Biomedical subjects

S P Macauley

Publications and source records attributed to S P Macauley.

8 recordsLinked to original sources

PubMed1998

Changes in the expression of extracellular matrix (ECM) and matrix metalloproteinases (MMP) of proliferating rat parotid acinar cells.

Tissue morphogenesis, development, and maintenance of function are mediated by signals generated through the composition of the extracellular matrix. The regulation of the composition of matrix is determined by enzymes specific for their degradation, the matrix metalloproteinases. Chronic injections of the beta-adrenergic receptor agonist, isoproterenol, result in a non-neoplastic hypertrophy and hyperplasia of the rat parotid gland. The activity of matrix metalloproteinases, as measured by gelatin zymography and enzymatic digestion of Azocoll substrates by gland lysates, decreased significantly (P < 0.05) following 24 hrs of agonist treatment, and slowly recovered to control values by 6 days of treatment. Daily administration of the broad-spectrum matrix metalloproteinase inhibitor Galardin for 3 days in combination with isoproterenol resulted in enhanced gland hypertrophy compared with that produced by isoproterenol alone. Given alone, Galardin also caused hypertrophy. The relative abundance of mRNA for the extracellular matrix molecules, collagens I and III and fibronectin, declined rapidly following the initiation of beta-agonist treatment in vivo, while laminin B1 and B2 mRNA levels increased initially before declining below control levels. These changes in patterns of mRNA levels also were observed in the concentrations of glandular protein when Western dot blot analysis of collagens I and III and laminin, respectively, was used. The importance of laminin, in vivo, was demonstrated by coinjection of anti-laminin antibody along with isoproterenol, which resulted in the inhibition of beta-agonist-induced parotid gland hypertrophy and hyperplasia. These data suggest that modulation of the ECM is associated with isoproterenol-induced salivary gland hypertrophy and hyperplasia. It is likely that this modulation of the ECM takes place through transcriptional regulation of some ECM genes and regulation of matrix-degrading enzyme activity.

Adrenergic beta-Agonists↗

PubMed1997

Extracellular-matrix gene expression during mouse submandibular gland development.

Early morphogenesis of mouse submandibular glands begins on late day 11 of fetal development when the epithelium begins to bud from the surrounding mandibular mesenchyme. Using total RNA collected from fetal BALB/c submandibular glands, steady-state levels of mRNA expression for extracellular matrix molecules were measured using quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR). By comparing the PCR amplification products of both the cellular mRNA and a synthetic template, pMATRIX, it was possible to measure the direct expression of collagens alpha2(I), alpha1(III), alpha1(IV), fibronectin, laminin B2, elastin and lysyl oxidase genes. There was an observed trend for an increasing concentration of collagen alpha2(I), collagen alpha1(III) and lysyl oxidase mRNA molecules per cell on day 16 of development. The relative abundance of elastin mRNA was detectable only on day 16. Fibronectin and laminin B2 were more constitutively present but had their highest copy number per cell on day 16. The presence of extracellular-matrix protein was confirmed by immunohistochemistry using day-16 fetal glands and adult glands. With the construction of the pMATRIX supertemplate and the advent of quantitative, competitive RT-PCR technology, it has been possible to measure small changes in the steady-state concentrations for extracellular-matrix mRNA during salivary gland development.

Animals↗

PubMed1996

Alterations in the secretory response of non-obese diabetic (NOD) mice to muscarinic receptor stimulation.

Salivary gland secretion in non-obese diabetic (NOD) and BALB/c mice was evaluated following stimulation with the muscarinic receptor agonist pilocarpine. Both saliva flow rates and total protein were similar in BALB/c and prediabetic NOD mice. With diabetes onset in NOD mice, the saliva flow rate and protein concentration were dramatically reduced. The level of cyclic AMP (cAMP) generated 10 min following agonist injection was similar for the parotid gland of BALB/c and prediabetic and diabetic NOD mice but was reduced in diabetic NOD mice. In the submandibular gland both prediabetic and diabetic NOD mice showed a reduced potential for the generation of cAMP compared with BALB/c mice. The parotid gland from NOD mice had elevated levels of basal and muscarinic receptor agonist (carbachol)-stimulated concentrations of inositol phosphate intermediates in in vitro assays compared with BALB/c animals, while in the submandibular gland of NOD mice, basal and agonist-stimulated concentrations of inositol phosphate intermediates were reduced relative to BALB/c. An evaluation of muscarinic receptor density showed a reduction with diabetes onset for the parotid gland, while a similar level was observed in prediabetic NOD and BALB/c mice. The receptor density was decreased for both the prediabetic and diabetic NOD submandibular glands compared with BALB/c animals. Sera from diabetic NOD but not BALB/c mice immunoprecipitated radiolabeled muscarinic receptor, indicating the presence of autoantibody to the receptor in diabetic mice. Thus, the reduction of muscarinic agonist response in NOD mice may be due, in part, to a generalized reduction in signal transduction components most evident in the prediabetic and diabetic submandibular gland and to a less certain degree in the parotid gland from diabetic NOD mice as a consequence of autoantibodies directed against cell surface antigens.

Animals↗

PubMed1996

Competitive RNA templates for detection and quantitation of growth factors, cytokines, extracellular matrix components and matrix metalloproteinases by RT-PCR.

Detection of low-abundance mRNAs by reverse transcription-polymerase chain reaction (RT-PCR) has become a standard technique to determine gene expression by tissues and cells in culture. The ability to determine relative or absolute copy number of specific mRNAs has been difficult due to inadequate internal standards to control for sample-to-sample variation. The use of a synthetic RNA standard with identical sequences to the PCR primers allows reproducible quantitation between samples and assays. By designing multi-sequence templates, several specific mRNAs can be quantitated using a single template. Addition of multiple templates to a single RT reaction allows the quantitation of a large number of targets from as little as 4 micrograms of total RNA. In this report, we present a series of seven primer/template systems to detect and quantitate 52 specific messages, including 26 growth factors and receptors, 8 extracellular matrix components, 10 matrix-modifying enzymes and their inhibitors and 8 cytokines.

Binding, Competitive↗

PubMed1995

Saliva and growth factors: the fountain of youth resides in us all.

The predominant focus of research dealing with saliva revolves around the role in the maintenance of oral health through a number of physiological and biological properties of constituent proteins. An ever-expanding literature exists indicating that the salivary glands additionally synthesize, and secrete into saliva, a wide range of growth factors. Animal studies with epidermal growth factor have provided evidence for a role in both oral and systemic health, through the promotion of wound healing rates. Thus, the ability to manipulate their rates of synthesis and absorption from saliva holds the potential to enhance tissue regeneration and homeostasis.

Animals↗

PubMed1994

Characterization of the synthesis and secretion of transforming growth factor-alpha from salivary glands and saliva.

Whole saliva collected from rat, mouse, and human sources was found to contain high concentrations of transforming growth factor-alpha (TGF alpha) when analyzed by RIA. The concentrations of TGF alpha in unstimulated human saliva (age, 30-45 yr; n = 10; 1.5 +/- 3.1 nM) was reduced with age (age, 55-70 yr; n = 10; 0.4 +/- 0.1 nM), but increased in oral pathologies manifested in xerostomia (age, 57-70; n = 6; 0.8 +/- 0.2 nM) and Paget's disease (age, 58-76; n = 8; 2.0 +/- 0.6 nM). Immunohistochemical localization of TGF alpha in the salivary glands of rats and mice revealed specific immunostaining of the granular ductal cells of the parotid and submandibular glands. Reverse transcription followed by polymerase chain reaction amplification of total RNA from the parotid and submandibular glands of rats and mice demonstrated the presence of TGF alpha mRNA, suggesting endogenous synthesis by the salivary glands. Thus, salivary glands appear to be an exocrine source for a second member of the epidermal growth factor-like growth factor family in the oral cavity.

Adult↗

PubMed1992

Characterization of the human lysyl oxidase gene locus.

Lysyl oxidase (EC 1.4.3.13) is a copper-dependent enzyme acting principally on collagen and elastin catalyzing the formation of aldehyde cross-links. It is also believed to possess a tumor suppressor activity as the anti-oncogene of ras. While rat, human, and mouse lysyl oxidase cDNAs have been cloned, little is known about the structure of the gene, its organization, or regulation. This paper describes the cloning of an intronic segment of the human lysyl oxidase gene. Sequence analysis defined the location of an intron that separates the prepro-coding segments from the segment encoding the catalytic domain. Genomic restriction mapping and gene copy number data established that multiple lysyl oxidase mRNA transcripts originate from a single gene and thus are products of alternative splicing. Northern analysis of adult and fetal fibroblast RNA showed a dominant approximately 4.3-kilobase lysyl oxidase mRNA transcript that varied in abundance as a function of cell line. These data are consistent with a complex mechanism regulating the expression of the lysyl oxidase gene.

Adult↗

PubMedDate not supplied

Effects of transforming growth factor-beta1 on extracellular matrix gene expression by human fibroblasts from a laryngeal stenotic lesion.

Transforming growth factor-beta1 appears to play important roles in normal wound healing by increasing synthesis of extracellular matrix components. However, the role of transforming growth factor-beta1 in the production of excessive scar tissue by fibroblasts from stenotic lesions of the larynx has not been evaluated. We examined the effect of transforming growth factor-beta1 on the steady-state messenger RNA levels of elastin, alpha2(l) procollagen, and lysyl oxidase (the enzyme that cross-links both of these structural proteins) in cell cultures of diploid human fibroblasts established from fetal skin, newborn foreskin, and an adult laryngeal stenotic lesion. Time-course and dose-response experiments demonstrated that treatment with 500 pmol/L transforming growth factor-beta1 for 20 hours induced maximal levels of mRNA for elastin (7- to 59-fold) and alpha2(l) procollagen (1.7- to 2.4-fold) in all three cultures of fibroblasts. Transforming growth factor-beta1 also increased levels of lysyl oxidase mRNA in fibroblasts cultured from newborn foreskin (2.4-fold) and a stenotic lesion (10-fold) but had minimal effects on the fibroblasts cultured from fetal skin (1.1-fold), which constitutively expressed high levels of lysyl oxidase mRNA. Furthermore, the fibroblast culture established from a laryngeal stenotic lesion responded with the highest fold-induction for all three mRNAs. Inhibition of mRNA synthesis by actinomycin D showed that transcription was required for transforming growth factor-beta1 induction of elastin, alpha2(l) procollagen, and lysyl oxidase mRNA in all three cultures of fibroblasts. Inhibition of protein synthesis by cycloheximide showed that translation was required for maximal induction by transforming growth factor-beta1 of elastin mRNA but had no observable effect on alpha2(l) procollagen mRNA in all three cultures of fibroblasts. In addition, translation was required for maximal induction of the lysyl oxidase mRNA by transforming growth factor-beta1 in the fibroblasts cultured from a stenotic lesion but not for fibroblast cultures established from fetal and adult skin. These results show that transforming growth factor-beta1 coordinately increases mRNA levels for the structural extracellular matrix proteins collagen and elastin, as well as for the cross-linking enzyme, lysyl oxidase. These data also support the hypothesis that transforming growth factor-beta1 may contribute to the formation of laryngeal stenotic lesions.

Journal Article↗