Biomedical subjects

T J Martin

Publications and source records attributed to T J Martin.

At least 73 records · Page 4Linked to original sources

PubMed1999

Calcitonin receptor antibodies in the identification of osteoclasts.

Osteoclasts are the cells responsible for bone resorption, and their number and rate of formation are critical in determining bone mass. To identify and quantify osteoclasts, as well as to study their formation in bone and in osteoclastogenic cultures, osteoclast-specific cell markers are required. Only the calcitonin receptor (CTR) expression unambiguously identifies osteoclasts and distinguishes them from macrophage polykaryons. However, present autoradiographic methods for CTR detection are cumbersome and time consuming. We have developed rabbit polyclonal antibodies specific for the C-terminal intracellular domain of the mouse and rat Cla CTR. These antibodies labeled HEK-293 cells stably transfected with CTR (but not untransfected HEK-293 cells). This labeling is abrogated by preabsorbing the antibodies with the recombinant antigen. The antibodies immunostained primary mouse and rat osteoclasts as well as osteoclasts in sections of mouse bone. Osteoclasts (both mononuclear and multinucleated) formed from mouse bone marrow or spleen cells cocultured with osteoblasts in the presence of 1,25 dihydroxyvitamin D3 and prostaglandin E2 were also specifically immunostained by the CTR antibodies. Cocultures incubated under conditions that did not allow osteoclastogenesis (i.e., omission of mediators or osteoblasts, or culture for less than 4 days) were not immunostained by CTR antibodies. Autoradiographic detection of 125I-labeled salmon calcitonin combined with CTR immunohistochemistry showed that both methods labeled the same cells. A CTR polyclonal antibody and monoclonal antibody F4/80 were used in combination to show immunofluorescence labeling of murine osteoclasts and macrophage populations, respectively, in marrow/osteoblast cocultures. These results indicate that simple and rapid CTR antibody-based methods can be used to identify osteoclasts, and can be used to characterize the antigenic profile of osteoclasts by using double immunofluorescence analysis.

Animals↗

PubMed1999

Osteoblasts/stromal cells stimulate osteoclast activation through expression of osteoclast differentiation factor/RANKL but not macrophage colony-stimulating factor: receptor activator of NF-kappa B ligand.

We previously reported that osteoblasts/stromal cells are essentially involved in the activation as well as differentiation of osteoclasts through a mechanism involving cell-to-cell contact between osteoblasts/stromal cells and osteoclast precursors/osteoclasts. Osteoclast differentiation factor (ODF, also called RANKL/OPGL/TRANCE) and macrophage colony-stimulating factor (M-CSF, also called CSF-1) are two essential factors produced by osteoblasts/stromal cells for osteoclastogenesis. In other words, osteoblasts/stromal cells were not necessary to generate osteoclasts from spleen cells in the presence of both ODF/RANKL and M-CSF. In the present study, we examined the precise roles of ODF/RANKL and M-CSF in the activation of osteoclasts induced by calvarial osteoblasts. Osteoclasts were formed in mouse bone marrow cultures on collagen gel-coated dishes in response to a soluble form of ODF/RANKL (sODF/sRANKL) and M-CSF, and recovered by collagenase digestion. When recovered osteoclasts were further cultured on plastic dishes, most of the osteoclasts spontaneously died within 24 h. Osteoclasts cultured for 24 h on dentine slices could not form resorption pits. Addition of sODF/sRANKL to the recovered osteoclasts markedly enhanced their survival and pit-forming activity. M-CSF similarly stimulated the survival of osteoclasts, but did not induce their pit-forming activity. When primary mouse osteoblasts were added to the recovered osteoclasts, resorption pits were formed on dentine slices. Bone-resorbing factors such as 1alpha,25-dihydroxyvitamin D3, parathyroid hormone, or prostaglandin E2 enhanced pit-forming activity of osteoclasts only in the presence of osteoblasts. M-CSF-deficient osteoblasts prepared from op/op mice similarly enhanced pit-forming activity of osteoclasts. The pit-forming activity of osteoclasts induced by sODF/sRANKL or osteoblasts was completely inhibited by simultaneous addition of osteoprotegerin/osteoclastogenesis inhibitory factor, a decoy receptor of ODF/RANKL. Primary osteoblasts constitutively expressed ODF/RANKL mRNA, and its level was upregulated by treatment with 1alpha,25-dihydroxyvitamin D3, parathyroid hormone, and prostaglandin E2. These results, obtained by using an assay system that unequivocally assesses osteoclast activation, suggest that ODF/RANKL but not M-CSF mediates osteoblast-induced pit-forming activity of osteoclasts, and that bone-resorbing factors stimulate osteoclast activation through upregulation of ODF/RANKL by osteoblasts/stromal cells.

Animals↗

PubMed1999

Localization of RANKL (receptor activator of NF kappa B ligand) mRNA and protein in skeletal and extraskeletal tissues.

RANKL (receptor activator of NFkappaB ligand) is a membrane-associated osteoblastic molecule, and along with macrophage-colony-stimulating factor, is crucial for osteoclast formation. RANKL is known to be strongly expressed in osteoblasts and lymphoid tissues. We have sought to determine the skeletal and extraskeletal sites of production of RANKL mRNA and protein using the techniques of in situ hybridization and immunohistochemistry. Expression of RANKL mRNA and protein were determined in the developmental progression of endochondral bone formation in mouse, intramembranous bone formation in a rabbit model (mRNA only), in human giant cell tumors of bone, and at extraskeletal sites in the mouse. RANKL mRNA was expressed in prehypertrophic and hypertrophic chondrocytes at day E15 embryonic mouse long bone, and its expression was maintained at these sites throughout development. In newborn and adult mice, high levels of RANKL mRNA were expressed in mesenchymal cells of the periosteum and in mature osteoblasts, while megakaryocytes within the marrow microenvironment expressed RANKL mRNA from 1 week of age. Immunohistochemical analysis revealed a similar localization pattern of RANKL protein at the sites described. In the intramembranous bone formation model, RANKL mRNA was expressed in mesenchymal cells and in actively synthesizing osteoblasts, but not in flattened lining osteoblasts or late osteocytes. Expression of RANKL mRNA and protein in osteoclasts was variable with those within resorption lacunae showing the strongest signal/staining. Likewise, expression varied in osteoclasts from giant cell tumor of bone with a minority of tartrate-resistant acid phosphatase-positive multinucleated cells having no detectable RANKL mRNA or protein. In extraskeletal tissues, RANKL mRNA and protein were detected in the brain, heart, kidney, skeletal muscle, and skin throughout mouse development, suggesting the possibility of several other functions of the molecule. RANKL was also developmentally regulated, as evidenced by its expression in the intestine, liver, and lung at E15 and newborn mouse but not in the adult.

Amino Acid Sequence↗

PubMed1999

A novel orthotopic model of breast cancer metastasis to bone.

Breast cancer affects approximately one woman in twelve and kills more women than any other cancer. If detected early, patients have a five year survival rate of 66%, but once metastatic disease has developed, there is no effective treatment. About 70% of patients with metastatic disease have bone involvement, while lungs and liver are the other common targets. Bone metastases cause severe pain, pathological fractures and hypercalcaemia and thus are a significant clinical problem. The development of new therapies for metastatic breast carcinoma depends on a better understanding of the mechanism of homing of the tumour cells to bone, liver and lungs and the factors required for their growth in these organs. Research on mechanisms of breast cancer metastasis, particularly to bone, has relied on in vitro studies or on tumour models in which the inoculation route is designed to promote delivery of tumour cells to a specific organ. Metastases in bone are achieved by inoculation into the right ventricle of the heart. To our knowledge there has been no report of a model of metastatic spread from the mammary gland to distant sites which reliably includes bone. In this paper, we describe our recent development of a novel murine model of metastatic breast carcinoma. The new model is unique in that the pattern of metastatic spread closely resembles that observed in human breast cancer. In particular, these murine breast tumours metastasise to bone from the primary breast site and cause hypercalcaemia, characteristics not normally found in murine tumours, but common in human disease. Furthermore, in a preliminary characterisation of this model, we show that secretion of parathyroid hormone-related protein, a role for which has been implicated in breast cancer spread to bone, correlates with metastasis to bone. This model therefore provides an excellent experimental system in which to investigate the factors that control metastatic spread of breast cancer to specific sites, particularly bone. The special advantage of this system is that it involves the whole metastasis process, beginning from the primary site. Existing models consider mechanisms that pertain to growth of tumour once the site has been reached. An understanding of the regulation of these factors by potential therapeutic agents could lead to improvement in therapies designed to combat metastatic disease. For the first time, this development will allow exploration of the molecular basis of site-specific metastasis of breast cancer to bone in a clinically relevant model.

Animals↗

PubMed1999

Subtilisin-like proprotein convertases, PACE4 and PC8, as well as furin, are endogenous proalbumin convertases in HepG2 cells.

Serum albumin is synthesized as a larger precursor form, proalbumin, which undergoes proteolytic processing at a dibasic site by a hepatic proprotein convertase within the secretory pathway to generate the mature form. Although furin, a member of the subtilisin-like proprotein convertase (SPC) family, was thought to be the only candidate hepatic convertase for proalbumin, SPC family members other than furin were recently suggested to also be involved in proalbumin processing. This study was designed to identify the endogenous proprotein convertases involved in proalbumin processing. Since human hepatoma HepG2 cells are highly differentiated and produce major plasma proteins, this cell line was used as a model for hepatocytes. Northern blot analysis revealed that PACE4, furin and PC8 of the SPC family were expressed in HepG2 cells as well as in the liver. Ribonuclease protection assay showed that PACE4A-II mRNA is the major transcript in HepG2 cells among the PACE4 isoforms. The coexpression studies showed that furin, PACE4A-II and PC8 were all able to convert proalbumin to albumin correctly. To elucidate the roles of these endogenous SPC family members in proalbumin processing, the antisense RNA for PACE4, furin and PC8 was stably expressed in HepG2 cells, respectively. The expression of each antisense RNA resulted in approximately 30% inhibition of endogenous proalbumin processing. We therefore concluded that PACE4 and PC8, as well as furin, are involved in the processing of proalbumin in HepG2 cells, and that these SPC family members are functionally redundant in this processing.

Furin↗

PubMed1999

IL-17 in synovial fluids from patients with rheumatoid arthritis is a potent stimulator of osteoclastogenesis.

IL-17 is a newly discovered T cell-derived cytokine whose role in osteoclast development has not been fully elucidated. Treatment of cocultures of mouse hemopoietic cells and primary osteoblasts with recombinant human IL-17 induced the formation of multinucleated cells, which satisfied major criteria of osteoclasts, including tartrate-resistant acid phosphatase activity, calcitonin receptors, and pit formation on dentine slices. Direct interaction between osteoclast progenitors and osteoblasts was required for IL-17-induced osteoclastogenesis, which was completely inhibited by adding indomethacin or NS398, a selective inhibitor of cyclooxgenase-2 (COX-2). Adding IL-17 increased prostaglandin E2 (PGE2) synthesis in cocultures of bone marrow cells and osteoblasts and in single cultures of osteoblasts, but not in single cultures of bone marrow cells. In addition, IL-17 dose-dependently induced expression of osteoclast differentiation factor (ODF) mRNA in osteoblasts. ODF is a membrane-associated protein that transduces an essential signal(s) to osteoclast progenitors for differentiation into osteoclasts. Osteoclastogenesis inhibitory factor (OCIF), a decoy receptor of ODF, completely inhibited IL-17-induced osteoclast differentiation in the cocultures. Levels of IL-17 in synovial fluids were significantly higher in rheumatoid arthritis (RA) patients than osteoarthritis (OA) patients. Anti-IL-17 antibody significantly inhibited osteoclast formation induced by culture media of RA synovial tissues. These findings suggest that IL-17 first acts on osteoblasts, which stimulates both COX-2-dependent PGE2 synthesis and ODF gene expression, which in turn induce differentiation of osteoclast progenitors into mature osteoclasts, and that IL-17 is a crucial cytokine for osteoclastic bone resorption in RA patients.

Animals↗

PubMed1999

Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families.

Osteoblasts/stromal cells are essentially involved in osteoclast differentiation and function through cell-to-cell contact (Fig. 8). Although many attempts have been made to elucidate the mechanism of the so-called "microenvironment provided by osteoblasts/stromal cells," (5-8) it has remained an open question until OPG and its binding molecule were cloned. The serial discovery of the new members of the TNF receptor-ligand family members has confirmed the idea that osteoclast differentiation and function are regulated by osteoblasts/stromal cells. RANKL, which has also been called ODF, TRANCE, or OPGL, is a member of the TNF ligand family. Expression of RANKL mRNA in osteoblasts/stromal cells is up-regulated by osteotropic factors such as 1 alpha, 25(OH)2D3, PTH, and IL-11. Osteoclast precursors express RANK, a TNF receptor family member, recognize RANKL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into pOCs in the presence of M-CSF. RANKL is also involved in the survival and fusion of pOCs and activation of mature osteoclasts. OPG, which has also been called OCIF or TR1, is a soluble receptor for RANKL and acts as a decoy receptor in the RANK-RANKL signaling system (Fig. 8). In conclusion, osteoblasts/stromal cells are involved in all of the processes of osteoclast development, such as differentiation, survival, fusion, and activation of osteoclasts (Fig. 8). Osteoblasts/stromal cells can now be replaced with RANKL and M-CSF in dealing with the whole life of osteoclasts. RANKL, RANK, and OPG are three key molecules that regulate osteoclast recruitment and function. Further studies on these key molecules will elucidate the molecular mechanism of the regulation of osteoclastic bone resorption. This line of studies will establish new ways to treat several metabolic bone diseases caused by abnormal osteoclast recruitment and functions such as osteopetrosis, osteoporosis, metastatic bone disease, Paget's disease, rheumatoid arthritis, and periodontal bone disease.

Animals↗

PubMed1999

Breast cancer cells interact with osteoblasts to support osteoclast formation.

Breast cancers commonly cause osteolytic metastases in bone, a process that is dependent upon osteoclast-mediated bone resorption. Recently the osteoclast differentiation factor (ODF), better termed RANKL (receptor activator of NF-kappaB ligand), expressed by osteoblasts has been cloned as well as its cognate signaling receptor, receptor activator of NFkappaB (RANK), and a secreted decoy receptor osteoprotegerin (OPG) that limits RANKL's biological action. We determined that the breast cancer cell lines MDA-MB-231, MCF-7, and T47D as well as primary breast cancers do not express RANKL but express OPG and RANK. MCF-7, MDA-MB-231, and T47D cells did not act as surrogate osteoblasts to support osteoclast formation in coculture experiments, a result consistent with the fact that they do not express RANKL. When MCF-7 cells overexpressing PTH-related protein (PTHrP) were added to cocultures of murine osteoblasts and hematopoietic cells, osteoclast formation resulted without the addition of any osteotropic agents; cocultures with MCF-7 or MCF-7 cells transfected with pcDNAIneo required exogenous agents for osteoclast formation. When MCF-7 cells overexpressing PTHrP were cultured with murine osteoblasts, osteoblastic RANKL messenger RNA (mRNA) levels were enhanced and osteoblastic OPG mRNA levels diminished; MCF-7 parental cells had no effect on RANKL or OPG mRNA levels when cultured with osteoblastic cells. Using a murine model of breast cancer metastasis to bone, we established that MCF-7 cells that overexpress PTHrP caused significantly more bone metastases, which were associated with increased osteoclast formation, elevated plasma PTHrP concentrations and hypercalcaemia compared with parental or empty vector controls.

Animals↗

PubMed1999

The role of the plasminogen system in bone resorption in vitro.

The plasminogen/plasmin proteolytic cascade plays an important role in extracellular matrix remodeling. The presence of the two plasminogen activators (PAs), tissue-type plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA), and their inhibitor type 1 (PAI-1) in bone cells, suggests a role in one or more aspects of bone resorption such as osteoclast formation, mineral dissolution, and degradation of the organic matrix. These different processes were assayed in vitro using cells derived from mice with either tPA (tPA-/-), uPA (uPA-/-), PAI-1 (PAI-1-/-) inactivation or with a combined inactivation (tPA-/-:uPA-/-) and compared with wild-type mice (WT). First, osteoclast formation, assessed by investigating the number and characteristics of tartrate-resistant acid phosphatase-positive multinucleated cells formed in cocultures of primary osteoblasts and bone marrow cells treated with 1alpha,25-dihydroxyvitamin D3, was not different between the different cell types. Second, dentine resorption, an assay for osteoclast activity, was not affected by the combined deficiency of both tPA and uPA. Finally, the ability to degrade nonmineralized bone-like matrix was however, significantly reduced in tPA-/-:uPA-/- cells compared with WT cells (28.1 +/- 0.6%, n = 6 vs. 56.4 +/- 3.1%, n = 6, respectively, p < 0.0001). Surprisingly, collagen proteolysis by bone cells was not dependent on the presence of plasmin as suggested by degradation assays performed on type I 3H-collagen films. Taken together, these data suggest that the plasminogen activator/plasmin system is not required for osteoclast formation, nor for the resorption of the mineral phase, but is involved in the removal of noncollagenous proteins present in the nonmineralized bone matrix.

Animals↗

PubMed1999

Calcitonin.

The peptide calcitonin (CT) was initially discovered in 1962 as a novel hypocalcemic hormone. This hypocalcemic response was principally due to a potent inhibitory action of CT on osteoclast mediated bone resorption and it is this action which underlies its widespread clinical use for the treatment of bone disorders, including Paget's disease, osteoporosis and hypercalcemia of malignancy. In this article we review the basic physiology of CT action, structure-function studies on CT peptides, cloning of CT receptors and the identification of isoforms of the receptor derived from alterative splicing of the receptor mRNA. We also review the state of understanding on CT receptor mediated signaling and receptor regulation, along with developing concepts of how CT peptides interact with the receptor, including how the receptors may interact with receptor activity modifying proteins to produce novel phenotypes. Finally, current therapeutic use is reviewed, and the potential for expanded use that may come with advances in delivery of peptides or CT mimetics.

Amino Acid Sequence↗

PubMed1999

Cancer-induced bone diseases--second international conference. 27-29 March 1999, Davos, Switzerland.

After the first successful meeting on this topic, held in London in 1997, 550 registrants from 39 countries gathered in Davos to consider basic and clinical aspects of cancer metastases to bone, and to review current and future therapies. The conference brought together basic scientists in bone cell biology and cancer with clinical oncologists involved in the management of patients exposed to this common complication of many cancers. A recurring theme of the meeting was the importance of continuing dialog among the bone and cancer biologists and the clinicians. The meeting consisted of 8 major lectures and 64 poster presentations, from which 18 were selected for oral presentation and discussion. Two industry-supported symposia were held in conjunction with the meeting to consider specific treatment modalities.

Journal Article↗

PubMed1998

Gene organization and alternative splicing of human prohormone convertase PC8.

The mammalian Ca2+-dependent serine protease prohormone convertase PC8 is expressed ubiquitously, being transcribed as 3.5, 4.3 and 6.0 kb mRNA isoforms in various tissues. To determine the origin of these various mRNA isoforms we report the characterization of the human PC8 gene, which has been previously localized to chromosome 11q23-24. Consisting of 16 exons, the human PC8 gene spans approx. 27 kb. A comparison of the position of intron-exon junctions of the human PC8 gene with the gene structures of previously reported prohormone convertase genes demonstrated a divergence of the human PC8 from the highly conserved nature of the gene organization of this enzyme family. The nucleotide sequence of the 5'-flanking region of the human PC8 is reported and possesses putative promoter elements characteristic of a GC-rich promoter. Further supporting the potential role of a GC-rich promoter element, multiple transcriptional initiation sites within a 200 bp region were demonstrated. We propose that the various mRNA isoforms of PC8 result from the inclusion of intronic sequences within transcripts.

Alternative Splicing↗

PubMed1998

Effects of beta-funaltrexamine on dose-effect curves for heroin self-administration in rats: comparison with alteration of [3H]DAMGO binding to rat brain sections.

These studies were undertaken to determine the effects of mu-opioid receptor depletion through irreversible alkylation on the dose-effect curve for heroin self-administration. Heroin maintained responding in rats with an inverted U-shaped dose-effect curve and administration of 10 nmol of beta-funaltrexamine i.c.v. (beta-FNA) significantly increased the ED50 on the ascending limb from 1.9 to 5.3 micrograms/infusion, and from 24.3 to 211.8 micrograms/infusion on the descending limb. Administration of saline i.c.v. produced no effect on heroin self-administration. Administration of 40 nmol of beta-FNA increased the ED50S from 5.1 to 33.9 and from 14.4 to 502.8 micrograms/infusion on the ascending and descending portions of heroin's dose-effect curve, respectively. beta-FNA (40 nmol, i.c.v.) had no effect on cocaine self-administration. [3H]DAMGO binding density was decreased in the caudate and nucleus accumbens by 29 or 54% 24 h after administration of 10 or 40 nmol of beta-FNA i.c.v., respectively. The effects of beta-FNA on heroin self-administration were completely overcome by increasing the dose of heroin however, as the shape and slope of the self-administration dose-effect curve was not different when higher doses of heroin were made available for self-administration compared to control data or saline administration. Therefore, there appear to be spare mu-opioid receptors for heroin for the production of its reinforcing effects in rats. Furthermore, the self-administration dose-effect curves returned to control values prior to the return of [3H]DAMGO binding, further suggesting that the full complement of mu-opioid receptors is not necessary for heroin to produce its reinforcing effects. These findings support the existence of spare mu-opioid receptors for heroin in maintaining self-administration in rats.

Analgesics, Opioid↗

PubMed1998

Anti-allodynic actions of intravenous opioids in the nerve injured rat: potential utility of heroin and dihydroetorphine against neuropathic pain.

Neuropathic pain has been suggested to be resistant to treatment with opiates. Such perceived lack of opioid responsiveness may be due to the dose-range over which specific opioid compounds have been studied as well as the efficacy of these compounds. Dihydroetorphine is a novel opiate that demonstrates significantly greater analgesic potency compared to morphine, and which also demonstrates diminished capacity for producing physical dependence in laboratory animals. The present study compared the intravenous (i.v.) efficacy, potency and duration of action of dihydroetorphine, fentanyl, heroin and morphine in producing anti-allodynic actions in a rat model of neuropathic pain (ligation of the L5/L6 nerve roots). All compounds produced significant anti-allodynic activity with dihydroetorphine being the most potent (A50 of 0.2 microg kg(-1), i.v.). Morphine was approximately 7440 times less potent than dihydroetorphine while heroin and fentanyl were approximately 163.5 and 6.9 times less potent in producing anti-allodynic actions. Dihydroetorphine also showed a maximal effect at 0.6 microg kg(-1) in all animals tested, while 100 microg kg(-1) was required for heroin to produce a maximal effect. Fentanyl and morphine did not elicit a maximum anti-allodynic response (74 and 76% maximum possible effect (%MPE), respectively). As expected, fentanyl showed a relatively brief duration of action (approximately 20 min at the highest tested dose), while dihydroetorphine and morphine demonstrated anti-allodynic actions for up to 45 min. Heroin had the longest duration of action, producing significant anti-allodynic effects for up to 90 min. These data show that dihydroetorphine and heroin produce potent and long-lasting anti-allodynic actions in this model. Additionally, in contrast to morphine and fentanyl, both dihydroetorphine and heroin were able to achieve a maximal response. The remarkable potency, maximal efficacy and duration of action of these compounds, particularly dihydroetorphine, suggests that these compounds may warrant further examination as potential therapeutic treatments for neuropathic pain states.

Analgesics, Opioid↗

PubMed1998

Effect of treatment of isolated systolic hypertension on left ventricular mass.

CONTEXT: Left ventricular (LV) hypertrophy is a common problem among elderly patients with isolated systolic hypertension (ISH), but the effect of treatment of ISH on LV mass is not known. OBJECTIVE: To assess the ability of antihypertensive drug treatment to reduce LV mass in ISH. DESIGN: Echocardiographic Substudy of the Systolic Hypertension in the Elderly Program (SHEP). PATIENTS: A total of 104 participants at the St Louis SHEP site who had interpretable baseline echocardiograms, 94 of whom had 3-year follow-up echocardiograms. INTERVENTION: The SHEP participants were randomized to placebo or active treatment with chlorthalidone (12.5-25 mg/d), with atenolol (25-50 mg/d) added if necessary to maintain goal blood pressure. MAIN OUTCOME MEASURE: Change in LV mass assessed by echocardiography. RESULTS: Minimum follow-up was 3 years. In the active treatment group, 91% and 80% of subjects were receiving treatment with chlorthalidone alone by the end of years 1 and 3, respectively. The LV mass index was 93 g/m2 in the active treatment group and 100 g/m2 in the placebo group (P<.001). The LV mass index declined by 13% (95% confidence interval, - 3% to - 23%) in the active treatment group compared with a 6% increase (95% confidence interval, - 3% to + 16%) in the placebo group over 3 years (P=.01). CONCLUSION: Treatment of ISH with a diuretic-based regimen reduces LV mass.

Aged↗

PubMed1998

Casein kinase 2 phosphorylation of recombinant rat osteopontin enhances adhesion of osteoclasts but not osteoblasts.

Osteopontin (OP) is a highly phosphorylated bone matrix protein and contains the RGD cell-binding motif, which mediates cell adhesion through integrin receptors that include alpha(v)beta3. Casein kinase 2 (CK2) is a factor-independent serine/threonine kinase, which may be the predominant physiologically relevant kinase for OP phosphorylation. This study was designed to examine the effects of unphosphorylated recombinant rat OP, and CK2-phosphorylated OP (P-OP), on the adhesion and function of mouse osteoclasts (OC) and osteoblast-like cells (UMR 201-10B and UMR 106-06) in vitro. OP significantly increased OC adhesion compared to plastic alone, and cell attachment was further increased at least twofold on OP phosphorylated with CK2. Attachment was dependent on the integrity of the RGD domain and was completely abolished in the presence of 1 mM RGD peptide. Neither CK2 phosphorylation of mutant OP, in which the RGD was converted to RGE or RAD, nor protein kinase C (PKC) phosphorylation of wild-type OP enhanced OC attachment. An antibody to the beta3 integrin subunit, but not anti-mouse CD44 antibody, specifically blocked the proportion of attachment due to phosphorylation of OP. Actin ring formation in OC was increased by plating cells onto OP, with no further increase by phosphorylation. Both OP and CK2-phosphorylated OP enhanced attachment of the two osteoblastic cell lines, compared to plastic, but in contrast to OCs, there was no significant difference with phosphorylation. Osteoblast attachment was totally blocked by 1 mM RGD peptide, but was not influenced by the beta3 integrin antibody. Plating of UMR 201-10B cells onto OP further increased retinoic acid-induced alkaline phosphatase expression. The results suggest that specific phosphorylation of OP is important for interaction with OCs, compared with osteoblastic cells, and that alternative integrins may be important in the interaction between osteoblastic cells and OP compared with OCs.

Actins↗

PubMed1998

Transcriptional and posttranscriptional regulation of osteopontin gene expression in preosteoblasts by retinoic acid.

This study examines the relative importance of transcriptional and posttranscriptional actions of retinoic acid (RA) in the regulation of osteopontin gene expression in a rat clonal preosteoblastic cell line, UMR 201. Nuclear run-on analysis demonstrated constitutive expression of the osteopontin gene which was increased by threefold after 4 hr treatment with 1 microM RA, returning to a basal level by 24 hr. However, Northern blot analysis, performed concurrently, showed that RA progressively increased the steady-state osteopontin mRNA level beginning 2 hr before any increase in gene transcription and peaking at 24 hr. There was no difference in osteopontin mRNA stability between control and RA-treated cells after gene transcription was inhibited with 5,6-dichloro-1-D-ribofuranosylbenzimidazole (DRB). Total RNA was obtained from cellular subfractions (nuclear matrix, nonmatrix chromatin, nuclear membrane, and cytoplasm) and reverse transcription-polymerase chain reaction (RT-PCR) performed with primers complementary to exons 3 and 4 of the mouse osteopontin gene. Unspliced PCR product, comprising the two exons and the intervening intron, was present in the nuclear matrix fractions of control and RA-treated cells. However, RA resulted in a time-dependent accumulation of mature osteopontin mRNA in all cellular subfractions, suggesting that the proficiency of nuclear processing of primary mRNA transcripts was greatly enhanced by RA. This action depended on de novo protein synthesis. These results demonstrate that the posttranscriptional action of RA is not unique to the regulation of alkaline phosphatase gene expression.

Alkaline Phosphatase↗

PubMed1998

Threshold perimetry of each eye with both eyes open in patients with monocular functional (nonorganic) and organic vision loss.

PURPOSE: To report a novel binocular perimetry test to identify monocular functional vision loss. METHODS: In a prospective study, 10 patients with monocular functional vision loss, 10 patients with monocular organic vision loss, and 10 normal subjects were tested using a Humphrey automated perimeter attachment that performs central threshold perimetry of both eyes in a single test. First, patients were tested after being told their "good" (unaffected) eye was being tested; this was followed by a second identical test said to be on the "bad" (affected) eye. Two measurements were calculated for each subject: the functional component (deltaF), defined as the difference (in dB) between the mean threshold of the first and second tests, and the organic component (deltaO), the difference (in dB) between the mean threshold of the unaffected and affected eyes. RESULTS: Patients with monocular functional vision loss produced lower thresholds when they thought their affected eye alone was being tested but little intereye difference (mean deltaF +/- SD = 17.35 +/- 7.50 dB; mean deltaO = 0.01 +/- 1.40 dB). Patients with monocular organic vision loss had little difference between tests and lower thresholds in their affected eye (mean deltaF = 0.84 +/- 1.15 dB; mean deltaO = 9.01 +/- 4.71 dB). Normal subjects demonstrated little intertest or intereye differences (mean deltaF = -0.15 +/- 0.78 dB; mean deltaO = -0.05 +/- 0.51 dB). CONCLUSION: This perimetry method effectively distinguished between normal subjects, patients with monocular functional vision loss, and patients with monocular organic vision loss.

Adolescent↗