LT（K63／R72）, a New Mutant of Escherichia coli Heat-labile Enterotoxin, Exhibits Characteristics More Similar to LT（K63） than LT（R72）

LT(K63), a non-toxic mutant and LT(R72), a low toxic mutant of E. coli heat-labile enterotoxin are frequently used mucosal adjuvants. In many cases, the adjuvanticity of LT(K63) is lower than that of LT(R72), but LT(K63), which induces a mixed Th1/Th2 response, exhibits a higher level of protection than LT(R72) which induces a polarized Th2-type response. To utilize the advantages of both adjuvants, a doublemutation LT(K63/R72) was generated and purified. The characterization results showed that there was no significant difference in production rate and immunogenicity between wild type LT and LT mutants. The results also showed that the toxicity and the trypsin sensitivity of LT(K63/R72) are between that of LT(K63)and LT(R72). Using HPLC, when samples in an OHpak SB-800 column were eluted by denatural buffer(TEAN containing 10 mg/ml SDS), we found the stability of LT(K63/R72) was higher than that of LT(R72)and lower than that of LT(K63). Through further analyzes, we found that LT(K63/R72) exhibits characteristics more closely related to LT(K63) than LT(R72).     

Orientation within the exosporium and structural stability of the collagen-like glycoprotein BclA of Bacillus anthracis

Bacillus anthracis spores, which cause anthrax, are enclosed by an exosporium consisting of a basal layer and an external hair-like nap. The filaments of the nap are composed of BclA, a glycoprotein containing distinct N-terminal (NTD) and C-terminal (CTD) domains separated by an extended collagen-like central region. In this study, we used immunogold electron microscopy to show that the CTD of BclA forms the distal end of each filament of the hair-like nap, indicating that the NTD is attached to the basal layer. Ten randomly chosen anti-BclA monoclonal antibodies, raised against spores or exosporium, reacted with the CTD, consistent with its exterior location. We showed that recombinant BclA (rBclA), encoded by the B. anthracis Sterne strain and synthesized in Escherichia coli, forms a collagen-like triple helix as judged by collagenase sensitivity and circular dichroism spectroscopy. In contrast, native BclA in spores was resistant to collagenase digestion. Thermal denaturation studies showed that the collagen-like region of rBclA exhibited a melting temperature (T(m)) of 37 degrees C, like mammalian collagen. However, rBclA trimers exhibited T(m) values of 84 degrees C and 95 degrees C in buffer with and without sodium dodecyl sulfate, respectively. CTD trimers exhibited the same T(m) values, indicating that the high temperature and detergent resistances of rBclA were due to strong CTD interactions. We observed that CTD trimers are resistant to many proteases and readily form large crystalline sheets. Structural data indicate that the CTD is composed of multiple beta strands. Taken together, our results suggest that BclA and particularly its CTD form a rugged shield around the spore.     

Increased expression of cystic fibrosis transmembrane conductance regulator in rat liver after common bile duct ligation

Liver disease associated with cystic fibrosis (CF) has been increasingly diagnosed and recognized as one of the major causes of death in CF during recent years. The autosomal-recessive disorder of CF results from mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) that encodes the CFTR protein. Due to its existence and multifunction in biliary epithelial, over- or less-expression of CFTR in the liver may play an important role in the development of CF liver disease (CFLD). The aim of current study is to investigate the expression of CFTR in the liver of common bile duct ligated (BDL) rats. After BDL, there was an increase in the abundance of CFTR mRNA and protein. Immunohistochemical staining also demonstrated an increased intensity of CFTR staining in the liver tissue section. In conclusion, there is an increased expression of CFTR in the liver after common BDL.     

MYBPC3 polymorphism is a modifier for expression of cardiac hypertrophy in patients with hypertrophic cardiomyopathy

Clinical phenotype of hypertrophic cardiomyopathy exhibits significant inter- and intra-familial heterogeneities. To test if MYBPC3 polymorphism could modify the expression of cardiac hypertrophy, 226 patients with hypertrophic cardiomyopathy and 226 age- and sex-matched controls were recruited according to the diagnostic criteria of WHO. Genotyping was completed by using PCR, restrictive enzyme digestion, and sequencing. Three polymorphisms of MYBPC3 were studied, only the GG genotype at 18443 in exon 30 associated with thicker left ventricular wall (25.2+/-5.9 mm) in patient group, not the AA and AG genotypes (19.0+/-5.0mm, P<0.001). After multiple regression analysis for adjustment of age and sex, the association remained. No difference was found in the genotype distribution between control and patients. Our results point out that GG genotype of MYBPC3 might be a genetic risk factor for the expression of cardiac hypertrophic phenotype in the patients with hypertrophic cardiomyopathy.     

Phosphatidylcholine-rich acceptors, but not native HDL or its apolipoproteins, mobilize cholesterol from cholesterol-rich insoluble components of human atherosclerotic plaques

To examine the potential of high density lipoproteins (HDL) to ameliorate atherosclerotic plaques in vivo, we examined the ability of native HDL, lipid-free HDL apolipoproteins (apo HDL), cholesterol-free discoidal reconstituted HDL (R-HDL) comprised of apo HDL and phosphatidylcholine (PC) and PC liposomes to release cholesterol from cholesterol-rich insoluble components of plaques (ICP) isolated from atherosclerotic human aorta. Isolated ICP had a free cholesterol (FC) to phospholipid (PL) mass ratio (0.8-3.1) and a sphingomyelin (SPM) to PC mass ratio (1.2-4.2) that exceeded those of plasma membranes of cultured cells. Surprisingly, native HDL and its apolipoproteins were not able to release cholesterol from ICP. However, R-HDL and PC liposomes were effectively released cholesterol from ICP. The release of ICP cholesterol by R-HDL was dose-dependent and accompanied by the transfer of > 8 x more PC in the reverse direction (i.e., from R-HDL to ICP), resulting in a marked enrichment of ICP with PC. Compared to R-HDL, PC liposomes were significantly less effective in releasing cholesterol from ICP but were somewhat more effective in enriching ICP with PC. Native HDL was minimally effective in enriching ICP with PC, but became effective after prior in vitro enrichment of HDL with PC from multilamellar PC liposomes. The enrichment of ICP with PC resulted in the dissolution of cholesterol crystals on ICP and allowed the removal of ICP cholesterol by apo HDL and plasma. Our study revealed that the removal of cholesterol from ICP in vivo will be possible through a change in the level, composition, and physical state of ICP lipids mediated by PC-enriched HDL.     

Pinning down phosphorylated tau and tauopathies

Neurofibrillary tangles (NFTs) are prominent neuronal lesions in a large subset of neurodegenerative diseases, including Alzheimer's disease (AD). NFTs are mainly composed of insoluble Tau that is hyperphosphorylated on many serine or threonine residues preceding proline (pSer/Thr-Pro). Tau hyperphosphorylation abolishes its biological function to bind microtubules and promotes microtubule assembly and precedes neurodegeneration. Not much is known about how tau is further regulated following phosphorylation. Notably, we have recently shown that phosphorylated Ser/Thr-Pro motifs exist in two distinct conformations. The conversion between two conformations in some proteins is catalyzed by the prolyl isomerase Pin1. Pin1 binds to tau phosphorylated specifically on the Thr231-Pro site and probably catalyzes cis/trans isomerization of pSer/Thr-Pro motif(s), thereby inducing conformational changes in tau. Such conformational changes can directly restore the ability of phosphorylated Tau to bind microtubules and promote microtubule assembly and/or facilitate tau dephosphorylation by its phosphatase PP2A, as PP2A activity is conformation-specific. Furthermore, Pin1 expression inversely correlates with the predicted neuronal vulnerability in normally aged brain and also with actual neurofibrillary degeneration in AD brain. Moreover, deletion of the gene encoding Pin1 in mice causes progressive age-dependent neuropathy characterized by motor and behavioral deficits, tau hyperphosphorylation, tau filament formation and neuronal degeneration. Distinct from all other mouse models where transgenic overexpression of specific proteins elicits tau-related pathologies, Pin1 is the first protein whose depletion causes age-dependent neurodegeneration and tau pathologies. Thus, Pin1 is pivotal in maintaining normal neuronal function and preventing age-dependent neurodegeneration. This could represent a promising interventive target to prevent neurodegenerative diseases.     

Distinct roles of the ERK pathway in modulating apoptosis of Ras-transformed and non-transformed cells induced by anticancer agent FR901228

Ectopic expression of oncogenic H-Ras in cells results in increases of cell susceptibility to the anticancer agent FR901228. Investigating the roles of Ras-induced pathways in FR901228-induced apoptosis, we have found that the phosphatidylinositol 3-kinase pathway plays an anti-apoptotic role, whereas the stress-activated protein kinase p38 pathway plays a pro-apoptotic role in FR901228-induced apoptosis. Interestingly, the extracellular signal-regulated kinase (ERK) pathway plays an anti-apoptotic role in non-transformed cells; however, it plays a pro-apoptotic role in Ras-transformed cells in response to FR901228 treatment. An essential role of the ERK pathway in regulating caspase-3 contents may contribute to its pro-apoptotic role in Ras-transformed cells.     

The characterization of plasma membrane Ca2+-ATPase in rich sphingomyelin-cholesterol domains

According to the raft hypothesis, sphingolipid-cholesterol (CHOL) microdomains are involved in numerous cellular functions. Here, we have prepared liposomes to simulate the lipid composition of rafts/caveolae using phosphatidylchone, sphingomyelin (SPM)-CHOL in vitro. Experiments of both 1,6-diphenyl-1,3,5-hexatriene and merocyanine-540 fluorescence showed that a phase transition from l(d) to l(o) can be observed clearly. In particular, we investigated the behavior of a membrane protein, plasma membrane Ca(2+)-ATPase (PMCA), in lipid rafts (l(o) phase). Three complementary approaches to characterize the physical appearance of PMCA were employed in the present study. Tryptophan intrinsic fluorescence increase, fluorescence quenching by both acrylamid and hypocrellin B decrease, and MIANS fluorescence decrease, indicate that the conformation of PMCA embedded in lipid l(o) phase is more compact than in lipid l(d) phase. Also, our results showed that PMCA activity decreased with the increase of SPM-CHOL content, in other words, with the increase of l(o) phase. This suggests that the specific domains containing high SPM-CHOL concentration are not a favorable place for PMCA activity. Finally, a possible explanation about PMCA molecules concentrated in caveolae/rafts was discussed.