Biomedical subjects

J Rosenbloom

Publications and source records attributed to J Rosenbloom.

At least 109 records · Page 6Linked to original sources

PubMed1990

Marfan syndrome: exclusion of genetic linkage to five genes coding for connective tissue components in the long arm of chromosome 2.

Marfan syndrome represents a heterogeneous connective tissue disease, the symptoms arising in several tissues and organs. The defective gene(s) behind this autosomal dominant condition has not been found despite considerable research. The main targets of the research have been the genes coding for connective tissue components. Several of the candidate genes suspected to be defective in Marfan syndrome are located on the long arm of chromosome 2. These genes include a cluster of two genes coding for fibrillar collagens COL3A1 and COL5A2, and a third member of the collagen gene family: COL6A3. Furthermore, genes for elastin (ELN) and fibronectin (FN) are also located in this area of chromosome 2. We studied this chromosomal area using restriction fragment length polymorphism (RFLP) linkage analysis in five Finnish Marfan families with affected members in three generations. In two point linkage analyses, Lod scores of -3.192 (theta = 0.1) to COL3A1, -1.683 (theta = 0) to COL6A3 and -2.664 (theta = 0.01) to FN were obtained, whereas the linkage analysis between elastin and the disease was non-informative (Lod score 0.444, theta = 0). With the multipoint linkage analysis that permits simultaneous examination of several loci and more efficient use of family data, we obtained an exclusion of all these loci as the site of the mutation leading to Marfan syndrome in these families.

Chromosomes, Human, Pair 2↗

PubMed1990

Production of recombinant human tropoelastin: characterization and demonstration of immunologic and chemotactic activity.

Tropoelastin cannot readily be prepared in quantity from natural sources and this has limited research in several important areas including structure/function relationships and fiber assembly. In order to eliminate this limitation, human tropoelastin has been expressed in a recombinant bacterial system and the protein has been highly purified. The size, amino acid composition, and sequence of the amino terminus of the recombinant tropoelastin (rTE) all agree with values predicted by the nucleotide sequence of the cDNA used in the expression vector. The rTE exhibits cross-reactivity with antibodies directed against a mixture of peptides derived from human elastin as well as antibody against a defined peptide located at the carboxy terminus of the protein. In addition, the rTE is chemotactic for fetal calf ligament fibroblasts. These results suggest that rTE could be a useful reagent for many types of studies.

Animals↗

PubMed1990

Immunocytochemical localization of enamelin proteins in developing bovine teeth.

Enamelins were localized at both the light and electron microscopic level using an antienamelin monoclonal antibody and indirect immunogold methods. Bovine fetal incisors (crown-rump length 17-30 cm) were preserved in Karnovsky's fixative and embedded in Epon. For light microscopy, 2 microM thick sections were immunostained by the indirect method using the monoclonal antibody and goat anti-mouse IgG linked to 5 nM gold particles, followed by silver enhancement to increase the sensitivity of the method. For electron microscopy, thin sections were immunostained (indirect) with the antienamelin monoclonal antibody and goat anti-mouse IgG linked to 5 or 15 nM gold. Control samples were treated with an unrelated monoclonal antibody. The localization of enamelins was confined in the light microscopic sections to the extracellular enamel matrix. No gold staining was observed in the ameloblasts or other enamel organ cells even though the gold-silver technique is extremely sensitive. Ultrastructurally, enamelin was localized in the enamel extracellular matrix and associated ameloblasts. Both the crystal-containing and granular matrix were positively stained, with most gold particles being closely associated with the crystals. Counting of gold particles indicated more than 4 times as many amelogeninas enamelin-reactive antigenic sites in similar regions. Decalcification did not increase immunostaining with the anti-enamelin antibody in the extracellular matrix. Within ameloblasts, the gold particles were associated with secretory granules and Golgi complexes. Thus it appears that enamelins are synthesized in ameloblasts and secreted into the extracellular matrix in a similar manner to amelogenins and are preferentially associated with matrix hydroxyapatite crystals. Transient levels of enamelins within the ameloblasts are apparently too low to be detected by light microscopy.

Ameloblasts↗

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↗

PubMed1990

Interferon-gamma reverses the stimulation of collagen but not fibronectin gene expression by transforming growth factor-beta in normal human fibroblasts.

It has recently become apparent that several cytokines and growth factors are capable of modulating fibroblast proliferation and biosynthetic activity. To understand the role of these factors in connective tissue regulation, we examined the effects of the simultaneous addition of interferon-gamma (IFN-gamma) and transforming growth factor-beta (TGF-beta) on normal human dermal fibroblast collagen and fibronectin production. In addition, in vitro transcription rates and steady-state mRNA levels for these molecules were determined by nuclear run-off assays and Northern and dot-blot hybridization using specific human cDNA probes. Treatment of cultures with TGF-beta caused stimulation of collagen and fibronectin production. Addition of IFN-gamma to the TGF-beta-treated cultures abrogated the stimulatory effects of TGF-beta on collagen production in a dose-dependent manner and resulted in a net inhibition of collagen production. In contrast, the increase in fibronectin synthesis induced by TGF-beta was augmented further by IFN-gamma. These changes in collagen and fibronectin production were accompanied by parallel changes in the steady-state mRNA levels for these proteins. The effects of TGF-beta plus IFN-gamma on fibronectin gene expression appeared to be mediated entirely by transcriptional mechanisms, whereas the effects on collagen gene expression resulted from a combination of transcriptional and post-transcriptional events.

Cells, Cultured↗

PubMed1990

Regulation of human lung fibroblast collagen production by recombinant interleukin-1, tumor necrosis factor, and interferon-gamma.

Several cytokines have been shown to modulate the synthesis of matrix molecules, but few reports have defined how the cytokines interact with one another in mediating these regulatory effects. To define the cytokine network regulating collagen production, we have studied the effects of interferon-gamma, interleukin-1, and tumor necrosis factor on collagen synthesis by cultured human lung fibroblasts. Confluent cells were treated with cytokines for 24 h in the absence of serum or in media containing 1% serum. In the absence of serum, IL-1 and TNF each dose-dependently stimulated types I and III collagen production, with a maximal 2-4-fold increase in collagen accumulation being observed. Cells treated with IL-1 and TNF in combination showed less stimulation than with either cytokine alone. IFN-gamma also diminished the stimulatory effects of both IL-1 and TNF. In contrast, in 1% serum IL-1 and TNF individually had relatively minor effects, while IFN-gamma inhibited collagen production. However, the combination of IL-1 and TNF inhibited collagen production. Generally, these effects were achieved through control of mRNA levels. These studies demonstrate the existence of a cytokine network regulating fibroblast collagen production and suggest that cytokine effects in vivo may vary depending upon the constellation of factors present in the tissue.

Adult↗

PubMed1990

A controlled trial of colchicine to reduce the elastase load in the lungs of cigarette smokers with chronic obstructive pulmonary disease.

Current data suggest that emphysema in smokers is caused at least in part by the unrestrained action of neutrophil elastase on pulmonary tissues. Since colchicine reduces the secretion of enzymes from stimulated neutrophils, we designed a clinical trial to determine if colchicine could reduce the elastase load in the lungs or several putative indicators of elastin destruction. We carried out a prospective, double-blind, randomized, and placebo-controlled clinical trial. Outpatients seeking treatment for chronic obstructive pulmonary disease at the University of Texas Health Center at Tyler who met specific criteria were recruited into the study. A group of 46 cigarette smokers between 45 and 75 yr of age with chronic obstructive pulmonary disease (COPD) were studied. Colchicine or placebo was given orally in disguised capsules, 0.6 mg three times per day. Volunteers were placed on a baseline bronchodilator regimen of Theodur orally and albuterol by inhalation. Blood, urine, and bronchoalveolar lavage fluids were obtained after 1 wk of stabilization. The patients were then randomized and treated for 14 days with colchicine, and the measurements were repeated. Modifications in plasma elastin peptides and neutrophil elastase-generated fibrinopeptide A, urinary desmosines, and bronchoalveolar lavage fluid neutrophils or neutrophil elastase were the indicators of success or failure of the treatment. Pre- and posttreatment measurements in each patient and the difference between colchicine-treated and placebo-treated groups were compared. There were no statistically significant differences in either of the two types of analyses in any of the variables. We conclude that variables related to elastase load in the lungs were not modified by colchicine treatment. If a drug can be identified that is successful in modifying one of these variables, it would then have to be tested in a large-scale clinical trial in which the rate of decline in the FEV1.0 or mortality would be measured. The data presented here may provide useful information about the variability of key measurements of elastase load in the lungs and the breakdown of elastin and may aid investigators in designing similar trials in the future.

Aged↗

PubMed1990

Cytokine networks in the regulation of inflammation and fibrosis in the lung.

To understand the processes regulating inflammation and fibrosis in the human lung, we characterized the effects of recombinant interleukin-1, tumor necrosis factor, and gamma interferon on fibroblast proliferation, collagen production, interleukin-1-alpha production, interleukin-1-beta production, and interleukin-6-production. These studies demonstrated the existence of complex cytokine networks by which inflammatory cells regulate fibroblast function and fibroblasts, in turn, feed back to regulate inflammatory cell function. They also demonstrated that, in this complex network, the effect of an individual cytokine varies with the state of activation of the target cell, the presence of other cytokines in the local microenvironment, and the ability of the target cell to produce bioactive autocoids such as prostaglandins. Aspects of this cytokine network are discussed and a testable hypothesis for granuloma and abscess formation is detailed.

Cell Division↗

PubMed1989

Analysis of the Actinobacillus actinomycetemcomitans leukotoxin gene. Delineation of unique features and comparison to homologous toxins.

Actinobacillus actinomycetemcomitans leukotoxin has been implicated as a virulence factor in human infections. To initiate delineation of leukotoxin structure/function relationships, molecular cloning of the leukotoxin gene was carried out. When an A. actinomycetemcomitans genomic DNA library in lambda EMBL3 was screened using a 1.3-kilobase pair restriction fragment containing a portion of the leukotoxin gene, 13 positive recombinants were identified. One recombinant, designated lambda OP8, containing a 16-kilobase pair insert was selected for detailed study. Lysates from lambda OP8, but not control lysates, exhibited leukotoxic activity with target cell specificity identical to the native toxin. Western blots identified the recombinant-produced toxin as a 125-kDa protein doublet identical in mobility to the native toxin. Restriction enzyme and extensive DNA analyses demonstrated that the leukotoxin gene showed strong homology to two other toxins produced by Escherichia coli and Pasteurella haemolytica. As in the other two species, the A. actinomycetemcomitans toxin is contained in a cluster of four genes in which the A gene encodes the toxin and the products of the B, C, and D genes are involved in posttranslational modification of the toxin and its membrane insertion and secretion. The target cell specificity of the A. actinomycetemcomitans toxin differs from the other two toxins and is restricted to human and some non-human primate cells of the monomyelocytic lineage. The A. actinomycetemcomitans leukotoxin is not secreted but remains associated with the bacterial membrane, possibly through a hydrophobic domain at the carboxyl terminus which distinguishes it from the E. coli and P. haemolytica toxins.

Amino Acid 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

Identification and expression of the Actinobacillus actinomycetemcomitans leukotoxin gene.

The leukotoxin produced by the oral bacterium Actinobacillus actinomycetemcomitans has been implicated in the pathogenesis of juvenile periodontitis. In order to elucidate the structure of the leukotoxin, molecular cloning of the leukotoxin gene was carried out. A DNA library of A. actinomycetemcomitans, strain JP2, was constructed by partial digestion of genomic DNA with Sau3AI and ligation of 0.5 to 5.0 kilobase pair fragments into the Bam HI site of the plasmid vector pENN-vrf. After transformation into E. coli RR1 (lambda cI857), the clones were screened for the production of A. actinomycetemcomitans leukotoxin with polyclonal antibody. Six immunoreactive clones were identified. The clones expressed proteins which ranged from 21-80 kilodaltons, and the clone designated pII-2, producing the largest protein was selected for further study. Antibodies eluted from immobilized pII-2 protein also recognized the native A. actinomycetemcomitans leukotoxin molecule indicating that both molecules shared at least one epitope. DNA sequence analysis demonstrated that there are regions of significant amino acid sequence homology between the cloned A. actinomycetemcomitans leukotoxin and two other cytolysins, Escherichia coli alpha-hemolysin and Pasteurella haemolytica leukotoxin, suggesting that a family of cytolysins may exist which share a common mechanism of killing but vary in their target cell specificity.

Actinobacillus↗

PubMed1989

Structure of the elastin gene and alternative splicing of elastin mRNA: implications for human disease.

The protein elastin is largely responsible for the elastic properties of vertebrate lungs, large blood vessels, and skin. The structure of the human, bovine, and chick elastin gene and protein monomer, tropoelastin, has recently been elucidated by using techniques of molecular biology. Extensive homology of amino acid sequence exists among the mammalian species and there is in addition strong conservation of nucleotide sequences in the 3' untranslated region of the gene. The translated exons are small and embedded in large expanses of introns. Sequences coding for the hydrophobic regions, responsible for the elastic properties of the molecule, and the alanine-lysine rich regions, responsible for crosslink formation between molecules, reside in separate exons and alternate for the most part in the elastin gene. S1 analyses and sequence analysis of cDNA and genomic clones have indicated that there is substantial alternative splicing of the primary elastin transcript. Variations in the structure of mRNAs resulting from alternative splicing could explain the existence of the multiple forms of tropoelastin observed electrophoretically in several species. Different kinds of splicing patterns could occur in human populations and may contribute to aging and pathological situations in the cardiovascular and pulmonary systems.

Aging↗

PubMed1989

Phylogenetic distribution of enamel proteins: immunohistochemical localization with monoclonal antibodies indicates the evolutionary appearance of enamelins prior to amelogenins.

The hard covering tissues, enamel or enameloid, of representative vertebrate teeth were immunohistochemically stained using specific monoclonal antibodies against bovine amelogenins and bovine enamelins in order to determine the phylogenetic distribution of enamelin and amelogenin proteins. Immunohistochemically, only enamelin proteins were present in lower vertebrate (shark, bony fish, and larval amphibian) teeth and dermal denticles. Both enamelin and amelogenin proteins were present in higher vertebrate (mammal, reptile, and adult amphibian) teeth. Large hydroxyapatite crystal size and high levels of mineralization, characteristics common to both enamel and enameloid, are probably due to the presence of the common protein enamelin. The evolution of enamel from enameloid in the tetrapods seems to have involved the development of the gene for amelogenin.

Amelogenin↗

PubMed1988

Cutis laxa: reduced elastin gene expression in skin fibroblast cultures as determined by hybridizations with a homologous cDNA and an exon 1-specific oligonucleotide.

Fibroblast cultures were established from six patients with cutis laxa, and elastin gene expression was analyzed by RNA hybridizations with a 2.5-kilobase human elastin cDNA or an exon 1-specific 35-base oligomer. Northern analyses using either probe detected mRNA transcripts of approximately 3.5 kilobases, and no qualitative difference between the control and cutis laxa mRNAs was detected. However, quantitation of the elastin mRNA abundance by slot blot hybridizations revealed markedly reduced levels in all cutis laxa cell strains. Assuming equal translational activity of the control and cutis laxa mRNAs, the reduced mRNA levels could result in diminished elastin production, providing an explanation for the paucity of elastic fibers in the skin and other tissues in cutis laxa.

Base Sequence↗

PubMed1988

Development of immunoreagents to ciliary zonules that react with protein components of elastic fiber microfibrils and with elastin-producing cells.

We describe the generation of a monoclonal antibody library to ocular zonule components and the characterization of three monoclonal antibodies: 1) one specific for microfibrillar associated glycoprotein (MAGP), a component of both ocular zonules and microfibrils of elastin fibers, 2) an antibody to an as yet unidentified 70,000 dalton antigen that is present in abundance in the extracellular matrix (ECM) of elastin-producing cells, and 3) an antibody reacting with the 67000 dalton subunit of the elastin receptor. The presence of antigenic determinants common to the ocular zonule and elastic fiber microfibrils suggests that zonules, which can be obtained in relatively pure form, can provide a valuable resource for characterizing proteins common to both microfibrillar structures.

Animals↗

PubMed1988

Cloning of full-length elastin cDNAs from a human skin fibroblast recombinant cDNA library: further elucidation of alternative splicing utilizing exon-specific oligonucleotides.

A human cDNA library was constructed utilizing RNA isolated from cultured skin fibroblasts. Recombinant clones containing elastin sequences were identified by plaque hybridizations with previously characterized human placental elastin cDNAs. Seven positive recombinant clones with inserts of approximately 3.2-2.2 kb were isolated. Characterization of the clones by restriction endonuclease analysis and dot-blot hybridizations with exon-specific synthetic oligonucleotides demonstrated considerable variability in the primary nucleotide sequence. Dideoxy nucleotide sequencing confirmed this finding. The variability is most likely a result of alternative splicing of exons from the primary elastin transcripts. The two largest clones contained approximately 1 kb of 3' untranslated sequence and approximately 2.2 kb of translated sequence encoding 730 amino acids. Six amino acids, encoded by exon 12A, have not been previously noted in human elastin cDNAs. In addition, these human skin fibroblast clones contained a 49 bp 5' untranslated sequence. These results demonstrate that there is considerable variability in the processed nucleotide sequence of the elastin mRNAs. These transcripts may code for isoforms of tropoelastin with different biologic properties.

Amino Acid Sequence↗

PubMed1988

Increased elastin mRNA levels associated with surgically induced intimal injury.

Quantitative levels of mRNAs coding for elastin, types I and III procollagen and gamma-actin were measured in porcine vascular material following balloon catheterization. A balloon catheter was introduced into the thoracic aorta and jugular vein of 3-6 week old pigs; following distention and six days of postoperative recovery, tissue samples were obtained for histopathology, electron microscopy, RNA extraction and mRNA quantitation. Using a series of mammalian cDNA clones and the procedure of slot blot hybridization, we have shown that elastin and types I and III procollagen mRNA levels rose significantly during the postoperative period following vascular distention. The increase correlated with an increase in the cell mass present in both the venous and arterial intimal layers. Changes in gamma-actin mRNA levels were also associated with this rapid proliferative response but in arterial tissue only.

Actins↗

PubMed1988

Isolation and characterization of human elastin cDNAs, and age-associated variation in elastin gene expression in cultured skin fibroblasts.

In attempts to isolate human elastin cDNAs, a human placental lambda gt11 cDNA library was screened with a 1.3 kilobase sheep genomic DNA subclone, corresponding to the 3'-end of the elastin mRNA. The four largest clones, the largest being approximately 3 kilobase, were characterized by Northern transfer analyses, restriction endonuclease digestions and dideoxy nucleotide sequencing. Northern transfer analyses of poly(A)+RNA revealed hybridization to mRNA transcripts in the region of 3.5 kilobase. Restriction endonuclease mapping and nucleotide sequencing demonstrated distinct domains characteristic of elastin, and identified areas of variability which apparently reflects alternative splicing of the primary elastin transcripts. To demonstrate the utilization of these cDNAs for studies on elastin gene expression in human cells, elastin mRNA was examined in fibroblast cultures established from the skin of several individuals of varying ages. Northern transfer analyses and slot blot hybridizations demonstrated that elastin gene expression is initiated early during fetal development, and continues at a relatively constant level through several decades. The lowest abundance of elastin mRNA was noted in the cell cultures established for the oldest individual studied (61-year-old female). Demonstration of elastin gene expression in cultured fibroblasts provides a system to study diseases affecting the elastic fibers.

Aging↗