Biomedical subjects

C Du

Publications and source records attributed to C Du.

At least 37 records · Page 2Linked to original sources

PubMed2001

Rhod-2 based measurements of intracellular calcium in the perfused mouse heart: cellular and subcellular localization and response to positive inotropy.

We have demonstrated a method of measuring intracellular calcium in the perfused mouse heart with the red fluorescent dye rhod-2. In Langendorff perfused isolated mouse hearts, rhod-2 is bolused through the perfusate, resulting in a 6.2+/-1.9-fold increase in fluorescence over background, and calcium transients with a transient amplitude to diastolic fluorescence ratio of 33+/-9%. Quantification of the relative amount of rhod-2 in the heart was done by taking the ratio of absorbance at 524 nm (rhod-2 sensitive) to 589 nm (rhod-2 insensitive). Maximal calcium saturated fluorescence was measured during tetanization of the heart with calcium chloride (20 mM) and cyclopiazonic acid (10 microM). Electron microscopy was used to determine the subcellular localization of rhod-2, by fixing rhod-2 in the heart with a carbodiimide compound, and then using a double antibody technique to stain rhod-2. These images demonstrated prominent cytosolic rhod-2 localization. Fluorescence and confocal fluorescence microscopy were consistent with the electron microscopy data. Endothelial cell uptake of rhod-2 was shown with fluorescence microscopy, though functional studies with bradykinin infusion (3 microM), which increases endothelial cell calcium, had no effects on mean fluorescence (N=4, p=NS), suggesting that endothelial uptake was small relative to total fluorescence. Calculated values of intracellular calcium were 686+/-237 nM at peak systole, and 360+/-101 nM in diastole, and with high perfusate calcium (3.5 mM) were 1199+/-215 and 544+/-53 nM, respectively. Thus, this appears a valid method of measuring cytosolic calcium in the perfused mouse heart, which will help determine the mechanisms of altered contractility in genetically engineered mice.

Animals↗

PubMed2001

The elongation domain of ELL is dispensable but its ELL-associated factor 1 interaction domain is essential for MLL-ELL-induced leukemogenesis.

The MLL-ELL chimeric gene is the product of the (11;19)(q23p13.1) translocation associated with de novo and therapy-related acute myeloid leukemias (AML). ELL is an RNA polymerase II elongation factor that interacts with the recently identified EAF1 (ELL associated factor 1) protein. EAF1 contains a limited region of homology with the transcriptional activation domains of three other genes fused to MLL in leukemias, AF4, LAF4, and AF5q31. Using an in vitro transformation assay of retrovirally transduced myeloid progenitors, we conducted a structure-function analysis of MLL-ELL. Whereas the elongation domain of ELL was dispensable, the EAF1 interaction domain of ELL was critical to the immortalizing properties of MLL-ELL in vitro. To confirm these results in vivo, we transplanted mice with bone marrow transduced with MLL fused to the minimal EAF1 interaction domain of ELL. These mice all developed AML, with a longer latency than mice transplanted with the wild-type MLL-ELL fusion. Based on these results, we generated a heterologous MLL-EAF1 fusion gene and analyzed its transforming potential. Strikingly, we found that MLL-EAF1 immortalized myeloid progenitors in the same manner as that of MLL-ELL. Furthermore, transplantation of bone marrow transduced with MLL-EAF1 induced AML with a shorter latency than mice transplanted with the MLL-ELL fusion. Taken together, these results indicate that the leukemic activity of MLL-ELL requires the EAF1 interaction domain of ELL, suggesting that the recruitment by MLL of a transactivation domain similar to that in EAF1 or the AF4/LAF4/AF5q31 family may be a critical common feature of multiple 11q23 translocations. In addition, these studies support a critical role for MLL partner genes and their protein-protein interactions in 11q23 leukemogenesis.

Amino Acid Sequence↗

PubMed2001

Ischemic dysfunction in transgenic mice expressing troponin I lacking protein kinase C phosphorylation sites.

To determine the in vivo functional significance of troponin I (TnI) protein kinase C (PKC) phosphorylation sites, we created a transgenic mouse expressing mutant TnI, in which PKC phosphorylation sites at serines-43 and -45 were replaced by alanine. When we used high-perfusate calcium as a PKC activator, developed pressures in transgenic (TG) perfused hearts were similar to wild-type (WT) hearts (P = not significant, NS), though there was a 35% and 32% decrease in peak-systolic intracellular calcium (P < 0.01) and diastolic calcium (P < 0.005), respectively. The calcium transient duration was prolonged in the TG mice also (12-27%, ANOVA, P < 0.01). During global ischemia, TG hearts developed ischemic contracture to a greater extent than WT hearts (41 +/- 18 vs. 69 +/- 10 mmHg, perfusate calcium 3.5 mM, P < 0.01). In conclusion, expression of mutant TnI lacking PKC phosphorylation sites results in a marked alteration in the calcium-pressure relationship, and thus susceptibility to ischemic contracture. The reduced intracellular calcium and prolonged calcium transients suggests that a potent feedback mechanism exists between the myofilament and the processes controlling calcium homeostasis.

Alanine↗

PubMed2001

Compensatory changes in Ca(2+) and myocardial O(2) consumption in beta-tropomyosin transgenic hearts.

Transgenic mice overexpressing beta-tropomyosin have increased myofilament Ca(2+) sensitivity that we hypothesized would result in altered relationships among pressure and heart rates, intracellular Ca(2+), and myocardial O(2) consumption. In perfused hearts from transgenic mice there was a marked negative force-frequency response between 6 and 10 Hz with a 30 +/- 3% reduction in peak-positive first derivative of pressure development over time (dP/dt) compared with 14 +/- 2% in wild-type mice (P < 0.001). At 8 Hz systolic pressures were normal, though peak systolic intracellular Ca(2+) was significantly reduced in transgenic mice versus wild type (726 +/- 61 vs. 936 +/- 67 nM, P < 0.05) indicating an alteration in the pressure-Ca(2+) relationship. Over a wide range of positive and negative inotropic interventions there were normal developed pressures, though marked elevations in myocardial O(2) consumption (15-54%). Because pressures are normal and intracellular Ca(2+) decreased and myocardial O(2) consumption increased, this suggests that these abnormalities are at least in part compensatory mechanisms to the altered myofilament function.

Animals↗

PubMed2001

Differential modulation of nicotinic acetylcholine receptor subtypes and synaptic transmission in chick sympathetic ganglia by PGE(2).

The diversity of neuronal nicotinic acetylcholine receptors (nAChRs) is likely an important factor in the modulation of synaptic transmission by acetylcholine and nicotine. We have tested whether postsynaptic nAChRs are modulated in a subtype-specific manner by prostaglandin E(2) (PGE(2)), a regulator of neuronal excitability in both the central and peripheral nervous systems, and examined the effects of PGE(2) on nicotinic transmission. Somatodendritic nAChRs in chick lumbar sympathetic ganglia include four nAChR subtypes distinguished on the basis of conductance and kinetic profile. Nanomolar PGE(2) applied to the extrapatch membrane differentially regulates opening probability (Po), frequency and the opening duration of each nAChR channel subtype in cell-attached patches. PGE(2) decreases the Po of the predominant nAChR subtype (36 pS) and significantly increases Po and open duration of the 23 pS subtype. The 23 pS subtype is gated by the alpha 7-selective agonist choline, and choline-gated currents are inhibited by alpha-bungarotoxin. To examine whether PGE(2) modulates nAChRs at synaptic sites, we studied the effects of PGE(2) on amplitude and decay of synaptic currents in visceral motoneuron-sympathetic neuron co-cultures. PGE(2) significantly decreases the amplitude of miniature excitatory postsynaptic currents (mEPSCs), consistent with the predominant inhibition by PGE(2) of all but the 23 pS subtype. The time constant of mEPSCs at PGE(2)-treated synapses is prolonged, which is also consistent with an increased contribution of the longer open duration of the 23 pS nAChR subtype with PGE(2) treatment. To examine the presynaptic effect of PGE(2), nanomolar nicotine was used. Nicotine induces facilitation of synaptic transmission by increasing mEPSC frequency, an action thought to involve presynaptic, alpha 7-containing nAChRs. In the presence of PGE(2), nicotine-induced synaptic facilitation persists. Thus the net effect of PGE(2) is to alter the profile of nAChRs contributing to synaptic transmission from larger conductance, briefer opening channels to smaller conductance, longer opening events. This subtype-specific modulation of nAChRs by PGE(2) may provide a mechanism for selective activation and suppression of synaptic pathways mediated by different nAChR subtype(s) at both pre- and postsynaptic sites.

Animals↗

PubMed2001

Stimulation of Th2 response by high doses of dehydroepiandrosterone in KLH-primed splenocytes.

Although dehydroepiandrosterone (DHEA) has long been considered as a precursor for steroid hormones, it has also been shown to have regulatory effects in immune homeostasis. We have examined the effect of high DHEA doses on T cell proliferation, differentiation, and cytokine secretion patterns following stimulation with mitogens and soluble antigens. DHEA profoundly inhibited T cell receptor-mediated T cell proliferation in the upstream of IL-2R signaling. Addition of DHEA to KLH-primed splenocytes stimulated Th2 response, indicated by an increase of IL-4 or a decrease of IFN-gamma production in the cultures. Further studies showed that DHEA enhanced IL-4, but inhibited IL-12-mediated T cell proliferation and IL-12 production in antigen-presenting cells (APCs). Our data demonstrated that supraphysiologic levels of DHEA favored Th2 immune responses in vitro by inhibition of IL-12 production from APCs and/or stimulation of Th2 proliferation during the interactions of T cells with APCs.

Adjuvants, Immunologic↗

PubMed2001

A novel mis-sense mutation (G1381A) in the G6PD gene identified in a Chinese man.

OBJECTIVE: To detect new mutations among 29 glucose-6-phosphate dehydrogenase (G6PD) deficient individuals from Yunnan province. METHODS: The nitroblue tetrazolium (NBT) method was used to screen G6PD deficient individuals. Mutation was identified by single strand conformation polymorphism (SSCP), amplification created restriction site (ACRS), amplification refractory mutation system (ARMS) and DNA sequencing. RESULTS: Among 29 cases, 18 cases of G1388A, 1 case of C1004A, and 1 case of G1381A were identified. Nine cases remained to be defined. The G1381A mutation is a novel mis-sense mutation, with a substitution of threonine for alanine (A461T). The resultant G6PD had reduced enzymatic activity. In addition, G1381A caused a restriction site of Stu I to disappear, providing a rapid method for the detection of this mutation. CONCLUSION: A novel mis-sense mutation G1381A was found. This mutation results in a substitution of threonine for alanine, producing enzyme with reduced activity. The loss of the Stu I restriction site offers a rapid method for the detection of this mutation.

Adult↗

PubMed2001

[Study on the dynamic changes of retinoblastoma gene of SO-Rb50 cell line].

PURPOSE: To study the dynamic changes of Retinoblastoma Gene of SO-Rb50 cell line. METHODS: 1) Southern blot hybridization was used to analyse the tumor cell DNA of 327th passage of SO-Rb50 cell line. 2) A promoter and 27 exons of Rb gene were screened exon-by-exon by using PCR-SSCP at 415th and 713th passages of SO-Rb50 cell line. 3) Three cell cloning strains named as MC2, MC3 and MC4 were isolated by single cell cloning technique from the SO-Rb50-775, and mutation of Rb gene were also screened exon-by-exon by using PCR-SSCP-HA in MC2-11, MC3-11, MC4-11 and MC3-138. RESULTS: The 3.5 Kb, 2.9 Kb and 1.0 Kb bands were deleted in the DNA of SO-Rb50-327 tumor cells, showing the deletion of Rb gene in SO-Rb50 cell line. Exon23 of 451th passage cells decreased one band; but exon 25 of 713th passage cells decreased two bands, indicating that exon 25 had new mutation. PCR-SSCP-HA analysis of exon24 showed that MC4-11 and MC3-138 had abnormal bands, but MC2-11 and MC3-11 weren't found mutation of Rb gene. This result suggested that new mutation occurred to exon24 of MC3 during a long-term culture in vitro. CONCLUSION: Retinoblastoma gene mutation of SO-Rb50 cell line had dynamic changes during a long-term culture.

Exons↗

PubMed2000

Amelioration of CR-EAE with lisofylline: effects on mRNA levels of IL-12 and IFN-gamma in the CNS.

CR-EAE is a Th1-mediated inflammatory autoimmune demyelinating disease of the CNS and serves as a model of human multiple sclerosis. Our previous studies have shown the protective effect of orally administered lisofylline in the prevention of active and passively induced acute EAE. In our present studies we have examined the efficacy and mechanism of action of lisofylline on CR-EAE. Lisofylline decreased the number and severity of paralytic attacks in mice with relapsing EAE. The reduction of clinical disease correlated with decreased levels of mRNA levels of IFN-gamma but not of mRNA levels of IL-12. These studies suggest that lisofylline may be an effective therapeutic for established Th1 mediated autoimmune disease and that it acts by blocking IL-12R signaling and not IL-12 production in vivo.

Animals↗

PubMed2000

Retrovirus-mediated gene transfer of MLL-ELL transforms primary myeloid progenitors and causes acute myeloid leukemias in mice.

The MLL-ELL fusion gene results from the translocation t(11;19)(q23;p13.1) that is associated with de novo and therapy-related acute myeloid leukemia. To study its transforming properties, we retrovirally transduced primary murine hematopoietic progenitors and assessed their growth properties both in vitro and in vivo. MLL-ELL increased the proliferation of myeloid colony-forming cells in methylcellulose cultures upon serial replating, whereas overexpression of ELL alone had no effect. We reconstituted lethally irradiated congenic mice with bone marrow progenitors transduced with MLL-ELL or the control MIE vector encoding the enhanced green fluorescent protein. When the peripheral blood of the mice was analyzed 11-13 weeks postreconstitution, we found that the engraftment of the MLL-ELL-transduced cells was superior to that of the MIE controls. At this time point, the contribution of the donor cells was normally distributed among the myeloid and nonmyeloid compartments. Although all of the MIE animals (n = 10) remained healthy for more than a year, all of the MLL-ELL mice (n = 20) succumbed to monoclonal or pauciclonal acute myeloid leukemias within 100-200 days. The leukemic cells were readily transplantable to secondary recipients and could be established as immortalized cell lines in liquid cultures. These studies demonstrate the enhancing effect of MLL-ELL on the proliferative potential of myeloid progenitors as well as its causal role in the genesis of acute myeloid leukemias.

Acute Disease↗

PubMed2000

Chromatin-related properties of CBP fused to MLL generate a myelodysplastic-like syndrome that evolves into myeloid leukemia.

As a result of the recurring translocation t(11;16) (q23;p13.3), MLL (mixed-lineage leukemia) is fused in frame to CBP (CREB binding protein). This translocation has been documented almost exclusively in cases of acute leukemia or myelodysplasia secondary to therapy with drugs that target DNA topo isomerase II. The minimal chimeric protein that is produced fuses MLL to the bromodomain, histone acetyltransferase (HAT) domain, EIA-binding domain and steroid-receptor coactivator binding domains of CBP. We show that transplantation of bone marrow retrovirally transduced with MLL-CBP induces myeloid leukemias in mice that are preceded by a long preleukemic phase similar to the myelodysplastic syndrome (MDS) seen in many t(11;16) patients but unusual for other MLL translocations. Structure-function analysis demonstrated that fusion of both the bromodomain and HAT domain of CBP to the amino portion of MLL is required for full in vitro transformation and is sufficient to induce the leukemic phenotype in vivo. This suggests that the leukemic effect of MLL-CBP results from the fusion of the chromatin association and modifying activities of CBP with the DNA binding activities of MLL.

Animals↗

PubMed2000

Structural and biochemical basis of apoptotic activation by Smac/DIABLO.

Apoptosis (programmed cell death), an essential process in the development and homeostasis of metazoans, is carried out by caspases. The mitochondrial protein Smac/DIABLO performs a critical function in apoptosis by eliminating the inhibitory effect of IAPs (inhibitor of apoptosis proteins) on caspases. Here we show that Smac/DIABLO promotes not only the proteolytic activation of procaspase-3 but also the enzymatic activity of mature caspase-3, both of which depend upon its ability to interact physically with IAPs. The crystal structure of Smac/DIABLO at 2.2 A resolution reveals that it homodimerizes through an extensive hydrophobic interface. Missense mutations inactivating this dimeric interface significantly compromise the function of Smac/DIABLO. As in the Drosophila proteins Reaper, Grim and Hid, the amino-terminal amino acids of Smac/DIABLO are indispensable for its function, and a seven-residue peptide derived from the amino terminus promotes procaspase-3 activation in vitro. These results establish an evolutionarily conserved structural and biochemical basis for the activation of apoptosis by Smac/DIABLO.

Amino Acid Sequence↗

PubMed2000

Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition.

We report here the identification of a novel protein, Smac, which promotes caspase activation in the cytochrome c/Apaf-1/caspase-9 pathway. Smac promotes caspase-9 activation by binding to inhibitor of apoptosis proteins, IAPs, and removing their inhibitory activity. Smac is normally a mitochondrial protein but is released into the cytosol when cells undergo apoptosis. Mitochondrial import and cleavage of its signal peptide are required for Smac to gain its apoptotic activity. Overexpression of Smac increases cells' sensitivity to apoptotic stimuli. Smac is the second mitochondrial protein, along with cytochrome c, that promotes apoptosis by activating caspases.

Adult↗

PubMed2000

Formation of calcium phosphate/collagen composites through mineralization of collagen matrix.

Several types of calcium phosphate/collagen composites, including noncrystalline calcium phosphate/collagen, poorly crystalline carbonate-apatite (PCCA)/collagen, and PCCA + tetracalcium phosphate/collagen composites, were prepared through the mineralization of collagen matrix. The type I collagen was presoaked with a PO(3-)(4) containing solution and then immersed in a Ca(2+) containing solution to allow mineral deposition. The solution of 0.56 M sodium dibasic phosphate (Na(2)HPO(4)) with a pH of nearly 14 was metastable and its crystallization produced Na(2)HPO(4) and sodium tripolyphosphate hexahydrate (Na(5)P(3)O(10)). 6H(2)O), leading to a controlled release of orthophosphate ions during the subsequent mineral precipitation. The development of the composites was investigated in detail. The mineral contributed up to 60-70% of the weight of the final composites. The strength and Young's modulus of the composites in tensile tests overlapped the lower range of values reported for bone. When implanted in muscle tissue, the composite showed biodegradability that was partly through a multinucleated giant cell mediated process. In a bone explant culture model it was observed that bone-derived cells deposited mineralizing collagenous matrix on the composite.

Animals↗

PubMed2000

Adult Apaf-1-deficient mice exhibit male infertility.

Release of cytochrome c from the mitochondria, and subsequent binding to apoptotic protease-activating factor-1 (Apaf-1), is a key trigger of apoptotic events. A complex composed of Apaf-1, dATP, and cytochrome c activates a series of cytoplasmic proteases called caspases, leading to apoptotic cell death. We have disrupted the Apaf-1 gene in the mouse. Like previous reports on this knockout model, we find that most Apaf-1 mutants die perinatally and frequently exhibit exencephaly and cranioschesis. We additionally find that the neural lesions that develop in the knockout are due to an excess of neural progenitor cells that manifests as early as embryonic day 9.5 in development. In contrast to previous reports on the Apaf-1 knockout mice, we find that 5% of the mutants successfully survive to adulthood. In these survivors, the brain develops normally, but in males, there is degeneration of spermatogonia resulting in the virtual absence of sperm. Thus, cytochrome c-mediated apoptosis is not absolutely required for normal neural development, but is essential for spermatogenesis. These findings strongly suggest that alternative apoptotic pathways work in conjunction with and parallel to Apaf-1 and can modify its effect on programmed cell death.

Adenosine Triphosphate↗

PubMed2000

Induction of interleukin-12/p40 by superantigens in macrophages is mediated by activation of nuclear factor-kappaB.

Multimerization of the MHC class II molecule by superantigens results in activation of cellular signal transduction pathways in macrophage and B cells. Here we show that superantigen staphylococcal enterotoxin B (SEB) induces IL-12/p40 secretion in macrophages. SEB-induced expression of the IL-12/p40 gene involves activation and nuclear translocation of nuclear factor kappaB (NF-kappaB). The NF-kappaB heterodimer bound to the NF-kappaB consensus sequence of the IL-12/p40 gene promoter is p50/C-Rel. Inhibition of PKC and PKA activation results in suppression of activation and translocation of NF-kappaB. We conclude that signals for IL-12/p40 gene transcription from MHC class II molecules follow activation of PKC and PKA, which in turn leads to the activation and translocation of NF-kappaB to the nucleus. Our study suggests that superantigens are capable of influencing the nature of the immune response by regulating cytokine production. Induction of IL-12 production by superantigens may therefore play a role in the regulation of Th 1-mediated immune response and autoimmune disease.

Animals↗

PubMed2000

DFF45/ICAD can be directly processed by granzyme B during the induction of apoptosis.

Granzyme B (GzmB) is a component of cytotoxic lymphocyte granules that can rapidly initiate apoptosis in target cells. While several procaspases are cleaved and activated by GzmB, the absolute requirement of caspase activation for GzmB-induced apoptosis is controversial. In this report, we demonstrate that GzmB can initiate apoptosis in the absence of caspase-3 activity by directly cleaving DFF45/ICAD to liberate activated DFF40/CAD. DFF45/ICAD cleavage occurs less efficiently in cells that lack caspase-3 activity, suggesting that the caspases normally amplify the GzmB death signal. DFF45/ICAD-deficient mouse embryo fibroblasts are partially resistant to GzmB-induced death, demonstrating the biological importance of DFF45/ICAD for GzmB-mediated apoptosis.

Animals↗

PubMed2000

Infection of immunosuppressed C57BL/6N adult mice with a single oocyst of Cryptosporidium parvum.

The present study was designed to determine the minimum number of Cryptosporidium parvum oocysts capable of producing patent infections in immunosuppressed C57BL/6N adult mice. Sixty-four female mice were divided into 6 groups of 8 mice each, except group 1 that contained 24 mice. Mice in groups 1-3 were immunosuppressed with dexamethasone and inoculated with 1, 5, and 10 oocysts per mouse, respectively. The accuracy of the inoculum size was microscopically confirmed. Mice in groups 4-6 served as controls: they received either only oocyst inoculation (group 4), or immunosuppression (group 5), or no treatments (group 6). Fecal oocyst shedding was monitored daily for each mouse using an indirect immunofluorescent assay. Parasite colonization in the terminal ileum of each mouse was evaluated histologically. Four of 24 mice in group 1 developed patent infections, with a prepatent period of approximately 6 days. All mice in groups 2 and 3 developed patent infections, with prepatent periods ranging from 4 to 7 days. Mice in groups 4-6 remained uninfected. Parasite colonization was observed in the terminal ilea of all mice in groups 1-3 that shed fecal oocysts. The present study experimentally demonstrates that a single viable oocyst can induce patent C. parvum infections in immunosuppressed C57BL/6N adult mice and indicates that this mouse model could be used for the parasite genotype or isolate cloning.

Animals↗