The boundaryless hospital--a commentary.
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Australia↗
Biomedical subjects
T J Martin
Publications and source records attributed to T J Martin.
In situ hybridization of parathyroid hormone-related protein in normal skin, skin tumors, and gynecological cancers using digoxigenin-labeled probes and antibody enhancement.
We describe a novel procedure for in situ hybridization that combines the use of digoxigenin-labeled oligonucleotide probes with an antibody enhancement step that can be performed on formalin-fixed, paraffin-embedded tissues. Addition of a second antibody enhances the visibility of parathyroid hormone-related protein (PTHrP) mRNA expression from barely to highly discernible and interpretable, with virtually no nonspecific background expression. This technique has allowed visualization of PTHrP mRNA in normal human skin and epithelium-derived tumors. PTHrP mRNA expression was confined to the basal and spinous keratinocyte layers of skin. There was strong hybridization in the spinous keratinocyte layer and a low level of hybridization in the basal layer. An extensive panel of positive and negative controls included poly d(T) probe to indicate total mRNA present in the sections. Squamous cell carcinomas and basal cell carcinomas of the skin, from pathology archives, were examined for the presence of PTHrP mRNA. The results reflected previous immunohistochemical studies, with every squamous cell carcinoma hybridizing strongly with the PTHrP probes. The basal cell carcinomas showed no expression of PTHrP mRNA, although the total mRNA signal was very strong. The localization of PTHrP mRNA in the tumors of the gynecological tract also reflected the immunohistochemical findings, with expression found in the squamous cell carcinomas but not in the adenocarcinomas. In situ hybridization with digoxigenin-labeled oligonucleotide probes and antibody enhancement has provided a sensitive, highly specific procedure for detection of PTHrP mRNA in tumors and normal tissue.
Antibodies, Monoclonal↗
Homologous regulation of the calcitonin receptor in mouse osteoclast-like cells and human breast cancer T47D cells.
Calcitonin (CT) down-regulates its receptor in several cancer cell lines, including T47D human breast cancer cells. Removal of CT results in the recovery of CT receptor (CTR) binding. However, homologous regulation of the CTR in osteoclasts is not well understood. To elucidate these phenomena in cells of the osteoclast lineage, mouse osteoblasts and bone marrow cells were cocultured on type 1 collagen gels. For the experiments, osteoclast-like cell (OCL)-enriched populations were subcultured from the collagen gels into multiwell dishes on days 7-8, and CT regulation of the CTR was determined and compared with that in T47D cells. When cells of either type were treated with CT for 1 h and then washed, binding capacity for [125I] salmon CT ([125I]sCT) was decreased dependent upon the preincubating concentration of CT. After removal of CT, the binding capacity in OCLs recovered toward the control level over 12 h. However, in contrast to that in T47D cells, recovery was transient, so that 24 h after removal of CT, the binding capacity in preincubated cells was strikingly reduced compared with that in control cells. This occurred even when the preincubating concentration of CT was too low to cause down-regulation of binding in 1 h. Scatchard analysis showed a decrease in receptor number in CT-treated compared with control OCLs 24 h after CT removal, with unchanged receptor affinity. By autoradiography, decreased CTR density on multinuclear OCLs was indicated. Preexposure of either OCLs or T47D cells to CT caused elevation of intracellular cAMP, which persisted for 6-12 h after removal of CT. In addition, there was desensitization to a rechallenge with CT, which, in T47D cells, recovered by 24-36 h. In contrast, OCLs showed incomplete recovery of desensitization. These data correlated with the results of semiquantitative reverse transcription-polymerase chain reaction studies; the CTR messenger RNA level was increased to about 150% of the control level in sCT-treated T47D cells 18-36 h after sCT removal; the level was markedly decreased to about 20% of the control value in sCT-treated OCLs 12-48 h after sCT removal and remained suppressed. This study suggests that CT-induced homologous down-regulation is a potential cause of the "escape" phenomenon, by producing a population of CT-resistant osteoclasts.
Animals↗
Regulation and regulatory role of the retinoids.
Retinoids regulate differentiation and cellular growth, exerting their physiological action by interacting with two families of nuclear receptors, the retinoic acid receptors (RARs), and the retinoid X receptors (RXRs), which regulate gene expression by forming transcriptionally active heterodimeric RAR/RXR or homodimeric RXR/RXR complexes on DNA. Although RAR/RXR heterodimers form preferentially in vitro and in vivo, it does not exclude the possibility that RXR/RXR homodimers may regulate a distinct signaling pathway. Synthetic retinoids that selectively activate or antagonize retinoic acid receptor isoforms promises to be useful tools for the elucidation of specific retinoid response pathways. Considerable progress has been made in understanding the molecular basis underlying limb bud formation, particularly the manner in which retinoic acid interacts with other signaling molecules to determine pattern formation. The phenotypic abnormalities observed in compound null mutants of retinoid receptors, recapitulating those described in the vitamin A deficiency syndrome, confirm the crucial function of endogenous retinoids in fetal development. However, the absence of phenotypic abnormalities in null mutants of individual RAR isoforms raises the possibility of functional redundancy among RAR subtypes and at the same time challenging the concept that the diverse effects of retinoids are related to the multiplicity of fnctionally distinct receptors.
Animals↗
Multiple sites of synthesis and action of parathyroid hormone-related protein.
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Animals↗
Some aspects of the anabolic action of parathyroid hormone.
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Animals↗
The plasminogen activator inhibitor system in bone cell function.
The plasminogen activator (PA)/plasmin pathway has been implicated in a variety of physiologic and pathologic processes that require tissue remodeling and cell motility. The pathway is highly regulated and results in the generation of the broad spectrum serine protease, plasmin, from the zymogen plasminogen. Urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) are produced by osteoblasts, as are the specific inhibitor plasminogen activator inhibitor-1 (PAI-1) and a cellular receptor for uPA. Little is known about regulation of the receptor, but the other 3 components of the pathway are regulated differentially by various osteotropic hormones and local factors. Several roles have been proposed for this pathway in bone. These involve proteolytic activation of procollagenase and latent growth factors, such as transforming growth factor beta (TGF beta) and insulin-like growth factor (IGF), as well as cell motility as a result of pericellular proteolysis. The roles of this pathway in bone remodeling may not be confined to proteolytic events, because uPA has been reported to act as a mitogen on osteoblast-like cells. This effect is independent of proteolytic activity but requires the growth factor domain of uPA to bind to uPA cellular receptors. If the plasminogen activator/plasmin pathway encompasses these roles, it would serve to couple formation and resorption of bone in a highly regulated manner.
Animals↗
Divergent structural requirements exist for calcitonin receptor binding specificity and adenylate cyclase activation.
The basis of the high potency of salmon calcitonin (sCT) in radioligand binding competition and cAMP accumulation studies with cloned calcitonin (CT) receptors from rats, pigs, and humans was examined using two sets of CT analogues, i.e., chimeric sCT/human CT (hCT) analogues and analogues of sCT with differing capacities to form an amphipathic alpha-helix. In competition for 125I-sCT binding the following relative specificities were observed for the chimeric peptides: rat C1a CT receptor, sCT > or = (1-16)hCT/(17-32)sCT (ACT-15) > (1-16)sCT/(17-32)hCT (ACT-27); rat C1b CT receptor, sCT >> ACT-15 > ACT-27; hCT receptor, sCT = ACT-15 > ACT-27; porcine CT receptor, sCT > ACT-27 > ACT-15. In contrast, in ligand-induced cAMP accumulation studies the relative efficacies were as follows: rat C1a CT receptor, sCT = ACT-15 > ACT-27; rat C1b CT receptor, sCT = ACT-15 > ACT-27; hCT receptor, sCT = ACT-15 > or = to ACT-27; porcine CT receptor, sCT = ACT-15 = ACT-27. The data demonstrate that residues present in the carboxyl-terminal half of sCT are more important for binding competition with the rat C1a, rat C1b, and human CT receptors, whereas residues in the amino-terminal half of sCT are more important for binding competition with the porcine CT receptor. Carboxyl-terminal sCT residues are also important for full potency in adenylate cyclase activation with the rat C1a and rat C1b CT receptors but are less important for activation via the hCT receptor. The disparity in the relative potencies of the peptides in studies of binding competition and cAMP accumulation is suggestive of significant differences in the relative affinities of the peptides for active and inactive conformations of the CT receptor. The use of sCT analogues with varying capacities to form alpha-helices also revealed divergence in the responses of different receptors. This was most apparent for the stimulation of cAMP production by the rat receptor isoforms C1a and C1b. In cells expressing the C1a receptor, the helical analogues sCT and des-Ser2-sCT were equipotent with [Gly8]-des-Leu19-sCT and des-1-amino-[Ala1,7,Gly8]-des-Leu19 sCT, analogues that have reduced or absent helical structure, respectively. In contrast, the nonhelical analogues were 100-1000-fold less potent than sCT and des-Ser2-sCT at the C1b receptor. In general, reduction in the ability of sCT analogues to form helix structures had a greater impact on the potency of the analogues in competition for 125I-sCT binding than in cAMP accumulation.(ABSTRACT TRUNCATED AT 400 WORDS)
Adenylyl Cyclases↗
The effects of intravenous heroin administration on extracellular nucleus accumbens dopamine concentrations as determined by in vivo microdialysis.
Dopaminergic innervations of the nucleus accumbens (NAcc) have a significant role in the brain processes underlying the reinforcing actions of abused drugs. In addition to the evidence from selective lesions and receptor antagonists, recent determinations of increased extracellular dopamine concentrations ([DA]e) in this structure during cocaine and ethanol self-administration have extended this notion. This study was undertaken to determine if the reinforcing effects of heroin were similarly correlated with changes in [DA]e in the NAcc using in vivo microdialysis. In the first experiment, naive rats were randomly divided into three groups and administered either two i.v. infusions of saline (n = 6) or heroin (5.4, 18 or 30 micrograms/infusion; n = 6, 8 and 6, respectively) 1 hr apart. [DA]e in the NAcc was significantly increased in a dose-dependent manner after response-independent heroin administration. In the second experiment, responding was engendered and maintained with 5.4, 18 or 30 micrograms/infusion of heroin (i.v.; n = 4, 5 and 5/group, respectively) under a fixed ratio 10 (FR10) schedule of reinforcement. After stable baselines of heroin intake were obtained, microdialysis samples were collected from the NAcc during the self-administration session. [DA]e did not significantly differ from baseline during self-administration in any of the groups. The increase in NAcc [DA]e following acute response-independent heroin administration is consistent with previously published reports. However, the latter results do not support the hypothesis that NAcc dopamine is critically involved in the processes underlying heroin self-administration. In summary, the results indicate that in vivo neurochemical data obtained from acute experimenter-administered heroin can not be equated with that obtained during heroin self-administration.
Animals↗
Left dural to right cavernous sinus fistula. A case report.
Direct carotid-cavernous sinus fistulas that present with signs and symptoms contralateral to the arterial supply of the fistulas are not uncommon. We present a thoroughly documented case of a dural-cavernous sinus fistula with symptoms exclusively contralateral to the arterial source, a rarer entity. The patient presented with a red, proptotic right eye and a history of transient horizontal diplopia and a "feeling of fullness" in that eye. Magnetic resonance imaging (MRI) of the brain and orbits performed at another hospital had shown no abnormalities. Carotid angiography performed on the right side was normal; carotid angiography performed on the left side showed a dural-cavernous sinus fistula, with shunting from branches of the left external carotid artery directly to the right cavernous sinus. Orbital duplex color-flow sonography showed reverse flow in a dilated right superior ophthalmic vein. This unusual manifestation of a dural-cavernous sinus fistula offers insight into the pathophysiology of arteriovenous fistulas involving the cavernous sinus, and is a reminder that bilateral injections are required when performing carotid angiography to characterize these disorders.
Angiography↗
Alkylation of mu opioid receptors by beta-funaltrexamine in vivo: comparison of the effects on in situ binding and heroin self-administration in rats.
Mu opioid receptors are known to be directly involved in the reinforcing effects of opiates; however, little is known regarding the relationship between alteration of mu opioid receptor binding and opiate reinforcement. Intracerebroventricular (i.c.v.) administration of beta-funaltrexamine (beta-FNA) has been shown to reduce the number of mu opioid receptors throughout the brain and can be used to address questions regarding the relationship of the density of these receptors to the pharmacological effects of opiates. The time course of the effects of beta-FNA on heroin self-administration was compared with the effects on mu opioid receptor binding. beta-FNA (40 nmol) or saline was administered i.c.v. to animals trained to self-administer either 18 or 60 micrograms/kg per infusion of heroin. The number of infusions decreased after beta-FNA administration but steadily returned to base-line levels approximately 10 days after beta-FNA treatment. The time course of the effects of beta-FNA on mu opioid receptor binding was determined in separate groups of animals. beta-FNA treatment decreased the number of [3H]D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin binding sites by 34 to 50% in rat brain sections; an effect that persisted for up to 18 days. The affinity was unaffected initially, but decreased in a linear manner from days 9 to 18 after beta-FNA administration. The return of heroin self-administration before the return of mu opioid receptor binding suggests that the recovery of mu opioid receptor function after beta-FNA treatment is more complex than merely synthesis of new receptors.
Alkylation↗
Regulation of plasminogen activator inhibitor-1 (PAI-1) expression by 1,25-dihydroxyvitamin D-3 in normal and malignant rat osteoblasts.
In rat calvarial osteoblast-like cells and in clonal osteogenic sarcoma cells (UMR 106-01), 1,25-dihydroxyvitamin D-3 (1,25(OH)2D3) enhanced plasminogen activator (PA) activity and decreased PA inhibitor-1 (PAI-1) production over the same concentration range. Steady-state levels of mRNA for PAI-1 were also decreased by 1,25(OH)2D3 in a dose-dependent manner, without significant effects on mRNA for either tissue-type PA (tPA) or urokinase-type PA (uPA). When protein synthesis was inhibited by cycloheximide treatment in UMR 106-01 cells, the action of 1,25(OH)2D3 on PAI-1 mRNA was abolished, as was observed previously with parathyroid hormone (PTH) treatment. In osteoblast-like cells however, 1,25(OH)2D3 and PTH actions differed, in that 1,25(OH)2D3 had no effect on either PAI-1 or uPA mRNA levels under conditions of protein synthesis inhibition, whereas PTH decreased PAI-1, and increased uPA mRNA. Identification of proteins involved in these actions may help to explain differences in molecular regulation by PTH and 1,25(OH)2D3, two agents which have similar actions on osteoblasts, but employ different signal transduction pathways.
Animals↗
Inhibition by FK506 of formyl peptide-induced neutrophil activation and associated protein synthesis.
The macrolide FK506 inhibited, by up to 50%, neutrophil migration and the production of the superoxide radical in response to the formyl peptide, formyl-methionyl-leucyl-phenylalanine (FMLP). The production of the superoxide radical in response to phorbol 12-myristate 13-acetate (PMA) was unaffected by FK506. The inhibition of neutrophil functions was accompanied by a partial reversal of FMLP-induced synthesis of cellular proteins, despite a rise in intracellular Ca2+. Neutrophils treated with FK506 demonstrated a small (average 23%) though significant decrease in formyl-peptide receptor numbers but receptor binding affinity was unaffected. The effects of FK506 on neutrophil activation appear to be analogous to those in T-lymphocytes. The incomplete inhibition, by FK506, of neutrophil responses suggests further that activation by FMLP is mediated via distinct multiple signalling pathways, including protein kinase activation and protein synthesis. The inability of FK506 to reduce FMLP-induced rises in cellular Ca2+ or PMA-induced activation of neutrophils suggests that its action is distal to Ca2+ mobilization and distinct from pathways relying on PKC activation. Thus the immunosuppressive effects of FK506 in vivo might be mediated through the inhibition of inflammatory cells other than lymphocytes and the drug therefore has therapeutic potential in a variety of inflammatory conditions. The drug also has potential in vitro for the characterization of signalling pathways from the plasma membrane to the nucleus.
Calcium↗
Novel action of retinoic acid. Stabilization of newly synthesized alkaline phosphatase transcripts.
Several observations led us to investigate the possibility that retinoic acid achieved its marked induction of alkaline phosphatase gene expression through a post-transcriptional effect in the nuclei of clonal rat pre-osteoblastic UMR 201 cells. The steady-state level of alkaline phosphatase mRNA was significantly stimulated by retinoic acid. Although nuclear run-on analysis showed that 10(-6) M retinoic acid caused an increase in alkaline phosphatase gene transcription, this was transient compared with the rise in alkaline phosphatase mRNA which continued to accumulate for many hours after retinoic acid stimulation of gene transcription had ceased. Moreover, the modest increase in transcriptional rate (approximately 2-fold) was not sufficient to account for the magnitude of the rise in mRNA level. In order, therefore, to examine the influence of retinoic acid on nuclear processing events, a cellular subfractionation method was applied. By nuclease protection analysis, and also by using reverse transcription-polymerase chain reaction, sequences corresponding to intron 2 and intron 4, respectively, were demonstrated specifically in the nuclear matrix fraction of both control and retinoic acid-treated cells. Mature (spliced) alkaline phosphatase mRNA accumulated in the non-matrix (DNase I/salt eluate, nuclear membrane) and cytoplasmic fractions of retinoic acid-treated cells at more than 100-fold greater levels than in control cells. This implies that nuclear processing of the primary RNA transcript occurred only in cells treated with retinoic acid. The post-transcriptional action of retinoic acid was inhibited by cotreatment with 10 micrograms/ml cycloheximide. Transforming growth factor beta (TGF beta) (1 ng/ml) did not influence whole cell alkaline phosphatase levels in UMR 201 cells. Nevertheless, TGF beta increased the transcriptional rate of the alkaline phosphatase gene. Although precursor mRNA was detected in the nuclear matrix fraction of TGF beta-treated cells, there was no evidence of further mRNA nuclear processing. The data are consistent with stabilization of nascent alkaline phosphatase mRNA chains by retinoic acid treatment and suggests that regulation of mRNA processing can be independent of gene transcription. This study demonstrates a novel post-transcriptional action of retinoic acid which plays an important, if not a dominant role, in determining the steady-state level of alkaline phosphatase mRNA.
Alkaline Phosphatase↗
Instrument for measuring relative brightness perception.
Neuro-ophthalmic disorders may cause a subjective difference in the brightness perceived by each eye. We developed a unique clinical device to measure this difference. The device consists of a disk equipped with graded neutral-density filters that fits into a View-Master stereo viewer (View-Master International Group, Inc., Portland, Oregon). This device provides simultaneous viewing of a stimulus target for each eye, and is administered in a random, forced-choice format that allows assessment of patient reliability. We administered this test to 37 control subjects and 84 patients with a variety of disorders. Thirty-six of the 37 control subjects (97%) and 51 of the 84 patients (61%) had no measured relative brightness deficit and no errors. Of the 34 subjects who had abnormal results, 21 (62%) isolated a relative brightness deficit within 0.2-log unit steps without error, and 28 (82%) did so within 0.4-log unit steps. This device proved to be a simple, reliable instrument for measuring relative brightness perception.
Adolescent↗
Parathyroid hormone-related protein and human papillomavirus in gynecological tumors.
The presence of parathyroid hormone-related protein (PTHrP) and human papillomavirus (HPV) in a series of gynecological tumors from 131 unselected patients was examined. PTHrP was localized immunohistochemically using a highly specific rabbit polyclonal anti-serum against PTHrP(1-16). The results confirmed that gynecological malignancies, although rarely associated with humoral hypercalcemia of malignancy (HHM), stained for PTHrP in a majority of the squamous-cell carcinomas (SCC) at all sites, but only in a minority of adenocarcinomas, and then in areas of squamous metaplasia. This included a series of endometrial tumors. Detection of HPV types was achieved using a polymerase-chain-reaction (PCR) detection system enabling the detection of HPV types 6, 11, 16, 18, 31, 33 and 45. PTHrP production was not directly related to HPV infection, but correlated with the type of tumor.
Adenocarcinoma↗
Carboxyl-terminal parathyroid hormone-related protein inhibits bone resorption by isolated chicken osteoclasts.
Carboxyl-terminal peptides from parathyroid hormone-related protein (PTHrP) have been studied for their effect on bone resorption by osteoclasts isolated from 15 day embryonic chickens. Basal bone resorption by chicken osteoclasts was directly inhibited by chicken and human PTHrP-(107-139) and the pentapeptide PTHrP-(107-111). The chicken and human analogs were equipotent. Both the number of resorption pits and the total area resorbed per bone slice were reduced by PTHrP-(107-139), but it did not alter the size of individual resorption pits. Resorption stimulated by hPTH-(1-34) in cocultures of chicken osteoclasts with osteoblasts was also inhibited by cPTHrP-(107-139) but required a concentration three orders of magnitude greater than that required to inhibit basal resorption in cocultures or cultures of isolated osteoclasts. The finding of resorption inhibitory activity by PTHrP-(107-139) in avian as well as mammalian species strengthens the hypothesis that carboxyl-terminal PTHrP may act as a paracrine regulator of bone cell activity.
Animals↗
In situ hybridization to show sequential expression of osteoblast gene markers during bone formation in vivo.
We investigated the sequence of expression of osteoblast gene markers during bone formation in vivo by in situ hybridization. Cylindrical lesions were induced in the femora of sheep with titanium analytic bone implants that allow removal of serial core samples to study bone formation. At 2 weeks (2W), granulation tissue made up of spindle-shaped cells had partially replaced the blood clot. Islands of osseous tissue, first noted in the periphery of the ingrowing tissue at 3W, became the predominant tissue by 6W. The surfaces of newly forming bone at 3W were apposed by cuboidal cells, which in some areas were several layers thick. By 6W, most of the cells lining bone trabeculae had assumed a flattened morphology. The temporal and spatial distribution of osteoblast gene markers was examined by in situ hybridization with nonradioactive digoxigenin probes for alpha 1(I) procollagen, alkaline phosphatase (ALP), osteopontin (OP), and bone Gla protein (BGP). The spindle-shaped cells in the granulation tissue expressed mRNA for alpha 1(I) procollagen, ALP, and OP but not BGP, suggesting that they may be osteoblast precursor cells. alpha 1(I) procollagen mRNA was strongly expressed by all cells on the surface of bone, with a peak intensity at 3W and then reducing sharply by 6W. Initially, only pockets of cuboidal cells on bone surfaces expressed ALP mRNA, with a peak intensity at 5W. Similarly, only a proportion of cuboidal cells expressed OP mRNA early in bone formation, but the number of cells expressing OP mRNA increased with time. Clumps of cuboidal cells expressed BGP mRNA only when bone was present, and the degree of expression increased with the amount of bone formed. This model allows the study of temporal and spatial sequence of gene expression in cells participating in osteogenesis. The temporal sequence is similar to that shown in vitro in other models of mineralization. The geographic localization of cells expressing mRNA for alpha 1(I) procollagen, ALP, OP, and BGP implies subspecialization of osteoblasts in bone formation.
Alkaline Phosphatase↗