Intracellular transport of cholesteryl esters from lysosomes to cytoplasm in macrophages

The mechanism through which nonmembranous lipid inclusion bodies consisting of cholesteryl esters accumulate in the cytoplasm was studied. Most lipid inclusion bodies in macrophages after 24 h incubation with anisotropic cholesteryl oleate liquid crystals were surrounded by a limiting membrane. The limiting membrane, however, could not be observed after further incubation for 48 h in the presence of esterastin, which is known to be an inhibitor of lipase and esterase. Under these conditions, the levels of hydrolysis and re-esterification of cholesteryl esters were less than 15% and 5% of the control ones, respectively. These results suggest that the inclusion bodies were transferred from lysosomes to the cytoplasm, with partial hydrolysis of cholesteryl esters, in addition to through the pathway via microsomes.     

Slow motile mutant in Salmonella typhimurium

Enomoto, Masatoshi (National Institute of Genetics, Misima, Japan). Slow motile mutant in Salmonella typhimurium. J. Bacteriol. 90:1696-1702. 1965.-A slow motile mutant, SJ399, was isolated from a wild-type strain of Salmonella typhimurium TM2. The mutant was as motile as the wild type in broth culture at 37 C. However, on semisolid medium it produced a much narrower swarming band than TM2. The motility of this mutant was hindered by the viscosity of semisolid medium. H antigenicity and morphological characters of flagella of the mutant were the same as those of the wild type. The motility phage, chi, responded differently to SJ399 and the wild type. Plaques of SJ399 were small and cloudy, whereas on the wild type they were large and clear. The efficiency of plating on SJ399 was 0.36 as compared with 1 with the wild type. Stained preparations revealed that the mutant had about one-third the number of flagella of the wild type. The reduction of the number of flagella also was ascertained by biochemical measurement of flagellar protein which was purified after deflagellation from cells. The content of flagellin in SJ399 was about 32% of that of the wild type. Phage P22-mediated transductions from SJ399 to nonflagellated (fla(-)) and paralyzed (mot(-)) mutants showed that the mutant SJ399 complements seven fla(-) and three mot(-) strains which are representative mutants of flagellation and motility cistrons, respectively. The mutation site of SJ399 was cotransduced with both motA and B cistrons. The two point cross tests between SJ399 and mot mutants revealed that the mutation site of SJ399 is located in the motB cistron. The insertion of the genetic region containing the mutation site of SJ399 to the motB cistron is discussed in relation to intracistronic complementation.     

A Soluble Form of LMIR5/CD300b Amplifies Lipopolysaccharide-Induced Lethal Inflammation in Sepsis

Leukocyte mono-Ig-like receptor 5 (LMIR5, also called CD300b) is an activating receptor expressed in myeloid cells. We have previously demonstrated that T cell Ig mucin 1 works as a ligand for LMIR5 in mouse ischemia/reperfusion injury of the kidneys. In this article, we show that LMIR5 is implicated in LPS-induced sepsis in mice. Notably, neutrophils constitutively released a soluble form of LMIR5 (sLMIR5) through proteolytic cleavage of surface LMIR5. Stimulation with TLR agonists augmented the release of sLMIR5. LPS administration or peritonitis induction increased serum levels of sLMIR5 in mice, which was substantially inhibited by neutrophil depletion. Thus, neutrophils were the main source of LPS-induced sLMIR5 in vivo. On the other hand, i.p. administration of LMIR5-Fc, a surrogate of sLMIR5, bound to resident macrophages (M) and stimulated transient inflammation in mice. Consistently, LMIR5-Fc induced in vitro cytokine production of peritoneal M via its unknown ligand. Interestingly, LMIR5 deficiency profoundly reduced systemic cytokine production and septic mortality in LPS-administered mice, although it did not affect in vitro cytokine production of LPS-stimulated peritoneal M. Importantly, the resistance of LMIR5-deficient mice to LPS- or peritonitis-induced septic death was decreased by LMIR5-Fc administration, implicating sLMIR5 in LPS responses in vivo. Collectively, neutrophil-derived sLMIR5 amplifies LPS-induced lethal inflammation.     

Saffold virus, a novel human Cardiovirus with unknown pathogenicity

Although cardioviruses have been thought to mainly infect rodents, a novel human cardiovirus, designated Saffold virus (SAFV), was identified in 2007. SAFV is grouped with Theiler-like rat virus and Theiler's murine encephalomyelitis virus (TMEV) in the species Theilovirus of the genus Cardiovirus of the family Picornaviridae. Eight genotypes of SAFV have now been identified. SAFV has been isolated from nasal and stool specimens from infants presenting with respiratory and gastrointestinal symptoms as well as from children with nonpolio acute flaccid paralysis; however, the relationship of SAFV to this symptomatology remains unclear. Of note, the virus has also been isolated from the cerebrospinal fluid specimens of patients with aseptic meningitis. This finding is of interest since TMEV is known to cause a multiple sclerosis-like syndrome in mice. The involvement of SAFV in various diseases (e.g., respiratory illness, gastrointestinal illness, neurological diseases, and type I diabetes) is presently under investigation. In order to clarify the pathogenicity of SAFV, additional epidemiological studies are required. Furthermore, identification of the SAFV cellular receptor will help establish an animal model for SAFV infection and help clarify the pathogenesis of SAFV-related diseases. In addition, investigation of the tissue-specific expression of the receptor may facilitate development of a novel picornavirus vector, which could be a useful tool in gene therapy for humans. The study of viral factors involved in viral pathogenicity using a reverse genetics technique will also be important.     

Tuning the geometries of a de novo blue copper protein by axial interactions

The axial interactions of Cu(2+) in type 1 copper proteins control the physical characteristics of the proteins. We tuned the geometries of a de novo designed blue copper protein with a four-helical bundle structure. The designed protein axially bound various ligands, such as chloride, phosphate, sulfate, acetate, azide, and imidazole, to Cu(2+), exhibiting a blue or green color. The UV-vis spectral bands were observed at approximately 600?nm and approximately 450?nm, with the A (~450)/A (~600) ratios between 0.14 and 1.58. The stronger axial interaction shifted the geometry of the type 1 copper site from trigonal planar geometry (blue copper) toward a tetrahedral-like geometry (green copper). Resonance Raman spectral analyses showed that the phosphate-bound type had the highest-strength Cu-S bond, similar to that of plastocyanin. The chloride-bound type exhibited features similar to those of stellacyanin and nitrite reductase, and the imidazole-bound type exhibited features similar to those of azurin M121E mutant.     

Differential gene expression and functional analysis implicate novel mechanisms in enteric nervous system precursor migration and neuritogenesis

Enteric nervous system (ENS) development requires complex interactions between migrating neural-crest-derived cells and the intestinal microenvironment. Although some molecules influencing ENS development are known, many aspects remain poorly understood. To identify additional molecules critical for ENS development, we used DNA microarray, quantitative real-time PCR and in situ hybridization to compare gene expression in E14 and P0 aganglionic or wild type mouse intestine. Eighty-three genes were identified with at least two-fold higher expression in wild type than aganglionic bowel. ENS expression was verified for 39 of 42 selected genes by in situ hybridization. Additionally, nine identified genes had higher levels in aganglionic bowel than in WT animals suggesting that intestinal innervation may influence gene expression in adjacent cells. Strikingly, many synaptic function genes were expressed at E14, a time when the ENS is not needed for survival. To test for developmental roles for these genes, we used pharmacologic inhibitors of Snap25 or vesicle-associated membrane protein (VAMP)/synaptobrevin and found reduced neural-crest-derived cell migration and decreased neurite extension from ENS precursors. These results provide an extensive set of ENS biomarkers, demonstrate a role for SNARE proteins in ENS development and highlight additional candidate genes that could modify Hirschsprung's disease penetrance.     

High phosphorylation of HBV core protein by two alpha-type CK2-activated cAMP-dependent protein kinases in vitro

Two alpha-type CK2-activated PKAs (CK2-aPKAIalpha and CK2-aPKAIIalpha) were biochemically characterized in vitro using GST-HBV core fusion protein (GST-Hcore) and GST-Hcore157B as phosphate acceptors. It was found that (i), in the absence of cAMP, these two CK2-aPKAs phosphorylated both Ser-170 and Ser-178 on GST-Hcore and Hcore157B; (ii) this phosphorylation was approx. 4-fold higher than their phosphorylation by cAMP-activated PKAs; and (iii) suramin effectively inhibited the phosphorylation of Hcore157B by CK2-aPKAIIalpha through its direct binding to Hcore157B in vitro. These results suggest that high phosphorylation of HBV-CP by two CK2-aPKAs, in the absence of cAMP, may be involved in the pregenomic RNA (pgRNA) encapsidation and DNA-replication in HBV-infected cells.