Medicine (St Vincent's) - Research Publications
Permanent URI for this collection
Search Results
1 - 10 of 27
-
ItemNo Preview AvailableImpaired cardiac contractility response to hemodynamic stress in S100A1-deficient mice(AMER SOC MICROBIOLOGY, 2002-04)Ca(2+) signaling plays a central role in cardiac contractility and adaptation to increased hemodynamic demand. We have generated mice with a targeted deletion of the S100A1 gene coding for the major cardiac isoform of the large multigenic S100 family of EF hand Ca(2+)-binding proteins. S100A1(-/-) mice have normal cardiac function under baseline conditions but have significantly reduced contraction rate and relaxation rate responses to beta-adrenergic stimulation that are associated with a reduced Ca(2+) sensitivity. In S100A1(-/-) mice, basal left-ventricular contractility deteriorated following 3-week pressure overload by thoracic aorta constriction despite a normal adaptive hypertrophy. Surprisingly, heterozygotes also had an impaired response to acute beta-adrenergic stimulation but maintained normal contractility in response to chronic pressure overload that coincided with S100A1 upregulation to wild-type levels. In contrast to other genetic models with impaired cardiac contractility, loss of S100A1 did not lead to cardiac hypertrophy or dilation in aged mice. The data demonstrate that high S100A1 protein levels are essential for the cardiac reserve and adaptation to acute and chronic hemodynamic stress in vivo.
-
ItemNo Preview AvailableGeneration and analysis of Siah2 mutant mice(AMER SOC MICROBIOLOGY, 2003-12)Siah proteins function as E3 ubiquitin ligase enzymes to target the degradation of diverse protein substrates. To characterize the physiological roles of Siah2, we have generated and analyzed Siah2 mutant mice. In contrast to Siah1a knockout mice, which are growth retarded and exhibit defects in spermatogenesis, Siah2 mutant mice are fertile and largely phenotypically normal. While previous studies implicate Siah2 in the regulation of TRAF2, Vav1, OBF-1, and DCC, we find that a variety of responses mediated by these proteins are unaffected by loss of Siah2. However, we have identified an expansion of myeloid progenitor cells in the bone marrow of Siah2 mutant mice. Consistent with this, we show that Siah2 mutant bone marrow produces more osteoclasts in vitro than wild-type bone marrow. The observation that combined Siah2 and Siah1a mutation causes embryonic and neonatal lethality demonstrates that the highly homologous Siah proteins have partially overlapping functions in vivo.
-
ItemInterleukin (IL)-6 induction of osteoclast differentiation depends on IL-6 receptors expressed on osteoblastic cells but not on osteoclast progenitors.(Rockefeller University Press, 1995-11-01)We reported that interleukin (IL) 6 alone cannot induce osteoclast formation in cocultures of mouse bone marrow and osteoblastic cells, but soluble IL-6 receptor (IL-6R) strikingly triggered osteoclast formation induced by IL-6. In this study, we examined the mechanism of osteoclast formation by IL-6 and related cytokines through the interaction between osteoblastic cells and osteoclast progenitors. When dexamethasone was added to the cocultures, IL-6 could stimulate osteoclast formation without the help of soluble IL-6R. Osteoblastic cells expressed a very low level of IL-6R mRNA, whereas fresh mouse spleen and bone marrow cells, both of which are considered to be osteoclast progenitors, constitutively expressed relatively high levels of IL-6R mRNA. Treatment of osteoblastic cells with dexamethasone induced a marked increase in the expression of IL-6R mRNA. By immunoblotting with antiphosphotyrosine antibody, IL-6 did not tyrosine-phosphorylate a protein with a molecular mass of 130 kD in osteoblastic cells but did so in dexamethasone-pretreated osteoblastic cells. Osteoblastic cells from transgenic mice constitutively expressing human IL-6R could support osteoclast development in the presence of human IL-6 alone in cocultures with normal spleen cells. In contrast, osteoclast progenitors in spleen cells from transgenic mice overexpressing human IL-6R were not able to differentiate into osteoclasts in response to IL-6 in cocultures with normal osteoblastic cells. These results clearly indicate that the ability of IL-6 to induce osteoclast differentiation depends on signal transduction mediated by IL-6R expressed on osteoblastic cells but not on osteoclast progenitors.
-
ItemSecretion of prostaglandins as bone-resorbing agents by renal cortical carcinoma in culture.(Springer Science and Business Media LLC, 1977-11)Fragments of human renal carcinoma tissue have been co-cultured with mouse calvaria. In 9/13 cases significant bone resorption occurred whilst in no case did control kidney cause significant resorption. When bone resorption did occur, it could be reduced by inclusion of indomethacin in the culture medium. In some cases when theophylline was included in culture medium to prevent cyclic AMP breakdown, there was enhancement of tumour-induced bone resorption. Control studies without tumour showed that none of the experimental treatments had a direct effect on bone. Radioimmunoassay of prostaglandin E (PGE) levels in pooled culture media showed that tumour fragments produced appreciable amounts of PGE, and that this production was lowered by indomethacin and increased by theophylline. It is concluded that the bone resorption induced by these tumours is due to a prostaglandin, and that prostaglandin production may be controlled by changes in cyclic AMP metabolism.
-
ItemAlternative promoter usage and mRNA splicing pathways for parathyroid hormone-related protein in normal tissues and tumours.(Springer Science and Business Media LLC, 1995-09)The parathyroid hormone-related protein (PTHrP) gene consists of nine exons and allows the production of multiple PTHrP mRNA species via the use of three promoters and 5' and 3' alternative splicing; as a result of 3' alternative splicing one of three protein isoforms may be produced. This organisation has potential for tissue-specific splicing patterns. We examined PTHrP mRNA expression and splicing patterns in a series of tumours and normal tissues, using the sensitive reverse transcription-polymerase chain reaction (RT-PCR) technique. Use of promoter 3 and mRNA specifying the 141 amino acid PTHrP isoform were detected in all samples. Transcripts encoding the 139 amino acid isoform were detected in all but two samples. Use of promoters 1 and 2 was less widespread as was detection of mRNA encoding the 173 amino acid isoform. While different PTHrP splicing patterns were observed between tumours, no tissue- or tumour-specific transcripts were detected. In comparing normal and tumour tissue from the same patient, an increase in the number of promoters utilised was observed in the tumour tissue. Furthermore, mRNA for the PTH/PTHrP receptor was detected in all samples, thus the PTHrP produced by these tumours may potentially act in an autocrine or paracrine fashion.
-
ItemTumor necrosis factor alpha stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL-RANK interaction.(Rockefeller University Press, 2000-01-17)Osteoclast differentiation factor (ODF, also called RANKL/TRANCE/OPGL) stimulates the differentiation of osteoclast progenitors of the monocyte/macrophage lineage into osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF, also called CSF-1). When mouse bone marrow cells were cultured with M-CSF, M-CSF-dependent bone marrow macrophages (M-BMM phi) appeared within 3 d. Tartrate-resistant acid phosphatase-positive osteoclasts were also formed when M-BMM phi were further cultured for 3 d with mouse tumor necrosis factor alpha (TNF-alpha) in the presence of M-CSF. Osteoclast formation induced by TNF-alpha was inhibited by the addition of respective antibodies against TNF receptor 1 (TNFR1) or TNFR2, but not by osteoclastogenesis inhibitory factor (OCIF, also called OPG, a decoy receptor of ODF/RANKL), nor the Fab fragment of anti-RANK (ODF/RANKL receptor) antibody. Experiments using M-BMM phi prepared from TNFR1- or TNFR2-deficient mice showed that both TNFR1- and TNFR2-induced signals were important for osteoclast formation induced by TNF-alpha. Osteoclasts induced by TNF-alpha formed resorption pits on dentine slices only in the presence of IL-1alpha. These results demonstrate that TNF-alpha stimulates osteoclast differentiation in the presence of M-CSF through a mechanism independent of the ODF/RANKL-RANK system. TNF-alpha together with IL-1alpha may play an important role in bone resorption of inflammatory bone diseases.
-
ItemCalcitonin-responsive adenylate cyclase in a calcitonin-producing human cancer cell line.(Springer Science and Business Media LLC, 1977-06)A calcitonin-responsive adenylate cyclase has been found in a cell line of a poorly differentiated bronchial carcinoma (BEN cells). The cells have previously been shown to secrete an immunoreactive form of calcitonin in culture. Salmon calcitonin (SCT), porcine calcitonin (PCT) and human calcitonin (CT-M) all stimulated adenylate cyclase activity in particulate preparations. CT-M sulphoxide had little effect. The concentrations of the calcitonins required for half the maximum activation of adenylate cyclase were 6-8, 18 and 90 nm respectively. SCT (30pm) and CT-M (60 pm) increased the intracellular concentration of cyclic AMP from 11-2+/-0-2 (s.e.) to 18-2+/-0-2 and 16-7+/-0-2 respectively over a 2-5-min period. SCT (labelled with 125I) bound to particulate preparations of Ben cells, and competition for binding occurred with unlabelled SCT and CT-M. The concentration of SCT required for half the maximum inhibition of [125I]SCT binding was 11 nm. CT-M sulphoxide inhibited only at high concentration (3 micron). The characteristics of the adenylate cyclase response to SCT did not change over the period between cell adhesion (after subculture) and confluence. However, pre-incubation of cells for 4 h with SCT (150 nm) abolished the subsequent adenylate cyclase response of particulate preparations to further hormone. The practical difficulties encountered in purifying and quantifying the large-mol.-wt. form of CT-M secreted by BEN cells has precluded direct investigation of the potential relationship between hormone secretion and the occurrence of the calcitonin receptor. This relationship is discussed in terms of its possible biological significance.
-
ItemLevels of expression of the mdr1 gene and glutathione S-transferase genes 2 and 3 and response to chemotherapy in multiple myeloma.(Springer Science and Business Media LLC, 1992-03)We have quantitated the levels of mRNAs in bone marrow samples from patients with multiple myeloma of the mdr1 gene (responsible for the Multidrug Resistance phenotype) and for two of the glutathione S-transferase gene, GST-2 and GST-3 (which can also inactivate a wide variety of cytotoxic drugs) and examined the relationship between the levels of expression of these genes and response to subsequent chemotherapy. From a total of 47 patients, 37 were treated with chemotherapy with 34 evaluable for response. Twenty-nine of the patients treated had not received any treatment prior to the marrow sampling while eight had previously received chemotherapy. Patients who failed to respond to initial chemotherapy had significantly higher levels of mdr1 than patients who responded (P = 0.01). In the total myeloma patient data set, mRNA levels for mdr1 and GST-2 were significantly correlated (Spearman rank correlation coefficient (r) = 0.54, P = 0.0004) as were expression levels of GST-2 with GST-3 (r = 0.43, P = 0.017). GST-3 and mdr1 levels were more weekly associated (r = 0.16, P = 0.4). These data would suggest a significant relationship between failure of chemotherapy in multiple myeloma patients and increases in expression of the mdr1 gene together with other genes whose products will generate additional mechanisms of resistance to chemotherapeutic agents.
-
ItemThe earliest step in B lineage differentiation from common lymphoid progenitors is critically dependent upon interleukin 7.(Rockefeller University Press, 2002-09-02)Little is known about the signals that promote early B lineage differentiation from common lymphoid progenitors (CLPs). Using a stromal-free culture system, we show that interleukin (IL)-7 is sufficient to promote the in vitro differentiation of CLPs into B220(+) CD19(+) B lineage progenitors. Consistent with current models of early B cell development, surface expression of B220 was initiated before CD19 and was accompanied by the loss of T lineage potential. To address whether IL-7 receptor (R) activity is essential for early B lineage development in vivo, we examined the frequencies of CLPs and downstream pre-pro- and pro-B cells in adult mice lacking either the alpha chain or the common gamma chain (gamma(c)) of the IL-7R. The data indicate that although gamma(c)(-/-) mice have normal frequencies of CLPs, both gamma(c)(-/-) and IL-7R(alpha)(-/-) mice lack detectable numbers of all downstream early B lineage precursors, including pre-pro-B cells. These findings challenge previous notions regarding the point in B cell development affected by the loss of IL-7R signaling and suggest that IL-7 plays a key and requisite role during the earliest phases of B cell development.
-
ItemLack of expression of Gp-130 makes pancreatic beta cell lines unresponsive to the IL-6 family of cytokines.(Hindawi Limited, 2001)Cytokine receptors from the IL-6 receptor family are comprised of ligand specific alpha chains and a common signalling chain, gp-130, which is also required for high affinity binding. A cDNA library generated from the beta-TC3 SV40 T-antigen transformed insulinoma cell line was screened for members of this receptor family potentially relevant to both beta cell development and autoimmunity. Degenerate oligonucleotide primers to a consensus region of these receptors were used and the IL-11 receptor alpha chain was identified. Despite confirmation of IL-11 receptor mRNA expression, iodinated bioactive IL-11 did not bind specifically to beta-TC3 cells and gp-130-dependent cytokines did not elicit signalling events in beta cell lines. This was explained by absence of gp-130 protein or mRNA in the beta cell lines tested and in primary islets. We conclude from these results that the previously recognised effects of IL-6 family member cytokines on pancreatic islets must be indirect via other non-beta cells within the islet, rather than due to direct effects on beta cells themselves.