Rho GTPase Cdc42 Is a Direct Interacting Partner of Adenomatous Polyposis Coli Protein and Can Alter Its Cellular Localization

Adenomatous Polyposis Coli (APC) is a tumor suppressor gene product involved in colon cancer. APC is a large multidomain molecule of 2843 amino acid residues and connects cell-cell adhesion, the F-actin/microtubule cytoskeleton and the nucleus. Here we show that Cdc42 interacts directly with the first three armadillo repeats of APC by yeast two-hybrid screens. We confirm the Cdc42-APC interaction using pulldown assays in vitro and FRET assays in vivo. Interestingly, Cdc42 interacts with APC at leading edge sites where F-actin is enriched. In contrast, Cdc42 interacts with the truncated mutant APC1–1638 in cellular puncta associated with the golgi-lysozome pathway in transfected CHO cells. In HCT116 and SW480 cells, Cdc42 induces the relocalization of endogenous APC and the mutant APC1–1338 to the plasma membrane and cellular puncta, respectively. Taken together, these data indicate that the Cdc42-APC interaction induces localization of both APC and mutant APC and may thus play a direct role in the functions of these proteins

Metal ion mediated inhibition of firefly bioluminescence: A possibility via a quaternary complex

256-267D(-) Luciferin, interacts with different metal ions to produce colourless soluble salts with absorption spectra broader, intense and red shifted as compared to those of the parent compound. The equilibrium constants for the luciferin-metal ion system vary in the order, depository divalent transition metal ions > alkali metal ions. The equilibrium constants for the ternary complexes formed between metal ions and a mixture of lucifcrin and luciferase are larger than that of binary complexes but vary in the same order. Steady state fluorometric titration's of luciferin further confirmed its complexation with metal ions. The single absorption maximum of firefly luciferase at 278 nm originating from tyrosine was split into a doublet in presence of transition metal ions. The absorption maximum at lower wavelength is attributed to the H-bond raptured free tyrosine denatured conformation of the luciferase while the longer wavelength band to tyrosine- transition metal ion complex. Difference spectra of luciferase metal ion complex yielded change in the molar extinction coefficients from which the number of tyrosine molecules exposed to aqueous solution by the perturbant metal ions are evaluated following the Donovan model. The number of tyrosine molecules exposed to the aqueous medium as a result of conformational change in the enzyme are 4, 3, 3, 2 and 3 by Hg2+,Mn2+, Co2+, Cd2+ and Cs+ respectively. The denaturation constants calculated for the luciferase-metal ion complexes vary between 0.152 and 0.570 and follow the order of Hg2+> Cs+> Cd2+> Co2+> Mn2+. Steady state fluorescence data reveal that the metal ions quench the fluorescence of enzyme by complexation with the side chain residues of the excited state tyro sine. Profound change in the UV CD spectrum of luciferin and luciferase in presence of metal ions was attributed to the conformational change in the substrate and enzyme. Thus the inhibition of luciferase activity in the firefly bioluminescence by metal ions is attributed to the quaternary complex formed between metal ion-luciferin-luciferase and ATP near or around the active site of the enzyme

Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera

10.1021/ac0624546Analytical Chemistry79124463-4470ANCH

Study of interaction of hypericin and its pharmaceutical preparation by fluorescence techniques

10.1117/1.3067726Journal of Biomedical Optics141-JBOP

Rif-mDia1 interaction is involved in filopodium formation independent of Cdc42 and rac effectors

10.1074/jbc.M110.182683Journal of Biological Chemistry2861513681-13694JBCH

mDia1 and WAVE2 proteins interact directly with IRSp53 in filopodia and are involved in filopodium formation

10.1074/jbc.M111.305102Journal of Biological Chemistry28774702-4714JBCH

Risk factors for urinary tract infections in renal allograft recipients: Experience of a tertiary care center in Hyderabad, South India

Renal transplantation is an effective and commonly performed procedure for end-stage renal disease. Urinary tract infections are a major cause of morbidity and mortality in renal transplant patients. As data on postrenal transplant urinary tract infections from the Indian subcontinent are limited, the present study was conducted to estimate the burden of urinary tract infections in this vulnerable group of patients. This was a prospective study on patients undergoing renal transplantation in 2014 at our tertiary hospital in South India with a follow-up of 2 years to evaluate the risk factors for urinary tract infections. The prevalence of urinary tract infections was 41.9% with a male preponderance of 76.9%. Mean age of the 31 patients was 32.4 ± 10.2 years (range: 16–55 years). Gram-negative bacilli were the most common isolates with Escherichia coli being the predominant pathogen (53.3%). All the infections occurred within 1 year of transplantation with delayed graft function (P < 0.001; confidence interval [CI]: 29.0–96.3) and prolonged hospital stay (P = 0.0281; CI: 42.1–99.6) being the significant risk factors for acquiring urinary tract infections. Carbapenemase production was noted in 33.3% of isolates and all the Gram-negative organisms isolated in the 1st month of transplantation were carbapenem-resistant (CR) E. coli. The high rate of carbapenem-resistant organisms in the early posttransplant period is a point of concern, especially with cadaver transplants. Infection control practices and catheter care need to be strictly monitored to minimize the risk for UTI in the immediate posttransplant period