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M L Brandi

Publications and source records attributed to M L Brandi.

At least 109 records · Page 6Linked to original sources
PubMed1995

Bidirectional regulation of osteoclast function by nitric oxide synthase isoforms.

Nitric oxide (NO) produces rapid osteoclast detachment and contraction in vitro, and this effect is accompanied by a profound inhibition of bone resorption. Work by others has confirmed these findings in vivo: inhibition of NO synthase [NOS; L-arginine, NADPH: oxygen oxidoreductase (NO-forming), EC 1.14.13.39] in normal rats is followed by increased bone resorption reflected by a marked loss in bone mineral density. In our present study, immunocytochemistry and Northern blotting show the presence of the constitutive calcium-sensitive NOS isoform (cNOS) in normal rat osteoclasts and in the human preosteoclast cell line (FLG 29.1). The inducible NOS isoform (iNOS) was also clearly demonstrable in the rat cells especially after treatment with gamma interferon (IFN-gamma) and bacterial wall products [lipopolysaccharide (LPS)], while a basal level of transcript was detected in the untreated human preosteoclast line. However NADPH-diaphorase activity was intense only in neonatal rat osteoclasts attached to bone, perhaps reflecting either enhancement of cNOS activity by calcium or increased amounts of the inducible isoform in activated osteoclasts in situ compared with isolated neonatal rat osteoclasts. These actively resorb devitalized bone but the untreated cells contain relatively low levels of NOS; they are extremely sensitive to inhibition by NO. The iNOS inhibitor aminoguanidine markedly enhances in vitro resorption by activated NOS-rich chick osteoclasts and by normal rat osteoclasts treated with LPS or IFN-gamma. In contrast, the nonselective NOS inhibitor NG-monomethyl-L-arginine inhibits resorption by untreated neonatal rat osteoclasts. Thus, osteoclast function may require intermittent calcium-stimulated increases in NO production by cNOS against a basal inhibitory background activity of the iNOS isoform. However, bone resorption depends on precursor replication and on the activity of the mature cells, and we found that the NO donor 3-morpholinosydnonimine (SIN-1) (50 microM) profoundly depressed replication in the human preosteoclast line. Taken together, these results strongly suggest that NO maintains a central control of bone resorption in both avian and mammalian species by exerting a powerful tonic restraint of osteoclast numbers and activity. The presence of NOS in human cells implies a similar function in man and that conventional views of calcium homoeostasis and skeletal metabolism will need substantial revision. Since NO also influences behavior of the osteoblast, the bone-forming cell, in vitro, a similar effect in vivo might imply a general influence on bone remodeling.

Adult↗
PubMed1995

RFLP analysis of human chromosome 11 region q13 in multiple symmetric lipomatosis and multiple endocrine neoplasia type 1-associated lipomas.

Six lipomas from patients affected by Multiple Symmetric Lipomatosis (MSL) and by Multiple Endocrine Neoplasia Type 1 (MEN 1) were analyzed for loss of heterozygosity on chromosome 11 region q12-13 using four RFLPs. Allelic loss for the D11S146 locus was found only in one visceral MEN 1-associated lipoma. Lipomas that exhibited a lack of allelic lesions were analyzed for an eventual abnormal amount or a defective function of the Gs protein by studying the Gs alpha subunit gene, codons 201 and 207, by PCR and TGGE techniques. All the samples were negative for activating mutations.

Alleles↗
PubMed1995

Biosynthesis of bone sialoprotein by a human osteoclast-like cell line (FLG 29.1).

Biosynthesis of bone sialoprotein (BSP) by a human osteoclastic cell line (FLG 29.1) during its differentiation induced by phorbol 12-myristate 13-acetate (TPA) was studied using metabolic radiolabeling experiments. The FLG 29.1 cells were metabolically radiolabeled with [3H]glucosamine and [35S]sulfate, and the labeled glycoproteins were analyzed by anion exchange chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation experiments. One of the major glycoproteins synthesized by the TPA-treated FLG 29.1 cells was sulfated, had an identical electrophoretic mobility to purified BSP, and could be immunoprecipitated with a specific antibody against human BSP (LF 6). Thus, this glycoprotein was tentatively identified as the BSP. Furthermore, mRNA for BSP was also detected in TPA-treated FLG 29.1 cells by RNA-polymerase chain reaction. Most BSP synthesized by FLG 29.1 cells remained cell-associated, and this is in contrast with those synthesized by osteoblasts, where the protein is rapidly released into the extracellular matrix. Immunocytochemistry using an anti-BSP antibody showed a prominent paranuclear (suggestive of Golgi apparatus) localization of BSP in the TPA-treated FLG 29.1 cells after permeabilization, while untreated cells were not significantly immunostained. Localization of BSP at the plasma membrane was also demonstrated in the TPA-treated FLG 29.1 cells by the fluorescence-activated cell sorting analysis. Since TPA has been demonstrated to induce expression of various osteoclastic characteristics in FLG 29.1 cells, induction of BSP expression by TPA suggests that the protein may play a role during the differentiation process of osteoclasts or in functions of differentiated osteoclasts.

Amino Acid Sequence↗
PubMed1995

Effects of endothelin-1 on Ca2+ signaling and secretion in parathyroid cells.

It has been previously reported that parathyroid cells express endothelin (ET) receptors and secrete ET-1 in an extracellular Ca2+ concentration ([Ca2+]e)-dependent manner. Here, we examined the effects of ET-1 on intracellular signaling and parathyroid hormone (PTH) secretion in dispersed bovine parathyroid (bPT) cells, which comprise several cell types including epithelial and endothelial cells, in two cell lines, the rat parathyroid epithelial (PT-r) and the bovine parathyroid endothelial (BPE-1) cells. An RNA-polymerase chain reaction analysis revealed that both ETA and ETB receptors are expressed in bovine parathyroid tissue and BPE-1 cells, and only the ETA receptor is expressed in PT-r cells. PT-r cells also expressed an inositol 1,4,5-trisphosphate (Ins[1,4,5]P3) receptor, and ionomycin induced an increase in the intracellular Ca2+ concentrations ([Ca2+]i) in a Ca(2+)-deficient medium, indicating the presence of an operative intracellular Ca2+ pool in these cells. In cells bathed in 1 mM [Ca2+]e, ET-1 induced a rapid and transient increase in the Ins(1,4,5)P3 production, which was associated with a similar profile of increase in [Ca2+]i and with a peak response of about 800 nM. No changes in the profile of [Ca2+]i responses were observed in ET-1-stimulated cells in the presence of Ca2+ channel blockers, or in Ca(2+)-deficient medium, indicating that Ca2+ mobilization was not associated with Ca2+ entry. Furthermore, a sustained stimulation with ET-1 induced a decrease in [Ca2+]i below the prestimulatory level in a large population of cells, and the percentage of the cell population that shows the sustained decrease of [Ca2+]i increased in higher ET-1 concentrations. [Ca2+]i in PT-r cells was also controlled by a [Ca2+]e-dependent mechanism that changed [Ca2+]i from 28 to 506 nM in a 0.1-3 mM concentration range with an EC50 of 1.2 mM, which is comparable to that reported for bPT cells. In the same range of [Ca2+]e, PTH secretion from bPT cells was inhibited with an IC50 of 1 mM, and ET-1 increased PTH release in a dose-dependent manner but without affecting the IC50 for the [Ca2+]e-dependent inhibition. Thus, the parathyroid epithelial cells appear to respond to ET-1 in a unique way, and the ET autocrine system can be regarded as a possible mechanism to modulate the sensitivity of [Ca2+]e-dependent PTH release.

Animals↗
PubMed1995

In vitro structural and functional relationships between preosteoclastic and bone endothelial cells: a juxtacrine model for migration and adhesion of osteoclast precursors.

The role of vascularization in the process of bone resorption has not been clarified. The interactions between vascular endothelium and osteoclast progenitors were analyzed using clonal cell lines of bone-derived endothelial and preosteoclastic cells. Insulin-like growth factor I is a major chemotactic stimulator of preosteoclastic cell migration mediated by bone endothelial cells. Osteoclast precursors rapidly adhered to bone endothelial monolayers. This phenomenon appeared to be cell-specific and mediated through the binding of vitronectin and fibronectin receptors to fibronectin. In addition, direct contact with bone endothelial cells induced osteoclast progenitors to differentiate into more mature elements, with the tendency to cluster together to form large multinucleated cells. These findings demonstrated specific in vitro interactions between bone endothelial cells and osteoclast progenitors, offering a new model for understanding the molecular mechanisms which direct the processes of osteoclast recruitment and ontogeny.

Animals↗
PubMed1995

Lack of allelic loss at the multiple endocrine neoplasia type 1 (MEN-1) gene locus in a pancreatic ductal (non-endocrine) adenocarcinoma of a patient with the MEN-1 syndrome.

The gene responsible for multiple endocrine neoplasia type I (MEN-1) syndrome has been mapped to chromosome 11q13. It appears to function as a tumour-suppressor gene analogous to that for retinoblastoma and allelic losses involving the wild-type of the MEN-1 allele have been found in parathyroid and pancreatic endocrine tumours of MEN-1 patients. No genetic information has been provided so far on non-endocrine malignancies that may occur in MEN-1 patients. A case of exocrine pancreatic adenocarcinoma presenting as the terminal event in a woman with a long standing history of MEN-1 syndrome and multiple endocrine tumours of the pancreas was investigated for possible allelic losses at the MEN-1 gene locus using restriction fragment length polymorphisms (RFLPs) closely linked to the MEN-1 gene and polymerase chain reaction (PCR) for D11S533 locus. No allelic losses were found in tumour tissue with two informative RFLPs (D11S97, D11S146) or with PCR analysis. These findings suggest that the MEN-1 gene does not confer a predisposition to develop tumours other than those that typify the syndrome.

Adenocarcinoma↗
PubMed1995

Interactions between ipriflavone and the estrogen receptor.

Estrogen replacement therapy is effective in the prevention of postmenopausal osteoporosis, and a direct action of 17-beta-estradiol (17 beta E2) on osteoblastic and osteoclastic cells has been demonstrated. The inhibition of bone resorption by ipriflavone (IP), an isoflavone derivative devoid of estrogenic properties but active in potentiating the effects of estrogen on bone tissue, has been shown in in vitro and in vivo studies and confirmed by clinical data. To investigate the molecular mechanisms that underlie IP effect, we studied the possible interactions of IP and its four main in vivo metabolites (I, II, III, and V) with the estrogen receptor (ER) in the human preosteoclastic cell line FLG 29.1, whose growth and function are modulated by the compound. In parallel experiments, the human breast cancer cell line MCF7 was also analyzed. IP binding sites were demonstrated in the nuclear fraction of FLG 29.1 cells. 17 beta E2 and other steroid compounds failed to displace IP binding to intact FLG 29.1 cells. Similarly, IP and metabolites I, III, and V were not able to displace 17 beta E2 binding to intact MCF7 cells, whereas metabolite II showed an IC50 of 61 nM. 17 beta E2 binding to FLG 29.1 cells was increased after preincubation with metabolites I, III, and V. IP and its metabolites did not induce ER-dependent gene expression in FLG 29.1 and MCF7 cells transfected with a reporter gene and an estrogen response element (ERE).(ABSTRACT TRUNCATED AT 250 WORDS)

Binding, Competitive↗
PubMed1995

P-glycoprotein is expressed in parathyroid epithelium and is regulated by calcium.

P-glycoprotein (Pgp), the multidrug resistance (mdr) gene product, has been described in normal tissues with diverse physiologic functions. A broad role as a transporter protein for toxins, hormones, and physiologic metabolites has been provisionally deduced, based on structural analysis and immunoanatomic localization. Recently, significant levels of Pgp have been demonstrated in endocrine and hormonally responsive tissues and tumors. We examined calcium-regulated, clonal parathyroid epithelial (PT-r) and endothelial cells (BPE-1) and frozen parathyroid tissue from normal human parathyroid, parathyroid hyperplasia, parathyroid adenoma, and parathyroid carcinoma for expression of the multidrug resistance gene (Mdr1) and Pgp utilizing Northern and Western analysis and immunohistochemistry. We also investigated the effect of extracellular calcium (eCa) on Pgp expression in PT-r cells at the molecular/cellular level. Immunohistochemistry, utilizing three murine monoclonal antibodies (MAbs)--C494, JSB-1, and C219--which recognize spatially distinct cytoplasmic epitopes of Pgp, revealed strong immunoreactivity in PT-r cells, normal parathyroid, and parathyroid hyperplasia, and weak immunostaining in parathyroid adenomas. BPE-1 cells, endothelial cells, and parathyroid carcinoma were negative. PT-r cells showed a single 130 kDa band (120 KDa after glycosidase treatment) on Western blot and a 4.6 kb transcript on Northern analysis, consistent with Pgp. Western and Northern blot analysis of PTr cells cultured in different eCa concentrations showed that eCa up-regulated Pgp expression.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Functional and structural interactions between osteoblastic and preosteoclastic cells in vitro.

Osteoblasts are involved in the bone resorption process by regulating osteoclast maturation and activity. In order to elucidate the mechanisms underlying osteoblast/preosteoclast cell interactions, we developed an in vitro model of co-cultured human clonal cell lines of osteoclast precursors (FLG 29.1) and osteoblastic cells (Saos-2), and evaluated the migratory, adhesive, cytochemical, morphological, and biochemical properties of the co-cultured cells. In Boyden chemotactic chambers, FLG 29.1 cells exhibited a marked migratory response toward the Saos-2 cells. Moreover, they preferentially adhered to the osteoblastic monolayer. Direct co-culture of the two cell types induced: (1) positive staining for tartrate-resistant acid phosphatase in FLG 29.1 cells; (2) a decrease of the alkaline phosphatase activity expressed by Saos-2 cells; (3) the appearance of typical ultrastructural features of mature osteoclasts in FLG 29.1 cells; (4) the release into the culture medium of granulocyte-macrophage colony stimulating factor. The addition of parathyroid hormone to the co-culture further potentiated the differentiation of the preosteoclasts, the cells tending to fuse into large multinucleated elements. These in vitro interactions between osteoblasts and osteoclast precursors offer a new model for studying the mechanisms that control osteoclastogenesis in bone tissue.

Adult↗
PubMed1995

Genetic screening to identify the gene carrier in Italian and German kindreds affected by multiple endocrine neoplasia type 1 (MEN 1) syndrome.

Multiple endocrine neoplasia type 1 (MEN 1) is an autosomal dominantly inherited disorder characterized by parathyroid hyperplasia, anterior pituitary adenomas and neoplasms of the endocrine cells of the gastroenteric tract. It has been established that also other tissues exhibit excessive proliferation associated to the MEN 1 syndrome: carcinoids (bronchial and intestinal), lipomas (visceral and cutaneous), thyroid adenomas and goiter, and adrenal gland cortex adenomas. The men 1 gene has been mapped by genetic studies to the long arm of human chromosome 11, region q12-13. Genetic analysis of families and tumoral deletion mapping made possible to narrow the men 1 region to a 5 cM interval on chromosome 11q12-13. Thirteen marker complexes (17 DNA probes) were found to be linked to the men 1 gene and they span a 14% meiotic recombination with the men 1 locus in the middle. We report a genetic study on 103 subjects from 7 collected MEN 1-kindreds, six Italian and one German, including 30 affected individuals. By linkage analysis to 9 DNA markers (10 DNA probes) of the chromosome region where the men 1 gene maps (11q12-13), we identified 10 mutant gene carriers. The predicted MEN 1 diagnosis was clinically confirmed for 2 of these identified carriers. A predictive accuracy of this genetic test can reach up to 99.5% when it is possible to exclude meiotic crossing-over between the analyzed DNA markers and the disease locus.

Adult↗
PubMed1995

Pheochromocytoma in multiple endocrine neoplasia type 2: European study. The Euromen Study Group.

OBJECTIVES: Pheochromocytoma (pheo) is the second component of the multiple endocrine neoplasia type 2 (MEN 2) syndrome. Clinical expression is sometimes poor, and chronology between medullary thyroid carcinoma (MTC) and pheo is not well evaluated. Therefore, a retrospective study was done in eight European countries in order to precise the main characteristics of pheo in MEN 2. SUBJECTS: Data from 300 MEN 2 patients with pheo (274 MEN 2 A and 26 MEN 2 B) were obtained from cases registered by the EuroMen study group, and collected by a medical standardized questionnaire. These cases occurred between 1969 and 1992. RESULTS: Mean age at diagnosis of pheo was 39.5 years (range 14-68 years) in MEN 2A and 32.4 years (range 15-41 years) in MEN 2B patients. Pheo occurred first in 25.1% of the cases (2-15 years before diagnosis of MTC) and after MTC in 40.2% (2-11 years). In other cases (34.7%), MTC and pheo were diagnosed at the same time. Involvement was bilateral in 67.8% of cases. Malignancy was only 4%. Thirty-nine deaths occurred in these 300 patients, 64.1% were linked in pheo, 23.1% to MTC and 12.8% to other causes. Surgery was unilateral in 39.7% of the cases and bilateral adrenalectomy was the first procedure in 48.4%. A bilateral adrenalectomy in two steps had to be done in 11.9% of cases. In conclusion, these results justify systematic and prolonged biochemical screening of pheo during follow-up of MTC and address some questions about the best mode of surgery.

Adolescent↗
PubMed1995

Osteolytic processes in human radicular cysts: morphological and biochemical results.

In an attempt to verify the nature of bone resorption processes on the bone surrounding radicular cysts, fragments of cysts with the adjacent bone tissue were studied by morphological, cytochemical and ultrastructural methods. Cyst fluid was analysed for its content of cytokines with osteolytic activity. The cyst wall exhibited several connective tissue extensions which penetrated the adjacent bone. Numerous multinucleated tartrate-resistant acid phosphatase (TRAP)-positive cells were seen at the tip of the intraosseous extensions of the cyst capsule and in direct contact with the bone tissue. Typical resorption lacunae were identified on the bone surface by scanning electron microscopy. Moreover, mononuclear TRAP-positive cells were seen within the cyst capsule. High levels of prostaglandin E2 (PGE2) and interleukin-6 (IL-6) were detected in the cyst fluids. In conclusion, active bone resorption may contribute significantly to the growth of these lesions within the jaws.

Acid Phosphatase↗
PubMed1995

Mutations in the human Ca(2+)-sensing-receptor gene that cause familial hypocalciuric hypercalcemia.

We report five novel mutations in the human Ca(2+)-sensing-receptor gene that cause familial hypocalciuric hypercalcemia (FHH) or neonatal severe hyperparathyroidism. Each gene defect is a missense mutation (228Arg-->Gln, 139Thr-->Met, 144Gly-->Glu, 63Arg-->Met, and 67Arg-->Cys) that encodes a nonconservative amino acid alteration. These mutations are each predicted to be in the Ca(2+)-sensing receptor's large extracellular domain. In three families with FHH linked to the Ca(2+)-sensing-receptor gene on chromosome 3 and in unrelated individuals probands with FHH, mutations were not detected in protein-coding sequences. On the basis of these data and previous analyses, we suggest that there are a wide range of mutations that cause FHH. Mutations that perturb the structure and function of the extracellular or transmembrane domains of the receptor and those that affect noncoding sequences of the Ca(2+)-sensing-receptor gene can cause FHH.

Calcium↗
PubMed1994

Calcium modulates the cyclin D1 expression in a rat parathyroid cell line.

We have used a rat epithelial parathyroid cell line (PT-r) to study the expression and regulation of D-type cyclins. In PT-r cells the cyclin D1 gene is the most abundantly expressed, being transcribed in at least two mRNAs whose levels oscillate during the cell cycle. We also screened a cDNA library prepared from PT-r cells with the human cyclin D1 probe and isolated its rat homologue. Cyclin D2 and D3 mRNAs are both represented in PT-r cells but the former one is only barely detectable. Moreover, the oscillation of cyclin D3 transcript is slightly delayed when compared to cyclin D1 and D2. Since extracellular calcium inhibits parathyroid cell proliferation, we looked for the effect of the ion on the expression of cyclin D genes in PT-r cells. Increasing amounts of calcium in the incubation medium reduced the expression of rat cyclin D1 and D2. The effect appears to be cell-specific and probably mediated through the inhibition of mitogenic signalling pathways.

Amino Acid Sequence↗
PubMed1994

Binding and bioeffects of Ipriflavone on a human preosteoclastic cell line.

Ipriflavone, a synthetic isoflavone derivative, reduces bone resorption by inhibiting osteoclasts activity. In order to evaluate the role of Ipriflavone on osteoclast growth and differentiation, we tested Ipriflavone and its four "in vivo" main metabolites (Metabolites I, II, III, and V) on a clonal population of human osteoclast precursor cells (FLG 29.1). Pharmacological doses of Ipriflavone and Metabolite III were able to inhibit cell proliferation and interleukin 6 release. In co-cultures of FLG 29.1 cells and osteoblastic (Saos-2) cells Ipriflavone at 1 microM dose inhibited the adhesion of FLG 29.1 cells to the osteoblastic monolayer and reduced the immunocytochemical reaction of the vitronectin receptor. Binding studies with tritiated Ipriflavone showed the presence of a single specific binding site, wtih a Kd of about 70 nM and a binding capacity of 8 fmol/10(6) cells. These results demonstrate a direct effect of Ipriflavone and of Metabolite III on the human osteoclast precursor cell line FLG 29.1.

Cell Adhesion↗
PubMed1994

Relaxin influences growth, differentiation and cell-cell adhesion of human breast-cancer cells in culture.

The effects of different concentrations of relaxin (RLX) on growth and differentiation of a human breast-cancer cell line (MCF-7) have been studied after various times of exposure. The cells were cultured for 4 and 7 days in the absence (control) and the presence of highly purified porcine RLX at concentrations of 10(-9) M and 10(-6) M. (3H)-Thymidine uptake assay was used to evaluate cell proliferation. Electron microscopy and immunocytochemistry for the cell-cell adhesion molecule E-cadherin were carried out to evaluate cell differentiation. Analysis of DNA changes associated with apoptosis was performed to clarify whether RLX induces active cell death in the MCF-7 cells. The findings obtained show that RLX, when applied at micromolar concentrations, or even at nanomolar concentrations for long exposure times, suppresses proliferation, stimulates differentiation, and enhances expression of the surface molecule E-cadherin. Growth inhibition is not accompanied by apoptosis. The results of this study show that RLX can be recognized as a novel agent active in influencing growth and differentiation of MCF-7 breast-cancer cells. When applied at appropriate concentrations and exposure times, the peptide has a growth-inhibitory action, thus reversing the growth-stimulatory effect exerted at low concentrations for short exposure times, and promotes differentiation and cell-cell adhesion. These last-mentioned properties might result in a decrease in invasiveness of breast adenocarcinoma cells.

Adenocarcinoma↗
PubMed1994

Characterization, regulation, and function of specific cell membrane receptors for insulin-like growth factor I on bone endothelial cells.

It is now widely accepted that insulin-like growth factor-I (IGF-I) has a local regulatory role in bone remodeling. IGF-I has also been demonstrated to regulate proliferation of bone-derived endothelial cells. Such studies suggest a role of IGF-I in skeletal angiogenesis. Using BBE cells, a bovine bone endothelial cell line, we characterized the kinetics and chemical properties of IGF-I receptors and examined the effect of IGF-I on bone endothelium migration. Two classes of binding sites with high affinity for IGF-I were detected by binding experiments on bone endothelial cells. Both competition analyses and cross-linking studies revealed the presence of type I IGF receptor in bone endothelial cells. Moreover, these cells produced and released authentic IGF-I into the medium, as evidenced by radioimmunoassay analyses of gel-filtered conditioned media. Both IGF-I binding capacity and release decreased either with increases in cell number or after treatment with 17 beta-estradiol (17 beta E2) and parathyroid hormone (PTH). Both hormones also inhibited chemotactic responses of bone endothelial cells to IGF-I. Taken together, these results strongly suggest that IGF-I, a growth factor that promotes the proliferation of various bone cell types, also induces growth and chemotactic responses in bone endothelium acting through the type I IGF receptor. This may be part of a generalized response of bone cells to IGF-I that facilitates cell migration.

Analysis of Variance↗