Genes responding to vernalization in hexaploid wheat

Genotype-specific gene expression in response to vernalization in common wheat was examined by the differential display method. Two near-isogenic lines of Vrn-A1 ( Vrn-A1 for the spring type and vrn-A1 for the winter type) were treated by vernalization of developing embryos in detached-ear cultures. This treatment was effective to promote vrn-A1 genotypes to head at a time equivalent to that of Vrn-A1. Differential cDNA fragments were isolated by the RT-PCR method from embryos subjected to vernalization treatments for 2- and 4-weeks at DAP10 and DAP20 stages, respectively. Among 110 differential cDNA fragments isolated, 48 were examined for their chromosomal locations and designated as wec ( wheat- embryo cold treatment) genes. Seven wec genes showed genotype-specific expression in response to vernalization. The statistical analysis utilizing two recombinant inbred lines showed that four wec genes were significantly associated with heading factors.     

Essential role of the adhesion receptor LFA-1 for T cell-dependent fulminant hepatitis

Viral hepatitis affects more than 2 billion people worldwide. In particular, no effective treatment exists to abrogate death and liver damage in fulminant hepatitis. Activation of T cells is an initial and critical event in the pathogenesis of liver damage in autoimmune and viral hepatitis. The precise molecular mechanisms that induce T cell-mediated hepatocyte injury remain largely unclear. In mice, T cell-dependent hepatitis and acute liver damage can be modeled using ConA. In this study, we examined the role of the adhesion receptor LFA-1 in ConA-induced acute hepatic damage using LFA-1(-/-) (CD11a) mice. Massive liver cell apoptosis and metabolic liver damage were observed in LFA-1(+/+) mice following ConA injection. By contrast, LFA-1(-/-) mice were completely resistant to ConA-induced hepatitis and none of the LFA-1(-/-) mice showed any hepatic damage. Whereas activated hepatic T cells remained in the liver in LFA-1(+/+) mice, activated T cells were rapidly cleared from the livers of LFA-1(-/-) mice. Mechanistically, T cells from LFA-1(-/-) mice showed markedly reduced cytotoxicity toward liver cells as a result of impaired, activation-dependent adhesion. Importantly, adoptive transfer of hepatic T cells from LFA-1(+/+) mice, but not from LFA-1(-/-) mice, sensitized LFA-1(-/-) mice to ConA-induced hepatitis. Thus, LFA-1 expression on T cells is necessary and sufficient for T cell-mediated liver damage in vivo. These results provide the first genetic evidence on an adhesion receptor, LFA-1, that has a crucial role in fulminant hepatitis. These genetic data identify LFA-1 as a potential key target for the treatment of T cell-mediated hepatitis and the prevention of liver damage.     

FGF10 maintains stem cell compartment in developing mouse incisors

Mouse incisors are regenerative tissues that grow continuously throughout life. The renewal of dental epithelium-producing enamel matrix and/or induction of dentin formation by mesenchymal cells is performed by stem cells that reside in cervical loop of the incisor apex. However, little is known about the mechanisms of stem cell compartment formation. Recently, a mouse incisor was used as a model to show that fibroblast growth factor (FGF) 10 regulates mitogenesis and fate decision of adult stem cells. To further illustrate the role of FGF10 in the formation of the stem cell compartment during tooth organogenesis, we have analyzed incisor development in Fgf10-deficient mice and have examined the effects of neutralizing anti-FGF10 antibody on the developing incisors in organ cultures. The incisor germs of FGF10-null mice proceeded to cap stage normally. However, at a later stage, the cervical loop was not formed. We found that the absence of the cervical loop was due to a divergence in Fgf10 and Fgf3 expression patterns at E16. Furthermore, we estimated the growth of dental epithelium from incisor explants of FGF10-null mice by organ culture. The dental epithelium of FGF10-null mice showed limited growth, although the epithelium of wild-type mice appeared to grow normally. In other experiments, a functional disorder of FGF10, caused by a neutralizing anti-FGF10 antibody, induced apoptosis in the cervical loop of developing mouse incisor cultures. However, recombinant human FGF10 protein rescued the cervical loop from apoptosis. Taken together, these results suggest that FGF10 is a survival factor that maintains the stem cell population in developing incisor germs.     

Purification of lactic acid dehydrogenase from crude bovine heart extract by pH-peak focusing counter-current chromatography

pH-peak focusing counter-current chromatography (CCC) was applied to the purification of lactic acid dehydrogenase (LDH) from a crude bovine heart extract using a cross-axis coil planet centrifuge (CPC). The experiment was performed with two sets of polymer phase systems composed of 16% (w/w) polyethylene glycol (PEG) 1000-12.5% (w/w) potassium phosphate buffer and 15% (w/w) PEG 1540-15% (w/w) ammonium sulfate each at various pH values. The best result was achieved from the PEG 1540-ammonium sulfate polymer phase system by adding a retainer (10 mM acetic acid) to the upper stationary phase and an eluter (100 mM sodium hydroxide) to the lower mobile phase. At a flow-rate of 0.5 ml/min, LDH was eluted as a sharp peak which was well resolved from other proteins. Collected fractions were analyzed by the LDH enzymatic activity and by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis to detect contaminated proteins. LDH was purified directly from crude bovine heart extract in a concentrated state.     

In situ alkylation with acrylamide for identification of cysteinyl residues in proteins during one- and two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis

Cysteinyl residues in proteins were alkylated with acrylamide during sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to yield a thioether derivative, cys-S-beta-propionamide (PAM cys). The process was termed in situ alkylation with acrylamide. Using this method, the recovery of PAM-cys peptides from bovine serum albumin (BSA) was 88.6% at 10 picomol in one-dimensional (1-D) SDS-PAGE and 97.1% at 50 picomol in two-dimensional (2-D) SDS-PAGE. The coverage of tryptic peptide of BSA in 1-D and 2-D SDS-PAGE was 83.7% and 81.1%, respectively. The advantages of in situ alkylation with acrylamide were the following: (i) cysteinyl peptides were effectively derived in a single PAM cys and then proteins were precisely identified using databases; (ii) marked reduction of salts compared with post alkylation, e.g., using carboxymethylamide (CAM), resulting in higher signal intensity and wider coverage of cysteinyl peptides from PAM cys, compared with those of CAM derivatives, in mass spectrometry peptide mapping; and (iii) shorter duration by excluding the processes of post alkylation and desalting before peptide mapping.     

Regulation of angiogenesis by the aging suppressor gene klotho

Advanced age is a major risk factor of peripheral artery disease. We examined the effects of the aging-suppressor gene klotho on angiogenesis in response to ischemia by introducing ischemic hindlimb model in mice heterozygously deficient for the klotho gene and in wild type mice. Blood flow recovery as assessed by laser doppler perfusion imaging and angiogenesis as assessed by density of PECAM-1/CD31-positive positive capillaries were markedly impaired in mice heterozygously deficient for the klotho gene (both <0.05). Our findings show that the aging-suppressor gene klotho affects angiogenesis and the possibility that age-related impairment of angiogenesis might be regulated by the klotho gene. Our results present a new possibility of therapeutic angiogenesis for patients of advanced age.     

Aggregation and neurotoxicity of mutant amyloid beta (A beta) peptides with proline replacement: importance of turn formation at positions 22 and 23

Aggregation of the amyloid beta peptides (A beta 1-42 and A beta 1-40) plays a pivotal role in pathogenesis of Alzheimer's disease. Although it is widely accepted that the aggregates of A betas mainly consist of beta-sheet structure, the precise aggregation mechanism remains unclear. To identify amino acid residues that are important for the beta-sheet formation, a series of proline-substituted mutants of A beta 1-42 peptides at positions 19-26 was synthesized in a highly pure form and their aggregation ability and neurotoxicity on PC12 cells were investigated. All proline-substituted A beta 1-42 mutants except for 22P- and 23P-A beta 1-42 were hard to aggregate and showed weaker cytotoxicity than wild-type A beta 1-42, suggesting that the residues at positions 19-21 and 24-26 are important for the beta-sheet formation. In contrast, 22P-A beta 1-42 extensively aggregated with stronger cytotoxicity than wild-type A beta 1-42. Since proline has a propensity for beta-turn structure as a Pro-X corner, these data implicate that beta-turn formation at positions 22 and 23 plays a crucial role in the aggregation and neurotoxicity of A beta peptides.     

Molecular organization of amyloid protofilament-like assembly of betabellin 15D: helical array of beta-sandwiches

Betabellin is a 32-residue peptide engineered to fold into a four-stranded antiparallel beta-sheet protein. Upon air oxidation, the betabellin peptides can fold and assemble into a disulfide-bridged homodimer, or beta-sandwich, of 64 residues. Recent biophysical and ultrastructural studies indicate that betabellin 15D (B15D) (a homodimer of HSLTAKIpkLTFSIAphTYTCAVpkYTAKVSH, where p = DPro, k = DLys, and h = DHis) forms unbranched, 35-A wide assemblies that resemble the protofilaments of amyloid fibers. In the present study, we have analyzed in detail the X-ray diffraction patterns of B15D prepared from acetonitrile. The fiber diffraction analysis indicated that the B15D fibril was composed of a double helix defined by the selection rule l = n + 7m (where l is even, and n and m are any integers), and having a 199-A period and pitch, 28-A rise per unit, and 10-A radius. This helical model is equivalent to a reverse-handed, single helix with half the period and defined by the selection rule l = -3n + 7m (where l is any integer). The asymmetric unit is the single B15D beta-sandwich molecule. These results suggest that the betabellin assembly that models the protofilaments of amyloid fibers is made up of discrete subunits on a helical array. Multiple intersheet hydrogen bonds in the axial direction and intersandwich polar interactions in the lateral direction stabilize the array.