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Biomedical subjects

J Rosenbloom

Publications and source records attributed to J Rosenbloom.

At least 91 records · Page 5Linked to original sources
PubMed1992

Rat somatotroph insulin-like growth factor-II (IGF-II) signaling: role of the IGF-I receptor.

The anterior pituitary contains insulin-like growth factor-II (IGF-II) and expresses abundant IGF-II-binding sites, but the IGF-II signaling pathway in the pituitary has not been defined. We, therefore, tested the effects of recombinant human IGF-II on pituitary function by assessing the GH responsiveness of the primary rat somatotroph to IGF-II. IGF-II (3.3 nM) suppressed GH secretion by 50%, similar to the effect elicited by equimolar doses of IGF-I. In contrast, a low concentration of IGF-II (0.2 nM) did not attenuate GH secretion, while a similar IGF-I dose was sufficient to produce 50% inhibition of basal GH secretion. Fifty percent competition for [125I]IGF-I binding by IGF-I and IGF-II in GC rat pituitary cells demonstrated a 14-fold lesser affinity of the IGF-II ligand for the IGF-I receptor compared to IGF-I; therefore, the binding affinity of IGF-II for the IGF-I receptor correlates with the concentration of IGF-II required for 50% GH inhibition. Transfected GH-secreting cell lines derived from GC cells overexpressing intact human IGF-I receptors exhibited enhanced responsiveness to IGF-II. In contrast, cells transfected with a truncated IGF-I receptor cDNA lacking the cytoplasmic receptor beta-subunit (952STOP) failed to transduce the IGF-II signal, indicating that functional IGF-I receptors are required for IGF-II signaling. In addition, a mutant IGF ligand, [Leu27]IGF-II, which selectively exhibits high affinity for the type II receptor, but only minimal binding to the IGF-I receptor, did not attenuate GH secretion. However, the analog [Arg54,Arg55]IGF-II, which exhibits high affinity to the IGF-I receptor, but no binding to the type II receptor, appropriately suppressed GH secretion. This unique model of somatotroph signaling provides evidence for IGF-II regulation of polypeptide hormone secretion mediated by the IGF-I receptor.

Animals↗
PubMed1991

Binding of elastin to Staphylococcus aureus.

Many pathogenic bacteria specifically bind to components of the extracellular matrix. In this study, we report the specific association of Staphylococcus aureus with elastin, a major structural component of elastic tissue. Competition assays in which the binding of radiolabeled tropoelastin was inhibited by excess unlabeled elastin peptides, but not by other proteins, established the specificity of the interaction. Kinetic studies showed that tropoelastin binding to the bacteria was rapid and saturable. Scatchard analysis of the equilibrium binding data indicated the presence of a single class of high affinity binding sites (KD approximately 4-7 nM) with approximately 1000 sites per organism. Protease susceptibility suggested that the elastin binding moiety on S. aureus was a protein, which was confirmed by the isolation of a 25-kDa elastin-binding protein from S. aureus extracts through affinity chromatography. Using a truncated form of tropoelastin, the bacterial binding domain on elastin was mapped to a 30-kDa fragment at the amino end of the molecule. Although the precise amino acid sequence recognized by the staphylococcal elastin receptor has not been characterized, it is clearly different from the region of tropoelastin that specifies binding to mammalian elastin receptors.

Amino Acid Sequence↗
PubMed1991

Identification of the leucine-rich amelogenin peptide (LRAP) as the translation product of an alternatively spliced transcript.

The polymerase chain reaction was used to amplify bovine tooth amelogenin cDNA, resulting in several products which were separated by agarose gel electrophoresis. Sequence determination of one of the products revealed that it encoded an amino acid sequence identical to that of a small leucine-rich amelogenin polypeptide (LRAP) previously characterized by protein sequencing. Comparison of the nucleotide sequence of this cDNA with that determined for the cloned bovine amelogenine gene strongly suggested that the LRAP transcript resulted from alternative splicing of the primary transcript of this gene, thus explaining the origin of the puzzling LRAP sequence. Analysis of the structure of LRAP suggests that the polypeptide might exhibit interesting properties relative to hydroxy apatite crystal formation.

Amelogenin↗
PubMed1991

Structure and expression of the bovine amelogenin gene.

In order to define further the mechanisms responsible for tooth amelogenin heterogeneity, seven bovine amelogenin cDNAs were sequenced. On the basis of these sequences, five of the cDNAs could be grouped into one class which differed appreciably in sequence from the second group of two cDNAs. Two overlapping bovine genomic clones were then isolated and shown by sequencing to contain six exons encoding the entire consensus sequence of the class I cDNA. Southern blot analysis of DNA from male and female animals using class I or class II specific oligonucleotide probes suggested that the class I gene sequence was located on the X chromosome while the class II sequence was located on the Y chromosome. Therefore, these results also suggest that the genes on the X and Y chromosomes are both transcribed. Furthermore, the results are consistent with alternative splicing of the class I primary transcript as a potential mechanism for generating amelogenin heterogeneity.

Amelogenin↗
PubMed1991

Structure and function of the B and D genes of the Actinobacillus actinomycetemcomitans leukotoxin complex.

The Actinobacillus actinomycetemcomitans leukotoxin gene complex, consisting of four genes, has been cloned and the sequence of the AaLtC and AaLtA genes reported. The present paper details the sequences of the AaLtB and AaLtD genes which, like AaLtC and AaLTA, are also homologues of genes found in other cytolytic toxin complexes of several other Gram-negative bacterial pathogens. When tested in a recombinant expression system, the AaLtB and/or AaLtD genes are required for the translocation and insertion of the A. actinomycetemcomitans leukotoxin (AaLtA) into the cell membrane of Escherichia coli.

Actinobacillus↗
PubMed1991

Regulation of elastin gene expression.

Recent isolation and characterization of cDNAs encompassing the full length of chicken, cow, and human elastin mRNA have led to the elucidation of the primary structure of the respective tropoelastins. Comparison of the tropoelastin from the different species has revealed that large segments of the sequence are conserved, but considerable variation also exists, ranging in extent from relatively small alterations, such as conservative amino acid substitutions, to large-scale deletions and insertions. Several distinct approaches have yielded compelling evidence of a single elastin gene per haploid genome. Analysis of the bovine and human elastin genes revealed that functionally distinct hydrophobic and cross-link domains of the protein are encoded in separate exons which alternate in the genes. The human gene contains 34 exons, the intron/exon ratio is unusually large (20:1), and the introns contain large amounts of repetitive sequences that may predispose to genetic instability. Comparison of the cDNA and genomic sequences has demonstrated that the primary transcript of both species is subject to considerable alternative splicing, which can account for the presence of multiple tropoelastin isoforms. It is likely that the conformation of elastin is, at least in part, that of a random coil, and therefore it might be expected that the stringency for conservation of the amino acid sequence would be less than that for other proteins with unique conformations. This suggests that functional elastin molecules that vary in their sequence and fitness may exist in the human population and be compatible with a normal life. Potentially though, these variations could have profound consequences on the properties of vital tissues found in the cardiovascular and pulmonary systems over the lifetime of the individual. Consequently, analysis of the structure of the elastin gene and its variation in what is regarded as the normal human population, rather than in those individuals with clearly heritable diseases, assumes greater importance. The 5'-flanking region of the gene is G + C rich and contains several SP-1 and AP2 binding sites, as well as putative glucocorticoid, cAMP, and TPA responsive elements, but no consensus TATA box or functional CAAT box. Primer extension and S1 mapping of the elastin mRNA indicated that transcription was initiated at multiple sites. Transfection experiments using promoter elements/reporter gene constructs demonstrated that the basic promoter element was found within region -128 to -1. In addition, three distinct up-regulatory and two down-regulatory regions were delineated. Taken together, these findings suggest that the regulation of elastin gene expression is complex and takes place at several levels.

Amino Acid Sequence↗
PubMed1991

Size distribution of human lung elastin-derived peptide antigens generated in vitro and in vivo.

The protease-antiprotease hypothesis of emphysema development suggests that degradation of elastin in the lung interstitium may give rise to abnormal quantities of circulating elastin-derived peptides (EDP) during periods of inflammation. Recent studies have shown a relationship between emphysema and high levels of EDP in human plasma. This report characterizes elastin digests on the basis of antigenicity, size, and method of preparation, as well as the size distribution of EDP found in the plasmas of nonsmokers, smokers, and emphysema patients. Gel filtration of elastin digests prepared by hydrolysis of human lung elastin using a low (1:500) ratio of neutrophil elastase to elastin generated a broad protein peak of approximately 70,000 daltons. In contrast, a high (1:25) ratio of neutrophil elastase to human lung elastin gave a broad protein peak, with a size distribution in the 10,000 to 30,000 dalton range. This digest showed distinct immunochemical properties. A polyclonal antibody directed against the low-ratio digest showed a minimum detection of 2 ng/ml for the homologous antigen but required 1,000 ng/ml of the high-ratio digest for detectable inhibition in an indirect ELISA assay. Gel filtration of plasmas from normal nonsmokers and the majority of normal smokers revealed a single immunoreactive EDP fraction of approximately 70,000 daltons. Plasmas from selected normal smokers and emphysema patients with high levels of circulating EDP (greater than 90 ng/ml) fractionated into a complex pattern of peptides in which the 70,000 dalton component represented 50% of the immunoreactive material and several lower molecular weight peptides represented the remaining circulating elastin antigens.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens↗
PubMed1991

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

A great deal of experimental evidence suggests that emphysema in smokers is caused by the action of neutrophil elastase on lung elastin. In order to test this hypothesis, it is necessary to find a drug that reduces the load of neutrophil elastase in the lungs of patients with emphysema. In a previous study we treated smokers with emphysema with colchicine, a drug that prevents neutrophil elastase secretion, to determine if it would reduce the elastase burden in the lungs. Colchicine was unable to reduce the elastase load while the stimulus of smoking continued. In this study we treated ex-smokers with emphysema to determine if colchicine could reduce the elastase burden in the lungs. The objective of the study was to determine if colchicine can reduce the elastase load and putative indicators of elastase load in the lungs of patients with chronic obstructive pulmonary disease. The study was a prospective, double-blind, randomized, and placebo-controlled clinical trial. The subjects were outpatients seeking treatment at the University of Texas Health Center at Tyler. We studied 16 ex-cigarette smokers between 45 and 75 yr of age with lung disease defined by FEV1 less than 70% of predicted but greater than 1.2 L whose airflow obstruction was less than 20% reversible with bronchodilators. Colchicine or placebo was taken by mouth in disguised capsules, 0.6 mg three times per day. Volunteers were placed on a baseline bronchodilator regimen of theodur by mouth and albuterol by inhalation. Blood, urine, and bronchoalveolar lavage fluids were obtained after 1 wk of stabilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
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↗