A novel array-based assay of in situ tissue transglutaminase activity in human umbilical vein endothelial cells

Transglutaminases (TGs), a family of calcium-dependent transamidating enzymes, are involved in functions such as apoptosis and inflammation and play a role in autoimmune diseases and neurodegenerative disorders. In this study, we describe a novel array-based approach to rapidly determine in situ TG activity in human umbilical vein endothelial cells and J82 human bladder carcinoma cells. Amine arrays were fabricated by immobilizing 3-aminopropyltrimethoxysilane on glass slides. The assay was specific and highly reproducible. The average coefficient of variation betweens spots was 2.6% (n = 3 arrays), and the average correlation coefficients between arrays and between arrays/reactions were 0.998 and 0.976, respectively (n = 3 arrays). The assay was successfully applied to detect changes in TG activity induced by maitotoxin and to analyze inhibition of the TG activation with cystamine and monodansyl cadaverine. In addition, the assay demonstrated that intracellular reactive oxygen species regulate the maitotoxin-induced activation of TG. Thus, the array-based in situ TG activity assay constitutes a rapid and high-throughput approach to investigating the roles of TGs in cell signaling.     

Regulatory role of GDP in the phosphoenzyme formation of guanine nucleotide: Specific forms of succinyl coenzyme a synthetase

We have previously shown that micromolar concentrations of GDP stimulate the GTP-mediated phosphorylation of p36, the subunit of succinyl-CoA synthetase (SCS), in lysates prepared fromDictyostelium discoideum. In this study, we report that this phenomenon represents an enhanced catalytic capacity of SCS to form the phosphoenzyme intermediate. Low concentrations of GDP stimulate phosphoenzyme formation by either GTP, or succinyl-CoA and P_i. Under these conditions GDP enhances the apparent rate of phosphoenzyme formation but does not significantly alter the fraction of phosphorylated enzyme. This effect is retained during purification of the protein and is also observed with purified pig heart SCS, indicating that GDP directly alters the enzyme to enhance its rate of phosphorylation. Under these conditions, GDP does not function at the catalytic site, implying an allosteric regulation of SCS.Abbreviations used SCS succinyl-CoA synthetase - P _i inorganic phosphate - NDP nucleotide diphosphate - NTP nucleotide triphosphate - PFK phosphofructokinase A-form; ADP-forming SCS; G-form; GDP-forming SCS     

Waist Circumference,Not Body Mass Index,Is Associated with Renal Function Decline in Korean Population: Hallym Aging Study

Background

Prospective investigation of obesity and renal function decline in Asia is sparse. We examined the associations of body mass index (BMI) and waist circumference (WC) with renal function decline in a prospective study of Korean population.

Methods

A total of 454 participants who had baseline estimated glomerular filtration rate (eGFR) levels of more than 60 mL/min/1.73 m² in Hallym Aging Study (HAS) were included and followed for 6 years. Renal function decline was defined as follows: (1) an eGFR decline ≥3 mL/min/1.73 m²/year (n = 82 cases); (2) an eGFR decrease of 20% or greater (n = 87 cases) at follow-up; (3) an eGFR decrease of 20% greater at follow-up or eGFR decline ≥3 mL/min/1.73 m²/year (n = 91 cases); and (4) an eGFR <60 mL/min/1.73 m² at follow-up (n = 54 cases). eGFR was determined based on the Modification of Diet in Renal Disease (MDRD) Study equation. Multivariate logistic regression model was used to determine the association between obesity and renal function decline.

Results

We found that central obesity was associated with faster renal function decline. Comparing WC of >95 cm in men or >90 cm in women with ≤90 cm in men or ≤85 cm in women, ORs (95% CIs) ranged from 2.31 (1.14–4.69) to 2.78 (1.19–6.50) for the 4 definitions of renal function decline (all p-values for trend <0.05). Waist-to-hip ratio (WHR) and waist-to-height ratio (WHtR) also was associated with renal function decline. There was no significant association of BMI with renal function decline.

Conclusions

Central obesity, but not BMI, is associated with faster renal function decline in Korean population. Our results provide important evidence that simple measurement of central fat deposition rather than BMI could predict decline in renal function in Korean population.     

Relationship between acrylamide concentration and enzymatic activity in an improved single fibrin zymogram gel system

Based on the zymography analysis, Bacillus sp. DJ-4 (screened from Doen-Jang, a Korean traditional fermented food) secretes seven extracellular fibrinolytic enzymes (EFEs; 68, 64, 55, 45, 33, 27, and 13 kDa) in culture broth. These seven EFEs were analyzed by newly applied SDSfibrin zymography combined with gradient polyacrylamide (SDS-FZGP). This improved gel system was used with a 5-20% acrylamide gradient in a fibrin zymogram gel for the separation of proteins with molecular masses from below 10 kDa to over 100 kDa on one gel plate. Using this system, high molecular weight bands (HMWBs) were clearly and sharply resolved. We also examined the relationship between an acrylamide concentration and the enzymatic activity of EFE using densitometric analysis.     

Free radical scavengers from the medicinal mushroom Inonotus xeranticus and their proposed biogenesis

New free radical scavengers, inoscavin D (1) and methylinoscavin D (2), were isolated from the methanolic extract of the fruiting bodies of Inonotus xeranticus (Hymenochaetaceae), along with the known compounds phelligridin D (3), 3,4-dihydroxybenzaldehyde (4), and 3,4-dihydroxybenzoic acid (5). Their structures were established by various spectroscopic analyses. Compounds 1 and 3 were proposed to be biosynthesized from the oxidative coupling of the precursor hispidin with 3,4-dihydroxybenzaldehyde and 3,4-dihydroxybenzoic acid, respectively. These compounds exhibited significant scavenging activity against the ABTS radical cation, and compounds 2 and 4 displayed moderate superoxide radical scavenging activity.