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Biomedical subjects

A Colosimo

Publications and source records attributed to A Colosimo.

At least 37 records · Page 2Linked to original sources
PubMed2000

Transfer and expression of foreign genes in mammalian cells.

The transfer of foreign genes into eukaryotic cells, in particular mammalian cells, has been essential to our understanding of the functional significance of genes and regulatory sequences as well as the development of gene therapy strategies. To this end, different mammalian expression vector systems have been designed. The choice of a particular expression system depends on the nature and purpose of the study and will involve selecting particular parameters of expression systems such as the type of promoter/enhancer sequences, the type of expression (transient versus stable) and the level of desired expression. In addition, the success of the study depends on efficient gene transfer. The purification of the expression vectors, as well as the transfer method, affects transfection efficiency. Numerous approaches have been developed to facilitate the transfer of genes into cells via physical, chemical or viral strategies. While these systems have all been effective in vitro they need to be optimized for individual cell types and, in particular, for in vivo transfection.

Animals↗
PubMed1999

Gene transfection efficiency of tracheal epithelial cells by DC-chol-DOPE/DNA complexes.

We evaluated the transfection efficiency of five different cationic liposome/plasmid DNA complexes, during the in vitro gene transfer into human epithelial tracheal cell lines. A dramatic correlation between the transfection efficiency and the charge ratio (positive charge of liposome to negative charge of DNA) has been found. DC-Chol-DOPE was found to be the most effective liposome formulation. Therefore, a morphological and structural analysis of DC-Chol-DOPE liposomes and DC-Chol-DOPE/DNA complexes, has been performed by transmission electron microscopy (TEM) and by confocal laser scanning microscopy (CLSM), respectively. The process of interaction between DC-Chol-DOPE/DNA complexes and human epithelial tracheal cells has been studied by CLSM. These results raise some issues for in vivo gene therapy.

Benzothiazoles↗
PubMed1999

Cellular uptake and delivery monitoring of liposome/DNA complexes during in vitro transfection of CFTR gene.

The cellular uptake and distribution of cationic liposomes Dc-Chol/DOPECFTR gene complexes were assessed by electronic and confocal laser scanner microscopy (CLSM) for the CFTR gene transfer to human adenocarcinoma and tracheal epithelial cell lines. Cationic lipid forms unilamellar and multilamellar vesicles capable of rapid and efficient transport of gene into target cells. The number of fluorescent complexes was increasing with time in cells up to 6 hours showing a punctate and homogeneous DNA distribution in the cytoplasmatic and nuclear compartments, including the nucleolus. No significant difference in the biochemical and cellular behavior was observed between the investigated system and other systems previously tested. This study adds new insights into the CFTR cationic liposome-mediated gene delivery.

Cell Line↗
PubMed1998

Structure and expression of the human ubiquitin fusion-degradation gene (UFD1L).

We report the genomic organization, RNA and protein expression patterns of the gene encoding for the human homolog of the yeast ubiquitin fusion-degradation protein-1 (UFD1L). This enzyme is involved in a ubiquitin-dependent proteolytic pathway (UFD), firstly described in yeast. The human UFD1L gene is organized into 12 exons ranging in size from 33 to 161 bp. Sequence analysis of the 5'-flanking region of the gene revealed a high GC content, multiple CCAAT-binding motifs, CREB, CFT, and AP-2 sites. RNA transcripts were detected in all tissues and cell lines examined, including thymus, thymocytes, T- and B-cells, fibroblasts, chorionic villi, and amniocytes. In Western blot, the UFD1L antibody demonstrated the presence of multiple protein isoforms in all the tested tissues. Expression profile and promoter characteristics suggest UFD1L is a housekeeping gene with implications in the pathogenesis of DiGeorge/velo-cardio-facial syndrome, due to 22q11.2 deletions.

Adaptor Proteins, Vesicular Transport↗
PubMed1998

Recurrence quantification analysis in structure-function relationships of proteins: an overview of a general methodology applied to the case of TEM-1 beta-lactamase.

Protein structure-function relationships have been increasingly scrutinized by a variety of correlational and information theoretic measures. In an effort to extend this methodology, a technique originally developed in non-linear science, recurrence quantification analysis, was combined with traditional principal components analysis to study a large number (56) of TEM-1 beta-lactamase mutants. The hydrophobicity profiles corresponding to the primary structure of 13 naturally occurring mutations partially impairing function, together with 43 artificial non-tolerated mutations were subjected to discriminant analysis, derived from the results of recurrence quantification analysis, coupled to a principal exponents extraction. Eleven (85%) of the naturally occurring mutants and 36 (84%) of the artificial mutants were correctly classified (p < 0.0001). We conclude that this technique may be useful in protein engineering and, in general, in structure-function studies of biopolymers.

Amino Acid Sequence↗
PubMed1998

A nonlinear explanation of aging-induced changes in heartbeat dynamics.

The possibility of computing a cardiac age on the basis of spectral analysis of healthy individual tachograms was confirmed and facilitated by the use of a nonlinear technique: recurrence quantification analysis. The age of 112 subjects was predicted by this technique in terms of a progressive increase in the deterministic character of the heartbeat. This result confirms the "random-walk" character of the heartbeat as predicted by the terminal dynamics paradigm, thus allowing for a simple and comprehensive model of the effect of aging on cardiac dynamics: as age progresses, heart rate dynamics become increasingly predictable (constrained) on a beat-to-beat basis. This implies a basically stochastic nature of heart rate dynamics, probably reflecting the continuous adjustments to an unpredictable internal environment.

Adult↗
PubMed1997

A study of oligonucleotide occurrence distributions in DNA coding segments.

In this paper we present a general strategy designed to study the occurrence frequency distributions of oligonucleotides in DNA coding segments and to deal with the problem of detecting possible patterns of genomic compositional inhomogeneities and disuniformities. Identifying specific tendencies or peculiar deviations in the distributions of the effective occurrence frequencies of oligonucleotides, with respect to what can be a priori expected, is of the greatest importance in biology. Differences between expected and actual distributions may in fact suggest or confirm the existence of specific biological mechanisms related to them. Similarly, a marked deviation in the occurrence frequency of an oligonucleotide may suggest that it belongs to the class of so-called "DNA signal (target) sequences". The approach we have elaborated is innovative in various aspects. Firstly, the analysis of the genomic data is carried out in the light of the observation that the distribution of the four nucleotides along the coding regions of the genoma is biased by the existence of a well-defined "reading frame". Secondly, the "experimental" numbers found by counting the occurrences of the various oligonucleotide sequences are appropriately corrected for the many kinds of mistakes and redundancies present in the available genetic Data Bases. A methodologically significant further improvement of our approach over the existing searching strategies is represented by the fact that, in order to decide whether or not the (corrected) "experimental" value of the occurrence frequency of a given oligonucleotide is within statistical expectations, a measure of the strength of the selective pressure, having acted on it in the course of the evolution, is assigned to the sequence, in a way that takes into account both the value of the "experimental" occurrence frequency of the sequence and the magnitude of the probability that this number might be the result of statistical fluctuations. If the strength of the selective pressure evaluated in this fashion turns out to be sufficiently large, the corresponding sequence will be considered to have an occurrence frequency beyond expectations and, hence, to be statistically and biologically interesting.

Algorithms↗
PubMed1997

Estimating a cardiac age by means of heart rate variability.

A data set of R-R intervals recorded for at least 15 min in 141 healthy individuals of different ages and under two different conditions ("resting" and "tilted" states) has been considered. The data have been subjected to spectral analysis by fast Fourier transform methods and considered in view of the possibility to work out a model in which the chronological and cardiac age could be compared. Understanding the results was greatly facilitated by 1) working out a number of derived variables from the original ones to highlight the presence of small but conceptually important variability factors; 2) extraction of the principal components from the original as well as from the derived variables to exclude redundancies and correlation effects; and 3) automatic clustering of the subjects in age classes, which allowed removal of individual variability within each class. The main conclusion is that, within the examined individuals, cardiac and chronological ages do not match for ages higher than approximately 50 years; this could reflect the presence of subtle (and difficult-to-envisage) biases in the data analysis or a real discrepancy. The latter hypothesis should be confirmed by similar observations in different systemic contexts. The use of a simple equation relating chronological and cardiac age, derived from a careful regression analysis on our data set and of general use for screening purposes, is demonstrated.

Adult↗
PubMed1996

Micro- and macrostructure of learning in active avoidance: a quantitative approach.

The dynamics of learning in the Active Avoidance test was analyzed at the trials level as well as at the level of daily sessions, each comprising numerous trials. The two scales (large scale for the sessions and small scale for the trials) were demonstrated to be mutually independent. The intermediate derived scales (blocks of trials) were found consistent among themselves and with small scale but independent of the large one. Moreover, the two extreme scales were kinetically discriminable. These results point to the existence of two independent mechanisms for large and small scale learning together with the need to postulate a consolidation process during the rest period.

Animals↗
PubMed1996

Non-invasive early prenatal molecular diagnosis using retrieved transcervical trophoblast cells.

Fetal DNA was recovered from 17 of 39 (44%) transcervical cell (TCC) samples obtained between 7 and 9 weeks of gestation by endocervical canal flushing. Trophoblast retrieval was adequate for polymerase chain reaction (PCR) amplification of Y chromosome-specific DNA sequences and detection of paternal-specific microsatellite alleles. The fetal sex predicted by PCR in TCCs was confirmed in all cases by karyotype analysis of chorionic villi at 10 weeks of gestation. The absence of the disease-associated paternal alleles in TCC samples from two pregnancies at risk for spinal muscular atrophy and myotonic dystrophy predicted unaffected fetuses in agreement with subsequent results on chorionic villi and newborns' leukocytes. A trisomy 21 fetus was diagnosed in TCCs using fluorescent in situ hybridization (FISH) and semi-quantitative PCR analysis of superoxide dismutase-1 (SOD 1). Present experience indicates that TCC sampling is a promising technique for early prenatal monitoring of Mendelian disorders and chromosome aneuploidy.

Adult↗
PubMed1996

Detection of eight beta-thalassemia mutations using a DNA enzyme immunoassay.

We describe the use of a polymerase chain reaction-based method followed by a DNA enzyme immunoassay for the simultaneous detection of the eight most common beta-thalassemia mutations in the Mediterranean population. The method is specific, sensitive, and easily applicable in routine clinical laboratories for the molecular diagnosis of beta-thalassemia patients and at risk couples.

Alleles↗
PubMed1996

cDNA characterization and chromosomal mapping of two human homologues of the Drosophila dishevelled polarity gene.

The Drosophila dishevelled gene (dsh) encodes a secreted glycoprotein, which regulates cell proliferation, acting as a transducer molecule for developmental processes, including segmentation and neuroblast specification. We have isolated and characterized cDNA clones from two different human dsh-homologous genes, designated as DVL-1 and DVL-3. DVL-1 and DVL-3 putative protein products show 64% amino acid identity. The DVL-1 product is 50% identical to dsh and 92% to a murine dsh homologue (Dvl-1). Both human DVL genes are widely expressed in fetal and adult tissues, including brain, lung, kidney, skeletal muscle and heart. DVL-1 locus maps to chromosome 1p36 and DVL-3 to chromosome 3q27. DVL-1 locus on chromosome 1 corresponds to the murine syntenic region where Dvl-1 is located. DVL-1 and DVL-3 are members of a human dsh-like gene family, which is probably involved in human development. Although the precise role of these genes in embryogenesis is only conjectural at present, the structural and evolutionary characteristics suggest that mutations at their loci may be involved in neural and heart developmental defects.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Human homologue sequences to the Drosophila dishevelled segment-polarity gene are deleted in the DiGeorge syndrome.

DiGeorge syndrome (DGS) is a developmental defect of some of the neural crest derivatives. Most DGS patients show haploinsufficiency due to interstitial deletions of the proximal long arm of chromosome 22. Deletions of 22q11 have also been reported with patients with the velocardio-facial syndrome and familial conotruncal heart defects. It has been suggested that the wide phenotype spectrum associated with 22q11 monosomy is a consequence of contiguous-gene deletions. We report the isolation of human cDNAs homologous to the Drosophila dishevelled (dsh) segment-polarity gene. Sequences homologous to the 3' UTR of these transcripts (DVL-22) were positioned within the DGS critical region and were found to be deleted in DGS patients. Human DVL mRNAs are expressed in several fetal and adult tissues, including the thymus and, at high levels, the heart. Two transcripts, 3.2 and 5kb, were detected, in northern blot analysis, with different expression patterns in the surveyed tissues when different cDNAs were used. The isolated cDNAs exhibit high amino acid homology with the mouse and Xenopus Dvl-1 gene, the only other vertebrate dsh homologues so far isolated. The pivotal role of dsh in fly development suggests an analogous key function in vertebrate embryogenesis of its homologue genes. Since DGS may be due to perturbation of differentiation mechanisms at decisive embryological stages, a Dsh-like gene in the small-region overlap (SRO) might be a candidate for the pathogenesis of this disorder.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Identification of multiple transcribed sequences from the spinal muscular atrophy region of human chromosome 5.

We report the isolation and characterization of novel expressed sequences from the spinal muscular atrophy (SMA) region on human chromosome 5q13. Based on the sequence homology studies these cDNAs were grouped in four classes, one of which shows extensive homologies with the beta-glucuronidase (BG) gene, differing in exon arrangement. The other cDNAs do not show any strong homology with known DNA sequences.

Base Sequence↗
PubMed1995

[Individual arterial pressure variability in newborn infants, adults, and the elderly].

The complexity of the cardiovascular function is related to a wide interindividual variability (VA) that changes with age. The aim of our study was to investigate the spontaneous blood pressure (BP) VA in 3 groups of 20 healthy subjects (S) each, 10 M and 10 F: Group I newborns 24 hours old; Group II 30-40 yrs; Group III 70-80 yrs. Each S underwent BP monitoring in a comfortable condition for 15'. Our data show an increase, with age, of both the average systolic and diastolic BP, paralleled by a simultaneous decrease of their standard deviations. Furthermore, the three distributions show a reasonably gaussian behaviour (maximum absolute value of the skewness was 0.21). These results emphasize the age-dependent reduction in functional flexibility of the cardiovascular system. A major pool of subjects is required to confirm our preliminary data.

Adult↗
PubMed1995

Variability in the arterial pressure of newborns as compared to that of adults and old men.

The systolic and diastolic blood pressure was monitored on healthy subjects of different ages, spanning from newborns (24-48 hs old, to elderly age (70-80 years old). Each subject was monitored for 15 minutes, and the pressure values collected every minute. This protocol allowed a statistical evaluation of both inter- and intraindividual variabilities among and within different class ages. As expected, increasing age is paralleled by a marked increase in the average values of systolic blood pressure; the trend of variabilities around the average, however, as traced by standard deviations, is just the opposite: a marked decrease going from newborns to adults is accompanied--in most cases--by a statistically significant difference in the same direction also between adults and old men. This can be rationalized, in the light of recent findings on the role of deterministic chaos in the functional behaviour of complex biological systems, in terms of decreased functional flexibility characteristic of the aging process.

Adult↗