Coronaviruses have large positive-strand RNA genomes that are 5' capped and 3' polyadenylated. The 5'-terminal two-thirds of the genome contain two open reading frames (ORFs), 1a and 1b, that ...together make up the viral replicase gene and encode two large polyproteins that are processed by viral proteases into 15-16 nonstructural proteins, most of them being involved in viral RNA synthesis. ORFs located in the 3'-terminal one-third of the genome encode structural and accessory proteins and are expressed from a set of 5' leader-containing subgenomic mRNAs that are synthesized by a process called discontinuous transcription. Coronavirus protein synthesis not only involves cap-dependent translation mechanisms but also employs regulatory mechanisms, such as ribosomal frameshifting. Coronavirus replication is known to affect cellular translation, involving activation of stress-induced signaling pathways, and employing viral proteins that affect cellular mRNA translation and RNA stability. This chapter describes our current understanding of the mechanisms involved in coronavirus mRNA translation and changes in host mRNA translation observed in coronavirus-infected cells.
Food-borne outbreaks caused by
Listeria monocytogenes have been recognized in US and European countries. Only sporadic cases, of neonatal listeriosis, have been reported in Japan. Since
L. ...monocytogenes has been often isolated from foods in Japan, food-borne outbreaks potentially could have occurred. In February 2001,
L. monocytogenes serotype 1/2b was isolated from a washed-type cheese during routine
Listeria monitoring of 123 domestic cheeses. Further samples from products and the environments at the plant that produced the contaminated cheese were examined for
L. monocytogenes.
L. monocytogenes serotype 1/2b was detected in 15 cheese samples, at most probable number that ranged from <
30 to 4.6
×
10
9/100 g, and in environmental samples. Studies with people who had consumed cheese from the plant revealed 86 persons who had been infected with
L. monocytogenes. Thirty-eight of those people had developed clinical symptoms of gastroenteritis or the common cold type after the consumption of cheese. Isolates from those patients exhibited the same serotype, pathogenicity for mice and HeLa cells, DNA fingerprinting patterns and PCR amplification patterns. From the epidemiological and genetic evidence, it appeared that the outbreak was caused by cheese. This is the first documented incidence of food-borne listeriosis in Japan.
Abstract Controlled invasion of extravillous trophoblast (EVT) is necessary for implantation and placentation. The serine protease HTRA3 is highly expressed in decidual cells in the late secretory ...phase of the menstrual cycle and throughout pregnancy. During the first trimester it is expressed in most trophoblast cell types, but not in the invading interstitial trophoblast. HTRA3 and its family members are down-regulated in a number of cancers and are proposed as tumour-suppressors. The current study investigated whether inhibiting HTRA3 in a first trimester trophoblast cell line expressing high levels of HTRA3 would alter invasion. HTR-8/SVneo (HTR-8, derived from first trimester placenta) and a number of choriocarcinoma cells (JEG-3, AC-1M88 and AC-1M32) were screened for HTRA3 expression. Only HTR-8 cells expressed high levels of HTRA3 mRNA, consistent with HTRA3 being down-regulated in cancer. Western blotting and immunofluorescence confirmed HTRA3 protein expression and localisation in HTR-8 cells. HTRA3 was detected in conditioned medium of HTR-8 cells, confirming its secretory nature. For functional studies, both long and short forms of recombinant human HTRA3, wild type and protease-inactive mutant (S305 A) were produced using wheat-germ cell-free technology. Both have a similar molecular size, but the mutants have negligible protease activity. In addition, the mutants significantly inhibited the wild type protease activity, supporting their dominant-negative inhibition and utility as specific inhibitors of the wild type protein. Inhibition of HTRA3 by exogenous addition of HTRA3 mutant resulted in a significant increase in HTR-8 cell invasion. These results strongly support the hypothesis that HTRA3 is an inhibitor of trophoblast invasion during placental development.
A gene encoding the resuscitation-promoting factor (Rpf) from
Salmonella Typhimurium LT2 was cloned and characterized. The amino acid sequence encoded by
S. Typhimurium LT2
rpf gene shares 24.2% ...homology with
Micrococcus luteus Rpf, which is secreted by growing cells, and required to resuscitate from viable but non-culturable (VNC) state. The
S. Typhimurium LT2
rpf gene is 696 bp long, and shared a conserved segment with
Salmonella enterica serovar Oranienburg (99.4%). Recombinant Rpf (rRpf) proteins of
S. Typhimurium LT2 after expression in
E. coli BL21 harboring the pET15-b plasmid was approximately 25 kDa. Since
S. Oranienburg cells are relatively quick to enter the VNC state just after incubating in the presence of 7% NaCl at 37 °C for 3 days, we evaluated the biological effect of rRpf by using
S. Oranienburg VNC cells. The rRpf not only promoted proliferation but also induced resuscitation of VNC cells to the culturable state in a dose-dependent manner. Therefore, rRpf may be useful for detection of bacterial contaminants present in the VNC form in food samples and the environment.
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