Biomedical subjects
Publications and source records attributed to P Izzo.
PSA has been measured out with highly sensitive method both in liquid of mammary cysts and in cytosol of neoplastic cells. Our data point out that dysplastic, metaplastic and anaplastic breast epithelial cells produce PSA. Such production is, probably, related to expression of hormonal receptors.
The expression of aldolase A L-type mRNA is increased in growth-arrested mouse NIH3T3 cells and remarkably down-regulated in actively proliferating cells. Treatment of proliferating cells with cycloheximide abolished the down-regulation of L-type mRNA expression, thus indicating that a protein factor acts as repressor in proliferating cells. Transient transfection experiments in NIH3T3 cells showed that a negative regulatory cis-element (NRE) is involved in the modulation of the transcriptional activity of the distal L promoter. The repressor, which is a protein of approximately 97 kDa, binds the murine aldolase A NRE, revealing a much more intense DNA-protein complex in proliferating NIH3T3 cells than in serum-deprived cells. Mutations in the negative regulatory cis-element showed that the GA-rich motif is required for protein binding and silencer function. We conclude that the expression of L-type mRNA is modulated by the interaction between a cell cycle-dependent DNA-binding protein and the murine aldolase A NRE.
A DNA region located at around -200 bp in the 5' flanking region (region D) of the human brain-type fructose-bisphosphate aldolase (aldolase C) gene has been analysed. We show by transient transfection assay and electrophoretic-mobility-shift assay (EMSA) that the binding of transcriptional activators to region D is much more efficient (80% versus 30%) in human neuroblastoma cells (SKNBE) than in the non-neuronal cell line A1251, which contains low levels of aldolase C mRNA. The sequence of region D, CAAGGTCA, is very similar to the AAAGGTCA motif present in the mouse steroid 21-hydroxylase gene; the latter motif binds nerve-growth-factor-induced B factor (NGFI-B), which is a member of the thyroid/steroid/retinoid nuclear receptor gene family. Competition experiments in EMSA and antibody-directed supershift experiments showed that NGFI-B is involved in the binding to region D of the human aldolase C gene. Furthermore, the regulation of the aldolase C gene (which is the second known target of NGFI-B) expression during development parallels that of NGFI-B.
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We screened the aldolase B gene in 14 unrelated Italian patients with hereditary fructose intolerance (HFI), and found two novel disease related mutations: a single nucleotide deletion in exon 2 (delta A20) that leads to an early stop codon, and a C-->T transition in exon 8 that substitutes an Arg with a Trp residue at codon 303 (R303W).
Transcription from the distal promoter (pL) of the human aldolase A gene is driven by both positive and negative cis-acting elements. With footprinting and gel mobility assays we defined: (i) the position of the negative regulatory cis-element (AldA-NRE), which spans a GA-rich sequence; (ii) the sequence of AldA-NRE, which is critical for protein specific binding and is homologous to other silencer-like motifs. Removal of the negative cis-element fully restores the expression of the distal promoter. Insertion of the AldA-NRE upstream from a heterologous promoter results in a 6-fold decrease in CAT reporter gene expression, suggesting that AldA-NRE might be involved in a more general type of mechanism that mediates repression of gene transcription.
Hunter disease, Mucopolysaccharidosis type II, is an X-linked recessive lysosomal storage disorder caused by a deficiency in iduronate sulfatase activity. We studied at molecular level a Neapolitan family with the disease. We report, in patient, the delta 139 mutation on the third exon of the gene, on female family members, the DNA analysis that allowed to assess or exclude their carrier status and on fetal DNA from a pregnancy of patient's mother, a prenatal diagnosis that resulted negative.
1. In order to assess the importance of the parabrachial nuclei in modulating cardiorespiratory activity, electric current or microinjections of glutamate were used to stimulate discrete regions of the parabrachial nuclei in anaesthetized rats. 2. Stimulation of cell bodies in the medial region of the parabrachial nuclei and in the Kölliker-Fuse nuclei, caused an expiratory facilitatory response. This consisted mainly of a decrease in respiratory rate as measured by observing phrenic nerve activity. 3. Stimulation of cell bodies in the lateral region of the parabrachial nuclei caused an inspiratory facilitatory response. This consisted mainly of an increase in respiratory rate. 4. At the majority of sites (16 out of 20) where changes in respiratory rate were elicited by glutamate injection or electrical stimulation an increase in blood pressure was observed. The coexistence of increases in blood pressure and heart rate indicates the presence of inhibition of the heart rate component of the baroreflex and/or an increase in cardiac sympathetic drive. 5. The expiratory facilitatory response was not evoked reflexly by the rise in blood pressure since it was still present after administration of guanethidine, which abolished the rise in blood pressure. 6. The interactions between the parabrachial nuclei and the medullary respiratory complex in eliciting these changes are discussed.
Two ubiquitous (L- and F-type) and one muscle-specific (M-type) mRNA species are generated by the human aldolase A gene. Despite the high degree of sequence similarities in the promoter region between human and rodents, no L-type mRNA expression has yet been found in the latter. Here we demonstrate that L-type aldolase A mRNA is expressed during the differentiation of mouse myogenic C2.7 and rat oligodendrocyte precursor CEINGE C13 cells. The L-type mRNA expression is increased during differentiation and is associated with cell-growth arrest caused by nocodazole treatment or serum deprivation in C2.7 and CEINGE C13 cells, respectively. The L-type aldolase A mRNA is correctly processed at the L1-L2 junction.
Direct sequencing and restriction enzyme digestion of the polymerase chain reaction (PCR) product encompassing the breakpoint were used to characterize the Sicilian (delta beta)(0)-thalassemia deletion in 33 unrelated Italian subjects. All cases showed the same sequencing features at the breakpoint region, suggesting a unique origin for this deletion in Italy. We also describe a one-step PCR assay for the rapid screening of homozygotes and carriers of Sicilian (delta beta) (0)-thalassemia by the simultaneous use of three specific oligonucleotides. This procedure could have an impact on genetic counseling of couples at risk for this type of thalassemia, and with respect to compound heterozygotes bearing a Sicilian chromosome.
Three major transcripts, differing in their 5'-untranslated regions, are produced from the human aldolase A gene by alternative usage of three promoters, designated distal, middle and proximal. We report that the genomic region (distal promoter) upstream from the first leader exon (exon L1) efficiently directs transcription of a reporter CAT gene after transient transfections in human hepatoma cells (Hep3B) in a fashion independent from the other two promoters of the same gene. The distal promoter region contains cis-acting elements that regulate transcription both positively and negatively.
We investigated the cis-acting sequences involved in the expression of the human aldolase C gene by transient transfections into human neuroblastoma cells (SKNBE). We demonstrate that 420 bp of the 5'-flanking DNA direct at high efficiency the transcription of the CAT reporter gene. A deletion between -420 bp and -164 bp causes a 60% decrease of CAT activity. Gel shift and DNase I footprinting analyses revealed four protected elements: A, B, C and D. Competition analyses indicate that Sp1 or factors sharing a similar sequence specificity bind to elements A and B, but not to elements C and D. Sequence analysis shows a half palindromic ERE motif (GGTCA), in elements B and D. Region D binds a transactivating factor which appears also essential to stabilize the initiation complex.
We investigated the molecular defects of the aldolase B gene in five unrelated patients affected by hereditary fructose intolerance. The techniques used were DNA amplification, direct sequencing and allele-specific oligonucleotide (ASO) hybridization. The most frequent substitutions found in the hereditary fructose intolerance alleles analysed were the A174D and the A149P mutations, which account for 50% and 30% of the alleles, respectively. In two unrelated families, we found a rare mutation, the MD delta 4 previously described only in one British family, which may be an important cause of the disease in Italy.
Congenital dyserythropoietic anaemia type II, or HEMPAS (hereditary erythroblastic multinuclearity with positive acidified serum lysis test) is a genetic disease caused by membrane disorganization of erythroid cells. The primary defect of this disease lies in the gene encoding enzyme(s) which is responsible for the biosynthesis of Asn-linked oligosaccharides chains of glycoproteins (Fukuda et al, 1990). In order to know whether this gene defect affects the glycosylation in the cells other than the erythroid cells, the carbohydrate structures of the transferrin isolated from the sera of HEMPAS patients were analysed. Fast atom bombardment mass spectrometry analysis showed the presence of high mannose type and hybrid type oligosaccharides in the HEMPAS transferrin which is in contrast to the complex-type oligosaccharides found in the normal transferrin. The results strongly suggest that biosynthesis of Asn-linked oligosaccharide chains in HEMPAS hepatocytes is disturbed. As a result, the serum glycoproteins with incompletely processed carbohydrates are circulating in the plasma in HEMPAS patients, but they must have been absorbed by the cells in the liver and the reticuloendothelial cells. Upon intravenous infusion into rats, as much as 30% of the HEMPAS transferrin was cleared from the plasma circulation. The majority of the HEMPAS transferrins was taken up by the liver, and transferrin was distributed both in the hepatocytes and the Kupffer cells. The presence of enormous amounts of aberrantly glycosylated serum glycoproteins may lead to the liver cirrhosis and secondary tissue siderosis seen in HEMPAS patients.
A young male from Apulia region (Southern Italy), heterozygous carrier of Hb-J Calabria (beta 64(E8) Gly-Asp), was described here. He showed mild polycythemia, and his whole blood dissociation curve for oxygen was clearly left-shifted, with a decreased p50. But these characteristics were not only due to the hyperaffinity of the abnormal pigment, accounting for about one third of the total Hb; in fact, also Hb-A, the main pigment present, had relative hyperaffinity. Nevertheless, this subject was completely symptomless, suggesting that the abnormal Hb was almost completely excluded from respiratory exchange and traveled always oxygenated also in the venous-capillary bed. However, the level reached by Hb-A furnished a satisfactory functional compensation against tissue hypoxia.