Biomedical subjects

Rong Guo

Publications and source records attributed to Rong Guo.

47 records · Page 3Linked to original sources

PubMed2003

[The biological activity of MHC classII transactivator ribonuclease P: a novel approach for hepatic transplantation rejection].

OBJECTIVE: This paper studied the effect of RNaseP against CIITA on repressing class II MHC (MHCII) expression. METHODS: It was constructed that M1-RNA with guide sequences (GS), recognizing the 629 site of CIITA (M1-629-GS), by PCR from pTK117 plasmid, then was cloned into psNAV (psNAV-M1-629-GS). CIITA target gene was obtained from Raji cell by RT-PCR, and then inserted into pGEM-7zf (+) (pGEM-800). psNAV-M1-629-GS and pGEM-800 were transcribed and then mixed up and incubated in vitro. Stable transfectants of hepatocyte with psNAV-M1-629-GS by nanometer were tested for MHCII induction by recombinant human interferon-gamma (IFN-gamma). mRNA abundance of CIITA was measured by RT-PCR. RESULTS: It showed that M1-629-GS could exclusively cleave pGEM-800 that formed a base pair with the GS. When induced with IFN-gamma, the expression of HLA-DR, -DP, -DQ on psNAV-M1-629-GS+ hepatocyte was (1.01+/-0.51)%, (4.37+/-1.28)%, (1.98+/-0.42)% respectively, was down-modulated 90.65%, 89.11% and 65.32% compared with control, while the mRNA content of CIITA reduced significantly (P<0.01). CONCLUSION: M1-629-GS could effectively repress MHCII expressing through cleaving CIITA mRNA. These results provided insight into the future application of it as a new nucleic acid drug against the rejection of hepatic transplantation.

Graft Rejection↗

PubMed2003

[The growth characteristics of mesenchymal stem/progenitor cells in human umbilical cord blood].

This study was done for investigating the frequency and proliferative feature of mesenchymal stem/progenitor cells (MSPC) in human umbilical cord blood (CB) and for searching a new seed cell for tissue engineering. Mononuclear cells was separated by Ficoll-Hypaque from cord blood and suspended in DMEM culture medium supplemented by 2% fetal bovine serum. The adherent CB cells were cultured and expanded at same medium. The results showed that the frequency of CB-MSPC was 0.5 x 10(-6) [(0.2 - 0.8) x 10(-6)]. The CB-MSPC showed a fibroblast-like morphology and retained their morphological feature at least after 20 sub-passages, and could extensively be expanded by about 1.3 x 10(7) times as much. The conclusion is that low serum DMEM culture could maintain the proliferation and differentiation potential of CB-MSPC. CB-MSPC might be a favorable seed cell for tissue engineering and regeneration.

Cell Adhesion↗

PubMed2003

[Major histocompatibility complex class II transactivator (CIITA) hammer- head ribozyme transfer inhibits major histocompatibility complex class II (MHC-II) expression in Jukart cells].

OBJECTIVE: Graft versus host disease (GVHD), a major cause of graft failure in allo-hematopoietic cell transplantation, was associated with the presence of major histocompatibility complex class II (MHCII), also called human leukocyte antigen (HLAII), on the tissues and organs of host. MHCII played a critical role in the induction of immune responses by presenting fragments of allo-antigenic peptides to CD(4)(+) T lymphocytes, then by resulting in CD(8)(+) T lymphocytes activation. Therefore, it was very important for compatibility of MHCII in allo-transplantation. But it was impossible to down-modulate MHCIIexpression directly. There were codominance and multiple allele for MHCII molecules which was owing to their complicated polymorphism, therefore it was difficult to repress every MHCII molecule expression. MHC class II transactivator (CIITA) was the major rate-limiting factor for both constitutive and inducible MHCIIexpression., and with rare exceptions, its expression paralleled that of MHCII transcripts. This study investigated the effect of anti-CIITA hammerhead ribozyme (Rz) on interferon (IFN)-gamma induced MHCII expression in Jukart cell line. METHODS: Three hammerhead Rz specific to 134, 218, 464 sites of CIITA gene were synthesized and named as Rz134, Rz218, Rz464, respectively. Then they were cloned into the EcoRI/BamHI of vector pGEM-7zf(+). CIITA target gene was obtained from Raji cell by RT-PCR, and then inserted also into the pGEM-7zf(+) plasmid. The above recombinant plasmids were screened out by sequence analysis. Hammerhead Rz and their target RNA were transcribed and then mixed up and incubated in vitro. The cleavage products were analyzed by PAGE and silver-staining. Rz464 was selected as the one with the highest activity, and then inserted into the plasmid with internal ribosome entry site-enhanced green fluorescent protein (pIRES2-EGFP), pRz464. Stable transfectants of Jukart cell line with pRz464 (pRz464-J) were tested for classic MHCII (HLA-DR, DP, DQ) induction by recombinant human IFN-gamma. mRNA abundance of CIITA was measured by RT-PCR. RESULTS: When induced with IFN-gamma, the expression of HLA-DR, DP, DQ in pRz464 positive (pRz464+) Jukart cells was 2.7%, 6.4% and 2.1%, respectively, and that in Jukart cells transfected by non-related ribozyme was 10.1%, 57.8% and 5.1%, respectively. Therefore, Rz464 suppressed IFN-gamma-induced up-regulation of HLA-DR, DP and DQ by 73.27%, 88.93% and 58.82%, respectively. Meanwhile, the mRNA content of CIITA was reduced significantly (P < 0.01). CONCLUSION: CIITA hammerhead ribozyme transfer inhibited MHC-II expression in Jukart cells. The above result provided insight into the future application of anti-CIITA hammerhead ribozyme for the antigen-specific tolerance induction and anti-GVHD treatment in the hematopoietic stem cell transplantation.

Cell Line, Tumor↗

PubMed2003

[Inhibition effect of CIITA anti-sense cDNA on the expression of MHCII molecules of Hela cells].

OBJECTIVE: To investigate the inhibiting effect of classII major histocompatibility complex transactivator (CIITA) anti-sense cDNA on MHCII molecules expression. METHODS: CIITA antisense cDNAs (arII1, arII2 and arII3) were obtained from Raji cell by RT-PCR, and then inserted into the pcDNA3.1B plasmid. The stable transfectants of Hela cell were tested for CIITA protein by immunohistochemistry, and classic MHCII molecules (HLA-DR, -DP, -DQ) induction through recombinant human IFN-gamma by flow cytometry. mRNAs of CIITA, invariant chain and classic MHCII molecules were detected by RT-PCR. RESULTS: The effect of arII2 was the best. When induced with IFN-gamma, the expression of CIITA protein was inhibited by 87.23%, and that of HLA-DR, -DP and -DQ was almost totally inhibited on arII2 positive Hela cells. The mRNA contents of CIITA, HLA-DR, -DP, -DQ and invariant chain were decreased significantly (P < 0.05). CONCLUSIONS: arII2 inhibits CIITA and thus the family of MHCIImolecules regulated by CIITA, therefore, it provides a novel approach to graft versus host disease study in hematopoietic stem cell transplantation.

DNA, Antisense↗

PubMed2003

[Protective effects of phosphoramidon on the experimental myocardial injury in rats].

OBJECTIVE: To investigate the protective effects of phosphoramidon on the experimental myocardial injury in rats. METHODS: Thirty-six Wistar rats were randomly divided into 3 groups of 12: the control group (Control), the isoproterenol group (ISO) and the protection group (PA). All rats were anesthetized after 24 hours and the blood was sampled from the carotid artery. The activities of creatine phosphokinase (CPK) and lactic dehydrogenase (LDH) in the serum and the contents of ET in the plasma were detected; then the hearts were isolated and the total layers of apical myocardium were solidified, embedded, sectioned, stained with HE and graded pathologically. RESULTS: The serum activities of CPK and LDH, and the plasmatic content of ET in the ISO group were significantly higher than those in the control group and PA group (P < 0.05); the degree of myocardial pathological damage in the ISO group was significantly higher than that in the control group (P < 0.01), while the degree of damage in the PA group was lower than that in the ISO group (P < 0.05). CONCLUSION: Endothelin is involved in the pathologic process of experimental myocardial injury in rats. Phosphoramidon can protect the myocardial injury in rats and its mechanism may be to inhibit ECE and reduce the endothelins production.

Animals↗

PubMed2002

Inhibition of MEPE cleavage by Phex.

X-linked hypophosphatemia (XLH) and the Hyp-mouse disease homolog are caused by inactivating mutations of Phex which results in the local accumulation of an unknown autocrine/paracrine factor in bone that inhibits mineralization of extracellular matrix. In these studies, we evaluated whether the matrix phosphoglycoprotein MEPE, which is increased in calvaria from Hyp mice, is a substrate for Phex. Using recombinant full-length Phex (rPhexWT) produced in Sf9 cells, we failed to observe Phex-dependent hydrolysis of recombinant human MEPE (rMEPE). Rather, we found that rPhex-WT inhibited cleavage of rMEPE by endogenous cathepsin-like enzyme activity present in Sf9 membrane. Sf9 membranes as well as purified cathepsin B cleaved MEPE into two major fragments of approximately 50 and approximately 42kDa. rPhexWT protein in Sf9 membrane fractions, co-incubation of rPhexWT and cathepsin B, and pre-treatment of Sf9 membranes with leupeptin prevented the hydrolysis of MEPE in vitro. The C-terminal domain of Phex was required for inhibition of MEPE cleavage, since the C-terminal deletion mutant rPhex (1-433) [rPhex3(')M] failed to inhibit Sf9-dependent metabolism of MEPE. Phex-dependent inhibition of MEPE degradation, however, did not require Phex enzymatic activity, since EDTA, an inhibitor of rPhex, failed to block rPhexWT inhibition of MEPE cleavage by Sf9 membranes. Since we were unable to identify interactions of Phex with MEPE or actions of Phex to metabolize cathepsin B, Phex may be acting to interfere with the actions of other enzymes that degrade extracellular matrix proteins. Although the molecular mechanism and biological relevance of non-enzymatic actions of Phex need to be established, these findings indicate that MEPE may be involved in the pathogenesis defective mineralization due to Phex deficiency in XLH and the Hyp-mouse.

Animals↗

PubMed2002

Reaction of the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate with human biofluids.

Using the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS), an oxidant was previously detected in the plasma of patients with renal failure and the synovial tissue of rheumatoid arthritis patients. This oxidant has been shown to react with DBNBS to give a 3-line electron paramagnetic resonance (EPR) spectrum, previously assigned to the DBNBS radical cation (DBNBS*(+). However, confusion has arisen as to whether this paramagnetic species is indeed DBNBS*(+) or, rather, the DBNBS sulfite radical adduct (DBNBS-SO(3)*(-)). In the present study, DBNBS*(+) (a(N)=1.32 mT) was distinguished from DBNBS-SO(3)*(-) (a(N)=1.32 mT with an additional splitting of a(H)=0.06 mT) by (a) using different EPR parameters, (b) determining the effect of addition of sulfite on the EPR spectrum resulting from the incubation of DBNBS with either human biofluids or the horseradish peroxidase (HRP)-hydrogen peroxide (H(2)O(2)) system, and (c) replacing DBNBS with its analogues (DBNBS-d(2,) DBNBS-15N and DBNBS-d(2)-15N) in the two systems.

Benzenesulfonates↗

PubMed2002

Expression, purification, crystallization and preliminary X-ray analysis of a DNA-binding protein from Methanococcus jannaschii.

A small DNA-binding protein of 87 amino-acid residues from the hyperthermophilic archaeon Methanococcus jannaschii (Mja10b) was cloned and overexpressed in Escherichia coli. The protein was crystallized and the crystals belong to the space group P6(1)22/P6(5)22, with unit-cell parameters a = b = 50.85, c = 124.02 A, alpha = beta = 90, gamma = 120 degrees. The crystals diffracted to a maximum resolution of 2.2 A at 100 K using Cu Kalpha radiation. The presence of one molecule per asymmetric unit gives a crystal volume per protein mass (V(M)) of 2.4 A(3) Da(-1) and a solvent content of 49% by volume. A full set of X-ray diffraction data was collected to 2.2 A from the native crystal.

Crystallography, X-Ray↗

PubMed2002

[A comparison between immunophenotypes of lymphoid cells from human umbilical cord blood and bone marrow and its significance].

To compare the expression of CD antigens on immune cells from umbilical cord blood (UCB) and bone marrow (BM) and analyze its clinical significance, the phenotypes of lymphoid cells and nucleated cells from 38 UCB and 10 BM samples were investigated by flow cytometry with double labeling monoclonal antibodies. The results showed that the immature lymphocytes (CD3(-) CD4(+)) were detected in UCB and higher than those in BM; cytotoxic T lymphocytes (CD3(+) CD16(+) CD56(+)) in UCB were significantly lower than those in BM. NK cells (CD3(-) CD16(+) CD56(+)) in UCB were higher than those in BM. The ratio of CD34(+) cells in nucleated cells of UCB was similar to that of BM, however, both the contents of myeloid (CD34(+) CD13(+) and CD34(+) HLA-DR(+)) and lymphoid (CD34(+) CD19(+)) progenitor cells in UCB were lower than those in BM. It is concluded that the immune cells in UCB possess immaturity, which might lead to mild GVHD after UCB transplantation. It is inferred from the higher ratio of NK cells in UCB, GVL will not decrease after UCB transplantation. The lower contents of myeloid and lymphoid progenitor cells in UCB probably accounted for the slow hematopoiesis and immune reconstitution following UCB transplantation.

Adult↗

PubMed2001

Overexpression of Phex in osteoblasts fails to rescue the Hyp mouse phenotype.

Inactivating mutations of Phex, a phosphate-regulating endopeptidase, cause hypophosphatemia and impaired mineralization in X-linked hypophosphatemia (XLH) and its mouse homologue, Hyp. Because Phex is predominantly expressed in bone and cultured osteoblasts from Hyp mice display an apparent intrinsic mineralization defect, it is thought that reduced expression of Phex in mature osteoblasts is the primary cause of XLH. To test this hypothesis, we studied both targeted expression of Phex to osteoblasts in vivo under the control of the mouse osteocalcin (OG2) promoter and retroviral mediated overexpression of Phex in Hyp-derived osteoblasts (TMOb-Hyp) in vitro. Targeted overexpression of Phex to osteoblasts of OG2 Phex transgenic Hyp mice normalized Phex endopeptidase activity in bone but failed to correct the hypophosphatemia, rickets, or osteomalacia. OG2 Phex transgenic Hyp mice did exhibit a small, but significant, increase in bone mineral density and dry ashed weight, suggesting a partial mineralization effect from restoration of Phex function in mature osteoblasts. Similarly, retroviral mediated overexpression of Phex in TMOb-Hyp osteoblasts restored Phex mRNA levels, protein expression, and endopeptidase activity but failed to correct their intrinsic mineralization defect. In addition, we failed to detect the Phex substrate FGF-23 in osteoblasts. Taken together, these in vivo and in vitro data indicate that expression of Phex in osteoblasts is not sufficient to rescue the Hyp phenotype and that other sites of Phex expression and/or additional factors are likely to be important in the pathogenesis of XLH.

Alkaline Phosphatase↗

PubMed2001

Fluorescence Quenching of Anthracene by N,N-Diethylaniline in the Sodium Dodecyl Sulfate/Benzyl Alcohol/Water System.

Photoinduced electron-transfer reaction of anthracene with N,N-diethylaniline (DEA) was studied in the SDS (sodium dodecyl sulfate)/BA (benzyl alcohol)/H(2)O system. In an oil/water microemulsion, only the excited anthracene located at the interface can be quenched by DEA. In a water/oil microemulsion, this quenching reaction occurs in the BA continuous phase. Besides being the quencher of the excited anthracene, DEA could also change the system's structure. Copyright 2001 Academic Press.

Journal Article↗