Kayoko Ookata

MBD3 and HDAC1, two components of the NuRD complex, are localized at Aurora-A-positive centrosomes in M phase

MBD3, a component of the histone deacetylase NuRD complex, contains the methyl-CpG-binding domain (MBD), yet does not possess appreciable mCpG-specific binding activity. The functional significance of MBD3 in the NuRD complex remains enigmatic, partly because of the limited availability of biochemical approaches, such as immunoprecipitation, to analyze MBD3. In this study, we stably expressed the FLAG-tagged version of MBD3 in HeLa cells. We found that MBD3-FLAG was incorporated into the NuRD complex, and the MBD3-FLAG-containing NuRD complex was efficiently immunoprecipitated by anti-FLAG antibodies. By exploiting this system, we found that MBD3 is phosphorylated in vivo in the late G2and early M phases. Moreover, we found that Aurora-A, a serine/threonine kinase active specifically in the late G2and early M phases, phosphorylates MBD3 in vitro, physically associates with MBD3 in vivo, and co-localizes with MBD3 at the centrosomes in the early M phase. Interestingly, HDAC1 is distributed at the centrosomes in a manner similar to MBD3. These results suggest the highly dynamic nature of the temporal and spatial distributions, as well as the biochemical modification, of the NuRD complex in M phase, probably through an interaction with kinases, including Aurora-A. These observations will contribute significantly to the elucidation of the yet-uncharacterized cell cycle-controlled functions of the NuRD complex.

Mechanism of development of ionocytes rich in vacuolar-type H+-ATPase in the skin of zebrafish larvae

Mitochondrion-rich cells (MRCs), or ionocytes, play a central role in aquatic species, maintaining body fluid ionic homeostasis by actively taking up or excreting ions. Since their first description in 1932 in eel gills, extensive morphological and physiological analyses have yielded important insights into ionocyte structure and function, but understanding the developmental pathway specifying these cells remains an ongoing challenge. We previously succeeded in identifying a key transcription factor, Foxi3a, in zebrafish larvae by database mining. In the present study, we analyzed a zebrafish mutant, quadro (quo), deficient in foxi1 gene expression and found that foxi1 is essential for development of an MRC subpopulation rich in vacuolar-type H(+)-ATPase (vH-MRC). foxi1 acts upstream of Delta-Notch signaling that determines sporadic distribution of vH-MRC and regulates foxi3a expression. Through gain- and loss-of-function assays and cell transplantation experiments, we further clarified that (1) the expression level of foxi3a is maintained by a positive feedback loop between foxi3a and its downstream gene gcm2 and (2) Foxi3a functions cell-autonomously in the specification of vH-MRC. These observations provide a better understanding of the differentiation and distribution of the vH-MRC subtype.

O2-filled swimbladder employs monocarboxylate transporters for the generation of O2 by lactate-induced root effect hemoglobin.

The swimbladder volume is regulated by O2 transfer between the luminal space and the blood In the swimbladder, lactic acid generation by anaerobic glycolysis in the gas gland epithelial cells and its recycling through the rete mirabile bundles of countercurrent capillaries are essential for local blood acidification and oxygen liberation from hemoglobin by the “Root effect.” While O2 generation is critical for fish flotation, the molecular mechanism of the secretion and recycling of lactic acid in this critical process is not clear. To clarify molecules that are involved in the blood acidification and visualize the route of lactic acid movement, we analyzed the expression of 17 members of the H+/monocarboxylate transporter (MCT) family in the fugu genome and found that only MCT1b and MCT4b are highly expressed in the fugu swimbladder. Electrophysiological analyses demonstrated that MCT1b is a high-affinity lactate transporter whereas MCT4b is a low-affinity/high-conductance lactate transporter. Immunohistochemistry demonstrated that (i) MCT4b expresses in gas gland cells together with the glycolytic enzyme GAPDH at high level and mediate lactic acid secretion by gas gland cells, and (ii) MCT1b expresses in arterial, but not venous, capillary endothelial cells in rete mirabile and mediates recycling of lactic acid in the rete mirabile by solute-specific transcellular transport. These results clarified the mechanism of the blood acidification in the swimbladder by spatially organized two lactic acid transporters MCT4b and MCT1b.

Monoclonal antibody (VOM2) specific for the luminal surface of the rat vomeronasal sensory epithelium

Monoclonal antibodies were raised and selected for reactivity with the luminal surface of the rat vomeronasal organ. Among the monoclonal antibodies generated, the one named VOM2 showed specific immunoreactivity within the luminal surface of the rat vomeronasal sensory epithelium. The VOM2 antigen appeared weakly on the luminal surface at postnatal day 14 (P14). After P21, VOM2 immunoreactivity as strong as that in the adult vomeronasal organ was observed. Immunofluorescence staining using VOM2 antibody showed no reactivity on the luminal surface of the adult mouse or hamster vomeronasal organ. An immunoblotting analysis showed that the VOM2 antigen was a protein with a molecular weight of 24,500.

Histological demonstration of glucose transporters, fructose-1,6-bisphosphatase, and glycogen in gas gland cells of the swimbladder: is a metabolic futile cycle operating?

Luminal surface of the swimbladder is covered by gas gland epithelial cells and is responsible for inflating the swimbladder by generating O(2) from Root-effect hemoglobin that releases O(2) under acidic conditions. Acidification of blood is achieved by lactic acid secreted from gas gland cells, which are poor in mitochondria but rich in the glycolytic activity. The acidic conditions are locally maintained by a countercurrent capillary system called rete mirabile. To understand the regulation of anaerobic metabolism of glucose in the gas gland cells, we analyzed the glucose transporter expressed there and the fate of ATP generated by glycolysis. The latter is important because the ATP should be immediately consumed otherwise it strongly inhibits the glycolysis rendering the cells unable to produce lactic acid anymore. Expression analyses of glucose transporter (glut) genes in the swimbladder of fugu (Takifugu rubripes) by RT-PCR and in situ hybridization demonstrated that glut1a and glut6 are expressed in gas gland cells. Immunohistochemical analyses of metabolic enzymes demonstrated that a gluconeogenesis enzyme fructose-1,6-bisphosphatase (Fbp1) and a glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (Gapdh) are highly expressed in gas gland cells. The simultaneous catalyses of glycolysis and gluconeogenesis reactions suggest the presence of a futile cycle in gas gland cells to maintain the levels of ATP low and to generate heat that helps reduce the solubility of O(2).

The active site titration of proteinases by using α2-macroglobulin and high-performance liquid chromatography

The active site titration for various proteinases relies on the development of optimal enzyme titrants for each proteinase, but these titrants are only available for a limited number of proteinases. We have described a new active site titration method applicable to various kinds of endoproteinases using small quantities of the enzymes. This method was carried out by using alpha 2-macroglobulin (alpha 2M) as a titrant and a high-performance liquid chromatography (HPLC) system. When the proteinase solution was treated with alpha 2M, the active proteinase was trapped by alpha 2M. In this reaction alpha 2M does not usually complex with inactive proteinase. After the reaction of proteinase with an excess of alpha 2M, the reaction mixture is applied to an HPLC gel column to separate the uncomplexed enzyme from the one complexed with alpha 2M. The active proteinase is complexed and eluted with alpha 2M, but the inactive proteinase is eluted at the original elution volume. The same amount of the enzyme was also applied to the column. From the decrease of the peak height at the elution position of the uncomplexed proteinase, we can estimate the ratio between enzymatically active proteinases and total proteinases. To test the usefulness of this method, we applied this method to chymotrypsin and trypsin whose activities were predetermined by conventional active site titration, and there was good agreement between both results. With this new method, we can estimate a proteinase activity with as little as 200 ng of the enzyme, a very small amount compared with those required in conventional methods.(ABSTRACT TRUNCATED AT 250 WORDS)

A recombinant bait region mutant of human α2-macroglobulin exhibiting an altered proteinase-inhibiting spectrum

Alpha 2-macroglobulin (α2M), a plasma glycoprotein produced in the liver, inhibits a variety of proteinases and thus considered to play important homeostatic roles in the body. This broad inhibitory spectrum has been explained by the trapping theory by which a proteinase recognizes a region of 25–30 amino acid peptide in α2M called bait region and cleaves it, leading to the conformational change of α2M, and to the subsequent entrapment and inhibition of the proteinase. We constructed α2M cDNAs with mutated DNA sequences in the bait region, and obtained recombinant CHO cell lines producing either wild type α2M, or mutant α2Ms, i.e., α2M/K692 and α2M/K696, each with substitution of Arg with Lys at codons 692 and 696, respectively. We tested if lysyl endopeptidase is not inhibited by wild type α2M, but could be inhibited by these engineered mutant α2Ms. Thus, recombinant α2M/K696 protein successfully inhibited lysyl endopeptidase activity, while recombinant α2M/K692 protein was not sensitive to lysyl endopeptidase, suggesting that not all bait region peptide bonds can equally be accessible and susceptible to proteinases. The present results not only provided the trapping theory with additional supportive evidence, but the first experimental evidence for the value of engineered α2M-derived proteinase inhibitor with an artificial proteinase inhibitory spectrum of potential industrial and/or therapeutic usefulness.