Abstract: The production of estradiol (E₂) by the ovary is vital to the development and maintenance of the female reproductive system.  E₂ synthesis is increased by follicle-stimulating hormone (FSH), which acts on G protein-coupled receptors in the ovary, increasing activity of a membrane-associated enzyme, adenylyl cyclase (AC).  Activated AC converts adenosine triphosphate (ATP) into adenosine 3’,5’ cyclic monophosphate (cyclic AMP), which activates signaling mechanisms that induce the expression of enzymes required for E₂ synthesis.  There are several isoforms of AC, and surprisingly little information is available regarding the AC isoforms expressed in the ovary.  Preliminary analysis of AC isoform mRNA levels suggests that ACIX may be a predominant isoform in the ovary.  The goal of this project is to examine the cell-specific regulation of ACIX protein levels in the rat ovary during follicle development, ovulation, and luteinization.  Immunoblot analysis will be used to detect the presence and regulated levels of ACIX protein in ovarian protein homogenates.  Immunocytochemistry will be used to determine the cellular localization and regulation of ACIX protein in the rat ovary. To begin these studies, brain tissue (positive control for ACIX expression) and ovarian tissue from immature, hormone-treated rats will be obtained.  Protein homogenates will be subjected to SDS-PAGE gel fractionation.  Protein will then be electrophoretically transferred to PVDF membranes, and immunoblot analysis of ACIX protein levels will be completed.  It is anticipated that immunoblot assessment with an ACIX antibody will reveal a predominant immunoreactive signal of about 161 kDa in rat brain and ovarian tissue, corresponding to the reported size of ACIX protein.  Subsequent immunocytochemical analysis will reveal the cell-specific expression and hormonal regulation of ACIX in the ovary.  The underlying hypothesis of these studies is that ACIX levels are increased by hormonal stimulation, contributing to hormone-induced cyclic AMP production.  Such findings will provide new information confirming the regulated expression of ACIX in the rat ovary, and provide the basis for determining potential roles of this important signaling molecule in ovarian functions.

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Construction of a cDNA of Sendai Virus, pRGV19 and Generation of a  Reverse Genetics Virus, RGV19 to Determine if M Gene Mutations in F1-R Play a Role in Systemic Infection

Wild-type Sendai virus causes a localized respiratory tract infection in mice, while a mutant, F1-R, causes a systemic infection (2).  Results from previous studies led us to hypothesize that mutations in the fusion (F) and matrix (M) genes are critical for the pantropic phenotype of F1-R.  To prove our hypothesis, a reverse genetics technique was used to generate viruses with various combinations of the mutations in the F and M genes of F1-R.  A reverse genetics virus that contains all six of the F1-R F mutations and both of the F1-R M mutations has recently been shown to cause a systemic infection in mice (1). Although we believe that the two mutations in M contribute to the pantropic phenotype of F1-R, we need to prove this by making a virus that has all of the F mutations and neither of the M mutations. Thus, the overall objective of the current study is to create another reverse genetics Sendai virus variant, RGV19, containing the six F mutations and neither of the M mutations of F1-R.  First, the RGV19 cDNA containing the desired mutations was generated by a 3-way ligation using two constructs from previous studies, pRS3Gg and pKS/Sev-19.  pRS3Gg contains the entire Sendai virus genome, and pKS/Sev-19 contains the desired six F mutations. Restriction digest analysis of a 3-way ligation product indicated that pRGV19 was successfully reconstructed.  Furthermore, sequencing of this construct revealed that the M gene contains the wild-type sequence and that the F gene contains the six F1-R F mutations. BSR-T7 cells were transfected with the pRGV19 construct and separate helper plasmids encoding the NP, P, and L proteins and infectious virions, RGV19, recovered. They will be used in a plaque assay and a multiple replication cycle assay in LLC-MK2 cells to determine whether the F protein of RGV19 has enhanced cleavability.  Mice will be infected and organs removed to identify presence of the virus and determine whether RGV19 has the ability to cause a systemic infection.

References

1.            Hou, X., Suquilanda, E., Zeledon, A., Kascinta, A., Moore, A., Seto, J.T., and McQueen, N.L.  2005.  Mutations in Sendai Virus Variant F1-R that Correlate with Plaque Formation in the Absence of Trypsin.  Medical Microbiology and Immunology.  194:  129-136.

2.            Ishida, N., and Homma, M.  1978.  Sendai Virus.  Adv. Virus Research.  23:  349-383.