Axle-less F1-ATPase rotates in the correct direction

F1-adenosine triphosphatase (ATPase) is an ATP-driven rotary molecular motor in which the central gamma subunit rotates inside a cylinder made of three alpha and three beta subunits alternately arranged. The rotor shaft, an antiparallel alpha-helical coiled coil of the amino and carboxyl termini of the gamma subunit, deeply penetrates the central cavity of the stator cylinder. We truncated the shaft step by step until the remaining rotor head would be outside the cavity and simply sat on the concave entrance of the stator orifice. All truncation mutants rotated in the correct direction, implying torque generation, although the average rotary speeds were low and short mutants exhibited moments of irregular motion. Neither a fixed pivot nor a rigid axle was needed for rotation of F1-ATPase.     

In vitro and in vivo studies of the growth inhibitory effect of a newly synthesized peptide, obiopeptide-1, on mice-bearing methylcholanthrene-induced murine tumours.

Novel synthetic Obiopeptide (Obi-1), Glycil-penta-Glutamate, as an immunoregulator, injected intramuscularly to methylcholanthrene (MC) induced tumour bearing mice at 30-100 micrograms per mouse weekly, significantly inhibited tumour growth, compared to that of the untreated control. Numbers of mononuclear cells (MNC) in the spleen obtained from Obi-1 treated and untreated mice were reduced to 64% and 10% of the normal levels, respectively. In histopathological findings, variations of MNC in the spleen was observed in colonies of large MNC and lymphocytes in Obi-1 treated red marrow, while untreated one showed only scattered number of small lymphocytes. A significant O2- production was noted in monolayers of normal macrophages incubated with 0.5 mg Obi-1/ml for 48 hr. Cytotoxicity of spleen MNC cultured with Obi-1 against MC-tumour, Meth A, P-815 and YAC-1 target cells showed less than 10% cytotoxic activity in almost all experiments.     

Generation of PDX‐1 mutant porcine blastocysts by introducing CRISPR/Cas9‐system into porcine zygotes via electroporation

Recently, we established the GEEP (“gene editing by electroporation of Cas9 protein”) method, in which the CRISPR/Cas9 system, consisting of a Cas9 protein and single guide RNA (sgRNA), is introduced into pig zygotes by electroporation and thus induces highly efficient targeted gene disruption. In this study, we examined the effects of sgRNA on the blastocyst formation of porcine embryos and evaluated their genome‐editing efficiency. To produce an animal model for diabetes, we targeted PDX‐1 (pancreas duodenum homeobox 1), a gene that is crucial for pancreas development during the fetal period and whose monoallelic disruption impairs insulin secretion. First, Cas9 protein with different sgRNAs that targeted distinct sites in the PDX‐1 exon 1 was introduced into in vitro‐fertilized zygotes by the GEEP method. Of the six sgRNAs tested, three sgRNAs (sgRNA1, 2, and 3) successfully modified PDX‐1 gene. The blastocyst formation rate of zygotes edited with sgRNA3 was significantly (p < 0.05) lower than that of control zygotes without the electroporation treatment. Our study indicates that the GEEP method can be successfully used to generate PDX‐1 mutant blastocysts, but the development and the efficiency of editing the genome of zygotes may be affected by the sgRNA used for CRISPR/Cas9 system.     

Fibrinonecrotic rhinitis caused by a concurrent infection of Fusobacterium necrophorum and Arcanobacterium pyogenes in a cow

An 8 year-old cow showing severe dyspnea and nasal mucosal necrosis immediately after parturition was subjected to pathological examination. The principal lesions were fibrinonecrotic rhinitis, necrotic bronchopneumonia and renal infarction. Fusobacterium necrophorum biotype A and Arcanobacterium pyogenes antigens were detected in the nasal and pulmonary lesions. These results suggest that the lesions were caused by a concurrent infection of the detected bacteria and that the pulmonary lesions were caused by the aspiration of infectious materials from the nasal ones. Mucosal coagulative necroses observed as the initial lesions in rhinitis were frequently associated with multiple thrombosis. The findings might suggest that thrombosis played an important role in the development of the nasal lesions.     

Lack of hepatocarcinogenicity of 2,2’-[1,2-ethanediylbis(oxymethylene)]bis-oxirane, 3-hydroxy-2-naphthoic acid,and acetoacetanilide in a medium-term rat liver bioassay

The carcinogenicity of 2,2’-[1,2-ethanediylbis(oxymethylene)]bis-oxirane (ethylene glycol diglycidyl ether; EGDE), 3-hydroxy-2-naphthoic acid (HNA), and acetoacetanilide (AAA) was investigated using a medium-term rat liver bioassay for an occupational safety assessment. F344 male rats were administered a single intraperitoneal injection of diethylnitrosamine (200 mg/kg body weight (bw)/day) and then starting 2 weeks later, they received EGDE at 6, 20, and 60 mg/kg bw/day, HNA at 20, 60, and 200 mg/kg bw/day, or AAA at 60, 200, and 600 mg/kg bw/day by oral gavage for 6 weeks. The animals in the positive control group received phenobarbital sodium solution (PB, 25 mg/kg bw/day) by oral gavage and those in the negative control group received a vehicle (water/corn oil) during the administration period of test substances in this model. All animals were subjected to two-thirds partial hepatectomy at week 3 and euthanized at week 8. Neither the number nor the area of hepatocellular foci positive for glutathione S-transferase placental form (GST-P) increased in any of the EGDE, HNA, or AAA treated groups. However, the number and area of GST-P-positive foci significantly increased in the positive control group treated with PB. The results indicate that EGDE, HNA, and AAA lack hepatocarcinogenicity in rats.     

Vitrification procedure decreases inositol 1,4,5‐trisphophate receptor expression,resulting in low fertility of pig oocytes

Although cryopreservation of mammalian oocytes is an important technology, it is well known that unfertilized oocytes, especially in pigs, are highly sensitive to low temperature and that cryopreserved oocytes show low fertility and developmental ability. The aim of the present study was to clarify why porcine in vitro matured (IVM) oocytes at the metaphase II (MII) stage showed low fertility and developmental ability after vitrification. In vitro matured cumulus oocyte complexes (COCs) were vitrified with Cryotop and then evaluated for fertility through in vitro fertilization (IVF). Although sperm‐penetrated oocytes were observed to some extent (30–40%), the rate of pronuclear formation was low (9%) and none of them progressed to the two‐cell stage. The results suggest that activation ability of cryopreserved oocytes was decreased by vitrification. We examined the localization and expression level of the type 1 inositol 1,4,5 trisphosphate receptor (IP₃R1), the channel responsible for Ca²⁺ release during IVF in porcine oocytes. Localization of IP₃R1 close to the plasma membrane and total expression level of IP₃R1 protein were both decreased by vitrification. In conclusion, our present study indicates that vitrified‐warmed porcine COCs showed a high survival rate but low fertility after IVF. This low fertility seems to be due to the decrease in IP₃R1 by the vitrification procedure.     

Generation of rats from vitrified oocytes with surrounding cumulus cells via in vitro fertilization with cryopreserved sperm

The objective of this study was to evaluate fertility and full‐term development of rat vitrified oocytes after in vitro fertilization (IVF) with cryopreserved sperm. Oocytes with or without surrounding cumulus cells were vitrified with 30% ethylene glycol + 0.5 mol/L sucrose + 20% fetal calf serum by using the Cryotop method. The warmed oocytes were co‐cultured with sperm. Although the denuded/vitrified oocytes were not fertilized, some of the oocytes vitrified with cumulus cells were fertilized (32.7%) after IVF with fresh sperm. When IVF was performed with cryopreserved sperm, vitrified or fresh oocytes with cumulus cells were fertilized (62.9% or 41.1%, respectively). In addition, to confirm the full‐term development of the vitrified oocytes with surrounding cumulus cells after IVF with cryopreserved sperm, 108 vitrified oocytes with two pronuclei (2PN) were transferred into eight pseudopregnant females, and eight pups were obtained from three recipients. The present work demonstrates that vitrified rat oocytes surrounded by cumulus cells can be fertilized in vitro with cryopreserved sperm, and that 2PN embryos derived from cryopreserved gametes can develop to term. To our knowledge, this is the first report of successful generation of rat offspring derived from vitrified oocytes that were fertilized in vitro with cryopreserved sperm.