Biomedical subjects
Publications and source records attributed to A Rinaldi.
Despite a fall in the proportion of CGs methylated, evidence has not been obtained for significant demethylation of prelabelled DNA when mouse erythroleukaemia cells are induced to differentiate. There is, however, a delay in the methylation of the DNA that is synthesised in the early period of induction, leading to its undermethylation by 30-50% and this may be a contributory cause of the observed fall in CG methylation.
DNA methylases from mouse and pea have been purified and characterized. Both are high molecular mass enzymes that show greater activity with hemimethylated than unmethylated substrate DNA. Both methylate cytosines in CpG preferentially, but not exclusively and show similar kinetics of methylation, which makes it difficult to saturate all possible sites on the DNA, but procedures are described that circumvent this problem.
An experimental model of cold induced brain oedema was carried out in 7 albino rabbits by topic application of fluid N2 on the skull to examine the favourable effects of an appropriate tailored craniodural opening both on the whole brain perfusion and on the local dynamics of the oedematous fluid. After positioning the animals in a stereotactic frame the following parameters were recorded: ICP, carotid blood pressure and flow velocity, EEG, EKG. Histological staining with Evan's blue was utilized to check fluid extravasation. In all the animals the lesion was followed both by an ICP increase and a reduction in the carotid blood flow velocity so indicating an increase in the cerebrovascular resistances. In 4 animals the removal of the craniodural flap over the lesion dramatically reduced the ICP values and normalized the blood flow velocity. No difference in the Evan's blue distribution was noticed between the two groups of animals.
The aims of this work are to describe the general and technical characteristics of a new device for the noninvasive monitoring of patients in intensive care and during general anaesthesia, and the results concerning the reliability of this method. An ultrasonic esophageal probe and an echo-Doppler device have been used to obtain continuous data of the aortic diameter and of blood velocity. Aortic output is calculated automatically. This method, together with other non-invasive monitoring techniques (blood pressure, heart rate, rhythm and cardiac conduction), gives on the one hand the data of aortic output, systemic peripheral resistance and stroke volume; on the other, through a computerized elaboration, the systolic time intervals (PEP pre-ejection period, LVET left ventricular ejection time, QS2 electromechanical systole, PEP/LVET ratio of PEP to LVET). The validation of STI data, has been obtained through 125 comparative measurements for each of the three parameters. The data obtained through the aortic velocity waveform in descending aorta (pulsed Doppler) have been compared with those obtained through the aortic pressure waveform (intra aortic catheter). The correlation was: PEP 0.92, LVET 0.95, QS2 0.93. The clinical application of this method supplies data concerning cardiac load, after-load and indirectly cardiac pre-load. This non-invasive procedure gives us a continuous measurement of hemo-dynamic situation, which allows the physician to plan and evaluate the therapeutical efficacy. Finally new pathologic events may be opportunely faced.
This article presents a general vision of a new non-invasive cardiovascular monitoring technique applied to patients during general anaesthesia. This method employs an esophageal eco-Doppler, able to supply a continuous measurement of the aortic diameter (eco-TM) and of the velocity of aortic blood flow (pulsed Doppler) for the calculation of aortic output; a monitor to survey the ECG and a noninvasive device for the measurement of blood pressure. The resulting data, opportunely processed by the computer, supply the values of aortic output, of systemic peripheral resistances and of stroke volume. The last two parameters are indexed with aortic output. Through the computer analysis of ECG and of the aortic velocity curve, it is possible to trace back the systolic time intervals and find out the left ventricular ejection time, the pre-ejection period and the electromechanical systole. All these values are indexed with heart rate. The validation of this method gave good results. The comparison between aortic output, measured by the eco-Doppler, and the value obtained with the thermodilution gave a correlation of r = 0.97, and comparing it with the one obtained with the electromagnetic ring, the correlation was r = 0.96. The aortic diameter was calculated with computerized tomography and the correlation between the two diameters resulted in 0.978. This method is quite useful because it supplies continuous parameters in an automatic and noninvasive way. Therefore it is possible, through a compete hemodynamic profile, to diagnose the variations and control the evolutions of the hemodynamic profile induced by the therapeutical treatments, on the basis of a correct physiopathological interpretation.
The still debated question of whether the expression of mental retardation in heterozygous carriers of the Martin-Bell syndrome is influenced by X inactivation has been investigated in a group of phase-known double heterozygotes for the FRA-X mutant and the G6PD Mediterranean variant. In these individuals, the number of somatic cells (fibroblasts or red cells) with an active FRA-X chromosome could be assessed through the G6PD phenotype at the single-cell level. The data reported indicate a significant inverse correlation between the IQ level (as measured by the Wechsler-Bellevue test) and the percentage of fibroblast cells with an FRA-X active chromosome. In contrast, no significant correlation was found when the IQ level and red cell data were compared, thus suggesting the occurrence of somatic selection against hematopoietic stem cells with an active FRA-X chromosome.
Cu-Amine oxidases (amine oxygen oxidoreductase deaminating, copper containing E.C. 1.4.3.6.) are found in all forms of life (1). They catalyze the following general reaction: R-CH2-NH2 + O2 + H2O----R-CHO + NH3 + H2O2. Cu-amine oxidases (Cu-AOs) have been extracted from different leguminosae: Pisum sativum (2-3), Lathyrus sativus (4), Lens esculenta (5), Vicia faba (6), Cicer arietinum (7), Glycine max (8) but not from Phaseolus vulgaris. Palavan and Galston (9), in a study of polyamine biosynthesis during developmental stages of Phaseolus vulgaris, did not detect diamine or polyamine oxidase activity in Phaseolus. The present paper describes the purification of Phaseolus vulgaris seedlings amine oxidase (PhSAO) and also compares the properties of this enzyme to the Lathyrus cicera enzyme (LcSAO), obtained with the same method of purification.
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A protein with NADH oxidase activity from the extreme thermophile Thermus aquaticus YT-1 was purified and characterised. The enzyme was found to have a relative molecular mass of 110,000 and be composed of two subunits of identical size. FAD was found to be present at a concentration of 0.7 mol/mol dimer and was required for activity. During the oxidation of NADH, oxygen uptake takes place with the production of hydrogen peroxide. The enzyme had, with the exception of a higher glutamic acid and tryptophan content, a similar amino acid composition as the NADH oxidase isolated from the mesophile Bacillus megaterium. Purified NADH oxidase was found to have a Km of 39 microM for beta-NADH and a Vmax of 4.68 mumol NADH mg-1 min-1 and was still active at 95 degrees C. Enzymatic activity was found to be independent of pH between 5.0 and 10.5.
Infection of rat embryo cells with herpes simplex virus type 2 caused undermethylation of host cell DNA synthesized during infection. DNA made prior to infection was not demethylated, but some of its degradation products, including methyl dCMP, were incorporated into viral DNA. The use of mutant virus showed that some viral DNA synthesis appears to be required for the inhibition of methylation. Inhibition of methylation cannot be explained by an absence of DNA methyltransferase as the activity of this enzyme did not change during the early period of infection. Inhibition of host cell DNA methylation may be an important step in the transformation of cells by herpesviruses, and various transformed cell lines tested showed reduced levels of DNA methylation.
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The dinucleotide CpG is deficient in (A + T)-rich regions of vertebrate DNA in both coding and non-coding sequences and there is a corresponding increase above expectation in the occurrence of TpG and CpA. By contrast in (G + C)-rich regions no deficiency of CpG is found. Such (G + C)-rich sequences, containing the expected number of CpG dinucleotides, alternate along the genome with (A + T)-rich sequences which have a lower than expected CpG content. The G + C content of vertebrate DNA can oscillate with a period of 150-200 bp and this may be a factor in positioning nucleosomes. The role of mutagenesis in loss of CpG and increase of A + T, particularly in non-coding regions, is discussed.
Although echinomycin is reported to intercalate and to bind to DNA at CG dinucleotides, the effects of the drug on DNA methylation in vitro and in vivo are much less apparent than are the effects on DNA synthesis and cell growth.
Fourteen families with X-linked mental retardation (XMR) have been studied clinically and cytogenetically. All affected males failed to show a fragile site (FS) on Xq of their peripheral lymphocytes. Five families may be considered examples of Renpenning syndrome while the remaining may be divided in two groups: one of seven (type I) and one of two (type II). The seven families of type I had some physical features of the Martin-Bell syndrome but with normal to large sized testes whence the name of X-linked MR with slight macroorchidism (XMR +/- MO). The two families of type II showed unremarkable facial appearance, mild to moderate degree of MR and a certain microorchidism whence the possible name of X-linked MR with different degree of microorchidism (XMR +/- MiO).
We have purified the DNA methylase from mouse ascites tumour cells to a specific activity of 11,500 units per mg protein using denatured Micrococcus luteus DNA as methyl acceptor. Methyl groups are transferred to cytosines almost exclusively in CpG dinucleotides. The purified enzyme contains two polypeptides of molecular mass 185 and 160 kDa, and an antiserum raised in a rabbit to the purified enzyme specifically reacts with these two proteins in crude extracts. The two proteins can be partially separated by affinity chromatography when activity is associated with the 185 kDa protein which can be proteolytically degraded to give polypeptides of 170 and later 100 and 50 kDa. Only the 185 kDa methylase is lost when cells are treated with azadeoxycytidine and this is the predominant form firmly bound in the nucleus of dividing cells. Antibody bound to the 185 kDa band in protein blots will itself bind native DNA methylase, which can be detected by its binding 14C-labelled, azacytosine-containing DNA.
The endogenous DNA methylase in nuclei isolated from growing mouse cells preferentially methylates DNA in micrococcal nuclease-resistant regions probably as a result of the location in these regions of the preponderance of hemimethylated sites. Added mouse ascites cell DNA methylase catalyses the methylation of exposed, nuclease-sensitive DNA in chromatin from growing or non-growing mouse or insect cells. The poor acceptor ability of nuclease-resistant regions in this situation is due to the presence of histone proteins which block de novo methylation. Transcriptionally active regions of chromatin are selectively methylated in vitro by either endogenous or added DNA methylase.
We previously proposed the hypothesis that the primary expression of the defect in X-linked Duchenne muscular dystrophy (DMD) occurred in the myoblast, or muscle precursor cell. This was based on the observation that the number of viable myoblasts obtained per gram DMD muscle tissue was greatly reduced and those that grew in culture had decreased proliferative capacity and an aberrant distended flat morphology. Here we test that hypothesis by determining whether the expression of the myoblast defect is X-linked. Muscle cells were obtained from five doubly heterozygous carriers of two X-linked loci, DMD and glucose-6-phosphate dehydrogenase (G6PD), and compared with those from five sex- and age-matched controls heterozygous for G6PD only. A total of 1,355 individual clones were determined to be muscle and evaluated at the single cell level for proliferative capacity, morphology, and G6PD isozyme expression. The results demonstrate that the proportion of defective myoblast clones is significantly increased in DMD carriers. However, since this cellular defect does not consistently segregate with a single G6PD phenotype in the myoblast clones derived from any of the carriers, it is unlikely to be the primary expression of the DMD mutant allele.