Cell surface expression and internalization of the murine erythroid AE1 anion exchanger tagged with an extracellular FLAG epitope

Anion exchanger 1 (AE1) is the most abundant integral membrane protein in red cells and is essential for maintaining red cell mechanical stability. However, the mechanism for the assembly of AE1 into the membrane skeletal network remains unknown. Several mutants of murine AE1 tagged with an N-terminal enhanced green fluorescent protein (EGFP) and/or an extracellular FLAG epitope inserted adjacent to the N-glycosylation site were prepared, and their expression was analyzed in HEK293 or COS-1 cells by immunofluorescence microscopy, biotinylation, and deglycosylation. The EGFP- and FLAG-tagged AE1 mutant, as well as the wild-type AE1, exhibited cell surface expression in transfected cells and showed a rapid internalization that appeared to occur through the early endosome into the Golgi apparatus. Interestingly, the form of the protein with an endoglycosidase H (endo H)-sensitive N-glycan was the major component of EGFP-tagged and wild-type AE1. By contrast, the polypeptide with an endo H-resistant oligosaccharide was the predominant form of FLAG-tagged AE1. These data demonstrate that the processing of N-glycan is not a prerequisite to cell surface expression of AE1 and suggest that the FLAG tag insertion altered the accessibility of the N-glycan to enzymes in the Golgi which facilitate processing of oligosaccharides. Although whether this structural alteration would affect the structural and functional properties of AE1 remains unknown, cell surface expression and endocytic internalization of FLAG-tagged AE1 mutants indicate that these mutants are suitable for studying the mechanisms of the assembly and plasma membrane insertion of AE1.     

Appearance of slow-reacting and complement-requiring neutralizing antibody in cattle infected with Akabane virus

Slow-reacting complement-requiring neutralizing (NT) antibody was detected in sera from cattle 2 weeks after infection with Akabane virus. Bovine sera obtained 3 or 4 weeks after infection contained slow-reacting noncomplement-requiring NT antibody. The slow-reacting complement-requiring NT antibody was sensitive to 2-mercaptoethanol (2-ME), whereas the slow-reacting noncomplement-requiring NT antibody was resistant to 2-ME. The initial phase may represent the IgM response and the later phase a change to IgG. A NT test was developed in which virus-serum mixtures were incubated at 4 degrees C for 48 h and then with complement at 37 degrees C for 60 min; this gave an improved sensitivity over the previous incubation at 37 degrees C for 60 min.     

Fate and persistence of residues on wheat used to explain efficacy differences between deltamethrin suspension concentrate and emulsifiable concentrate formulations

To help explain the increased residual activity of a deltamethrin suspension concentrate (SC) formulation against grasshoppers, the persistence, location and nature of residues from the SC and an emulsifiable concentrate (EC) formulation have been compared. Wheat plants were sprayed in a cabinet sprayer at 7.6 g ha^?1 for the EC and 5.6 g ha^?1 for the SC, then weathered outdoors for 16 days. With the SC formulation, residues were more persistent, more residues were located on the exterior of the leaves, and less deltamethrin was converted to inactive isomers. Eight days after spraying, the exterior deltamethrin residues were 0.13 μg plant^?1 from the SC versus 0.06 μg plant^?1 from the EC. Thus, more residual deltamethrin is available to grasshoppers with the SC formulation.     

Factorial models to estimate isoleucine requirements for broilers

The objective of this work was to determine the efficiency of utilization (EU) and produce factorial models for optimal isoleucine (Ile) intake. Six dose–response trials were carried out, three for males and three for females, with 640 Ross 308 in each studied phase. The initial (1–14 days), grower (15–28 days) and finisher (29–42 days) phases were evaluated to cover the growing phase of the broiler chicken. In total, eight treatments were randomly distributed to four replicates of 20 birds each. The treatments consisted of seven crescent levels of Ile and one counter proof to ensure that Ile was the first limiting amino acid in the diet. Dilution technique was applied to produce the levels of Ile and keep the amino acid ratio with lysine. The EU was determined to account for whole body or partitioned for feather‐free body (Bff) and feather. Two distinct factorial models were adjusted, M1 and M2. The M2 model was evaluated for one or two EU, being denominated as M2 and M3. When the efficiency was partitioned, the values of 53% and 69% for feather and Bff were determined. The optimal Ile intake estimated for each model were of 275, 908, 1,412 mg of Ile/bird/day (M1); 258, 829, 1,321 mg of Ile/bird/day (M2); and 284, 835, 1,288 mg of Ile/bird/day (M3) for initial, grower and finisher phases respectively. The EU partitioned for feather‐free body and feather reduced the biased of the model M3. Overall, higher values of Ile intake are estimated when model M1 is used, which may be the difference in account for body weight gain (M1) or only protein gain (M2 and M3) to estimate the amount of amino acid required for broiler.     

Treatment with the MAPK kinase inhibitor U0126 during the first two hours of in vitro maturation improves bovine oocyte developmental competence

This study examined the effects of treatment with U0126, which inhibits MAPK by inhibiting MAPK kinase, during the first 2 hr of in vitro maturation on bovine developmental competence and on gap junction (GAPJ) communication between the oocyte and cumulus cells. The percentage of oocytes developing to the blastocyst stage in the group treated with 5 μM U0126 (28%) was significantly higher than that in controls (15%, p < .05), while that in the group treated with 10 μM U0126 (18%) was not. Breakdown of the GAPJs was delayed in the group treated with 5 μM U0126 when compared to controls, as estimated by immunohistochemical examination of connexin 43, which is a primary constituent of the GAPJs. These results indicate that treatment with 5 μM U0126 during in vitro maturation delays GAPJ breakdown and improves bovine oocyte developmental competence.     

Antinephritis and radical scavenging activity of prenylflavonoids

Antinephritis activity of 5 prenylflavonoids similar to glabridin (1-5), isolated from Morus alba, Artocarpus communis, Glycyrrhiza uralensis and G. inflata, was evaluated in mice with glomerular disease (Masugi-nephritis). Oral administrations of artonin E (2) or licochalcone A (4) for 10 days (30 mg kg(-1) day(-1)) reduced the amount of urinary protein excretion compared to nephritic mice. ESR spectroscopy demonstrated that morusin (1) and licorisoflavan A (5) increased the radical intensity of sodium ascorbate by about two times. Morusin, licoricidin (3), licochalcone A and licorisoflavan A showed weak scavenging activity against superoxide anion radical.     

Restricted localization of claudin-16 at the tight junction in the thick ascending limb of Henle's loop together with claudins 3, 4, and 10 in bovine nephrons

Claudin-16 is one of the tight junction protein claudins and has been shown to contribute to reabsorption of divalent cations in the human kidney. In cattle, total deficiency of claudin-16 causes severe renal tubular dysplasia without aberrant metabolic changes of divalent cations, suggesting that bovine claudin-16 has some roles in renal tubule formation and paracellular transport that are somewhat different from those expected from the pathology of human disease. As the first step to clarify these roles, we examined the expression and distribution of claudin-16 and several other major claudin subtypes, claudins 1-4 and 10, in bovine renal tubular segments by immunofluorescence microscopy. Claudin-16 was exclusively distributed to the tight junction in the tubular segment positive for Tamm-Horsfall glycoprotein, the thick ascending limb (TAL) of Henle's loop, and was found colocalized with claudins 3, 4, and 10. This study also demonstrates that bovine kidneys possess segment-specific expression patterns for claudins 2-4 and 10 that are different from those reported for mice. Particularly, distribution of claudin-4 in the TAL and distal convoluted tubules was characteristic of bovine nephrons as were differences in the expression patterns of claudins 2 and 3. These findings demonstrate that the total lack of claudin-16 in the TAL segment is the sole cause of renal tubular dysplasia in cattle and suggest that the tight junctions in distinct tubular segments including the TAL have barrier functions in paracellular permeability that are different among animal species.     

Manipulation of fish germ cell: visualization, cryopreservation and transplantation

Germ-cell transplantation has many applications in biology and animal husbandry, including investigating the complex processes of germ-cell development and differentiation, producing transgenic animals by genetically modifying germline cells, and creating broodstock systems in which a target species can be produced from a surrogate parent. The germ-cell transplantation technique was initially established in chickens using primordial germ cells (PGCs), and was subsequently extended to mice using spermatogonial stem cells. Recently, we developed the first germ-cell transplantation system in lower vertebrates using fish PGCs and spermatogonia. During mammalian germ-cell transplantation, donor spermatogonial stem cells are introduced into the seminiferous tubules of the recipient testes. By contrast, in the fish germ-cell transplantation system, donor cells are microinjected into the peritoneal cavities of newly hatched embryos; this allows the donor germ cells to migrate towards, and subsequently colonize, the recipient genital ridges. The recipient embryos have immature immune systems, so the donor germ cells can survive and even differentiate into mature gametes in their allogeneic gonads, ultimately leading to the production of normal offspring. In addition, implanted spermatogonia can successfully differentiate into sperm and eggs, respectively, in male and female recipients. The results of transplantation studies in fish are improving our understanding of the development of germ-cell systems during vertebrate evolution.     

Pigment pattern formation by contact-dependent depolarization

Although recent experimental studies have suggested that the interactions among the pigment cells play a key role in the skin pattern formation, details of the mechanism remain largely unknown. By using an in vitro cell culture system, we have detected interactions between the two pigment cell types, melanophores and xanthophores, in the zebrafish skin. During primary culture, the melanophore membrane transiently depolarizes when contacted with the dendrites of a xanthophore. This depolarization triggers melanophore migration to avoid further contact with the xanthophores. Cell depolarization and repulsive movement were not observed in pigment cells with the jaguar mutant, which shows defective segregation of melanophores and xanthophores. The depolarization-repulsion of wild-type pigment cells may explain the pigment cell behaviors generating the stripe pattern of zebrafish.