Developmental capacity of vitrified immature porcine oocytes following ICSI: effects of cytochalasin B and cryoprotectants

In the present study, effects of concentration and pretreatment time of cytochalasin B (CB), and of two types of cryoprotectant solutions on the nuclear maturation of vitrified-warmed porcine oocytes were examined. Also, the developmental capacity of vitrified immature porcine oocytes following intracytoplasmic sperm injection (ICSI) was investigated. The nuclear maturation rate (46.8%) of the vitrified-warmed oocytes treated with 7.5 microg/mL CB for 30 min was significantly higher (P < 0.05) than those (13.9-39.2%) of the vitrified-warmed oocytes treated with 0, 2.5, or 5.0 microg/mL CB for 10 or 30 min. Additionally, the nuclear maturation rate of oocytes treated with CB and vitrified in ethylene glycol (EG) (37.1%) was significantly higher (P < 0.05) than that of EG + dimethyl sulfoxide (Me(2)SO) (23.9%). However, no significant differences were observed in the cleavage and blastocyst development rates among the control (45.2 and 20.0%, respectively), the EG group (37.8 and 13.5%, respectively) and the EG + Me(2)SO group (39.3 and 14.3%, respectively). These results demonstrated that: (1) pretreatment with 7.5 microg/mL CB was beneficial for the vitrification of immature porcine oocytes; (2) the combination of EG and Me(2)SO as a cryoprotectant was not advantageous for in vitro maturation (IVM) of vitrified immature porcine oocytes; and (3) vitrified-warmed porcine oocytes matured after IVM, developed to the blastocyst stage without distinct differences compared to fresh oocytes following ICSI.     

The kinase inhibitor fasudil (HA-1077) reduces intimal hyperplasia through inhibiting migration and enhancing cell loss of vascular smooth muscle cells.

Smooth muscle cell (SMC) migration plays an important role in restenosis after angioplasty. Myosin phosphorylation is necessary for cell migration. Fasudil is an inhibitor of protein kinases, including myosin light chain kinase and Rho associated kinase, thereby inhibiting myosin phosphorylation, and it has been clinically used to prevent vasospasm following subarachnoid hemorrage. Based on these findings, we examined the anti-migrative action of fasudil. In SMC (SM-3), fasudil (1-100 microM) inhibited SMC migration in a dose-dependent manner (p < 0.001). Fasudil suppressed actin stress fiber formation dose dependently. In rabbit carotid artery, fasudil (10 mg/kg/day) markedly reduced intimal hyperplasia 14 days following balloon injury. Cell kinetic study showed that fasudil did not affect proliferation but enhanced cell loss in the media after injury. We concluded that fasudil reduced neointimal formation after balloon injury through both inhibiting migration and enhancing cell loss of medial SMC.     

Application of photo-crosslinkable resin prepolymers to entrap microbial cells. Effects of increased cell-entrapping gel hydrophobicity on the hydrocortisone Δ1-Dehydrogenation

Summary Acetone-dried cells of Arthrobacter simplex, whose steroid ¹ activity had been previously induced, were entrapped by the use of photo-crosslinkable resin prepolymers. When the hydrophobicity of the cell-entrapping gel was increased by mixing a hydrophobic prepolymer (main chain component; polypropyleneglycol) with a hydrophilic prepolymer (main chain component; polypropyleneglycol) with a hydrophilic prepolymer (main chain component; polyethyleneglycol) (up to 30%), the hydrocortisone to prednisolone conversion rate of the immobilized cells increased significantly, attaining approximately 20% of that of the free cells. A 10% addition of organic solvents, such as methanol, to the aqueous reaction mixture enhanced the solubility of the substrate greatly and to a lesser degree the reaction rate of the immobilized cells. The presence of an electron acceptor, phenazine methosulfate or 2,6-dichlorophenolindophenol, stimulated the steroid conversion of the entrapped as well as the free cells. The stability of the entrapped cells over repeated reactions was improved by immobilization.     

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by recombinant bacteria expressing the PHA synthase gene phaC1 from Pseudomonas sp. 61-3

Pseudomonas sp. 61-3 accumulated a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer consisting of 3-hydroxyalkanoate (3HA) units of 4–12 carbon atoms. The genes encoding β-ketothiolase (PhbA_Re) and NADPH-dependent acetoacetyl-CoA reductase (PhbB_Re) from Ralstoniaeutropha were expressed under the control of promoters for Pseudomonas sp. 61-3 pha locus or R. eutropha phb operon together with phaC1 _Ps gene (PHA synthase 1 gene) from Pseudomonas sp. 61-3 in PHA-negative mutants P. putida GPp104 and R. eutropha PHB⁻4 to produce copolyesters [P(3HB-co-3HA)] consisting of 3HB and medium-chain-length 3HA units of 6–12 carbon atoms. The introduction of the three genes into GPp104 strain conferred the ability to synthesize P(3HB-co-3HA) with relatively high 3HB compositions (up to 49 mol%) from gluconate and alkanoates, although 3HB units were not incorporated at all or at a very low fraction (3 mol%) into copolyesters by the strain carrying phaC1 _Ps gene only. In addition, recombinant strains of R. eutropha PHB⁻4 produced P(3HB-co-3HA) with higher 3HB fractions from alkanoates and plant oils than those from recombinant GPp104 strains. One of the recombinant strains, R. eutropha PHB⁻4/pJKSc46-pha, in which all the genes introduced were expressed under the control of the native promoter for Pseudomonas sp. 61-3 pha locus, accumulated P(3HB-co-3HA) copolyester with a very high 3HB fraction (85 mol%) from palm oil. The nuclear magnetic resonance analyses showed that the copolyesters obtained here were random copolymers of 3HB and 3HA units. Received: 12 July 1999 / Received revision: 1 October 1999 / Accepted: 2 October 1999     

Hearing dysfunction in heterozygous MitfMi‐wh/+ mice,a model for Waardenburg syndrome type 2 and Tietz syndrome

The human deafness‐pigmentation syndromes, Waardenburg syndrome (WS) type 2a, and Tietz syndrome are characterized by profound deafness but only partial cutaneous pigmentary abnormalities. Both syndromes are caused by mutations in MITF. To illuminate differences between cutaneous and otic melanocytes in these syndromes, their development and survival in heterozygous Microphthalmia‐White (Mitf^Mi‐wh/+) mice were studied and hearing function of these mice characterized. Mitf^Mi‐wh/+ mice have a profound hearing deficit, characterized by elevated auditory brainstem response thresholds, reduced distortion product otoacoustic emissions, absent endocochlear potential, loss of outer hair cells, and stria vascularis abnormalities. Mitf^Mi‐wh/+ embryos have fewer melanoblasts during embryonic development than their wild‐type littermates. Although cochlear melanocytes are present at birth, they disappear from the Mitf^Mi‐wh/+ cochlea between P1 and P7. These findings may provide insight into the mechanism of melanocyte and hearing loss in human deafness‐pigmentation syndromes such as WS and Tietz syndrome and illustrate differences between otic and follicular melanocytes.     

A New Isoflavone with Antifungal Activity from Immature Fruits of Lupinus luteus

A new isoflavone having antifungal activity was isolated from immature fruits of Lupinus luteus (Leguminosae), and named luteone. The structure was shown to be 5,7,2′,4′-tetrahy-droxy-6-(3,3-dimethylallyl)-isoflavone by degradative and spectroscopic studies.     

RELATIONSHIPS AMONG MORPHOLOGICAL STATUS, STEROID HORMONES, AND POST-THAWING VIABILITY OF FROZEN SPERMATOZOA OF MALE MINKE WHALES (BALAENOPTERA ACUTOROSTRATA)

Spermatozoa from 21 mature minke whales ( Balaenoptera acutorostrata ) taken in the Antarctic Ocean for Japanese research were recovered from vasa deferentia, diluted 1:9 in a Tris-based diluent, and frozen at - 80°C on board the vessel. After a period ranging from 45 to 125 d, the samples were transferred to liquid nitrogen and transported to the laboratory. After thawing at 37°C the motility (percentage of motile spermatozoa), vitality (proportion of live spermatozoa), and sperm concentration were determined for each sample. These values were tested for correlations with morphological measurements (body size, body weight, testis weight) and serum concentrations of progesterone (Pd), estradiol-17β (E2), and testosterone (T). Ten of 21 samples had motile spermatozoa (2%-40%). Although no motile spermatozoa were observed in 1.1 samples, all sperm samples were examined by eosinnigrosin staining and showed vitality levels of 3%44%. It was found that the motility (Y = 0.54) and vitality (r = 0.53) of the spermatozoa were significantly (P < 0.01) correlated with the E₂ levels (8.50 ± 1.80 pg/ml). Serum T levels (0.07 ± 0.02 ngml) were significantly correlated with the E₂ levels (r = 0.58, P < 0.01>, but sperm concentrations were not correlated with either Ea or T levels. The present study demonstrates that spermatozoa of minke whales can be successfully cryopreserved.     

PMab-219: A monoclonal antibody for the immunohistochemical analysis of horse podoplanin

Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, and bovine podoplanin (PDPN), a lymphatic endothelial cell marker, have been established in our previous studies. However, mAbs against horse PDPN (horPDPN), which are useful for immunohistochemical analysis, remain to be developed. In the present study, mice were immunized with horPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/horPDPN), and hybridomas producing mAbs against horPDPN were screened using flow cytometry. One of the mAbs, PMab-219 (IgG_2a, kappa), specifically detected CHO/horPDPN cells via flow cytometry and recognized horPDPN protein using Western blotting. Furthermore, PMab-219 strongly stained CHO/horPDPN via immunohistochemistry. These findings suggest that PMab-219 is useful for investigating the function of horPDPN.     

High-level secretory production of phospholipase A1 by Saccharomyces cerevisiae and Aspergillus oryzae

Phospholipase A1 (PLA1) is a hydrolytic enzyme that catalyzes the removal of the acyl group from position 1 of lecithin to form lysolecithin. The PLA1 gene, which had been cloned from Aspergillus oryzae, was expressed in Saccharomyces cerevisiae and A. oryzae. Through the modification of the medium composition and the feeding conditions of substrate, the production level of PLA1 by S. cerevisiae was increased to a level fivefold higher than that indicated in a previous report. In the case of A. oryzae, introduction of multicopies of PLA1 expression units, and the morphological change from the pellet form to the filamentous form were effective for the enhancement of PLA1 production. We succeeded in producing 3,500 U/ml of PLA1 using an industrial-scale fermentor.