Immobilization of glucose oxidase on sepharose by UV-initiated graft copolymerization

The performance of a new method of enzyme immobilization based on photochemically initiated direct graft copolymerization was recently investigated. The immobilization reaction can be carried out in a simple way and by carefully selecting the reaction conditions, the enzyme-graft copolymer can be obtained as the main reaction product. Coupling efficiency of glucose oxidase has been found to depend only on the amount of photocatalyst (FeCl(3)) fixed on Sepharose used as polysaccharide support. Small quantities of glycidymethacrylate (GMA) (0.25 g/g dry Sepharose) are sufficient but necessary to achieve the best enzyme coupling efficiency (20-40%). Enzyme immobilization occurs very rapidly and the entire reaction occurs within 60 min. Reaction patterns and physicochemical characteristics of the obtained enzyme-graft copolymers exclude the glucose oxidase entrapment: therefore a covalent attachment mechanism may be proposed. The kinetic parameters of immobilized glucose oxidase (K(m)' = 2.0 x 10(-2)M) are quite similar to those of free enzyme (K(m) = 1.93 x 10(-2)M), and no diffusion limitation phenomena are evidenced in samples having different enzyme or polymer content. Lyophilization, thermostability, and long-term continuous operation also have been investigated. The advantages of this method over that using vinylenzyme copolymerization are discussed.     

Enzymatic preparation of 20 beta-hydroxysteroids in a two-phase system.

The behavior of 20 beta-hydroxysteroid dehydrogenase in a two-phase system consisting of an emulsion of water with an immiscible organic solvent was investigated. The effect of several organic solvents on the stability, activity, and kinetic profile of the enzyme was considered. The most suitable system for carrying out the enzymatic reaction proved to be water-butyl acetate. The production of high quantitied of 20 beta-hydroxysteroids in 100% yield using catalytic amount of cofactor was achieved by coupling the 20 beta-hydroxysteroid dehydrogenase-and the alcohol dehydrogenase-catalyzed reactions.     

Diffusion of carbon dioxide through lipid bilayer membranes. Effects of carbonic anhydrase, bicarbonate, and unstirred layers

Diffusion of (14)C-labeled CO(2) was measured through lipid bilayer membranes composed of egg lecithin and cholesterol (1:1 mol ratio) dissolved in n-decane. The results indicate that CO(2), but not HCO(3-), crosses the membrane and that different steps in the transport process are rate limiting under different conditions. In one series of experiments we studied one-way fluxes between identical solutions at constant pCO(2) but differing [HCO(3-)] and pH. In the absence of carbonic anhydrase (CA) the diffusion of CO(2) through the aqueous unstirred layers is rate limiting because the uncatalyzed hydration-dehydration of CO(2) is too slow to permit the high [HCO(3-)] to facilitate tracer diffusion through the unstirred layers. Addition of CA (ca. 1 mg/ml) to both bathing solutions causes a 10-100-fold stimulation of the CO(2) flux, which is proportional to [HCO(3-)] over the pH range 7-8. In the presence of CA the hydration- dehydration reaction is so fast that CO(2) transport across the entire system is rate limited by diffusion of HCO(3-) through unstirred layers. However, in the presence of CA when the ratio [HCO(3-) + CO(3=)]:[CO(2)] more than 1,000 (pH 9-10) the CO(2) flux reaches a maximum value. Under these conditions the diffusion of CO(2) through the membrane becomes rate limiting, which allows us to estimate a permeability coefficient of the membrane to CO(2) of 0.35 cm s(-1). In a second series of experiments we studied the effects of CA and buffer concentration on the net flux of CO(2). CA stimulates the net CO(2) flux in well buffered, but no in unbuffered, solutions. The buffer provides a proton source on the upstream side of the membrane and proton sink on the downstream side, thus allowing HCO(3-) to facilitate the net transport of CO(2) through the unstirred layers.     

Effects of deep freezing on the energy metabolism of bovine spermatozoa during in vitro capacitation: A cytochemical approach

In order to study the effects of deep freezing on the energy metabolism of bovine spermatozoa, a cytochemical quantitative study was carried out by a microdensitometric method on cytochrome oxidase and lactate dehydrogenase (LDH) activities. These were evaluated in situ on individual frozen-thawed bull spermatozoa collected at different times during in vitro capacitation. The results showed that in bull spermatozoa both the initiation of motility and capacity to fertilize eggs were associated with the anaerobic rather than aerobic glycolysis. The freezing-thawing processes and storage in liquid nitrogen induced a general enhancement of both the enzymatic activities examined. The high ionic strength treatment gave rise to a significant but reversible decrease in both the cytochrome oxidase and LDH activities in the fresh as well as in the frozen-stored sperm. The findings, based on cytochemical observations of energy metabolism of spermatozoa and evaluated during in vitro capacitation, suggest that the respiration and the anaerobic glycolysis of spermatozoa seem to be slightly impaired by the freezing-thawing and storage processes.     

Evaluation of newborn canine viability by means of umbilical vein lactate measurement, apgar score and uterine tocodynamometry

Newborn viability evaluation and early detection of fetal distress could contribute to reducing mortality at birth in canine species. High neonatal mortality rate in dogs is reported subsequent to complicated or uncomplicated whelping. Umbilical vein lactate and tocodynamometry could provide valuable clinical information to the obstetricians so that appropriate medical and surgical treatments or oxygen and warm administration can be properly and timely applied to mother and newborn pup. In humans, the fetal lactate level represents an objective indicator of fetal distress and a valid predictor of babies' survival. Fetal acidosis recognition by umbilical lactate (UL) measurement, APGAR score classification, and uterine activity monitoring during labour, can represent an advanced system in the evaluation of the canine newborn patient. The purpose of this study was to correlate UL levels with canine neonatal morbidity and mortality within 48 h of birth. We evaluated the relationship among neonatal parameters at birth (mucous membrane color, heart and respiratory rate, reflex irritability, mobility, suckling and vocalization, UL, weight, and temperature) with labour characteristics (uterine contractions recorded by the tocodynamometric system of Whelpwise™ Veterinary Perinatal Specialties®, delivery time, and pup presentation), in view to predict pup viability. We considered also vaginal parturition versus elective and emergency Caesarean section, and uterotonic drugs influence on delivery. Umbilical lactate concentration proved to be useful to predict canine neonatal mortality within 48 h of birth (P < 0.05). We identified 5 mmol/L of vein umbilical lactate concentration as the cut off value, allowing us to distinguish between healthy and distressed pups. Higher values of UL were related with distressed pups, whereas lower values characterized vigorous pups. Lactate concentrations lower than 5 mmol/L and APGAR scores higher than 9, related to mean delivery time of 105 min with effective uterine contractions (10 mm of Hg of strength or more, frequency from 4 to 12 contractions per hour, and 2-5 min in duration), should be considered good prognostic factors in canine labour and neonatology.     

Prevalence of methotrexate intolerance in rheumatoid arthritis and psoriatic arthritis

Introduction

The aim of this study was to determine the prevalence of gastrointestinal and behavioural symptoms occurring before (anticipatory/associative) and after methotrexate (MTX) administration, termed MTX intolerance, in rheumatoid (RA) and psoriatic arthritis (PsA).

Methods

Methotrexate Intolerance Severity Score (MISS), previously validated in juvenile idiopathic arthritis patients, was used to determine MTX intolerance prevalence in 291 RA/PsA patients. The MISS consisted of four domains: abdominal pain, nausea, vomiting and behavioural symptoms, occurring upon, prior to (anticipatory) and when thinking of MTX (associative). MTX intolerance was defined as ≥6 on the MISS with ≥1 point on anticipatory and/or associative and/or behavioural items.

Results

A total of 123 patients (42.3%) experienced at least one gastrointestinal adverse effect. The prevalence of MTX intolerance was 11%. MTX intolerance prevalence was higher in patients on parenteral (20.6%) than on oral MTX (6.2%) (p < 0.001).

Conclusion

Besides well-known gastrointestinal symptoms after MTX, RA and PsA patients experienced these symptoms also before MTX intake. RA and PsA patients on MTX should be closely monitored with the MISS for early detection of MTX intolerance, in order to intervene timely and avoid discontinuation of an effective treatment.     

A New Microarray Substrate for Ultra-Sensitive Genotyping of KRAS and BRAF Gene Variants in Colorectal Cancer

Molecular diagnostics of human cancers may increase accuracy in prognosis, facilitate the selection of the optimal therapeutic regimen, improve patient outcome, reduce costs of treatment and favour development of personalized approaches to patient care. Moreover sensitivity and specificity are fundamental characteristics of any diagnostic method. We developed a highly sensitive microarray for the detection of common KRAS and BRAF oncogenic mutations. In colorectal cancer, KRAS and BRAF mutations have been shown to identify a cluster of patients that does not respond to anti-EGFR therapies; the identification of these mutations is therefore clinically extremely important. To verify the technical characteristics of the microarray system for the correct identification of the KRAS mutational status at the two hotspot codons 12 and 13 and of the BRAF^V600E mutation in colorectal tumor, we selected 75 samples previously characterized by conventional and CO-amplification at Lower Denaturation temperature-PCR (COLD-PCR) followed by High Resolution Melting analysis and direct sequencing. Among these samples, 60 were collected during surgery and immediately steeped in RNAlater while the 15 remainders were formalin-fixed and paraffin-embedded (FFPE) tissues. The detection limit of the proposed method was different for the 7 KRAS mutations tested and for the V600E BRAF mutation. In particular, the microarray system has been able to detect a minimum of about 0.01% of mutated alleles in a background of wild-type DNA. A blind validation displayed complete concordance of results. The excellent agreement of the results showed that the new microarray substrate is highly specific in assigning the correct genotype without any enrichment strategy.