Differential expression of LHRH-receptor in bovine nasal tissue and its role in deslorelin delivery

Deslorelin, a luteinizing hormone releasing hormone (LHRH) agonist, is transported via the LHRH-receptor (LHRH-R) and exhibits regional variation as follows: inferior turbinate posterior (ITP)>medium turbinate posterior (MTP)>medium turbinate anterior (MTA) of the bovine nasal mucosa. Differential LHRH-R expression in various regions of the nose is a potential explanation for regional variation in deslorelin transport. Thus, the objective was to determine whether LHRH-R expression exhibits regional variation in bovine nasal mucosa. LHRH-R density (B(max)) and affinity constant (K(d)) were determined by saturation experiments using 0.5mg tissue in the presence of increasing amounts of I(125)-deslorelin (100-100,000 cpm) at 4 degrees C for 4h. The 50% inhibitory concentration (IC(50)) was determined by competition experiments using various amounts of unlabelled deslorelin (0.01-3000 ng) at 4 degrees C for 4h. LHRH-R mRNA and protein expressions were determined using real-time PCR and Western blot analysis, respectively. LHRH-R B(max) and K(d) varied between the regions of excised bovine nasal mucosa: ITP>MTP>MTA. The inhibition experiments yielded two IC(50) concentrations which exhibited trends similar to B(max) and K(d). Real-time PCR and Western blot analysis indicated that LHRH-R expression exhibits similar trends: ITP>MTP>MTA. We identified two deslorelin binding sites in the nasal tissues, with high affinity sites representing approximately 60-70% of the total sites available. In summary, regional differences in nasal deslorelin transport correlate with regional differences in LHRH-R expression, with LHRH-R expression, peptide binding, and transport being the highest in the inferior turbinate posterior region of the nose.     

Potent and selective HIV-1 ribonuclease H inhibitors based on a 1-hydroxy-1,8-naphthyridin-2(1H)-one scaffold

Optimization studies using an HIV RNase H active site inhibitor containing a 1-hydroxy-1,8-naphthyridin-2(1H)-one core identified 4-position substituents that provided several potent and selective inhibitors. The best compound was potent and selective in biochemical assays (IC₅₀ = 0.045 μM, HIV RT RNase H; 13 μM, HIV RT-polymerase; 24 μM, HIV integrase) and showed antiviral efficacy in a single-cycle viral replication assay in P4-2 cells (IC₅₀ = 0.19 μM) with a modest window with respect to cytotoxicity (CC₅₀ = 3.3 μM).     

Discovery of potent sulfonamide P4-capped ketoamide second generation inhibitors of hepatitis C virus NS3 serine protease with favorable pharmacokinetic profiles in preclinical species

Hepatitis is a disease characterized by inflammation of the liver, usually producing swelling and, in many cases, permanent damage to liver tissues. Viral hepatitis C (HCV), a small (+)-RNA virus, infects chronically 3% of the world’s population. Boceprevir, SCH 503034, (1) our first generation HCV inhibitor, has already established proof-of- concept and is currently in late stage (phase III) clinical trials. In view of the positive data from our first generation compound, further work aimed at optimizing its overall profile was undertaken. Herein, we report that extension of our earlier inhibitor to the P₄ pocket by introducing a new sulfonamide moiety and optimization of the P1/P₁′ capping led to the discovery of a novel series of inhibitors of the HCV NS3 serine protease. Optimization of the P₁ residue significantly improved potency and selectivity. The combination of optimal moieties led to the discovery of compound 47 which, in addition to being a potent inhibitor of HCV subgenomic RNA replication, was also found to have good PK profile in rat, dog and monkey.     

Targeting efflux pumps—In vitro investigations with acridone derivatives and identification of a lead molecule for MDR modulation

To search multi drug resistance modulators, acridones carrying hydroxyl amine substituent at N-10 and COOH/Cl at C-4 were investigated for their interactions with the three components of efflux pump viz. P-gp, ATP, and Mg²⁺. Experimental and theoretical results indicated that the compounds with COOH group at C-4 interact with P-gp and Mg²⁺ while other set of compounds with Cl at C-4 interact with ATP and Mg²⁺. Spot assay and R6G influx/efflux assay of compound 3 using Candida albicans showed decrease in the fungal growth and efflux of R6G, respectively, in presence of compound 3 suggesting the suitability of this compound for MDR modulation.     

Variable domain I of nematode CLEs directs post-translational targeting of CLE peptides to the extracellular space

Effector proteins expressed in the esophageal gland cells of cyst nematodes are delivered into plant cells through a hollow, protrusible stylet. Although evidence indicates that effector proteins function in the cytoplasm of the syncytium,^1–3 technical constraints have made it difficult to directly determine where nematode effector proteins are initially delivered: cytoplasm, extracellular space, or both. Recently, we demonstrated that soybean cyst nematode CLE (HgCLE) propeptides are delivered to the cytoplasm of syncytial cells. Genetic and biochemical analyses indicate that the variable domain (VD) sequence is then required for targeting cytoplasmically delivered nematode CLEs to the apoplast where they function as ligand mimics of endogenous plant CLE peptides.⁴ The fact that nematode CLEs are targeted through the gland cell secretory pathway and delivered as mature propeptides into plant cells makes it impossible for these proteins to be subsequently delivered to the extracellular space via co-translational translocation through the endoplasmic reticulum (ER) secretory pathway of the host cell. However, when expressed in transgenic plants, if the mature nematode CLE propeptide harbored a functional cryptic signal peptide, it could possibly traffic to the apoplast through the ER secretory pathway by co-translational translocation. Here, we present evidence that VDI, the N-terminal sequence of the VD of HgCLE2,⁴ is sufficient for trafficking CLE peptides to the apoplast and that trafficking is indeed through an alternative pathway other than co-translational translocation.Key words: cyst nematode, effector, CLE, variable domain, trafficking, endoplasmic reticulum, co-translational translocation, post-translational     

Two-stage designs for gene-disease association studies with sample size constraints

Gene-disease association studies based on case-control designs may often be used to identify candidate polymorphisms (markers) conferring disease risk. If a large number of markers are studied, genotyping all markers on all samples is inefficient in resource utilization. Here, we propose an alternative two-stage method to identify disease-susceptibility markers. In the first stage all markers are evaluated on a fraction of the available subjects. The most promising markers are then evaluated on the remaining individuals in Stage 2. This approach can be cost effective since markers unlikely to be associated with the disease can be eliminated in the first stage. Using simulations we show that, when the markers are independent and when they are correlated, the two-stage approach provides a substantial reduction in the total number of marker evaluations for a minimal loss of power. The power of the two-stage approach is evaluated when a single marker is associated with the disease, and in the presence of multiple disease-susceptibility markers. As a general guideline, the simulations over a wide range of parametric configurations indicate that evaluating all the markers on 50% of the individuals in Stage 1 and evaluating the most promising 10% of the markers on the remaining individuals in Stage 2 provides near-optimal power while resulting in a 45% decrease in the total number of marker evaluations.     

Physicochemical and saccharide-binding studies on the galactose-specific seed lectin from Trichosanthes cucumerina

Physicochemical and saccharide-binding studies have been performed on Trichosanthes cucumerina seed lectin (TCSL). The agglutination activity of TCSL is highest in the pH range 8.0-11.0, whereas below pH 7.0 it decreases quite rapidly, which is consistent with the involvement of imidazole side chains of His residues, which titrate in this pH range, in the sugar-binding activity of the lectin. The lectin activity is unaffected between 0 and 60 degrees C, but a sharp decline occurs at higher temperatures. Isoelectric focusing studies show that TCSL has three isoforms with pI values of 5.3, 6.2, and 7.1, with the isoform of pI 6.2 being the most abundant. Circular dichroism spectroscopic studies reveal that TCSL contains about 28.4% beta-sheet, 10.6% beta-turns, 7% polyproline type 2 structure, with the remainder comprising unordered structure; the alpha-helix content is negligible. Binding of 4-methylumbelliferyl-beta-D-galactopyranoside (MeUmbbetaGal) to TCSL results in a significant increase in the fluorescence intensity of the ligand, and this change has been used to obtain the association constant for the interaction. At 25 degrees C, the association constant, K(a), for the TCSL-MeUmbbetaGal interaction was determined as 6.9 x 10(4)M(-1). Binding of nonfluorescent, inhibitory sugars was studied by monitoring their ability to reverse the fluorescence changes observed when MeUmbbetaGal was titrated with TCSL.     

Inhibition of NF-kappaB sensitizes A431 cells to epidermal growth factor-induced apoptosis,whereas its activation by ectopic expression of RelA confers resistance

Epidermal growth factor (EGF) is a well known mitogen, but it paradoxically induces apoptosis in cells that overexpress its receptor. We demonstrate for the first time that the EGF-induced apoptosis is accelerated if NF-kappaB is inactivated. To inactivate NF-kappaB, human epidermoid carcinoma cells (A431) that overexpress EGF receptor were stably transfected with an IkappaB-alpha double mutant construct. Under the NF-kappaB-inactivated condition, A431 cells were more sensitive to EGF with decreased cell viability and increased externalization of phosphatidylserine on the cell surface, DNA fragmentation, and activation of caspases (3 and 8 but not 9), typical features of apoptosis. These results were further supported by the potentiation of the growth inhibitory effects of EGF by chemical inhibitors of NF-kappaB (curcumin and sodium salicylate) and the protective role of RelA evidenced by the resistance of A431-RelA cells (stably transfected with RelA) to EGF-induced apoptosis. EGF treatment or ectopic expression of RelA in A431 cells induced DNA binding activity of NF-kappaB (p50 and RelA) and the expression of c-IAP1, a downstream target of NF-kappaB. A431-RelA cells exhibited spontaneous phosphorylation of Akt (a downstream target of phosphatidylinositol 3-kinase and regulator of NF-kappaB) and EGF treatment stimulated it further. Blocking this basal Akt phosphorylation with LY294002, an inhibitor of phosphatidylinositol 3-kinase, did not affect their viability but blocking of EGF-induced phosphorylation of Akt sensitized the otherwise resistant A431-RelA cells to EGF-mediated growth inhibition. Our results favor an anti-apoptotic role for NF-kappaB in the regulation of EGF-induced apoptosis.     

Novel route to the synthesis of peptides containing 2-amino-1'-hydroxymethyl ketones and their application as cathepsin K inhibitors

Cathepsin K is highly expressed in human osteoclasts, and is implicated in bone resorption. This makes it an attractive target for the treatment of osteoporosis. Peptides containing 2-amino-1'-hydroxymethyl ketones and 2-amino-1'-alkoxymethyl ketones were discovered as potent inhibitors of cathepsin K. A novel synthetic route was devised to facilitate rapid elucidation of the SAR of these inhibitors. The synthesis and SAR of hydroxymethyl ketones are presented.     

Posters. P1 A 2‐year audit of thyroid fine needle aspiration cytology

Introduction Fine needle aspiration (FNA) cytology of the thyroid is a well‐established test in the clinical work‐up of patients with solitary nodules of the thyroid. Thyroid FNA does however have limitations and audit of diagnostic performance is important. Methods The histopathology archives of the Royal Victoria Hospital were searched for all thyroid resections and the histopathological diagnosis was correlated with the pre‐operative cytological diagnosis, where available. Special emphasis was placed on the accuracy of tumour diagnosis. Results A total of 173 cases were identified during the 2‐year period, of these 93 had available pre‐operative FNA. A total of 57 tumours were identified. A small number (six of 57) of significant discrepancies were identified. These included a malignant lymphoma diagnosed as Hashimoto's thyroiditis, a metastasis which the FNA had suggested was a medullary carcinoma and an insular carcinoma diagnosed as medullary carcinoma on FNA. False positives included a colloid cyst diagnosed as suspicious of malignancy and a cytological diagnosis of papillary carcinoma not confirmed on histology. Discussion At present, the majority of thyroid FNAs in our clinics are performed by surgeons and material is not routinely available for immunocytochemistry. In spite of these limitations, there were few major discrepancies. These might be reduced if pathologist aspirators were able to perform FNAs and collect material for further studies, where necessary. This would allow identification of medullary carcinomas and malignant lymphomas. Conclusion FNA of thyroid lesions is a useful investigation in our clinical setting, however, some areas of potential for improvement have been identified.