Cloning and nucleotide sequence of the mono- and diacylglycerol lipase gene (mdlB) of Aspergillus oryzae

Abstract Aspergillus oryzae IFO4202 produces at least two extracellular lipolytic enzymes L1 and L2 (cutinase, and mono- and diacylglycerol lipase, respectively). Southern hybridization of restriction enzyme-digested genomic DNA fragments with 23mer oligonucleotides synthesized according to the amino acid sequence of the L2 as probe suggested the presence of the L2 gene (tentatively designated as mdlB ) and an additional weakly hybridizing region. A fragment containing the genomic mdlB gene was cloned in Escherichia coli . Nucleotide sequencing of the fragment revealed an open reading frame, comprising 1021 nucleotides, which contains two introns (51 and 52 nucleotides). Putative polyadenylation signals were found 182 and 287 bp downstream of the stop codon. The deduced amino acid sequence of the mdlB gene corresponds to 306 amino acid residues including a leader sequence of 28 amino acids and is highly similar to that of the mdlA gene of Penicillium camembertii . Three residues presumed to form the catalytic triad (serine, aspartic acid and histidine) of lipases were also conserved.     

Enhanced plasma ghrelin levels in rats with streptozotocin-induced diabetes

Human saliva, which contains nitrite, is normally mixed with gastric juice, which contains ascorbic acid (AA). When saliva was mixed with an acidic buffer in the presence of 0.1 mM AA, rapid nitric oxide formation and oxygen uptake were observed. The oxygen uptake was due to the oxidation of nitric oxide, which was formed by AA-dependent reduction of nitrite under acidic conditions, by molecular oxygen. A salivary component SCN⁻ enhanced the nitric oxide formation and oxygen uptake by the AA/nitrite system. The oxygen uptake by the AA/nitrite/SCN⁻ system was also observed in an acidic buffer solution. These results suggest that oxygen is normally taken up in the stomach when saliva and gastric juice are mixed.     

Settlement of Planulae of the Moon Jellyfish Aurelia aurita onto Hydrophilic Polycarbonate Plates Modified by Atmospheric Plasma Treatment

It has been reported that planula larvae of some jellyfish prefer artificial substrates for settlement. This research focused on the relationship between the settlement of planulae and the wettability of artificial substrate surfaces. We used atmospheric plasmas to change the wettability of the surfaces of polycarbonate (PC) plates because plasma treatment has no chemical side effects. The treatment made the surfaces hydrophilic, as evidenced by the decrease of contact angle from 85° to 35°. X-ray photoelectron spectroscopy revealed that the change of wettability of the PC plates could be attributed to N₂, which was probably ionized in the air above the plates. Scanning electron microscopy revealed no difference in the surface morphology of the plates before and after plasma treatment. Results of bioassays using treated PC plates showed that planulae tended to preferentially settle on hydrophobic surfaces.     

Transcellobiosylation Reactions Catalyzed by Different Exoglucanase Components of a Trichoderma viride Cellulase in Aqueous Organic Solvent

The contribution of three exoglucanases from a commercial Trichoderma viride cellulase to transcellobiosylation, and the tolerance of these enzymes to acetonitrile co-solvent were studied. The enzymatic reactions were performed with p-nitrophenyl-β-d-cellobioside as the starting substrate. Among these enzymes, the least anionic exoglucanase (Exo I) showed the highest transcellobiosylation activity and acetonitrile tolerance. Exo I retained considerable activity even in 30% MeCN/water and produced p-nitrophenyl-β-d-cellotetraoside at about 1.5% conversion from the initial substrate in 30% MeCN/water. The residual activity of Exo I after incubation in MeCN/water mixture was almost identical to that of the crude cellulase and a considerable amount of the transcellobiosylation properties of the crude cellulase seemed to be attributable to this Exo I component.     

Global overexpression of divalent metal transporter 1 delays crocidolite-induced mesothelial carcinogenesis in male mice

Abstract

Exposure to asbestos fiber is central to mesothelial carcinogenesis, for which iron overload in or near mesothelial cells is a key pathogenic mechanism. Alternatively, iron chelation therapy with deferasirox or regular phlebotomy was significantly preventive against crocidolite-induced mesothelial carcinogenesis in rats. However, the role of iron transporters during asbestos-induced carcinogenesis remains elusive. Here, we studied the role of divalent metal transporter 1 (DMT1; Slc11a2), which is a Fe(II) transporter, that is present not only on the apical plasma membrane of duodenal cells but also on the lysosomal membrane of every cell, in crocidolite-induced mesothelial carcinogenesis using DMT1 transgenic (DMT1Tg) mice. DMT1Tg mice show mucosal block of iron absorption without cancer susceptibility under normal diet. We unexpectedly found that superoxide production was significantly decreased upon stimulation with crocidolite both in neutrophils and macrophages of DMT1Tg mice, and the macrophage surface revealed higher iron content 1?h after contact with crocidolite. Intraperitoneal injection of 3?mg crocidolite ultimately induced malignant mesothelioma in ～50% of both wild-type and DMT1Tg mice (23/47 and 14/28, respectively); this effect was marginally (p?=?0.069) delayed in DMT1Tg mice, promoting survival. The promotional effect of nitrilotriacetic acid was limited, and the liver showed significantly higher iron content both in DMT1Tg mice and after crocidolite exposure. The results indicate that global DMT1 overexpression causes decreased superoxide generation upon stimulation in inflammatory cells, which presumably delayed the promotional stage of crocidolite-induced mesothelial carcinogenesis. DMT1Tg mice with low-stamina inflammatory cells may be helpful to evaluate the involvement of inflammation in various pathologies.     

Spatial and temporal progress of programmed cell death in the developing starchy endosperm of rice

Programmed cell death (PCD) is the genetically regulated disassembly of cells, and occurs in the endosperm of cereals during seed maturation. Since PCD determines the lifetime of cells, it can affect endosperm growth and, therefore, cereal yield. However, the features and mechanisms of PCD in the developing starchy endosperm in the Poaceae remain unclear. In the present study, we investigated the characteristics of PCD in developing starchy endosperm of rice (Oryza sativa L.) by fluorescence microscopy, focusing on the spatial and temporal progress of PCD-associated responses. Cell death commenced in the central region of starchy endosperm, and then spread to the peripheral region. PCD-associated responses, such as mitochondrial membrane permeabilization and activation of the protease that cleaves the amino acid sequence VEID, showed similar spatial patterns to that of cell death, but preceded cell death. Degradation of nuclear DNA could not be detected in developing starchy endosperm by the TUNEL assay. These results indicated that PCD in developing starchy endosperm of rice proceeds via a highly organized pattern. In addition, these results suggested that PCD in developing starchy endosperm of rice is characterized by the involvement of mitochondrial signaling and the activity of a caspase-like protease that cleaves the VEID sequence.     

Optically Detected Structural Change in the N-Terminal Region of?the?Voltage-Sensor Domain

The voltage-sensor domain (VSD) is a functional module that undergoes structural transitions in response to membrane potential changes and regulates its effectors, thereby playing a crucial role in amplifying and decoding membrane electrical signals. Ion-conductive pore and phosphoinositide phosphatase are the downstream effectors of voltage-gated channels and the voltage-sensing phosphatase, respectively. It is known that upon transition, the VSD generally acts on the region C-terminal to S4. However, whether the VSD also induces any structural changes in the N-terminal region of S1 has not been addressed directly. Here, we report the existence of such an N-terminal effect. We used two distinct optical reporters—one based on the Förster resonance energy transfer between a pair of fluorescent proteins, and the other based on fluorophore-labeled HaloTag—and studied the behavior of these reporters placed at the N-terminal end of the monomeric VSD derived from voltage-sensing phosphatase. We found that both of these reporters were affected by the VSD transition, generating voltage-dependent fluorescence readouts. We also observed that whereas the voltage dependencies of the N- and C-terminal effects appear to be tightly coupled, the local structural rearrangements reflect the way in which the VSD is loaded, demonstrating the flexible nature of the VSD.     

Lung Surfactant Levels are Regulated by Ig-Hepta/GPR116 by Monitoring Surfactant Protein D

Lung surfactant is a complex mixture of lipids and proteins, which is secreted from the alveolar type II epithelial cell and coats the surface of alveoli as a thin layer. It plays a crucial role in the prevention of alveolar collapse through its ability to reduce surface tension. Under normal conditions, surfactant homeostasis is maintained by balancing its release and the uptake by the type II cell for recycling and the internalization by alveolar macrophages for degradation. Little is known about how the surfactant pool is monitored and regulated. Here we show, by an analysis of gene-targeted mice exhibiting massive accumulation of surfactant, that Ig-Hepta/GPR116, an orphan receptor, is expressed on the type II cell and sensing the amount of surfactant by monitoring one of its protein components, surfactant protein D, and its deletion results in a pulmonary alveolar proteinosis and emphysema-like pathology. By a coexpression experiment with Sp-D and the extracellular region of Ig-Hepta/GPR116 followed by immunoprecipitation, we identified Sp-D as the ligand of Ig-Hepta/GPR116. Analyses of surfactant metabolism in Ig-Hepta^+/+ and Ig-Hepta^−/− mice by using radioactive tracers indicated that the Ig-Hepta/GPR116 signaling system exerts attenuating effects on (i) balanced synthesis of surfactant lipids and proteins and (ii) surfactant secretion, and (iii) a stimulating effect on recycling (uptake) in response to elevated levels of Sp-D in alveolar space.