An assessment of oral health needs of the community served by West Central Community Health Centres.
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Community Health Centers↗
Biomedical subjects
J Rosenbloom
Publications and source records attributed to J Rosenbloom.
Microfibril-associated glycoprotein binds to the carboxyl-terminal domain of tropoelastin and is a substrate for transglutaminase.
Microfibril-associated glycoprotein (MAGP) is an integral component of microfibrillar structures that play a critical role in the organization of elastic fibers in the extracellular matrix. To study possible molecular interactions between MAGP and other elastic fiber components, we have generated native MAGP using a baculovirus expression system and tested its ability to associate with tropoelastin and fibrillin. MAGP produced by SF9 cells underwent processing similar to the mammalian protein, including correct cleavage of the signal peptide and sulfation of tyrosine residues. When tested in solid-phase binding assays, native MAGP specifically bound to tropoelastin but not fibrillin-1. Binding to tropoelastin was divalent cation-independent and was completely blocked by reduction and alkylation of either protein. Antibody inhibition studies indicated that the carboxyl terminus of tropoelastin mediated its interaction with MAGP. In addition to binding to elastin, MAGP was also a substrate for transglutaminase, which might explain its propensity to form high molecular weight aggregates that cannot be dissociated with reduction or denaturation. Together, the results of this study provide new insights into the functional relationship between microfibrillar proteins and have important implications for understanding elastic fiber assembly.
Amino Acid Sequence↗
Structure of the human gene encoding the associated microfibrillar protein (MFAP1) and localization to chromosome 15q15-q21.
Microfibrils with 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. To extend our understanding of the proteins composing these microfibrils, the cDNA and gene encoding the human associated microfibril protein (MFAP1) have been cloned and characterized. The coding portion is contained in 9 exons, and the sequence is very homologous to the previously described chick cDNA, but does not appear to share homology or domain motifs with any other known protein. Interestingly, the gene has been localized to chromosome 15q15-q21 by somatic hybrid cell and chromosome in situ analyses. This is the same chromosomal region to which the fibrillin gene, FBN1, known to be defective in the Marfan syndrome, has been mapped. MFAP1 is a candidate gene for heritable diseases affecting microfibrils.
Amino Acid Sequence↗
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↗
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↗
Regulation of amelogenin gene expression during tooth development.
The amelogenins are the predominant matrix proteins in developing enamel and are crucial for proper enamel mineralization. Transgenic mice were constructed in order to identify the segment of the amelogenin gene required for specific expression in enamel organ cells. A 3.5 kb fragment of the bovine X-chromosomal amelogenin gene that includes a TATA box, the transcription initiation site, and 32 bp of exon 1 was linked to the beta galactosidase gene and injected into fertilized mouse eggs. Newborn transgene positive mice expressed beta galactosidase activity in developing teeth treated with the chromogenic substrate Xgal. Foci of ameloblasts were positive in newborn mice; stain intensity and number of positive ameloblasts increased in 1-day and 2-day postnatal mice. Some of the adjacent stratum intermedium cells also were positive in the later stages. Targeting of the transgene to the enamel organ was specific; the only other cells observed to be positive were macrophages, which have endogenous beta galactosidase activity.
Ameloblasts↗
Developmental regulation of aortic elastin gene expression involves disruption of an IGF-I sensitive repressor complex.
Nuclear proteins were isolated from different aged chick embryonic aorta and examined by gel mobility shift and footprint analyses using the -195 to +2 bp region of the human elastin gene. A major developmental change in DNA/protein complexes involved the loss of binding complexes present initially in the 8- to 11-day nuclear extracts. Both chemical and enzymatic footprinting demonstrated that the deprotected binding complexes corresponded to positions -167 to -137 bp within the elastin promoter fragment. Gel shift competition assays confirmed the identity of the specific DNA sequences affected. These results demonstrate that increased elastogenesis during aortic embryogenesis involves the derepression of a negative element functionality previously shown to be affected by IGF-I in vitro.
Animals↗
Insulin-like growth factor-I regulates transcription of the elastin gene.
Neonatal rat aortic smooth muscle cell cultures were used to investigate the mechanisms by which insulin-like growth factor-I (IGF-I) up-regulates aortic elastogenesis. The addition of IGF-I (50 ng/ml) to quiescent smooth muscle cell cultures resulted in a 5-fold increase in the steady-state levels of tropoelastin mRNA beginning between 2 and 4 h and reaching maximal levels at 8 h. Addition of cycloheximide blocked the effect of IGF-I. Nuclear run-on transcription analyses of nuclei isolated from IGF-I-treated cells showed increased synthesis of new tropoelastin transcripts indicating that transcriptional activation is a major component of IGF-I up-regulation. Transient transfections with deletion constructs containing different portions of the elastin 5'-upstream region localized the IGF-I-responsive area to sequences between -195 and -136 base pairs and further showed that this region contains a negative element. Gel retardation assays using nuclear proteins extracted from control and IGF-I-treated cells demonstrated that IGF-I treatment results in the loss of binding complexes. Footprint analyses of specific binding complexes affected by IGF-I show the deprotection of two closely positioned sequences spanning positions -165 to -137 base pairs. These results suggest that IGF-I up-regulation of elastogenesis involves the abrogation of a negative element functionality.
Animals↗
Oxidation, cross-linking, and insolubilization of recombinant tropoelastin by purified lysyl oxidase.
The use of recombinant human tropoelastin (rTE) and selected variants thereof as substrates for the assay of lysyl oxidase activity in vitro was explored. The possibility was also assessed that an insoluble elastin-like product could be generated from this elastin precursor in the absence of other macromolecules found associated with elastin in vivo. rTE was more efficiently oxidized by lysyl oxidase than the insoluble chick aorta elastin substrate conventionally used. Anionic amphiphilic elastin ligands strongly inhibited rTE oxidation consistent with the importance of electrostatic enzyme-substrate interactions previously noted with the insoluble elastin substrate. An rTE variant, rTE delta 26A, lacking the hydrophilic sequence coded by exon 26A, was a less effective substrate than rTE, largely due to an increase in Km, while the kinetic parameters for the oxidation of rTE delta 36, lacking the C-terminal polybasic sequence coded by exon 36, were quite similar to those for rTE. Incubation of rTE delta 26A with lysyl oxidase not only resulted in the generation of peptidyl alpha-aminoadipic-delta-semialdehyde and lysine-derived cross-linkages, but also yielded a product insoluble in hot 0.1 N NaOH, consistent with the properties of insoluble elastin. Thus, oxidation, cross-linking and insolubilization of elastin substrates by lysyl oxidase can occur in the absence of other macromolecules implicated as being involved in this process in vivo, although such macromolecules may be essential to obtain the proper alignment between tropoelastin units for specifically placed cross-linkages and optimally functional elastic fibers.
Amino Acid Sequence↗
Identification of antigenic epitopes in type IV collagen by use of synthetic peptides.
Peptides representing potential antigenic regions of the NC-1 and 7-S domains of the human alpha 1 and alpha 2, and bovine alpha 3 chains of type IV collagen were synthesized either chemically or by the recombinant DNA technique and tested by ELISA using antibodies raised in rabbits against the whole type IV collagen or the NC-1 domain. Sera from patients with Goodpasture syndrome (GP) or with acute poststreptococcal glomerulonephritis (APSGN) were also tested. The location of antigenic determinants was predicted from the primary and secondary structure of the chains, that is, aromaticity, hydrophilicity and presence of beta-turns. All synthetic peptides reacted with the antiserum to type IV collagen (anti-Col IV). Whereas all peptides arising from the NC-1 domain reacted with anti-NC-1, intact 7-S or peptides of the alpha 1 or alpha 2 chain of the 7-S domain did not react. However intact 7-S reacted with anti-Col IV. Two synthetic peptides from the NC-1 domain of alpha 1, (a.a. 71-90 and a.a. 176-190), one from the alpha 2 (a.a. 70-83) and four from the alpha 3 chain (a.a. 72-89, a.a. 104-117, a.a. 133-145, a.a. 185-203) reacted with anti-NC-1 and anti-COL IV. The above peptides, except alpha 3 (72-89) and alpha 3 (185-203), were tested and found to be reactive with sera from patients with GP.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Intrasubject test-retest variability in clinical electrocochleography.
Seventeen healthy volunteers without a history of auditory or vestibular difficulty and with normal screening audiometry had bilateral (34 ears) electrocochleography performed repeatedly at 1-week intervals. Each subject had from four to seven electrocochleograms performed on each ear. Measurements were made in the external auditory canal using a gold-foil "TIPtrode." Amplitude and latency for the summating potential (SP) and action potential (AP) were measured and SP/AP ratios were calculated. Averages and standard deviations for amplitude, latency, and SP/AP ratios were computed. The average SP/AP ratio was 0.22 with a standard deviation of 0.06. The range was 0.04 to 0.50. The average difference between the highest and lowest SP/AP ratio for a given individual was 0.155 (i.e., the range 0.03 to 0.31) with a standard deviation of 0.07. These normative data are useful in assessing the results of dehydration electrocochleography and in establishing how much test-retest variation is required to suggest pathology.
Action Potentials↗
Extracellular matrix 4: the elastic fiber.
The elastic properties of many tissues such as the lung, dermis, and large blood vessels are due to the presence of elastic fibers in the extracellular space. These fibers have been shown by biochemical and ultrastructural analysis to be composed of two distinct components, a more abundant amorphous component and a 10-12 nm microfibrillar component, which is located primarily around the periphery of the amorphous component. The protein elastin makes up the highly insoluble amorphous component and is responsible for the elastic properties. Elastin is found throughout the vertebrate kingdom and possesses an unusual chemical composition rich in glycine, proline, and hydrophobic amino acids, consonant with its characteristic physical properties. The 72-kDa biosynthetic precursor, tropoelastin, is secreted into the extracellular space where it becomes highly cross-linked into a rubber-like network through the activity of the copper-requiring enzyme lysyl oxidase. Analysis of the elastin gene has demonstrated that hydrophobic and cross-linking domains are encoded in separate exons and that there is significant alternative splicing, resulting in multiple isoforms of tropoelastin. The elastin gene promoter contains many potential binding sites for various modulating factors indicative of a complex pattern of transcriptional regulation. The microfibrils contain several proteins, including fibrillin, and probably act as an organizing scaffold in the formation of the elastin network. There appears to be a fibrillin gene family in which each protein contains multiple repeats of a motif previously found in epidermal growth factor and a second motif observed in transforming growth factor beta 1-binding protein. Mutations in the fibrillin gene located on human chromosome 15 have been strongly implicated as the cause of the Marfan syndrome.
Amino Acid Sequence↗
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↗
Bovine amelogenin message heterogeneity: alternative splicing and Y-chromosomal gene transcription.
The amelogenins are the most abundant proteins in developing tooth enamel. Previous analyses have demonstrated that transcriptionally active genes encoding the proteins are located on both the bovine X and the bovine Y chromosomes. We report here the cloning and sequence analysis of the Y-chromosomal gene and corresponding cDNA. The Y-specific mRNA encodes a translation product in which a 21 amino acid domain has been deleted, relative to the X-specific amelogenin, resulting in loss of a structure tentatively described as a beta-spiral. There are also 13 single amino acid differences compared to the X-specific amelogenin. In addition, we have cloned and sequenced an X-chromosomal alternatively spliced amelogenin cDNA that encodes a 43 amino acid amelogenin primary translation product. Hydrophobicity analysis indicates that all analyzed amelogenin proteins have a mean hydrophilic character and the two peptides translated from alternatively spliced messages have significant increases in percentage of hydrophobic amino acids.
Amelogenin↗
Molecular cloning of human macrophage capping protein cDNA. A unique member of the gelsolin/villin family expressed primarily in macrophages.
Macrophage capping protein (MCP) is a Ca(2+)-sensitive protein which reversibly blocks the barbed ends of actin filaments but does not sever preformed actin filaments. The human cDNA for MCP has been cloned and sequenced. The derived amino acid sequence predicts a polypeptide of 38.4 kDa. Human MCP expressed in Escherichia coli using a pET12a vector was functionally identical to the native protein purified from rabbit alveolar macrophages with respect to Ca2+ sensitivity and ability to block monomer exchange at the barbed end of actin filaments. Sequence comparison with other actin-binding protein sequences indicates that MCP is a member of the gelsolin/villin family of barbed end blocking proteins. Unlike gelsolin, this protein has a limited tissue distribution being detected primarily in macrophages where it was abundant, representing 0.9-1% of the total cytoplasmic protein. Northern blot analysis of U937 and HL60 cells differentiated to macrophage-like cells demonstrated that MCP message increases to 2.6 and greater than 7 times initial levels, respectively. Human MCP displays a 93% amino acid sequence identity with two recently described mouse proteins, gCap39 and Mbh1. Its abundance in macrophages and the corresponding increases in mRNA levels upon promyelocyte and monocyte development into macrophages indicate that MCP may play an important role in macrophage function.
Actins↗
Electron immunocytochemical localization of Actinobacillus actinomycetemcomitans leukotoxin.
The Actinobacillus actinomycetemcomitans leukotoxin was localized in A. actinomycetemcomitans bacteria using an electron immunocytochemical thin-section labeling method. An immuno-dot blot procedure was initially used to ascertain the optimal specimen fixation. This consisted of a periodate-lysine-paraformaldehyde (2%) fixative in a phosphate buffer followed by embedding in LR White. Affinity-purified toxin was used to produce a monospecific polyclonal antibody. The reaction sites were visualized with a colloidal gold-tagged reporter antibody. The leukotoxin was found to be localized either in the cell envelope and/or in membranous vesicles on the outer surface of the bacterial cell. These results support previous observations indicating the same location.
Aggregatibacter actinomycetemcomitans↗
IGF-I regulation of elastogenesis: comparison of aortic and lung cells.
Rat neonatal aortic smooth muscle and pulmonary fibroblast cell cultures were exposed to different amounts of insulin-like growth factor-I (IGF-I, 1-100 ng/ml of medium) for 24 h. Aortic smooth muscle cells exhibited an increase in both steady-state levels of tropoelastin mRNA and soluble elastin with increasing amounts of IGF-I, suggesting that the growth factor is acting by increasing transcription or transcript stability. In contrast, pulmonary fibroblast cultures did not exhibit an elastogenic response to IGF-I because neither the steady-state levels of tropoelastin mRNA nor soluble elastin were affected. Transient transfection of the two cell cultures with a chimeric construct containing 500 bp of the elastin gene 5'-flanking region fused to the chloramphenicol acetyltransferase reporter gene showed that reporter activity was increased threefold in smooth muscle cells treated with IGF-I, whereas activity remains essentially the same in control and growth factor-treated pulmonary fibroblast cells. Receptor binding analyses revealed that both cell types possess the type I IGF-I receptor. Therefore, the lack of an elastogenic response in the lung cells cannot be attributed to lack of the appropriate receptor. These data, obtained in vitro with cell types that are principal producers of lung and aortic elastin, agree with results obtained in vivo. This agreement suggests that the regulation of elastin gene expression varies among cells derived from different tissues and furthermore provides model systems to investigate differential regulation of the elastin gene.
Animals↗
Specificity and sensitivity of the assay for elastin-derived peptides in chronic obstructive pulmonary disease.
One prediction of the protease-antiprotease hypothesis of chronic obstructive pulmonary disease (COPD) pathogenesis is the appearance of elastin-derived degradation products in the plasmas of affected patients that are due to the breakdown of alveolar interstitial elastin by an excess of elastolytic activity in the lung. We previously demonstrated a significant elevation of plasma elastin-derived peptides (EDP) in subjects with COPD in comparison with asymptomatic smokers with normal spirometry or normal nonsmokers. To better determine the selectivity of the assay for EDP as a marker of COPD, we measured plasma EDP levels in different patient populations. These included subjects with COPD, subjects with diseases that may involve accelerated elastolysis (pneumonia, atherosclerotic vascular disease, and inflammatory arthritis), subjects with diseases hypothesized to involve pulmonary inflammation without elastolysis (asthma and acute tracheobronchitis), asymptomatic smokers with normal spirometry, and healthy, nonsmoking subjects. Mean plasma EDP levels in subjects with COPD were elevated above those in all other subjects (p less than 0.01). The prospective analyses of specificity and sensitivity of plasma EDP levels as markers of COPD gave values of 91 and 65%, respectively. Utilizing receiver operating characteristic curve analysis to assess the diagnostic and screening performance of plasma EDP as a test for COPD (perfect test equals an area under the curve of 1.0), the area under the curve was 0.87, which compares favorably with many widely used clinical tests. These data demonstrate that the assay for plasma EDP is a quantitative, easily measured, and highly specific marker for subjects with COPD.
Adult↗