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T J Martin

Publications and source records attributed to T J Martin.

At least 235 records · Page 13Linked to original sources
PubMed1991

Structure-activity relationships in calcitonin gene-related peptide: cyclic AMP response in a preosteoblast cell line (KS-4).

Synthetic analogs of chicken and human CGRP were used to define structure-function relationships in a murine osteoblast precursor cell line, KS-4, that exhibits a substantial cyclic AMP response to CGRP but no response to calcitonin. Human CGRP had 40% of the activity of chicken CGRP, but [Asp-14]-human CGRP was equipotent with chicken CGRP. Similar observations were made previously for calcium- and phosphate-lowering effects. Des-1-Ala,deamino CGRP compounds of both human and chicken origin were three-fold more potent than the respective native CGRPs. The [4-F-Phe)-37]-chicken CGRP analog had a four-fold enhanced activity. In all of the 13 analogs either truncated amino-terminally or missing the (2-7)-disulfide bridge, biologic activity was greatly reduced (0.1% of chicken CGRP), as it was in chicken CGRP-(1-36)-OH, which lacks the C-terminal amino acid. The same analog has been shown to retain a hypocalcemic effect, which is most likely mediated through calcitonin receptors. In confirmation of their antagonistic effect reported in liver, analogs that lack the N-terminal ring structure exhibited significant antagonistic activity. The data illustrate the specificity of CGRP receptors in promoting cyclic AMP formation in osteoblast-like cells. Such CGRP analogs will be useful in investigating structure-function relationships of CGRP.

Animals↗
PubMed1991

Lack of effects of neutralization of parathyroid hormone-related protein on calcium homeostasis in neonatal mice.

Large quantities of parathyroid hormone-related protein (PTHrP) are present in the milk of various species. It has been suggested that PTHrP may play a role in neonatal calcium homeostasis. In the present study we evaluated the effect of neutralization of amino-terminal PTHrP activity by passive immunization in 1-day-old mouse pups. Neutralization of amino-terminal PTHrP activity had no significant effect on serum calcium or whole-body calcium content in the neonatal mice. In additional studies, we demonstrated that subcutaneous administration of PTHrP-(1-34) increased serum calcium, whereas oral administration had no significant effect in 3-day-old pups. The studies therefore demonstrate that the amino terminus of PTHrP may not play a significant role in neonatal calcium homeostasis. Local effects of PTHrP cannot be excluded by the results of the present study.

Administration, Oral↗
PubMed1991

Regulation of osteoblast proliferation by leukemia inhibitory factor.

We recently showed that leukemia inhibitory factor (LIF) stimulates 45Ca release from neonatal mouse calvariae in vitro and that it increases DNA and protein synthesis in this model. To elucidate further the actions of LIF on bone we now report the effects of this cytokine on DNA synthesis and cell proliferation in isolated fetal rat osteoblasts and in the osteogenic sarcoma cell line, UMR-106. In both actively growing and growth-arrested rat osteoblasts, LIF stimulated [3H]thymidine incorporation in a dose-dependent manner. The increase in DNA synthesis was time dependent, was associated with an increase in the number of osteoblasts, and was not blocked by indomethacin. LIF-treated cells showed reduced [3H]thymidine incorporation in comparison with control, as they approached confluence, possibly because of the increased cell density in the LIF-treated cultures. In UMR-106 cells, treatment with LIF inhibited [3H]thymidine incorporation in both actively growing and growth-arrested cultures. The effect was dose dependent and sustained with time. There was a corresponding decrease in cell numbers. It is concluded that although LIF causes an early stimulation of proliferation in isolated osteoblasts, it has opposing effects on UMR-106 cells. It is not possible to determine which of these effects is more relevant to the actions of LIF in vivo. The demonstration of a LIF effect on both these cell types, however, provides further evidence that this cytokine acts directly on osteoblasts.

Animals↗
PubMed1991

Transforming growth factor beta inhibits plasminogen activator (PA) activity and stimulates production of urokinase-type PA, PA inhibitor-1 mRNA, and protein in rat osteoblast-like cells.

Transforming growth factor beta (TGF beta) treatment of rat osteoblast-rich calvarial cells or of the clonal osteogenic sarcoma cells, UMR 106-01, resulted in dose-dependent inhibition of plasminogen activator (PA) activity, and increased production of 3.2 kb mRNA and protein for PA inhibitor -1 (PAI-1). Although tissue-type PA (tPA) protein was not measured, TGF beta did not influence production of mRNA for tPA. Production of 2.3 kb mRNA for urokinase-type PA (uPA) was also increased by TGF beta in a dose-dependent manner. The effects of TGF beta on synthesis of mRNA for PAI-1 and uPA were maintained when protein synthesis was inhibited, and were abolished by inhibition of RNA synthesis. Although uPA had not been detected previously as a product of rat osteoblasts, treatment of lysates of osteoblast-like cells with plasmin yielded a band of PA activity on reverse fibrin autography, corresponding to a low Mr form of uPA. Untreated conditioned media from normal osteoblasts or UMR 106-01 cells contained no significant TGF beta activity, but activity could be detected in acidified medium. Treatment of conditioned media with plasmin resulted in activation of approximately 50% of the TGF beta detectable in acidified media. The results identify several effects of TGF beta on the PA-PA inhibitor system in osteoblasts. Net regulation of tPA activity through the stimulatory actions of several calciotropic hormones and the promotion of PAI-1 formation by TGF beta could determine the amount of osteoblast-derived TGF beta activated locally in bone. Stimulation of osteoblast production of mRNA for uPA could reflect effects on the synthesis of sc-uPA, a precursor for the active form of the enzyme.

Animals↗
PubMed1991

Effects of leukemia inhibitory factor on bone resorption and DNA synthesis in neonatal mouse calvaria.

Leukemia inhibitory factor (LIF) is a recently characterized cytokine which has been shown to regulate cell growth and differentiation in a variety of tissues. We have shown that LIF stimulates bone resorption and DNA synthesis in bone organ culture and, in vivo, LIF has been shown to have marked effects on bone remodeling. The present study examines further the dose-response, time course and mechanisms of action of LIF in neonatal mouse calvaria. 45Ca release was significantly increased by LIF at concentrations of 10-5,000 U/ml, and its stimulation of bone resorption increased with time from 24 to 96 hours. These concentrations of LIF also increased DNA synthesis at 24 hours. At 72 hours, low concentrations of LIF produced less marked stimulation of [3H]-thymidine incorporation, and 5,000 u/ml actually inhibited DNA synthesis at both this time point and at 96 hours. The effect of LIF on 45Ca release was partially inhibited when DNA synthesis was blocked by hydroxyurea (50 microM). The resorptive effect of supramaximal concentrations of LIF was not additive to that of parathyroid hormone, 1,25-dihydroxyvitamin D3, prostaglandin E2, or transforming growth factor-beta. Although LIF-stimulated resorption is at least partially dependent on DNA synthesis, these results suggest that there are different mechanisms involved in mediating LIF's effects on bone resorption and DNA synthesis. The demonstration of effects of LIF at low concentrations indicates that this cytokine may be involved in the physiological regulation of bone metabolism in vivo.

Animals↗
PubMed1991

Circumvention of doxorubicin resistance in multi-drug resistant human leukaemia and lung cancer cells by the pure antioestrogen ICI 164384.

ICI 164384, a new steroidal antioestrogen, entirely devoid of oestrogenic activity, modulates doxorubicin resistance in vitro. At non-cytotoxic concentrations, ICI 164384 potentiated the cytotoxicity of doxorubicin in a dose-dependent manner in both the classical multi-drug resistant (MDR) human leukaemia cell lines CEM/VLB 100 and CEM/VLB 1000 and the human small cell lung cancer cell line H69 LX4. ICI 164384 had no effect on the two respective parental cell lines, CEM/CCRF and H69 P. None of these cell lines expressed the oestrogen receptor. In comparative studies at concentrations ranging from 1.25 to 10 mumols/l, ICI 164384 was significantly more effective (1.2-6-fold) than tamoxifen in reducing the IC50 of doxorubicin in the CEM/VLB 100 line. In resistant cells, ICI 164384 increased 3H-daunomycin accumulation in a dose-dependent manner and was significantly more effective than tamoxifen at concentrations ranging from 2.5 to 10 mumol/l. ICI 164384 reduced the efflux of daunomycin from resistant cells more effectively than tamoxifen. These studies suggest that ICI 164384 is an effective modulator of MDR.

Carcinoma, Small Cell↗
PubMed1991

Synthesis of 2-exo- and 2-endo-mecamylamine analogues. Structure-activity relationships for nicotinic antagonism in the central nervous system.

Nine analogues of mecamylamine (2) which differ in the number and substitution pattern of methyl groups, were prepared. In four of these analogues the amine functionality is in an endo orientation. Enantiomers of 2-endo- and 2-exo-N-methylfenchylamine (25 and 26, respectively) were also prepared. The hydrochloride salts of these compounds were tested for nicotinic antagonism relative to mecamylamine in vivo and none was found to be as potent as mecamylamine, although a broad range of activity was observed. In general, methyl substituents at the C1, C2, and C7 positions of the mecamylamine structure do not appear to be significant for antagonistic activity. Methyl substituents at C3, however, appear to be very important for activity. Three sets of enantiomers of N-methylfenchylamine analogues, 28-30, possessing structural features of mecamylamine and nicotine were also prepared. These compounds were inactive as antagonists. Only a small degree of stereoselectivity was elicited in this series, less than that seen with enantiomers of nicotine. Antagonists with the exo N-methylamine functionality are slightly more active than the endo isomers. The extent to which structural modification might change lipophilicities was estimated through calculated partition coefficients; such changes alone appeared insufficient to explain differences in activities of the analogues. Lastly, a tolerance for a tertiary (dimethyl) amine functionality was demonstrated in addition to the lack of tolerance for bulkier substituents at C3 or on the nitrogen.

Analgesia↗
PubMed1991

Role of the fetal parathyroid glands and parathyroid hormone-related protein in the regulation of placental transport of calcium, magnesium and inorganic phosphate.

The plasma Ca concentration of the fetus is maintained higher than maternal levels by active placental transport. Ca, Mg and PO4 accumulation by the fetus is mainly associated with skeletal growth. The fetal parathyroid glands are essential for maintenance of elevated plasma Ca, which is necessary for the stimulation of fetal osteoblasts and mineralization of cartilage and osteoid. Fetal thyroparathyroidectomy (TxPTx) results in a decreased activity of the placental Ca pump. The presence of a parathyroid hormone-related protein (PTHrP) has been demonstrated in fetal parathyroid glands and placental tissue. Extracts of fetal parathyroid glands and purified PTHrP, as well as recombinant PTHrP (1-84, 1-108 and 1-141), stimulate Ca and Mg but not PO4 transport across the placenta of TxPTx-ized fetuses perfused with autologous blood in the absence of the fetus. Parathyroid hormone (PTH) and the N-terminal region of PTHrP do not stimulate placental Ca and Mg transport. It is concluded that a mid-molecule region of this novel hormone may be required to stimulate placental Ca transfer and contribute to the regulation of fetal Ca homeostasis.

Animals↗
PubMed1991

Production of calcitonin gene related peptide, calcitonin and PTH-related protein by a prostatic adenocarcinoma.

PTH and calcitonin are the two major hormones controlling calcium metabolism. Recently two new substances related to these hormones have been isolated: calcitonin gene related peptide (CGRP) and PTH-related protein (PTHrP). CGRP is a potent vasodilator and stimulant of intestinal secretion while PTHrP is probably the agent responsible for humoral hypercalcaemia of malignancy. We report here a patient with a prostatic tumour presenting with vasodilation, diarrhoea and hypercalcaemia. Our investigations revealed that the primary prostatic and liver secondary tumour contained CGRP, calcitonin and PTHrP. Most of the immunoreactive CGRP in the tumour and plasma co-eluted with the biologically active form of CGRP. The circulating levels of CGRP correlated with the presence of the diarrhoea. PTHrP concentration in the tumours was one of the highest reported for any tumour although previous studies may have utilized less than optimal extraction procedures. The somatostatin analogue, octreotide (SMS 201-995), did not reduce the plasma CGRP or the diarrhoea, a finding similar to that seen in patients with medullary thyroid carcinoma and high plasma CGRP. The hypercalcaemia was also unaffected by octreotide administration. This is the first report of a prostatic tumour associated with over-production of calcitonin, PTHrP and CGRP. The major life-threatening effects of this unusual case of prostatic carcinoma were diarrhoea and hypercalcaemia. Both these effects could be tentatively ascribed to newly discovered substances, CGRP and PTHrP. With the greater availability of assays to measure CGRP and PTHrP in plasma, a detailed examination of the incidence of over-production of these substances in various cancers will be possible.

Adenocarcinoma↗
PubMed1991

Parathyroid hormone-related protein of malignancy: immunohistochemical and biochemical studies in normocalcaemic and hypercalcaemic patients with cancer.

Immunohistochemical staining for parathyroid hormone-related protein was performed in 27 tumours from 19 normocalcaemic and eight hypercalcaemic patients with cancer. All the tumours from hypercalcaemic patients stained positively for the protein, as did 17 tumours from normocalcaemic patients. Only hypercalcaemic patients had biochemical evidence of increased bone resorption and abnormalities of renal tubular reabsorption of calcium and phosphate, consistent with the presence of parathyroid hormone-related protein. While tumour mass was higher in hypercalcaemic patients, only one of the initially normocalcaemic patients with positively staining tumours subsequently went on to develop hypercalcaemia and more advanced disease. These data confirm the importance of parathyroid hormone-related protein as a mediator of humoral hypercalcaemia in patients with solid tumours and suggest that low tumour mass may be one reason why serum calcium values are not increased in all patients with tumours containing parathyroid hormone-related protein. None the less normocalcaemia, despite tumour progression in patients whose tumours stained positively for parathyroid hormone-related protein, suggests that other factors may also be important, such as differences in the rate of secretion of the protein by different tumours, or the production of different forms of parathyroid hormone-related protein with varying biological effects.

Humans↗
PubMed1991

Growth hormone (GH) receptors in clonal osteoblast-like cells mediate a mitogenic response to GH.

GH receptors have not hitherto been demonstrated or characterized on osteoblasts. In this study we report the characterization of functional GH receptors on the clonal rat osteoblast-like cell line UMR 106.06. The receptors have a typical somatogenic specificity, with high affinity for human GH, 10-fold lower affinity for rat GH, and very poor affinity for rat PRL. The affinity for rat GH is 1.2 +/- 0.4 x 10(9) M-1, and there are approximately 9000 receptors per cell. GH binding increased over several hours when incubations were carried out in serum free minimal essential medium, but binding reduced rapidly when incubations were carried out in Tris-NaCl-Mg++, HEPES, or bicarbonate buffer, suggesting a critical dependence of receptor expression on nutritional factors. Rat GH stimulated proliferation of UMR 106.06 cells in a dose-dependent fashion with a maximum 43 +/- 2% stimulation above control and half-maximal effect at a final hormone concentration of 15 +/- 3 ng/ml. A proliferative response was not observed at low cell density, suggestive of a requirement for a threshold concentration of autocrine mediators or density-dependent receptor expression. A monoclonal antibody (MAb 263) which blocks GH binding to a subset (type 1) of rat hepatic GH receptors did not block binding to osteoblast GH receptors, and did not block the proliferative response. Thus, the proliferative response appears to be mediated by a class of GH receptors not blocked by MAb 263 and possibly related to the type 2 hepatic GH receptors. RNA was extracted from UMR 106.06 cells and a second osteoblast-like cell line UMR 201. Hybridization to a 32P-labeled complementary DNA probe to the rabbit hepatic growth hormone receptor revealed two major labelled bands (3.5 and 1.2 kilobases) and one minor band (2.4 kilobases) in both cell types. In summary, GH receptors are present in clonal osteoblast-like cells, and the receptors mediate a proliferative response to GH. The UMR 106.06 cells provide a valuable model system for studying the mechanism of GH action.

Animals↗
PubMed1991

Ontogeny of parathyroid hormone-related protein in the ovine parathyroid gland.

PTH-related protein (PTHrP) has been implicated in calcium regulation during fetal life. In this study the ontogeny of PTHrP was examined in ovine parathyroid glands. Immunohistochemical techniques, Western blot analysis, and a RIA with antisera raised against synthetic fragments of human (h) PTHrP (i.e. 1-34, 1-40, 50-69, and 107-141) were used to detect the presence of immunoreactive PTHrP in parathyroid glands from fetal and neonatal lambs and maternal ewes. Positive immunostaining for PTHrP was observed in fetal (from 116 days of gestation) and lamb (up to 180 days post birth) but not maternal parathyroid glands with the PTHrP(50-69) antiserum. Fetal and lamb parathyroid glands consisted entirely of one cell type in which PTHrP immunoreactivity to PTHrP(50-69) antiserum was found. In contrast, immunoreactivity to PTHrP could not be detected in sections of fetal, lamb, or maternal parathyroid glands with antisera raised against PTHrP(1-34) or PTHrP(107-141). However, PTHrP immunoreactivity in urea/acid extracts of newborn lamb parathyroid glands could be detected by Western blot analysis and RIA with antisera raised against the N-terminal portion of PTHrP. Western blot analysis with the PTHrP(1-34) antisera revealed that urea/acid extracts of newborn lamb parathyroid glands contained a substance with a mol wt of 14.4K, which corresponded in size to that of hPTHrP(1-84). Newborn lamb parathyroid glands contained 0.35 ng PTHrP/micrograms extract, whereas maternal parathyroid glands contained only 0.035 ng PTHrP/micrograms extract when tested in a RIA employing recombinant hPTHrP(1-84) as standard and an antibody raised against hPTHrP(1-40). The detection of immunoreactive PTHrP in the developing ovine parathyroid gland provides further evidence to support the suggestion that PTHrP produced in the parathyroid gland is involved in the normal hormonal regulation of calcium metabolism in the mammalian fetus and neonate.

Animals↗
PubMed1991

A carboxyl-terminal peptide from the parathyroid hormone-related protein inhibits bone resorption by osteoclasts.

PTH-related protein (PTHrP) interacts, via its amino-terminal 34 residues, with PTH receptors on osteoblasts to stimulate osteoclastic bone resorption indirectly. We now report that mature hPTHrP-(1-141) (EC50, approximately 10(-11) M) and a carboxyl-terminal fragment, PTHrP-(107-139) (EC50, approximately 10(-15) M), are potent inhibitors of resorption in an isolated rat osteoclast bone resorption assay, whereas hPTHrP-(1-108) and hPTHrP-(1-34) are inactive in this respect. PTHrP-(107-139) also inhibits resorption in a rat long bone organ culture system and reduces osteoclast spreading. PTHrP-(107-139) does not act on osteoclasts via a cAMP signal transduction mechanism, but its effects may be mediated by protein kinase-C. This previously unrecognized action of PTHrP, to inhibit osteoclastic bone resorption directly, indicates that PTHrP may be a precursor of multiple biologically active peptides with differing physiological functions.

Amino Acid Sequence↗
PubMed1991

A potent inhibitor of osteoclastic bone resorption within a highly conserved pentapeptide region of parathyroid hormone-related protein; PTHrP[107-111].

We have recently shown that a carboxyl-terminal sequence of parathyroid hormone-related protein, PTHrP[107-139], is a potent direct inhibitor of osteoclastic bone resorption. We now report that this bone resorption inhibitory activity, which we have called osteostatin, is entirely contained within the highly conserved pentapeptide PTHrP[107-111]. Our results indicate that processing at residue 106 and a free amino terminus is required for full activity of the peptide. The retroinverted peptide is considerably less potent than the parent peptide. The retention of full biological activity in such a short fragment was unexpected. This data provides the basis for the development of further analogs with potential therapeutic use in disorders associated with increased osteoclastic bone resorption.

Amino Acid Sequence↗
PubMed1991

Parathyroid hormone-related protein: elevated levels in both humoral hypercalcemia of malignancy and hypercalcemia complicating metastatic breast cancer.

A RIA for PTH-related protein (PTHrP) is described, using a polyclonal goat antiserum against synthetic PTHrP-(1-40) and recombinant PTHrP-(1-84) as standard. The detection limit is 2 pmol/L, and intra- and interassay coefficients of variation are 4.8% and 13.6%, respectively. This assay does not detect PTH even at concentrations of up to 2000 pmol/L. Cross-reactivity studies using various synthetic PTHrP peptides localize the antibody-binding epitope between residues 20 and 29. Hypercalcemic patients with a range of solid tumors and no evidence of bone metastases on radionuclide scanning (n = 27) all had detectable PTHrP levels (range, 2.8-51.2 pmol/L). Of 17 patients with solid tumors (other than breast) and bone metastases, 11 (64%) also had detectable PTHrP levels (range, 4.9-47.5 pmol/L). Twenty samples from breast cancer patients with hypercalcemia, 19 with evidence of bone metastases, and 1 with a negative bone scan were assayed, and detectable PTHrP levels were found in 13 (65%; range, 3.8-61.6 pmol/L). Patients with squamous cell carcinomata and normal serum calcium levels (n = 11) had no detectable PTHrP or levels close to the detection limit of the assay (range, less than 2 to 3.7 pmol/L). Plasma levels in normal volunteers were below the detection limit of the assay in all but 1 of 38 normal subjects. Patients with chronic renal failure on hemodialysis (n = 18) and patients with primary hyperparathyroidism (n = 14) all had undetectable PTHrP in this assay. This assay allows positive identification of patients with PTHrP-mediated hypercalcemia and, therefore, should be useful in the clinical investigation of the hypercalcemic patient. Furthermore, it has allowed detection of circulating PTHrP in hypercalcemic breast cancer patients with bone metastases, indicating a significant role for PTHrP in this disease.

Adult↗
PubMed1991

Parathyroid hormone-related protein in milk and its correlation with bovine milk calcium.

Parathyroid hormone-related protein (PTHrP) has been shown to be present in milk of various mammals. We have assayed PTHrP in milk of various species by radioimmunoassay and estimated the molecular weights by Western blot analysis. PTHrP concentrations in bovine, ovine and human milk were 59.2 +/- 18.5, 74.1 +/- 35.0 and 36.6 +/- 20.7 micrograms/l (mean +/- S.D.) respectively, in pooled samples collected at various stages of lactation. PTHrP in mammalian milk was found to exist in two forms with molecular weights of 17.5 kDa and 21.5 kDa approximating those of PTHrP(1-108) and (1-141) respectively. In comparison, marsupial milk PTHrP appeared as a single low molecular weight form of 14.4 kDa which approximated to that of PTHrP(1-84). We performed a longitudinal study measuring the concentration of PTHrP in milk throughout lactation in cows, and found it to increase with the duration of lactation (r = 0.669, n = 91). We further examined the relationship between the concentration of PTHrP and total calcium in bovine milk, and the differences between these constituents in milk from Friesian and Jersey cows. PTHrP concentrations correlated positively with total milk calcium (r = 0.346, n = 105). The mean milk concentration of PTHrP of the Jerseys was significantly higher than that of the Friesians (52.6 +/- 5.4 micrograms/l compared with 41.0 +/- 4.8 micrograms/l, P less than 0.01), as was the mean milk calcium concentration (30.5 +/- 3.0 mmol/l compared with 26.7 +/- 2.7 mmol/l, P less than 0.01). We therefore postulate that production of PTHrP by the mammary gland may be associated with calcium transport from blood to milk. Also PTHrP may play a role in the development of milk fever in Jerseys which are predisposed to this condition.

Animals↗
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↗