Biomedical subjects

Y Yuan

Publications and source records attributed to Y Yuan.

At least 37 records · Page 2Linked to original sources

PubMed2001

Hypoxia-inducible factor 2alpha binds to cobalt in vitro.

The hypoxia-inducible factor (HIF) activates the expression of genes that contain a hypoxia response element (HRE). The alpha subunit of the HIF transcription factors is degraded by proteasome pathways during normoxia, but stabilized under hypoxic conditions. It has previously been established that cobalt causes accumulation of HIF-2alpha and HIF-1alpha. However, little is known about the mechanism by which cobalt mimics hypoxia and stabilizes these transcription factors. We show here that cobalt binds directly to HIF-2alpha in vitro with a high affinity and in an oxygen-dependent manner. We found that HIF-2alpha, which had been stabilized with a proteasome inhibitor, could bind to cobalt, whereas hypoxia-stabilized HIF-2alpha could not. Mutations within the oxygen-dependent degradation domain of HIF-2alpha prevented cobalt binding and led to accumulation of HIF-2alpha during normoxia. This suggests that transition metal such as iron may play a role in regulation of HIF-2alpha in vivo.

Amino Acid Sequence↗

PubMed2001

Interaction with BRCA2 suggests a role for filamin-1 (hsFLNa) in DNA damage response.

The BRCA2 tumor suppressor plays significant roles in DNA damage response. The human actin binding protein filamin-1 (hsFLNa, also known as ABP-280) participates in orthogonal actin network, cellular stress responses, signal transduction, and cell migration. Through a yeast two-hybrid system, an in vitro binding assay, and in vivo co-immunoprecipitations, we identified an interaction between BRCA2 and hsFLNa. The hsFLNa binding domain of BRCA2 was mapped to an internal conserved region, and the BRCA2-interacting domain of hsFLNa was mapped to its C terminus. Although hsFLNa is known for its cytoplasmic functions in cell migration and signal transduction, some hsFLNa resides in the nucleus, raising the possibility that it participates in DNA damage response through a nuclear interaction with BRCA2. Lack of hsFLNa renders a human melanoma cell line (M2) more sensitive to several genotoxic agents including gamma irradiation, bleomycin, and ultraviolet-c light. These results suggest that BRCA2/hsFLNa interaction may serve to connect cytoskeletal signal transduction to DNA damage response pathways.

BRCA2 Protein↗

PubMed2001

AML1-ETO expression is directly involved in the development of acute myeloid leukemia in the presence of additional mutations.

The t(8;21) is one of the most frequent chromosomal abnormalities associated with acute myeloid leukemia (AML). The translocation, which involves the AML1 gene on chromosome 21 and the ETO gene on chromosome 8, generates an AML1-ETO fusion transcription factor. To examine the effect of the AML1-ETO fusion protein on leukemogenesis, we made transgenic mice in which expression of AML1-ETO is under the control of the human MRP8 promoter (hMRP8-AML1-ETO). AML1-ETO is specifically expressed in myeloid cells, including common myeloid progenitors of hMRP8-AML1-ETO transgenic mice. The transgenic mice were healthy during their life spans, suggesting that AML1-ETO alone is not sufficient for leukemogenesis. However, after treatment of newborn hMRP8-AML1-ETO transgenic mice and their wild-type littermates with a strong DNA-alkylating mutagen, N-ethyl-N-nitrosourea, 55% of transgenic mice developed AML and the other 45% of transgenic mice and all of the wild-type littermates developed acute T lymphoblastic leukemia. Our results provide direct evidence that AML1-ETO is critical for causing myeloid leukemia, but one or more additional mutations are required for leukemogenesis. The hMRP8-AML1-ETO-transgenic mice provide an excellent model that can be used to isolate additional genetic events and to further understand the molecular pathogenesis of AML1-ETO-related leukemia.

Animals↗

PubMed2001

Hypermethylation leads to silencing of the SYK gene in human breast cancer.

A number of cancer-associated genes have been shown to be inactivated by hypermethylation of CpG islands during breast tumorigenesis. SYK, a candidate tumor suppressor, has been found not expressed in a subset of breast cancer cell lines, but the mechanism by which SYK is silenced is unclear. In this study, we examined the 5' CpG island methylation status of the SYK gene in breast cancer cell lines and primary breast cancer tissues. We found SYK 5' CpG hypermethylation in 30% (6/20) of breast cancer cell lines, and the aberrant methylation status was strongly associated with loss of SYK gene expression. Treatment of cells with a methylation inhibitor, 5-aza-2'-deoxycytidine, led to a reactivation of SYK expression in SYK-negative cells, as detected by reverse transcription-PCR. Using methylation-specific PCR, we demonstrated that SYK is hypermethylated in 32% (12/37) of unselected breast tumors, whereas all of the matched neighboring normal breast tissues exhibited unmethylated DNA status. We concluded that SYK is frequently inactivated through an epigenetic pathway in breast cancer. Because SYK has been shown to function as a tumor suppressor, and its loss of expression in breast cancer has been correlated with tumor invasiveness, the aberrant SYK methylation is responsible for the loss of expression and may consequently play a permissive role for tumor aggressiveness.

Breast Neoplasms↗

PubMed2001

[Changes of somatostatin receptor of pancreas and effectiveness of octreotide on acute necrotizng pancreatitis in mice].

OBJECTIVE: Changes of somamtostatin receptor (SSTR) of pancreas, pancreatic blood flow and its relationship to metabolism of eicosanoids were investigated in order to elucidate the effectiveness of octreotide, an analogue of somatostatin, in acute necrotizng pancreatitis (ANP). METHODS: Rats with ANP were triggered with sodium taurocholate via pancreatobillary duct; SSTR was detected by radioligand binding assay (RBA) with (125)I-somatostatiin-14; in Situ hybridization was employed in analysis of SSTR2 mRNA of pancreas; pancreatic blood flow was determined with tissue blood flowmeter and metabolites of eicosanoids were analyzed with radioimmunoassay. RESULTS: The level of SSTR of pancreas was 110 +/- 58 fmol/mg.protein in normal rats. Significant decrease of SSTR was shown at 3, 6, 12 hours after onset of ANP and of the signals of SSTR2 mRNA by in situ hybridization as well. Pancreatic blood flow was reduced while thromboxin-2 was increased significantly in the course of ANP. But in the group of ANP treated with octreotide, both the decrease of pancreatic blood flow and abnormal metabolism of eicosanoids were corrected, and the pathologic damages were reduced. CONCLUSION: SSTR of pancreas is significantly reduced in ANP. Therefore, correction of abnormal metabolism of eicosanoids and improvement of pancreatic microcirculation may be the major mechanism of somatostatin analogues in treatment of ANP while inhibition of pancreatic enzymes via receptors may play a minor role.

Animals↗

PubMed2001

Cloning, expression, and characterization of a human inosine triphosphate pyrophosphatase encoded by the itpa gene.

ITP and dITP exist in all cells. dITP is potentially mutagenic, and the levels of these nucleotides are controlled by inosine triphosphate pyrophosphatase (EC ). Here we report the cloning, expression, and characterization of a 21.5-kDa human inosine triphosphate pyrophosphatase (hITPase), an enzyme whose activity has been reported in many animal tissues and studied in populations but whose protein sequence has not been determined before. At the optimal pH of 10.0, recombinant hITPase hydrolyzed ITP, dITP, and xanthosine 5'-triphosphate to their respective monophosphates whereas activity with other nucleoside triphosphates was low. K(m) values for ITP, dITP, and xanthosine 5'-triphosphate were 0.51, 0.31, and 0.57 mm, respectively, and k(cat) values were 580, 360, and 640 s(-1), respectively. A divalent cation was absolutely required for activity. The gene encoding the hITPase cDNA sequence was localized by radiation hybrid mapping to chromosome 20p in the interval D20S113-D20S97, the same interval in which the ITPA inosine triphosphatase gene was previously localized. A BLAST search revealed the existence of many similar sequences in organisms ranging from bacteria to mammals. The function of this ubiquitous protein family is proposed to be the elimination of minor potentially mutagenic or clastogenic purine nucleoside triphosphates from the cell.

Amino Acid Sequence↗

PubMed2001

Expression and permeation properties of the K(+) channel Kir7.1 in the retinal pigment epithelium.

Bovine Kir7.1 clones were obtained from a retinal pigment epithelium (RPE)-subtracted cDNA library. Human RPE cDNA library screening resulted in clones encoding full-length human Kir7.1. Northern blot analysis indicated that bovine Kir7.1 is highly expressed in the RPE. Human Kir7.1 channels were expressed in Xenopus oocytes and studied using the two-electrode voltage-clamp technique. The macroscopic Kir7.1 conductance exhibited mild inward rectification and an inverse dependence on extracellular K+ concentration ([K+]o). The selectivity sequence based on permeability ratios was K+ (1.0) approximately Rb+ (0.89) > Cs+ (0.013) > Na+ (0.003) approximately Li+ (0.001) and the sequence based on conductance ratios was Rb+ (9.5) >> K+ (1.0) > Na+ (0.458) > Cs+ (0.331) > Li+ (0.139). Non-stationary noise analysis of Rb+ currents in cell-attached patches yielded a unitary conductance for Kir7.1 of approximately 2 pS. In whole-cell recordings from freshly isolated bovine RPE cells, the predominant current was a mild inwardly rectifying K+ current that exhibited an inverse dependence of conductance on [K+]o. The selectivity sequence based on permeability ratios was K+ (1.0) approximately Rb+ (0.89) > Cs+ (0.021) > Na+ (0.003) approximately Li+ (0.002) and the sequence based on conductance ratios was Rb+ (8.9) >> K+ (1.0) > Na+ (0.59) > Cs+ (0.23) > Li+ (0.08). In cell-attached recordings with Rb+ in the pipette, inwardly rectifying currents were observed in nine of 12 patches of RPE apical membrane but in only one of 13 basolateral membrane patches. Non-stationary noise analysis of Rb+ currents in cell-attached apical membrane patches yielded a unitary conductance for RPE Kir of approximately 2 pS. On the basis of this molecular and electrophysiological evidence, we conclude that Kir7.1 channel subunits comprise the K+ conductance of the RPE apical membrane.

Animals↗

PubMed2001

A preliminary model for predicting heavy metal contaminant loading from an urban catchment.

The toxicity of heavy metals to biota in urban catchments has been regarded as a very important non-point source pollution issue. Numerous studies on heavy metal pollution in urban receiving waters have found that metal transport by surface runoff is closely correlated to the partitioning of the metal forms between dissolved and particulate phases, where sediment plays an important role in the transport process. Sediment cycling on urban streets, metal binding form, and rainfall character in the catchment area are considered to be the key factors for metal transport. A preliminary model is developed based on these considerations. Starting from classical build-up and wash-off processes for the suspended sediment (SS) on the urban impervious surface, the model links the transport of suspended sediment to the transport of metal species. Monitoring data from a small highway catchment were used in the model development. A total of 47 rain events over 1 year were monitored intensively at short time intervals (5-10 min) for hydrological data, rainfall intensity, and stormwater quality. In developing the model, lead was used for the metal load prediction, as it has been a common fuel additive for urban transportation. Agreement between model results and monitoring data indicates that the model can be used in predicting metal load from impervious urban areas, such as streets and roadways, on a long-term basis.

Animals↗

PubMed2001

Inhibition of breast and brain cancer cell growth by BCCIPalpha, an evolutionarily conserved nuclear protein that interacts with BRCA2.

BRCA2 is a tumor suppressor gene involved in mammary tumorigenesis. Although important functions have been assigned to a few conserved domains of BRCA2, little is known about the longest internal conserved domain encoded by exons 14-24. We identified a novel protein, designated BCCIPalpha, that interacts with part of the internal conserved region of human BRCA2. Human BCCIP represents a family of proteins that are evolutionarily conserved, and contain three distinct domains: an N-terminus acidic domain (NAD) of 30-60 amino acids, an internal conserved domain (ICD) of 180-220 amino acids, and a C-terminus variable domain (CVD) of 30-60 amino acids. The N-terminal half of the human BCCIP ICD shares moderate homology with regions of calmodulin and M-calpain, suggesting that BCCIP may also bind Ca. Human cells express both a longer, BCCIPalpha, and a shorter, BCCIPbeta, form of the protein, which differ in their CVD. BCCIP is a nuclear protein highly expressed in testis. Although BCCIPbeta expression is relatively consistent in cancer cells, the expression of BCCIPalpha varies in cancer cell lines. The BCCIPalpha gene is located at chromosome 10q25.3-26.2, a region frequently altered in brain and other cancers. Furthermore, expression of BCCIPalpha inhibits breast and brain cancer cell growth, but fails to inhibit HT1080 cells and a non-transformed human skin fibroblast. These results suggest that BCCIPalpha is an important cofactor for BRCA2 in tumor suppression.

Amino Acid Sequence↗

PubMed2001

Surface-renewable cobalt(II) hexacyanoferrate-modified graphite organosilicate electrode and its electrocatalytic oxidation of thiosulfate.

Cobalt(II) hexacyanoferrate (CoHCF) was deposited on graphite powder by an in situ chemical deposition procedure and then dispersed into methyltrimethoxysilane-derived gels to prepare a surface-renewable CoHCF-modified electrode. The electrochemical behavior of the modified electrode in different supporting electrolyte solutions was characterized by cyclic voltammetry. In addition, square-wave voltammetry was employed to investigate the pNa-dependent electrochemical behavior of the electrode. The CoHCF-modified electrode showed a high electrocatalytic activity toward thiosulfate oxidation and could thus be used as an amperometric thiosulfate sensor.

Journal Article↗

PubMed2001

Effects of leukaemia inhibitory factor on endometrial receptivity and its hormonal regulation in rabbits.

The effects of hormones on production of leukaemia inhibitory factor (LIF) and the uterine receptivity in rabbits were studied. In ovariectomised rabbits, LIF protein was not detected in control but upregulated by progesterone alone. Oestrogen had a slightly negative effect when the rabbits were treated with both oestrogen and progesterone. Mifepristone (Mi) inhibited the progesterone-stimulated production of LIF in rabbit uterus. The transfer of embryos to LIF-treated recipients significantly increased pregnancy rate (70%) and implantation rate (27%) as compared with control (pregnancy rate=40% and implantation rate=17%). The transfer of embryos to LIF and mifepristone-treated recipients significantly decreased pregnancy rate (30%) and implantation rate (9%). The results indicated that LIF protein had a beneficial effect on uterine receptivity and mifepristone prevented this effect.

Animal Population Groups↗

PubMed2001

Comparative study on the immunogenicity between Hsp70 DNA vaccine and Hsp65 DNA vaccine in human Mycobacterium tuberculosis.

The BALB/c mice were immunized with Hsp70 DNA and Hsp65 DNA vaccines in human Mycobacterium tuberculosis. Eight weeks after immunization, the eyeballs were removed, blood and spleen taken, and intraperitoneal macrophages were harvested. The lymphocytic stimulating index (SI) was used to measure the cellular proliferating ability and NO release to measure the phagocytic activity of the macrophages. With ELISA kit, the levels of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) in serum and the splenic lymphocytic cultured supernatant were detected. The results showed that after the mice were immunized with 100 micrograms/mouse of Hsp70 DNA vaccine intramuscularly, the splenic lymphocytic proliferating ability in the mice was significantly increased as compared with that in the control group, vector group and Hsp65 DNA vaccine group (P < 0.01); The contents of NO in the intraperitoneal macrophages of the mice were significantly lower than in the control group and Hsp65 DNA vaccine group (P < 0.01); The levels of serum IL-2 in the mice were significantly higher than in the control group, but there was no statistical difference between Hsp65 DNA group and vector group (P > 0.05); The contents of serum IFN-gamma in the mice were significantly higher than in the control group, but significantly lower than in the Hsp65 DNA vaccine group (P < 0.05). It was indicated that immunization with Hsp70 DNA vaccine could obviously enhance the immune response, but its intensity seemed inferior to Hsp65 DNA vaccine. The anti-infection mechanisms and clinical use in the future of the vaccines of Hsp70 DNA and Hsp65 DNA are worth further studying.

Animals↗

PubMed2001

Effects of DDPH on HECCM-induced proliferation and immunophenotypes of the pulmonary vascular pericytes.

In order to study the effects of 1-(2, 6-dimethylphenoxy)-2-(3, 4-dimethoxyphenylethylamino) propane hydrochloride (DDPH) on proliferation and immunophenotypes of newborn rat pulmonary vascular pericytes induced by hypoxic endothelial cell conditioned medium (HECCM) from porcine pulmonary arteries, the cultured pericytes were divided into 4 groups according to the endothelial cell conditioned medium (ECCM) used: normoxic ECCM (NECCM) group, NECCM + DDPH group, HECCM group and HECCM + DDPH group. Cell culture, immunocytochemical staining, image analysis and flow cytometric method were used to investigate the effects of HECCM and DDPH on the expression of alpha-smooth muscle actin (alpha-SM-Actin) antigen, CD34 antigen, S-100 antigen and proliferating cell nuclear antigen (PCNA) and cell cycle in pericytes. The results showed that the alpha-SM-Actin antigen in the pericytes in HECCM group was stronger positively expressed than in the other three groups, but CD34 antigen and S-100 antigen were negatively expressed. The expression of alpha-SM-Actin antigen, CD34 antigen and S-100 antigen was positive in the groups of NECCM, NECCM + DDPH and HECCM + DDPH; The expression of alpha-SM-Actin and PCNA in HECCM group was 1.32 times (P < 0.01) and 1.24 times (P < 0.05) that in NECCM group, 1.30 times (P < 0.01) and 1.21 times (P < 0.05) that in HECCM + DDPH group, respectively. The percentage of the cells in the GO-G1 phase in the HECCM group was lower by 11.7% and 9.1%, in S phase higher by 5.6% and 4.2%, in G2-M phase higher by 6.1% and 4.9% than in the groups of NECCM, HECCM + DDPH, respectively. The inhibitory rate of DDPH on the increased alpha-SM-Actin and PCNA syntheses in pericytes induced by HECCM were 23.4% and 17.1% respectively. The inhibitory rate on the increased pericytes from GO-G1 phase to S phase was 8.3%. These results suggest that DDPH can directly inhibit pericytes from proliferation and immunophenotypical transformation of smooth muscle-like cells induced by HECCM.

Animals↗

PubMed2001

The molecular basis of O2-sensing and hypoxia tolerance in pheochromocytoma cells.

Hypoxia is a common environmental stimulus. However, very little is known about the mechanisms by which cells sense and respond to changes in oxygen. Our laboratory has utilized the PC12 cell line in order to study the biophysical and molecular response to hypoxia. The current review summarizes our results. We demonstrate that the O2-sensitive K(+) channel, Kv1.2, is present in PC12 cells and plays a critical role in the hypoxia-induced depolarization of PC12 cells. Previous studies have shown that PC12 cells secrete a variety of autocrine/paracrine factors, including dopamine, norepinephrine, and adenosine during hypoxia. We investigated the mechanisms by which adenosine modulates cell function and the effect of chronic hypoxia on this modulation. Finally, we present results identifying the mitogen- and stress-activated protein kinases (MAPKs and SAPKs) as hypoxia-regulated protein kinases. Specifically, we show that p38 and an isoform, p38gamma, are activated by hypoxia. In addition, our results demonstrate that the p42/p44 MAPK protein kinases are activated by hypoxia. We further show that p42/p44 MAPK is critical for the hypoxia-induced transactivation of endothelial PAS-domain protein 1 (EPAS1), a hypoxia-inducible transcription factor. Together, these results provide greater insight into the mechanisms by which cells sense and adapt to hypoxia.

Animals↗

PubMed2001

Identification of hypoxia-responsive genes in a dopaminergic cell line by subtractive cDNA libraries and microarray analysis.

Transplantation of dopamine-secreting cells harvested from fetal mesencephalon directly into the striatum has had limited success as a therapy for Parkinson's disease. A major problem is that the majority of the cells die during the first 3 weeks following transplantation. Hypoxia in the tissue surrounding the graft is a potential cause of the cell death. We have used subtractive cDNA libraries and microarray analysis to identify the gene expression profile that regulates tolerance to hypoxia. An improved understanding of the molecular basis of hypoxia-tolerance may allow investigators to engineer cells that can survive in the hypoxic environment of the brain parenchyma following transplantation.

Journal Article↗

PubMed2001

Effects and mechanisms of somatostatin analogs on apoptosis of pancreatic acinar cells in acute pancreatitis in mice.

AIM: To investigate the effects of somatostatin analogs (SSa) on apoptosis of pancreatic acinar cells and apoptosis-regulated gene bax, and p53 in treating acute pancreatitis in mice. METHODS: In cerulein-induced pancreatitis, with or without treatment of somatostatin, analogs (Octreotide) in CD-1 (BALB/c x DBetaAlpha/1) mice, apoptosis of pancreatic acinar cells was detected by using the TdT-mediated dUTP nick-end labeling (TUNEL) method, and the expression of apoptosis-regulated gene bax and p53 was determined by using the streptavidin-peroxidase immunohistochemical technique and the RT-PCR method, respectively. RESULTS: On HE staining, acinar cells in the pancreas showed pyknotic nuclei and the formation of apoptotic bodies, which are the typical morphological features of apoptosis. Regarding TUNEL use, the apoptotic index of pancreatic acinar cells in the non-treated group at 5 and 14 h after induction of acute pancreatitis was significantly lower than those of the SSa-treated group, respectively (P < 0.01). On immunohistochemistry and RT-PCR, there was an expression of neither bax nor p53 in normal pancreatic tissues. The expression of bax in the SSa-treated group at 5 and 14 h after treatment of SSa was markedly higher than those of the non-treated group, respectively (P < 0.01), but there was no significant difference in the expression of p53 between the SSa-treated group and the non-treated group. CONCLUSIONS: The induction of apoptosis in pancreatic acinar cells injury to reduce inflammatory reaction might be one of the mechanisms of SSa in treating acute pancreatitis in mice, and the mechanisms of apoptosis probably correlated with the expression of apoptosis-regulated gene bax, but have no relationship with the expression of p53.

Acute Disease↗

PubMed2001

Reduction of functional N-methyl-D-aspartate receptors in neurons by RNase P-mediated cleavage of the NR1 mRNA.

One approach to studying the functional role of individual NMDA receptor subunits involves the reduction in the abundance of the protein subunit in neurons. We have pursued a strategy to achieve this goal that involves the use of a small guide RNA which can lead to the destruction of the mRNA for a specific receptor subunit. We designed a small RNA molecule, termed 'external guide sequence' (EGS), which binds to the NR1 mRNA and directs the endonuclease RNase P to cleave the target message. This EGS has exquisite specificity and directed the RNase P-dependent cleavage at the targeted location within the NR1 mRNA. To improve the efficiency of this EGS, an in vitro evolution strategy was employed which led to a second generation EGS that was 10 times more potent than the parent molecule. We constructed an expression cassette by flanking the EGS with self-cleaving ribozymes and this permitted generation of the specified EGS RNA sequence from any promoter. Using a recombinant Herpes simplex virus (HSV), we expressed the EGS in neurons and showed the potency of the EGS to reduce NR1 protein within neurons. In an excitotoxicity assay, we showed that expression of the EGS in cortical neurons is neuroprotective. Our results demonstrate the utility of EGSs to reduce the expression of any gene (and potentially any splice variant) in neurons.

Animals↗

PubMed2001

In vitro uptake of SCH 27899 (evernimicin) by rat alveolar macrophages.

The in vitro uptake of [(14)C]evernimicin ([(14)C]SCH 27899) by primary cultures of rat alveolar macrophages and hepatocytes was determined. Both cell populations exhibited linear rates of uptake. However, the initial rate of drug uptake by alveolar macrophages was about threefold higher than that by hepatocytes. These findings demonstrate that [(14)C]evernimicin is taken up by rat alveolar macrophages, supporting the likelihood that the drug is able to reach sites of infection.

Aminoglycosides↗