Establishing gene Amelogenin as sex-specific marker in yak by genomic approach.

Yak, an economically important bovine species considered as lifeline of the Himalaya. Indeed, this gigantic bovine is neglected because of the scientific intervention for its conservation as well as research documentation for a long time. Amelogenin is an essential protein for tooth enamel which eutherian mammals contain two copies in both X and Y chromosome each. In bovine, the deletion of a fragment of the nucleotide sequence in Y chromosome copy of exon 6 made Amelogenin an excellent sex-specific marker. Thus, an attempt was made to use the gene as an advanced molecular marker of sexing of the yak to improve breeding strategies and reproduction. The present study confirmed that the polymerase chain reaction amplification of the Amelogenin gene with a unique primer is useful in sex identification of the yak. The test is further refined with qPCR validation by quantifying the DNA copy number of the Amelogenin gene in male and female. We observed a high level of sequence polymorphisms of AMELX and AMELY in yak considered as novel identification. These tests can be further extended into several other specialized fields including forensics, meat production and processing, and quality control.

Characterization and in vitro expression of non-structural 1 protein of canine parvovirus (CPV-2) in mammalian cell line.

Parvoviruses are small, 260-A-diameter, icosahedral, non-enveloped, single-stranded DNA viruses with a genome of approximately 5 kb. Non structural protein, (NS-1) is especially relevant, being both essential for virus replication and the main factor responsible for virus pathogenicity and cytotoxicity. This protein has also been reported to possess the property of killing of transformed cells. The present study was carried out to clone, characterize and express the NS-1 gene of canine parvovirus. NS-1 complete CDS 2020bp was amplified, cloned into eukaryotic expression vector pcDNA 3.1(+), sequenced and characterized by in vitro expression analysis. Functional activity of recombinant construct, pcDNA.cpv.NS-1, was evaluated by RT-PCR and flow cytometry for the expression of NS-1 specific mRNA and NS-1 protein, respectively, in transfected HeLa cells. This recombinant plasmid may serve as an important tool to evaluate the apoptotic potential of NS-1 protein of canine parvovirus in cultured HeLa cells.

Structure-activity relationship studies of gonadotropin-releasing hormone antagonists containing S-aryl/alkyl norcysteines and their oxidized derivatives.

A series of acyline analogues incorporating l- and d-isomers of S-arylated/alkylated norcysteines [Ncy(R), where R is 2-naphthyl, methyl, and isopropyl] at positions 1, 4, 7, and 10 were synthesized. Some of these analogues were mono- and dioxidized to sulfoxides and sulfones. All of the analogues of acyline were screened for the antagonism of the GnRH-induced response in a reporter gene assay in HEK-293 cells expressing the human GnRH receptor. Nine of the analogues (9, 11, 15, 16, 17, 19, 20, 21, and 22) had antagonistic potency (IC50 < 2 nM) similar to that of acyline (IC50 = 0.52 nM) in this assay. Selected analogues (9, 11, 15, 16, 19, and 21) were tested in vitro for their antagonism at the rat GnRH-R in a reporter gene assay as well as in an in vivo intact male rat assay. Analogues 9 and 15 were the most potent in suppressing testosterone levels.

Novel analogues of degarelix incorporating hydroxy-, methoxy-, and pegylated-urea moieties at positions 3, 5, 6 and the N-terminus. Part III.

Novel degarelix (Fe200486) analogues were screened for antagonism of GnRH-induced response (IC(50)) in a reporter gene assay. Inhibition of luteinizing hormone release over time was measured in the castrated male rat. N(omega)-Hydroxy- and N(omega)-methoxy-carbamoylation of Dab and Dap at position 3 (3-6), and N(omega)-hydroxy-,N(omega)-methoxy-carbamoylation and pegylation of 4Aph at positions 5 and 6 (7-10, 15-17, 22-25) were carried out. Modulation of hydrophobicity was achieved using different acylating groups at the N-terminus (11-14, 18-21, 26-28). Analogues 8, 15-17, 22, and 23 were equipotent to acyline (IC(50) = 0.69 nM) and degarelix (IC(50) = 0.58 nM) in vitro. Analogues 7, 17, and 23 were shorter acting than acyline, when 9, 11, 13, 15, 16, and 22 were longer acting. Only 9 and 14 were inactive at releasing histamine. No analogue exhibited a duration of action comparable to that of degarelix. Analogues with shorter and longer retention times on HPLC (a measure of hydrophilicity) than degarelix were identified.

Iterative approach to the discovery of novel degarelix analogues: substitutions at positions 3, 7, and 8. Part II.

Degarelix (FE200486, Ac-d-2Nal(1)-d-4Cpa(2)-d-3Pal(3)-Ser(4)-4Aph(l-Hor)(5)-d-4Aph(Cbm)(6)-Leu(7)-ILys(8)-Pro(9)-d-Ala(10)-NH(2)) is a potent and very long acting antagonist of gonadotropin-releasing hormone (GnRH) after subcutaneous administration in mammals including humans. Analogues of degarelix were synthesized, characterized, and screened for the antagonism of GnRH-induced response in a reporter gene assay in HEK-293 cells expressing the human GnRH receptor. The duration of action was also determined in the castrated male rat assay to measure the extent (efficacy and duration of action) of inhibition of luteinizing hormone (LH) release. Structurally, this series of analogues has novel substitutions at positions 3, 7, and 8 and N(alpha)-methylation at positions 6, 7, and 8 in the structure of degarelix. These substitutions were designed to probe the spatial limitations of the receptor's cavity and to map the steric and ionic boundaries. Some functional groups were introduced that were hypothesized to influence the phamacokinetic properties of the analogues such as bioavailability, solubility, intra- or intermolecular hydrogen bond forming capacity, and ability to bind carrier proteins. Substitutions at positions 3 ([N(beta)-(2-pyridyl-methyl)d-Dap(3)]degarelix, IC(50) = 2.71 nM) (5), 7 ([Pra(7)]degarelix, IC(50) = 2.11 nM) (16), and 8 ([N(delta)-(IGly)Orn(8)]degarelix, IC(50) = 1.38 nM) (20) and N-methylation ([N(alpha)-methyl-Leu(7)]degarelix, IC(50) = 1.47 nM) (32) yielded analogues that were equipotent to degarelix (2) in vitro (IC(50) = 1.64 nM) but shorter acting in vivo. Out of the 33 novel analogues tested for the duration of action in this series, two analogues ([N(epsilon)-cyclohexyl-Lys(8)]degarelix, IC(50) = 1.50 nM) (23) and ([N(beta)-(IbetaAla)Dap(8)]degarelix, IC(50) = 1.98 nM) (26) had antagonist potencies and duration of action similar to that of azaline B {inhibited LH (>80%) release for >72 h after sc injection to castrated male rats at a standard dose of 50 mug/rat in 5% mannitol}. Under similar conditions analogues ([N(gamma)-(IGly)Dab(8)]degarelix, IC(50) = 1.56 nM) (21) and ([IOrn(8)]degarelix, IC(50) = 1.72 nM) (18) had a longer duration of action {inhibited LH (>96 h) release} than azaline B; however they were shorter acting than degarelix. Hydrophilicity of these analogues, a potential measure of their ability to be formulated for sustained release, was determined using RP-HPLC at neutral pH yielding analogues with shorter as well as longer retention times. No correlation was found between retention times and antagonist potency or duration of action.

Synthesis, in vivo and in vitro biological activity of novel azaline B analogs.

Several azaline B analogs (2-10) were synthesized and evaluated for their ability to antagonize GnRH in vitro and for duration of action in inhibiting luteinizing hormone secretion in a castrated male rat assay in vivo. Analogs, 8 (IC(50) = 1.85 nM), and 9 (IC(50) = 1.78 nM), are equipotent with azaline B (1, IC(50) = 1.36 nM) in vitro. Whereas 9 is short acting, 8 is as long acting as azaline B. Other analogs have IC(50) greater than 2.0 nM and are all short acting.

Novel gonadotropin-releasing hormone antagonists with substitutions at position 5.

Gonadotropin-releasing hormone (GnRH) antagonists with high potency and improved duration of action are needed for potential clinical applications. We synthesized four new antagonists (2-5) of GnRH homologues to Azaline B (1), with a common core sequence of [Aph(X)5, D-Aph(Cbm)6]Azaline B. In these analogs, (X) contains hydrophobic aromatic moieties (like homoveratoyl in 2, homovanillyl in 3, 2,5-dimethoxyphenylacetyl in 4, and 3,5-dimethoxyphenylacetyl in 5) designed to improve the duration of action over that of Azaline B. These analogs were tested in vitro for their ability to antagonize the GnRH receptor and in vivo for duration of action in a castrated male rat assay. Analogs 2, 4, and 5 were potent in vitro, but were found to be short acting in vivo. However, analog 3 [Aph(Hvn)5,D-Aph(Cbm)6]Azaline B is a potent human GnRH receptor antagonist in vitro (IC50 1.47 nM) and exhibits a longer duration of action than azaline B.

Six novel gonadotropin-releasing hormones are encoded as triplets on each of two genes in the protochordate, Ciona intestinalis.

GnRH is the key regulator of the reproductive axis in vertebrates, but little is known about GnRH before the origin of vertebrates. We have identified two genes encoding GnRH in a protochordate, Ciona intestinalis, thought to be related to the ancestral animal that gave rise to vertebrates. Each gene, Ci-gnrh1 and Ci-gnrh2, encodes in tandem three GnRH peptides, each of which is unique compared with known forms. Ci-gnrh1 encodes three peptides and contains no introns, whereas Ci-gnrh2 encodes three more peptides but has two introns. This is the first report in which more than one GnRH peptide is encoded on a single gene. The Ciona genes reveal consensus promoter elements that are conserved compared with human GNRH1. Both tunicate genes are expressed as mRNA early and throughout development, measured at the stages of four-cell, gastrulation, tail release, and tail resorption. In a closely related tunicate species, Ciona savignyi, we used in silico analysis to identify two similar genes encoding six peptides, only one of which is unique compared with C. intestinalis. Immunohistochemistry showed that at least one GnRH peptide was in the nerve net that surrounds the dorsal strand. Synthetic forms of the seven novel tunicate peptides induced release of gametes in adult tunicates. In contrast, the peptides did not activate the human GnRH-I receptor or cause release of LH in a rat pituitary cell assay. These data provide insight into the structural evolution of the GnRH peptides and their genes and show a functional role for GnRH in tunicate spawning.

CD8(+) T cell tolerance to a tumor-associated antigen is maintained at the level of expansion rather than effector function.

CD8+ T cell tolerance to self-proteins prevents autoimmunity but represents an obstacle to generating T cell responses to tumor-associated antigens. We have made a T cell receptor (TCR) transgenic mouse specific for a tumor antigen and crossed TCR-TG mice to transgenic mice expressing the tumor antigen in hepatocytes (gag-TG). TCRxgag mice showed no signs of autoimmunity despite persistence of high avidity transgenic CD8+ T cells in the periphery. Peripheral CD8+ T cells expressed phenotypic markers consistent with antigen encounter in vivo and had upregulated the antiapoptotic molecule Bcl-2. TCRxgag cells failed to proliferate in response to antigen but demonstrated cytolytic activity and the ability to produce interferon gamma. This split tolerance was accompanied by inhibition of Ca(2+) flux, ERK1/2, and Jun kinase phosphorylation, and a block in both interleukin 2 production and response to exogenous interleukin 2. The data suggest that proliferation and expression of specific effector functions characteristic of reactive cells are not necessarily linked in CD8+ T cell tolerance.