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Biomedical subjects

M T Gillespie

Publications and source records attributed to M T Gillespie.

At least 73 records · Page 4Linked to original sources
PubMed1994

Dexamethasone regulation of parathyroid hormone-related protein (PTHrP) expression in a squamous cancer cell line.

Dexamethasone regulation of PTHrP expression has been studied in an epidermal squamous cancer cell line COLO 16, which secretes immunoreactive PTHrP into conditioned medium. Dexamethasone was found to suppress PTHrP expression in a time- and dose-dependent manner, which was reversible upon removal of dexamethasone. The half-maximal effective concentration of dexamethasone was 1 nM and an effect of dexamethasone on PTHrP mRNA was first observed after 2 h of treatment, with maximal inhibition by 6 h. Dexamethasone action on PTHrP expression was steroid specific since progestin, 5alpha-dihydroxytestosterone and oestrogen did not regulate PTHrP expression in COLO 16 cells. The gluocorticoid/progesterone receptor antagonist RU486 inhibited the dexamethasone effect, indicating glucocorticoid receptor-mediated regulation of PTHrP expression. The half-life of PTHrP mRNA in COLO 16 cells was approximately 120 min and was not altered by treatment of cells with dexamethasone. Nuclear run-on assays revealed that dexamethasone reduced PTHrP gene transcription in COLO 16 cells. Transient transfection assays with a series of reporter gene constructs encompassing 3.5 kb of the 5' end of the PTHrP gene failed to identify a region of the gene responsible for glucocorticoid down-regulation. PCR of reverse-transcribed RNA from COLO 16 cells revealed that dexamethasone down-regulated transcripts driven from all three promoters (i.e., the TATA promoters 5' to exons I and IV and the GC-rich promoter 5' to exon III) of the human PTHrP gene.

Carcinoma, Squamous Cell↗
PubMed1994

The parathyroid hormone-related protein gene and its expression.

Parathyroid hormone-related protein (PTHrP) was originally identified as a tumor product, but it is now established that PTHrP is expressed in many tissues where it exerts paracrine functions. Three potential isoforms of PTHrP, 139, 141 and 173 amino acids in length, have been described and these isoforms result from alternative splicing of the PTHrP gene. The gene is composed of nine exons of which only two are invariant in PTHrP transcripts. The other seven exons may be represented in the PTHrP mRNA complement as a result of alternative splicing, which allows for the production of up to 15 transcripts. Three spatially-distinct promoters, two TATA and one GC-rich region, are responsible for transcription of the gene and these appear to be differentially regulated. The PTHrP gene contains nucleotide sequence motifs in common with members of the immediate-early response gene family, as well as other hallmark features which include induction by growth factors, serum or cycloheximide and relatively short-lived mRNA.

Animals↗
PubMed1993

Transforming growth factor beta stimulation of parathyroid hormone-related protein (PTHrP): a paracrine regulator?

The regulation of PTHrP expression and production by transforming growth factor-beta (TGF beta) has been investigated in an epidermal squamous cancer cell line COLO 16. TGF beta 1 treatment increased steady-state levels of PTHrP mRNA and concentrations of PTHrP immunoreactivity in conditioned medium in a time- and dose-dependent manner with a half-maximal effect at 40 pM. An effect of TGF beta 1 on PTHrP mRNA was observed first after 4 h treatment and continued to increase up to 48 h with a concomitant increase in PTHrP immunoreactivity in the culture medium. TGF beta 1 was found to stabilize PTHrP mRNA as assessed by actinomycin C1 experiments. In addition, a direct effect of TGF beta to increase PTHrP transcription was indicated by nuclear run-on and transient transfection experiments using a CAT promoter/expression construct encompassing the region -1100 bp to -20 bp from the initiating AUG of the human PTHrP gene. The conditioned medium from COLO 16 cells was also shown to contain both latent and active TGF beta at concentrations of 160 pM and 16 pM, respectively, in 72 h conditioned medium. A neutralizing antibody to TGF beta 1 (and TGF beta 2) decreased the level of immunoassayable PTHrP in the medium.

Base Sequence↗
PubMed1993

Identification of brain isoforms of the rat calcitonin receptor.

Two rat calcitonin receptor isoforms have been identified by cDNA cloning from a hypothalamic library. The clones, C1a and C1b, specified proteins of 478 and 515 amino acids, respectively. The clones were identical, except that the C1b sequence encoded a 37-amino acid insert in the second extracellular domain, which conferred altered ligand recognition. Compared to the C1a receptor, expressed C1b receptors exhibited decreased affinity for porcine CT, relative to salmon CT, and negligible affinity for human CT. Clone C1b mRNA was predominately expressed in the brain, whereas mRNA for the C1a clone was present in both brain and peripheral tissues. Both receptors were able to couple functionally to adenylate cyclase. Thus, clone C1b represents a novel brain isoform of the CT receptor with different affinity for CT analogs resulting from an altered second extracellular domain.

Adenylyl Cyclases↗
PubMed1992

Substrate specificity and energetics of antiseptic and disinfectant resistance in Staphylococcus aureus.

Clinical isolates of Staphylococcus aureus carry various antiseptic and disinfectant resistance determinants (qac genes) on a variety of plasmids. The biochemistry and specificity of these resistance genes in S. aureus is the subject of this report. The qac genes were separated into two families on the basis of resistance profiles and DNA homology. Isotopic and fluorimetric assays demonstrated that the qac genes encode efflux systems that rely on proton motive force.

Anti-Infective Agents, Local↗
PubMed1992

Localization of parathyroid hormone-related protein mRNA expression in breast cancer and metastatic lesions by in situ hybridization.

Parathyroid hormone-related protein (PTHrP) has been identified immunohistochemically in 60% of breast carcinoma and in 92% of breast cancer metastases in bone. To establish whether the localization of the PTHrP antigen reflects protein synthesis and also to investigate the role of PTHrP in metastatic disease, as part of an ongoing study, we used in situ hybridization to study the localization of PTHrP mRNA in a retrospective series of primary breast tumors and their metastatic lesions. Paraffin sections of 17 primary and 26 metastatic lesions, 11 of which were in bone, were available for the study: 10 of the 17 (59%) primary lesions, 8 of 11 (73%) breast cancer metastases to bone, and 3 of 15 (20%) metastases to non-bone sites showed specific localization of PTHrP mRNA. These findings establish that PTHrP is commonly synthesized by primary breast cancers and support previous immunohistochemical studies reporting a higher incidence of PTHrP-positive tumor cells in skeletal metastases than in nonskeletal metastases.

Breast Neoplasms↗
PubMed1992

Production of parathyroid hormone-related protein by the rat mammary gland in pregnancy and lactation.

Production of parathyroid hormone-related protein by the rat mammary gland in pregnancy and lactation. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E1077-E1085, 1992.--Production of parathyroid hormone-related protein (PTHrP) by the mammary gland of Sprague-Dawley rats has been examined using immunohistochemistry and in situ hybridization to detect PTHrP and PTHrP mRNA, respectively. PTHrP and PTHrP mRNA could be demonstrated in nests of epithelial cells of the developing mammary gland at day 14 of pregnancy and in the epithelial secretory cells lining the alveoli during the latter stages of pregnancy and during lactation. A specific radioimmunoassay was also used to measure the concentration of PTHrP secreted in the milk throughout lactation. The concentration of PTHrP in milk was relatively low initially but increased during the latter stages of lactation, whereas calcium concentrations remained virtually constant throughout lactation. No correlation was found between the concentrations of calcium and PTHrP in rat milk. These results show that PTHrP is present in rat milk and also in mammary tissue before parturition, and therefore it may assist in the development of the mammary gland during pregnancy.

Animals↗
PubMed1992

Oestrogen enhancement of the myometrial response to exogenous parathyroid hormone-related protein (PTHrP), and tissue localization of endogenous PTHrP and its mRNA in the virgin rat uterus.

Classical pharmacological studies have shown that oestrogen dominance in humans and other animals can increase the responsiveness of the uterus to many locally acting peptides. Parathyroid hormone-related protein (PTHrP) has been shown to be expressed in the pregnant and non-pregnant rat uterus and exogenous PTHrP is known to relax uterine contraction in vitro. We investigated whether oestrogen dominance can influence the responsiveness of the uterine horn to PTHrP, and further studied the localization of PTHrP mRNA and protein in the rat uterine horn using in-situ hybridization and immunohistochemistry. Exogenous PTHrP(1-34) inhibited spontaneous and electrically induced contractions in uteri isolated from non-cycling rats. Pretreatment of non-cycling rats with oestradiol-17 beta increased uterine sensitivity to PTHrP: EC50 values for inhibition of spontaneous contractions by PTHrP were 0.33 nmol/l, 1.1 nmol/l, 2.6 nmol/l and 7800 nmol/l in uteri from animals treated for 2 days with oestradiol-17 beta alone, 2 days with oestradiol-17 beta + 1 day progesterone, 1 day with oestradiol-17 beta alone and in untreated rats respectively. Similar EC50 values were obtained for electrically stimulated uteri. In agreement with these findings, uterine horns from cycling rats in pro-oestrous and oestrous phases of the cycle showed a higher responsiveness to PTHrP(1-34) when compared with uterine horns taken from rats in metoestrus and dioestrus. PTHrP mRNA and protein were detected in the endometrial epithelium lining of the lumen and the endometrial glands, as well as in the myometrium of rats which were either pretreated for 2 days with oestradiol-17 beta or untreated. This study suggests that PTHrP may act in an autocrine and/or paracrine manner to modulate uterine motility and function.

Animals↗
PubMed1991

4',4'' adenyltransferase activity on conjugative plasmids isolated from Staphylococcus aureus is encoded on an integrated copy of pUB110.

In staphylococci, linked resistance to the aminoglycosides kanamycin, neomycin, paromomycin, and tobramycin (KmNmPmTmr) is generally mediated by an aadD determinant which encodes production of an adenyltransferase aminoglycoside modifying enzyme, AAD(4',4''). The aadD resistance determinant is located on small multicopy plasmids such as pUB110, and has also been found on large multiresistance plasmids and on the chromosome in some strains. Examination of two conjugative plasmids from strains of Staphylococcus aureus isolated in North America indicated that the aadD determinant on these plasmids is located on an integrated copy of pUB110. The integrated pUB110 is flanked by direct repeats of the staphylococcal insertion sequence IS257. Analysis of the conjugative plasmid pSK41 showed an 8-bp duplication of the pUB110 sequence immediately adjacent to flanking IS257 elements, suggesting that integration of pUB110 was mediated by IS257.

Aminoglycosides↗
PubMed1991

Parathyroid hormone-related protein: biochemistry and molecular biology.

This article critically reviews the current state of knowledge regarding the recently identified and cloned novel hormone parathyroid hormone-related protein (PTHrP). PTHrP is produced by tumors associated with the syndrome of humoral hypercalcemia of malignancy giving rise to the parathyroid hormone (PTH)-like symptoms characteristic of the syndrome. Areas that will be reviewed include identification, purification and cloning, localization, actions, and significance of PTHrP in cancers and normal physiology. The structure and regulation of the PTHrP gene that may be ancestrally related to the PTH gene will also be discussed. Studies in vivo and in vitro with synthetic and recombinant PTHrP sequences and antibodies developed against them have established that the PTH-like actions of PTHrP are mediated via the N-terminal sequences, which show some limited sequence homology with PTH. Evidence for PTH and non-PTH-like actions of PTHrP in normal physiology, which implicate a role for PTHrP in fetal and neonatal development, is also presented.

Amino Acid Sequence↗
PubMed1990

Typing of methicillin-resistant Staphylococcus aureus by antibiotic resistance phenotypes.

The identification of new epidemic strains of methicillin-resistant Staphylococcus aureus is essential for rapid, effective infection control. We have developed a typing method which uses antibiotic sensitivity patterns to differentiate methicillin-resistant S. aureus and which is faster and more cost-effective than biochemical analysis or bacteriophage typing. Characterisation of phenotypes which are chromosomally-encoded, plasmid- or chromosomally-encoded or exclusively plasmid-mediated has enabled us to separate Australian strains of methicillin-resistant S. aureus into 11 classes, representatives of which were indistinguishable by bacteriophage type, or plasmid profile alone. The value of this procedure is thus clearly shown.

Acriflavine↗
PubMed1990

Molecular analysis of a gentamicin resistance transposonlike element on plasmids isolated from North American Staphylococcus aureus strains.

Plasmid-encoded resistance to the aminoglycosides gentamicin (Gm), tobramycin (Tm), and kanamycin (Km) (GmTmKmr) in strains of Staphylococcus aureus isolated in Australia and North America appears to be mediated by one resistance determinant. In Australian isolates, this determinant is flanked by inverted copies of a 1.3-kb insertion sequence, IS256, thereby forming a composite transposon, Tn4001. Analysis of two conjugative plasmids and a related nonconjugative plasmid from strains of S. aureus isolated in North America showed that the GmTmKmr determinant on these plasmids is also flanked by inverted repeats. In the nonconjugative plasmid, these repeats include only 425 bp of IS256 immediately adjacent to the GmTmKmr region and identical to that on Tn4001. This truncated Tn4001 element is flanked by copies of the insertion element IS257, and together these elements form a truncated Tn4001-IS257 hybrid transposonlike structure. A third copy of IS257 was located 418 bp from the hybrid structure. The truncated Tn4001 and three repeats of IS257 were present at a conserved site on the plasmids studied. Four additional copies of IS257 were identified on the two conjugative plasmids. These elements flank determinants for resistance to the aminoglycosides neomycin and paromomycin and to ethidium bromide and quaternary ammonium compounds, as well as the region involved in conjugative plasmid transfer.

Amino Acid Sequence↗
PubMed1989

Structure of the 5' flanking region of the gene encoding human parathyroid-hormone-related protein (PTHrP).

We have characterized a human genomic clone that contains the 5' coding and 5' flanking sequences of the human parathyroid hormone-related protein gene (PTHrP). The 5' end of the gene contains three exons separated by two small introns of 60 and 165 bp, respectively. The coding region of the PTHrP gene exhibits significant structural homology to the human parathyroid hormone gene (PTH), including the position of at least two introns. However, there is no significant nucleotide sequence homology to the PTH gene within the intragenic region nor in the flanking genomic sequences. The PTHrP gene has been localized, by chromosomal in situ hybridization to bands p11 or p12, on human chromosome 12. Analysis of the 5'-noncoding DNA reveals a complex, putative regulatory region, with multiple potential transcription start points. Nucleotide sequence analysis shows the position of one consensus TATA sequence, at -514 bp, from the start of translation whereas the other regulatory domain is located at least 1 kb further 5' to this consensus TATA sequence. Evidence from the structure of a number of cDNA clones, as well as S1 nuclease and primer extension studies supports the hypothesis that the PTHrP gene contains at least two mRNA transcription start points that define two putative regulatory domains. The result of expression from these different promoters combined with an alternative splicing event would be to produce multiple forms of PTHrP mRNA that differ in the 5'-untranslated region. This analysis of the human PTHrP gene is the first report of a PTHrP gene for any species.

Base Sequence↗
PubMed1989

Parathyroid hormone-related protein: isolation, molecular cloning, and mechanism of action.

Many factors, such as interleukin 1, TGF alpha, tumor necrosis factor alpha and beta, and PGs, have been implicated in etiological roles in HHM (Martin and Mundy, 1987). Much interest in the past has also centered upon the likelihood of ectopic secretion of PTH in this condition. We have purified a protein (PTHrP) implicated in HHM from a human lung cancer cell line (BEN). Full-length cDNA clones have been isolated and were found to encode a prepropeptide of 36 amino acids and a mature protein of 141 amino acids. Eight of the first 13 amino acids were identical with human PTH, although antisera directed to the NH2 terminus of PTHrP do not recognize PTH; this homology is not maintained in the remainder of the molecule. PTHrP therefore represents a previously unrecognized hormone, possibly related to the PTH gene by a gene duplication mechanism. In support of this notion, the PTHrP gene has been localized to the short arm of chromosome 12; it is believed that chromosome 11, containing the PTH gene, and chromosome 12 are evolutionarily related. In addition, the human PTHrP gene has been isolated, characterized, and shown to have a similar intron--exon organization as the PTH gene. It is possible that the original ancestral gene is indeed the PTHrP gene; resolution of this question awaits studies in lower species. Peptides synthesized to the predicted protein sequence have enabled detailed structure-function studies that have identified NH 2-terminal sequences to be responsible for the biological effects of the molecule. Antibodies raised against the various synthetic peptides have led to the immunohistochemical localization of PTHrP in many human squamous cell carcinomas as well as in a subpopulation of keratinocytes of normal skin. The availability of these antibodies has opened the way for the development of a radioimmunoassay to detect PTHrP in the sera of cancer patients at risk of developing hypercalcemia. The recent characterization of PTHrP-like activity in the ovine fetus suggests some physiological function for PTHrP. It is possible that PTHrP, as the fetal counterpart of PTH, has the role of maintaining the maternal-fetal calcium gradient. The isolation and characterization of PTHrP have added to our understanding of the mechanisms of hypercalcemia and may contribute to the understanding of other metabolic bone diseases, such as osteoporosis and Paget's disease. Finally, and perhaps most importantly, PTHrP may play a hitherto unrecognized role in normal cell physiology.

Amino Acid Sequence↗