Mutation scanning of the p53 tumor suppressor gene in renal and liver transplant patients in Hungary.

INTRODUCTION: The increased incidence of malignancies among transplanted patients is well known. Abnormal function of the p53 tumor suppressor gene has been reported in more than half of all tumors. The aim of our study was to detect point mutations of p53 gene in transplanted patients because the presence of mutations may be a predictive factor for tumor development. An earlier diagnosis can help to develop new strategies for immunosuppressive therapies. METHODS: Three point mutations were chosen based on the literature: exon5-codon175, exon7-codon248, exon8-codon273. Genomic DNA from the plasma of 60 liver, 362 renal transplants, and 45 nontransplanted patients with different tumors and 20 suspected healthy patients were analyzed with a real-time PCR method using the Roche LightCycler. The mutations were evaluated by melting curve analysis. RESULTS: We elaborated a special protocol for scanning the above mentioned p53 point mutations, which were proved by sequencing as well. Among 487 patients, 486 showed a wild-type genotype. The only patient carrying a mutation at codon 273 (heterozygous) was a liver transplant patient, who developed pancreas carcinoma and had already died. CONCLUSION: Our data suggest that mutations of the targeted codons in leukocyte DNA seem to be rare, but a mutation could be lethal. The evaluated three point mutations of p53 gene were not predictive for tumor development.

Prenatal diagnosis of steroid 21-hydroxylase deficiency by allele-specific amplification.

OBJECTIVE: Congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency (21-OHD) is the most common cause of ambiguous genitalia in females at birth. Here, we report the first prenatal diagnosis of 21-OHD by DNA analysis in Hungary. METHODS: Allele-specific amplification (ASA) of the DNA obtained by chorionic villus sampling was performed. RESULTS: The fetus had a homozygous nonsense mutation (Gln318Stop), suggesting a salt-wasting phenotype. Dexamethasone treatment of the mother was started on the 8th gestational week and, as the fetus was an affected female, it was continued until term. The newborn had normal external genitalia at birth, and severe salt-wasting crisis and postnatal virilization was prevented by mineralo- and glucocorticoid replacement therapy. CONCLUSION: 21-OHD was genotyped by ASA, and virilization of the fetus was prevented by antenatal dexamethasone therapy.

Ultra-thin-layer agarose gel electrophoresis II. Separation of DNA fragments on composite agarose-linear polymer matrices.

The effect of hydrophilic linear polymer additives (non-cross-linked polyacrylamide, hydroxyethyl cellulose and polyethylene oxide) on the migration behavior of double stranded DNA molecules, ranging from 200-1000 base pairs, were studied in ultra-thin-layer agarose gel electrophoresis. The detection sensitivity was found to be less than 0.1 ng/band using To-Pro-3 fluorophore labeling and fiber optic bundle-based scanning detection system with a 640 nm red diode laser. Among the various polymers investigated, addition of linear polyacrylamide resulted in the best separation performance (steepest Ferguson plots), while composite gels with hydroxyethylcellulose and polyethylene oxide still exhibited adequate resolving power. Using the composite matrices of 1% agarose-linear polyacrylamide (0.5-3%), 1% agarose-hydroxyethylcellulose (0.2-1%) and 1% agarose-polyethylene oxide (0.2-1%), the mechanism of the separation was found to be in the Ogston sieving regime. Activation energy curves were also plotted based on the slopes of the Arrhenius plots of the various composite matrices, and exhibited decreasing characteristics for the agarose-linear polyacrylamide composite matrix and increasing characteristics for the agarose-hydroxyethylcellulose and agarose-polyethylene oxide composite matrices.

Human dopamine D4 receptor allele genotyping by ultrathin agarose gel electrophoresis with To-Pro-3 complexation.

Growing evidence shows the correlation between the allelic type of the dopamine D4 receptor and the human novelty-seeking personality trait. A sensitive, ultrathin agarose gel electrophoresis-based, high-throughput screening method was developed for genotyping the dopamine D4 receptor (D4DR) exon III 48 base pair repeat polymorphism. The efficiency of the method was increased by reamplification nested polymerase chain reaction (PCR) - of the 48 base pair repeat containing the PCR product with internal primers. The nested PCR fragments were analyzed by ultrathin layer agarose gel electrophoresis with an automated real-time laser-induced fluorescent detection system. Noncovalent affinity complexation was accomplished during the separation process by the addition of a very low concentration of intercalation dye, To-Pro-3 (2 nM) to the gel buffer system. This resulted in instant fluorescent labeling of the migrating PCR fragments. This method can readily facilitate genetic association studies between dopamine receptor genotypes and some human behavioral and neuropsychiatric disorders.

Ultra-thin-layer agarose gel electrophoresis. I. Effect of the gel concentration and temperature on the separation of DNA fragments.

A novel, rapid and efficient separation method is described for the analysis of double stranded (ds) DNA fragments in the form of horizontal ultra-thin-layer agarose gel electrophoresis. This separation technique combines the multilane, high-throughput separation format of agarose slab gel electrophoresis with the excellent performance of capillary electrophoresis. The electrophoretic separation of the fluorophore (Cy5)-labeled dsDNA molecules were imaged in real time by a scanning laser-induced fluorescence/avalanche photodiode detection system. Effects of the gel concentration (Ferguson plot) and separation temperature (Arrhenius plot) on the migration characteristics of the DNA fragments are discussed. An important genotyping application is also shown by characterizing the polymorphic region (2 X or 4 X 48 base pair repeats) of the dopamine D4 receptor gene (D4DR, exon III region) for ten individuals, using PCR technology with Cy5-labeled primers and ultra-thin-layer agarose gel electrophoresis.

Activation of deoxycytidine kinase during inhibition of DNA synthesis by 2-chloro-2'-deoxyadenosine (Cladribine) in human lymphocytes.

Deoxycytidine kinase (dCK, EC.2.7.1.74), a key enzyme in intracellular metabolism of many antileukemic drugs, was shown to be activated during treatment of lymphocytes by 2-chloro-2'-deoxyadenosine (Cl-dAdo, cladribine), a potent inhibitor of DNA synthesis. While 5-[3H]-thymidine (TdR) incorporation into DNA was decreased by 80-90%, dCK activity was doubled as a consequence of incubating the cells with 1 microM 2-chloro-2'-deoxyadenosine. Thymidine kinase (dTK, EC.2.7.1.21) activity was slightly decreased under the same conditions, similarly to 5-[3H]-thymidine incorporation. dCK activation could not be prevented by cycloheximide, and neither the amount of dCK protein nor its mRNA level was increased after 2-chloro-2'-deoxyadenosine treatment. These results suggest a post-translational activation of dCK protein during inhibition of DNA synthesis.

Effects of buprenorphine on cardiovascular and pulmonary function in clinically normal horses and horses with chronic obstructive pulmonary disease.

OBJECTIVES: To evaluate the cardiovascular and respiratory effects of buprenorphine administered intravenously in clinically normal horses and horses with chronic obstructive pulmonary disease (COPD). ANIMALS: 5 clinically normal horses and 5 horses with COPD that were in partial clinical remission (period A) or were having an acute attack of airway obstruction (period B). PROCEDURES: Pulmonary function testing, arterial blood gas analysis, and arterial blood pressure measurements were performed before and after a single intravenous bolus of buprenorphine (3 microg/kg of body weight). Respiratory rate (f), tidal volume (VT), expiratory-to-inspiratory time ratio (TE/TI), minute expiratory ventilation (VE), maximal change in transpulmonary pressure (deltaPL), dynamic compliance (Cdyn), and pulmonary resistance (RL) were calculated with a pulmonary function computer. Heart rate (HR) and systolic (SABP), diastolic (DABP), and mean arterial blood pressures (MABP) were measured. RESULTS: At baseline, COPD horses in period A had decreased Cdyn and increased f, VE, PL, and HR, whereas COPD horses in period B had decreased TE/TI and Cdyn, arterial blood pH, and PO2, and increased f, VE, deltaPL, and RL, compared with clinically normal horses. After drug administration, SABP, DABP, and MABP increased in all horses, f and VE increased in clinically normal horses, and PaO2 decreased within 60 minutes in horses with COPD. CONCLUSION AND CLINICAL RELEVANCE: Buprenorphine can induce excitement in unsedated horses or horses that do not have signs of pain, but does not seem to induce severe respiratory depression or adverse cardiovascular effects in clinically normal horses or those with COPD.

Real-time detection of allele-specific polymerase chain reaction products by automated ultra-thin-layer agarose gel electrophoresis.

Ultra-thin-layer agarose gel electrophoresis, a novel combination of agarose slab gel electrophoresis and capillary gel electrophoresis was introduced in conjunction with laser-induced fluorescence (LIF) scanning detection for the analysis of polymerase chain reaction (PCR) products. Allele-specific fragments, amplified from genomic DNA of patients with congenital adrenal hyperplasia (most often caused by mutations of 21-hydroxylase gene, CYP-21), were used as a model system to investigate the applicability, sensitivity and resolving power of the method. The allele-specific products were generated by PCR and separated by ultra-thin-layer agarose gel electrophoresis. The double-stranded DNA fragments were easily visualized in real-time via complexation during the separation process by the intercalator dye TO-PRO-3 which was part of the separation gel-buffer system. In this way, the migrating dsDNA-dye complexes were detected in real-time by a scanning LIF detection system with sub-nanogram sensitivity. The system employs a 632-nm solid-state laser and an avalanche photodiode detector scanning to the separation platform by means of a fiber bundle system. Automated ultra-thin-layer agarose gel electrophoresis with 'on the fly' TO-PRO-3 staining of dsDNA fragments and LIF detection system proved to be a very fast, high-throughput separation method for individual or multiplexed PCR products, with excellent sensitivity.

Post-mortem stability of membrane-associated sialidase activity in brain.

The post-mortem changes in membrane-associated sialidase (N-acetylneuraminosyl glycohydrolase, EC 3.2.1.18) were examined in rat brain obtained and stored in a manner which paralleled neuropathological handling of human brains. When whole brains were held at 4 degrees C in covered containers for varying periods of time (0.67 h), sialidase activity toward endogenous membrane substrate was elevated. This elevated activity was maximal at 8 h of storage and decreased thereafter. The apparent decrease in enzyme activation from 8 to 67 h of storage was not due to a reduction of activity, but was the result of depletion of endogenous membrane substrate, since activity toward exogenous ganglioside remained elevated. The changes were due to whole brain storage at 4 degrees C, and not a result of being stored at -80 degrees C. The post-mortem activation of sialidase was not due to the expression of a new form of the enzyme, since it displayed characteristics similar to those reported previously: (a) membrane gangliosides being the preferred native substrate, and (b) ganglioside GM1 and lactosylceramide being the major hydrolytic products. The results underscore the importance of post-mortem storage conditions when analyzing complex carbohydrates of brain.

Neurochemical changes in white matter. Aged human brain and Alzheimer's disease.

We report a reduction in isolatable myelin in white matter from regional areas of aged human brain. This decrease was most prominent in association subcortex of Alzheimer's material. We also found structural changes in myelin lipids. These changes involved an increase in unsaturated acyl chains and suggest an age-related instability of subcortical white matter. This specific chemical change in myelin glycosphingolipids has been found in all regional areas of normal aged and Alzheimer's brain material. This remains an age-related molecular change that seems unrelated to the pathophysiology of senile dementia of the Alzheimer's type.

Neurochemical studies in aging brain. I. Structural changes in myelin lipids.

Age-related changes in the lipid chemical composition and structure of myelin and myelin subfractions have been studied in the rat brain. Myelin subfractions with a content ranging from the more mature and compact lamellae to fractions containing earlier membrane fractions were isolated from mature, old, and aged animal groups. We found changes in cholesterol-phospholipid ratios and an increasing degree of unsaturation in the longest acyl chains of major myelin glycosphingolipids. These changes suggest that increased fluidity and decreased stability occurs with age in myelin.

Pyrithiamine-induced thiamine deficiency: effects on rat myelination.

Pyrithiamine was administered to newborn rats throughout the vulnerable period for myelinogenesis. A major metabolic defect was produced in the cerebral activities of the thiamine-dependent enzymes, transketolase and pyruvate decarboxylase. In spite of a defect in carbohydrate metabolism which is lethal in adult rats, overall development, and myelination as indicated by biochemical and morphological criteria, proceeded at an essentially normal rate. These findings indicate alternative metabolic pathways may be operational in newborn rat brain enabling it to circumvent major blockage in thiamine-dependent reactions.

Reversal of delayed myelinogenesis in experimental hypothyroidism.

Neonatal hypothyroidism has been shown to induce delayed myelinogenesis in the developing rat brain. This delay in myelin formation is most prominent during the critical 3 - 4 week postnatal period and the heavy myelin fraction is more selectively affected. The experimental animals, however, continue active myelin formation and by 6 weeks post natum, they do not differ significantly from littermate controls. Myelin protein analysis by SDS acrylamide gels and densitometer methods revealed a decreasing proportion of high molecular weight protein and no significant change in either proteolipid or basic protein in developing myelin. The data suggest that neonatal hypothyroidism induces a reversible delay without qualitative change in myelinogenesis.

Globoid leukidystrophy. I. Clinical and enzymatic studies.

In a complementary clinical and biochemical study of patients with globoid leukodystrophy (GLD), cases differed from the classic phenotype of Krabbe disease and suggested a broader spectrum of clinical presentations. In terms of pathogenesis, the advanced development achieved before symptom onset suggested normal early maturation and myelination. Enzyme studies were carried out on white blood cells from the patients, their siblings, parents, and normal agematched controls. These studies utilized galactosyl ceramide of brain origin and a new assay technique. We found a specific deficit in cerebrosidase activity in leukocyte preparations from patients with GLD and intermediate levels of activity in their parents. These findings confirm prior reports and indicate an autosomal recessive mode of genetic expression.

Globid leukodystrophy. II. Ultrastructure and chemical pathology.

A chemical-pathoologic study of globoid leukodystrophy (GLD) was made that compared myelin isolated from maximally involved central white matter with myelin from noninvolved U fiber areas. The results were compared to analyses of myelin from normal, age-matched control human brains. Myelin from U fiber areas contained relatively greater amounts of a lighter myelin fraction and this lighter myelin differed in protein and lipid composition from the heavier myelin fraction. The data have been interpreted to suggest that the metabolic error in GLD results in the formation of an unstable compact or heavier myelin and that a subsequent process of disintegration selectively affects this component.

Myelination of brain in experimental hypothyroidism. An electron-microscopic and biochemical study of purified myelin isolates.

Myelin development has been studied in the neonatally hypothyroid rat brain by isolation and characterization of purified myelin preparations. The hormone deficiency results in a suppression of compact myelin formation and the persistence of a lighter possibly pro-myelin fraction. The appearance of myelin basic protein was markedly delayed in the hormone-deficient animals and this effect on the basic protein moiety may be responsible for the delayed myelinogenesis.

A comparative study of myelin fractions from metachromatic and globoid leukodystrophies.

Myelin was isolated post mortem from brains of patients with metachromatic and globoid forms of leukodystrophy. In the leukodystrophies, isolates were obtained from central white matter and subcortical "U" fiber areas. Myelin also was obtained from age-matched human controls, and the method yielded light and heavy myelin fractions. In both disorders, the pathologic process affected the heavy isolates to a relatively greater degree. Comparative chemical analyses were made of the myelin isolates, and we concluded that a similar pathogenesis--the production of an unstable membrane in later stages of myelin formation--affects both disorders.