A novel process for mutation detection using uracil DNA-glycosylase

A novel process is presented for the detection of known mutations and polymorphisms in DNA. This process, termed glycosylase mediated polymorphism detection (GMPD) involves amplification of the target DNA using three normal dNTPs and a fourth modified dNTP, whose base is a substrate for a specific DNA-glycosylase once incorporated into the DNA. The work described here utilises uracil DNA-glycosylase as the specific glycosylase and dUTP as the modified dNTP. Primers are designed so that during extension, the position of the first uracil incorporated into the extended primers differs depending on whether a mutation is present or absent. Subsequent glycosylase excision of the uracil residues followed by cleavage of the apyrimidinic sites allows detection of the mutation in the amplified fragment as a fragment length polymorphism. Variation in the sizes of the fragment length polymorphisms generated, can be readily achieved through the use of inosine bases in place of adenine bases in the upper and/or lower primers. The GMPD process is also adaptable to solid phase analysis. The use of the process for detection of mutations in the RYR1 and CFTR genes is demonstrated. Overall, the simplicity, specificity, versatility and flexibility of the GMPD process make it an attractive candidate for both small and large scale application in mutation detection and genome analysis.     

Pharmacokinetics and pharmacodynamics of cisatracurium after a short infusion in patients under propofol anesthesia

Fourteen patients, ASA physical status I or II, were recruited to assess the pharmacokinetic-pharmacodynamic relationship of cisatracurium under nitrous oxide/sufentanil/propofol anesthesia. The electromyographic response of the abductor digiti minimi muscle was recorded on train-of-four stimulation of the ulnar nerve. A 0.1-mg/kg dose of cisatracurium was given as an infusion over 5 min. Arterial plasma concentrations of cisatracurium and its major metabolites were measured by using high-performance liquid chromatography. A nontraditional two-compartment pharmacokinetic model with elimination from central and peripheral compartments was used. The elimination rate constant from the peripheral compartment was fixed to the in vitro rate of degradation of cisatracurium in human plasma (0.0237 min(-1)). The mean terminal half-life of cisatracurium was 23.9+/-3.3 min, and its total clearance averaged 3.7+/-0.8 mL x min(-1) x kg(-1). Using this model, the volume of distribution at steady state was significantly increased compared with that obtained when central elimination only was assumed (0.118+/-0.027 vs 0.089+/-0.017 L/kg). The effect-plasma equilibration rate constant was 0.054+/-0.013 min(-1). The 50% effective concentration (153+/-33 ng/mL) was 56% higher than that reported in patients anesthetized with volatile anesthetics, which suggests that, compared with inhaled anesthetics, a cisatracurium neuromuscular block is less enhanced by propofol. IMPLICATIONS: The drug concentration-effect relationship of the muscle relaxant cisatracurium has been characterized under balanced and isoflurane anesthesia. Because propofol is now widely used as an IV anesthetic, it is important to characterize the biological fate and the concentration-effect relationship of cisatracurium under propofol anesthesia as well.     

Experimental study of therapeutical properties and safety of king crab collagenase-containing ointment

The effects of ointment containing king crab (Paralithodes camtschatica) collagenase on intact skin, thermal, and pyonecrotic wounds were studied in rats by using hematological, biochemical, immunological, and morphological methods. The ointment for the skin and viscera was shown to be safe. It is highly effective in debriding the infected wounds. Different concentrations of collagenase were tested. The concentration of collagenase was recommended to be 0.2 mg/g ointment for use.     

Effects of carbontetrachloride and chenodeoxycholic acid on hepatic monooxygenase system, lipid peroxidation and some immunologic parameters in rats

BACKGROUND: Nutritional characteristics of the mediterranean diet, with a high intake of complex carbohydrates, fibre, monounsatured fatty acids and vegetables, are related to a lower prevalence of some nutritional associated diseases. The aim of our study was to perform a longitudinal analysis of the evolution of food intake in a mediterranean population in order to observe its influence on the energy and nutritional intake and their balance. The latter could have some effects on health status. METHODS: Dietary intake was evaluated using the 24 hours recall method in a representative sample (n = 941, age range = 10-69) of a Reus population. This longitudinal study consisted of 70% of the samples studied in 1983 using identical methodology. RESULTS: During this decade (1983-1993), energy intake decreased significantly 180 kcal/day for men and 158 kcal/day for women, carbohydrates being the main cause for this drop (132 and 84 kcal/day less for men and women, respectively). Protein intake decreased significantly in both sexes, 5.6% for men and 8.0% for women. However, the evolution of fat intake was different for men (no changes) and for women (a significant decrease of 5.7%). Saturated and monounsatured fatty acids did not show significant changes in this decade. Cholesterol intake decreased significantly in both sexes. Energy percents obtained from lipids, saturated and monounsaturated fatty acids significantly increased. However, in absolute values very little changes in fat intake in both sexes were observed. CONCLUSIONS: The dietary pattern evolved to a lower energy intake with an increment of the percentage of dietary lipids, but this feature was did not reflect a greater fat intake in absolute values. Moreover, the main characteristics of the typical mediterranean diet (which is basically different to the usual diet of other non mediterranean european countries mainly due to its richness in monounsaturated fatty acids) did not change in the period analyzed.     

The time window of apoptosis: a new component in the therapeutic strategy for cardiovascular remodeling

APOPTOSIS AND EXPERIMENTAL HYPERTROPHY: Apoptosis (programmed cell death) is a physiological counterpart of cell replication with shared as well as specific pathways. Our initial studies have demonstrated increased apoptosis in the heart, kidney and brain of spontaneously hypertensive rats (SHR) and mice, persisting in cultured vascular smooth muscle cells. In these target organs of hypertension, programmed cell death paralleled the well known hypertrophy/hyperplasia. We also observed that the two processes can be dissociated in time, as in experimental hypertrophy of the heart induced by pressure overload. In this context, only a short-lived apoptotic window precedes the overt development of cardiac hypertrophy. EFFECTS IN HYPERTENSION: We now propose that a more prolonged apoptotic window is present in hypertension. Apoptosis seems to be significantly reduced during the first weeks of life in SHR, possibly contributing to the early cardiac hyperplasia. However, increased apoptosis is clearly evident thereafter throughout the development of hypertension and fades below the levels observed in normotensive animals after the age of 24 weeks. ANTIHYPERTENSIVE THERAPY AND APOPTOSIS: In addition to the apoptotic windows that suggest a dynamic regulation of this process in disease states, antihypertensive therapy with angiotensin converting enzyme inhibitors, angiotensin II receptor antagonists and calcium channel blockers can also modify the contribution of apoptosis, independently of the blood pressure fall. We propose that the presence of apoptotic windows and the involvement of this biological process as a primary or secondary event in cardiovascular remodeling should be taken into account when designing innovative therapeutic approaches.     

Progress on development of the Nimbus-University of Pittsburgh axial flow left ventricular assist system

Nimbus Inc. (Rancho Cordova, CA) and the University of Pittsburgh have completed the second year of development of a totally implanted axial flow blood pump under the National Institutes of Health Innovative Ventricular Assist System Program. The focus this year has been on completing pump hydraulic development and addressing the development of the other key system components. Having demonstrated satisfactory pump hydraulic and biocompatibility performance, pump development has focused on design features that improve pump manufacturability. A controller featuring full redundancy has been designed and is in the breadboard test phase. Initial printed circuit layout of this circuit has shown it to be appropriately sized at 5 x 6 cm to be compatible with implantation. A completely implantable system requires the use of a transcutaneous energy transformer system (TETS) and a diagnostic telemetry system. The TETS power circuitry has been redesigned incorporating an improved, more reliable operating topography. A telemetry circuit is undergoing characterization testing. Closed loop speed control algorithms are being tested in vitro and in vivo with good success. Eleven in vivo tests were conducted with durations from 1 to 195 days. Endurance pumps have passed the 6 month interval with minimal bearing wear. All aspects of the program continue to function under formal quality assurance.     

Use of photo-anchoring of DNA probes for fluorescent in situ hybridization

A possibility was investigated to use photo-crosslinking DNA probes for fluorescent in situ hybridization (FISH). DNA probes were modified by incorporating photonucleotides in these, containing a photoreactive group (tetrafluorobenzazid) and capable of making covalent bonds with the examined DNA, when irradiated in 300-330 nm region. The photonucleotide was incorporated into the probe either by nick-translation, or upon elongation of the hybridized probe by the Kljonow fragment. It has been shown that the DNA probe, cross-linking to a chromosome as a result of covalent bonds, is not removed from the place of hybridization under consequent denaturating washing, which makes it possible to carry out the following DNA hybridization with selective conservation of signals obtained due to previous hybridization. This peculiarity of photo-linking DNA probes makes it possible to use them for the two-step DNA hybridization. To demonstrate this, preparations of human chromosomes were investigated. On the first step, chromosomal DNA was hybridized by means of DNA probe having nucleotide sequences of centromeric regions of chromosomes 13 and 21, the probe being linked to chromosomal DNA by the photonucleotide. Following the denaturation treatment of the preparation, and after the second chromosomal DNA hybridization with cosmid DNA, containing chromosome 13 DNA nucleotide sequence, the signal in chromosome 13 centromeric region was retained to serve a marker of this chromosome, thus fascilitating its easier identification following the hybridization of its DNA with cosmic DNA. The denaturation stability of photo-crosslinking probes opens some new possibilities in technology of DNA in situ hybridization.