ABSTRACT
Peroxisomes are thought to be formed by division of pre-existing peroxisomes after the import of newly synthesized proteins. However, it has been recently suggested that the endoplasmic reticulum (ER) provides an alternative de novo mechanism for peroxisome biogenesis in some cells. To test a possible role of the ER-Golgi transit in peroxisome biogenesis in mammalian cells, we evaluated the biogenesis of three peroxisomal membrane proteins (PMPs): ALDRP (adrenoleukodystrophy related protein), PMP70 and Pex3p in CHO cells. We constructed chimeric genes encoding these PMPs and green fluorescent protein (GFP), and transiently transfected them to wild type and mutant CHO cells, in which normal peroxisomes were replaced by peroxisomal membrane ghosts. The expressed proteins were targeted to peroxisomes and peroxisomal ghosts correctly in the presence or absence of Brefeldin A (BFA), a drug known to block the ER-Golgi transit. Furthermore, low temperature did not disturb the targeting of Pex3p-GFP to peroxisomes. We also constructed two chimeric proteins of PMPs containing an ER retention signal "DEKKMP": GFP-ALDRP-DEKKMP and myc- Pex3p-DEKKMP. These proteins were mostly targeted to peroxisomes. No colocalization with an ER maker was found. These results suggest that the classical ER-Golgi pathway does not play a major role in the biogenesis of mammalian PMPs.
Subject(s)
Animals , Cricetinae , Endoplasmic Reticulum/physiology , Golgi Apparatus/physiology , Mutation , Membrane Proteins/metabolism , Peroxisomes/metabolism , CHO Cells , Cricetulus , Endoplasmic Reticulum/metabolism , Membrane Proteins/geneticsABSTRACT
We have cloned and sequenced ten Helicobacter pylori genes from a Chilean strain (CH-CTX1) including: a cytotoxin VacA fragment, a CagA fragment (A17), a species-specific protein (TsaA), urease subunits (UreA, UreB), a flagellin subunit (FlaB), heat shock proteins (HspA and HspB), adhesin (HpaA) and a lipoprotein (Lpp20). We compared their deduced amino acid sequences with the corresponding sequences from three unrelated H. pylori strains, including fully sequenced strains 26695(UK) and J99(USA), and found that eight of them (UreA, UreB, FlaB, HspA, HspB, Lpp20, TsaA and HpaA) presented more than 97.3% identity. In contrast, VacA partial sequence showed lower identity values (93.2-94.9%). Moreover, we found major differences in the A17 region respect to the number and arrangement of the internal repeated elements when sequences from different strains were aligned. The A17 regions from strains CH-CTX1 and 26695 are very similar (91.8% identity) but lacked 6 repeated elements when compared to the Australian strains ATCC 43526 and NCTC 11637. The CCUG 17874 A17 region showed the largest deletion involving 9 repeats. A17 size differences between strains CCUG 17874 and CH-CTX1 were verified by PCR and polypeptide size. Such differences may explain variations in virulence among H. pylori strains as well as diversity in serum immunoreactivity.
Subject(s)
Bacterial Proteins , Cloning, Molecular , Genetic Variation , Helicobacter pylori , Alleles , Amino Acid Sequence , Base Sequence , Blotting, Western , DNA Primers , Genes , Helicobacter pylori , Polymerase Chain Reaction , VirulenceABSTRACT
Intra-oviductal administration of RNA obtained from oviducts of estradiol-treated rats resulted in accelerated egg transport (Rios et al., 1997). It is probable that estradiol-induced messenger RNA (mRNA) entered oviductal cells and was translated into the proteins involved in accelerated egg transport. In order to test this interpretation we deposited in vivo 50 micrograms of pure beta-galactosidase (beta-gal) mRNA, 50 micrograms of pure DNA from the reporter gene beta-gal under SV40 promoter or the vehicle (control oviducts) into the oviductal lumen of rats. Twenty four hours later the beta-gal activity was assayed in oviductal tissue homogenates using o-nitrophenyl-beta-D-galactopyranoside as a substrate. The administration of beta-gal mRNA and pSVBgal plasmid increased beta-gal activity by 71% and 142%, respectively, over the control oviducts. These results indicate that naked DNA and mRNA coding for beta-gal can enter oviductal cells and be translated into an active enzyme. They are consistent with the interpretation that embryo transport acceleration caused by the injection of estradiol-induced RNA in the oviduct involves translation of the injected mRNA
Subject(s)
Animals , Female , Rats , beta-Galactosidase , Fallopian Tubes , Ovum Transport , RNA, Messenger , beta-Galactosidase , Fallopian Tubes , Gene Expression , Rats, Sprague-DawleyABSTRACT
La proteinosis alveolar (PA), es una enfermedad pulmonar difusa caracterizada por el depósito de material fosfolipídico en los espacios alveolares, con conservación de la arquitectura pulmonar y sin reacción inflamatoria. La etiología es desconocida, sin embargo, actualmente, se han demostrado alteraciones anatómicas y funcionales de los macrófagos alveolares. Las evolución de la enfermedad es crónica y en muchas ocasiones indistinguible de enfermedades intersticiales difusas, en algunos casos el diagnóstico es siguerido por su asociación con enfermedades infecciosas (micosis). El diagnóstico definitivo siempre es histopatológico. No existe un tratamiento curativo, sin embargo, en la catualidad se ha utilizado el lavado pulmonar total, como una alternativa con buenos resultados en la mayoría de los casos. El presente reporte es con motivo de 2 casos estudiados en el Instituto con diferencias en su presentación clínica, radiológica, funcional y evolución final
Subject(s)
Humans , Male , Female , Adult , Middle Aged , Pulmonary Alveolar Proteinosis/etiology , Bronchoalveolar Lavage Fluid/therapy , Diagnosis, Differential , Macrophages/immunology , Pulmonary Alveolar Proteinosis/diagnosis , Pulmonary Alveolar Proteinosis/physiopathologyABSTRACT
Mediante técnicas de ingeniería genética se ha logrado obtener líneas estables de células animales que sintetizan y secretan eficientemente partículas de antígeno de superficie del virus de la hepatitis B. Estas partículas presentan al microscopio electrónico un tamaño de 22 nm y son estructuralmente e inmunogénicamente similares a las obtenidas de plasma de enfermos crónicos, por lo que constituyen una excelente fuente de antígeno para la elaboración de una vacuna contra el virus