RANKL directly induces bone morphogenetic protein-2 expression in RANK-expressing POS-1 osteosarcoma cells

International audienceThe POS-1 murine model of osteolytic osteosarcoma was used to elucidate the molecular and cellular mechanisms involved in the development of primary bone tumors and associated lung metastasis. The POS-1 cell line is derived from an osteosarcoma tumor which develops spontaneously in C3H mice. The POS-1 cell line was characterized in vitro by mineralization capacity and expression of bone markers by semi-quantitative RT-PCR, compared to primary osteoblasts and bone marrow cells. POS-1 cells showed no mineralization capacity and exhibited an undifferentiated phenotype, expressing both osteoblastic and unexpected osteoclastic markers (TRAP, cathepsin K and RANK). Thereby, experiments were performed to determine whether RANK was functional, by studying the biological activity of murine RANKL through the receptor RANK expressed on POS-1 cells. Results revealed a RANKL-induced increase in ERK phosphorylation, as well as BMP-2 induction at the mRNA and protein levels, and a decrease of POS-1 cell proliferation in the presence of 10 ng/ml RANKL. BMP-2 induction is dependent on the ERK 1/2 signal transduction pathway, as its expression is abolished in the presence of UO126, a specific synthetic inhibitor of the ERK 1/2 pathway. Moreover, a 2-fold molar excess of soluble RANK blocks the RANKL-induced BMP-2 expression, demonstrating that the biological effects of RANKL observed in POS-1 cells are mediated by RANK. This is the first report describing a functional RANK expressed on osteosarcoma cells, as shown by its ability to induce signal transduction pathways and biological activity when stimulated by RANKL

LF : a language for reliable embedded systems

Thesis (MSc)--University of Stellenbosch, 2001.ENGLISH ABSTRACT: Computer-aided verification techniques, such as model checking, are often considered essential to produce highly reliable software systems. Modern model checkers generally require models to be written in eSP-like notations. Unfortunately, such systems are usually implemented using conventional imperative programming languages. Translating the one paradigm into the other is a difficult and error prone process. If one were to program in a process-oriented language from the outset, the chasm between implementation and model could be bridged more readily. This would lead to more accurate models and ultimately more reliable software. This thesis covers the definition of a process-oriented language targeted specifically towards embedded systems and the implementation of a suitable compiler and run-time system. The language, LF, is for the most part an extension of the language Joyce, which was defined by Brinch Hansen. Both LF and Joyce have features which I believe make them easier to use than other esp based languages such as occam. An example of this is a selective communication primitive which allows for both input and output guards which is not supported in occam. The efficiency of the implementation is important. The language was therefore designed to be expressive, but constructs which are expensive to implement were avoided. Security, however, was the overriding consideration in the design of the language and runtime system. The compiler produces native code. Most other esp derived languages are either interpreted or execute as tasks on host operating systems. Arguably this is because most implementations of esp and derivations thereof are for academic purposes only. LF is intended to be an implementation language. The performance of the implementation is evaluated in terms of practical metries such as the time needed to complete communication operations and the average time needed to service an interrupt.AFRIKAANSE OPSOMMING: Rekenaar ondersteunde verifikasietegnieke soos programmodellering, is onontbeerlik in die ontwikkeling van hoogs betroubare programmatuur. In die algemeen, aanvaar programme wat modelle toets eSP-agtige notasie as toevoer. Die meeste programme word egter in meer konvensionele imperatiewe programmeertale ontwikkel. Die vertaling vanuit die een paradigma na die ander is 'n moelike proses, wat baie ruimte laat vir foute. Indien daar uit die staanspoor in 'n proses gebaseerde taal geprogrammeer word, sou die verwydering tussen model en program makliker oorbrug kon word. Dit lei tot akkurater modelle en uiteindelik tot betroubaarder programmatuur. Die tesis ondersoek die definisie van 'n proses gebaseerde taal, wat gemik is op ingebedde programmatuur. Verder word die implementasie van 'n toepaslike vertaler en looptyd omgewing ook bespreek. Die taal, LF, is grotendeels gebaseer op Joyce, wat deur Brinch Hansen ontwikkel is. Joyce en op sy beurt LF, is verbeterings op ander esp verwante tale soos occam. 'n Voorbeeld hiervan is 'n selektiewe kommunikasieprimitief wat die gebruik van beide toevoer- en afvoerwagte ondersteun. Omdat 'n effektiewe implementasie nagestreef word, is die taalontwerp om so nadruklik moontlik te wees, sonder om strukture in te sluit wat oneffektief is om te implementeer. Sekuriteit was egter die oorheersende oorweging in die ontwerp van die taal en looptyd omgewing. Die vertaler lewer masjienkode, terwyl die meeste ander implementasies van eSP-agtige tale geinterpreteer word of ondersteun word as prosesse op 'n geskikte bedryfstelsel- die meeste eSP-agtige tale word slegs vir akademiese doeleindes aangewend. LF is by uitstek ontwerp as implementasie taal. Die evaluasie van die stelsel se werkverrigting is gedoen aan die hand van praktiese maatstawwe soos die tyd wat benodig word vir kommunikasie, sowel as die gemiddelde tyd benodig vir die hantering van onderbrekings

Improving the PCR protocol to amplify a repetitive DNA sequence

Although PCR-based techniques have become an essential tool in the field of molecular and genetic research, the amplification of repetitive DNA sequences is limited. This is due to the truncated nature of the amplified sequences, which are also prone to errors during DNA polymerase-based amplification. The complex structure of repetitive DNA can form hairpin loops, which promote dissociation of the polymerase from the template, impairing complete amplification, and leading to the formation of incomplete fragments that serve as megaprimers. These megaprimers anneal with other sequences, generating unexpected fragments in each PCR cycle. Our gene model, MaSp1, is 1037-bp long, with 68% GC content, and its amino acid sequence is characterized by poly-alanine-glycine motifs, which represent the repetitive codon consensus. We describe the amplification of the MaSp1 gene through minor changes in the PCR program. The results show that a denaturation temperature of 98°C is the key determinant in the amplification of the MaSp1 partial gene sequence

Clinicopathological and toxicological aspects of poisoning by the clomazone herbicide in sheep

AbstractClomazone (2-[(2-chlorophenyl)methyl]-4,4-dimethyl-3-isoxazolidinone) is a herbicide which has been widely used in southern Brazil in the rice cultivation and can be toxic for humans and animals. This study reports the first outbreak poisoning due to clomazone in a flock of 103 sheep, 20 of which showed mainly neurological and respiratory signs. Clomazone was detected in soil and vegetation samples and in the liver, kidney and muscles of poisoned animals. The poisoning was experimentally reproduced in three sheep by the administration of a 134mgkg body weight dose of clomazone. In both the natural and experimental cases, the clinical signs included tachypnea, anorexia, somnolence, weakness and ataxia. Macroscopically, there were no significant changes. Histologically, vacuolization in the white matter, perineuronal vacuoles and congestion of the leptomeningeal and brain vessels were observed. Ultrastructurally, the vacuolar lesions in the brain corresponded to swelling of the dendrites and astrocytic processes. It is concluded that clomazone causes toxic neuropathy in sheep