Chirag A. Patel

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) receptor specific peptide analogues for PET imaging of breast cancer: In vitro/in vivo evaluation.

Vasoactive intestinal peptide and pituitary adenylate cyclase activating peptide have high affinity for VPAC1, VPAC2 and PAC1 receptors overexpressed on human cancer cells. Four potent analogues of these peptides, TP3939, TP3982, TP4200 and TP3805 were labeled with (64)Cu and evaluated ex vivo and in vivo to asses their biological activity and receptor specificity. The ultimate goal is to utilize (64)Cu analogues for positron emission tomography (PET) imaging of breast cancers in humans. Radiochemical purity of each analogue was >92%. The muscle relaxivity assay revealed IC(50) to be 5.3x10(-8) M, 4.4x10(-8) M, 8.1x10(-8) M, 8.1x10(-9) M and Kd values determined by receptor specific cell binding assays were 3.3 nM, 0.33 nM, 0.2 nM and 0.72 nM for TP3805, TP3939, TP3982, and TP4200 respectively. The receptor affinity, using human breast cancer tissues, was 10.93 times greater than normal breast tissues. RT-PCR confirmed increased VPAC1 receptor expression on human breast tumor cells over normal cells and corroborated with autoradiography data. The blood clearance was rapid and in vivo translocation of (64)Cu to plasma protein was <15%. Data demonstrate that these analogues are potent, have uncompromised biological activity and are worthy of further evaluation for accurate PET imaging of human breast cancers and in determining malignant and benign lesions.

RhoA Prenylation Inhibitor Produces Relaxation of Tonic Smooth Muscle of Internal Anal Sphincter

RhoA prenylation is a critical step for the translocation of RhoA to the membrane and its activation in response to agonist-induced sustained contraction of the smooth muscle. However, the effect and role of RhoA prenylation in the spontaneously tonic smooth muscle, such as internal anal sphincter (IAS), is not known. Present studies determined RhoA prenylation and its association with the basal tone in the IAS before and after the RhoA prenylation inhibitor, geranylgeranyl transferase inhibitor GGTI-297 [N-4-[2(R)-amino-3-mercaptopropyl]amino-2-naphthylbenzoyl-(l)-leucine,TFA]. Western blot analyses of cytosolic and membrane fractions determined the effects of RhoA prenylation inhibition on the cellular distribution of the RhoA. Additional studies were performed to determine the relationship between RhoA prenylation and Rho kinase (ROCK) activity. GGTI-297 decreased prenylation of RhoA, decreased ROCK activity, and caused a corresponding fall in the IAS tone. These inhibitory effects following RhoA prenylation blockade were demonstrated to be directly on the spontaneously contracted IAS smooth muscle cells. Western blot analysis revealed high levels of RhoA in the IAS smooth muscle cellular membrane in the basal state, and GGTI-297 shifted the RhoA localization to the cytosol. RhoA prenylation may play an important role in the translocation of RhoA to the smooth muscle cell membrane leading to its activation and for the maintenance of basal tone in the IAS.

Hinderin, a five-domains protein including coiled-coil motifs that binds to SMC3

BackgroundThe structural maintenance of chromosome proteins SMC1 and SMC3 play an important role in the maintenance of chromosomal integrity by preventing the premature separation of the sister chromatids at the onset of anaphase. The two proteins are constitutive components of the multimeric complex cohesin and form dimers by interacting at their central globular regions.ResultsIn order to identify proteins that by binding to SMC3 may interfere with the protein dimerization process, a human cDNA library was screened by the yeast two-hybrid system by using the hinge region of SMC3 as bait. This has lead to the identification of Hinderin, a novel five domains protein including two coiled-coil motifs and sharing a strikingly structural similarity to the SMC family of proteins. Hinderin is ubiquitously expressed in human tissues. Orthologue forms of the protein are present in other vertebrates but not in lower organisms. A mapping of the interaction sites revealed that the N- and C-terminal globular domains mediate the binding of Hinderin to SMC3. Hinderin/SMC3 complexes could be recovered by immunoprecipitation from cell lysates using an anti-SMC3 antibody, thus demonstrating that the two proteins interact in vivo. On the contrary, Hinderin did not interact with SMC1. In vivo the rate of SMC1/SMC3 interaction was decreased by the ectopic expression of Hinderin.ConclusionsHinderin is a novel binding partner of SMC3. Based on its ability to modulate SMC1/SMC3 interaction we postulate that Hinderin affects the availability of SMC3 to engage in the formation of multimeric protein complexes.