Production of propeptide-directed antibody: its application to the purification of proalbumin and analysis of proalbumin processing

Antibodies which specifically recognize rat proalbumin, but not mature serum-albumin, were raised. Propeptide (NH2-Arg-Gly-Val-Phe-Arg-Arg) with an additional cysteine residue at the carboxyl terminus was conjugated to ovalbumin through either sulfide or amino group and the conjugates were injected into rabbits. Monospecific antibodies were purified on a propeptide-coupled affinity column and further immobilized as an affinity support, allowing us to purify proalbumin in a single step from rat liver microsomes. The antibodies were also used to analyze the proteolytic processing of proalbumin in primary cultured rat hepatocytes.     

Cl-]i modulation of Ca2+-regulated exocytosis in ACh-stimulated antral mucous cells of guinea pig

The effects of intracellular Cl- concentration ([Cl-]i) on acetylcholine (ACh)-stimulated exocytosis were studied in guinea pig antral mucous cells by video microscopy. ACh activated Ca2+-regulated exocytosis (an initial phase followed by a sustained phase). Bumetanide (20 microM) or a Cl- -free (NO3-) solution enhanced it; in contrast, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, a Cl- channel blocker) decreased it and eliminated the enhancement induced by bumetanide or NO3- solution. ACh and Ca2+ dose-response studies demonstrated that NO3- solution does not shift their dose-response curves, and ATP depletion studies by dinitrophenol or anoxia demonstrated that exposure of NO3- solution prior to ATP depletion induced an enhanced initial phase followed by a sustained phase, whereas exposure of NO3- solution after ATP depletion induced only a sustained phase. Intracellular Ca2+ concentration ([Ca2+]i) measurements showed that bumetanide and NO3- solution enhanced the ACh-stimulated [Ca2+]i increase. Measurements of [Cl-]i revealed that ACh decreases [Cl-]i and that bumetanide and NO3- solution decreased [Cl-]i and enhanced the ACh-evoked [Cl-]i decrease; in contrast, NPPB increased [Cl-]i and inhibited the [Cl-]i decrease induced by ACh, bumetanide, or NO3- solution. These suggest that [Cl-]i modulates [Ca2+]i increase and ATP-dependent priming. In conclusion, a decrease in [Cl-]i accelerates ATP-dependent priming and [Ca2+]i increase, which enhance Ca2+-regulated exocytosis in ACh-stimulated antral mucous cells.     

Macrophage inflammatory protein-1α (MIP-1α) enhances a receptor activator of nuclear factor κB ligand (RANKL) expression in mouse bone marrow stromal cells and osteoblasts through MAPK and PI3K/Akt pathways

Osteolytic lesions are rapidly progressive during the terminal stages of myeloma, and the bone pain or bone fracture that occurs at these lesions decreases the patients’ quality of life to a notable degree. In relation to the etiology of this bone destruction, it has been reported recently that MIP-1α, produced in large amounts in myeloma patients, acts indirectly on osteoclastic precursor cells, and activates osteoclasts by way of bone-marrow stromal cells or osteoblasts, although the details of this process remain obscure. In the present study, our group investigated the mechanism by which RANKL expression is induced by MIP-1α and the effects of MIP-1α on the activation of osteoclasts. RANKL mRNA and RANKL protein expressions increased in both ST2 cells and MC3T3–E1 cells in a MIP-1α concentration-dependent manner. RANKL mRNA expression began to increase at 1 h after the addition of MIP-1α; the increase became remarkable at 2 h, and continuous expression was observed subsequently. Both ST2 and MC3T3-E1 cells showed similar levels of increased RANKL protein expression at 1, 2, and 3 days after the addition of MIP-1α. After the addition of MIP-1α, the amount of phosphorylated ERK1/2 and Akt protein expressions showed an increase, as compared to the corresponding amount in the control group. On the other hand, the amount of phosphorylated p38MAPK protein expression showed a decrease from the amount in the control group after the addition of MIP-1α. U0126 (a MEK1/2 inhibitor) or LY294002 (a PI3K inhibitor) was added to ST2 and MC3T3-E1 cells, and was found to inhibit RANKL mRNA and RANKL protein expression in these cells. When SB203580, a p38MAPK inhibitor, was added, RANKL mRNA and RANKL protein expression were increased in these cells. MIP-1α was found to promote osteoclastic differentiation of C7 cells, an osteoclastic precursor cell line, in a MIP-1α concentration-dependent manner. MIP-1α promoted differentiation into osteoclasts more extensively in C7 cells incubated together with ST2 and MC3T3-E1 cells than in C7 cells incubated alone. These results suggested that MIP-1α directly acts on the osteoclastic precursor cells and induces osteoclastic differentiation. This substance also indirectly induces osteoclastic differentiation through the promotion of RANKL expression in bone-marrow stromal cells and osteoblasts. The findings of this investigation suggested that activation of the MEK/ERK and the PI3K/Akt pathways and inhibition of p38MAPK pathway were involved in RANKL expression induced by MIP-1α in bone-marrow stromal cells and osteoblasts. This finding may be useful in the development of an osteoclastic inhibitor that targets intracellular signaling factors.     

Estimation of the embryotoxic effect of CBZ using an ES cell differentiation system

Carbamazepine (CBZ) is one of the most commonly prescribed antiepileptic drugs (AEDs). However, a higher rate of congenital anomalies has been found in infants of mothers treated with CBZ during early pregnancy. Here, we characterize the effects of CBZ using a mouse ES cell differentiation system. The analysis of tissue-specific gene markers showed that CBZ induced early endodermal and mesodermal differentiation but inhibited differentiation of later stages. CBZ also induced ectodermal development, and there was evidence of neural differentiation as ES cells with an immature neuronal phenotype were observed. In contrast, valproic acid (VPA), another anticonvulsant drug, was previously shown to be able to induce ES cells to differentiate into neurons with a mature appearance. CBZ was less cytotoxic to ES cells than VPA. The in vitro ES cell assay system has the potential to provide a rapid and accurate approach for estimating the in vivo embryotoxicity of therapeutic drugs.     

Transforming activity of purinergic receptor P2Y, G-protein coupled, 2 revealed by retroviral expression screening

Colorectal cancer (CRC) is one of the leading causes of cancer death in humans. In order to identify novel cancer-promoting genes in CRC, we here constructed a retroviral cDNA expression library from a CRC cell line RKO, and used it for a focus formation assay with mouse 3T3 fibroblasts, leading to the identification of 42 independent cDNAs. One of such cDNAs turned out to encode purinergic receptor P2Y, G-protein coupled, 2 (P2RY2). The oncogenic potential of P2RY2 was confirmed in vitro with the focus formation assay as well as soft agar-growth assay, and also in vivo with a tumorigenicity assay in nude mice. While our P2RY2 cDNA encodes a protein with two amino-acid substitutions compared to the reported one, we have confirmed that the wild-type P2RY2 has a strong transforming potential as well. These results indicate an unexpected role of P2RY2 in the carcinogenesis of human cancers.     

Genomic characterization of the filamentous integrative bacteriophages {phi}RSS1 and {phi}RSM1, which infect Ralstonia solanacearum

The genomic DNA sequences were determined for two filamentous integrative bacteriophages, phiRSS1 and phiRSM1, of the phytopathogen Ralstonia solanacearum. The 6,662-base sequence of phiRSS1 contained 11 open reading frames (ORFs). In the databases, this sequence showed high homology (95% identity) to the circular double-stranded DNA plasmid pJTPS1 (6,633 bp) isolated from a spontaneously occurring avirulent mutant of R. solanacearum. Two major differences between the two sequences were observed within phiRSS1 ORF7, corresponding to pIII, a minor coat protein required for host adsorption, and at the phiRSS1 intergenic (IG) region. The 9,004-base sequence of phiRSM1 showed 12 ORFs located on the same strand (plus strand) and 2 ORFs on the opposite strand. Compared with Ff-type phages, two insertions are obvious in the phiRSM1 replication module. Genomic DNA fragments containing the phiRSM integration junctions were cloned and sequenced from phiRSM lysogenic strain R. solanacearum MAFF211270. The att core sequence was identified as 5'-TGGCGGAGAGGGT-3', corresponding to the 3' end of the serine tRNA (UCG) gene. Interestingly, ORF14, located next to the attP site on the phiRSM1 genome, showed high amino acid sequence homology with bacterial DNA recombinases and resolvases, different from XerCD recombinases. attP of phiRSS1 is within a sequence element of the IG region.     

Release of the lipopolysaccharide deacylase PagL from latency compensates for a lack of lipopolysaccharide aminoarabinose modification-dependent resistance to the antimicrobial peptide polymyxin B in Salmonella enterica

Salmonella enterica modifies its lipopolysaccharide (LPS), including the lipid A portion, to adapt to its environments. The lipid A 3-O-deacylase PagL exhibits latency; deacylation of lipid A is not usually observed in vivo despite the expression of PagL, which is under the control of a two-component regulatory system, PhoP-PhoQ. In contrast, PagL is released from latency in pmrA and pmrE mutants, both of which are deficient in aminoarabinose-modified lipid A, although the biological significance of this is not clear. The attachment of aminoarabinose to lipid A decreases the net anionic charge at the membrane's surface and reduces electrostatic repulsion between neighboring LPS molecules, leading to increases in bacterial resistance to cationic antimicrobial peptides, including polymyxin B. Here we examined the effects of the release of PagL from latency on resistance to polymyxin B. The pmrA pagL and pmrE pagL double mutants were more susceptible to polymyxin B than were the parental pmrA and pmrE mutants, respectively. Furthermore, introduction of the PagL expression plasmid into the pmrA pagL double mutant increased the resistance to polymyxin B. In addition, PagL-dependent deacylation of lipid A was observed in a mutant in which lipid A could not be modified with phosphoethanolamine, which partly contributes to the PmrA-dependent resistance to polymyxin B. These results, taken together, suggest that the release of PagL from latency compensates for the loss of resistance to polymyxin B that is due to a lack of other modifications to LPS.     

Regulation of 20-hydroxyecdysone on the larval pigmentation and the expression of melanin synthesis enzymes and yellow gene of the swallowtail butterfly, Papilio xuthus

The swallowtail butterfly, Papilio xuthus, changes its larval body pattern dramatically during the fourth ecdysis. Cuticular pigmentation occurs with precise timing just before ecdysis. We previously found that the cuticular pigmentation was regulated by three melanin synthesis genes, tyrosine hydroxylase (TH), dopa decarboxylase (DDC), and ebony. We discovered that yellow is strongly expressed in the presumptive black markings earlier than TH and DDC. Because the ecdysis is triggered by 20-hydroxyecdysone (20E), the effects of 20E on the pigmentation and expression of the melanin synthesis genes were examined. Here, we established a method for the topical application of 20E to molting specimens, so that 20E has only a partial effect, resulting in successful ecdysis. When we applied 20E during the mid-phase of the molting period, when the 20E titer is declining, cuticular pigmentation was completely inhibited. The cessation of hormonal treatments caused delayed pigmentation. yellow expression was promoted by a high titer of 20E, whereas the expression of TH, DDC, and ebony was suppressed, suggesting that a decline in the 20E concentration is necessary for the induction of the expression of the latter three genes. These results indicate that cuticular pigmentation is controlled by the exposure to 20E and its removal.     

斑马鱼总RNA提取和纯化

采取异硫氰酸胍法从斑马鱼的3个样品中提取和纯化RNA,结果表明,RNAA260分别为0、587、0．719、0．151.A260/A280比值分别为1．89、1．92、1．82。电泳条带也清晰可见,可以用于进一步的分子生物学研究。