, is a potent inhibitor of breast cancer cell growth both in vivo and in vitro. We have shown that MCF-7 cells treated with 100 nM 1,25(OH)2D3 exhibit characteristic apoptotic morphology (pyknotic nuclei, chromatin and cytoplasmic condensation, nuclear matrix protein reorganization) within 48 h. In the experiments reported here, we examined the interactions between 1,25(OH)2D3 and the antiestrogen 4-hydroxytamoxifen (TAM), which also induces apoptosis in MCF-7 cells. Our data suggest that TAM significantly potentiates the reduction in cell number induced by 1,25(OH)2D3 alone. Combined treatment with 1,25(OH)2D3 and TAM enhances the degree of apoptosis assessed using morphological markers that identify chromatin and nuclear matrix protein condensation. We have selected a subclone of MCF-7 cells resistant to 1,25(OH)2D3 (MCF-7D3Res). These cells express the vitamin D receptor and exhibit doubling times comparable to the parental MCF-7 cells, even when grown in 100 mM 1,25(OH)2D3. Treatment of both parental and resistant MCF-7 cells with TAM induces apoptosis and clusterin. These data emphasize that apoptosis can be induced in MCF-7 cells either by activation of vitamin-D-mediated signalling or disruption of estrogen-dependent signalling.Key words: apoptosis, breast, vitamin D, antiestrogen, gene expression, clusterin.
Apoptotic cell death in the limb and its relationship to pattern formation
Authors: Zakeri Zahra F. , Ahuja Harleen Singh
Published: December 2009

Detection of cell death throughout embryogenesis demonstrates its importance in the normal form and function of the organism. We have examined cellular death during normal limb development by use of markers that display the morphology of cell death, the presence of phagocytic cells, and lysosomal activity. In addition in situ labeling confirms fragmentation of DNA in the mammalian limb. By these criteria, cell death in the developing limb can be categorized as type 1 or apoptotic cell death. However, the signal(s) responsible for cellular destruction and activation of phagocytosis by neighboring cells or recruited macrophages remain to be identified. The decision for cellular fate during development and regulation of it once the decision is made are key questions. To address the specific question of what determines that one cell will die while its neighbor survives, we have used compounds, such as retinoic acid (RA), that have been shown to alter the pattern of normal development. We and others have shown that RA does indeed alter the pattern of cell death to the extent of inducing malformations in the limb. The mouse mutant Hammertoe (Hm) provides an abnormal system in which the pattern of cell death is specifically altered in the inter-digital regions of the limb. Our preliminary data suggest that RA can also introduce cell death between digits 2, 3, 4, and 5 of the Hm mutant where there was no cell death to begin with. Our observations of the effect of RA on mutant limbs suggest that a direct relationship between RA and cell death does exist and that this interaction may be required for correct pattern formation. The alteration in the pattern of cell death in the mutant mouse is of great interest, since it would provide a rare example of specific correction of a birth defect by direct intercession against the physiological effect of the mutation.Key words: cell death, limb, pattern formation, retinoic acid, limb deformity mutant.
Mitochondrial stress protein actions during chemically induced renal proximal tubule cell death
Authors: Bruschi S. A. , Lindsay J. G.
Published: December 2009

We have previously shown that the potent mammalian nephrotoxicant tetrafluoroethyl-L-cysteine (TFEC) covalently modifies a select group of mitochondrial proteins prior to cell death. More recently we have identified these adducted proteins as subunits of mitochondrial dehydrogenase multienzyme complexes, which are involved in key regulatory steps of cellular respiration. Most importantly the E2 and E3 subunits of α-ketoglutarate dehydrogenase are adducted. We report here that the consequence of adduction is the formation of tertiary complexes between dehydrogenase subunits and the mitochondrial heat shock protein 60 (HSP60) and a HSP70 homolog (mortalin/PBP74). Thus, adduction perturbs protein structural integrity sufficiently to allow for mitochondrial stress protein recognition. These data also suggest that, in our mammalian system, HSP60 appears to act in the identification and maintenance of protein integrity, as has been previously established for simpler eukaryotic systems.Key words: cell death, adducts, stress proteins.
Interaction of transferrin with rat alveolar macrophages
Authors: Janicka Maria , Regoeczi Erwin , Bolyos Maria , Hu Wei-Li
Published: December 2009

Binding of rat transferrin to isolated alveolar macrophages was investigated in the 0.125 nM to 2 μM range. Computer analysis of the data revealed two classes of binding sites, a small number (<1000 exposed/cell) having high affinity (dissociation constant (Kd), 3.4 nM) and a large number (approximately 4 × 106/cell) having low affinity (Kd48 μM). Measurements with a monoclonal antibody to the rat transferrin (rTf) receptor yielded values in the same range as the high-affinity sites derived from studies of ligand binding. Binding to the low-affinity sites at pH 5.8 was nearly one order of magnitude stronger than that at pH 7.3. Bovine lactoferrin (12 μM), cationized bovine serum albumin (14 μM), L-arginine (50 mM), and L-lysine (50 mM) did not compete against rTf binding to the low-affinity sites. Removal of an average of 2.6 × 108sialyl residues from each cell did not affect binding. Heparan sulphate proteoglycan purified from alveolar macrophages bound strongly to immobilized rTf, thus raising the possibility that the low-affinity interaction of transferrin with these cells may be mediated, at least in part, by this glycosaminoglycan.Key words: heparan sulphate proteoglycan, macrophage, transferrin, transferrin receptor.
Signaling in new directions
Authors: Samiei Haleh Vahidi
Published: December 2009

Many laboratories, using a variety of organisms, have contributed to deciphering the identity and the order of the components leading from ligand-bound receptor tyrosine kinases to various intracellular events, including changes in gene expression. The gaps have only been filled recently. This minireview summarizes the findings and points out the degree of conservation of the same pathway in distant organisms, both at the molecular level and in terms of the consecutive steps. The review also looks at points at which this pathway might be diverging and points onto which other pathways might be converging. These interactions are not always clear cut, and understanding them will be the challenge for the future.Key words: signal transduction, receptor tyrosine kinase, RAS, RAF, MAP kinase.
Enzymatic characterization of purified recombinant human renin
Authors: Pilote L. , McKercher G. , Thibeault D. , Lamarre D.
Published: December 2009

Renin is a highly specific aspartyl protease of the renin–angiotensin system initially synthesized as preprorenin. Recombinant human prorenin was produced in cell factories from stably transfected DAMP cells, a dog epithelial cell line. The equivalent of 10–15 mg of recombinant human renin was secreted in the supernatant from each cell factory. Following a single affinity chromatography step using a renin inhibitor as the ligand, a 181-fold purification was achieved with 81% recovery of the renin activity. This highly pure recombinant enzyme having a specific activity of 3.44 mg angiotensin I∙mg protein−1∙h−1was used for kinetic analysis. The kinetic parameters were determined with the natural substrate angiotensinogen and a tetradecapeptide substrate corresponding to the amino terminus of angiotensinogen, Asp1–Asn14, at their respective optimum pH of 5.5 and 6.8. Although there was a six-fold increase in both Kmand kcatvalues for the peptidic substrate (13.3 μM and 8.1 s−1, respectively), when compared with values for the natural substrate (2.04 μM and 1.41 s−1), the catalytic efficiency (0.69 μM−1∙s−1) of the enzyme for both substrates was the same. However, the kcat/Kmvalue with angiotensinogen at the physiological pH 7.4 was 30% lower than that observed at the optimum pH 5.5. The recombinant human renin displayed similar optimum pH and kinetic parameters with angiotensinogen and the tetradecapeptide substrate when compared with human kidney renin.Key words: recombinant human renin, aspartyl protease, enzyme kinetics.
Hypothyroidism prolongs and increasesmdxmuscle precursor proliferation and delays myotube formation in normal and dystrophic limb muscle
Authors: McIntosh L.M. , Anderson J. E.
Published: December 2009

Hypothyroidism (induced by 8 weeks of oral 0.05% propylthiouracil) heightened the phenotype of mdx mouse dystrophin-deficient myopathy to more closely resemble human Duchenne muscular dystrophy. Muscle repair after crush injury to the tibialis anterior muscle (TA) in hypothyroid mdx mice showed decreased myotube formation and delayed debris removal. To investigate whether reduced muscle precursor cell proliferation can account for the effects of hypothyroidism on repair from injury, immunocytochemistry for neural cell adhesion molecule (NCAM) on muscle precursor cells and autoradiography to detect DNA synthesis were performed in control and mdx TA. The proportions of labelled polymorphonuclear leukocyte nuclei (PMN), myotube nuclei (MN), and total mononuclear cell nuclei (TLN, the majority being muscle precursors) were counted in defined areas of regenerating TA after 2 and 4 days recovery. MN and the numbers of activated satellite cell nuclei on intact fibers were counted in surviving areas. In the same muscle, earlier phases of regeneration were observed in areas distal than proximal to the injury. At 2 days of regeneration, labelled PMN were increased in treated compared with untreated mdx TA. In distal areas at 4 days, fewer muscle precursors had recently fused to myotubes in treated than in untreated mdx TA. In proximal areas at 4 days (relatively late in repair), TLN data suggested that muscle precursor proliferation was greater in hypothyroid compared with untreated mdx TA. NCAM immunostaining was consistent with proliferation data and confirmed that there were more muscle precursors in mdx than in control regenerating muscle. These results suggest that hypothyroidism prolongs and increases the phase of replication by mdx muscle precursors and delays precursor fusion into myotubes in regeneration.Key words: hypothyroidism, mdx mouse, regeneration, muscle precursor cell, autoradiography.
Molecular genetics of the parasitic protozoanLeishmania
Authors: Wong Alexander Kin-Choi
Published: December 2009

Leishmania is a member of the family Trypanosomatidae, which causes debilitating diseases in humans. Over the past several years, researchers have applied molecular genetic techniques to study extensively the biology of this parasitic protozoan. Many aspects of Leishmania biology are found to be unique when compared with the higher eukaryotes. This minireview highlights some recent developments in the molecular genetic analysis of this fascinating organism.Key words: Leishmania, protozoan, molecular genetics, disease.
Expression ofTrichoderma reeseiandTrichoderma viridexylanases inEscherichia coli
Authors: Sung Wing L. , Luk Catherine K. , Chan Benedict , Wakarchuk Warren , Yaguchi Makoto , Campbell Robert , Willick Gordon , Ishikawa Kazuhiko , Zahab Diana M.
Published: December 2009

Synthetic genes encoding the 190 amino acid Trichoderma reesei xylanase II (TrX) and the closely related Trichoderma viride xylanases have been synthesized in a two-step procedure. Initially, a partial gene encoding amino acids 92–190 was constructed in fusion with the N-terminal half of the Bacillus circulons xylanase (BcX). The remaining BcX gene sequence was replaced during the assembly of the coding sequence for amino acids 1–91. Expression of the synthetic genes in Escherichia coli yielded recombinant xylanases with specific activity generally identical with the natural TrX. However, the recombinant TrX showed thermostability and temperature optimum lower than those of the natural TrX, thus indicating that the posttranslational modifications of the latter in its fungal host are essential to its greater stability. A mutation N19K further decreased the thermostability of the recombinant TrX.Key words: xylanase, thermostability.
Probucol, but not MaxEPA fish oil, inhibits mononuclear cell adhesion to the aortic intima in the rat model of atherosclerosis
Authors: Barbeau Michele L. , Whitman Stewart C. , Rogers Kem A.
Published: December 2009

We have examined the influence of both dietary fish oil and probucol on monocyte adhesion to the aortic endothelium of rats fed an atherogenic diet for 2 weeks. All rats were fed a low-fat diet supplemented with 4% cholesterol, 1% cholic acid, and 0.5% 2-thiouracil. In addition to the atherogenic diet, group 1 (FO; n = 20) received a dietary supplement of the fish oil concentrate MaxEPA (5% w/w); group 2 (CO; n = 20) received a supplement of a control oil with the same polyunsaturated–monounsaturated–saturated fatty acid ratio as MaxEPA; and group 3 (PR; n = 20) received both the control oil supplement (5% w/w) and a 1% (w/w) supplement of probucol. Analysis of blood samples taken at 2 weeks revealed that both fish oil and probucol lowered total plasma cholesterol by 30% compared with the CO group. In addition, fish oil supplementation caused a significant decrease in cholesterol contained in the VLDL fraction while probucol supplementation significantly lowered cholesterol in the HDL fraction. Analysis of mononuclear cell adhesion to the aortic endothelium in vivo revealed that, while fish oil had no significant effect, probucol reduced adhesion by 40%. The results of this study suggest that probucol, but not fish oil, may inhibit the initiation of lesion formation in the rat model of atherosclerosis.Key words: rat, hypercholesterolemia, monocyte adhesion, fish oil, probucol.
Probing transmembrane mechanical coupling and cytomechanics using magnetic twisting cytometry
Authors: Wang Ning , Ingber Donald E.
Published: December 2009

We recently developed a magnetic twisting cytometry technique that allows us to apply controlled mechanical stresses to specific cell surface receptors using ligand-coated ferromagnetic microbeads and to simultaneously measure the mechanical response in living cells. Using this technique, we have previously shown the following: (i) β1 integrin receptors mediate mechanical force transfer across the cell surface and to the cytoskeleton, whereas other transmembrane receptors (e.g., scavenger receptors) do not; (ii) cytoskeletal stiffness increases in direct proportion to the level of stress applied to integrins; and (iii) the slope of this linear stiffening response differs depending on the shape of the cell. We now show that different integrins (β1, αVβ3, αV, α5, α2) and other transmembrane receptors (scavenger receptor, platelet endothelial cell adhesion molecule) differ in their ability to mediate force transfer across the cell surface. In addition, the linear stiffening behavior previously observed in endothelial cells was found to be shared by other cell types. Finally, we demonstrate that dynamic changes in cell shape that occur during both cell spreading and retraction are accompanied by coordinate changes in cytoskeletal stiffness. Taken together, these results suggest that the magnetic twisting cytometry technique may be a powerful and versatile tool for studies analyzing the molecular basis of transmembrane mechanical coupling to the cytoskeleton as well as dynamic relations between changes in cytoskeletal structure and alterations in cell form and function.Key words: integrins, mechanical stress, magnetic beads, cytoskeleton, cell shape.
Mechanical basis of cell shape: investigations with the scanning acoustic microscope
Authors: Bereiter-Hahn Jürgen , Karl Llonka , Lüers Holger , Vöth Monika
Published: December 2009

The shape of cells during interphase in sparse cultures often resembles that of fried eggs. XTH-2 cells, which have been derived from tadpole heart endothelia, provide a typical example of this type of shape. To understand the physical basis of this shape, the cytoskeleton of these cells has been investigated in detail. Subcellular elasticity data have been achieved by scanning acoustic microscopy (SAM). Their changes were observed during treatment of the cells with microtubule-disrupting agents (colcemid and low temperature), and shape generation in giant cells produced by electro-fusion was observed with SAM, revealing the role of the nucleus as a force centering organelle. From these observations combined with well-documented observations on cellular dynamics described in the literature, a model is developed explaining the fried-egg shape of cells by means of interacting forces and fluxes (cortical flow, bulk flow of cytoplasm, microtubule-mediated transport of cytoplasm) of cytoplasm. The model also allows the comprehension of the increase of tension in cells treated with colcemid.Key words: cell shape, elasticity, grant cells, microtubules, acoustic microscopy.
Dynamic reciprocity revisited: a continuous, bidirectional flow of information between cells and the extracellular matrix regulates mammary epithelial cell function
Authors: Roskelley C. D. , Bissell M. J.
Published: December 2009

Interactions between cells and the extracellular matrix (ECM) generate two classes of signals, mechanical and biochemical. In the case of the mammary epithelial cell, both are required to initiate ECM-dependent expression of the abundant milk protein β-casein. Mechanical signals induce a cellular rounding, while functional biochemical signals are associated with an increase in tyrosine phosphorylation. These individual components are part of a complex signalling hierarchy that leads to the emergence of the fully functional lactational phenotype. Interestingly, both the assembly and disassembly of this hierarchy, which occur cyclically in vivo, are constantly modulated by dynamic and reciprocal interactions that take place within a functional unit composed of both the cell and the ECM.Key words: mammary epithelium, differentiation, extracellular matrix, casein.
Modeling of molecular mechanisms of cell adhesion
Authors: Skierczynski B. A. , Skalak R. , Chien S.
Published: December 2009

Cellular adhesion is a process of great importance in biology. We present a simple model of the adhesion process in which the molecular mechanisms involve a receptor, a ligand, and the cytoskeleton of the cell. Based on the energetic consideration of the process, we propose a molecular interpretation of the existing experimental data. The model suggests that the interaction of the receptor and (or) receptor-ligand with the cytoskeleton can have important influence on the formation and strength of the adhesion complex as well as on the subsequent interaction with different ligands. When conformational changes take place during the adhesion process, the characterization of the adhesion bonds based on chemical kinetics alone seems to be incomplete and must be supplemented by parameters, describing the functionality of the complex, i.e., change of the affinity for different ligands, as in the signal transduction, or the strength of the bond, as in the adhesion process.Key words: adhesion, ligand–receptor interaction, modeling, signal transduction, conformational changes.
A new model for the research into rhythmic contraction activity of cardiomyocytes in vitro
Authors: Korohoda Włodzimierz , Jurkiewicz Anna , Figiel Izabela , Czyż Jarosław
Published: December 2009

Heart cells continue to contract rhythmically after isolation and in culture in vitro. We describe a model of heart preparation in vitro that permits quantitative research on the frequency of contractions of cardiomyocytes. The chick embryo heart explants placed on a network of elastic glass fibers continued beating for months, recorded and analyzed with the methods of computer-assisted image analysis. The efficacy of this experimental model for the screening of effects of various agents on the frequency of contractions was examined by following the effects of nifedipine, caffeine, ethanol, and benzamide. The reversibility of the effects and the reproducibility of results were demonstrated quantitatively. The significance of a mechanical elastic load provided by glass fibers for the preservation of long-lasting contractile activity of cardiomyocytes is discussed and the common occurrence of oscillatory contraction processes in various eucaryotic cells is noted.Key words: heart, contractility, benzamide, caffeine, nifedipine, ethanol, image analysis, cardiomyocytes.
Response of single, pairs, and clusters of epithelial cells to substratum topography
Authors: Oakley C. , Brunette D. M.
Published: December 2009

Cells cultured on grooved substrata change their shape, orientation, and direction of locomotion in response to substratum topography, a phenomenon called contact or topographic guidance. Porcine epithelial cells (E-cells) spread on micromachined grooved or smooth control surfaces were examined by epifluorescence and confocal microscopy to determine area, cell shape, and orientation in conjunction with distributions and orientations of actin filaments and microtubules. Single cells, cells within a pair or cluster, and pairs or clusters considered as a unit were compared. As expected, cell contact increased cell spreading, but surprisingly, increased cell contact influenced cell shape on smooth and grooved surfaces and increased alignment of cells spread on grooves. Both actin filaments and microtubules aligned initially and most consistently along the walls and ridge–groove edges. Single E-cells displayed the least variability of aligned cytoskeletal patterns. E-cells within clusters displayed the most variability as local topographic effects on the cytoskeleton could be overridden by adjacent cell contact. Overall, contact guidance of E-cells was neither synonymous with nor contingent upon an elliptical morphology oriented to the topography. E-cells also differed from fibroblasts in their response to cell contact and in their lack of a relationship between cell polarity and locomotion.Key words: microtubules, actin, topographic guidance, micromachined substrata.
Modeling the structure of the ribosome
Authors: Easterwood Thomas R. , Harvey Stephen C.
Published: December 2009

Considering the size and complexity of the ribosome and the growing body of data from a wide range of experiments on ribosomal structure, it is becoming increasingly important to develop tools that facilitate the development of reliable models for the ribosome. We use a combination of manual and computer-based approaches for building and refining models of the ribosome and other RNA–protein complexes. Our methods are aimed at determining the range of models compatible with the data, making quantitative statements about the positional uncertainties (resolution) of different regions, identifying conflicts in the data, establishing which regions of the ribosome need further experimental exploration, and, where possible, predicting the outcome of future experiments. Our previous low-resolution model for the small subunit of the Escherichia coli ribosome is briefly reviewed, along with progress on atomic resolution modeling of the mRNA–tRNA complex and its interaction with the decoding site of the 16S RNA.Key words: molecular models, 30S subunit, 16S decoding site, Escherichia coli, tRNA–mRNA complex.
Getting closer to an understanding of the three-dimensional structure of ribosomal RNA
Authors: Mueller Florian , Döring Thomas , Erdemir Tarik , Greuer Barbara , Jünke Nicole , Osswald Monika , Rinke-Appel Jutta , Stade Katrin , Thamm Sabine , Brimacombe Richard
Published: December 2009

Two experimentally unrelated approaches are converging to give a first low-resolution solution to the question of the three-dimensional organization of the ribosomal RNA from Escherichia coli. The first of these is the continued use of biochemical techniques, such as cross-linking, that provide information on the relative locations of different regions of the RNA. In particular, recent data identifying RNA regions that are juxtaposed to functional ligands such as mRNA or tRNA have been used to construct improved topographical models for the 16S and 23S RNA. The second approach is the application of high-resolution reconstruction techniques from electron micrographs of ribosomes in vitreous ice. These methods have reached a level of resolution at which individual helical elements of the ribosomal RNA begin to be discernible. The electron microscopic data are currently being used in our laboratory to refine the biochemically derived topographical RNA models.Key words: site-directed cross-linking, functional sites in ribosomal RNA, electron microscopy, three-dimensional reconstruction, model fitting.
Trans-acting factors in yeast pre-rRNA and pre-snoRNA processing
Authors: Lafontaine Denis , Tollervey David
Published: December 2009

The major intermediates in the pathway of pre-rRNA processing in yeast and other eukaryotes were originally identified by biochemical analyses. However, as a result of the analysis of the effects of mutations in trans-acting factors, the yeast pre-rRNA processing pathway is now characterized in far more detail than that of other eukaryotes. These analyses have led to the identification of processing sites and intermediates that were either too close in size or too short lived to be detected by biochemical analyses alone. In addition, it was generally unclear whether pre-rRNA processing steps were endonucleolytic or exonucleolytic; analyses of trans-acting factors is now revealing a complex mixture of endonucleolytic and exonucleolytic processing steps. Many of the small nucleolar RNAs (snoRNAs) are excised from larger precursors. Analyses of trans-acting factors are also revealing details of pre-snoRNA processing in yeast. Interestingly, factors involved in pre-snoRNA processing turn out to be components that also function in pre-rRNA processing, suggesting a potential mechanism for the coregulation of rRNA and snoRNA synthesis. In general, very little is known about the regulation of pre-rRNA processing steps. The best candidate for a system regulating specific pre-rRNA processing reactions has recently been revealed by the analysis of a yeast pre-RNA methylase. Here we will review recent data on the trans-acting factors involved in yeast ribosome synthesis and discuss how these analyses have contributed to our current view of this complex process.Key words: RNA processing, ribosome biogenesis, rRNA, exonuclease, methylation, yeast.
Small nucleolar RNA
Authors: Gerbi Susan A.
Published: December 2009

A growing list of small nucleolar RNAs (snoRNAs) has been characterized in eukaryotes. They are transcribed by RNA polymerase II or III; some snoRNAs are encoded in the introns of other genes. The nonintronic polymerase II transcribed snoRNAs receive a trimethylguanosine cap, probably in the nucleus, and move to the nucleolus. snoRNAs are complexed with proteins, sometimes including fibrillarin. Localization and maintenance in the nucleolus of some snoRNAs requires the presence of initial precursor rRNA (pre-rRNA). Many snoRNAs have conserved sequence boxes C and D and a 3′ terminal stem; the roles of these features are discussed. Functional assays done for a few snoRNAs indicate their roles in rRNA processing for cleavage of the external and internal transcribed spacers (ETS and ITS). U3 is the most abundant snoRNA and is needed for cleavage of ETS1 and ITS1; experimental results on U3 binding sites in pre-rRNA are reviewed. 18S rRNA production also needs U14, U22, and snR30 snoRNAs, whereas U8 snoRNA is needed for 5.8S and 28S rRNA production. Other snoRNAs that are complementary to 18S or 28S rRNA might act as chaperones to mediate RNA folding. Whether snoRNAs join together in a large rRNA processing complex (the "processome") is not yet clear. It has been hypothesized that such complexes could anchor the ends of loops in pre-rRNA containing 18S or 28S rRNA, thereby replacing base-paired stems found in pre-rRNA of prokaryotes.Key words: RNA processing, small nucleolar RNAs, nucleolus, ribosome biogenesis, rRNA processing complex.
A proposal for the conformation of loop E in Escherichia coli 5S rRNA
Authors: Dallas Anne , Rycyna Robert , Moore Peter
Published: December 2009

A proposal is advanced for the conformation of the loop E region of prokaryotic 5S rRNAs based on spectroscopic data obtained from pAD3 RNA, a construct that includes helix IV, helix V, and loops D and E from Escherichia coli 5S rRNA. Even though loop E juxtaposes bases that cannot form Watson–Crick base pairs, it resembles an A-form double helix; its nucleotides relate to each other spectroscopically in a helix-like way and are in the anti conformation. The ends of loop E, which is palindromic, have the same conformation. Working in from either end towards the center of the loop, a closing GC is followed by a side-by-side GA and then by a reversed Hoogsteen AU, a pattern resembling that found at one end of eukaryotic loop E. The center of the loop consists of three nucleotide pairs, which appear to be an asymmetric GG pair, a Watson–Crick-like AG, and a GU stabilized by a single hydrogen bond.Key words: 5S rRNA, loop E, NMR, bulged G motif, prokaryote.
An oligonucleotide analog approach to the decoding region of 16S rRNA
Authors: Stern Seth , Purohit Prakash
Published: December 2009

Despite the passage of about 30 years since the discovery of the translational activities of ribosomes and the outlining of the roles of the large and small subunits, the actual molecular basis for the mRNA decoding activities of the small subunit has remained essentially obscure. In this paper, we describe a new approach using oligonucleotide analogs of 16S ribosomal RNA, in which the small ribosomal subunit is effectively deconstructed into a smaller more experimentally tractable form. Specifically, we review the results of experiments using an oligonucleotide analog of the decoding region of 16S ribosomal RNA, suggesting that the decoding region is the functional core of the small subunit, that it contacts both mRNA codons and tRNA anticodons, and that it mediates and probably enhances codon–anticodon base pairing, that is, decoding.Key words: translation, ribosome, 30S, 16S, RNA, decoding, antibiotic.
Peptidyl transferase and beyond
Authors: Wower Jacek , Wower Iwona K. , Kirillov Stanislav V. , Rosen Kirill V. , Zimmermann Robert A. , Hixson Stephen S.
Published: December 2009

The peptidyl transferase center of the Escherichia coli ribosome encompasses a number of 50S-subunit proteins as well as several specific segments of the 23S rRNA. Although our knowledge of the role that both ribosomal proteins and 23S rRNA play in peptide bond formation has steadily increased, the location, organization, and molecular structure of the peptidyl transferase center remain poorly defined. Over the past 10 years, we have developed a variety of photoaffinity reagents and strategies for investigating the topography of tRNA binding sites on the ribosome. In particular, we have used the photoreactive tRNA probes to delineate ribosomal components in proximity to the 3′ end of tRNA at the A, P, and E sites. In this article, we describe recent experiments from our laboratory which focus on the identification of segments of the 23S rRNA at or near the peptidyl transferase center and on the functional role of L27, the 50S-subunit protein most frequently labeled from the acceptor end of A- and P-site tRNAs. In addition, we discuss how these results contribute to a better understanding of the structure, organization, and function of the peptidyl transferase center.Key words: peptidyl transferase, ribosome, tRNA, photoreactive nucleos/tides, crosslinking.
Translational bypassing: a new reading alternative of the genetic code
Authors: Groisman Irina , Engelberg-Kulka Hanna
Published: December 2009

The translation of the genetic code, once thought to be rigid, has been found to be quite flexible, and several alternatives in its reading have been described. An unusual alternative is translational bypassing, a frameshift event where the transition from frame 0 to another frame occurs by translational bypassing of an extended region of the mRNA sequence rather than by slippage past a single nucleotide, as has been described for most examples of frameshifting. Translational bypassing has been characterized in two cases, T4 gene 60 coding for a topoisomerase subunit and in a trpR–lac′Z fusion. The latter was discovered in our laboratory, and the unique bypass mechanism is investigated further in this study. Using a trpR+1–lac′Z fusion system, we show that the Gln codon at the beginning of lacZ end at the 3′ side of the gap is required for bypassing to occur. The Gln codon is part of an mRNA segment that can (potentially) base pair with a segment at the 5′ and of Escherichia coli 16S rRNA. A model of trpR+1–lac′Z bypassing is suggested in which the untranslated region of the mRNA is looped out through base pairing between a segment in the 5′ end of the 16S rRNA and two sites in the mRNA. Translational bypassing is a newly discovered mechanism of gene expression, and trpR is the first cellular gene identified in which such a mechanism could operate. The understanding of this mechanism and its associated signals may be considered a paradigm for the expression of other genes by this alternative reading of the genetic code.Key words: genetic code, translation, frameshifting, trpR.
Comparison of functional peptide encoded in theEscherichia coli23S rRNA with other peptides involved incis-regulation of translation
Authors: Tenson Tanel , Mankin Alexander
Published: December 2009

A new approach for studying functional rRNA fragments has been developed based on using a plasmid library expressing random fragments of rRNA. A 34 nucleotide long fragment of Escherichia coli 23S rRNA has been identified that renders cells resistant to erythromycin, when expressed in vivo. The rRNA fragment contains a five codon long open reading frame, initiating at GUG and terminating at UAA, with a Shine–Dalgarno sequence located at an appropriate distance from the initiator codon. Translation of this mini-gene is required for the observed erythromycin resistance. Experiments with in vitro translated, or synthetic, peptide indicate the ribosome as a likely target for the action of the identified rRNA-encoded peptide, which apparently remains associated with the ribosome after completion of its translation. The known properties of the rRNA-encoded peptide are compared with information about other functionally active short peptides that can be involved in regulation of translation.Key words: ribosome, rRNA, peptide, translation, erythromycin.
Termination of translation in eukaryotes
Authors: Kisselev Lev L. , Frolova Lyudmila Yu.
Published: December 2009

Termination of translation is governed in ribosomes by polypeptide chain release factors (pRF and eRF in prokaryotes and eukaryotes, respectively). In prokaryotes, three pRF have been identified and sequenced, while in eukaryotes, only a single eRF has been identified to date. Recently, we have characterized a highly conserved protein family called eRF1. At least, human and Xenopus laevis proteins from this family are active as eRFs in the in vitro assay with any of the three stop codons. No structural similarity has been revealed between any of the three pRFs and eRF1 family. Furthermore, GTP-binding motifs have not been revealed, although translation termination in eukaryotes is a GTP-dependent process. We have demonstrated that in eukaryotes a second eRF exists in addition to eRF1, called eRF3. The eRF3 family has two features in common: presence of GTP-binding motifs and high conservation of the C-terminal domain structure. The C-terminal domain of the X. laevis eRF3 has no RF activity although it stimulates the eRF1 activity considerably at low concentration of the stop codons, conferring GTP dependence to the termination reaction. Without eRF3, the eRF1 activity is entirely GTP independent. Some features of X. laevis eRF3 (C-terminal domain) resemble those of pRF3. The newly identified eRF1 and eRF3 are structurally conserved and distinct from the respective pRF1/2 and pRF3 proteins, pointing to the possibility of different evolution of translation termination machinery in prokaryotes and eukaryotes. Bipartition of the translation termination apparatus probably provides high rate and accuracy of translation termination.Key words: higher eukaryotic polypeptide chain release factors, translation termination, protein biosynthesis.
Translational termination efficiency in both bacteria and mammals is regulated by the base following the stop codon
Authors: Tate Warren P. , Poole Elizabeth S. , Horsfield Julie A. , Mannering Sally A. , Brown Chris M. , Moffat John G. , Dalphin Mark E. , McCaughan Kim K. , Major Louise L. , Wilson Daniel N.
Published: December 2009

The translational stop signal and polypeptide release factor (RF) complexed with Escherichia coli ribosomes have been shown to be in close physical contact by site-directed photochemical cross-linking experiments. The RF has a protease-sensitive site in a highly conserved exposed loop that is proposed to interact with the peptidyltransferase center of the ribosome. Loss of peptidyl–tRNA hydrolysis activity and enhanced codon–ribosome binding by the cleaved RF is consistent with a model whereby the RF spans the decoding and peptidyltransferase centers of the ribosome with domains of the RF linked by conformational coupling. The cross-link between the stop signal and RF at the ribosomal decoding site is influenced by the base following the termination codon. This base determines the efficiency with which the stop signal is decoded by the RF in both mammalian and bacterial systems in vivo. The wide range of efficiencies correlates with the frequency with which the signals occur at natural termination sites, with rarely used weak signals often found at recoding sites and strong signals found in highly expressed genes. Stop signals are found at some recoding sites in viruses where −1 frame-shifting occurs, but the generally accepted mechanism of simultaneous slippage from the A and P sites does not explain their presence here. The HIV-1 gag-pol −1 frame shifting site has been used to show that stop signals significantly influence frame-shifting efficiency on prokaryotic ribosomes by a RF-mediated mechanism. These data can be explained by an E/P site simultaneous slippage mechanism whereby the stop codon actually enters the ribosomal A site and can influence the event.Key words: translational stop signal, decoding, release factor, frame-shifting.
Regulation of theEscherichia coliS10 ribosomal protein operon by heterologous L4 ribosomal proteins
Authors: Zengel Janice M. , Vorozheikina Dariya , Li Xiao , Lindahl Lasse
Published: December 2009

We have cloned the L4 ribosomal protein genes from Morganella morganii and Haemophilus influenza. The sequences of these genes were compared with published sequences for Escherichia coli, Yersinia pseudotuberculosis, and Bacillus stearothermophilus. All five of these L4 genes were expressed in E. coli and shown to function as repressors of both transcription and translation of the E. coli S10 operon. Possible implications for regulation of r-protein synthesis in species other than E. coli are discussed.Key words: ribosomes, autogenous control, r-protein L4, phylogeny.
Regulation of translation termination: conserved structural motifs in bacterial and eukaryotic polypeptide release factors
Authors: Nakamura Yoshikazu , Ito Koichi , Matsumura Kiyoyuki , Kawazu Yoichi , Ebihara Kanae
Published: December 2009

Translation termination requires codon-dependent polypeptide release factors. The mechanism of stop codon recognition by release factors is unknown and holds considerable interest since it entails protein–RNA recognition rather than the well-understood mRNA–tRNA interaction in codon–anticodon pairing. Bacteria have two codon-specific release factors and our picture of prokaryotic translation is changing because a third factor, which stimulates the other two, has now been found. Moreover, a highly conserved eukaryotic protein family possessing properties of polypeptide release factor has now been sought. This review summarizes our current understanding of the structural and functional organization of release factors as well as our recent findings of highly conserved structural motifs in bacterial and eukaryotic polypeptide release factors.Key words: translation termination, stop codon recognition, peptide chain release factors, seven-domain model.
Recognition determinants for proteins and antibiotics within 23S rRNA
Authors: Douthwaite Stephen , Voldborg Bjørn , Hansen Lykke Haastrup , Rosendahl Gunnar , Vester Birte
Published: December 2009

Ribosomal RNAs fold into phylogenetically conserved secondary and tertiary structures that determine their function in protein synthesis. We have investigated Escherichia coli 23S rRNA to identify structural elements that interact with antibiotic and protein ligands. Using a combination of molecular genetic and biochemical probing techniques, we have concentrated on regions of the rRNA that are connected with specific functions. These are located in different domains within the 23S rRNA and include the ribosomal GTPase-associated center in domain II, which contains the binding sites for r-proteins L10-(L12)4and L11 and is inhibited by interaction with the antibiotic thiostrepton. The peptidyltransferase center within domain V is inhibited by macrolide, lincosamide, and streptogramin B antibiotics, which interact with the rRNA around nucleotide A2058. Drug resistance is conferred by mutations here and by modification of A2058 by ErmE methyltransferase. ErmE recognizes a conserved motif displayed in the primary and secondary structure of the peptidyl transferase loop. Within domain VI of the rRNA, the α-sarcin stem–loop is associated with elongation factor binding and is the target site for ribotoxins including the N-glycosidase ribosome-inactivating proteins ricin and pokeweed antiviral protein (PAP). The orientations of the 23S rRNA domains are constrained by tertiary interactions, including a pseudoknot in domain II and long-range base pairings in the center of the molecule that bring domains II and V closer together. The phenotypic effects of mutations in these regions have been investigated by expressing 23S rRNA from plasmids. Allele-specific priming sites have been introduced close to these structures in the rRNA to enable us to study the molecular events there.Key words: rRNA tertiary structure, rRNA–antibiotic interaction, r-protein binding, Erm methyltransferase, rRNA modification.
Cotranslational folding of proteins
Authors: Kolb Vyacheslav A. , Makeyev Eugeny V. , Kommer Aigar , Spirin Alexander S.
Published: December 2009

Many unfolded polypeptides are capable of refolding into their native structure upon the removal of the denaturant. However, the folding of the mature protein during renaturation does not accurately reflect the folding process of nascent proteins in the interior of the cell. This view resulted from the discovery of molecular chaperones known to modulate protein folding. Recent publications discussing the possible role and mechanisms of chaperone action suggest that folding in vivo may be a posttranslational process. Here we discuss data that indicate the final native structure and biological activity can be attainted by nascent protein on the ribosome, thus supporting the cotranslational folding hypothesis.Key words: nacent peptide, globin, luciferase, folding.
Repeat-induced point mutations ofHSP80 gene ofNeurospora crassa: methylation of duplicated DNA sequences in the vegetative state
Authors: Vijayaraghavan Y. , Kapoor M.
Published: December 2009

The process of repeat-induced point mutations (RIP) was used to disrupt the gene encoding the 80-kDa heat-inducible protein of Neurospora crassa. Germinated conidia of the wild-type recipient strain were electrotransformed with a plasmid containing a 7-kb fragment harbouring the complete hsp80 gene sequence. Some of the transformants with a duplication of hsp80 gene sequence showed extensive methylation of these sequences even in vegetatively growing cells. The presence of an extra gene copy in transformants of this type resulted in a marked reduction in the expression of this gene. Progeny of a cross of one such transformant, showing methylation of hsp80, was analyzed by Southern blot and Northern blot hybridization to examine the relationship between methylation and the accumulation of hsp80 mRNA under hyperthermia. In addition, HSP80 polypeptide levels were monitored in stressed and unstressed cells by immunoblot analysis using polyclonal anti-HSP80 IgG preparations. A correlation between the extent of RIP and expression of this gene was observed in the progeny isolates.Key words: 80-kDa heat shock protein, RIP, DNA methylation, expression, HSP.
Multiple pathways for denaturation of horse plasma gelsolin
Authors: Koepf Edward K. , Burtnick Leslie D.
Published: December 2009

Gelsolin purified from horse plasma carries a surface charge distribution that greatly influences how the protein unfolds, aggregates, or precipitates as a function of temperature or concentration of chemical denaturant. Modification of gelsolin with fluorescein isothiocyanate replaces positive charges on amine groups with bulky, negatively charged fluorescein moieties. This postpones thermally induced precipitation by about 10 °C [Koepf, E.K., and Burtnick, L.D. 1993. Eur. J. Biochem. 212: 713–718]. Interaction with cations such as Ca2+or guanidinium+also alters the surface charge on gelsolin. This affects the structure of the protein in solution, modifies the pathway for unfolding, and moderates the onset of precipitation induced by chemical denaturants or heat. Denaturation of gelsolin is not interpretable in terms of a simple two-state cooperative mechanism. The pathway to a denatured state and intermediate structures present along the way depend upon the agent used to unfold the protein.Key words: gelsolin; denaturation, chemical, thermal, circular dichroism.
De novo design of α-helical proteins: basic research to medical applications
Authors: Hodges Robert S.
Published: December 2009

The two-stranded α-helical coiled-coil is a universal dimerization domain used by nature in a diverse group of proteins. The simplicity of the coiled-coil structure makes it an ideal model system to use in understanding the fundamentals of protein folding and stability and in testing the principles of de novo design. The issues that must be addressed in the de novo design of coiled-coils for use in research and medical applications are (i) controlling parallel versus antiparallel orientation of the polypeptide chains, (ii) controlling the number of helical strands in the assembly (iii) maximizing stability of homodimers or heterodimers in the shortest possible chain length that may require the engineering of covalent constraints, and (iv) the ability to have selective heterodimerization without homodimerization, which requires a balancing of selectivity versus affinity of the dimerization strands. Examples of our initial inroads in using this de novo design motif in various applications include: heterodimer technology for the detection and purification of recombinant peptides and proteins; a universal dimerization domain for biosensors; a two-stage targeting and delivery system; and coiled-coils as templates for combinatorial helical libraries for basic research and drug discovery and as synthetic carrier molecules. The universality of this dimerization motif in nature suggests an endless number of possibilities for its use in de novo design, limited only by the creativity of peptide–protein engineers.Key words: de novo design of proteins, α-helical coiled-coils, protein folding, protein stability, dimerization domain, dimerization motif.