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B Saitta

Publications and source records attributed to B Saitta.

At least 19 recordsLinked to original sources
PubMed1996

Presence of different collagens and collagen mRNAs during embryogenesis and in adult tissues of the sea urchin Paracentrotus lividus.

The presence of four different collagen genes had been previously described in the sea urchin genome and four different cDNAs had been cloned and sequenced. Two of them code for 140 and 300 KDaltons proteins, belonging to the fibrillar collagens, and the other two families code for two type IV collagens with a molecular weight of about 210 KDaltons. In this paper immunological evidence is provided for the presence in the developing P. lividus sea urchin embryo of at least seven major collagen proteins. Western blot analyses, carried out by means of specific polyclonal antibodies, show a series of collagenase sensitive bands, with molecular weights ranging from 55 to 200 KDaltons, which are present from eggs to plutei. Northern blot analyses show the presence of the previously described 6 and 9 Kb RNA bands from oocytes till plutei; in the later stages two other collagen RNAs are detected. The presence of two sets of genes coding for the 6 Kb mRNAs, differentially expressed during development, is also discussed. Immunofluorescence histological analyses show the location of collagen in gonads, oocytes, eggs, embryos and adult tissues.

Animals↗
PubMed1995

Characterization of the human alpha 1(VI) collagen promoter and its comparison with human alpha 2(VI) promoters.

From a human cosmid library, we isolated a clone (5B) with an insert of 32 kb, encoding the amino-terminal and the 5'-end flanking region of the alpha 1(VI) collagen gene. Exon 1 was found to be 194 bp and contain the 5' untranslated region plus 97 bp coding sequence. Exon 2 consists of 130 bp, a size that is conserved across the chicken and mouse species. S1-nuclease-protection assays and primer-extension analysis, using mRNA from human dermal fibroblasts, show the presence of multiple transcription start sites located in a region of approximately 20 nucleotides. Canonical TATA and CAAT boxes, as found in the chicken and mouse alpha 1 promoters, were absent in the human alpha 1(VI) promoter. The promoter region from positions -1 to -190, is a polypyrimidine/polypurine-rich region containing 12 CCCTCCCC (CT element consensus) sequences and has multiple potential binding sites for the Sp1, and AP2 transcription factors. These regulatory proteins bind to the alpha 2(VI) promoters [Saitta, B. & Chu, M.-L. (1994) Eur. J. Biochem. 223, 675-682]. To test the transcriptional activity of the alpha 1 promoter, transient transfection experiments of the DNA constructs were performed in human dermal fibroblasts and in human fibrosarcoma (HT1080) cell lines. The DNA constructs drive the expression of the chloramphenicol acetyl transferase (CAT) gene. The results show strong CAT activity for the constructs at positions -1700, -298 and -257, while low activity was found for the constructs at positions -4400, -142 and -5 when transfected in fibroblasts. The experiments also identified positive and negative regulatory regions in the alpha 1(VI) promoter CAT constructs when transfected in fibroblasts, but did not identify them in the fibrosarcoma cells.

Animals↗
PubMed1995

Head to tail organization of the human COL6A1 and COL6A2 genes by fiber-FISH.

Two type VI collagen genes, COL6A1 and COL6A2, both map to 21q22.3, but the order, distance, and organization of these two genes relative to one another were not known. Recently developed high-resolution fluorescence in situ hybridization (FISH) techniques have great potential to facilitate the construction of fine-resolution maps of telomeric regions where gene density is high. Here we have determined the distance separating the COL6A1 and COL6A2 genes (150 kb), the size of the COL6A1 gene (29 kb); and the 5'-3' orientation of these genes (5' COL6A1 3'-5' COL6A2 3') using fiber-FISH.

Chromosome Mapping↗
PubMed1995

Myocardial technetium-99m sestamibi single-photon emission tomography as a prognostic tool in coronary artery disease: multivariate analysis in a long-term prospective study.

To date several studies have evaluated the accuracy of thallium-201 myocardial scan in risk stratification of coronary artery disease (CAD), while reports using technetium-99m methoxyisobutylisonitrile (MIBI), a tracer particularly suited to single-photon emission tomographic (SPET) imaging, are lacking. To rectify this omission, a prospective study was started in 1988 and at present 176 consecutive, and thus unselected, patients have been enrolled. All of them have been submitted to stress-rest MIBI SPET for the diagnosis or evaluation of CAD; 147 patients (121 males and 26 females, aged 53 +/- 9 years) have completed a surveillance period of at least 36 months following the scintigraphic study (range 36-60 months, mean 43). Sixty-one patients had a documented previous myocardial infarction. The mean pre-test likelihood of CAD was 44% in the patients without prior infarction. The main anamnestic, clinical, EKG and scintigraphic findings were evaluated and statistically correlated with the incidence of ensuing cardiac events using both univariate (chi-square test) and multivariate analysis (logistic regression model). Twenty-nine patients suffered from a cardiac event during the follow-up period (i.e. three cardiac deaths, six myocardial infarctions and 20 cases of unstable angina). Statistical multivariate analysis identified MIBI scan as the only highly significant and independent prognostic predictor [P = 0.006, relative risk (RR) = 17.62]. In detail, the most important scintigraphic parameters were the presence of a reversible defect (P = 0.0089, RR = 5.11) and the extension of the stress perfusion defect (P = 0.0255, RR = 3.27). The presence of typical angina proved to be a slightly significant predictor (P = 0.051, RR = 2.45), while no other examined parameter showed a significant correlation with a bad prognosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Angina, Unstable↗
PubMed1995

Usefulness of 99mTc-MIBI stress myocardial SPECT bull's-eye quantification in coronary artery disease.

99mTc-methoxy-isobutyl isonitrile (MIBI) myocardial SPECT quantification performed using a Bull's-eye polar map, was evaluated and compared with visual analysis in 120 patients with proven or suspected CAD. The study series comprised 106 men and 14 women, age 37-75 years (mean 51 +/- 6), 68 of whom had had a prior myocardial infarction. Coronary angiography was taken as the gold standard: one-vessel disease was present in 24 patients, two-vessel disease in 39, and three-vessel disease in 44, whereas no significant stenosis was documented in 13 cases. Forty age-matched subjects (26 men, 14 women), with less than a 5% chance of having CAD, were enrolled to establish the normal database for males and females. ROC analysis was used to calculate the optimal thresholds for the definition of the disease extension in each vascular territory of the Bull's-eye polar map: 10% for LAD, 8% for LCX, and 20% for RCA territory. The sensitivity/specificity ratio of the scintigraphy was: 75/82% with the visual and 78/74% with the quantitative analysis for LAD; 60/90% with visual and 72/64% with visual and 70/62% with quantitative analysis for RCA territory. The sensitivity/specificity ratios for the CAD diagnosis were similar with the visual and the Bull's-eye analysis in 92/61% and 93/61% respectively. Bull's-eye analysis agreed with visual analysis in 296/360 vessels. Two and three-vessel disease were most frequently observed using the Bull's-eye approach.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Two promoters control the transcription of the human alpha 2(VI) collagen gene.

Our previous studies have demonstrated that the human alpha 2(VI) collagen gene produces four mRNA species with different 5'-untranslated regions [Saitta, B., Timpl, R. & Chu, M.-L. (1992) J. Biol. Chem. 267, 6188-6196]. The major mRNA species initiates from exon 1, located at the most 5' end, whereas three minor mRNAs start from an alternative exon, 1A, located 657 bp downstream of exon 1. In this study, we have investigated whether or not these different mRNAs are transcribed from two separate promoters. DNA fragments preceding exons 1 and 1A were fused with a reporter gene for chloramphenicol acetyl transferase (CAT) and transfected into human dermal fibroblasts and fibrosarcoma HT1080 cells. Strong CAT activity in both cell types was observed using a construct containing DNA from nucleotide -502 to + 115 preceding exon 1. The CAT activity of a construct containing nucleotide +514 to +894 preceding exon 1A was almost as high as that of the former construct, indicating the presence of two promoters, P1 and P2, preceding exons 1 and 1A, respectively. Transient transfection assays also identified positive and negative regulatory regions for the P1 promoter, located from nucleotide -2152 to -1384 and from nucleotide -1383 to -503, respectively. A negative regulatory region located at nucleotide +116 to +513 was found for the P2 promoter. This region strongly inhibits the P2 promoter in dermal fibroblasts, and thus may be responsible for the low expression of the endogenous exon-1A-containing mRNAs in these cells. Footprinting analysis of the two promoters with purified Sp1 protein and AP2 protein extract showed several sites of DNA-protein interaction. The specificity of these sites was confirmed by competition experiments using consensus Sp1 and AP2 oligonucleotides. The results thus demonstrate that the human alpha 2(VI) collagen gene contains two promoters, which are regulated by positive and negative cis-acting DNA elements and trans-acting factors.

Base Sequence↗
PubMed1994

Effects of exercise-induced alterations of cardiac physiology on nuclear perfusion imaging.

The effects produced on nuclear perfusion images by exercise-induced changes in the heart and respiration rate and possible transient myocardial stunning are not well understood. In this study we attempted to indirectly evaluate the potential artifacts created by exercise-induced changes in cardiac physiology. Twenty patients with prior myocardial infarction and suspected peri-infarct ischemia were studied by 99mTc-MIBI imaging. Two SPECT perfusion studies were performed after 99mTc-MIBI administration at rest. The first acquisition was carried out 90 minutes after injection of the tracer. Immediately afterwards, the patients underwent a stress test followed by a second acquisition (exercise stress superimposed on rest tracer distribution). A standard stress perfusion scan was also performed 48 hours later. Superimposed exercise stress produced artifactual defects in the resting distribution of the tracer in 15 out of 20 patients (68 of 360 segments). Standard stress images demonstrated concordant defects in 48 of these segments, indicating the concomitant presence of ischemia and stunning. This study indicates that exercise-induced changes in cardiac physiology may result in artifactual perfusion defects in scintigraphic images acquired shortly after the stress.

Adult↗
PubMed1993

Dopamine receptor SPET imaging in Parkinson's disease: a [123I]-IBZM and [99mTc]-HM-PAO study.

Single photon emission tomography (SPET) with the novel ligand [123I]-IBZM was used to image central dopamine D2 receptors in Parkinson's disease (PD) patients. The aim was to assess basal ganglia (BG) receptor densities in relation to the response to L-dopa therapy. To better characterize the clinical potential of [123I]-IBZM SPET, each patient underwent a second study with the regional perfusion tracer [99mTc]-HM-PAO. Tracer activity ratios were calculated for caudate and putamen with mean activity over the cerebellar hemispheres as internal standard. In PD patients we found a significant decline of mean caudate [123I]-IBZM activity, as compared with age-matched control subjects. However, when patients were grouped according to their therapeutic behavior, the [123I]-IBZM uptake in BG ganglia regions of the PD patient group with a poor and fluctuating response to L-dopa was significantly reduced from mean values of patients with a sustained response to L-dopa therapy. [99mTc]-HM-PAO caudate and putamen uptake indexes in PD were similar to control values, even in patients with deteriorated therapeutic response. Our results indicate that BG D2 receptor alterations in PD may contribute to the altered response to L-dopa.

Adult↗
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↗