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Biomedical subjects

A Sidoli

Publications and source records attributed to A Sidoli.

At least 37 records · Page 2Linked to original sources
PubMed1991

A form of familial hypobetalipoproteinaemia not due to a mutation in the apolipoprotein B gene.

Familial hypobetalipoproteinaemia (FHBL) is a dominant disorder of lipoprotein metabolism characterized by levels of apolipoprotein B-carrying lipoproteins (VLDL, IDL and LDL) which are 50% of the normal levels in the heterozygotes and almost absent in the homozygotes. Several reports have recently shown that the underlying defect in FHBL involves different mutations in the apo B gene which lead to reduced levels of apo B mRNA or to the production of truncated forms of apo B having either a lower synthetic rate or a higher catabolic rate than normal apo B. We here present a three-generation family with several FHBL members in which the linkage analysis shows absence of co-segregation between apo B gene alleles and the hypocholesterolaemic phenotype. We conclude that a dominantly transmitted mutation in a gene other than that for apo B is responsible for the low plasma cholesterol levels.

Adult↗
PubMed1990

Detection of beta-nerve growth factor mRNA in the human fetal brain.

Nerve growth factor (NGF) is a trophic molecule recently demonstrated to interact with different structures in the central nervous system. The expression of the beta-NGF mRNA from human fetal cortices at the 15-16th week of gestational age has been demonstrated and quantitated by polymerase chain reaction amplification of the specific cDNA. beta-NGF mRNA expression in the human brain coincides with the period of active differentiation and synaptogenesis, suggesting that the trophic agent plays a role in the cerebral development.

Base Sequence↗
PubMed1990

DNA based diagnostic tests: recombinant DNA and cardiovascular disease risk factors.

Advances in molecular biology and medical biotechnology are continuously creating exciting possibilities for DNA based diagnostics. It is now possible by simple procedures to detect polymorphic DNA markers, structural variants and regulatory mutants of human genes, allowing detailed genotyping of patients. The innovative combination of immunoenzymatic techniques, monoclonal antibodies and recombinant tracer proteins, results in new DNA based tests for the determination of important biochemical parameters, in order to define more precisely the phenotype and hence assess the individual risk. The application of these technologies to the analysis of dyslipidemias, atherosclerosis and cardiovascular diseases may not only lead to a better understanding of the molecular and genetic basis of these pathologies, but also to their early recognition and better management.

Base Sequence↗
PubMed1990

Human apolipoprotein A-I gene promoter polymorphism: association with hyperalphalipoproteinemia.

An apolipoprotein A-I gene promoter polymorphism, due to an adenine (A) to guanine (G) transition 78 base pairs upstream from the transcription initiation site, was studied by amplification of the corresponding region of the apoA-I gene, DNA sequencing, and allele-specific oligonucleotide hybridization. The frequency of the polymorphism was studied on female and male individuals classified into three groups according to the high density lipoprotein (HDL) cholesterol concentration. The allelic frequencies for the A polymorphism were 0.10, 0.14, 0.27 in women and 0.08, 0.17, 0.14 in men in the lowest, in the intermediate (between 10th and 90th percentile), and the highest decile of HDL cholesterol levels, respectively. Statistical analysis showed a significant difference of allelic frequencies between the highest and the lowest deciles (P less than 0.006) and between the highest and the intermediate deciles of HDL cholesterol in women (P less than 0.04) but not in men. As the sequences surrounding the polymorphism are known to be involved in transcription modulation, it is possible that the A-G transition polymorphism may have an influence on apoA-I synthesis and, in consequence, on the HDL cholesterol levels in women.

Adult↗
PubMed1990

RIECA: an innovative immuno enzymatic competition assay for apolipoproteins AI and B determination.

The authors describe the production, via recombinant DNA technology, of bifunctional polypeptides for immunoenzymatic assays. These molecules contain an apolipoprotein moiety fused to an active beta-galactosidase enzyme, and are used as tracers in competition assays with specific monoclonal antibodies. The final colorimetric result is inversely correlated with the apolipoproteins plasma values. This technology, named RIECA (Recombinant Immunoenzymatic Competition Assay) is very accurate and flexible and may be applied to a wide range of diagnostic interest.

Antibodies, Monoclonal↗
PubMed1989

Erythrocyte adducin differential properties in the normotensive and hypertensive rats of the Milan strain. Characterization of spleen adducin m-RNA.

Previous studies have shown that erythrocytes from the Milan hypertensive strain of rats (MHS) differ from erythrocytes from the control normotensive strain (MNS). These differences are determined within the stem cells, are genetically associated with the development of hypertension, and are similar to those found between the tubular cells of the two strains. Moreover they seem to be dependent upon the presence of the membrane skeleton proteins. In this paper we describe our studies aimed at identifying some precise protein difference between the membrane skeletons of the two strains, which may cause the cellular differences described above. Milan hypertensive strain and MNS rats were immunized with ghost or membrane skeleton extracts prepared from the other or their own strains. Only MHS rats immunized with MNS ghost or membrane skeleton extracts produced an antibody against a 105 KD protein in about 95% of the animals. This protein has been identified with the recently described cytoskeletal protein adducin on the following bases: the protein binds calmodulin (CaM) and protein kinase C (PKc) in a Ca2+ dependent way. It also binds phosphatidylserine, is the substrate of exogenous PKc, and finally it is purified by high salt extraction of Triton-X100 insoluble erythrocyte cytoskeletons followed by affinity chromatography on CaM-sepharose. Using this antibody the isolation from a mouse spleen library, the characterization and sequencing of a partial cDNA clone coding for this protein has been carried out. In conclusion adducin may be considered a very useful tool to test the hypothesis that the cellular differences between MHS and MNS may be caused by a difference in a membrane skeleton protein.

Amino Acid Sequence↗
PubMed1988

Characterization of genetic markers in the 3' end of the apo B gene and their use in family and population studies.

The 3' end of the apo B gene is highly polymorphic. Two point mutations in the coding sequence of the gene create EcoRI (E+, E-) and XbaI (X+, X-) RFLPs. The two loci are in random association and the frequency of the four haplotypes, E+X+, E+X-, E-X+ and E-X- in the normolipidaemic population are 0.68, 0.30, 0.02 and 0.00, respectively. Although the polymorphic nucleotide underlying the EcoRI RFLP creates an amino acid substitution in the apo B protein (Glu----Lys) in a region close to a putative LDL-receptor recognition site(s), we find no statistically significant difference in the frequency of the apo BGlu and apo BLys alleles in hyperlipidaemic patients (familial hypercholesterolaemia, type IIA with no tendon xanthomas, IIB and probably IV) and the normolipidaemic population. In contrast, we confirm previous findings, that the X+ allele is in linkage disequilibrium with a genetic locus that predisposes to the development of higher fasting plasma triglyceride levels than the X- allele. We have characterized a highly polymorphic region immediately 3' to the apo B gene. At least 5 alleles of this locus exist in the population and our family studies show it should be an extremely informative locus to use in studies where polymorphic or mutant apo B alleles are suspected to underly certain forms of familial hyperlipidaemia. DNA sequence analysis of this highly polymorphic locus shows that the variation is entirely attributable to the number of times the simple repeating sequence 5'-TTTATAATTAAAATATTTATAATTAAATAT-3' is present.

Adult↗
PubMed1986

Expression of the human apolipoprotein AI gene fused to the E. coli gene for beta-galactosidase.

The human apoAI gene was expressed in E. coli by in-frame fusion to a modified beta-galactosidase gene present in plasmid pUR291. The fused beta-galactosidase-apoAI gene product was expressed at a high level and was recognized by an anti-human apoAI antiserum. Besides the fused protein, at least one degradation product having an Mr similar to that of beta-galactosidase was present in high amounts in bacterial extracts. These results and those of a pulse-chase experiment indicate that degradation took place only in the apoAI moiety of the chimeric protein.

Apolipoprotein A-I↗
PubMed1985

Molecular cloning of human LDL apolipoprotein B cDNA. Evidence for more than one gene per haploid genome.

We have isolated an apolipoprotein B (apo B) clone (pXB1) from a human liver cDNA expression library, by immunoselection with a polyclonal antibody to human low density lipoprotein. pXB1 was used to isolate 3 clones (pB2, pB3 and pB4), containing cDNA inserts spanning a region of 3.75 kbp, from a second human liver cDNA library. We report the sequence of 1359 nucleotides at the 3' end of the pB4 cDNA insert and the amino acids encoded by this sequence. The cDNA inserts of pBX1 and pB2 overlapped the sequenced portion of pB4. pB2 contained an EcoR1 restriction site (resulting in a Glu-Lys replacement) which is not present in pB3 or pB4 and pB3 contained an MspI site not present in pB2 or pB4. Since all 3 clones were derived from the mRNA of a single human liver, we suggest that the human haploid genome contains more than one functional apo B gene. Labelled probes spanning almost the whole of the pB4 cDNA insert hybridized with RNA from human liver and small intestine, showing that the apo B mRNAs from these two tissues have nucleotide sequences in common. The nucleotide sequence in human liver apo B mRNA is probably longer than 12 kb, showing that the MW of monomeric apo B is at least 350kd. Clone pB4 hybridized with mRNA of similar length in rabbit liver and small intestine. These results raise the possibility that the low MW apo B synthesized in the intestine (B-48) and the high MW apo B synthesized in the liver (B-100) are translated from the same mRNA. The expression products of fragments of pB4 cloned into an expression vector were blotted with monoclonal antibodies to human LDL. The results suggest that the cDNA insert in pB4 encodes a part of apo B common to B-48 and B-100 and a region close to the recognition site for the LDL receptor.

Animals↗
PubMed1985

Organization of the human transferrin gene: direct evidence that it originated by gene duplication.

We present the characterization of two overlapping human transferrin genomic clones isolated from a liver DNA library. The two clones represent a total length of 24 kilobase pairs and code for 70% of the protein. The organization of this gene region was elucidated by restriction mapping and DNA sequencing. It contains 12 exons, ranging from 33 to 181 base pairs, separated by introns of 0.7-4.9 kilobase pairs. This gene can be divided into two unequal parts corresponding to the known domains of the protein. Each part is essentially composed of an equal number of exons; introns interrupt the coding sequences, creating homologous exons of similar size in each moiety. Moreover, the pattern of intron interruption of the codon sequence is identical for all the analyzed homologous exon pairs. Comparison with the organization of the ovotransferrin gene shows an identical exon size distribution. These data confirm, at the gene level, the hypothesis that transferrins originated by a gene-duplication event. A model accounting for the origin of the human transferrin gene is presented.

Amino Acid Sequence↗
PubMed1984

Human apolipoproteins AI, AII, CII and CIII. cDNA sequences and mRNA abundance.

The structure and function of the genes encoding the polypeptide components of plasma lipoproteins are of interest because of the central role they play in the regulation of lipid metabolism. We have now completed our previous studies on the human apoAI gene and furthermore isolated and sequenced cDNA clones for apoAII , CII and CIII. The nucleotide sequences show the signal peptides of apoAII , CII and CIII to be 18, 22 and 20 amino acids in length, respectively, and in addition that prepro apoAII bears a classical propeptide structure of 5 amino acids. The amino acid homology detected between apoCII and pro- apoAI is discussed, as is the gene arrangement of the 5' non-coding region of apoAI mRNA. The relative liver mRNA levels of the 4 apolipoproteins analysed in this study have been estimated and compared with their corresponding plasma products. The data reported here provide an essential basis for further studies of structural and functional alleles of apo AI, AII, CII and CIII genes.

Amino Acid Sequence↗
PubMed1984

DNA-dependent ATPases in Bacillus subtilis mutants and in competent cells.

Three DNA-dependent ATPases (gamma phosphohydrolases) can be isolated from Bacillus subtilis cells. We studied these enzymes in a number of mutants deficient in recombination or repair functions (rec, uvr) and in competent cells. The recA mutant studied had lower ATPase II activity, while competent cells had higher ATPase I activity, in comparison with the parental strain not brought to competence.

Adenosine Triphosphatases↗
PubMed1984

Fibroblast interferon from a human amniotic cell line: a strategy for rapid molecular cloning.

A strategy for rapid cloning and identification of cDNA clones was employed to obtain fragments of the gene for human fibroblast (beta) interferon. A human amniotic cell line (UAC), widely used for interferon assay, yielded high titers of beta-interferon and was used as a source for cDNA synthesis. Recombinant clones were screened by a combination of hybridization conditions: (a) comparing clones that hybridized with radioactive cDNA from induced UAC cells vs cDNA from cells not induced for beta-IFN; (b) using a synthetic oligonucleotide proble complementary to a region of the beta-IFN gene. Among clones selected on the basis of their positive response to both screening procedure, one (4F7) was sequenced and shown to be highly homologous to the beta-IFN gene. This clone was used as a probe to screen a human chromosomal DNA library, yielding the entire chromosomal beta-IFN gene. A "Northern" hybridization analysis of UAC mRNA from uninduced and induced cells revealed the existence of a single molecular species of mRNA for beta-IFN with the expected molecular weight.

Amnion↗