Activity of the glycosylating enzyme, core 2 GlcNAc (beta1,6) transferase, is higher in polymorphonuclear leukocytes from diabetic patients compared with age-matched control subjects: relevance to capillary occlusion in diabetic retinopathy

The exact mechanism for capillary occlusion in diabetic retinopathy is still unclear, but increased leukocyte-endothelial cell adhesion has been implicated. We examined the possibility that posttranslational modification of surface O-glycans by increased activity of core 2 transferase (UDP-Glc:Galbeta1-3GalNAcalphaRbeta-N-acetylglucoaminyltr ansferase) is responsible for increased adhesion of leukocytes to vascular endothelium in diabetes. The mean activity of core 2 transferase in polymorphonuclear leukocytes isolated from type 1 and type 2 diabetic patients was higher compared with age-matched control subjects (1,638 +/- 91 [n = 42] vs. 249 +/- 35 pmol x h(-1) x mg(-1) protein [n = 24], P = 0.00013; 1,459 +/- 194 [n = 58] vs. 334 +/- 86 [n = 11], P = 0.01). As a group, diabetic patients with retinopathy had significantly higher mean activity of core 2 transferase compared with individuals with no retinopathy. There was a significant association between enzyme activity and severity of retinopathy in type 1 and type 2 diabetic patients. There was a strong correlation between activity of core 2 transferase and extent of leukocyte adhesion to cultured retinal capillary endothelial cells for diabetic patients but not for age-matched control subjects. Results from transfection experiments using human myelocytic cell line (U937) demonstrated a direct relationship between increased activity of core 2 transferase and increased binding to cultured endothelial cells. There was no relationship between activity of core 2 transferase and HbA(1c) (P = 0.8314), serum advanced glycation end product levels (P = 0.4159), age of the patient (P = 0.7896), and duration of diabetes (P = 0.3307). On the basis that branched O-glycans formed by the action of core 2 transferase participate in leukocyte adhesion, the present data suggest the involvement of this enzyme in increased leukocyte-endothelial cell adhesion and the pathogenesis of capillary occlusion in diabetic retinopathy.

Evaluation of a new commercial enzyme-linked immunosorbent assay for the detection of porcine antibodies against Trichinella spp

Trichinellosis is a zoonotic disease that is caused by the nematode Trichinella spp. Both European Union regulations and guidelines from the World Organization for Animal Health foresee the possibility of conducting serological surveillance for Trichinella spp. A newly developed commercial enzyme-linked immunosorbent assay (ELISA) was evaluated against 2 existing diagnostic techniques: an in-house ELISA and an in-house Western blot. A total of 875 Trichinella larva-negative samples of pigs and 93 Trichinella larva-positive samples of both naturally and experimentally infected pigs were included in the study. Bayesian modeling techniques were used to correct for the absence of a perfect reference test. The sensitivity and specificity of the commercial ELISA was 97.1-97.8% and 99.5-99.8%, respectively. Sensitivity analysis demonstrated high stability in the models. In a serological surveillance system, ELISA-positive samples should be tested by a confirmatory test. The Western blot is a suitable test for this purpose. With the use of the results of the models, the sensitivity and specificity of a test protocol in both ELISA and Western blot were 95.9% and 99.9%, respectively. The high sensitivity and specificity were achieved with a lower limit of detection than that of the routine artificial digestion test, suggesting that serological surveillance is a valuable alternative in surveillance for Trichinella spp. in pig production.

Second Annual Kansas State–Nebraska Biochemical Engineering Symposium (April 29, 1972)

This booklet contains abstracts of papers presented at a biochemical engineering symposium conducted at the University of Nebraska-Lincoln on April 29, 1972. This was the second annual symposium on this subject, the first having been held at Kansas State University on June 4, 1971. It is expected that future symposia will alternate between the two campuses. ContentsS.H. Lin, Kansas State University, "Enzyme Reaction in a Tubular Reactor with Laminar Flow" Gregory C. Martin, University of Nebraska, "Estimation of Parameters in Population Models for Schizosaccharomyces pombe from Chemostat Data" Jaiprakash S. Shastry and Prakash N. Mishra, Kansas State University, "Immobilized Enzymes: Analysis of Ultrafiltration Reactors" Mark D. Young, University of Nebraska, "Modelling Unsteady-State Two-Species Data Using Ramkrishna's Staling Model" G.C.Y. Chu, Kansas State University, "Optimization of Step Aeration Waste Treatment Systems Using EVOP" Shinji Goto, University of Nebraska, "Growth of the Blue-Green Alga Microcytis aeruginosa under Defined Conditions" Prakash N. Mishra and Thomas M.C. Kuo, Kansas State University, "Digital Computer Simulation of the Activated Sludge System: Effect of Primary Clarifier on System Performance" Mark D. Young, University of Nebraska, "Aerobic Fermentation of Paunch Liquor"

Proceedings of the Ninth Annual Biochemical Engineering Symposium

This report presents the proceedings of the Biochemical Engineering Symposium held at Kansas State University, April 28, 1979. Since a number of the contributions will be published in detail elsewhere, only brief reports of each contribution are included here. Requests for further information on work at Iowa State University should be directed to Dr. Peter J. Reilly; at Colorado State University to Drs. V. G. Murphy and A. R. Moreira, and at Kansas State University to Drs. L. T. Fan and L. E. Erickson. ContentProperties of a Homogeneous Xylobiohydrolase from Aspergillus niger, Mary M. Frederick, Iowa State University Kinetic Studies on the Enzymatic Hydrolysis of Cellulose–Absorption and Desorption of Cellulase onto Cellulose and the Behavior of Absorbed Cellulase, Yong-Hyun Lee and L. T. Fan, Kansas State University Properties of a Homogeneous Endo-Xylanase from Aspergillus niger, Ricardo Fournier A., Iowa State University Solid State Fermentation of Manure Fibers, D. C. Ulmer, Colorado State University Analysis and Consistency of Experimental Data for Microbial Growth on Renewable Resources, B. 0. Solomon, Kansas State University Biochemical Mechanisms of Enzyme Regulation, Frederick A. Blum, Colorado State University An Evaluation of Cellulose Pretreatments for Enzymatic Hydrolysis, David H. Beardmore, Kansas State University Use of Immobilized 8-Amylase/Glucoamylase Mixtures to Produce High Maltose Syrups, Carol G. Bohnenkamp, Iowa State University Effect of Viscosity on Bubble Behavior in an Airlift Fermentor, Vasanti Deshpande, Kansas State University

Angiotensin I-converting enzyme-gene-polymorphism: relationship to albumin excretion and blood pressure in pediatric patients with type-I-diabetes mellitus

The purpose of this study was the evaluation of a predictive genetic marker for nephropathy and hypertension in patients with type-I-diabetes mellitus (IDDM). The study was performed on 247 pediatric patients with IDDM. The mean age was 15.5 years (range 3.1-29.3), the mean duration of diabetes was 7.6 years (range 0.1-25.7). Age-related blood pressure and nocturnal albumin excretion rate were compared with the insertion/deletion-(I/D) polymorphism of the angiotensin-I converting enzyme gene. The genotype distribution did not differ significantly between IDDM patients (ID 48%, D 28%, I 24%) and the control group (ID 44%, D 37%, I 19%). Neither in the entire group, nor in patients with IDDM for more than 5 years, was a correlation found bet-ween allele distribution and albumin excretion rate. No correlation was found between genotype and blood pressure. When patients with a chronological age above 12 years were analysed separately, the genotype distribution between the groups with normal and elevated blood pressure showed no significant difference. The previously reported association of the I/D-polymorphism with nephropathy could not be confirmed in this study. The development of microalbuminuria, nephropathy and hypertension will be followed in our pediatric patients.

Dimerization of β-site β-amyloid precursor protein-cleaving enzyme

Cleavage of the beta-amyloid precursor protein (APP) by the aspartyl protease beta-site APP-cleaving enzyme (BACE) is the first step in the generation of the amyloid beta-peptide, which is deposited in the brain of Alzheimer's disease patients. Whereas the subsequent cleavage by gamma-secretase was shown to originate from the cooperation of a multicomponent complex, it is currently unknown whether in a cellular environment BACE is enzymatically active as a monomer or in concert with other proteins. Using blue native gel electrophoresis we found that endogenous and overexpressed BACE has a molecular mass of 140 kDa instead of the expected mass of 70 kDa under denaturing conditions. This suggests that under native conditions BACE exists as a homodimer. Homodimerization was confirmed by co-immunoprecipitation of full-length BACE carrying different epitope tags. In contrast, the soluble active BACE ectodomain was exclusively present as a monomer both under native and denaturing conditions. A domain analysis revealed that the BACE ectodomain dimerized as long as it was attached to the membrane, whereas the cytoplasmic domain and the transmembrane domain were dispensable for dimerization. By adding a KKXX-endoplasmic reticulum retention signal to BACE, we demonstrate that dimerization of BACE occurs already before full maturation and pro-peptide cleavage. Furthermore, kinetic analysis of the purified native BACE dimer revealed a higher affinity and turnover rate in comparison to the monomeric soluble BACE. Dimerization of BACE might, thus, facilitate binding and cleavage of physiological substrates.