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Biomedical subjects

M M Bashir

Publications and source records attributed to M M Bashir.

At least 19 recordsLinked to original sources
PubMed2001

Development of stratum intermedium and its role as a Sonic hedgehog-signaling structure during odontogenesis.

Stratum intermedium is a transient and subtle epithelial structure closely associated with inner dental epithelium in tooth germs. Little is known about its development and roles. To facilitate analysis, we used bovine tooth germs, predicting that they may contain a more conspicuous stratum intermedium. Indeed, early bell stage bovine tooth germs already displayed an obvious stratum intermedium with a typical multilayered organization and flanking the enamel knot. Strikingly, with further development, the cuspally located stratum intermedium underwent thinning and involution, whereas a multilayered stratum intermedium formed at successive sites along the cusp-to-cervix axis of odontogenesis. In situ hybridization and immunohistochemistry showed that stratum intermedium produces the signaling molecule Sonic hedgehog (Shh). Maximal Shh expression was invariably seen in its thickest multilayered portions. Shh was also produced by inner dental epithelium; expression was not constant but varied with development and cytodifferentiation of ameloblasts along the cusp-to-cervix axis. Interestingly, maximal Shh expression in inner dental epithelium did not coincide with that in stratum intermedium. Both stratum intermedium and inner dental epithelium expressed the Shh receptor Patched2 (Ptch2), an indication of autocrine signaling loops. Shh protein, but not RNA, was present in underlying dental mesenchyme, probably resulting from gradual diffusion from epithelial layers and reflecting paracrine loops of action. To analyze the regulation of Shh expression, epithelial and mesenchymal layers were separated and maintained in organ culture. Shh expression decreased over time, but was maintained in unoperated specimens. Our data show for the first time that stratum intermedium is a highly regulated and Shh-expressing structure. Given its dynamic and apparently interactive properties, stratum intermedium may help orchestrate progression of odontogenesis from cusp to cervix.

Animals↗
PubMed1998

Molecular cloning and characterization of the bovine and human tuftelin genes.

The bovine tuftelin gene was cloned and its structure determined by DNA sequence analysis and comparison to bovine tuftelin cDNA. The analyses demonstrated that the cDNA contains a 1014 bp open reading frame encoding a protein of 338 residues with a calculated molecular weight of 38,630 kDa and an isoelectric point of 5.85. Although similar, these results differ from those previously published [Deutsch et al. (1991) J. Biol. Chem. 266, 16021-16028] which contained a different conceptual amino acid sequence for the carboxy terminal region and identification of a different termination codon because of the absence of a single guanine residue in the published sequence. The protein does not appear to share homology or domain motifs with any other known protein. The bovine gene consists of 13 exons ranging in size from 66 to 1531 bp, the latter containing the encoded carboxy terminal and 3' untranslated regions. These exons are embedded in greater than 28 kbp of genomic DNA and codons are generally not divided at exon/intron borders. Sequence analysis of the cDNA and products produced by reverse transcriptase/polymerase chain reaction demonstrated that exons 2, 5 and 6 are alternatively spliced. The 3' portion of the human gene was also isolated and characterized by DNA sequencing, which demonstrated agreement between the bovine and human sequences in the segment in question. The difference between the presently reported sequence and that of the previously published one suggests the possibility of an unusual type of polymorphism which would result in markedly different amino acid sequences at the carboxy terminal region of the protein. The human tuftelin gene was localized to chromosome 1q21 by in situ hybridization.

Alternative Splicing↗
PubMed1997

Molecular cloning and characterization of the bovine tuftelin gene.

The bovine tuftelin gene was cloned and its structure determined by DNA sequence analysis and comparison to that of bovine tuftelin cDNA. The analyses demonstrated that the cDNA contains a 1014-bp open reading frame encoding a protein of 338 residues with a calculated mol. wt of 38,630 and an isoelectric point of 5.85. These results differ from those previously published, (1991) which contained a different conceptual amino acid sequence for the carboxy terminal region and identified a different termination codon. The protein does not appear to share homology or domain motifs with any other known protein. The gene consists of 13 exons ranging in size from 66 to 1531 bp, the latter containing the encoded carboxyterminal and 3' untranslated regions. The exons are embedded in more than 28 kbp of genomic DNA. Codons are generally not divided at exon/intron borders. Several alternatively spliced transcripts were identified by DNA sequence analysis of the isolated products produced by reverse transcriptase/polymerase chain reaction.

Alternative Splicing↗
PubMed1997

Stabilization of elastin mRNA by TGF-beta: initial characterization of signaling pathway.

The cytokine transforming growth factor-beta (TGF-beta) has multiple effects on a wide variety of cell types. These effects include modulation of growth and regulation of gene transcription. In a few instances, TGF-beta has also been shown to regulate gene expression posttranscriptionally by altering message stability, but the pathway by which this activity is executed remains largely unknown. In the present work, we demonstrate that TGF-beta 1 has no effect on transcription of the elastin gene in cultured human fetal lung fibroblasts, but does stabilize elastin messenger RNA (mRNA), leading to a dramatic increase in the steady-state level of elastin mRNA. A corresponding increase in production of tropoelastin accompanies the increase in elastin mRNA. Through the use of specific inhibitors, we demonstrate that phosphatidylcholine (PC)-specific phospholipase C (PLC) and protein kinase C (PKC) are involved in mediating the elastin message stabilization. In contrast, G proteins and extracellularly regulated kinases do not appear to be involved. These results suggest that although the TGF-beta signaling pathway leading to message stabilization shares components with that modulating transcription, the message-stabilization pathway also contains diverse other elements.

Bridged-Ring Compounds↗
PubMed1996

Insulin-like growth factor-I regulates transcription of the elastin gene through a putative retinoblastoma control element. A role for Sp3 acting as a repressor of elastin gene transcription.

Previous studies have demonstrated that insulin-like growth factor-I (IGF-I) increases elastin gene transcription in aortic smooth muscle cells and that this up-regulation is accompanied by a loss of protein binding to the proximal promoter. Sp1 has been identified as one of the factors whose binding is lost, and in the present study we show that Sp3 binding is also abrogated by IGF-I, but in a selected manner. In functional analyses using Drosophila SL-2 cells, Sp1 expression can drive transcription from the elastin proximal promoter, while co-expression of Sp3 results in a repression of Sp1 activity. Footprint and gel shift analyses position the IGF-I responsive sequences to a putative retinoblastoma control element (RCE). Mutation of the putative RCE sequence as assessed by transient transfection of smooth muscle cells results in an increase in reporter activity equal in magnitude to that conferred by IGF-I on the wild type promoter. Together these results support the hypothesis that IGF-I-mediated increase in elastin transcription occurs via a mechanism of derepression involving the abrogation of a repressor that appears to be Sp3 binding to the RCE.

Animals↗
PubMed1996

Analysis of the human gene encoding latent transforming growth factor-beta-binding protein-2.

Transforming growth factor (TGF)-beta is secreted as an inactive complex, which frequently contains a large molecular weight binding protein designated latent TGF-beta-binding protein (LTBP). Recently, the LTBPs have been shown to be a gene family that contains three known members and exhibits a multidomain structure containing cysteine-rich motifs that are also found in the fibrillin gene family. The present work seeks to characterize the gene encoding LTBP-2 and to compare its features to that of the other LTBPs and to the fibrillins. Human fibroblast libraries were used to isolate cDNA encoding LTBP-2 which was then used to identify LTBP-2 transcripts and to isolate the corresponding LTBP-2 gene. The cloned cDNA encodes a 195 kDa protein containing 20 epidermal growth factor (EGF)-like repeats, three repeats containing eight cysteines, and one segment that appears to be a hybrid of the two. Single exons encode EGF repeats while the eight-cysteine repeats are encoded in two exons. Northern analysis identified two transcripts of 7.5 and 9.0 kb, with the presently analyzed cDNA probably corresponding to the 7.5 transcript. Phylogenetic sequence comparisons demonstrated that LTBP-3 is more similar to LTBP-1 than LTBP-2, while LTBP-2 shows the most similarity to the fibrillins. These analyses suggest that LTBP-1 diverged from LTBP-3, and that LTBP-2 diverged from LTBP-1. Within the fibrillin family, fibrillin-1 is nearest to the LTBPs. While the domain structure of LTBP-2 is similar to that of the other LTBPs, LTBP-2 possesses unique regions that make it the largest member of the LTBP family. LTBP-2 may have dual functions as a member of the TGF-beta latent complex and as a structural component of microfibrils.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Molecular cloning of the microfibrillar protein MFAP3 and assignment of the gene to human chromosome 5q32-q33.2.

Microfibrils having a diameter of 10-12 nm, found either in association with elastin or independently, are an important component of the extracellular matrix of many tissues, but characterization of these microfibrils is incomplete. To further our understanding of the gene structure of proteins composing the microfibrils and to identify their chromosomal location, we have cloned and characterized another microfibril protein, designated microfibril-associated protein-3 (MFAP3). The human gene encoding MFAP3 has a very simple structure, containing only two translated exons encoding a protein of 362 amino acids. Monospecific antibodies prepared against the recombinantly expressed protein reacted with the microfibrils found in ocular zonules. MFAP3 does not appear to share homology with any other known protein. The gene was found to be located on chromosome 5q32-q33.2, near the locus 5q21-q31 reported for the fibrillin gene, FBN2, which has been linked to congenital contractural arachnodactyly. MFAP3 is a candidate gene for heritable diseases affecting microfibrils.

Amino Acid Sequence↗
PubMed1995

Skin elastic fibres: regulation of human elastin promoter activity in transgenic mice.

Elastic fibres form an extracellular network which provides elasticity and resilience to tissues such as the skin. To study the regulation of human elastin gene expression, we have developed a line of transgenic mice which harbour 5.2 kb of human elastin gene promoter region in their genome. This promoter is linked to the chloramphenicol acetyltransferase (CAT) reporter gene which allows determination of the expression of human elastin promoter in different tissues. The highest CAT activity was found in the lungs and aorta, tissues rich in elastin, while lower levels were detected in a variety of other tissues, including skin. Assay of CAT activity in the lungs of fetal and newborn animals revealed high activity which progressively declined during the postnatal period up to six months. Thus, there was evidence of tissue-specific and developmentally regulated expression of the human elastin promoter activity in these mice. These animals were then used to examine the expression of the elastin gene by a variety of factors which have previously shown to alter elastin gene expression, as determined at the mRNA or protein levels. First, injection of transforming growth factor beta 1 (100 ng) subcutaneously into the transgenic animals resulted in a time-dependent elevation of the promoter activity up to 10-fold after a single injection. Secondly, enhancement of the human elastin promoter activity by interleukin 1 beta injected subcutaneously resulted in an approximately 10-fold elevation of the CAT activity. Finally, subcutaneous injection of these animals with triamcinolone acetonide or dexamethasone, two glucocorticosteroids in clinical use, resulted in marked enhancement of human elastin promoter activity. Similar changes were noted in fibroblast cultures established from the transgenic animals. These data indicate that the 5.2 kb upstream segment of the human elastin gene contains cis-elements which allow tissue-specific and developmentally regulated expression of the human elastin promoter. Furthermore, this segment of the gene contains responsive elements to a variety of cytokines and pharmacological agents. Collectively, these data indicate that elastin gene expression in the skin in vivo can be regulated at the transcriptional level.

Animals↗
PubMed1995

Structure of the elastin gene.

The isolation and characterization of cDNAs encompassing the full length of chicken, cow, rat and human elastin mRNA have led to the elucidation of the primary structure of the respective tropoelastins. Large segments of the sequence are conserved but there are also considerable variations which range in extent from relatively small alterations, such as conservative amino acid substitutions, to variation in the length of hydrophobic segments and largescale deletions and insertions. In general, smaller differences are found among mammalian tropoelastins and greater ones between chicken and mammalian tropoelastins. Although only a single elastin gene is found per haploid genome, the primary transcript is subject to considerable alternative splicing, resulting in multiple tropoelastin isoforms. Functionally distinct hydrophobic and cross-link domains of the protein are encoded in separate exons which alternate in the gene. The introns of the human gene are rich in Alu repetitive sequences, which may be the site of recombinational events, and there are also several dinucleotide repeats, which may exhibit polymorphism and, therefore, be effective genetic markers. The 5' flanking region is G+C rich and contains potential binding sites for numerous modulating factors, but no TATA box or functional CAAT box. The basic promoter is contained within a 136 bp segment and transcription is initiated at multiple sites. These findings suggest that the regulation of elastin gene expression is complex and takes place at several levels.

Alternative Splicing↗
PubMed1994

Tissue-specific and developmentally regulated expression of human elastin promoter activity in transgenic mice.

We have recently cloned the entire human elastin gene, including approximately 5.2 kilobases of the 5'-flanking sequences. To examine tissue-specific expression of the elastin gene, we have developed a transgenic mouse line that expresses the human elastin promoter linked to the chloramphenicol acetyltransferase (CAT) reporter gene. Assay of CAT activity in different tissues revealed the highest expression in the lungs and aorta, while lower levels were detected in the kidneys, heart, brain, and skin; this distribution parallels the accumulation of elastin in developing animals. Comparison of CAT activity in the lungs of fetal (15-day gestation) and newborn (5-day postnatal) animals revealed significantly (approximately 4-fold) higher activity in the fetal tissue. The relatively high activity in the lungs progressively declined during the postnatal period up to 6 months. The promoter activity in the aorta remained constant from 5 days to 3 months and then gradually declined, while in the skin, the activity peaked at 3 months, returning thereafter to the control (5-day) level. Thus, there is evidence for developmentally regulated, tissue-specific expression of the elastin promoter in vivo as tested in these transgenic mice.

Aging↗
PubMed1994

Microfibril-associated glycoprotein: characterization of the bovine gene and of the recombinantly expressed protein.

Microfibrils having a diameter of 10-12 nm, found either in association with elastin or independently, are an important component of the extracellular matrix of many tissues. Because isolation of native proteins composing the microfibrils has proven difficult, information on structure/function relationships is limited. In order to extend our understanding of the 31-kDa microfibril-associated glycoprotein (MAGP), the bovine gene has been cloned and characterized and the protein has been expressed in a eukaryotic system. The compact coding portion of the gene is contained in 4.5 kbp of genomic DNA and does not appear to share any domain motifs with other known proteins. The size, amino acid composition, and sequence of the amino terminus of the secreted recombinant protein (rMAGP) all agree with values predicted by the nucleotide sequence of the cDNA used in the expression vector. The rMAGP reacts with a monospecific antibody prepared against a defined amino acid sequence of the natural molecule and reacts specifically with recombinantly produced tropoelastin, suggesting that rMAGP will be a useful reagent with which to study its interaction with other extracellular matrix components.

Amino Acid Sequence↗
PubMed1992

Tumor necrosis factor-alpha down-regulates human elastin gene expression. Evidence for the role of AP-1 in the suppression of promoter activity.

Cytokine modulation of elastin gene expression was examined by assay of elastin mRNA abundance and by transient transfections of cultured human skin fibroblasts and rat aortic smooth muscle cells with elastin promoter/reporter gene (chloramphenicol acetyltransferase, CAT) constructs. Incubation of cells with human recombinant tumor necrosis factor-alpha (TNF-alpha) markedly suppressed the elastin mRNA levels in a time- and dose-dependent manner by up to 91%. TNF-alpha also suppressed the expression of the elastin promoter/CAT construct by up to 70% in transiently transfected cells, indicating regulation at the transcriptional level. This suppression was temporally preceded by rapid and transient up-regulation of c-jun and c-fos genes. The down-regulatory effect of TNF-alpha on elastin promoter activity was abolished by co-transfections with a synthetic double-stranded AP-1 oligomer. Furthermore, co-transfection of the elastin promoter construct with c-jun and c-fos expression plasmids resulted in a marked decrease in the promoter activity. Elucidation of the cis-regulatory elements in the elastin promoter by 5' deletion construct analysis implicated a region -290 to -198 containing one AP-1 binding site. The functional role of this AP-1 site was further tested by gel retardation assays which indicated formation of a DNA-protein complex specific for TNF-alpha treated cells. This complex could be partially dissociated by a competing oligomer containing the consensus AP-1 binding site. These observations suggest that the inhibitory effects of TNF-alpha on elastin gene expression involve the transcription factor AP-1. Interferon-gamma also suppressed the elastin gene expression at the mRNA level by approximately 52%, but it had no effect on the elastin promoter activity, suggesting post-transcriptional mechanisms. These results indicate that mediators released from inflammatory cells can modulate elastin gene expression, and such modulation may play a role in diseases characterized by altered accumulation of elastic fibers in tissues.

Adult↗
PubMed1990

Deletion analyses of 5'-flanking region of the human elastin gene. Delineation of functional promoter and regulatory cis-elements.

To delineate cis-acting regulatory elements of the human elastin gene, several elastin promoter region/chloramphenicol acetyltransferase reporter gene constructs were developed. The spectrum of inserts, spanning from -2260 to +2, was shown to contain several SP-1 and AP2 binding sites, as well as putative glucocorticoid, cAMP, and 12-O-tetradecanoylphorbol-13-acetate responsive elements. Assay of promoter activity in transient transfections of rat aortic smooth muscle cells, human skin fibroblasts, HT-1080 human fibrosarcoma cells, HeLa cells, or mouse NIH-3T3 cells allowed delineation of several functional subregions within 2.26 kilobases of the 5'-flanking DNA. The results suggest that the basic promoter element resides within the region -128 to -1, and the 5'-flanking DNA contains several functional regulatory subregions. Also, the regulatory function of three putative SP-1 binding sites was demonstrated by transfections with a plasmid devoid of such sequences. These findings attest to the complexity of transcriptional regulation of the elastin gene.

Animals↗
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

Characterization of the complete human elastin gene. Delineation of unusual features in the 5'-flanking region.

Genomic clones encompassing the entire human elastin gene, including 11 kilobases flanking the ATG translation initiation codon, have been obtained and characterized by restriction enzyme analysis and extensive DNA sequencing. These analyses demonstrated that functionally distinct hydrophobic and cross-linking domains of the protein are segregated into separate exons throughout the gene. All exons are multiples of three nucleotides, and exon:intron borders always split codons in the same way which permits cassette-like alternative splicing. The 5'-flanking region lacks a canonical TATA sequence, is G + C-rich, and contains several SP1 binding sites and an AP2 binding site. Primer extension and S1 nuclease protection experiments indicate that transcription is initiated at multiple sites in the gene.

Amino Acid Sequence↗
PubMed1989

Structure of the bovine elastin gene and S1 nuclease analysis of alternative splicing of elastin mRNA in the bovine nuchal ligament.

Genomic clones encompassing all the translated sequences, the 3' untranslated sequence, and 1 kb flanking the ATG translation initiation codon of bovine tropoelastin have been obtained and characterized by restriction enzyme analysis and extensive DNA sequencing. These analyses demonstrated that functionally distinct hydrophobic and cross-linking domains of the protein are segregated into separate exons throughout the gene. The putative promoter region lacks a TATA box, has an extremely high G+C content, and contains several SP1 binding sites. Comprehensive S1 analyses using probes covering the entire mRNA and RNA isolated from the nuchal ligament of bovine fetuses of different ages, neonate calves, and adult cows demonstrated that while only a single exon is alternatively spliced at high frequency, many exons are alternatively spliced at limited, variable frequencies. The results also suggest that such limited splicing is increased in the adult tissue relative to fetal and neonate tissues.

Amino Acid Sequence↗
PubMed1988

The reciprocal partners of both the t(14; 18) and the t(11; 14) translocations involved in B-cell neoplasms are rearranged by the same mechanism.

Both the t(14; 18) and the t(11; 14) chromosomal translocations are associated with human B-cell neoplasms. Sequence analysis of the regions on chromosomes 18q- and 11q- involved in the joining of chromosomes 14 and 18 and of chromosomes 11 and 14 during their respective translocations indicates the involvement of the DH region. There is a deletion of the region between the involved D segment and the JH region, and extranucleotides are present at the joining sites on the 18q- and on the 11q- chromosomes. We have previously reported that the joining region on chromosome 14q+ involved in both the t(11; 14) and the t(14; 18) translocations are structurally identical. We now report that the joining regions on chromosomes 11q- and 18q- involved in these two different translocations are also identical. Thus, these translocations arose by the same mechanisms. The deletions of the region between the involved segment and the JH region must likely occur during either a D-J recombination event on the 18q- and 11q- chromosomes following the translocations or, alternatively, during the D-J recombination event that results in the translocation.

B-Lymphocytes↗