Biomedical subjects
Publications and source records attributed to Q Zhao.
The field of antisense therapeutics has attracted great interest during the past decade. A large body of literature has recently appeared in which the antisense mechanism is claimed to be involved and a number of human clinical trials are underway. Questions regarding the specificity of action and side effects of antisense phosphorothioate oligonucleotides have arisen simultaneously.
Hemorphins extraction by organic solvents from a hemoglobin peptic hydrolysate was investigated. About thirteen solvents were used and only the aliphatic alcohols displayed selectivity for hemorphins. The resulting extracted phases, analyzed by SE-HPLC, RP-HPLC, and mass spectrometry, proved 1-butanol and 2-butanol to be the best extracting solvents towards hemorphins. The opioid activity test was carried out following each step of extraction and the obtained results were in agreement with a purification.
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Oligonucleotides are promising therapeutic agents for the prevention or treatment of a variety of diseases. The therapeutic potential of oligonucleotide therapy depends greatly on the bioavailability of oligonucleotides to their target cells and organs. We previously reported the pharmacokinetics and distribution of phosphorothioate oligonucleotide in mice using [35S]-labeled oligonucleotide ([35S]-oligo). To extend this study, we administered 30 mg/kg of fluorescent-labeled oligonucleotide (FITC-oligo) to mice and examined oligonucleotide distribution by measuring the fluorescence intensity in various cells and tissues using flow cytometry. Following FITC-oligo administration, fluorescence was detected in all the tissues examined. In terms of the fluorescent intensity, accumulation was greatest in liver and kidney, intermediate in spleen and bone marrow, and very low in peripheral blood mononuclear cells (PBMC). At 4 hours after administration, the level of oligonucleotide uptake in PBMC, spleen lymphocytes, and bone marrow cells revealed the following pattern: monocytes/macrophages > B cells > T cells. Confocal microscopy detected intracellular fluorescence in PBMC prepared under the same conditions as those for flow cytometry. These studies provide a rationale for designing cell targets for antisense therapeutics.
A Pseudomonas aeruginosa strain carrying an insertion of an omega Hg interposon in the mexB gene (mexB::omega Hg; strain K879) produced markedly reduced but still detectable levels of OprM, the product of the third gene of the mexAB-oprM multidrug efflux operon. By using a lacZ transcriptional fusion vector, promoter activity likely responsible for OprM expression in the mexB::omega Hg mutant was identified upstream of oprM. Introduction of the oprM gene, but not the mexAB genes, into a P. aeruginosa multidrug-susceptible delta mexAB-oprM mutant increased resistance to quinolones, cephalosporins, erythromycin, and tetracycline. A delta mexAB-oprM strain carrying the oprM gene accumulated markedly less antibiotic than the deletion strain without oprM. Antibiotic accumulation by the MexAB- OprM+ strain was markedly enhanced upon treatment of cells with the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), indicating that MexAB-independent OprM function likely involves an efflux process. Moreover, pretreatment of cells with CCCP prior to the accumulation assay abrogated any differences in accumulation levels between the MexAB- OprM+ and MexAB- OprM- strains, indicating that reduced drug accumulation by the OprM+ strain (in the absence of CCCP) cannot be due to OprM-mediated reduction in outer membrane permeability. It appears, therefore, the OprM can be expressed and function in a drug efflux capacity independent of MexAB.
TonB couples the energized state of the cytoplasmic membrane to the operation of outer membrane receptors responsible for Fe(III) siderophore uptake across the outer membrane of gram-negative bacteria. A tonB mutant of Pseudomonas aeruginosa deficient in iron siderophore uptake was shown in the present study to be hypersusceptible to a wide variety of antibiotics, reminiscent of the phenotype of mutants defective in the mexAB-oprM antibiotic efflux operon. This was not related to influences of a tonB mutation on the iron status of the cell, and indeed, intrinsic antibiotic susceptibility and mexAB-oprM expression were unaffected by iron levels in the growth medium. The presence of tonB on a multicopy plasmid increased the level of resistance of a MexAB-OprM+ strain but not that of a MexAB-OprM- strain to a variety of antimicrobial agents. mexAB-oprM expression was not, however, altered in a tonB deletion mutant, indicating that any influence of TonB on MexAB-OprM-mediated multidrug resistance was at the level of pump activity. Consistent with this, drug accumulation assays revealed that the tonB deletion mutant exhibited decreased levels of drug efflux. Still, the multidrug resistance of a nalB strain was not wholly abrogated by a tonB mutation, indicating that it is likely not an essential component of the efflux apparatus. Similarly, elimination of tonB from an nfxB strain only partially compromised MexCD-OprJ-mediated multidrug resistance. Intriguingly, the drug susceptibility of a mexAB-oprM deletion strain was increased following deletion of tonB, suggesting that TonB may also influence antibiotic resistance mediated by determinants other than MexAB-OprM (and MexCD-OprJ). Thus, TonB plays an important role in both intrinsic and acquired antibiotic resistance in P. aeruginosa.
The gut and liver share a common embryological origin. The gene encoding the gut hormone neurotensin/neuromedin N (NT/N) is expressed in the adult small bowel, and NT/N is transiently expressed in the fetal liver, suppressed in the adult liver, and reexpressed in certain liver cancers. In our present study, we found that the NT/N gene was expressed at high levels in the human hepatoma cell line Hep 3B but was not expressed in Hep G2 cells. To further determine the mechanisms regulating NT/N expression, we performed Southern blotting and gene cloning techniques. Neither alteration nor mutation of the NT/N gene was responsible for this differential NT/N expression pattern. Human NT/N promoter constructs were transfected into either Hep 3B or Hep G2. Both cell lines supported NT/N transcription, indicating that the absence of NT/N expression in Hep G2 cells was due to mechanisms other than the absence of positive transcription factors. The role of DNA methylation was next assessed. Methylation of NT/N promoter constructs in vitro resulted in a 67-fold reduction in promoter activity, whereas treatment with the demethylating agent 5-azacytidine induced NT/N expression in Hep G2 cells, thus suggesting that DNA methylation plays a role in the expression of the gut endocrine gene NT/N. Defining the mechanisms regulating NT/N expression in these hepatic-derived cell lines will provide not only a better understanding of cell-specific and developmental regulation of a gut endocrine gene but also possible insight into liver cell lineage patterns and the derivation of certain hepatocellular cancers.
Pulmonary inflammation has been observed in humans and in many animal species after ozone exposure. Inflammatory cell accumulation involves local synthesis of chemokines, including neutrophil chemoattractants such as macrophage inflammatory protein-2 (MIP-2), and monocyte chemoattractants, such as monocyte chemoattractant protein-1 (MCP-1). To better understand the mechanism of ozone-induced inflammation, we exposed mice and rats to ozone for 3 h and measured MIP-2 and MCP-1 gene expression. In C57BL/6 mice, steady-state mRNA levels for MCP-1 in the lung increased at 0.6 parts/million (ppm) ozone and were maximal at 2.0 ppm ozone. After exposure to 2 ppm ozone, MIP-2 mRNA levels peaked at 4 h postexposure, whereas MCP-1 mRNA levels peaked at 24 h postexposure. Neutrophils and monocytes recovered in bronchoalveolar lavage fluid peaked at 24 and 72 h, respectively. The accumulation of monocytes was thus delayed relative to that of neutrophils, consistent with the sequential expression of the corresponding chemokines. The role of MCP-1 in monocyte accumulation was evaluated in greater detail in rats. Ozone caused an increase in monocyte chemotactic activity in bronchoalveolar fluid that was inhibited by an antibody directed against MCP-1. Ozone-induced MCP-1 mRNA levels were higher in lavage cells than in whole lung tissue, indicating that lavage cells are an important source of MCP-1. In these cells, nuclear factor-kappa B, a nuclear transcription factor implicated in MCP-1 gene regulation, was also activated 20-24 h after ozone exposure. These findings indicate that monocyte accumulation subsequent to acute lung injury can be mediated through MCP-1 and that nuclear factor-kappa B may play a role in ozone-induced MCP-1 gene expression.
Acute lung inflammation is complicated by altered pulmonary surfactant phospholipid and protein composition. The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) and the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) inhibit expression of surfactant-associated proteins A and B (SP-A and SP-B), both important for normal surfactant function. The transcription factor nuclear factor-kappa B (NF-kappa B) frequently mediates regulation of gene expression by TPA and TNF-alpha. In the present study, electrophoretic mobility shift assays (EM-SAs) and pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappa B activation, were utilized to determine the role of NF-kappa B activation in TPA and TNF-alpha inhibition of the surfactant proteins in NCI-H441 cells. Pentoxifylline (PTX), which inhibits TNF-alpha cellular effects without preventing NF-kappa B activation, was also tested. By EMSA, TPA and TNF-alpha increased nuclear NF-kappa B binding activity in temporally distinct patterns. PDTC decreased TPA- and TNF-alpha-induced NF-kappa B binding activity but did not limit their inhibition of SP-A and SP-B mRNAs. PDTC independently decreased both SP-A and SP-B mRNAs. PTX partially reversed TNF-alpha-but not TPA-mediated inhibition of SP-A and SP-B mRNAs without altering NF-kappa B binding. The effects of PDTC and PTX on NF-kappa B and the surfactant proteins suggest that NF-kappa B activation does not mediate TPA or TNF-alpha inhibition of SP-A and SP-B mRNA accumulation.
A conjugate with specific binding to the epidermal growth factor receptor, EGFR, and of interest for clinical tests was prepared using mouse epidermal growth factor, mEGF, and dextran. The mEGF was first coupled to dextran by reductive amination in which the free amino group on the N-terminal of mEGF was reacted with the aldehyde group on the reductive end of the dextran chain. The end-end coupled intermediate was further activated by the cyanopyridinium agent CDAP and tyrosines introduced to the dextran part of the conjugate. The mEGF-dextran-tyrosine conjugate was, with high efficiency, iodinated with the chloramine-T method. Approximately 25-35% of the radioactivity could be removed from the conjugate after exposure to protease K while 65-75% of the radioactivity could be removed after exposure to dextranase. Thus, the largest amount of the iodine was on the dextran part of the conjugate. The iodinated mEGF-dextran-tyrosine had EGFR specific binding since the binding to an EGFR rich human glioma cell line could be displaced by an excess of non-radioactive mEGF. The conjugate was to a large extent internalized in these cells and the administrated radioactivity was thereby retained inside the cells for at least up to 50 h.
OBJECTIVE: To evaluate the accuracy of CT-guided stereotactic biopsy in making correct pathological diagnosis and choosing corresponding management of brain tumors. METHODS: From 1991 to 1995, CT-guided stereotactic biopsy was performed in 310 patients with intracerebral lesions which were deep-seated or located in certain main functional areas. The patients were 198 men and 112 women. Their ages ranged from 4.5 to 70 years (average: 39.3 years). The lesions were located in the deep cerebrum (74 patients), the sellar area (62), the basal ganglion (51), the posterior part of the third ventricle (38), other intraventricleular area (21), the cerebellum (17) and the brain stem (9), and intracranial multiple lesions were found in 38 patients. RESULTS: Brain tumors were diagnosed pathologically in 266 patients (85.8%); inflammatory process in 25 (8.1%), other lesions in 8 (2.6%) and uncertain cases were 11 (3.6%). The overall positive rate of biopsy was 96.4% and the positive rate for brain tumor was 85.8%. Intracranial hematomas after biopsy were found in 5 patients (1.6%). There were no deaths induced by the biopsy or other serious complications. CONCLUSIONS: The results suggest that CT-guided stereotactic biopsy is a reliable method for histopathological diagnosis of brain tumors and it is also of great help in selecting appropriate management.
AIM: To study mRNA level of cyclophilin in the white blood cells (WBC) of the renal allograft recipients (RAR) and its correlation with ciclosporin concentration in whole blood. METHODS: The cyclophilin mRNA levels and beta-actin as controls in the WBC of 47 RAR were measured by quantitative reverse transcription polymerase chain reaction. The blood ciclosporin assay utilized monoclonal antibody fluorescence polarization immunoassay. RESULTS: With the increase of ciclosporin concentration in whole blood (from 62 micrograms.L-1 to 678 micrograms.L-1), relative cyclophilin mRNA level in the WBC of RAR decreased nonlinearly (from 1.1 to 0.03, r = 0.8195). CONCLUSION: There was a negative correlation between the mRNA level of cyclophilin in the WBC of RAR and the ciclosporin concentration in whole blood.
AIM: To study the genetic basis of N-acetylatransferase polymorphism in Chinese. METHODS: Genotypes in 120 healthy Han volunteers from 19 provinces of China were assayed. The 3 common mutant alleles (M1, M2, M3) and one normal wild-type (WT) allele of the N-acetyltransferase (NAT2) gene were detected by allele-specific polymerase chain reaction technique. RESULTS: The NAT2 allele frequencies in 120 Chinese (WT = 0.625, M1 = 0.0458, M2 = 0.188, M3 = 0.142) were different (P < 0.01). The NAT2 genotype distribution for all detected combinations of NAT2 alleles in 120 Chinese subjects was consisitent with Hardy-Weinberg equilibrium (chi 2 = 7.27, nu = 8, 0.7 > P > 0.5). Fifty subjects (41.7%) were homozygous wildtypes, 50 subjects (41.7%) were heterozygous mutants, and 20 subjects (16.7%) were homozygous mutants. CONCLUSION: The lower frequency of mutant M1 allele compared with that of Caucasians explains the low frequency of slow acylators in Chinese.
OBJECTIVE: To investigate the level of acute phase protein level (APP) synthesized in hepatocytes in pathogenic process of multiple organ dysfunction syndrome (MODS) and the effect of Dachengqi Decoction (DCQD) on it. METHODS: The levels of C-reactive protein (CRP), alpha 1-acid glycoprotein (alpha 1-AGP), alpha 1-antitrypsin total (alpha 1-AT), alpha 2-macroglobulin (alpha 2-MG), ceruloplasmine (Cp), hepatoglobin (Hp) and transferrin (Tf) synthesis were determined in 32 cases of MODS patients and rat models of MODS induced by acute infective peritonitis and mesenterial artery ischemia-reperfusion. RESULTS: The serum APP level in MODS patients was increased and higher than that in the control group (P < 0.01). After 3 days of DCQD treatment, it reduced significantly (P < 0.05). Serum APP level in MODS model was also increased significantly, and after 3 days of DCQD treatment, it reduced significantly (P < 0.05). The APP secreting level of rat hepatocytes cultured in vitro raised after stimulated and activated by lipopolysacchride, which was reduced after cultured with rabbits' serum containing different concentration of effective ingredients of DCQD. The APP inhibiting effect of DCQD was dose dependent. CONCLUSIONS: Purgation therapy with DCQD could reduce the stimulation of pathogenic factors on hepatocytes, protect the tissues and organs from injury caused by excessive inflammatory reaction.
In order to find out the effects of microgravity on the distribution of Ca2+ in asparagus seedlings, tropic reaction of rhizome, the response of cells and organizations, as well as the distribution and transportation in organization under simulated microgravity condition were observed by means of 45CaCl2 labelled autoradiography. The results showed that the distribution of calcium cations highly correlated with gravity. The absorption, distribution and transportation of calcium were greatly affected by gravity.
OBJECTIVE: To study a case of living related liver transplantation. METHOD: The patient was a 10-year-old girl who suffered from congenital diffuse intrahepatic cholangiectasis, recurrent cholangitis and hepatocirrhosis. The donor was the patient's father aged 40. The left lateral lobe of donor's liver was cut and grafted to the patient. Intensive care and treatment as well as follow-up were given after operation. RESULT: For the donor, the operation lasted 400 min, with 410 ml bleeding and 300 g liver removed. The recovery was satisfactory. For the recipient, the operation lasted 652 min, with 1665 ml bleeding, 80 min non-liver stage, 0 min graft hot ischemic stage, and 137 min cold ischemic stage. Immunosuppressive therapy was given using cyclosporin A, azathioprine and adrenocortical hormones. There was an acute rejection 11 days after operation, which was controlled using hormone impulsive therapy. The patient has been surviving 7 months with normal liver function. CONCLUSION: The living related liver transplantation is feasible under modern surgical conditions. It is demonstrated that perfect postoperative management is the key for the successful liver transplantation.
OBJECTIVE: To increase the effect of epilepsy surgery, we use multiple subpial transection (MST) clinically on the basis of experimental study. METHOD: We analyzed the seizure type and course of 116 intractable epilepsy patients treated with MST and surgical technique were modified. RESULT: 100 patients were followed up for 1 to 5 years. Complete control of seizure was obtained in 62 patients (62%), significant reduction (over 75%) in 20 (20%), reduction (over 50%) in 12 (12%), and no change in 6 (6%). The total effective rate was 94%, and the significant effective rate was 82%. No functional defect was found in any patients. CONCLUSION: MST is a effective in surgical treatment of intractable epilepsy and can replace conventional methods. Combined bilateral frontal lobe MST and anterior callosotomy is a good way to treat intractable generalized epilepsy.