Novel Rana keratin genes and their expression during larval to adult epidermal conversion in bullfrog tadpoles

The conversion of the larval to adult epidermis during metamorphosis of tadpoles of bullfrog, Rana catesbeiana, was investigated utilizing newly cloned Rana keratin cDNAs as probes. Rana larval keratin (RLK) cDNA (rlk) was cloned using highly specific antisera against Xenopus larval keratin (XLK). Tail skin proteins of bullfrog tadpoles were separated by 2-dimensional gel electrophoresis and subjected to Western blot analysis with anti-XLK antisera. The Rana antigen detected by this method was sequenced and identified as a type II keratin. We cloned rlk from tadpole skin by PCR utilizing primers designed from these peptide sequences of RLK. RLK predicted by nucleotide sequences of rlk was a 549 amino acid -long type II keratin. Subtractive cloning between the body and the tail skin of bullfrog tadpole yielded a cDNA (rak) of Rana adult keratin (RAK). RAK was a 433 amino acid-long type I keratin. We also cloned a Rana keratin 8 (RK8) cDNA (rk8) from bullfrog tadpole epidermis. RK8 was 502 amino acid-long and homologous to cytokeratin 8. Northern blot analyses and in situ hybridization experiments showed that rlk was actively expressed through prometamorphosis in larva-specific epidermal cells called skein cells and became completely inactive at the climax stage of metamorphosis and in the adult skin. RAK mRNA was expressed in basal cells of the tadpole epidermis and germinative cells in the adult epidermis. The expression of rlk and rak was down- and up-regulated by thyroid hormone (TH), respectively. In contrast, there was no change in the expression of RK8 during spontaneous and TH-induced metamorphosis. RK8 mRNA was exclusively expressed in apical cells of the larval epidermis. These patterns of keratin gene expression indicated that the expression of keratin genes is differently regulated by TH depending on the type of larval epidermal cells. The present study demonstrated the usefulness of these genes for the study of molecular mechanism of postembryonic epidermal development and differentiation.     

Rotenoids and flavonoids with anti-invasion of HT1080, anti-proliferation of U937, and differentiation-inducing activity in HL-60 from Erycibe expansa

Principal rotenoids (deguelin, tephrosin, rotenone, and 12a-hydroxyrotenone) (3-30microM) isolated from the stems of Erycibe expansa significantly inhibited invasion of human fibrosarcoma HT1080 cells through Matrigel-coated filters and release of proMMPs-2 and 9. In addition, deguelin and tephrosin showed differentiation-inducing activity in human promyelocytic leukemia HL-60 cells. Furthermore, effects of various constituents isolated from the ethyl acetate-soluble fraction on proliferation of human leukemia U937 cells were examined. As a result, most of isoflavones and several flavans as well as rotenoids showed moderate or substantial anti-proliferative activities.     

Mass spectrometry meets the challenge of understanding the complexity of the lipoproteome: recent findings regarding proteins involved in dyslipidemia and cardiovascular disease

Despite the fact that link between dyslipidemia and atherosclerosis was made over 100 years ago, atherosclerosis remains a major cause of morbidity and mortality worldwide. Major efforts focus towards understanding lipid metabolism, particularly by studying its particle compartments in circulation: the lipoproteins. In recent years, mass spectrometry has played an integral role in the deep sequencing of the lipoproteome and in metabolism studies conducted in vivo. This review highlights the path of lipoprotein research towards state-of-the-art mass spectrometry with special emphasis on the method of selected reaction monitoring and its impact on apolipoprotein metabolism studies. Also presented is what is expected for the lipoprotein field with the recent advent of high resolution/accurate mass parallel reaction monitoring mass spectrometry. The benefits of high resolution/accurate mass measurements are demonstrated by example instrument workflows and by detailing a novel method to quantify very low levels of circulating proprotein convertase subtilisin-kexin type 9 in rabbit. It is anticipated that future clinical studies or clinical trials aimed to treat dyslipidemia by manipulating key regulatory proteins will benefit from the new and exciting opportunities afforded by the latest technologies in mass spectrometry.     

Disruption of Sept6, a fusion partner gene of MLL, does not affect ontogeny, leukemogenesis induced by MLL-SEPT6, or phenotype induced by the loss of Sept4

Septins are evolutionarily conserved GTP-binding proteins that can heteropolymerize into filaments. Recent studies have revealed that septins are involved in not only diverse normal cellular processes but also the pathogenesis of various diseases, including cancer. SEPT6 is ubiquitously expressed in tissues and one of the fusion partner genes of MLL in the 11q23 translocations implicated in acute leukemia. However, the roles of this septin in vivo remain elusive. We have developed Sept6-deficient mice that exhibited neither gross abnormalities, changes in cytokinesis, nor spontaneous malignancy. Sept6 deficiency did not cause any quantitative changes in any of the septins evaluated in this study, nor did it cause any additional changes in the Sept4-deficient mice. Even the depletion of Sept11, a close homolog of Sept6, did not affect the Sept6-null cells in vitro, thus implying a high degree of redundancy in the septin system. Furthermore, a loss of Sept6 did not alter the phenotype of myeloproliferative disease induced by MLL-SEPT6, thus suggesting that Sept6 does not function as a tumor suppressor. To our knowledge, this is the first report demonstrating that a disruption of the translocation partner gene of MLL in 11q23 translocation does not contribute to leukemogenesis by the MLL fusion gene.     

The formation of primordial germ cells from germline cells in spherical embryos derived from the blastodisc of 2-cell embryos in goldfish, Carassius auratus

The property of primordial germ cells (PGCs) in fragmented goldfish embryos was investigated. When 1- and 2- cell embryos were cut at several perpendicular levels at the animal-vegetal axis, cells expressing vas mRNA were observed in the resultant embryos derived from all kinds of animal fragments. Blastodisc fragments from the 1- to 2-cell stage developed to spherical embryos containing yolk body with a yolk syncytial layer (YSL). Germ ring and no tail expression were not observed in the spherical embryo. When the spherical embryo labeled with tracer dye or GFP-nos1 3'UTR mRNA was transplanted onto the animal part of the blastoderm in a host embryo at the blastula stage, PGCs of spherical embryo origin were detected around the gonadal ridges in the resultant embryos which developed normally. These results suggest that small animal fragments should contain factors sufficient for PGC differentiation and that PGCs differentiate without mesoderm induction, since mesoderm is not induced in a spherical embryo.     

Structural analysis of the sheath of a sheathed bacterium, Leptothrix cholodnii

Leptothrix cholodnii is an aerobic sheath-forming bacterium often found in oligotrophic and metal-rich aquatic environments. The sheath of this bacterium was isolated by selectively lysing the cells. Glycine and cysteine were the major amino acids of the sheath. The sheath was readily dissolved in hydrazine, and a polysaccharide substituted with cysteine was recovered from the solution. Galactosamine, glucosamine and galacturonic acid were detected in the hydrazinolysate by gas liquid chromatography analysis. FAB-MS analysis of the hydrazinolysate suggested a sugar sequence of HexN-GalA-HexN-HexN. Methylation linkage analysis revealed the presence of 4-linked GalA, 3-linked HexN and 4-linked HexN. The sulfhydryl groups of the sheath were used for labeling with the fluorogenic reagent, 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). The labeled sheath (ABD-sheath) was partially hydrolyzed and three fluorescent fragments were purified by HPLC. One of them was identified as ABD-cysteine. The second one was found to be the ABD-cysteine tetramer. Another fragment was indicated to be a pentasaccharide substituted with ABD-cysteine by nuclear magnetic resonance (NMR) analysis. It can be assumed that the polysaccharide and peptide moieties of the sheath are connected by a cysteine residue. NMR analysis of the hydrazinolysate revealed that the polysaccharide moiety of the sheath was constructed from a pentasaccharide repeating unit containing 2-amino-2-deoxygalacturonic acid (GalNA), as shown below. -->4)-alpha-GalNA-(1-->4)-alpha-D-GalN(p)-(1-->4)-alpha-D-GalA(p)-(1-->4)-beta-D-GlcN(p)-(1-->3)-beta-D-GalN(p)-(1-->.     

Dual DNA recognition codes of a short peptide derived from the basic leucine zipper protein EmBP1

Sequence-specific DNA binding of short peptide dimers derived from a plant basic leucine zipper protein EmBP1 was studied. A homodimer of the EmBP1 basic region peptide recognized a palindromic DNA sequence, and a heterodimer of EmBP1 and GCN4 basic region peptides targets a non-palindromic DNA sequence when a beta-cyclodextrin/adamantane complex is utilized as a dimerization domain. A homodimer of the EmBP1 basic region peptide binds the native EmBP1 binding 5'-GCCACGTGGC-3' and the native GCN4 binding 5'-ATGACGTCAT-3' sequences with almost equal affinity in the alpha-helical conformation, indicating that the basic region of EmBP1 by itself has a dual recognition codes for the DNA sequences. The GCN4 basic region peptide binds 5'-ATGAC-3' in the alpha-helical conformation, but it neither shows affinity nor helix formation with 5'-GCCAC-3'. Because native EmBP1 forms 100 times more stable complex with 5'-GCCACGTGGC-3' over 5'-ATGACGTCAT-3', our results suggest that the sequence-selectivity of native EmBP1 is dictated by the structure of leucine zipper dimerization domain including the hinge region spanning between the basic region and the leucine zipper.     

Input of orexin/hypocretin neurons revealed by a genetically encoded tracer in mice

The finding of orexin/hypocretin deficiency in narcolepsy patients suggests that this hypothalamic neuropeptide plays a crucial role in regulating sleep/wakefulness states. However, very little is known about the synaptic input of orexin/hypocretin-producing neurons (orexin neurons). We applied a transgenic method to map upstream neuronal populations that have synaptic connections to orexin neurons and revealed that orexin neurons receive input from several brain areas. These include the amygdala, basal forebrain cholinergic neurons, GABAergic neurons in the preoptic area, and serotonergic neurons in the median/paramedian raphe nuclei. Monoamine-containing groups that are innervated by orexin neurons do not receive reciprocal connections, while cholinergic neurons in the basal forebrain have reciprocal connections, which might be important for consolidating wakefulness. Electrophysiological study showed that carbachol excites almost one-third of orexin neurons and inhibits a small population of orexin neurons. These neuroanatomical findings provide important insights into the neural pathways that regulate sleep/wakefulness states.