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Biomedical subjects

Yan Hu

Publications and source records attributed to Yan Hu.

At least 37 records · Page 2Linked to original sources
PubMed2005

Cloning and expression of earthworm fibrinolytic enzyme PM(246) in Pichia pastoris.

We have cloned, expressed, and purified a novel earthworm fibrinolytic enzyme (EFE) of Lumbricus rubellus in Pichia pastoris. Its cDNA sequence revealed a 747bp region containing an intact ORF that encodes a protein of 246 amino acid residues, designated as EFE PM(246). While EFE PM(246) is distinct, its cDNA shows a high degree of sequence homologies with four other EFE cDNAs registered in GenBank. The recombinant EFE PM(246) was active, showing a fibrinolytic activity of 7.5 x 10(6)U/L in basal salts medium, a higher fibrinolytic activity than those produced in other expression systems. The recombinant EFE PM(246) expressed in basal salts medium was purified by a three-step purification procedure with a recovery rate of about 20%. This is the first report detailing the successful purification of a genetically engineered earthworm fibrinolytic enzyme. The main physiochemical features of the EFE PM(246), including temperature stability, pH resistance, and sensitivity to some protein inhibitors, were also characterized.

Amino Acid Sequence↗
PubMed2005

CXCR3 and its ligands participate in the host response to Bordetella bronchiseptica infection of the mouse respiratory tract but are not required for clearance of bacteria from the lung.

Intranasal inoculation of mice with Bordetella bronchiseptica produces a transient pneumonia that is cleared over several weeks in a process known to require both neutrophils and lymphocytes. In this study, we evaluated the roles of the chemokines MIG (CXCL9), IP-10 (CXCL10), and I-TAC (CXCL11) and their common receptor, CXCR3. Following bacterial inoculation, message expression of interleukin-1 (IL-1), IL-6, and the neutrophil-attracting chemokines KC, LIX, and MIP-2 was rapidly induced, with maximal expression found at 6 h. In contrast, message expression of gamma interferon, MIG, IP-10, and I-TAC peaked at 2 days. Expression of all of these chemokines and cytokines returned to near baseline by 5 days, despite the persistence of high levels of live bacteria at this time. Induced MIG, IP-10, and I-TAC protein expression was localized in areas of inflammation at 2 to 3 days and was temporally associated with increased levels of CXCR3(+) lymphocytes in bronchoalveolar lavage fluid. There was no increase in mortality in mice lacking CXCR3. However, the clearance of bacteria from the lung and trachea was delayed, and the recruitment of lymphocytes and NK cells was slightly decreased, for CXCR3(-/-) mice relative to CXCR3(+/+) mice. We conclude that the CXCR3 receptor-ligand system contributes to pulmonary host defense in B. bronchiseptica infection by recruiting lymphocytes and NK cells into the lung.

Animals↗
PubMed2005

[Expression of cyclooxygenase-2 in fetal membranes of premature delivery].

OBJECTIVE: To investigate the effect of cyclooxygenase-2 (COX-2) in pathogenesis of premature delivery. METHODS: A cross-sectional study was conducted with 30 women in the following categories: preterm delivery in labor with intact membranes( n = 8), the term in labor with intact membranes (n = 10), and the term not in labor (control group, n = 12). The expressions of COX-2 in different areas of the fetal membranes were examined by immunohistochemical assay. RESULTS: The immuoreactivity of COX-2 was found in epithelial cells and stromal cells in amniotic and chorion membranes. Expression of COX-2 in different areas of the fetal membranes varied markedly in preterm delivery in labor, term in labor and the control group. Immunohistochemical scores(IH score) of COX-2 in the cervical and corpus fetal membranes in the preterm delivery in labor group were higher than those in the term in labor group and the control group(P < 0.05). There was no difference in COX-2 expression between the cervical and corpus fetal membranes among the three groups (P > 0.05). CONCLUSION: COX-2 may play an important role in the pathogenesis of premature labor.

Cross-Sectional Studies↗
PubMed2005

[Prokaryotic expression and purification of human immunodeficiency virus p24 antigen].

OBJECTIVE: To express and purify the HIV p24 gene in E. coli cells, and identify p24 antigen activity. METHODS: The full length gene fragment of HIV p24 was amplified by PCR and inserted into the pRSET vector in order to construct the pRSET-p24 recombined vector. After transforming into E. coil, the purified p24 protein was prepared by metal-ligand affinity chromatography (IMAC). The accuracy of inserted gene and activity, specificity of HIV p24 proteins were detected by two enzymes digestion technology, SDS-PAGE, Western Blot (WB) and ELISA. RESULTS: The length of the HIV p24 gene fragment was 690 bp after digesting the recombinant plasmid T-p24 and pRSET-p24 with BamH I and Hind III. The expressed proteins had a single expected band of about 24 x 10(3) in SDS-PAGE. The specificity and activity of p24 protein were tested by WB and ELISA. CONCLUSION: The HIV p24 sequence from HIV-1 gene plasmid hasbeen expressed in E. coil. This protein possessed good specificity and activity.

Blotting, Western↗
PubMed2005

[Effects of different nitrogenous compounds on growth and nodulation of Abrus cantoniensis].

OBJECTIVE: The research aimed at the effects of different nitrogenous compounds on growth and nodulation of Abrus cantoniensis. METHOD: After the seedlings of the herb were inoculated with rhizobia in potted culture, they were supplied with nutrition solutions which contained the three nitrogenous compounds, KNO3, NH4NO3, (NH4)2SO4 of different nitrogen concentration. The growth and nodulation of seedlings was determined after 70 days. RESULT: Different nitrogenous compounds were able to enhance the vegetable growth of seedlings variously. The effect of (NH4)2SO4 and NH4NO3 on growth was better than that of KNO3. Seedlings nodulation was obviously inhibited by these nitrogenous compounds. Their inhibitory effects ranked NH4NO3 > (NH4)2SO4 > KNO3. The treatments of KNO3 and the lower concentration treatments of NH4NO3 and (NH4)2 SO4 didn't inhibit the nodulation of seedlings, but the higher concentration treatment of NH4NO3 and (NH4)2SO4 severely inhibited nodulation or even made a no formation of nodule. CONCLUSION: The results showed that ammonium nitrogen the higher inhibitory ability to the nodulation of seedlings of A. cantoniensis than nitrate nitrogen. Therefore, the application of ammonium nitrogen fertilizer should be controlled in culture of the herb, which is in favor of increasing the function of biological nitrogen fixation and the quality of the medicinal materials of A. cantoniensis.

Abrus↗
PubMed2005

[Study on the compliance and safety of the oral antifungal agents for the treatment of onychomycosis].

OBJECTIVE: To explore the rate of compliance, influencing factors and the safety of patients with onychomycosis under treatment of oral antifungal agents. METHODS: According to the scoring clinical index of onychomycosis (SCIO), 330 patients with onychomycosis, their target nail's integral of the SCIO were calculated and randomly divided into three groups under the baseline of the SCIO integral range. Patients were treated with intermittent pulse itraconazole (A group), continuous terbinafine (B group) and intermittent terbinafine (C group) respectively. Self-administered questionnaire was applied in the survey on every onychomycosis patient. RESULTS: The average rate of compliance was 55.15%. The cure rate for those compliance with doctors' order was 89.01%, while it was only 30.41% for those noncompliant patients The overall non-compliant rate was 44.85%. Among the noncompliant ones, 29.73% were worried about the side effects of medicine, 22.30% thought that they had already been cured, 15.54% was due to economic reasons and 12.16% could not bear the side effects of medicine. It was found that the compliant rates were significantly correlated to ageing, position of the target nails, the integral of the SCIO and the therapy scheme (P < 0.05), while no significant correlations were seen between male and female, culture degree and course (P > 0.1). The frequency of adverse incident of A, B, C groups were 22.73%, 21.43%, 23.15% respectively, but without statistical significance (P > 0.1). Majority of the adverse incidents happened during the first month of therapy but were mild and reversible. CONCLUSION: Our results showed that the overall compliance was low which exerted a significant influence on the curative effect of onychomycosis patients. Factors as ageing, position of the target nail, integral of the SCIO and the therapy scheme had an influence on the compliant rate. When treating onychomycosis with oral itraconazole, the results seemed to be just as safe as when using terbinafine.

Administration, Oral↗
PubMed2004

Targeted knockdown of the RNA-binding protein CRD-BP promotes cell proliferation via an insulin-like growth factor II-dependent pathway in human K562 leukemia cells.

The c-myc mRNA coding region determinant-binding protein (CRD-BP) was first identified as a masking protein that stabilizes c-myc mRNA in a cell-free mRNA degradation system. Thus, CRD-BP is thought to promote cell proliferation by maintaining c-Myc at critical levels. CRD-BP also appears to be an oncofetal protein, based upon its expression during mammalian development and in some tumors. By using K562 leukemia cells as a model, we show that CRD-BP gene silencing by RNA interference significantly promoted proliferation, indicating an inhibitory effect of CRD-BP on proliferation. Unexpectedly, CRD-BP knockdown had no discernible effect on c-myc mRNA levels. CRD-BP is also known as insulin-like growth factor II (IGF-II) mRNA-binding protein-1. It has been reported to repress translation of a luciferase reporter mRNA containing an IGF-II 5'-untranslated region known as leader 3 but not one containing IGF-II leader 4. CRD-BP knockdown markedly increased IGF-II mRNA and protein levels but did not alter translation of luciferase reporter mRNAs containing 5'-untranslated regions consisting of either IGF-II leader 3 or leader 4. Addition of antibody against IGF-II to cell cultures inhibited the proliferative effect of CRD-BP knockdown, suggesting that regulation of IGF-II gene expression, rather than c-myc mRNA levels, mediates the proliferative effect of CRD-BP knockdown. Thus, we have identified a dominant function for CRD-BP in cell proliferation of human K562 cells, involving a possible IGF-II-dependent mechanism that appears independent of its ability to serve as a c-myc mRNA masking protein.

5' Untranslated Regions↗
PubMed2004

IL-12 pretreatments enhance IFN-alpha-induced Janus kinase-STAT signaling and potentiate the antitumor effects of IFN-alpha in a murine model of malignant melanoma.

IFN-alpha 2b (IFN-alpha) has been used to treat patients with metastatic malignant melanoma and patients rendered disease-free via surgery but at high risk for recurrence. We hypothesized that IL-12 pretreatments would result in endogenous IFN-gamma production, and that this, in turn, would up-regulate levels of Janus kinase-STAT signaling intermediates and lead to increased expression of genes regulated by IFN-alpha. Treatment of PBMCs with IL-12 stimulated a significant and dose-dependent production of IFN-gamma. Pretreatment of PBMCs and tumor cells with IFN-gamma-containing supernatants from IL-12-stimulated PBMCs led to up-regulation of STAT1, STAT2, and IFN regulatory factor 9 (IRF9) and potentiated IFN-alpha-induced STAT signaling within PBMCs and tumor cells. These effects were abrogated by neutralization of IFN-gamma in the PBMC supernatants with an anti-IFN-gamma Ab. Pretreatment of HT144 melanoma cells and PBMCs with IFN-gamma or IFN-gamma-containing supernatants enhanced the actions of IFN-alpha at the transcriptional level, as measured by real-time RT PCR analysis of the IFN-stimulated gene 15. Experiments in wild-type C57BL/6 and IFN-gamma receptor knockout (B6.129S7-Ifngr(tm1Agt)) mice demonstrated that a regimen of IL-12 pretreatment, followed by IFN-alpha, could cure mice of i.p. B16F1 melanoma tumors (p < 0.007), whereas mice treated with either agent alone or PBS succumbed to fatal tumor burden. However, this treatment regimen did not significantly prolong the survival of IFN-gamma-deficient (B6.129S7-Ifng(tm1Ts)) mice compared with mice treated with IFN-alpha alone. These results suggest that the response to IFN-alpha immunotherapy can be significantly enhanced by IL-12 pretreatment, and this effect is dependent upon endogenous IFN-gamma production and its actions on melanoma cells.

Animals↗
PubMed2004

RNA interference of achaete-scute homolog 1 in mouse prostate neuroendocrine cells reveals its gene targets and DNA binding sites.

We have previously characterized a transgenic mouse model (CR2-TAg) of metastatic prostate cancer arising in the neuroendocrine (NE) cell lineage. Biomarkers of NE differentiation in this model are expressed in conventional adenocarcinoma of the prostate with NE features. To further characterize the pathways that control NE proliferation, differentiation, and survival, we established prostate NE cancer (PNEC) cell lines from CR2-TAg prostate tumors and metastases. GeneChip analyses of cell lines harvested at different passages, and as xenografted tumors, indicated that PNECs express consistent features ex vivo and in vivo and share a remarkable degree of similarity with primary CR2-TAg prostate NE tumors. PNECs express mAsh1, a basic helix-loop-helix (bHLH) transcription factor essential for NE cell differentiation in other tissues. RNA interference knockdown of mAsh1, GeneChip comparisons of treated and control cell populations, and a computational analysis of down-regulated genes identified 12 transcriptional motifs enriched in the gene set. Affected genes, including Adcy9, Hes6, Iapp1, Ndrg4, c-Myb, and Mesdc2, are enriched for a palindromic E-box motif, CAGCTG, indicating that it is a physiologically relevant mAsh1 binding site. The enrichment of a c-Myb binding site and the finding that c-Myb is down-regulated by mAsh1 RNA interference suggest that mAsh1 and c-Myb are in the same signaling pathway. Our data indicate that mAsh1 negatively regulates the cell cycle (e.g., via enhanced Cdkn2d, Bub1 expression), promotes differentiation (e.g., through effects on cAMP), and enhances survival by inhibiting apoptosis. PNEC cell lines should be generally useful for genetic and/or pharmacologic studies of the regulation of NE cell proliferation, differentiation, and tumorigenesis.

Animals↗
PubMed2004

Widespread correction of lysosomal storage following intrahepatic injection of a recombinant adeno-associated virus in the adult MPS VII mouse.

Mucopolysaccharidosis type VII is a lysosomal storage disease caused by deficiency of the acid hydrolase beta-glucuronidase. MPS VII mice develop progressive lysosomal accumulation of glycosaminoglycans within multiple organs, including the brain. Using this animal model, we investigated whether gene transfer mediated by a recombinant adeno-associated virus (rAAV) type 2 vector is capable of reversing the progression of storage in adult mice. We engineered an rAAV2 vector to carry the murine beta-glucuronidase cDNA under the transcriptional direction of the human elongation factor-1alpha promoter. Intrahepatic administration of this vector in adult MPS VII mice resulted in stable hepatic beta-glucuronidase expression (473 +/- 254% of that found in wild-type mouse liver) for at least 1 year postinjection. There was widespread distribution of vector genomes and beta-glucuronidase within extrahepatic organs. The level of enzyme activity was sufficient to reduce lysosomal storage within the liver, spleen, kidney, heart, lung, and brain. Within selected regions of the brain, neuronal, glial, and perivascular cells had histopathologic evidence of reduced storage. Also, brain alpha-galactosidase and beta-hexosaminidase enzyme levels, secondarily elevated by the storage abnormality, were normalized. These data demonstrate that peripheral administration of an rAAV2 vector in adult MPS VII mice can lead to transgene expression levels sufficient for improvements in both the peripheral and the central manifestations of this disease.

Adenoviridae↗
PubMed2004

Innate versus adaptive immunity in Candida albicans infection.

Candida albicans is a common opportunistic pathogen, causing both superficial and systemic infection. Clinical observations indicate that mucocutaneous infections are commonly associated with defective cell-mediated immune responses, whereas systemic infection is more frequently seen in patients with deficiencies in neutrophil number or function. Analysis of mechanisms of host resistance against gastrointestinal and oral infection in mouse models has demonstrated an absolute dependence on CD4(+) T cells, although clearance also involves phagocytic cells. Both IL-12 and TNF-alpha appear to be important mediators, but mouse strain-dependent variations in susceptibility to infection may be related to T-cell enhancement of production of phagocytic cells by the bone marrow. In murine systemic infection, the role of innate and adaptive responses is less well defined. Studies in immunodeficient and T-cell-depleted mice suggest that clearance of the yeast may be predominantly a function of the innate response, whereas the adaptive response may either limit tissue damage or have the potential to cause immunopathology, depending on the host genetic context in which the infection takes place.

Animals↗
PubMed2004

[The expression and activity detection of a variant N protein of SARS-CoV].

AIM: To construct an expression vector pGEX-2T/N, and to express the fusion protein consisting of N protein of SARS-CoV and GST in E.coli. METHODS: The N region gene of SARS-CoV was cloned by RT-PCR. The expression vector was constructed by DNA recombination. The recombinant plasmid was transformed into E.coli BL21(DE3). The expression of the fusion protein was detected by Western blot. RESULTS: (1) As compared with the sequences in GenBank, 20 bp were deleted in DNA sequence of the cloned N protein. (2) The fusion protein GST-N was soluble. Western blot analysis showed that the reaction of GST-N to anti-SARS-CoV sera was positive. CONCLUSION: The pGEX-2T/N has been constructed and expressed in the form of fusion protein GST-N successfully, which lays the foundation for further study of SARS-CoV N protein.

Base Sequence↗
PubMed2004

[Effects of hydrocortisone sodium succinate on sodium current in human and guinea pig cardiac myocytes].

AIM: To study the effects of hydrocortisone sodium succinate on sodium current in human atrial myocytes and in guinea pig ventricular myocytes. METHODS: Single cardiac myocytes were isolated by enzyme. The effects of hydrocortisone sodium succinate on sodium current (INa) were assessed by applying whole-cell patch clamp techniques. RESULTS: Hydrocortisone sodium succinate (1, 3, 10 micromol x L(-1)) was shown to inhibit INa of both human atrial myocytes and guinea pig ventricular myocytes in concentration dependent manner and the IC50 were 6.97 and 8.74 micromol x L(-1), respectively. The inhibition effects acted quickly (1-3 min) and the maximal activating voltage of INa was not changed in both human and guinea pig cardiac myocytes. CONCLUSION: Hydrocortisone sodium succinate can exhibit inhibitory effects on INa in both human and guinea pig cardiac myocytes, and its inhibitory effects act rapidly, which are not consistent with genomic effects, so there may be nongenomic effects.

Adolescent↗
PubMed2004

[Preparation and characterization of the monoclonal antibody against HIV-1 p24 antigen].

AIM: To establish hybridoma cells secreting monoclonal antibodies (mAbs) against HIV-1 p24 antigen and characterize their properties. METHODS: BALB/c mice were immunized with purified p24 protein and then the from splenocytes immunized mice were fused with Sp2/0 cells. Monoclonal hybridoma cell lines were obtained by limiting dilution, HAT and HT-selective culture. The specificity of mAbs was identified by Dot blot and indirect ELISA. RESULTS: We had established two hybridoma cell lines secreting mAbs against HIV-1 p24 antigen. The titers of two mAbs in ascitic fluid were 1 x 10(-5) and 1.7 x 10(4)-1.8 x 10(4) mol/L, respectively. Both mAbs belonged to IgG1. Indirect ELISA detection demonstrated that both mAbs had no cross-reactivities with other viral antigens, such as HBcAg, HCV RNA positive sera and HIVgp41. CONCLUSION: Two hybridoma cell lines secreting mAbs against HIV-1 p24 antigen have been established successfully, which lays the foundation for detecting HIV-1 p24 antigen.

Animals↗
PubMed2004

[Study on the response of specific antibodies against SARS-CoV in patients infected with SARS].

OBJECTIVE: To study the response of specific antibodies against severe acute respiratory syndrome (SARS)-CoV in patients infected with SARS. METHODS: IgM-capture, indirect and antigen-sandwiched enzyme linked immunosorbent assay (ELISA) were used to detect the SARS-CoV specific IgM, IgG and total antibodies in sera of clinical SARS patients or non-SARS individuals. RESULTS: The positive rates of IgM, IgG and total antibodies to SARS-CoV in 146 sera of SARS patients collected in different phases of the disease were 61.64%, 53.43% and 69.86%, respectively. The earliest detectable days after onset of the disease for IgM and IgG to SRAS-CoV were 7 and 12 days, respectively. The specific IgM disappeared as early as 42 days after the onset of SARS. Of 70 sera from hepatitis A patients, 2 showed false positive results, while 127 sera from other patients were all negative, detected by the 3 methods. Serum from one medical worker who had been close contact to SARS patients was positive for anti-SARS-CoV IgG and total antibodies. These 3 methods used for detection were all not influenced by rheumatoid factor (RF). CONCLUSION: All of the three methods were specific and sensitive for the detection of specific antibodies to SARS-CoV, and useful for epidemiological research and clinical diagnosis, but not for early diagnosis of SARS.

Antibodies, Viral↗
PubMed2003

DNA methylation-related chromatin remodeling in activity-dependent BDNF gene regulation.

In conjunction with histone modifications, DNA methylation plays critical roles in gene silencing through chromatin remodeling. Changes in DNA methylation perturb neuronal function, and mutations in a methyl-CpG-binding protein, MeCP2, are associated with Rett syndrome. We report that increased synthesis of brain-derived neurotrophic factor (BDNF) in neurons after depolarization correlates with a decrease in CpG methylation within the regulatory region of the Bdnf gene. Moreover, increased Bdnf transcription involves dissociation of the MeCP2-histone deacetylase-mSin3A repression complex from its promoter. Our findings suggest that DNA methylation-related chromatin remodeling is important for activity-dependent gene regulation that may be critical for neural plasticity.

Animals↗
PubMed2003

The antitumor effects of IFN-alpha are abrogated in a STAT1-deficient mouse.

IFN-alpha activates the signal transducer and activator of transcription (STAT) family of proteins; however, it is unknown whether IFN-alpha exerts its antitumor actions primarily through a direct effect on malignant cells or by stimulating the immune system. To investigate the contribution of STAT1 signaling within the tumor, we generated a STAT1-deficient melanoma cell line, AGS-1. We reconstituted STAT1 into AGS-1 cells by retroviral gene transfer. The resulting cell line (AGS-1STAT1) showed normal regulation of IFN-alpha-stimulated genes (e.g., H2k, ISG-54) as compared with AGS-1 cells infected with the empty vector (AGS-1MSCV). However, mice challenged with the AGS-1, AGS-1STAT1, and AGS-1MSCV cell lines exhibited nearly identical survival in response to IFN-alpha treatment, indicating that restored STAT1 signaling within the tumor did not augment the antitumor activity of IFN-alpha. In contrast, STAT1-/- mice could not utilize exogenous IFN-alpha to inhibit the growth of STAT1+/+ melanoma cells in either an intraperitoneal tumor model or in the adjuvant setting. The survival of tumor-bearing STAT1-/- mice was identical regardless of treatment (IFN-alpha or PBS). Additional cell depletion studies demonstrated that NK cells mediated the antitumor effects of IFN-alpha. Thus, STAT1-mediated gene regulation within immune effectors was necessary for mediating the antitumor effects of IFN-alpha in this experimental system.

Animals↗