Micronucleus induction in mouse peripheral reticulocytes by 7,12-dimethylbenz[a]anthracene.

Micronucleus assays using mouse peripheral blood stained vitally on acridine orange (AO)-coated slides were evaluated at two laboratories with 7,12-dimethylbenz[a]anthracene (DMBA) and compared with the standard bone marrow assay. DMBA was administered by single intraperitoneal injection to CD-1 mice at doses ranging from 5 to 80 mg/kg, then 5 microliters of peripheral blood was sampled from a tail vein at 24, 48, 72, 96, and 120 h after treatment. Similar incidences of micronucleated young erythrocytes were observed in peripheral blood reticulocytes and bone marrow polychromatic erythrocytes. The dose response of micronucleated reticulocytes was delayed compared to that of micronucleated polychromatic erythrocytes. The dose-response curves after treatment with DMBA differed depending on the sampling times, which revealed the difficulty of obtaining accurate dose-response relations in the micronucleus assay. The present result demonstrated that the simple and rapid AO supravital staining method is a valuable and easier method for obtaining dose- and time-response data for quantification of micronucleus induction by chemicals.     

Proof of plasmatic imbibition in rat musculocutaneous grafts: enzymatic proof using peroxidase.

At present, the concept of plasmatic imbibition is generally accepted. That is, observation of weight change or pigmentation methods of the grafts were established to support it. In the present study, we investigated plasmatic imbibition in rat musculocutaneous grafts histologically using peroxidase, which is one of the reductases. Tissue pigmentation by peroxidase became an insoluble sediment that indicated the sites of peroxidase activity. The entire contact surface of the graft with the recipient bed was stained brown within a few minutes after the operation. At 30 minutes, the panniculus carnosus and dermis were stained dark brown diffusely, extending toward the epidermis. This condition continued until the sixth day at least. As a result, peroxidase that was dissolved with the exudate between the graft and recipient bed was imbibed into the musculocutaneous graft.     

The alpha 1-adrenergic photoaffinity probe [125I]arylazidoprazosin binds to a specific peptide of P-glycoprotein in multidrug-resistant cells

Much evidence suggests that P-glycoprotein (P-gp) confers multidrug-resistance (MDR) in tumor cells by energy-dependent efflux of hydrophobic cytotoxic agents. In this study, we have used the alpha 1-adrenergic photoaffinity probe, [125I]arylazidoprazosin ([125I]AAP), and identified P-gp as a specific acceptor for prazosin. Drugs to which MDR cells are resistant, including vincristine, vinblastine, doxorubicin, actinomycin D and colchicine as well as agents reversing MDR, including verapamil, nicardipine, prenylamine, diltiazem, trifluoperazine, dibucaine, reserpine, monensin, and progesterone, differentially reduced [125I]AAP photolabeling of P-gp. We also analyzed the influence of alpha 2-adrenergic drugs and dopaminergic drugs on [125I]AAP photolabeling of P-gp. Limited proteolysis of [125I]AAP photolabeled P-gp with Staphylococcus aureus V8 protease revealed that prazosin binds to a single 8 kDa fragment of P-gp.     

Biochemical characterization of the submicrosomal membrane of the rat brain. Selective solubilization of the components of the light smooth-surfaced membrane by lysophosphatidylcholine

The light smooth-surfaced membrane, one of the three membrane fractions derived from the rat brain microsomal fraction, was fractionated into its soluble and insoluble parts by the use of lysophosphatidylcholine and the chemical composition of these was investigated. Under the condition whereby the maximal amount of the membrane protein was solubilized by lysophosphatidylcholine (0.5% lysophosphatidylcholine at 37 degrees C for 10 min), the insoluble residue, which accounted for approximately 30% of the membrane protein, was ultracentrifugally homogeneous and showed a granular structure under the electron microscope. The lipid composition of the soluble and insoluble fractions, as well as their protein composition, revealed a preferential and limited solubilization of the constituents of the membrane by lysophosphatidylcholine.     

Impact of Noncovalent Sulfur–Fluorine Interaction Position on Properties,Structures, and Photovoltaic Performance in Naphthobisthiadiazole‐Based Semiconducting Polymers

Controlling the energetics and backbone order of semiconducting polymers is essential for the performance improvement of polymer‐based solar cells. The use of fluorine as the substituent for the backbone is known to effectively deepen the molecular orbital energy levels and coplanarize the backbone by noncovalent interactions with sulfur of the thiophene ring. In this work, novel semiconducting polymers are designed and synthesized based on difluoronaphthobisthiadiazole (FNTz) as a new family of naphthobisthiadiazole (NTz)–quaterthiophene copolymer systems, which are one of the highest performing polymers in solar cells. The effect of the fluorination position on the energetics and backbone order is systematically studied. It is found that the dependence of the solar cell fill factor on the active layer thickness is very sensitive to the fluorination position. It is thus further investigated and discussed how the structural features of the polymers influence the photovoltaic parameters as well as the diode characteristics and bimolecular recombination. Further, the polymer with fluorine on both the naphthobisthiadiazole and quaterthiophene moieties exhibits a quite high power conversion efficiency of 10.8% in solar cells in combination with a fullerene. It is believed that the results would offer new insights into the development of semiconducting polymers.     

Rheb (Ras Homologue Enriched in Brain)-dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Activation Becomes Indispensable for Cardiac Hypertrophic Growth after Early Postnatal Period

Cardiomyocytes proliferate during fetal life but lose their ability to proliferate soon after birth and further increases in cardiac mass are achieved through an increase in cell size or hypertrophy. Mammalian target of rapamycin complex 1 (mTORC1) is critical for cell growth and proliferation. Rheb (Ras homologue enriched in brain) is one of the most important upstream regulators of mTORC1. Here, we attempted to clarify the role of Rheb in the heart using cardiac-specific Rheb-deficient mice (Rheb^−/−). Rheb^−/− mice died from postnatal day 8 to 10. The heart-to-body weight ratio, an index of cardiomyocyte hypertrophy, in Rheb^−/− was lower than that in the control (Rheb^+/+) at postnatal day 8. The cell surface area of cardiomyocytes isolated from the mouse hearts increased from postnatal days 5 to 8 in Rheb^+/+ mice but not in Rheb^−/− mice. Ultrastructural analysis indicated that sarcomere maturation was impaired in Rheb^−/− hearts during the neonatal period. Rheb^−/− hearts exhibited no difference in the phosphorylation level of S6 or 4E-BP1, downstream of mTORC1 at postnatal day 3 but showed attenuation at postnatal day 5 or 8 compared with the control. Polysome analysis revealed that the mRNA translation activity decreased in Rheb^−/− hearts at postnatal day 8. Furthermore, ablation of eukaryotic initiation factor 4E-binding protein 1 in Rheb^−/− mice improved mRNA translation, cardiac hypertrophic growth, sarcomere maturation, and survival. Thus, Rheb-dependent mTORC1 activation becomes essential for cardiomyocyte hypertrophic growth after early postnatal period.     

Purification and Properties of a Thermo-labile Antigen from Baker’s Yeast Cells

A thermo-labile antigen (TLA) on the yeast cell surface was isolated from a yeast cell autolyzate and purified to a homogeneous state by chromatography on an immunoadsorbent affinity column. The molecular weight of TLA was about 1.45 x 10⁵ on SDS-polyacrylamide gel electrophoresis and about 1.5 x l0⁵ on gel chromatography on Sephadex G-200. The TLA contained 74.5% protein and 25.5% sugar. It was characterized by high contents of glycine, glutamic acid, serine and aspartic acid. Half-cystine, methionine, histidine and arginine were not found. The sugar moiety was composed of galactose, mannose, N-acetylglucosamine and fucose. The antigenic determinant of TLA was distinct from that of cell wall mannan in the Ouchterlony immunodiffusion test. No precipitin line against anti-TLA serum was observed, when TLA was heated at 90°C for 10 min. Oxidation with periodate had little effect on antigenicity, but digestion with Pronase or treatment with protein denaturants resulted in loss of the antigenicity. These results suggest that the protein moiety plays an important role as the antigenic determinant of TLA. Moreover, the antiserum specific to TLA agglutinated fresh yeast cells, and the distribution of TLA was apparent on the yeast cell surface by immunofluorescence staining. These findings suggest that TLA molecules were exposed on the outer surface of the yeast cell wall.     

Possible requirement of intercellular adhesion molecule-1 for invasion of gingival epithelial cells by Treponema medium

Oral treponemes are members of the spirochete family of bacteria associated with periodontal diseases. In the present study, we demonstrate that intercellular adhesion molecule-1 (ICAM-1) on human gingival epithelial cells (HGEC) contributed to the invasion of Treponema medium, a medium-sized oral Treponema, into those cells. The quantity of T. medium in HGEC was found to peak at 2 h after inoculation and then decreased gradually. Immunofluorescence microscopy findings showed that the bacteria were colocalized with ICAM-1 on HGEC. Furthermore, knockdown of ICAM-1 in HGEC resulted in inhibition of T. medium invasion by RNA interference, whereas that of Toll-like receptor 2 did not. These results suggest that ICAM-1 may be required for the invasion of T. medium into HGEC, and they indicate that the molecule plays a principal role in the primary stages of the development and progression of chronic periodontitis.     

Disruption of phospholipase B gene, PLB1, increases the survival of baker's yeast Torulaspora delbrueckii

Abstract An uracil auxotrophic mutant of baker's yeast Torulaspora delbrueckii , which is resitant to 5-fluoro-orotic acid, was complemented by transformation with YEp24 which harbors 2 μm origin and URA3 derived from Saccharomyces cerevisiae . The phospholipase B in T. delbrueckii cells is active in both acidic and alkaline conditions. However, activity of phospholipase B gene ( PLB1 ) in cells of disruption mutant ( plbI : : URA3 ) was lost in both conditions, which indicates that all phospholipase B activity is encoded by a single gene (or a single polypeptide) in these yeast cells. Over-expression of PLB1 with YEp plasmid vector in T. delbrueckii cells showed ∼ 2.5-fold increase in phospholipase B activity, comparing with that in wild-type cells. Cells of plb1 Δ mutant showed increased survival when cells of plb1 Δ mutant and wild-type strain were incubated in water at 30 °C. Cells of PLB1 -over-expressed strain died rapidly even during the cultivation period, indicating that phospholipase B activity may be a determinant for the survival of this yeast.     

Na(+)-coupled transport of L-carnitine via high-affinity carnitine transporter OCTN2 and its subcellular localization in kidney

The mechanism of Na(+)-dependent transport of L-carnitine via the carnitine/organic cation transporter OCTN2 and the subcellular localization of OCTN2 in kidney were studied. Using plasma membrane vesicles prepared from HEK293 cells that were stably transfected with human OCTN2, transport of L-carnitine via human OCTN2 was characterized. Uptake of L-[(3)H]carnitine by the OCTN2-expressing membrane vesicles was significantly increased in the presence of an inwardly directed Na(+) gradient, with an overshoot, while such transient uphill transport was not observed in membrane vesicles from cells that were mock transfected with expression vector pcDNA3 alone. The uptake of L-[(3)H]carnitine was specifically dependent on Na(+) and the osmolarity effect showed that Na(+) significantly influenced the transport rather than the binding. Changes of inorganic anions in the extravesicular medium and of membrane potential by valinomycin altered the initial uptake activity of L-carnitine by OCTN2. In addition, the fluxes of L-carnitine and Na(+) were coupled with 1:1 stoichiometry. Accordingly, it was clarified that Na(+) is coupled with flux of L-carnitine and the flux is an electrogenic process. Furthermore, OCTN2 was localized on the apical membrane of renal tubular epithelial cells. These results clarified that OCTN2 is important for the concentrative reabsorption of L-carnitine after glomerular filtration in the kidney.