Biomedical subjects
Publications and source records attributed to Z Qu.
In this paper, the determination of trace lead in foods by optical temperature control GFAAS was studied. The method was certified by the analysis of the national standard reference materials.
The determination of trace mercury using the self-developed circulating enrichment apparatus with cold vapour atomic absorption spectrometry was investigated. Various experimental conditions were discussed and optimized. The effect of interferences was examined. The developed method is sensitive, accurate, simple and available and has already been used in the analysis of various kinds of food samples and national standard reference materials, and the results obtained were satisfactory.
OBJECTIVE: To construct retroviral vector carrying rapidly selective marker. METHODS: The recombination retroviral vector GCGFPPXSN was constructed by cloning the green fluorescent protein (GFP) cDNA into the retroviral vector containing putative internal ribosome entry sites GCXPXSN and transferred in ecotropic packaging cell line PE501 by electroporation method. The supernatants of the PE501GCGFPPXSN were used to infect the amphotropic packaging cell line PA317. The G418 resistant clones were selected in 4 weeks and were detectable by fluorescence microscopy or by fluorescence-activated cell sorting(FACS). RESULTS: A recombination retroviral vector GCGFPPXSN carrying rapidly selective marker GFP was constructed. GFP expression in packaging cell line PA317-GCGFPPXSN transferred by GCGFPPXSN was detected by fluorescence microscopy of FACS. PA317-GCGFPPXSN grew to a titer of 1.2 x 10(5) cpu/ml. By cocultivating retroviral vector producing cells and T lymphocytes, expression of GFP was observed in T lymphocytes 2 days after the end of the cocultivation. T lymphocytes expressing GFP were separated. CONCLUSION: The mammalian cell can be efficient gene transfected by retroviral vector carrying GFP. The use of GFP for cell marking represents an important advantage over conventional strategies which typically involve the use of neomycin resistance. GFP, in fact, allows a rapid in vitro selection of transduced cell by FACS. The selection requires only two-day culture with this retroviral vector, compared with 10-14 day culture with a classical retroviral vector.
PURPOSE: To compare the mRNA expression of growth factor receptors in cultured human trabecular meshwork (HTM) cells with ex vivo HTM tissues and to determine whether HTM cells generate a physiologic response after exposure to exogenous growth factors. METHODS: The reverse transcription-polymerase chain reaction (RT-PCR) method was used to detect the expression of various growth factor receptor mRNAs using early passaged, cultured HTM cells from donors of several ages. RT-PCR on ex vivo HTM tissues from healthy donors and donors with glaucoma were also used to compare and contrast mRNA expression with cell culture results. After the exogenous administration of growth factors, cell proliferation and extracellular acidification rate studies were used to measure the functional responses of HTM cells to growth factors. RESULTS: Amplification products of the expected size for 15 growth factor receptors were detected in cultured HTM cells and in ex vivo HTM tissues. The administration of exogenous growth factors showed that (a) hepatocyte growth factor (HGF), epidermal growth factor (EGF), insulinlike growth factor (IGF)-1, tumor necrosis factor (TNF) alpha, platelet-derived growth factor (PDGF)-AA, PDGF-BB, PDGF-AB, and basic fibroblast growth factor (FGF-2) stimulated cell proliferation, whereas FGF-1 (acidic), transforming growth factor (TGF) alpha, interleukin (IL)-1alpha, nerve growth factor (NGF), and FGF-7 (keratinocyte growth factor [KGF]) had no significant influence on cell proliferation; (b) TGF-beta isoforms significantly inhibited EGF-stimulated trabecular meshwork cell proliferation; and (c) FGF-1 (acidic), TGF-alpha, EGF, IL-1alpha, IL-1beta, HGF, TNF-alpha, PDGF-AA, and IGF-1 significantly stimulated extracellular acidification, whereas FGF-2 (basic), FGF-7 (KGF), TGF-beta1-beta3 and NGF had no significant influence on extracellular acidification. CONCLUSIONS: These studies show that mRNA for numerous growth factor receptors can be detected in cultured HTM cells and in ex vivo HTM tissues. They also show that many of the receptors are functional, because exogenous growth factor administration elicits a physiologic response. In vivo, these receptors may be activated by growth factors present within the aqueous humor (aquecrine/paracrine) or by growth factors synthesized and released locally by trabecular meshwork cells themselves (autocrine). Specific growth factors acting through high-affinity receptors may be involved in maintaining the normal microenvironment of the HTM and also may be involved in the pathogenesis of primary open-angle glaucoma.
BACKGROUND: LL2 is a murine immunoglobulin (Ig)G2a-kappa anti-B-cell monoclonal antibody with proven targeting and therapeutic efficacy in the management of non-Hodgkin's lymphoma (NHL). The authors had previously generated a humanized LL2 (hLL2) that demonstrated binding properties identical to those of LL2. Nevertheless, the productivity of the cell line was insufficient for large-scale production of the antibody for clinical studies. Therefore, the authors chose an amplifiable system for the generation of hLL2. METHODS: The hLL2 sequences were ligated into the expression vector pdHL2, which has a dhfr amplifiable gene, and were incorporated into the SP2/0 cells by electroporation. A methotrexate (MTX) resistant clone producing hLL2 was identified. Stepwise increases in MTX concentrations, from 0.1 to 5 microM, and subcloning of the cells by limiting dilution were performed. RESULTS: By amplifying the dhfr and hLL2 genes with stepwise increases in the MTX concentration, the antibody production was enhanced from its original 1.4 to 70 +/- 5 mg per liter of culture media. Subsequent subcloning further improved the productivity. Immunoreactivity of the antibody was conserved, as proven by enzyme-linked immunosorbent assay and cell-binding assays. By isoelectrofocusing, the isoelectric point (pI) of the antibody was measured at approximately 9.6. The productivity of the clone was not affected by culture conditions or storage of the cells in liquid nitrogen. CONCLUSIONS: By means of gene amplification, the authors have generated a high-producing hLL2-IgG clone suitable for production of the quantity of antibody necessary for clinical diagnostic and therapeutic trials of NHL patients.
An efficient modified adeno system to express foreign genes to the central nervous system was developed recently. This modified recombinant adenoviral vectors can be used successfully to deliver lacZ to the hypothalamic suprachiasmatic nucleus (SCN) which is composed of mammalian clock pacemaker. The expression of lacZ in the primary culture of SCN was dose-dependent and higher enough in nearly 100% of these cells. We also showed that viral toxicity and lacZ overexpression had no serious effects on the rhythmic expression of arginine vasopressin (AVP) release from the SCN cell culture.
The propagation of electrical excitation in a ring of cells described by the Noble, Beeler-Reuter, Luo-Rudy I, and third-order simplified mathematical models is studied using computer simulation. For each of the models it is shown that after transition from steady-state circulation to quasiperiodicity achieved by shortening the ring length (RL), the action potential duration (APD) restitution curve becomes a double-valued function and is located below the original (that of an isolated cell) APD restitution curve. The distributions of APD and diastolic interval along a ring for the entire range of RL corresponding to quasiperiodic oscillations remain periodic with the period slightly different from two RLs. The sigmoidal shape of the original APD restitution curve determines the appearance of the second steady-state circulation region for short RLs. For all the models and the wide variety of their original APD restitution curves, no transition from quasiperiodicity to chaos was observed.
IFN beta-1b reduces the frequency of major multiple sclerosis attacks by 50 percent. Serial MRI scanning over the course of the clinical trial that led to approval of the agent revealed a significant lessening both in disease activity and in accumulating burden of disease in IFN beta-1b-treated patients compared to placebo-treated controls. The mechanism by which IFN beta-1b exerts its beneficial effect in multiple sclerosis is unknown. T suppressor cell function fails during MS attacks and is persistently subnormal in multiple sclerosis patients with progressive disease. IFN beta-1b partially restores suppressor function in multiple sclerosis patients. IFN beta-1b also inhibits release of lymphotoxin, tumor necrosis factor, and interferon gamma, at least in vitro. All three cytokines are toxic to oligodendrocytes. In contrast; production of transforming growth factor beta-1 (TGF beta 1) is increased by IFN beta-1b. TGF beta 1 is an immunosuppressive cytokine. All of the above listed actions of IFN beta-1b could contribute to its beneficial effect. Perhaps all do.
A double staining method combining Ulex europaeus agglutinin I lectin (UEA-I) and collagen type IV staining was used to determine the capillary density and the number of capillaries relative to different fibre types in human skeletal muscles. The result of this combined staining was compared with that of other staining methods including amylase-periodic acid Schiff (PAS), UEA-I, anti-collagen type IV and anti-von Willebrand factor. Muscle biopsy specimens, 12 from M. vastus lateralis and 6 from M. soleus, were obtained from 18 healthy young men. Compared with amylase-PAS staining, double staining showed a larger number of capillaries surrounding type I (+9.6%), type IIA (+8.6%) and type IIB (+11.6%) fibres in the M. vastus lateralis specimens (P < 0.001 for all differences). The capillary to fibre ratio (cap fibre-1) and the capillary density (cap.mm-2) were 8.3% (P < 0.002) and 7.9% (P < 0.001) larger, respectively. In the M. soleus specimens, cap.fibre-1 and cap.mm-2 were 7.4 and 9.9% larger, respectively, by double staining compared with PAS staining. Further comparisons showed that the cap.fibre-1 and cap.mm-2 obtained with double staining were similar to the values determined by the UEA-I staining, but greater than that measured by the collagen type IV method. The double staining gave a more marked stain of capillaries and revealed muscle fibre borders clearly, which is an advantage in studies that require comparisons between serial sections using computerised image analyses. It is concluded that the double staining method is superior to either the UEA-I, collagen type IV or the traditional amylase-PAS staining methods in analysing capillary density of normal human skeletal muscle.
Dietary protein restriction decreases plasma concentrations of insulin-like growth factor-I (IGF-I) and reduces IGF-I mRNA levels in the liver. In addition to the actions of systemic IGF-I, locally produced IGF-I is thought to mediate autocrine and paracrine growth effects in the colon. The objectives of the present study were to investigate the IGF-I pathway in the colon and liver of adult rats under conditions of dietary protein restriction, surgical stress, and dietary protein repletion. Two groups of rats were placed on either a 20% or 2% casein diet for 19 days. Two additional groups of rats underwent gastrostomy after a 2% casein diet for 2 weeks, and then were either kept on the 2% casein diet or changed to a 20% casein diet until day 19. Dietary protein restriction reduced plasma concentrations of IGF-I and IGF-binding proteins (IGFBPs) and hepatic IGF-I mRNA content, while increasing colonic IGF-I receptor mRNA. Gastrostomy in protein-depleted animals had no effect on hepatic IGF-I mRNA, but led to a marked increase in colonic IGF-I mRNA levels. Dietary protein repletion resulted in a decrease in colonic IGF-I receptor mRNA. The distinct effects of dietary protein depletion and operative stress on the IGF pathway in the colon as compared with the liver may serve to maintain the level of IGF-I signaling in the colon by autocrine or paracrine mechanisms under these conditions.
The purpose of this study was to determine the effects of insulin-like growth factor-I (IGF-I) on glucose metabolism in normal and tumor necrosis factor (TNF)-treated rats under euglycemic and hyperinsulinemic conditions. During a hyperinsulinemic clamp (10 mU/kg.min), rats further received either saline or IGF-I (3.33 micrograms/kg.min) infusion for 2 hours. Glucose kinetics were determined with [3H-3]-glucose. Glucose utilization in peripheral tissues was examined by glucose uptake using [14C-2]-deoxyglucose (14C-DG) and by glycogen content in select tissues. The results showed that TNF infusion significantly decreased the rate of glucose infusion required to maintain euglycemia. TNF decreased glycogen content significantly in liver and marginally in abdominis muscle. TNF also decreased glucose uptake in muscle, although the decrease was only statistically significant compared with IGF-I infusion. In addition, TNF significantly reduced plasma IGF-I concentration. However, during hyperinsulinemic and euglycemic conditions, exogenous IGF-I significantly increased glucose uptake in muscle and glycogen storage in the liver and abdominis muscle in both saline- and TNF-treated groups. IGF-I normalized each of the effects of TNF in the rats, including those on plasma IGF-I, glucose uptake in muscle, and glycogen content in liver and abdominis muscle. These data suggest that under hyperinsulinemic and euglycemic conditions, TNF-treated rats, although resistant to insulin, have a normal response to IGF-I, indicating that the TNF-induced defect in the insulin pathway may not be a step in the IGF-I pathway.
The sequences were determined of RNAs 3 and 4 of a Chinese isolate (Y) of rice stripe tenuivirus (RStV) and were compared with those of two RStV isolates (M and T) from Japan. Both RNAs of the Y isolate were longer than those of the M and T isolates. There was almost complete conservation in the 5' and 3' non-coding regions for each RNA between the isolates. The analogous ambisense coding regions for each isolate were exactly the same size and the sequences were highly conserved. The major differences were in the intergenic regions, the sizes of which accounted for the differences in size of each RNA of the three isolates. There were no obvious patterns of differences in comparisons of the two RNA over the three isolates. The significance of the similarities and differences in sequences of isolates of RStV separated by more than 3500 km is discussed.
Two humanized antibody mutants, hLL2HCN1 and hLL2HCN5, engineered with CH1 domain-appended carbohydrates (CHOs) were generated to facilitate site-specific conjugation of radionuclides and anti-cancer drugs to antibodies. Such site-specific conjugation may minimize the incidence of immunoreactivity perturbation as is often observed with random conjugation. Since the compositions and structures of CHOs are important in determining the chemistry, efficiency, and extent of conjugation, the sequences of the CH1-appended CHOs were determined by exoglycosidase digestions and fluorophore-assisted CHO electrophoresis (FACE). The CHO species attached at HCN1 and HCN5 sites in hLL2HCN1 and hLL2HCN5, respectively, were distinct from each other, heterogeneous, and extensively processed. All of these CHOs were core-fucosylated complex-type oligosaccharides and contained Gal (galactose) and GlcNAc (N-acetylglucosamine) residues in the outer branches. Some of the outer branches were composed of Gal alpha1-3Galbeta1-4GlcNAc structure, also known as alpha-galactosyl epitope. Most of the CHOs were sialylated. While all HCN1-CHOs were biantennary, the majority of HCN5-CHOs (>60%) were triantennary. The CH1-appended CHOs have favorable structural characteristics suitable for site-specific conjugation. For efficient conjugation of large drug complexes, hLL2HCN5 is preferable to hLL2HCN1 because the attached CHO is larger in size and more remotely positioned from the V region. The effects of the alpha-galactosyl epitope found in these CHOs on the immunological properties of the immunoconjugates as efficient cancer diagnostics and therapeutics are being studied.
Insulin resistance is a common feature in first-degree relatives of NIDDM patients. To explore the mechanism(s) behind this condition in more detail, a percutaneous muscle biopsy (vastus lateralis) was performed in 25 first-degree relatives of NIDDM patients and 21 control subjects to examine muscle fiber composition and capillary density. Insulin-stimulated glucose disposal (Rd) was determined employing a hyperinsulinemic-(insulin infusion rate 0.6 mU x kg[-1] x min[-1]) euglycemic clamp. Rd (5.76 +/- 0.35 vs. 8.06 +/- 0.36 mg x kg lean body weight [LBW]-1 x min[-], P < 0.001) and estimated VO2max (49.3 +/- 2.8 vs. 57.2 +/- 3.5 mg x kg LBW[-1] x min[-1], 0.05 < P < 0.10) were decreased in the relatives. The number of type IIb fibers (29.5 +/- 2.5 vs. 21.0 +/- 2.8%, P < 0.05) was increased in the relatives, whereas no significant differences were found in other fiber types or capillary density between the groups. Correlations were observed between number of type I fibers (positive), number of type IIb fibers (negative), and capillary density (positive) versus Rd as well as estimated VO2max (P < 0.05). In a multiple linear regression analysis with Rd as a dependent variable, estimated VO2max, family history of NIDDM, and number of type IIb fibers (P < 0.001, r2 = 0.64) significantly determined the level of Rd, whereas capillary density did not. In conclusion, insulin-resistant first-degree relatives of NIDDM patients are characterized by an increased number of type IIb muscle fibers. Whether this finding reflects a reduced physical activity level and fitness in the relatives or is of primary genetic origin remains to be determined.
Basic fibroblast growth factor (bFGF) is a potent inducer of growth and proliferation for many cell types involved in wound healing. Although bFGF has previously been identified in lung tissue, its role in the pathogenesis of pulmonary fibrosis is unknown. We investigated the distribution of bFGF after bleomycin-induced lung injury in the rat in hope of learning how bFGF might participate in the process of lung injury, repair and fibrosis. Increased immunoreactive bFGF was found in the extracellular matrix after bleomycin and co-localized to a marker of active cell proliferation. This suggests that bFGF may participate in directing cell proliferation following lung injury. In addition, a marked increase in the number of mast cells with strong reactivity for bFGF was found at days 14 and 21 after bleomycin. These cells may represent a source of bFGF during the fibroproliferative stage after lung injury.
OBJECTIVE: To study the relationship between the intercellular adhesion molecule 1 (ICAM-1) mRNA expression of hepatocellular carcinoma (HCC) and its invasion and metastasis. METHODS: 34 cases of HCC and their pericarcinomatous tissues were studied on cryostat sections by in situ hybridization to investigate ICAM-1 mRNA expression. RESULTS: The ICAM-1 mRNA expression was stronger in the carcinomatous tissues of 19/21 invasive cases than that in the pericarcinomatous tissues, while in the 13 non-invasive cases, only 4 exhibited weak ICAM-1 mRNA expression. CONCLUSION: ICAM-1 mRNA expression may correlate with increased risk of invasion and metastasis of HCC.
OBJECTIVE: To explore an approach to the reduction of activation induced apoptosis of T cell so as to maximize the number of CTL and enhance the tumor cytotoxicity. METHODS: CD3-induced Jurkat cell line was used as the activated T cell apoptosis model,and the antisense Fas cDNA was introduced into Jurkat cells with retroviral vector. RESULTS: The expression level of Fas protein in Jurkat cells transducted with the retroviral vector was decreased. Apoptosis was reduced in antisense Fas-transfected Jurkat cells after anti-CD3 or anti-Fas treatment. CONCLUSION: Antisense blocking of Fas expression can partially inhibit Jurkat cell apoptosis induced by anti-CD3 or anti Fas.
The expression of MHC class II molecules is normally restricted to antigen presenting cells. Aberrant expression of class II molecules, however, was detected in the thyrocytes of autoimmune thyroid diseases. We attempted to regulate the expression of HLA-DR molecules in thyroid carcinoma cells by expressing the exogenous poly(ADP-ribose) synthetase gene. We transfected a metal inducible expression plasmid capable of expressing poly(ADP-ribose) synthetase gene into thyroid carcinoma 8505C cells and the transformants, treated with metal and IFN-gamma, were separated by Magnetic Cell Separation. The activity of the synthetase was increased in the HLA-DR-enriched transformants as compared with that in control or the HLA-DR+ transformants. RNA blot analysis and flow cytometric analysis revealed that the IFN-gamma-inducible expression of HLA-DR molecules was depressed by the induction of exogenous poly(ADP-ribose) synthetase gene. This result indicates that HLA-DR expression was correlated with the level of poly(ADP-ribose) synthetase in human thyroid carcinoma cells. Furthermore we examined the level of poly(ADP-ribose) synthetase in patients with autoimmune thyroid diseases. We observed a significant decrease in poly(ADP-ribose) synthetase in the patients. Taken together with the previous observation, the decrease in poly(ADP-ribose) synthetase is closely linked to the aberrant expression of HLA-DR molecules in some autoimmune thyroid diseases.