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Biomedical subjects

M Pines

Publications and source records attributed to M Pines.

At least 19 recordsLinked to original sources
PubMed1995

Probing the bimolecular interactions of parathyroid hormone and the human parathyroid hormone/parathyroid hormone-related protein receptor. 2. Cloning, characterization, and photoaffinity labeling of the recombinant human receptor.

Parathyroid hormone (PTH) acts to regulate calcium homeostasis by interacting with a G-protein-coupled receptor that also binds PTH-related protein (PTHrP). In this report we describe the cloning, characterization, and biological activity of the cloned human (h) PTH/PTHrP receptor (Rc) and cross-linking of a benzophenone-substituted PTH analog, [Nle8,18,Lys13(epsilon-pBZ2),L-2-Nal23,Tyr34]bPTH(1-34 )NH2(K13), to cells endogenously expressing the Rc and cells transiently or stably transfected with the human Rc. A full-length cDNA clone was isolated and fully sequenced from a human kidney cDNA library. Northern blot analysis of normal human tissues revealed a limited tissue distribution: a single transcript of approximately 2.3 kb was detected in kidney, lung, placenta, and liver. In human embryonic kidney cells (HEK-293, clone C-21) stably transfected with hPTH/PTHrP Rc, a single 85-90 kDa Rc-hormone complex was formed after photolysis in the presence of K13. This covalent cross-linking reaction was specifically inhibited by excess quantities of biologically active 1-34 analogs of bovine (b) PTH or hPTHrP but not by C-terminal and midregion PTH peptides. Photoincorporation of 125I-labeled K13 into the Rc occurred with high efficiency (60-70%), approximately an order of magnitude greater than that achieved with conventional aryl azide cross-linking reagents. These results support the feasibility of our approach for specifically cross-linking a tagged PTH analog to the Rc, as a first step in the effort to identify directly the amino acid residues that constitute the Rc binding site.

Affinity Labels↗
PubMed1995

Synthesis and phosphorylation of osteopontin by avian epiphyseal growth-plate chondrocytes as affected by differentiation.

The regulation of synthesis and phosphorylation of osteopontin in relation to avian epiphyseal growth-plate chondrocyte differentiation was studied in situ and in culture. Osteopontin gene expression was evaluated in the tibia growth-plate of 3-week-old chickens by in situ hybridization. The gene was expressed mainly at the lower hypertrophic zone where cartilage matrix is calcified and endochondral bone formation is initiated. Within the hypertrophic region, a poorly labeled area separated the layer of osteopontin-positive hypertrophic chondrocytes from those associated with endochondral bone formation. In culture, proliferative chondrocytes show no alkaline phosphatase activity in contrast to ascorbic acid-treated chondrocytes which display the enzyme activity. Chondrocytes not treated with ascorbic acid, exhibited lower levels of osteopontin mRNA than the treated cells. The phorbol ester TPA--an activator of protein kinase C--and to a lesser extent FGF but not EGF, stimulated osteopontin gene expression. Chondrocytes secreted low levels of phosphorylated osteopontin to the medium. EGF treatment resulted in the appearance of phosphorylated osteopontin in the medium, without affecting the synthesis of other proteins. FGF and TGF beta, but not IGF-I or IGF-II, also caused phosphorylation of osteopontin. Ascorbic acid-treated chondrocytes secreted higher levels of phosphorylated osteopontin than the non-treated cells, but addition of FGF or TPA did not stimulate osteopontin phosphorylation any further. Parathyroid hormone caused a dose-dependent attenuation of osteopontin phosphorylation and inhibited the EGF-dependent osteopontin phosphorylation. The results suggest that osteopontin gene expression and phosphorylation in chondrocytes are regulated by separate mechanisms. The response to the various controlling agents varies with the state of differentiation. Both processes--the synthesis and phosphorylation of osteopontin--are under the control of local growth factors which are involved in bone growth and calcification.

Alkaline Phosphatase↗
PubMed1995

Osteopontin gene expression and alkaline phosphatase activity in avian tibial dyschondroplasia.

Osteopontin (OPN) gene expression and alkaline phosphatase activity were evaluated in the epiphyseal growth plates of normal chickens and in diet-induced tibial dyschdroplasia (TD)-afflicted chickens. In the normal growth plate, OPN gene was expressed by a) cells of the subperichondrial zone surrounding the articular cartilage, b) a narrow layer of hypertrophic chondrocytes at the hypertrophic zone, and c) lower hypertrophic chondrocytes at the zone of matrix calcification and endochondral bone formation. The latter two layers were separated by OPN-negative chondrocytes. Osteopontin gene was not expressed throughout the zone of articular cartilage in the nonhypertrophic or upper hypertrophic portions of the growth plate cartilage. Only at sites of calcification of the lower hypertrophic zone was the expression of the OPN gene associated with alkaline phosphatase activity. In all TD lesions, regardless of the induction procedure, the layer of chondrocytes of the lower hypertrophic zone expressing the OPN gene and the layer of OPN-negative cells separating the two areas of OPN-expressing cells were grossly enlarged. This resulted in a wide discontinuity between the chondrocytes of the lower hypertrophic zone expressing the OPN gene and the cells expressing the OPN gene that are associated with mineralization. In TD, no alkaline phosphatase activity was detected within the growth plate cartilage, but normal OPN gene expression was observed at the subperichondrium zone and at the zone of endochondral bone formation. The results of this study suggest that in the epiphyseal growth plate, OPN expression is not restricted to sites of bone calcification.

Alkaline Phosphatase↗
PubMed1994

Generation and characterization of human kidney cell lines stably expressing recombinant human PTH/PTHrP receptor: lack of interaction with a C-terminal human PTH peptide.

Parathyroid hormone (PTH) exerts its biological action by binding to membrane-bound, G-protein coupled receptors expressed predominantly in bone and kidney. In this study, we describe the production and characterization of a panel of cell lines, derived from a human embryonic kidney cell line (HEK-293), each of which stably express different amounts of the recombinant human PTH/parathyroid hormone-related protein (PTHrP) receptor (Rc). A total of 52 distinct clones displaying different levels of PTH-responsive cAMP production were analyzed; three clones were chosen for more detailed evaluation. These clones (and the receptor-lacking parental cell line) were examined for PTH binding, PTH-stimulated cyclic AMP accumulation and PTH/PTHrP Rc mRNA expression. Receptor-positive clones display a spectrum of PTH-responsiveness that correlates with receptor number/cell and level of receptor mRNA present. The interaction of a C-terminal hPTH-(52-84) peptide with the stably expressed human receptor was examined in cells expressing the highest amount of Rc (> 400,000 Rc/cell). There was no direct binding of hPTH-(52-84) or specific competition versus radiolabeled PTH-(1-34). However, competition versus radiolabeled PTH-(1-34) was observed with bPTH-(1-34), hPTH-(1-84) and hPTHrP-(1-34). These data suggest that hPTH-(52-84) does not interact with the only known form of the human PTH/PTHrP Rc. Therefore, the reported effects of PTH-(52-84) in other systems must be via an alternate (as yet unidentified) mechanism(s). The expression of various amounts of the human PTH/PTHrP Rc in a human target cell background should facilitate characterization of the ligand-binding properties and physiological signal transduction mechanism of the Rc.

Cell Line↗
PubMed1993

Growth hormone receptors in avian epiphyseal growth-plate chondrocytes.

Growth hormone receptor (GH-R) gene expression was evaluated in avian growth-plate cartilage by Northern blot and hybridization using the avian GH-R probe. A single transcript of approximately 5.2 kb was demonstrated in cultured growth-plate chondrocytes as well as in growth-plate extracts. GH receptor gene expression was inhibited by chicken GH (cGH) in a dose- and time-dependent manner. Chicken GH was more potent in down-regulating the GH-R gene expression than hGH, but on the other hand cGH exhibited a lower affinity to avian chondrocytes receptor than did the human hormone. Addition of ascorbic acid to the culture media caused cell differentiation: induction of alkaline phosphatase activity and attenuation of collagen type II gene expression. No differences in the GH-R gene expression were observed in the nondifferentiated cells compared with the differentiated cells. Chicken GH did not form any complex with the purified hGH binding protein (hGHBP), did not bind to human lymphocytes GH receptor, and did not affect Nb2 cell proliferation. These systems represent somatogenic and lactogenic types of GH receptors, respectively. In summary, avian growth-plate chondrocytes in situ and in culture exhibit GH-R and these receptors are capable of binding GH. Thus, the failure of GH to affect avian chondrocytes' proliferation was not due to either the absence of receptors on the cell membrane or to a lack in its binding activity, but rather may be due to events farther downstream.

Alkaline Phosphatase↗
PubMed1992

Differential post-receptor responses of adenylate cyclase in white and brown adipose tissue membranes of rats fed high-energy diets.

1. Adenylate cyclase activity was determined in membranes of white and brown adipose tissue (WAT and BAT, respectively) from rats fed a high-energy diet (EXP group) vs those fed a nutritionally balanced one (CON group). 2. The isoproterenol- and guanine nucleotide-induced adenylate cyclase activity in WAT membranes of EXP rats was lower than that in CON rats. 3. Relative adenylate cyclase activity in like treated BAT membranes was higher in EXP than in CON rats. 4. It is concluded that feeding high-energy diets to rats induces similar post-receptor modifications of adenylate cyclase as found in genetic obese rodents.

Adenylyl Cyclases↗
PubMed1992

Group-analytic psychotherapy.

Group analysis is a flexible and effective method of providing group psychotherapy, thereby promoting maturation and symptom relief. Its metapsychology remains incompletely worked out but provides potentially exciting interfaces with biological and physical sciences, through Foulkes's (1973) seminal concept of the group matrix.

Humans↗
PubMed1992

Tibia development in turkeys and chickens as affected by early-age feed restriction.

Tibia development was studied by histomorphometry in male chickens and turkeys fed ad libitum throughout the growing period, or subjected to an early-age (1-week) severe feed restriction for 6 and 14 days, respectively. In turkeys, the rates of longitudinal bone growth and epiphyseal width growth, both dependent on the activity of chondrocytes, proceeded rapidly and reached maxima at early ages of 24 and 11 days, respectively. Moreover, longitudinal bone growth and the growth of epiphyseal width were reduced during early-age feed restriction, in both chickens and turkeys. On the other hand, the rate of growth of metaphyseal width, determined by activity of bone cells such as osteoclasts and osteoblasts, reached in turkeys a maximum at the relatively late age of 70 days, and was not influenced by feed restriction. Rhythmic variation characterized the temporal behavior of the height of the hypertrophic and non-hypertrophic zones of the growth plate, the trabecular width, and the osteoclasts number at the subchondral region. Rhythmicity appeared similar in both ad libitum-fed and early-age feed-restricted birds of both species. The height of the non-hypertrophic epiphyseal cartilage appeared not to change, whereas the hypertrophic zone decreased with age and early-age feed restriction, demonstrating the importance of cartilage cell hypertrophy as a main determinant of longitudinal bone growth. Sexual maturation was accompanied by the disappearance of both hypertrophic and non-hypertrophic zones of the epiphyseal growth plate and the end of bone elongation. Trabecular width at the end of bone elongation. Trabecular width at the subchondral region increased while osteoclast number decreased with age, reflecting the net increase in bone mass, but were not affected significantly by early age feed restriction. Compensatory growth of the early-age feed-restricted birds, resulted in the complete recovery of affected bone variables within a few weeks of resumed ad libitum feed intake.

Aging↗
PubMed1991

Natriuretic peptide receptors regulate endothelin synthesis and release from parathyroid cells.

Cloned rat parathyroid cells (PTr cell line) that produce parathyroid hormone-related peptide plus endothelin 1 and primary cultures of human parathyroid cells were tested for growth and differentiation responses to atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). High- and low-affinity binding sites for ANP were found on PTr cells; BNP appeared to bind to the same receptors with similar affinities. Either ANP or BNP stimulated production of cGMP and caused a 30% decrease in Na(+)-K(+)-Cl- cotransport. Each peptide increased synthesis and secretion of endothelin 1 by PTr cells in a dose-dependent fashion, but cell growth was not affected. Human parathyroid cells (normal and pathological) also responded to ANP or BNP with an increase in cGMP production. The finding of receptors for natriuretic hormones on parathyroid cells with consequent effects on release of endothelin 1 might be of relevance in understanding the clinical association between hyperparathyroidism and hypertension.

Animals↗
PubMed1991

Growth hormone and insulin-like growth factor I regulate collagen gene expression and extracellular collagen in cultures of avian skin fibroblasts.

Avian skin fibroblasts were isolated, cultured and incubated with [3H]proline for 24 h. The cells exported radiolabeled collagenase-digestible (CDP) and non-collagenase-digestible (NCDP) proteins into the medium. Human, bovine and avian growth hormone (GH) as well as insulin-like growth factor I (IGF-I) attenuated the appearance of [3H]CDP in the medium without affecting [3H]NCDP. The appearance of [3H]CDP was not affected by prolactin. The effects of GH and IGF-I were enhanced by increasing concentrations of fetal calf serum (FCS). A synergism was observed between GH and IGF-I in their effect on CDP. Each peptide, at an ineffective concentration, increased the sensitivity of the cells to the other peptide. Collagenase activity in the medium was enhanced by IGF-I, but not modified by GH, FCS, or by their interaction with IGF-I. GH and IGF-I inhibition of type I procollagen gene expression was demonstrated with the aid of probes containing sequences corresponding to the mRNAs for avian alpha I and alpha II chains. The results suggest that GH and IGF-I cooperate in regulating collagen synthesis, but collagen degradation is affected by IGF-I and not by GH.

Animals↗
PubMed1991

Epidermal growth factor receptor gene expression in avian epiphyseal growth-plate cartilage cells: effect of serum, parathyroid hormone and atrial natriuretic peptide.

Avian chondrocytes and fibroblasts, derived from epiphyseal growth-plate and skin, respectively, were cultured in vitro. In chondrocytes, epidermal growth factor (EGF) caused a dose-dependent stimulation of proliferation. EGF receptor mRNA was not detected with the v-erb B probe in chondrocytes cultured in the presence of 5% fetal calf serum (FCS). In the absence of FCS in the medium, a time-dependent increase in the level of EGF receptor mRNA was observed. Parallel changes were also observed in the level of EGF receptor, as demonstrated by immunofluorescence using antibodies directed against avian EGF receptor. In avian fibroblasts, EGF receptor mRNA and EGF receptor levels were not affected by FCS. Furthermore, FCS did not affect the level of thyroid hormone receptor mRNA (using v-erb A as a probe) in either chondrocytes or fibroblasts. Parathyroid hormone (PTH), which acts as a mitogen in avian chondrocytes attenuated--whereas atrial natriuretic peptide (ANP), a suppressor of chondrocyte proliferation, enhanced--EGF receptor mRNA. The present results show that avian growth-plate chondrocytes respond to EGF and bear EGF receptors. The levels of EGF mRNA and EGF receptor are inversely related to cell proliferation. The results also support previous suggestions that PTH and ANP play important roles in chondrocyte proliferation, possibly through their effect on the synthesis of the EGF receptor.

Animals↗
PubMed1991

Increased skin tearing in broilers and reduced collagen synthesis in skin in vivo and in vitro in response to the coccidiostat halofuginone.

In vivo and in vitro experiments were conducted in an effort to elucidate the mechanism of suppression by halofuginone of skin strength in broilers. In the in vivo study, halofuginone was included at concentrations of 0, 1.5, 3, and 6 mg/kg of diet, corresponding to 0, 50, 100, and 200%, respectively, of the amount recommended for use as a coccidiostat. Each dietary treatment was given to 260 female broiler day-old chickens. Skin tearing was evaluated at the processing plant. Skin collagen and Kjeldahl-nitrogen were determined chemically. At the age of 7 wk, BW and feed efficiency were affected only in birds consuming the diet containing the highest concentration of the drug. Skin tearing increased but skin collagen concentration decreased in a dose-dependent manner. Fibroblasts were obtained by collagenase digestion from chicken skin and cultured. The cultured cells were incubated with various concentrations of halofuginone, monensin, and nicarbazin, and [3H]proline incorporation was evaluated in collagenase-digestible (representing mostly collagen) and nondigestible proteins exported by the cells into the medium. Halofuginone, at a concentration as low as 10(-11) M, inhibited incorporation of [3H]proline into collagenase-digestible proteins, but did not affect incorporation of [3H]proline into collagenase-nondigestible proteins. Even at concentrations as high as 10(-9) M, neither monensin nor nicarbazin affected collagenase-digestible proteins. The in vitro results suggest that halofuginone specifically inhibits collagen synthesis by skin fibroblasts. Results of both in vivo and in vitro trials suggest that the increase of skin tearing during processing, induced by halofuginone, is caused by direct suppression of skin collagen synthesis.

Animals↗
PubMed1991

The role of the growth plate in longitudinal bone growth.

The epiphyseal growth plate is the main site of longitudinal growth of the long bones. At this site, cartilage is formed by the proliferation and hypertrophy of cells and synthesis of the typical extracellular matrix. The formed cartilage is then calcified, degraded, and replaced by osseous tissue. Proliferation and differentiation of cartilage cells (i.e., chondrocytes) as studied mostly in culture, is regulated by various endocrine, paracrine, and autocrine agents such as growth hormone, insulin-like growth factor-I (IGF-I), transforming growth factor (TGE-beta), and vitamin D metabolites (1,25-dihydroxycholecalciferol and 24,25-dihydroxycholecalciferol). Avian chondrocyte proliferation is enhanced by agents which use adenosine 3':5'-cyclic monophosphate as a second messenger, such as parathyroid hormone or prostaglandin-E2, and is depressed by guanosine 3':5'-cyclic monophosphate agonists, such as atrial natriuretic peptide. Several of the regulating agents also affect synthesis of the main extracellular components (i.e., collagen and proteoglycans) and their transfer to the extracellular space. Cartilage calcification involves matrix vesicles secreted by the chondrocytes at a specific stage. Calcification probably involves some initial nucleation agent and participation of phosphatases. During sexual maturation, the growth plate closes by an unknown mechanism and longitudinal bone growth ceases. Disorders in the metabolism of the controlling agents or the cellular responses in growth plate may lead to several deformities classified as dysplasias. In poultry, this class of disorders is represented by chondrodystrophy and dyschondroplasia.

Animals↗
PubMed1991

Skin tearing in broilers in relation to skin collagen: effect of sex, strain, and diet.

The relationship between skin tearing and collagen in broilers was investigated in two trials in which strain and sex, and strain and diet served as factorial-arranged variables, respectively. In the first trial, males and females of three strains were examined. Both skin tearing and skin collagen were significantly influenced by strain and sex without any significant strain by sex interaction. Skin collagen, expressed as a fraction of fresh skin protein (N x 6.25) was lower and skin tearing was higher in females than in males, particularly in the most susceptible strain. In the second trial, the effects of supplementary protein or methionine and of a low-density diet were tested in females of two strains that differed in their susceptibility to skin tearing. High dietary protein reduced skin tearing and increased skin collagen. The significant diet by strain interaction resulted from the more pronounced response of the susceptible strain. Neither supplementary methionine nor feeding of low-dietary-density diet significantly affected skin tearing or skin collagen.

Animals↗
PubMed1990

Proliferation of bovine undifferentiated mammary epithelial cells in vitro is modulated by G-proteins.

Several cAMP-elevating agents such as cholera toxin (CT), forskolin and 3-isobutyl-1-methylxanthine (IBMX) exhibited weak mitogenic activity on bovine undifferentiated mammary epithelial cells in three-dimensional collagen culture. CT and IBMX strongly synergized with epidermal growth factor (EGF), insulin-like growth factor I (IGF-I) or both, but not with 10% fetal calf serum (FCS). Permeable cAMP analogs also synergized with IGF-I. Other hormones such as ovine prolactin, bovine growth hormone, estrogen or progesterone were not mitogenic and not synergistic with EGF, IGF-I, CT and FCS. Pertussis toxin (PT) reduced the DNA synthesis in cells cultured in the basal medium and attenuated 40-90% of the mitogenic activity stimulated by 10% FCS. PT inhibition of DNA synthesis was accompanied by ADP-ribosylation of 40 kDa and 41 kDa membrane proteins. The 41 kDa protein cross-reacted with antibodies that recognize the Gi-protein of the adenylate cyclase system, indicating the involvement of the latter in the mitogenic process. The nature of the second protein remains unknown. The present results suggest that the mitogenesis of normal mammary epithelial cells which is stimulated by IGF-I, EGF and other factors found in FCS is mediated through both cAMP-dependent and independent pathways. These pathways include PT-sensitive GTP-binding proteins.

1-Methyl-3-isobutylxanthine↗
PubMed1990

Cyclic AMP-dependent inhibition of collagen synthesis in avian epiphyseal cartilage cells: effect of chicken and human parathyroid hormone and parathyroid hormone-related peptide.

Avian cartilage cells derived from epiphyseal growth-plate and avian skin fibroblasts were cultured in vitro. Production of cAMP by cartilage cells was stimulated by the synthetic fragments (1-34) of chicken (cPTH), human (hPTH) parathyroid hormone and by parathyroid hormone-related peptide (PTHrP). The enhancement of cAMP production by any of the peptides could be blocked by the parathyroid hormone analogue (3-34)PTH, suggesting interaction with PTH specific receptors. When incubated with [3H]proline, both cell types released radiolabelled collagenase-digestible and non-digestible proteins into the medium. cPTH, hPTH, PTHrP, forskolin, prostaglandin E2 (PGE2) and 8-bromo cAMP inhibited collagen production in cartilage cells with only minor effects on non-collagenase digestible proteins. No effect of cAMP on collagen production by fibroblasts was observed. The present results provide additional evidence that avian growth-plate cartilage cells are targets for PTH, and are first to demonstrate the response of a non-mammalian system to mammalian PTHrP. The data suggest that collagen production by epiphyseal growth-plate cartilage cells is inhibited by PTH and that this inhibition is mediated by cAMP.

Animals↗