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Biomedical subjects

S L Cheng

Publications and source records attributed to S L Cheng.

At least 73 records · Page 4Linked to original sources
PubMed1994

Detection of the readthrough protein of barley yellow dwarf virus.

The single open reading frame (ORF) 5 encoding the 50-kDa protein of barley yellow dwarf virus PAV-IL (BYDV-PAV-IL) was expressed in bacteria, purified, and used as an immunogen/antigen to produce/screen antibodies specific to the 50-kDa protein. Two monoclonal antibodies (MAb PAV-IL-22 kDa and MAb PAV-IL-50 kDa) raised against BYDV-PAV-IL could specifically detect the presence of the 72-kDa readthrough protein in extracts from the BYDV-infected leaf tissue. The results suggest that ORF 5 (50-kDa protein) is translated by readthrough of ORF 3 (22-kDa coat protein) to produce the 72-kDa protein. The readthrough protein is thought to be a structural protein on the external surface of BYDV.

Antibodies, Viral↗
PubMed1994

Stimulation of human osteoblast differentiation and function by ipriflavone and its metabolites.

Ipriflavone (IP), an isoflavone derivative, has been shown to interfere with bone remodeling by inhibiting bone resorption and perhaps stimulating bone formation. In this study, we have analyzed the effect of IP and its metabolites on the differentiation and function of human osteoblastic cells. Bone marrow stromal osteoprogenitor cells (BMC) and trabecular bone osteoblasts (HOB) were isolated from human donors. The former can be induced to differentiate by treatment with dexamethasone, whereas the latter represent a more differentiated osteoblast. Incubation of BMC with metabolite III (10(-5) M) for 1 week induced modest but significant changes of alkaline phosphatase activity. Though both IP and metabolite III stimulated the expression of bone sialoprotein mRNA, a protein involved in cell attachment to the matrix, only metabolite III increased the steady-state level of decorin mRNA, a collagen fibrillogenesis-regulating proteoglycan. Metabolites III and V, but not the other isoflavones, increased the expression of type I collagen mRNA in HOB, whereas no detectable changes were observed in BMC cells with any of the experimental compounds. In HOB, an increased abundance of osteopontin and bone sialoprotein mRNA were also obtained after 1-week treatment with IP or metabolite V. No appreciable effects of IP or its metabolites were seen on osteocalcin expression and synthesis by either cell type. Finally, IP consistently increased the amount of 45Ca incorporated into the cell layer by BMC, and stimulated mineralization of both BMC and HOB, assessed by von Kossa staining. Thus, IP and its metabolites regulate the differentiation and biosynthetic properties of human bone-forming cells by enhancing the expression of some important matrix proteins and facilitating the mineralization process.

Alkaline Phosphatase↗
PubMed1994

Differentiation of human bone marrow osteogenic stromal cells in vitro: induction of the osteoblast phenotype by dexamethasone.

Human bone marrow stromal cells were examined for their osteogenic potential in an in vitro cell culture system. Dexamethasone (Dex) treatment induced morphological transformation of these cells from an elongated to a more cuboidal shape, increased their alkaline phosphatase activity and cAMP responses to PTH and prostaglandin E2, and was essential for mineralization of the extracellular matrix. Dex-induced differentiation of human bone marrow stromal cells was apparent after 2-3 days of treatment and reached a maximum at 7-14 days, as judged by alkaline phosphatase activity, although induction of osteocalcin by 1,25-dihydroxyvitamin D3 was attenuated by Dex. Withdrawal of Dex resulted in an enhancement of the 1,25-dihydroxyvitamin D3-induced secretion of osteocalcin, whereas alkaline phosphatase activity and the cAMP response to PTH remained at prewithdrawal levels. The steady state mRNA level of osteonectin was not affected by Dex. Our results, which demonstrate that Dex conditions the differentiation of human bone marrow osteogenic stromal cells into osteoblast-like cells, support the hypothesis of a permissive effect of glucocorticoids in ensuring an adequate supply of mature osteoblast populations. Furthermore, the established human bone marrow stromal cell culture provides a good model of an in vitro system to study the regulation of differentiation of human bone osteoprogenitor cells.

Adult↗
PubMed1994

The effect of glucocorticoid on the synthesis of biglycan and decorin in human osteoblasts and bone marrow stromal cells.

We have previously demonstrated that glucocorticoids induce differentiation of human bone marrow stromal cells (BMSC) into cells expressing mature osteoblast phenotype. As glucocorticoids have marked effects on extracellular matrix protein synthesis in bone, and because proteoglycans are important components of bone matrix and may condition the differentiation and biological activities of osteoblasts, we studied the effects of dexamethasone (Dex) on the synthesis of small proteoglycans [decorin (DCN) and biglycan (BGN)] in adult human BMSC and human osteoblasts (HOB). First passaged HOB and BMSC were treated with either ethanol or 10(-7) M Dex for 7 days. After treatment, the cells were metabolically labeled with either [35S]SO4 alone or [35S]SO4 and [3H]leucine together for 24 h. Conditioned media were collected, and cell layers were extracted with 4 M guanidine HCl. The extracts and conditioned media were subjected to gel filtration and ion exchange chromatography. Fractions containing radiolabeled proteoglycans were analyzed either directly or after immunoprecipitation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Dex treatment resulted in a dramatic increase in DCN and an associated decrease in BGN in both the conditioned medium and the cell layer of HOB cultures. In Dex-treated BMSC cultures, BGN was decreased in both the conditioned medium and cell layer, whereas DCN was stimulated in the majority of cultures. Northern blot analysis indicated that steady state messenger RNA (mRNA) concentration of DCN was increased by Dex in all of the HOB cultures and in seven of eight BMSC cultures analyzed. The steady state mRNA level of BGN was decreased by Dex in both HOB and BMSC cultures. The regulation of DCN and BGN mRNA by Dex in both HOB and BMSC (when responsive) was dose dependent. Time-course analysis indicated that as little as 1 day of treatment with Dex was sufficient to decrease BGN mRNA and increase DCN mRNA (when observed) levels in BMSC; the regulation spanned a 4-week interval, during which the extracellular matrix of BMSC was mineralized. The effect of Dex on the steady state mRNA levels of DCN and BGN in HOB was also apparent after 1 day of treatment. These accumulated results suggest that Dex modulates the synthesis of small proteoglycans in both human bone marrow stromal osteoprogenitor cells and mature osteoblasts.

Biglycan↗
PubMed1994

Absence of a direct anabolic effect of 17 beta-estradiol on normal human bone marrow stromal cells.

The effects of 17 beta-estradiol on the proliferation and differentiation of cultured normal human bone marrow stromal cells were investigated. Treatment of 17 beta-estradiol at the concentration of 10(-6) approximately 10(-10) M for either 48 hours or 7 days did not affect [3H] thymidine incorporation. 17 beta-estradiol (10(-8)M) treatment for 4 or 7 days also failed to stimulate alkaline phosphatase activity. Similarly, incubation with 17 beta-estradiol (10(-8) M) for 48 hours did not increase the incorporation of [3H] proline into collagenase digestible protein and noncollagen proteins and secretion of IGF-I and IGF binding proteins in human bone marrow stromal cells. Present data indicate that 17 beta-estradiol does not have a direct effect on cultured normal human bone marrow stromal cells. With previous findings that estradiol elicits few effects on normal human osteoblasts, our results strongly suggest that estrogen does not have a direct anabolic effect on normal human osteoblast lineage. Therefore, the in vivo estrogen effects may be entirely through an antiresorptive mechanism or, if any anabolic role of estrogen is present, it must be indirect and mediated by other hormones or local factors.

Bone Marrow↗
PubMed1993

A comparison of the strength and stability of six techniques of cerclage wire fixation for fractures.

An in vitro comparison of six methods of cerclage wire fixation was performed using 1-mm-diameter stainless steel wire. A testing apparatus was designed to simulate the tensile forces on a cerclage wire around a femoral shaft fracture. The hairpin cerclage knot that we describe generated significantly higher initial compressive force (p < 0.001) and had significantly higher tensile strength (p < 0.02) compared with the other five methods.

Bone Wires↗
PubMed1993

Treponema denticola (ex Brumpt 1925) sp. nov., nom. rev., and identification of new spirochete isolates from periodontal pockets.

Standard growth and isolation methods were used to obtain five new treponema strains in pure culture from deep periodontal pockets. The strains were identified to the species level by various methods, including agglutination, immunofluorescence, a dot blot immunoassay, electron microscopy, gas-liquid chromatography of metabolic volatile fatty acids, and DNA hybridization. Two isolates were strains of Treponema socranskii; the other three were strains of "Treponema denticola," a species described in 1925 by Brumpt. Because no type strain was designated for this species previously, the name was not included on the Approved Lists of Bacterial Names and has no current nomenclatural standing. We propose that Treponema denticola (ex Brumpt) sp. nov., nom. rev. is a valid and distinct species of the genus Treponema and designate strain ATCC 35405 as the type strain and strains ATCC 33520 and ATCC 35404 as reference strains. T. denticola appears to be the species that is most frequently isolated from periodontal pockets. Unless new isolation and cultivation techniques are introduced, it appears that present technology can yield only isolates belonging to the currently described oral anaerobic spirochete species and that there is little chance of isolating the larger treponemes.

Agglutination Tests↗
PubMed1992

Regulation of ornithine decarboxylase by parathyroid hormone in osteoblastic cell systems.

Parathyroid hormone (PTH) has been shown to induce osteoblastic activity via a complex signal transduction process which is mediated either by cAMP or cytosolic calcium ([Ca2+]i), or a combination thereof. One of the PTH functions in osteoblasts is the induction of ornithine decarboxylase (ODC) activity. We have analyzed the second messengers involved in this process. 8-Bromo cAMP, a cAMP derivative, enhanced ODC activity in UMR106-01 osteoblastic cell system. The calcium ionophore A23187 and the protein kinase stimulator phorbol-12-myristate 13-acetate did not alter ODC activity. ODC activity was increased by bPTH-(1-34), PGE1, and PGE2 which stimulated both cAMP and [Ca2+]i. In contrast, PTH-(2-34), propionyl bPTH-(2-34), bPTH-(3-34), bPTH-(7-34), and PGF2 alpha, which only enhanced [Ca2+]i but not cAMP, had no effect on ODC activity. Thus, the stimulation of ODC in UMR106 cells by PTH appeared to be mediated primarily via the cAMP signal transduction pathway, and the mere increase in intracellular calcium could not account for the stimulation of ODC activity. ODC mRNA level was found to be increased by PTH treatment. Therefore, translation of ODC may be stimulated by PTH. Moreover, PTH also stimulated ODC antizyme activity, suggesting that the ODC degradation rate was increased.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

Structure-function relationship of parathyroid hormone: activation of phospholipase-C, protein kinase-A and -C in osteosarcoma cells.

Recent evidence indicates that after PTh interaction with its receptor, both protein kinase-A (PKA) and protein kinase-C (PKC) are activated. To investigate the relationship between PTH structure and protein kinase stimulation, we have analyzed the effects of synthetic PTH fragments on PKA and PKC in the rat osteogenic sarcoma cells, UMR 106-01. Activation of PKA by 10(-7) M bovine (b) PTH-(1-34) was maximal (2.7-fold of control) at 5 min and remained elevated 15 min after hormone exposure. bPTH-(2-34), at equimolar doses, also stimulated PKA, but with a lower potency (1.4-fold of control), whereas propionyl bPTH-(2-34) [pbPTH-(2-34)], bPTH-(3-34), [Tyr34]bPTH-(7-34) amide [bPTH-(7-34)], and bPTH-(30-34) were ineffective. On the other hand, translocation of PKC activity from the cytosol to the membrane after exposure to bPTH-(1-34) was transient, with a peak at 1 min (1.9-fold of control), and returned to basal levels after 5 min. Other fragments, bPTH-(2-34), pbPTH-(2-34), bPTH-(3-34), and bPTH-(7-34), were also active on PKC, with relative potencies of 81%, 67%, 62%, and 51% of bPTH-(1-34), respectively, whereas bPTH-(30-34) was inactive. bPTH-(1-34), bPTH-(2-34), pbPTH-(2-34), and bPTH-(3-34) also induced inositol 1,4,5-trisphosphate production, with a potency order of 1.6-, 1.6-, 1.5-, and 1.6-fold over the control value, respectively, thus indicating activation of phospholipase-C. Neither bPTH-(7-34) nor bPTH-(30-34) caused a statistically significant increase in inositol 1,4,5-trisphosphate production. These results demonstrate that PTH signal transduction through the two different pathways can be dissociated; while activation of the cAMP/PKA system requires amino acids 1 and 2, the phospholipase-C/PKC system is coupled to a longer domain of the hormone's N-terminus.

Animals↗
PubMed1991

Regulation of plasminogen activator and plasminogen activator inhibitor production by growth factors and cytokines in rat calvarial cells.

Fetal rat osteoblast-enriched calvarial cells were used to study the effects of various growth factors and cytokines on plasminogen activator (PA) and plasminogen activator inhibitor (PAI) activities and the possible relationship of these effects to bone resorption. Confluent cultures were exposed to various factors under serum-free conditions, and levels of PA and PAI activities were examined in both conditioned medium (CM) and cell layer using the 125I-fibrin plate assay, fibrin zymogram, and reverse fibrin zymogram. According to the 125I-fibrin plate assay or zymogram, incubation of cells with acidic fibroblast growth factor (aFGF), basic FGF (bFGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) elevated the PA activity in the CM as well as in the cell layer extract. Incubation with interleukin 1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha), and insulin-like growth factor I (IGF-I) produced no change in PA activity in either CM or cell layer. Addition of transforming growth factor beta (TGF beta) to calvarial cells resulted in nearly undetectable PA activity in CM with the fibrin plate assay but increased PA activity on the fibrin zymogram after PAI was separated from PA by SDS-PAGE. A reverse fibrin zymogram indicated that PAI activity was greatly enhanced in TGF beta-treated CM. TGF beta treatment also increased PA activity in the cell layer of calvarial cells. Treatment of calvarial cells with bFGF and PDGF slightly increased PAI secretion into medium. This increase, however, was not as dramatic as the increase of PA induced by these two agents. IL-1 alpha and TNF alpha did not change PAI concentration in CM. No detectable PAI activity was found in the cell layer in control and treated groups. The PA found in the CM and cell layer of rat calvarial cells was the urokinase type; the PAI stimulated by TGF beta was the endothelial cell type, PAI-1. The regulation of PA activity by growth factors and cytokines did not correlate with their resorption-stimulating activities. Thus, PA secreted by osteoblasts may not be the only factor involved in the initiation of bone resorption. Delineation of the function of PA and PAI in the physiology of bone tissue awaits further studies.

Animals↗
PubMed1991

Dissociation of second messenger activation by parathyroid hormone fragments in osteosarcoma cells.

PTH activates multiple second messengers in its target cells, but the level at which the hormonal signal splits into different pathways is still unknown. To achieve insights on this issue, we have studied the structure-function relationship of PTH by analyzing the effects of bovine PTH-(1-34) [bPTH-(1-34)] and PTH fragments truncated at the N-terminus on the intracellular calcium concentration [( Ca2+]i) and cAMP production in the rat osteogenic sarcoma cell line UMR 106-01. [Ca2+]i was measured in single cells using fura-2. When exposed to 10(-7) M bPTH-(1-34), 20% of the cells responded with a transient increase in [Ca2+]i of variable amplitude. Equimolar doses of bPTH-(2-34), propionyl bPTH-(2-34) [(pbPTH-(2-34)], and bPTH-(3-34) also transiently increased [Ca2+]i, whereas both [tyrosine34]bPTH-(7-34) amide [bPTH-(7-34)] and bPTH-(30-34) were ineffective. The amplitude of the [Ca2+] i transients was dose-dependent, with threshold concentrations of 10(-10) M for bPTH-(1-34) and bPTH-(2-34), and 10(-9) M for bPTH-(3-34). The response rate to the active peptides ranged between 10-30%, without a clear dose-relatedness. A second addition of 10(-7) M bPTH-(1-34) to cells prestimulated with equimolar doses of bPTH-(2-34), pbPTH-(2-34), or bPTH-(3-34) produced another transient, whereas after exposure to 10(-7) M bPTH-(1-34), the cells were completely desensitized to a second homologous stimulation, suggesting that the binding affinity of the truncated peptides for the PTH receptor is lower than that of the intact bPTH-(1-34) fragment. In addition, both bPTH-(1-34) and bPTH-(2-34) dose-dependently stimulated cAMP production, but the former was more potent (ED50 = 10(-9) vs. 10(-7) M, respectively). On the contrary, pbPTH-(2-34), bPTH-(3-34), and bPTH-(7-34) had no effect on cAMP. Pretreating the cells with pertussis toxin to enhance cAMP responses via inhibition of Gi potentiated the effect of bPTH-(1-34) and bPTH-(2-34) and disclosed weak but detectable agonist action of pbPTH-(2-34). These results indicate that specific domains of the PTH molecule are linked to activation of different second messenger pathways; while the first two amino acids are indispensable for activating the cAMP system, generation of the [Ca2+]i signal appears to involve a longer domain, including the amino acid residue in position 3.

Animals↗
PubMed1990

Characterization and hormonal modulation of IL-1 binding in neonatal mouse osteoblastlike cells.

Considerable evidence indicates that interleukin-1 (IL-1) is a potent regulator of bone cell activity. Consequently, we studied its binding to neonatal mouse osteoblastlike cells. Purified, labeled recombinant IL-1 alpha bound specifically to neonatal mouse osteoblastlike cells with a dissociation constant of 30-200 pM at 22 degrees C. There were 3000-15,000 receptors per cell. IL-1 bound to cell surfaces at 4 degrees C was rapidly internalized when the temperature was raised to 37 degrees C. Receptor specificity was confirmed by demonstrating that, among a series of 11 polypeptides, only IL-1 inhibited 125I-IL-1 binding. Treatment of surface-bound 125I-IL-1 alpha with a bivalent water-soluble cross-linker identified a membrane peptide of Mr 70,000 cross-linked to IL-1. The apparent IL-1 receptor was solubilized from a plasma membrane-enriched fraction using 3-[(3-cholamidopropyldimethylammonio]-1-propanesulfonate (CHAP). The resulting material exhibited specific IL-1 binding. Preincubation of cells with IL-1, retinoic acid, transforming growth factor beta (TGF-beta), or phorbol ester caused a reduction in apparent receptor numbers per cell, while preincubation with epidermal growth factor (EGF), dexamethasone, or parathyroid hormone (PTH) increased receptor numbers per cell. Preincubation with insulin, vitamin D, prostaglandin E2 (PGE2), interferon-gamma (IFN-gamma), and 17 beta-estradiol had no effect. These results suggest that specific, high-affinity IL-1 receptors are present on osteoblastlike cells and that the receptor number can be modified by various osteotropic agents. Regulation of bone cell IL-1 receptors may contribute to the control of bone remodeling.

Animals↗
PubMed1989

In vitro susceptibilities of oral bacterial isolates to spiramycin.

Four hundred strains of oral bacteria were tested for their susceptibility to spiramycin. Actinobacillus actinomycetemcomitans and most species of Lactobacillus were resistant to the antibiotic. All strains of cariogenic Streptococcus mutans and most strains of bacterial species implicated in adult chronic periodontitis (Bacteroides gingivalis, B. intermedius, and Treponema denticola) were susceptible to spiramycin.

Bacteria↗
PubMed1989

Monokines produced by macrophages stimulate the growth of osteoblasts.

We have previously reported that the J774A.1 macrophage-like tumor cell line produces two potent monokines which stimulate the growth of osteoblasts and chondrocytes. These growth factors, which have an affinity for heparin-agarose, have been termed HEP I (a 30 Kd PDGF-like molecule) and HEP II (an approximately 20 Kd molecule), respectively, based on their elution profile. Unlike HEP I, HEP II does not stimulate the growth of fibroblasts. Extensive biological and chromatographic studies disclosed that HEP II appears to be a unique bone cell mitogen unlike any known growth factor, including the FGFs, IL-1s, and TNFs, EGF, IGF-I and -II, TGF-beta, beta 2 microglobulin, G-CSF, CSF-1 and GM-CSF. To characterize more fully the effects of the macrophage-derived monokines on osteoblast growth and function, clones were derived from calvaria explant cultures. Two clones, SDFRC-2.05 and SDFRC-3, were developed and found to exhibit osteoblastic characteristics, including high levels of alkaline phosphatase, synthesis of type I but not type III collagen, and an increased intracellular cAMP production in response to PTH. The SDFRC-3 cells exhibited a polygonal morphology like that of the explant-derived cells while SDFRC-2.05 cells exhibited a more fibroblastic morphology. When tested on the explant cultures and clones, HEP I and HEP II were found to stimulate DNA synthesis and increase protein per culture, but decreased alkaline phosphatase activity. Clone SDFRC-3 was found to be more responsive to HEP II than clone SDFRC-2.05. Both monokines were found to be more potent mitogens for bone cells than TGF-beta. HEP II, but not HEP I or TGF-beta, induced a transformation of bone cells from a polygonal to a fibroblastic morphology, suggesting the induction of migration prior to proliferation. Thus, macrophages may be responsible not only for bone repair but also for ensuring the linkage of bone formation to resorption during physiological remodeling.

Animals↗
PubMed1987

J774A.1 macrophage cell line produces PDGF-like and non-PDGF-like growth factors for bone cells.

In light of evidence that macrophages participate in the local regulation of bone remodeling, we have examined the production of peptide stimulators of bone cell growth and specialization by the J774A.1 macrophage cell line. Cultured J774A.1 cells secrete growth-promoting activities which have an affinity for heparin. The first partially purified material, termed HEP I, appears to contain platelet-derived growth factor (PDGF)-like activity. It has a molecular weight of about 30,000 daltons, inhibits the binding of labeled PDGF to its receptors, reacts with polyclonal anti-human PDGF antibody, and exhibits mitogenic activity for osteoblasts, which is partially blocked by anti-PDGF antisera. Like PDGF, HEP I is active in a wide variety of mesenchyme-derived cells, including osteoblasts, chondrocytes, smooth muscle cells, fibroblasts, 3T3 cells and NRK cells. The J774A.1 cells contain mRNA, which hybridizes to a v-sis DNA probe, suggesting that they express the c-sis gene, which contains the code for a PDGF-like protein. The second factor, HEP II, has an approximate molecular weight of 20,000 daltons and possesses substantial mitogenic activity for osteoblasts, chondrocytes, and smooth muscle cells, but is not mitogenic for fibroblasts, 3T3 cells, and NRK cells. HEP II appears to be a unique bone cell mitogen, which is distinct from the growth factors presently known. Neither HEP I nor HEP II contained interleukin 1, a macrophage product known to promote bone resorption and perhaps the growth and activity of osteoblasts.

Animals↗
PubMed1986

Comparison of rabbit epididymal androgen binding protein and serum testosterone estradiol binding globulin--II. Immunological properties.

Purified rabbit epididymal androgen binding protein and serum testosterone estradiol binding globulin have been immunologically compared. A comparison using the steady state gel method of Ritzen et al. indicated immunological cross-reactivity. In order to further compare their immunological properties we developed a radioimmunoassay for both rbABP and rbTeBG using specific antisera directed against each. When these assays were compared, the extent or completeness of displacement proved to be the only parameter that was significantly different. This data obtained with homologous and heterologous radioimmunoassays is consistent with the idea that these two proteins contain minor antigenic determinants which are distinct.

Androgen-Binding Protein↗