Determination of Copper Content in o-Diphenol Oxidase by Neutron Activation Analysis

A polyphenol oxidase (o-diphenol oxidase) [o-diphenol: O₂ oxidoreductase E. C. 1.10. 3.1] from sweet potato named component II_b was highly purified. The copper content of this enzyme was measured by neutron activation analysis. Samples were analyzed with or without chemical separation after neutron irradiation. The copper content of the enzyme was determined to be 0.27%, and the minimum molecular weight of this enzyme was caluculated to be 23,500.     

Divergence times in the termite genus Macrotermes (Isoptera: Termitidae)

The evolution of fungus-growing termites is supposed to have started in the African rain forests with multiple invasions of semi-arid habitats as well as multiple invasions of the Oriental region. We used sequences of the mitochondrial COII gene and Bayesian dating to investigate the time frame of the evolution of Macrotermes, an important genus of fungus-growing termites. We found that the genus Macrotermes consists of at least 6 distantly related clades. Furthermore, the COII sequences suggested some cryptic diversity within the analysed African Macrotermes species. The dates calculated with the COII data using a fossilized termite mound to calibrate the clock were in good agreement with dates calculated with COI sequences using the split between Locusta and Chortippus as calibration point which supports the consistency of the calibration points. The clades from the Oriental region dated back to the early Tertiary. These estimates of divergence times suggested that Macrotermes invaded Asia during periods with humid climates. For Africa, many speciation events predated the Pleistocene and fall in range of 6-23 million years ago. These estimates suggest that savannah-adapted African clades radiated with the spread of the semi-arid ecosystems during the Miocene. Apparently, events during the Pleistocene were of little importance for speciation within the genus Macrotermes. However, further investigations are necessary to increase the number of taxa for phylogenetic analysis.     

Correction of a CD55 mutation to quantify the efficiency of targeted knock-in via flow cytometry

Molecular Biology Reports - Targeted knock-in assisted by the CRISPR/Cas9 system is an advanced technology with promising applications in various research fields including medical and agricultural...     

Silencing of TBC1D15 promotes RhoA activation and membrane blebbing

Membrane blebs are round-shaped dynamic membrane protrusions that occur under many physiological conditions. Membrane bleb production is primarily controlled by actin cytoskeletal rearrangements mediated by RhoA. Tre2–Bub2–Cdc16 (TBC) domain-containing proteins are negative regulators of the Rab family of small GTPases and contain a highly conserved TBC domain. In this report, we show that the expression of TBC1D15 is associated with the activity of RhoA and the production of membrane blebs. Depletion of TBC1D15 induced activation of RhoA and membrane blebbing, which was abolished by the addition of an inhibitor for RhoA signaling. In addition, we show that TBC1D15 is required for the accumulation of RhoA at the equatorial cortex for the ingression of the cytokinetic furrow during cytokinesis. Our results demonstrate a novel role for TBC1D15 in the regulation of RhoA during membrane blebbing and cytokinesis.     

Morphological and functional characterization of a novel Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase-immunoreactive,follicle-like structure on the gill septum of Japanese banded houndshark, <Emphasis Type="Italic">Triakis scyllium</Emphasis>

In teleost fishes, it is well-established that the gill serves as an important ionoregulatory organ in addition to its primary function of respiratory gas exchange. In elasmobranch fish, however, the ionoregulatory function of the gills is still poorly understood. Although mitochondria-rich (MR) cells have also been found in elasmobranch fish, these cells are considered to function primarily in acid-base regulation. In this study, we found a novel aggregate structure made up of cells with basolaterally-expressed Na⁺/K⁺-ATPase (NKA), in addition to NKA-immunoreactive MR cells that have already been described in the gill filament and lamella. The cell aggregates, named follicularly-arranged NKA-rich cells (follicular NRCs), were found exclusively in the epithelial lining of the venous web in the cavernous region of the filament and the inter-filamental space of the gill septum. The follicular NRCs form a single-layered follicular structure with a large lumen leading to the external environment. The follicular NRCs were characterized by: (i) well-developed microvilli on the apical membrane, (ii) less prominent infoldings of the basolateral membrane and (iii) typical junction structures including deep tight junction between cells. In addition, large numbers of vesicles were observed in the cytoplasm and some of them were fused to the lateral membrane. The follicular NRCs expressed Na⁺/H⁺ exchanger 3 and Ca²⁺ transporter 1. The follicular NRCs thus have the characteristics of absorptive ionoregulatory cells and this suggests that the elasmobranch gill probably contributes more importantly to body fluid homeostasis than previously thought.     

Neoplastic transformation and induction of H+,K+-adenosine triphosphatase by N-methyl-N′-nitro-N-nitrosoguanidine in the gastric epithelial RGM-1 cell line

N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) induces gastric cancer in animal models. We established an MNNG-induced mutant of the rat murine RGM-1 gastric epithelial cell line, which we named RGK-1, that could be used as an in vitro model of gastric cancer. This cell line showed signs of neoplasia and transformation, in that it lost contact inhibition and formed tumors in nude mice. The mutant cells also expressed parietal cell-specific H⁺,K⁺-adenosine triphosphatase (H⁺,K⁺-ATPase), which parent RGM-1 did not. The results suggested that parent RGM-1 cells were gastric progenitor cells. This mutant RGK-1 cell line will contribute to future investigation on gastric carcinogenesis and to the development of other pathophysiologic fields.     

Microtubule-dependent microtubule nucleation based on recruitment of gamma-tubulin in higher plants

Despite the absence of a conspicuous microtubule-organizing centre, microtubules in plant cells at interphase are present in the cell cortex as a well oriented array. A recent report suggests that microtubule nucleation sites for the array are capable of associating with and dissociating from the cortex. Here, we show that nucleation requires extant cortical microtubules, onto which cytosolic gamma-tubulin is recruited. In both living cells and the cell-free system, microtubules are nucleated as branches on the extant cortical microtubules. The branch points contain gamma-tubulin, which is abundant in the cytoplasm, and microtubule nucleation in the cell-free system is prevented by inhibiting gamma-tubulin function with a specific antibody. When isolated plasma membrane with microtubules is exposed to purified neuro-tubulin, no microtubules are nucleated. However, when the membrane is exposed to a cytosolic extract, gamma-tubulin binds microtubules on the membrane, and after a subsequent incubation in neuro-tubulin, microtubules are nucleated on the pre-existing microtubules. We propose that a cytoplasmic gamma-tubulin complex shuttles between the cytoplasm and the side of a cortical microtubule, and has nucleation activity only when bound to the microtubule.     

Effect of sucrose on ascorbate level and expression of genes involved in the ascorbate biosynthesis and recycling pathway in harvested broccoli florets

The relationship between sucrose (Suc) and ascorbate (AA) metabolism was investigated in harvested broccoli (Brassica oleracea L. var. italica) florets. Decreases in both Suc and AA content were observed in broccoli florets 48 h after all the leaves were excised, but none were observed when the plants were kept intact or with leaves attached in a room at 20 degrees C. In harvested broccoli plants without leaves and roots, continuous absorption of a 10% (w/v) Suc solution from the cut surface of the stem suppressed the degreening of sepals and the loss of AA content in florets. The expression of the genes related to AA metabolism in chloroplasts and its biosynthesis were up-regulated by Suc feeding in broccoli florets. These data suggest that a decline in Suc leads to considerable damage not only to AA biosynthesis but also to the hydrogen peroxide-scavenging system in chloroplasts. In addition, the cessation of the Suc supply from leaves can be the main factor of AA degradation in harvested broccoli florets.