Isolation and analysis of the human MEKA gene encoding a retina-specific protein

We have isolated and sequenced a human gene encoding photoreceptor cell-specific MEKA protein. The protein coding region was separated into three exons, which encoded 246 amino acid residues with a molecular weight of 28,311. The coding region of the human gene exhibited the homologies of 90.7% nucleotides and 88.5% amino acids with those of the bovine cDNA. Southern blot analysis revealed that the human MEKA gene has a single copy number. However, the anti-bovine MEKA stained both rod and cone photoreceptor cells in the human retina.     

Purification and characterization of two distinct Ca2+-activated proteinases from hearts of hypertensive rats

Two distinct Ca2+-activated proteinases were purified and characterized from hearts of hypertensive rats. Ca2+-activated proteinases I and II, having low and high Ca2+ requirements, respectively, were first separated by DEAE-cellulose chromatography. The enzymes were then purified individually by different column procedures: chromatography on phenyl-Sepharose, then Sephadex G-200 for proteinase I and reactive-red agarose for proteinase II. The apparent molecular weight of purified proteinase I was 125 000 and that for purified proteinase II was 110 000. Both enzymes are heterodimers made up of a larger catalytic subunit and a smaller subunit devoid of proteinase activity. Ca2+ concentrations for half-maximal activation were 5 microM for proteinase I and 200 microM for proteinase II. Both enzymes were inhibited by sulfhydryl-modifying agents, but exhibited different characteristics in the auto-digestion reaction in the presence of Ca2+. Proteinases I and II were also purified from hearts of normotensive rats and shown to be identical to their respective counterparts from hearts of hypertensive rats. However, proteinase II activity in hypertensive rat hearts was significantly elevated as compared to controls.     

Effects of monoclonal antibodies to bovine and Paracoccus denitrificans cytochromes c on reactions with oxidase, reductase and peroxidase

The effects of monoclonal antibodies to bovine and Paracoccus denitrificans cytochromes c (Kuo, L.M. and Davies, H.C. (1983) Mol. Immunol. 20, 827-838) in the reactions of the cytochromes c with cytochrome c oxidase, reductase and peroxidase were studied. Spectrophotometric assays were employed, under conditions where binding of cytochrome c to the enzymes appears to be rate-limiting. Less than stoichiometric amounts of antibodies to P. denitrificans cytochrome c added to the cytochrome rendered some of it nonoxidizable or nonreducible by the P. denitrificans membrane-bound electron transport system and decreased the rate constant with the remaining cytochrome c. The antibodies appear to affect both electron transport reactions (blocking effects) with the oxidase and reductase and binding effects (effects on rate constants) and to distinguish between the two. Different ratios of antibody site to cytochrome c gave different extents of blocking of the reductase as compared with the oxidase reaction. Differences were also apparent in the effect of these antibodies on the reaction of yeast peroxidase and the oxidase with the P. denitrificans cytochrome c. Antibodies to bovine and P. denitrificans cytochromes c had considerably less effect on the reactions of the bovine cytochrome with bovine oxidase and reductase. One antibody was inhibitory to the oxidase reaction with bovine cytochrome c, but not to that with the reductase. Also, an antibody which inhibited the oxidase reaction had no effect on the reaction with yeast peroxidase. The data give evidence that the interaction areas on cytochrome c for oxidase and reductase and peroxidase are not identical, although they may be nearby.     

Identification of a retina-specific MEKA protein as a 33 K protein

A photoreceptor-specific MEKA protein was purified from bovine retinal soluble fraction. The purified sample was eluted as a single peak of 74 kDa protein from a Superose column, which was dissolved into three components, MEKA protein (32 kDa), beta-(36 kDa) and gamma-(10 kDa) subunits of transducin on a SDS-PAGE. From several lines of evidence, we concluded that MEKA protein is identical with a 33k phosphoprotein reported by Lee et al (1).     

Clinical applications of fluorescence in situ hybridization

We review here the application of fluorescence in situ hybridization with chromosome-specific probes to chromosome classification and to detection of changes in chromosome number or structure associated with genetic disease. Information is presented on probe types that are available for disease detection. We discuss the application of these probes to detection of numerical aberrations important for prenatal diagnosis and to detection and characterization of numerical and structural aberrations in metaphase spreads and in interphase nuclei to facilitate tumor diagnosis.     

A dual-therapy approach for the treatment of biofilm-mediated Salmonella gallbladder carriage

Asymptomatic carriage of Salmonella Typhi continues to facilitate the transmission of typhoid fever, resulting in 14 million new infections and 136,000 fatalities each year. Asymptomatic chronic carriage of S. Typhi is facilitated by the formation of biofilms on gallstones that protect the bacteria from environmental insults and immune system clearance. Here, we identified two unique small molecules capable of both inhibiting Salmonella biofilm growth and disrupting pre-formed biofilm structures without affecting bacterial viability. In a mouse model of chronic gallbladder Salmonella carriage, treatment with either compound reduced bacterial burden in the gallbladder by 1–2 logs resulting in bacterial dissemination to peripheral organs that was associated with increased mortality. Co-administration of either compound with ciprofloxacin not only enhanced compound efficacy in the gallbladder by a further 1–1.5 logs for a total of 3–4.5 log reduction, but also prevented bacterial dissemination to peripheral organs. These data suggest a dual-therapy approach targeting both biofilm and planktonic populations can be further developed as a safe and efficient treatment of biofilm-mediated chronic S. Typhi infections.     

Thiazolidinediones and Risk of Long-Term Dialysis in Diabetic Patients with Advanced Chronic Kidney Disease: A Nationwide Cohort Study

Thiazolidinediones (TZDs) reduce urinary albumin excretion and proteinuria in diabetic nephropathy. The effect of TZDs on hard renal outcome in diabetic patients with chronic kidney disease (CKD) is unknown. We investigate the association of TZDs and risk of long-term dialysis or death in diabetic patients with advanced CKD. The nationwide population-based cohort study was conducted using Taiwan’s National Health Insurance Research Database. From January 2000 to June 2009, 12350 diabetic patients with advanced CKD (serum creatinine levels greater than 6 mg/dL but not yet receiving renal replacement therapy) were selected for the study. We used multivariable Cox regression models and a propensity score-based matching technique to estimate hazard ratios (HRs) for development of long-term dialysis and the composite outcome of long-term dialysis or death for TZD users (n=1224) as compared to nonusers (n=11126). During a median follow-up of 6 months, 8270 (67.0%) patients required long-term dialysis and 2593 (21.0%) patients died before starting long-term dialysis. Using propensity score matched analysis, we found TZD users were associated with a lower risk for long-term dialysis (HR, 0.80; 95% confidence interval [CI], 0.74-0.86) and the composite outcome of long-term dialysis or death (HR, 0.85; 95% CI, 0.80-0.91). The results were consistent across most patient subgroups. Use of TZDs among diabetic patients with advanced CKD was associated with lower risk for progression to end-stage renal disease necessitating long-term dialysis or death. Further randomized controlled studies are required to validate this association.