Association of 16S and 23S ribosomal RNAs to form a bimolecular complex

Association of the 30S and 50S subunits to generate the 70S ribosomes of Escherichia coli has long been known but the mechanism of this interaction remains obscure. Light-scattering studies indicate that naked 16S and 23S RNAs can also associate under conditions similar to those required for the assembly of ribosomes from the constituent RNAs and proteins. The RNA-RNA association also takes place in the presence of ethanol, which promotes folding of 16S and 23S RNAs into specific compact structures with the morphological features of 30S and 50S ribosomes, respectively. Equimolar amounts of the two RNAs are involved in the association. The formation of a stoichiometric complex was shown by light scattering, sucrose density gradient centrifugation, and composite polyacrylamide/agarose gel electrophoresis. The presence of the two species of RNA in the complex was also shown by gel electrophoresis. The association of naked 16S and 23S RNAs suggests that RNA-RNA interaction may play an important role in the association of 30S and 50S subunits

Transport in bacteriophage P22-infected Salmonella typhimurium

There was rapid efflux of L-leucine, L-phenylalanine, and α-methyl-D-glucoside after infection of Salmonella typhimurium with the clear plaque mutant C1 of phage P22. The efflux was similar to that observed with cyanide or arsenate treatment except that there was partial recovery in the case of phage infection and almost complete recovery under the condition of lysogeny. There was no efflux after infection with the temperature-sensitive mutant ts16C1 at nonpermissive temperature. Superinfection of superinfection exclusion negative lysogen (sie A - sie B-) with C1 led to efflux, whereas the efflux was much less on superinfection of sie A+ Sie B+ lysogen. These results indicate that an effective injection process is enough to cause depression in the cellular transport processes

Interconversion of tight and loose couple 50 S ribosomes and translocation in protein synthesis

On incubation of 50 S ribosomes, isolated from either tight couple (TC) or loose couple (LC) 70 S ribosomes, with elongation factor G (EG-G) and guanosine 5'-triphosphate, a mixture of TC and LC 50 S ribosomes is formed. There is almost complete conversion of LC 50 S ribosomes to TC 50 S ribosomes on treatment with EF-G, GTP, and fusidic acid. Similarly, TC 50 S ribosomes are converted to LC 50 S ribosomes, although partially, by treatment with EF-G and a GTP analogue like guanyl-5'-yl methylenediphosphate (GMP-P(CH2)P) or guanyl-5'-yl imidodiphosphate (GMP-P(NH)P) and including a polymer of 5'-uridylic acid (poly(U] in the incubation mixture. Furthermore, LC 23 S RNA isolated from LC 50 S ribosomes is converted to TC 23 S RNA on heat treatment, but similar treatment does not affect TC 23 S RNA. The interconversion was followed by several physical and biological characteristics of TC and LC 50 S ribosomes, like association capacities with 30 S ribosomes before and after kethoxal treatment, susceptibility to RNase I and polyphenylalanine-synthesizing capacity in association with 30 S ribosomes, as well as thermal denaturation profiles, circular dichroic spectra, and association capacity of isolated 23 S RNAs. These data strongly support the proposition that TC and LC 50 S ribosomes are the products of translocation during protein synthesis. The conformational change of 23 S RNA induced by EF-G and GTP is most probably responsible for the interconversion, and L7/L12 proteins play an important role in the process. A two-site model based on kethoxal data has also been proposed to explain the tightness and looseness of 70 S couples

Magnus force and acoustic Stewart-Tolman effect in type II superconductors

At zero magnetic field we have observed an electromagnetic radiation from superconductors subjected by a transverse elastic wave. This radiation has an inertial origin, and is a manifestation of the acoustic Stewart-Tolman effect. The effect is used for implementing a method of measurement of an effective Magnus force in type II superconductors. The method does not require the flux flow regime and allows to investigate this force for almost the whole range of the existence of the mixed state. We have studied behavior of the gyroscopic force in nonmagnetic borocarbides and Nb. It is found that in borocarbides the sign of the gyroscopic force in the mixed state is the same as in the normal state, and its value (counted for one vortex of unit length) has only a weak dependence on the magnetic field. In Nb the change of sign of the gyroscopic force under the transition from the normal to the mixed state is observed.Comment: 4 pages, 5 figure

Baculovirus vector-mediated expression of heterologous genes in insect cells

The baculovirus expression system employing Autagrapha californica nuclear polyhidrosis virus and Spodoptera frugiperda insect cells in culture has proved very popular for high level expression of heterologous genes: In this system, transcription of the foreign gene is usually driven by the hyperactive and temporally regulated polyhedrin gene promoter. Replacement of the polyhedrin gene, which encodes a 29-kDa occlusion protein (non-essential for viral replication), with a gene of interest leads to an occlusion negative phenotype which serves as a visual marker to select for recombinant viruses. Simultaneous expression of multiple genes can also be achieved. The heterologous proteins synthesized in this system are antigenically, immunologically and functionally identical in most respects to their native counterparts. This mini-review will aim at summarizing the potentials and utility of the baculovirus expression vector system and will address some important questions relating to the biology of this system

MiR223-3p promotes synthetic lethality in BRCA1-deficient cancers

Defects in DNA repair give rise to genomic instability, leading to neoplasia. Cancer cells defective in one DNA repair pathway can become reliant on remaining repair pathways for survival and proliferation. This attribute of cancer cells can be exploited therapeutically, by inhibiting the remaining repair pathway, a process termed synthetic lethality. This process underlies the mechanism of the Poly-ADP ribose polymerase-1 (PARP1) inhibitors in clinical use, which target BRCA1 deficient cancers, which is indispensable for homologous recombination (HR) DNA repair. HR is the major repair pathway for stressed replication forks, but when BRCA1 is deficient, stressed forks are repaired by back-up pathways such as alternative nonhomologous end-joining (aNHEJ). Unlike HR, aNHEJ is nonconservative, and can mediate chromosomal translocations. In this study we have found that miR223-3p decreases expression of PARP1, CtIP, and Pso4, each of which are aNHEJ components. In most cells, high levels of microRNA (miR) 223-3p repress aNHEJ, decreasing the risk of chromosomal translocations. Deletion of the miR223 locus in mice increases PARP1 levels in hematopoietic cells and enhances their risk of unprovoked chromosomal translocations. We also discovered that cancer cells deficient in BRCA1 or its obligate partner BRCA1-Associated Protein-1 (BAP1) routinely repress miR223-3p to permit repair of stressed replication forks via aNHEJ. Reconstituting the expression of miR223-3p in BRCA1- and BAP1-deficient cancer cells results in reduced repair of stressed replication forks and synthetic lethality. Thus, miR223-3p is a negative regulator of the aNHEJ DNA repair and represents a therapeutic pathway for BRCA1- or BAP1-deficient cancers

Elevated expression of artemis in human fibroblast cells is associated with cellular radiosensitivity and increased apoptosis

Copyright @ 2012 Nature Publishing GroupThis article has been made available through the Brunel Open Access Publishing Fund.Background: The objective of this study was to determine the molecular mechanism(s) responsible for cellular radiosensitivity in two human fibroblast cell lines 84BR and 175BR derived from two cancer patients. Methods: Clonogenic assays were performed following exposure to increasing doses of gamma radiation to confirm radiosensitivity. γ-H2AX foci assays were used to determine the efficiency of DNA double strand break (DSB) repair in cells. Quantitative-PCR (Q-PCR) established the expression levels of key DNA DSB repair proteins. Imaging flow cytometry using Annexin V-FITC was used to compare artemis expression and apoptosis in cells. Results: Clonogenic cellular hypersensitivity in the 84BR and 175BR cell lines was associated with a defect in DNA DSB repair measured by the γ-H2AX foci assay. Q-PCR analysis and imaging flow cytometry revealed a two-fold overexpression of the artemis DNA repair gene which was associated with an increased level of apoptosis in the cells before and after radiation exposure. Over-expression of normal artemis protein in a normal immortalised fibroblast cell line NB1-Tert resulted in increased radiosensitivity and apoptosis. Conclusion: We conclude elevated expression of artemis is associated with higher levels of DNA DSB, radiosensitivity and elevated apoptosis in two radio-hypersensitive cell lines. These data reveal a potentially novel mechanism responsible for radiosensitivity and show that increased artemis expression in cells can result in either radiation resistance or enhanced sensitivity.This work was supported in part by The Vidal Sassoon Foundation USA. This article is made available through the Brunel Open Access Publishing Fund

Improvement in medication education in a pediatric subspecialty practice

<p>Abstract</p> <p>Background</p> <p>The purpose of this study was to measure the impact of an educational intervention on parents of children taking methotrexate (MTX) for juvenile idiopathic arthritis (JIA).</p> <p>Methods</p> <p>This study was conducted using a pre- and postsurvey design. The parents of 100 children with JIA taking MTX for at least 2 months were surveyed during a routine office visit. The parents completed an initial questionnaire regarding the safe use, adverse effects, and guidelines for monitoring the toxicity of MTX. An educational intervention was then administered, and an identical follow-up questionnaire was given during the next office visit. Statistical analysis using a paired <it>t</it>-test (critical <it>P </it>value < 0.05) was performed on individuals who answered both questionnaires.</p> <p>Results</p> <p>There were 100 responses to the initial questionnaire and 67 responses to the follow-up questionnaire. The mean length of time between surveys was 2.9 ± 0.9 months. In those who completed both questionnaires, the overall correct score increased significantly from 75.8% to 93.4%, respectively (<it>P </it>< 0.0001). Individuals scored the lowest (49%) on the question that addressed MTX's impact on pregnancy and fertility.</p> <p>Conclusions</p> <p>MTX knowledge may be less than expected in the parents of children with JIA. Brief educational interventions in the pediatric subspecialty practice can significantly affect a family's understanding of their child's medications.</p

Abschätzung der Auswirkungen der Krautfäule auf den Bio-Kartoffelbau in verschiedenen Europäischen Ländern, sowie Inventar der angewendeten Anbau- und Pflanzenschutzstrategien

Problemstellung/Ziele Die Kraut- und Knollenfäule (Phytophthora infestans) ist die Krankheit, die im biologischen (aber auch konventionellen) Kartoffelanbau in Europa die größten Probleme verursacht. Unter günstigen klimatischen Bedingungen breitet sich die Krankheit sehr rasch aus und kann große Ertragsausfälle verursachen. Innerhalb von Europa variiert der durch P. infestans verursachte wirtschaftliche Schaden stark zwischen den Regionen. Dies hängt von verschiedenen Faktoren ab, aber in biologisch bewirtschafteten Anbausystemen immt man an, dass die klimatischen Bedingungen, die verwendeten Sorten sowie die agronomischen Maßnahmen wie Bodenbearbeitung oder die Verwendung von Pflanzenschutzmitteln eine wichtige Rolle spielen. Die Reduktion oder das Verbot des Kupfereinsatzes im biologischen Kartoffelanbau wird deshalb auch unterschiedliche Auswirkungen in den verschiedenen Europäischen Regionen haben. Als Teilprojekt des EU-finanzierten Projektes Blight-MOP (QLRT 31065) wurde eine detaillierte Studie der ökonomischen und gesetzlichen Rahmenbedingungen und ein Inventar der Anbausysteme in 7 Ländern durchgeführt, um (i) Ein Inventar der derzeitigen Anbautechniken zu erstellen, (ii) die Auswirkungen von P. infestans auf Erträge und Wirtschaftlichkeit zu evaluieren und die Auswirkungen eines Kupferverbotes abzuschätzen und um (iii) Pflanzenschutzstrategien von Bioproduzenten zu identifizieren, die bereits jetzt ohne den Einsatz von Kupfer auskommen. Fazit: Diese Betriebsanalyse weist darauf hin, dass eine Optimierung der Einzelmassnahmen und die regionsspezfische Integration von Massnahmen zu einer substanziellen Verbesserung des Anbauerfolges führen können. Die Betriebsanalyse zeigt auch, dass Kupfer bislang eine wichtige Rolle bei der Ertragsbildung gespielt hat. Ein Kupferverbot ohne Angebot von praxistauglichen Alternativlösungen (wie sie innerhalb von Blight-MOP und anderen Projekten erarbeitet werden) würde demnach zu einer starken Destabilisierung des biologischen Kartoffelanbaues und vermutlich zu einer Angebotsvernappung führen. Andere Teilprojekte des Blight-MOP Projektes zielen auf die Verbesserung von anbautechnischen Massnahmen und die Entwicklung von Ersatzprodukten für Kupfer ab

Endonuclease EEPD1 Is a Gatekeeper for Repair of Stressed Replication Forks

Replication is not as continuous as once thought, with DNA damage frequently stalling replication forks. Aberrant repair of stressed replication forks can result in cell death or genome instability and resulting transformation to malignancy. Stressed replication forks are most commonly repaired via homologous recombination (HR), which begins with 5' end resection, mediated by exonuclease complexes, one of which contains Exo1. However, Exo1 requires free 5'-DNA ends upon which to act, and these are not commonly present in non-reversed stalled replication forks. To generate a free 5' end, stalled replication forks must therefore be cleaved. Although several candidate endonucleases have been implicated in cleavage of stalled replication forks to permit end resection, the identity of such an endonuclease remains elusive. Here we show that the 5'-endonuclease EEPD1 cleaves replication forks at the junction between the lagging parental strand and the unreplicated DNA parental double strands. This cleavage creates the structure that Exo1 requires for 5' end resection and HR initiation. We observed that EEPD1 and Exo1 interact constitutively, and Exo1 repairs stalled replication forks poorly without EEPD1. Thus, EEPD1 performs a gatekeeper function for replication fork repair by mediating the fork cleavage that permits initiation of HR-mediated repair and restart of stressed forks