Two atypical ANGUSTIFOLIA without a plant-specific C-terminus regulate gametophore and sporophyte shapes in the moss Physcomitrium (Physcomitrella) patens

ANGUSTIFOLIA (AN) is a plant-specific subfamily of the CtBP/BARS/AN family, characterized by a plant-specific C-terminal domain of approximately 200 amino acids. Previously, we revealed that double knockout (DKO) lines of Physcomitrium (Physcomitrella) patens ANGUSTIFOLIA genes (PpAN1-1 and PpAN1-2) show defects in gametophore height and the lengths of the seta and foot region of sporophytes, by reduced cell elongation. In addition to two canonical ANs, the genome of P. patens has two atypical ANs without a coding region for a plant-specific C-terminus (PpAN2-1 and PpAN2-2); these were investigated in this study. Similar to PpAN1s, both promoters of the PpAN2 genes were highly active in the stems of haploid gametophores and in the middle-to-basal region of young diploid sporophytes that develop into the seta and foot. Analyses of PpAN2-1/2-2 DKO and PpAN quadruple knockout (QKO) lines implied that these four AN genes have partially redundant functions to regulate cell elongation in their expression regions. Transgenic strains harboring P. patens α-tubulin fused to green fluorescent protein, which were generated from a QKO line, showed that the orientation of the microtubules in the gametophore tips in the PpAN QKO lines was unchanged from the wild-type and PpAN1-1/1-2 DKO plants. In addition to both PpAN2-1 and PpAN2-2, short Arabidopsis AN without the C-terminus of 200 amino acids could rescue the Arabidopsis thaliana an-1 phenotypes, implying AN activity is dependent on the N-terminal regions.     

Hypothalamic neuronal histamine signaling in the estrogen deficiency-induced obesity

Menopause is one of the triggers that induce obesity. Estradiol (E2), corticotropin-releasing hormone (CRH), and hypothalamic neuronal histamine are anorexigenic substances within the hypothalamus. This study examined the interactions among E2, CRH, and histamine during the regulation of feeding behavior and obesity in rodents. Food intake was measured in rats after the treatment of E2, α-fluoromethyl histidine, a specific suicide inhibitor of histidine decarboxylase that depletes hypothalamic neuronal histamine, or CRH antagonist. We measured food intake and body weight in wild-type mice or mice with targeted disruption of the histamine receptors (H1-R) knockout (H1KO mice). Furthermore, we investigated CRH content and histamine turnover in the hypothalamus after the E2 treatment or ovariectomy (OVX). We used immunohistochemical staining for estrogen receptors (ERs) in the histamine neurons. The E2-induced suppression of feeding was partially attenuated in rats pre-treated with α-fluoromethyl histidine or CRH antagonist and in H1KO mice. E2 treatment increased CRH content and histamine turnover in the hypothalamus. OVX increased food intake and body weight, and decreased CRH content and histamine turnover in the hypothalamus. In addition, E2 replacement reversed the OVX-induced changes in food intake and body weight in wild-type mice but not in H1KO mice. Immunohistochemical analysis revealed ERs were expressed on histamine neurons and western blotting analysis and pre-absorption study confirmed the specificity of ER antiserum we used. These results indicate that CRH and hypothalamic neuronal histamine mediate the suppressive effects of E2 on feeding behavior and body weight.     

Apolipoprotein A-IV interacts synergistically with melanocortins to reduce food intake

Apolipoprotein (apo) A-IV is an anorexigenic gastrointestinal peptide that is also synthesized in the hypothalamus. The goal of these experiments was to determine whether apo A-IV interacts with the central melanocortin (MC) system in the control of feeding. The third ventricular (i3vt) administration of a subthreshold dose of apo A-IV (0.5 microg) potentiated i3vt MC-induced (metallothionein-II, 0.03 nmol) suppression of 30-min feeding in Long-Evans rats. A subthreshold dose of the MC antagonist (SHU9119, 0.1 nmol, i3vt) completely attenuated the anorectic effect of i3vt apo A-IV (1.5 microg). The i3vt apo A-IV significantly elevated the expression of c-Fos in neurons of the paraventricular nucleus of the hypothalamus, but not in the arcuate nucleus or median eminence. In addition, c-Fos expression was not colocalized with proopiomelanocortin-positive neurons. These data support a synergistic interaction between apo A-IV and melanocortins that reduces food intake by acting downstream of the arcuate.     

Osteoprotegerin protects endothelial cells against apoptotic cell death induced by Porphyromonas gingivalis cysteine proteinases

Osteoprotegerin (OPG) is a key regulator of osteoclastogenesis during the progression of periodontitis. Recent reports suggest that osteoprotegerin may also prevent arterial calcification and contribute to endothelial cell survival. To determine whether the vascular functions of osteoprotegerin are involved in periodontitis, we examined whether osteoprotegerin contributed to the survival of endothelial cells damaged by Porphyromonas gingivalis cysteine proteinases (gingipains). Gingipain proteinases cleave a broad range of host proteins, and are important virulence factors of P. gingivalis, a major causative bacterium of adult periodontitis. Human microvascular endothelial cells (HMVEC) were exposed to activated gingipain extracts from P. gingivalis 381, with and without pretreatment with osteoprotegerin. Cell viability was quantified by the tetrazolium (WST-8) reduction assay, and apoptosis was examined using Hoechst 33342 nuclear staining. After 16 h of treatment with activated gingipain extracts, HMVEC showed near-complete detachment from the tissue culture dish, and apoptosis was evident by 24 h. Pretreatment of HMVEC with osteoprotegerin reduced the extent of both cellular detachment and apoptotic cell death. Our results indicated that osteoprotegerin pretreatment protected HMVEC against detachment and apoptotic cell death induced by gingipain-active bacterial cell extracts. These results also suggest that osteoprotegerin may function as a survival factor for endothelial cells during periodontitis.     

Affinity chromatography of chymotrypsin on a sepharose derivative coupled with a chymostatin analogue

A Sepharose derivative coupled with a chymostatin analogue, Gly-Gly-L-Leu-L-phenylalaninal (Pheal), was prepared. A number of native and chemically modified proteases were applied on a column of the adsorbent. Bovine chymotrypsins [EC 3.4.21.1] and Streptomyces griseus protease B were adsorbed strongly at pH 8.2. The affinities of these enzymes under various conditions were measured quantitatively by frontal chromatography in terms of the dissociation constant (Kd) of the enzyme-immobilized ligand complex. The pH dependence of the Kd value of alpha-chymotrypsin was consistent with that of the inhibition constant (Ki) of the enzyme for a corresponding soluble peptide aldehyde. Anhydro-chymotrypsin, in which the active site Ser-195 is converted to dehydroalanine, was not adsorbed. Ser-195 proved to be essential for the binding. The frontal chromatography method also gave the amount of the immobilized ligand that can interact with the enzyme. It was extremely small compared with the amount of the immobilized ligand determined by amino acid analysis. This was explained on the basis of the structural features of the agarose gel.     

Characterization of Antigenic Polypeptides Recognized by Monoclonal Antibodies Specific to the Myoplasm of Eggs of the Ascidian Ciona intestinalis

The determinants responsible for the differentiation of ascidian larval muscle cells are thought to be contained within the egg myoplasm. To analyze the macromolecules composing the myoplasm, several hybridoma cell lines which secrete monoclonal antibodies specific to myoplasmic components of Ciona eggs have been established (17). In the present investigation, seven of these myoplasm-specific antigens were characterized according to their molecular features and distribution patterns within the egg cytoplasm. Four of the seven antigenic polypeptides were shown to be components of the cortical cytoplasm, two were related to mitochondria, and one is likely to be a yolk protein. An antigen recognized by IIG6B2 antibody, which inhibited muscle development when injected into fertilized eggs, was a single polypeptide of relative molecular mass about 40,000 and isoelectric point about 5. The antigen was designated myoplasmin-C1 after its characteristic localization. The IIF9E9 antigen was a single 35-kDa polypeptide related to mitochondria and was thus designated myoplasmin-M1. The other five antibodies recognized two or more spots by immunoblotting analysis using two-dimensional gel electrophoresis. All of these myoplasm-specific antigens, except for the IIH10D6 antigen, are likely to be produced by the oocyte itself. Synthesis of IIH10D6 antigen seems to be associated with test cells.     

Affinity chromatography of trypsin and related enzymes. IV. Quantitative comparison of affinity adsorbents containing various arginine peptides

In order to study the mechanism of substrate binding of trypsin by affinity chromatography, we synthesized various L-arginine-terminated oligopeptides having different chain length and amino acid sequences, and immobilized them on agarose gel. The interaction of beta-trypsin with these adsorbents was studied by a quantitative affinity chromatographic procedure which gave the dissociation constant (Kd) of the trypsin-immobilized ligand complex. This procedure proved to be very useful and to give information equivalent to that obtained by kinetic procedures. The contribution of the amino acid residue at P2 of the ligands to the affinity was studied by using tripeptide (Gly-X-Arg) Sepharoses, and alanine was found to be more effective than glycine or valine. This conclusion was supported by a kinetic experiment in which Ki values of the corresponding soluble tripeptides (Ac-Gly-X-Arg) were determined. A significant decrease in Kd was observed when the ligand was elongated from dipeptide to tripeptide. However, Kd decreased only slightly when the ligand was elongated further. This suggests that a tripeptide is sufficiently long as a ligand. On the basis of these results, the mode of substrate binding of trypsin is discussed.