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B Saitta

Publications and source records attributed to B Saitta.

At least 37 records · Page 2Linked to original sources
PubMed1992

Human alpha 2(VI) collagen gene. Heterogeneity at the 5'-untranslated region generated by an alternate exon.

Cosmid clones containing the 5' region of the human alpha 2(VI) collagen gene have been isolated and characterized. DNA sequencing indicates that the signal peptide and the amino-globular domain are encoded by four exons of 142, 596, 21, and 66 base pairs (bp). However, S1 nuclease and primer extension analyses show that the transcription start site is not present in the 142-bp exon. Two different 5' cDNA clones are generated by the anchored polymerase chain reaction. Using the 5' cDNA clones as probes, two untranslated exons (1, 1A) are found 12 kilobase pairs upstream of the first coding exon. These two exons are alternatively used in human fibroblasts, and most transcripts contain exon 1 sequence. Exon 1 shows, by primer extension and S1 nuclease protection assay, two major and several minor transcription start sites. The promoter region contains a canonical TATA box, seven GGGCGG sequences, two possible CAAT boxes, and two sequences resembling AP2 binding sites. Exon 1A contains three alternative splice donor sites and is located 650 bp downstream of exon 1. The most 3' splice donor site of exon 1A is found within an Alu repeat sequence. Exon 1A is preceded by five GGGCGG sequences and one resembling the AP2 binding site although neither TATA or CAAT boxes are found. Two additional GGGCGG sequences are located at the beginning of exon 1A. This study establishes that the human alpha 2(VI) collagen gene is 36 kilobase pairs long and contains 30 exons. The 5'-untranslated and promoter regions are significantly different from the corresponding segments of the chicken gene. The human gene produces by alternative processing multiple mRNAs differing in the 5'-untranslated region as well as the 3'-coding and noncoding sequences.

Amino Acid Sequence↗
PubMed1991

Human alpha 3(VI) collagen gene. Characterization of exons coding for the amino-terminal globular domain and alternative splicing in normal and tumor cells.

We recently reported the isolation and sequencing of human cDNA clones corresponding to the alpha 3 chain of type VI collagen (Chu, M.-L., Zhang, R.-Z., Pan, T.-c., Stokes, D., Conway, D., Kuo, H.-J., Glanville, R., Mayer, U., Mann, K., Deutzmann, R., and Timpl, R. (1990) EMBO J. 9, 385-393). The study indicates that the amino-terminal globular domain of the alpha 3(VI) chain consists of nine repetitive subdomains of approximately 200 amino acid residues (N1-N9) and the gene appeared to undergo alternative splicing since some clones lacked regions encoding the N9 and part of the N3 subdomains. In the present study, we report the exon structure for the region encoding the amino-terminal globular domain of the human alpha 3(VI) chain. The nine repetitive subdomains are encoded by 10 exons spanning 26 kilobase pairs of genomic DNA. Eight of the repetitive subdomains (N2-N9) were found to be encoded by separate exons of approximately 600 base pairs each. The only exception is the N1 subdomain which is encoded by two exons of 417 and 146 base pairs. Characterization of the exon/intron structure showed that the cDNA variants were the result of splicing out of exon 9 (encoding the N9 subdomain) and part of exon 3 (encoding the N3 subdomain). Nuclease S1 analysis and the polymerase chain reaction demonstrated that exon 7 (N7 subdomain) was also subject to alternative splicing in normal skin fibroblasts. Examination of these splicing events by nuclease S1 analysis in normal fibroblasts, three different human tumor cell lines, and several human tissues showed that splicing out of exon 9 is much more efficient in normal as compared to tumor cells.

Adult↗
PubMed1991

The exon organization of the triple-helical coding regions of the human alpha 1(VI) and alpha 2(VI) collagen genes is highly similar.

The alpha 1(VI) and alpha 2(VI) chains, two of the three constituent chains of type VI collagen, are highly similar in size and domain structure. They are encoded by single-copy genes residing in close proximity on human chromosome 21. To study the evolution of the type VI collagen genes, we have isolated and characterized genomic clones coding for the triple-helical domains of the human alpha 1(VI) and alpha 2(VI) chains, which consist of 336 and 335 amino acid residues, respectively. Nucleotide sequencing indicates that, in both genes, the exons are multiples of 9 bp in length (including 27, 36, 45, 54, 63, and 90 bp) except for those encoding for regions with triple-helical interruptions. In addition, the introns are positioned between complete codons. The most predominant exon size is 63 bp, instead of 54 bp as seen in the fibrillar collagen genes. Of particular interest is the finding that the exon structures of the alpha 1(VI) and alpha 2(VI) genes are almost identical. A significant deviation is that a segment of 30 amino acid residues is encoded by two exons of 54 and 36 bp in the alpha 1(VI) gene, but by a single exon of 90 bp in the alpha 2(VI) gene. The exon arrangement therefore provides further evidence that the two genes have evolved from tandem gene duplication. Furthermore, comparison with the previously reported gene structure of the chick alpha 2(VI) chain indicates that the exon structure for the triple-helical domain of the alpha 2(VI) collagen is strictly conserved between human and chicken.

Amino Acid Sequence↗
PubMed1991

Human clathrin heavy chain (CLTC): partial molecular cloning, expression, and mapping of the gene to human chromosome 17q11-qter.

The nucleotide sequence of a 916-bp human cDNA clone isolated from a human colon lambda gt11 cDNA library was determined. Sequence analysis showed this cDNA to have 88% homology to the nucleotide sequence of the heavy chain of rat clathrin. The deduced amino acid sequence was 98.7% identical to the rat sequence, a change of only four amino acids. The mRNA identified in both human and rat cells with the human clathrin clone revealed transcripts of approximately 6.5 kb, which is consistent with the predicted 180 kDa molecular weight of the clathrin heavy chain. Southern analysis of human/rodent somatic cell hybrids localized the human clathrin heavy chain gene (CLTC) to chromosome 17. Additional analyses using panels of human/rodent somatic cell hybrids with specific chromosomal translocations and deletions mapped the human clathrin heavy chain gene locus to 17q11-qter.

Amino Acid Sequence↗
PubMed1991

Human nidogen gene: structural and functional characterization of the 5'-flanking region.

Nidogen is a sulfated multifunctional glycoprotein present in basement membranes. In this study, we have cloned the 5'-flanking region of the human nidogen gene. Initially, an approximately 35-kb DNA clone (NCos4) was isolated from a human cosmid genomic library. Southern hybridization of EcoRI-digested NCos4 allowed isolation of a 3.7-kb fragment, which was shown to contain a portion of intron 1, the entire exon 1, and approximately 0.9 kb of 5'-flanking sequences of the nidogen gene. Nucleotide sequencing of the 5'-flanking DNA revealed the presence of two canonic CCAAT consensus sequences in the antisense strand and a potential variant of the TATA motif, TATTT, in the sense strand. One putative AP-2 and six putative SP1 binding sites were also present. To test the functional promoter activity of the 5'-flanking genomic DNA, two nidogen promoter/CAT reporter gene constructs, with the promoter segment spanning from -864 to -1 and from -534 to -1, respectively, were developed and analyzed in transient transfections of human and mouse cell cultures. Both constructs showed clearly detectable promoter activity, and the activity of the larger construct could be up-regulated by 12-O-tetradecanoyl phorbol 13-acetate up to 2.5 times. The results indicate that the nidogen promoter/CAT gene constructs developed in this study provide a means to examine the transcriptional regulation of nidogen gene expression in human diseases of the basement membrane zone.

Amino Acid Sequence↗
PubMed1991

Proposal of a modified scintigraphic method to evaluate duodenogastroesophageal reflux.

Hepatobiliary scintigraphy with 99mTc-HIDA offers a noninvasive method to detect duodenogastric reflux. Biliary reflux was graded using the persistence rather than the intensity of the radioactive refluxate: Grade 0 was considered the absence of reflux, minimal reflux, or reflux in the first 10-15 min; Grade 1 was repetitive reflux lasting less than 10 min; Grade 2 was persistent reflux; and Grade 3 was reflux up to the esophagus. Twenty-five patients with foregut symptoms were studied and results were compared to 24-hr gastric pH monitoring. Scintigraphy and pH monitoring agreed in 15 out of 25 patients (60%), but no correlation was found with the endoscopic findings. The rationale for this approach is based on pathophysiologic evidence that damage to gastric and/or esophageal mucosa is mainly related to the prolonged contact time with duodenal contents. This technique seems to allow a complete functional evaluation of the esophagogastroduodenal tract without causing adjunctive irradiation or discomfort to the patient.

Adult↗
PubMed1990

Alternative splicing of the human alpha 2(VI) collagen gene generates multiple mRNA transcripts which predict three protein variants with distinct carboxyl termini.

We recently reported the isolation and sequencing of two classes of human alpha 2(VI) collagen cDNA clones which share common sequences for the first two-thirds of the molecule but contain a different sequence of either 607 or 887 base pairs at their 3' ends (Chu, M.-L., Pan, T.-C., Conway, D., Kuo, H.-J., Glanville, R. W., Timpl, R., Mann, K., and Deutzmann, R. (1989) EMBO J. 8, 1939-1946). In the present study, we report the sequence of another cDNA clone, which is identical to one class of the previously isolated cDNAs except for a 293-base pair insertion between the common and variable regions. Together, the different classes of cDNAs, referred to as the alpha 2C2, alpha 2C2a, and alpha 2C2a' predict three variant alpha 2 chains of type VI collagen with carboxyl globular domains of 429, 328, and 238 amino acid residues, respectively. In order to explore the mechanisms by which the variations are generated, we isolated and characterized the 3' end of the human alpha 2(VI) collagen gene. The carboxyl globular domain was found to be encoded by six exons which appear to delineate its structural subdomains. The exon/intron arrangement clearly demonstrated that the cDNA variants arose from alternative splicing events by mutually exclusive utilization of the last two exons in conjunction with the selective usage of an internal splice acceptor site in the penultimate exon. The presence of the corresponding mature mRNA transcripts (3.2-3.5 kilobase pairs (kb] in human fibroblasts was shown by Northern blot hybridization, S1 nuclease protection assay, and the polymerase chain reaction. The results indicated that the alpha 2C2 mRNA is the major species, whereas the alpha 2C2a and alpha 2C2a' are the minor forms. Northern blot hybridization also revealed an alpha 2(VI) collagen mRNA of 6.0 kb. This mRNA retained a 2.3-kb intron located between the two alternatively spliced exons and predicted a translational product that is the same as the alpha 2C2a variant.

Amino Acid Sequence↗
PubMed1990

Regulation of elastin gene expression: evidence for functional promoter activity in the 5'-flanking region of the human gene.

Analysis of nucleotide sequences in the 5'-flanking region of the human elastin gene has revealed several unusual features, suggesting that regulation of elastin gene expression is complex. To identify any cis-acting regulatory promoter elements, a 35-kb fragment of DNA (CosE) was isolated from a human genomic cosmid library by hybridizations with a human elastin cDNA. Southern blots of EcoRI digests of CosE DNA, utilizing a 5'-end labeled 21-mer oligonucleotide corresponding to the signal sequence of elastin, revealed the presence of a single 7.8-kb genomic fragment. Partial dideoxynucleotide sequencing of this EcoRI genomic subclone revealed that it extended approximately 2.5 kb 3' of the translation initiation site (ATG), encompassing exon 1 and a portion of the first intron, while the remaining DNA encompassed the 5'-flanking region. Exonuclease III digestion (3'----5') was performed to remove sequences of the first intron and first exon, including the ATG site. One clone, approximately 5 kb in size, had the 3' end located 14 bp upstream of the ATG site. A 462-bp 3' portion of this 5-kb fragment was subcloned into a Bluescript/CAT chimeric plasmid (pBS0CAT) to generate an elastin gene promoter/CAT reporter gene construct (pEP6CAT). Transient transfection experiments with pEP6CAT using human skin fibroblasts, human HT-1080, mouse NIH-3T3, or freshly isolated neonatal rat aortic smooth muscle cells revealed significant CAT activity in each cell line. These results suggest that the 5'-flanking region of the elastin gene contains the cis-acting regulatory elements necessary for transcription. The chimeric plasmid pEP6CAT provides a means to study the transcriptional control of elastin gene expression by exogenous affector molecules, as well as in human dermatologic diseases.

Base Sequence↗
PubMed1989

Isolation of a putative collagen-like gene from the sea urchin Paracentrotus lividus.

Using a Caenorhabditis elegans collagen probe we have isolated a 17.6 kb clone from a Paracentrotus lividus genomic library. Sequencing of nearly 2.6 kb identified five open reading frames flanked at both sides by splice site consensus sequences and coding for ninety-five uninterrupted Gly-X-Y repeats. Interestingly, three of the putative exons exhibit sizes which are identical to those featured by vertebrate fibrillar collagen genes, namely 54 bp and 99 bp. Hybridization of the Gly-X-Y encoding sequences to RNA extracted from different developmental stages identified a specific 6 kb transcript, which appears first at mid-gastrula, greatly increases at prism and then progressively accumulates until pluteus stage. Based on these data, we conclude that the genomic clone is likely to code for a developmentally regulated mRNA whose expression coincides with the reported time of appearance of collagenous molecules in the sea urchin embryo.

Amino Acid Sequence↗
PubMed1989

Single-photon emission computed tomography studies with 99mTc-hexamethylpropyleneamine oxime in dementia: effects of acute administration of L-acetylcarnitine.

The acute effects of intravenously administered L-acetylcarnitine (LAC) were evaluated with single-photon emission computed tomography (SPECT) and 99mTc-hexamethylpropyleneamine oxime in 30 demented patients (21 with a clinical diagnosis of Alzheimer's dementia and 9 with mixed-type dementia). Two SPECT scans were performed: in basal conditions, and 30 min after the administration of 500, 1,000, 1,500, or 2,000 mg LAC intravenously. Tracer activity ratios were determined in 10 pairs of cerebellar, cortical and subcortical regions. After administration of the lowest dose of LAC, no changes from the basal values were observed in any of the regions examined. The higher doses of the drug significantly elevated the tracer activity in cortical regions, particularly in the parietal lobe, which showed an impaired regional cerebral blood flow in the basal study. These effects of LAC and their relation with the cholinomimetic properties of the drug are discussed.

Acetylcarnitine↗
PubMed1989

Scintigraphy of the parathyroid glands with 201TI: experience with 250 operated patients.

Parathyroid scintigraphy confirmed its validity in the preoperative localization of enlarged parathyroids, showing a sensitivity of 82% in a series of 250 patients suffering from primary hyperparathyroidism and successfully operated on. The glands better visualized were in an ectopic site or they were completely or partially outside the thyroid so that they were easily visible without employing digital image subtraction. This is nevertheless necessary to visualize parathyroids in a retrothyroid site but some problems arise, related not only to movements of the patient but also to the instrumentation to perform a correct image subtraction.

Adenoma↗
PubMed1988

[99mTc]-HM-PAO SPECT in Parkinson's disease.

Thirty-six patients affected by Parkinson's disease were studied using single photon emission computed tomography (SPECT) and [99mTc]-HM-PAO as a tracer. The scanning procedure was performed 16-24 h after discontinuation of specific therapy. Tracer activity ratios were determined in 10 pairs of cerebellar, cortical, and subcortical regions. Data were compared with those of 10 age-matched controls. Most of the regions examined did not show any relevant change between parkinsonian and control subjects. Notably, mean activity in striatal regions were similar in the two groups. Increased activity in caudate-putamen was found in patients who were on chronic DOPA therapy. Side-to-side asymmetries in the basal ganglia increased with the severity of the disease. Significant reductions of tracer uptake, from control values, were observed bilaterally in the parietal cortex. These deficits were more pronounced in patients with mental deterioration and in subjects who had been chronically treated with anticholinergic drugs. Parietal perfusion deficits in parkinsonian patients resemble those described in Alzheimer's dementia. These findings suggest that the heterogeneous alterations of regional cerebral blood flow (rCBF) in parkinsonian patients reflect the multifactorial pathophysiology of the disease.

Adult↗
PubMed1985

alpha alpha alpha anti-4.2 Haplotype and heterozygous beta null thalassemia in a Sicilian family.

The presence of the alpha alpha alpha anti-4.2 haplotype and heterozygous beta null thalassemia in a Sicilian family is described. These findings confirm the presence in Italy of a leftward deletion (-alpha 4.2) and indicate that this may not be rare. Furthermore, although the beta thalassemia determinant in this family has a severe expression, the interaction with the triplicated alpha gene does not necessarily express itself as thalassemia intermedia.

Adult↗