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Cell shrinkage is a hallmark of the apoptotic mode of programmed cell death, but it is as yet unclear whether a reduction in cell volume is a primary activation signal of apoptosis. Here we studied the effect of an acute elevation of osmolarity (NaCl or sucrose additions, final osmolarity 687 mosmol l −1 ) on NIH 3T3 fibroblasts to identify components involved in the signal transduction from shrinkage to apoptosis. After 1.5 h the activity of caspase-3 started to increase followed after 3 h by the appearance of many apoptotic-like bodies. The caspase-3 activity increase was greatly enhanced in cells expressing a constitutively active G protein, Rac (RacV 12 A 3 cell), indicating that Rac acts upstream to caspase-3 activation. The stress-activated protein kinase, p38, was significantly activated by phosphorylation within 30 min after induction of osmotic shrinkage, the phosphorylation being accelerated in fibroblasts overexpressing Rac. Conversely, the activation of the extracellular signal-regulated kinase (Erk1/2) was initially significantly decreased. Subsequent to activation of p38, p53 was activated through serine-15 phosphorylation, and active p53 was translocated from the cytosol to the nucleus. Inhibition of p38 in Rac cells reduced the activation of both p53 and caspase-3. After 60 min in hypertonic medium the rate constants for K + and taurine efflux were increased, particular in Rac cells. We suggest the following sequence of events in the cell shrinkage-induced apoptotic response: cellular shrinkage activates Rac, with activation of p38, followed by phosphorylation and nuclear translocation of p53, resulting in permeability increases and caspase-3 activation.

BackgroundMycoplasma pneumoniae is a common cause of community-acquired pneumonia in older children. Pulmonary and extra-pulmonary symptoms associated with M. pneumoniae infection are reported. M. pneumoniae is mainly epidemic in Denmark with the recurrence every 4-7th year.AimsRetrospectively, to describe the epidemiology and clinical features, in infants and children, during the M. pneumoniae epidemic in 2010 and 2011.MethodsAll children under the age of 16 that were tested for M. pneumoniae during the period 01.02.2010–31.01.2012 were included. Medical charts, as well as radiological findings, were reviewed for all children with M. pneumoniae. A post-hoc analysis of viral co-infections was done on part of the cohort.Results134 of 746 children were tested positive for M. pneumoniae by PCR or serology. Positive tests were found in 65% of children seven years and older, in 30% of 2-6-year-olds and 4% of infants (less than two years of age). Viral co-infection was found in 27% of the tested samples. The clinical presentation was a cough, asthma-like symptoms and low-grade fever. Extra-pulmonary symptoms were common and presented as nausea/vomiting by 33% of the children and skin manifestations by 25%. 84% of the children had a chest x-ray taken, and there were positive radiological findings in 94% of these.ConclusionM. pneumoniae also affected infants and young children and symptoms were similar to infections with respiratory viruses, but severe LRTI were also seen. During an up-coming epidemic, assessment of extra-pulmonary manifestations can be helpful when diagnosing M. pneumoniae infections.

Modulation of translation initiation efficiency on classical swine fever virus (CSFV) RNA can be achieved by targeted mutations within the internal ribosome entry site (IRES). In this study, cDNAs corresponding to the wild-type (wt) or mutant forms of the IRES of CSFV strain Paderborn were amplified and inserted into dicistronic reporter plasmids encoding Fluc and Rluc under the control of a T7 promoter. The mutations were within domains II, IIId 1 , and IIIf of the IRES. The plasmids were transfected into baby hamster kidney (BHK) cells infected with recombinant vaccinia virus vTF7-3, which expresses the T7 RNA polymerase. IRES mutants with different levels of IRES activity were identified and then introduced by homologous recombination into bacterial artificial chromosomes (BACs) containing CSFV Paderborn cDNA downstream of a T7 promoter. From the wt and mutant BACs, full-length CSFV RNA transcripts were produced in vitro and electroporated into porcine PK15 cells. Rescued mutant viruses were obtained from RNAs that contained mutations within domain IIIf which retained more than 75% of the wt translation efficiency. Sequencing of cDNA generated from these rescued viruses verified the maintenance of the introduced changes within the IRES. The growth characteristics of each rescued mutant virus were compared to those of the wt virus. It was shown that viable mutant viruses with reduced translation initiation efficiency can be designed and generated and that viruses containing mutations within domain IIIf of the IRES have reduced growth in cell culture compared to the wt virus. C lassical swine fever virus (CSFV), the causative agent of the highly contagious pig disease classical swine fever (CSF), belongs to the pestivirus genus within the family Flaviviridae. Its positive-sense single-stranded RNA genome (ca. 12.3 kb) contains one long open reading frame (ORF) encoding structural and nonstructural proteins within a single polyprotein (33). The CSFV RNA is infectious, as it functions as an mRNA to produce the structural and nonstructural proteins necessary for virus replication and formation. The ORF is flanked by untranslated regions (UTRs) termed the 5= UTR (373 nucleotides [nt]) and 3= UTR (ca. 228 nt) (9). The UTRs within the CSFV RNA are distinct from those of most eukaryotic mRNAs. There is no 5=-terminal cap structure; instead, the 5= UTR harbors an internal ribosome entry site (IRES) (40, 47), while the 3= UTR lacks a poly(A) tail but contains a variable AU-rich region and a conserved region (9, 54). Previous studies (6, 9, 11) have shown that the CSFV 5= UTR contains a number of structural elements, including two small stem-loops (domains Ia and Ib) plus the IRES, which comprises a single large stem-loop (domain II) and a complex, domain III, which contains the subdomains IIIa, IIIb, IIIc, IIId 1 , IIId 2 , and IIIe plus IIIf (the pseudoknot structure). The organization of motifs and domains within the IRES is conserved among some groups of viruses belonging to the Flaviviridae family, including hepatit...

Friis MB, Vorum KG, Lambert IH. Volume-sensitive NADPH oxidase activity and taurine efflux in NIH3T3 mouse fibroblasts. Am J Physiol Cell Physiol 294: C1552-C1565, 2008. First published April 16, 2008 doi:10.1152/ajpcell.00571.2007.-Reactive oxygen species (ROS) are produced in NIH3T3 fibroblasts during hypotonic stress, and H2O2 potentiates the concomitant release of the organic osmolyte taurine (Lambert IH. J Membr Biol 192: 19 -32, 2003). The increase in ROS production [5-(and-6)-carboxy-2Ј, 7Ј-dichlorodihydrofluorescein diacetate fluorescence] is detectable after a reduction in the extracellular osmolarity from 335 mosM (isotonic) to 300 mosM and reaches a maximal value after a reduction to 260 mosM. The swelling-induced ROS production is reduced by the flavoprotein inhibitor diphenylene iodonium chloride (25 M) but is unaffected by the nitric oxide synthase inhibitor N -nitro-L-arginine methyl ester, indicating that the volume-sensitive ROS production is NADPH oxidase dependent. NIH3T3 cells express the NADPH oxidase components: p22 phox , a NOX4 isotype; p47 phox ; and p67 phox (real-time PCR). Exposure to the Ca 2ϩ -mobilizing agonist ATP (10 M) potentiates the release of taurine but has no effect on ROS production under hypotonic conditions. On the other hand, addition of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 100 nM) or the lipid messenger lysophosphatidic acid (LPA, 10 nM) potentiates the swelling-induced taurine release as well as the ROS production. Overexpression of Rac1 or p47 phox or p47 phox knockdown [small interfering (si)RNA] had no effect on the swelling-induced ROS production or taurine release. NOX4 knockdown (siRNA) impairs the increase in the ROS production and the concomitant taurine release following osmotic exposure. It is suggested that a NOX4 isotype plus p22 phox account for the swelling-induced increase in the ROS production in NIH3T3 cells and that the oxidase activity is potentiated by PKC and LPA but not by Ca 2ϩ . organic osmolytes; NOX4; lysophospholipids; arachidonic acid mobilization; adenosine triphosphate; calcium REACTIVE OXYGEN SPECIES (ROS), e.g., superoxides, hydroxyl radicals, and H 2 O 2 , are produced in a variety of cells upon ligand stimulation, during stress, cell proliferation, and apoptosis (9,16,59,62,63). Within recent years, it has been shown that ROS are produced also upon osmotic stress in various cell types (35,51,71) and that ROS are involved in the swellinginduced activation and inactivation of the volume-sensitive release pathway for the organic osmolyte taurine in, e.g., NIH3T3 fibroblasts (35, 38) and in the activity of the volumesensitive, outwardly rectifying Cl Ϫ channel in liver cells (71). Activation of the volume-sensitive taurine release pathway in NIH3T3 fibroblasts involves specific phospholipase A 2 (PLA 2 ) (40,41), and it appears that the swelling-induced ROS production involves a NADPH oxidase, which is activated at a step downstream to the PLA 2 activation (35). Colston and coworkers (13) similarly ...