Heterospecific expression of misrepair-enhancing activity of mucAB in Escherichia coli and Bacillus subtilis.

Enterobacterial plasmid genes mucAB, which possess error-prone repair activity, were cloned and sequenced independently of a sequence previously determined (K.L. Perry, S.J. Elledge, B.B. Mitchell, L. Marsh, and G.C. Walker, Proc. Natl. Acad. Sci. USA 82:4331-4335, 1985). The survival- and mutation-enhancing activities of mucAB ligated to the MLSr promoter of a Bacillus subtilis plasmid in the shuttle vector pTE22R were expressed in B. subtilis as well as in Escherichia coli after mutagenic treatment. mucAB fragments with 5' deletions of various lengths up to the base sequence encoding Ala-26-Gly-27, the putative RecA-mediated cleavage site of the MucA protein, showed mutation-enhancing activity for noninducible lexA3 E. coli when ligated to the MLSr promoter in frame. This activity was lost by extending the deletion downstream. The formations of MucA and MucB proteins in B. subtilis and E. coli were demonstrated by Western blot (immunoblot) analysis. MucA cleavage in Rec+ B. subtilis was observed only after treatment with an alkylating agent and was not observed in RecA- and RecE- strains, whereas in E. coli cleavage was observed in Rec+ cells after treatment with either mitomycin C or an alkylating agent but was not detected in RecA- cells. Common activity of B. subtilis Rec and E. coli RecA in the induction of mutants is suggested.     

Discovery and SAR of a novel series of Natriuretic Peptide Receptor-A (NPR-A) agonists

Novel thienopyrimidine compounds 2 and 3 were discovered from high-throughput screening as Natriuretic Peptide Receptor A (NPR-A) agonists. Scaffold hopping of a thienopyrimidine ring to a quinazoline ring, introduction of the basic functional group and optimization of the substituent on the 6-position of the benzene ring of quinazoline led to improved agonistic activity. We discovered compound 48, which showed potent agonistic activity for NPR-A with an EC₅₀ value of 0.073 μM, indicating 350-fold potency compared to the hit compound 3.     

Zebrafish β-adrenergic receptor mRNA expression and control of pigmentation

Beta adrenergic receptors (β-ARs) are members of the G-protein-coupled receptor superfamily and mediate various physiological processes in many species. The expression patterns and functions of β-ARs in zebrafish are, however, largely unknown. We have identified zebrafish β-AR orthologs, which we have designated as adrb1, adrb2a, adrb2b, adrb3a and adrb3b. adrb1 was found to be expressed in the heart and brain. Expression of adrb2a predominated in the brain and skin, whereas adrb2b was found to be highly expressed in muscle, pancreas and liver. Both adrb3a and adrb3b were exclusively expressed in blood. Knock-down of these β-ARs by morpholino oligonucleotides revealed a functional importance of adrb2a in pigmentation. Expression of atp5a1 and atp5b, genes that encode subunits of F1F0-ATPase, which is known to be involved in pigmentation, was significantly increased by knock-down of adrb2a. Our data suggest that adrb2a may regulate pigmentation, partly by modulating F1F0-ATPase.     

Growth factor combination for chondrogenic induction from human mesenchymal stem cell

During the last decade, many strategies for cartilage engineering have been emerging. Stem cell induction is one of the possible approaches for cartilage engineering. The mesenchymal stem cells (MSCs) with their pluripotency and availability have been demonstrated to be an attractive cell source. It needs the stimulation with cell growth factors to make the multipluripotent MSCs differentiate into chondrogenic lineage. We have shown particular patterns of in vitro chondrogenesis induction on human bone marrow MSCs (hBMSCs) by cycling the growth factors. The pellet cultures of hBMSCs were prepared for chondrogenic induction. Growth factors: TGF-beta3, BMP-6, and IGF-1 were used in combination for cell induction. Gene expression, histology, immunohistology, and real-time PCR methods were measured on days 21 after cell induction. As shown by histology and immunohistology, the induced cells have shown the feature of chondrocytes in their morphology and extracellular matrix in both inducing patterns of combination and cycling induction. Moreover, the real-time PCR assay has shown the expression of gene markers of chondrogenesis, collagen type II and aggrecan. This study has demonstrated that cartilage tissue can be created from bone marrow mesenchymal stem cells. Interestingly, the combined growth factors TGF-beta3 and BMP-6 or TGF-beta3 and IGF-1 were more effective for chondrogenesis induction as shown by the real-time PCR assay. The combination of these growth factors may be the important key for in vitro chondrogenesis induction.     

Changes in the ovarian dynamics and endocrine profiles in goats treated with a progesterone antagonist during the early luteal phase of the estrous cycle

The aim of the present study was to determine the physiological role of endogenous progesterone in the regulation of ovarian dynamics, gonadotropin and progesterone secretion during the early luteal phase in the goat. Cycling Shiba goats received subcutaneously a vehicle (control group, n=5) or 50 mg of RU486 (RU486 group, n=4) daily from 1 to 7 days after ovulation (day 0) determined by transrectal ultrasonography. Ovarian dynamics were monitored by the ultrasonography and blood samples were collected daily until the subsequent ovulation for analysis of progesterone, luteinizing hormone (LH) and follicle stimulating hormone (FSH) secretion. Blood samples were also collected at 10 min intervals for 6 h on day 3 and day 7 for the analysis of pulsatile patterns of LH and FSH. The LH pulse frequency was significantly (P<0.05) higher in the RU486 group than in the control group on day 7 (4.8+/-1.1 pulses/6 h versus 1.2+/-0.4 pulses/6 h). The shape of the FSH pulses was unclear on day 3 and day 7 in both groups and the overall means of FSH concentration for 6 h on day 3 and day 7 were not significantly different between the RU486 and the control groups. The pattern of daily FSH concentrations showed a wave-like fluctuation in both groups. There was no significant difference in the inter-peak intervals of the wave-like pattern of daily FSH secretion between the RU486 and the control groups (4.1+/-0.6 days versus 4.5+/-0.6 days). The maximum diameter of the largest follicle that grew from day 1 to day 7 in the RU486 group tended to be greater than that in control goats (6.4+/-0.8 mm versus 5.0+/-0.8 mm, P=0.050), whereas no significant difference was detected in the size of the corpus luteum and progesterone concentrations between the control and RU486 groups on almost all days during the treatment period. These results indicate that the rise of the progesterone concentration suppresses the pulsatile LH secretion and follicular growth, whereas progesterone has no physiological role in the regulation of FSH secretion and luteal function during the early luteal phase of the estrous cycle in goats.     

Entrapment of microbial cells and organelles with hydrophilic urethane prepolymers

Summary Microbial cells and cellular organelles were immobilized by mixing aqueous suspensions of the biocatalysts with water-miscible urethane prepolymers. Thus immobilized preparations of acetone-dried cells of Arthrobacter simplex and thawed cells of Nocardia rhodocrous showed appreciable {ie351-1} activities in the transformation of hydrocortisone into prednisolone and 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione, respectively. The activities of catalase and alcohol oxidase were observed in the immobilized peroxisomes (microbodies) of a methanol-grown yeast Kloeckera sp. No. 2201. Yeast mitochondria entrapped with the prepolymer showed adenylate kinase activity. These results indicate the usefulness of the urethane prepolymers as convenient materials for entrapment of not only enzymes, but also organelles and microbial cells.     

Gene delivery to embryonic stem cells

Since the establishment of embryonic stem (ES) cells and the identification of tissue-specific stem cells, researchers have made great strides in the analysis of the natural biology of such stem cells for the development of therapeutic applications. Specifically, ES cells are capable of differentiating into all of the cell types that constitute the whole body. Thus, ES cell research promises new type of treatments and possible cures for a variety of debilitating diseases and injuries. The potential medical benefits obtained from stem cell technology are compelling and stem cell research sees a bright future. Control of the growth and differentiation of stem cells is a critical tool in the fields of regenerative medicine, tissue engineering, drug discovery, and toxicity testing. Toward such a goal, we present here an overview of gene delivery in ES cells, covering the following topics: significance of gene delivery in ES cells, stable versus transient gene delivery, cytotoxicity, suspension versus adherent cells, expertise, time, cost, viral vectors for gene transduction (lentiviruses, adenoviruses, and adeno-associated viruses, chemical methods for gene delivery, and mechanical or physical gene delivery methods (electroporation, nucleofection, microinjection, and nuclear transfer).