Biomedical subjects
Publications and source records attributed to H Xie.
The geniposide and paeoniflorin in Jiaowei Xiaoyao Pills were separated and determined by HPLC, with a C18 column and a mixture of CH3CN-0.1% H3PO4(13:87) as mobile phase. The method is simple, rapid and accurate. The components have a good linearity, and the average recovery with RSD(n = 5) is 101.81% + 2.38% for geniposide and 98.75% + 1.95% for paeoniflorin.
OBJECTIVE: In order to investigate whether cord blood transplantation could induce severe graft versus host disease (GVHD) similar to that in bone marrow transplantation. METHODS: The frequency of allospecific IL-2-secreting helper T lymphocyte (AIHTL) in 17 cord blood and 14 adult peripherial blood samples were detected by limiting dilution assay. RESULTS: No statistically significant difference between the cord blood and adult peripheral blood AIHTLs was revealed. CONCLUSION: Alloreactivity of cord blood was the same as that of adult blood. HLA typing and powerful immunosuppressive therapy should be adopted to reduce the incidence of GVHD in cord blood transplantation.
Dried Lycium Barbarum L. is pretreated simply through shivering, homogenizing and centrifuging, and then the extract is passed through a cation-exchange column to exclude the interference of other amino acids. The effluent is derivatized prior to high performance liquid chromatographic separation on a Zorbax-C8 column. The derivatization reagents consist of A and B (1:9, V/V). A is 4% OPA solution in methanol (W/V); B is urea:sodium phosphate buffer (pH 6.8) = 1:3 (W/V). The mobile phase is methanol:0.01 mol/L sodium acetate (pH 6.8) = 35:65 (V/V). The ultraviolet-visible detector is at 330 nm. 0.1-1.0 mmol/L taurine can be determined quantitatively. The recovery is 100.31% +/- 1.98%. Coefficient of variation is 1.94%.
The levels of R- and S-mephenytoin in human urine were determined by chiral capillary gas chromatography with nitrogen-phosphorus detector. The conditions of the chromatography and detection included a chiral capillary column (Chirasil-Val, 25 m x 0.25 mm i.d., Alltech), column temperature (T) 190 degrees C, injector T 220 degrees C, detector T 240 degrees C, and the flow-rates of 0.85, 3.5 and 120 mL/min for nitrogen, hydrogen and air respectively. Based on the above conditions, the satisfactory separation of R- and S-mephenytoin was gained, and other interference from urine sample was not found. The linear curves for both tested compounds ranged between 12.5 and 2500 microg/L, with a minimum detectable concentration of about 6 microg/L. Because of its simplicity, rapidity, sensitivity and accuracy, this method has been extensively used for testing metabolic ability of S-mephenytoin polymorphic oxidation in the Chinese populations and for the determination of the activity of hepatic drug-metabolizing enzyme CYP2C19 using mephenytoin S/R ratio of the urine samples of subjects as an indicator.
The frequency of micronuclei (MN) in cells of the nasal mucosa, oral mucosa and in lymphocytes was evaluated for 25 students in anatomy classes exposed to formaldehyde (FA) over an 8-week period. Each student served as his or her own control. The time-weighted average concentration (TWA) of formaldehyde in anatomical laboratories and in students' dormitories was 0.508 +/- 0.299 mg/m3 and 0.012 +/- 0.0025 mg/m3, respectively. A higher frequency of micronuclei was observed in nasal and oral exfoliative cells after formaldehyde exposure (3.85 +/- 1.48 vs 1.20 +/- 0.676 and 0.857 +/- 0.558 vs 0.568 +/- 0.317, paired-t test: P < 0.001 and P < 0.01, respectively). No significant increase in the frequency of lymphocyte micronuclei was found after formaldehyde exposure (P > 0.05). The present study shows that nasal mucosa cells exposed through respiration are the chief target of FA-induced genotoxic effects.
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IA-2 is a transmembrane protein tyrosine phosphatase isolated recently from a human insulinoma subtraction library. Its expression in normal human tissues is restricted primarily to the pancreatic islets and brain. In this report, we describe the expression of IA-2 mRNA in a panel consisting of 20 lung tumor cell lines with neuroendocrine and non-neuroendocrine phenotype and 17 non-lung tumor cell lines. IA-2 mRNA was detected in 8 of 11 neuroendocrine small cell lung carcinomas, 4 of 4 non-small cell lung carcinomas with neuroendocrine phenotype, and 11 of 12 non-lung neuroendocrine tumor cell lines. In contrast, IA-2 mRNA was not detected in five non-neuroendocrine lung carcinomas, nor in a panel of other non-neuroendocrine tumor cell lines. The expression pattern of IA-2 mRNA suggests that IA-2 may represent a new marker for neuroendocrine differentiation In human lung cancer cells and perhaps other neuroendocrine tumors.
We previously reported that cross-linking surface immunoglobulin (sIg) leads to induction of the transcription factor CREB in B lymphocytes through phosphorylation at Ser133, despite the lack of an increase in cAMP. Further, cAMP-raising agents fail to induce CREB Ser133 phosphorylation and CRE-dependent gene expression in these cells, which differs sharply from the situation in PC12 rat pheochromocytoma cells where CREB responds to elevation of cAMP through the activity of protein kinase A. In this study, we characterized the signal transduction pathways leading from sIg engagement to CREB activation. By using specific inhibitors for protein kinase C (PKC), Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), and protein kinase A (PKA), we found that anti-Ig-induced CREB Ser133 phosphorylation depends on PKC, but does not require activation of PKA or CaM kinase II. The differential responsiveness of CREB to forskolin in PC12 cells and BAL-17 B cells may relate to the more marked elevation of cAMP in the former as opposed to the latter; however, high concentrations of dbcAMP which should readily enter B cells and artificially increase cAMP levels still failed to induce CREB Ser133 phosphorylation, even in conjunction with a phosphodiesterase inhibitor. Taken together, the cAMP/PKA pathway does not appear to be as active a contributor to CREB phosphorylation in B lymphocytes as in PC12 cells, and does not appear to be involved in sIg-induced, PKC-dependent, CREB activation.
A novel cDNA, IA-2beta, was isolated from a mouse neonatal brain library. The predicted protein sequence revealed an extracellular domain, a transmembrane region, and an intracellular domain. The intracellular domain is 376 amino acids long and 74% identical to the intracellular domain of IA-2, a major autoantigen in insulin-dependent diabetes mellitus (IDDM). A partial sequence of the extracellular domain of IA-2beta indicates that it differs substantially (only 26% identical) from that of IA-2. Both molecules are expressed in islets and brain tissue. Forty-six percent (23 of 50) of the IDDM sera but none of the sera from normal controls (0 of 50) immunoprecipitated the intracellular domain of IA-2beta. Competitive inhibition experiments showed that IDDM sera have autoantibodies that recognize both common and distinct determinants on IA-2 and IA-2beta. Many IDDM sera are known to immunoprecipitate 37-kDa and 40-kDa tryptic fragments from islet cells, but the identity of the precursor protein(s) has remained elusive. The current study shows that treatment of recombinant IA-2beta and IA-2 with trypsin yields a 37-kDa fragment and a 40-kDa fragment, respectively, and that these fragments can be immunoprecipitated with diabetic sera. Absorption of diabetic sera with unlabeled recombinant IA-2 or IA-2beta, prior to incubation with radiolabeled 37-kDa and 40-kDa tryptic fragments derived from insulinoma or glucagonoma cells, blocks the immunoprecipitation of both of these radiolabeled tryptic fragments. We conclude that IA-2beta and IA-2 are the precursors of the 37-kDa and 40-kDa islet cell autoantigens, respectively, and that both IA-2 and IA-2beta are major autoantigens in IDDM.
It would be desirable to develop antiviral agents that can be targeted to liver to enhance their antiviral effects and reduce nonhepatic toxicity. 2',3'-Dideoxyguanosine (ddG) has been found to be a potent and selective antihepatitis B agent both in vitro and in vivo. To evaluate ddG and its liver-targeted analog, we synthesized a series of phosphatidyl-ddGs and incubated them with 2.2.15 cells, which chronically produce hepatitis B virus. 1,2-Dipalmitoylphosphatidyl-dideoxyguanosine (DPP-ddG) inhibited the production of hepatitis B virus (HBV) DNA in the culture medium by 90% at 4.5 mumol/L versus 9.1 mumol/L for ddG, while the liposome vehicle itself had no effect. To compare the efficacy of free ddG with its lipid prodrug in vivo, we treated woodchucks that were experimentally infected with woodchuck hepatitis virus (WHV) for 4 weeks by intraperitoneal injection of 2.6 mumol/kg/d of free ddG or liposomes containing 2.6 mumol/kg/d of DPP-ddG. Liposomal DPP-ddG reduced serum WHV DNA by 23- to 46-fold at the end of the fourth week, while free ddG reduced serum WHV DNA by 2.2- to 10.4-fold. Treatment with small unilamellar liposomes containing DPP-ddG is substantially more effective than free ddG in reducing WHV-DNA levels in serum in WHV-infected woodchucks. The data suggest that the use of lipid prodrugs to target the liver may be useful in enhancing antiviral therapy of hepatitis.
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A new insulin sensitivity test (IST) is described using a modified euglycemic clamp in cats and rats. The IST uses the amount of glucose required to be infused to maintain euglycemia over a 30-min period in rats and 60 min in cats following a bolus administration of insulin as the index of insulin sensitivity. Glucose levels are determined at short time intervals (2-5 min), and variable glucose infusion is used to hold glucose levels within a few percentage points of the basal pre-test glucose level. A new blood sampling procedure is described that allows each IST to be carried out using a total of only 0.5 mL of blood. The IST is sensitive and allows clear insulin dose effects to be demonstrated with 100 mU/kg requiring 355.0 +/- 14.3 mg/kg over 30 min and 50 mU/kg requiring 198.7 +/- 11.1 mg/kg. Five consecutive tests were reproducibly carried out (%CV = 3.0 +/- 0.5) over a 12-hr period in the cat with insulin, glucagon, and glucose levels remaining stable prior to each IST. Glucagon and norepinephrine plasma concentrations do not change significantly during the IST. The IST is sufficiently sensitive to allow demonstration of dose-response relationships for atropine-induced insulin resistance. The IST is thus sensitive, reproducible, and able to demonstrate acute insulin resistance in anesthetized cats and rats. The test is demonstrated in fed (rats) and fasted (cats) state.
Protein disulfide isomerase (PDI) catalyzes protein folding and thiol-disulfide interchange reactions. The enzyme is localized in the lumen of endoplasmic reticulum (ER) and is abundant in secretory cells of various tissues. In this study we describe the isolation and characterization from human pancreas of a new protein, PDIp, that is structurally and functionally related to PDIs. PDIp cDNA is 1,659 bp in length and predicts a protein with an open reading frame of 511 amino acids. PDIp amino acid sequence shows 46% identity and 66% similarity to that of human PDI. PDIp possesses two thioredoxin-like active sites (WCGHCQ and WCTHCK) and an endoplasmic reticulum retention signal sequence, KEEL, at the carboxyl terminus. Northern analysis of normal human tissues and various human tumor cell lines revealed PDIp mRNA (2.0 kb) expression only in the normal pancreas. Recombinant PDIp protein catalyzed reductive cleavage of insulin and renaturation of reduced RNaseA. Somatic cell genetics and fluorescence in situ hybridization localized the PDIp gene to the short arm of human chromosome 16. It is concluded that PDIp is a new member of the PDI family and is highly expressed in human pancreas.
We recently demonstrated that epidermal growth factor receptor (EGFR)-mediated signaling of cell motility and mitogenesis diverge at the immediate post-receptor level. How these two mutually exclusive cell responses cross-communicate is not known. We investigated a possible role for a phospholipase C (PLC)-dependent feedback mechanism that attenuates EGF-induced mitogenesis. Inhibition of PLC gamma activation by U73122 (1 microM) augmented the EGF-induced [3H]thymidine incorporation by 23-55% in two transduced NR6 fibroblast lines expressing motility-responsive EGFR; increased cell division and mitosis was observed in parallel. The time dependence of this increase revealed that it was due to an increase in maximal incorporation and not a foreshortened cell cycle. Motility-responsive cell lines expressing a dominant-negative PLC gamma fragment (PLCz) also demonstrated augmented mitogenic responses by 25-68% when compared with control cells. PLCz- or U73122-augmented mitogenesis was not observed in three non-PLC gamma activating, nonmotility-responsive EGFR-expressing cell lines. Protein kinase C (PKC), which may be activated by PLC-generated second messengers, has been proposed as mediating feedback attenuation due to its capacity to phosphorylate EGFR and inhibit the receptor's tyrosine kinase activity. Inhibition of PKC by Calphostin C (0.05 microM) resulted in a 57% augmentation in the fold of EGF-induced thymidine incorporation. To further establish PKC's role in this feedback attenuation mechanism, an EGFR point mutation, in which the PKC target threonine654 was replaced by alanine, was expressed. Cells expressing these PKC-resistant EGFR constructs demonstrated EGF-induced motility comparable to cells expressing the threonine-containing EGFR. However, when these cells were treated with U73122 or Calphostin C, the mitogenic responses are not enhanced. These findings suggest a model in which PKC activation subsequent to triggering of motility-associated PLC gamma activity attenuates the EGFR mitogenic response.
The objective was to determine the site of insulin resistance produced by intraportal atropine or surgical hepatic denervation. A modified euglycemic clamp was used in fasted cats to test the acute effect of insulin (100 mU/kg) on arteriovenous glucose gradients across the hindlimbs (mainly reflecting skeletal muscle), the guts (all organs draining into the portal vein), and the liver. Responses to insulin were determined before and after hepatic denervation and after 3 mg/kg intraportal atropine. The interventions were done in random order. Responses after either intervention were similar and were not potentiated by the combined treatment. Regional insulin resistance was assessed by comparing the change in glucose gradients in response to insulin before and after treatments. Hepatic and gut responses to insulin were unaltered, but hindlimb responses were significantly impaired after denervation or atropine. We speculate that the hepatic parasympathetic nerves regulate release of a liver-generated factor that selectively controls insulin effectiveness in skeletal muscle. This mechanism may be involved with insulin resistance in non-insulin-dependent diabetes and chronic liver disease.
The role of hepatic parasympathetic nerves in insulin effectiveness was evaluated in fed anesthetized rats. Insulin sensitivity was assessed using a modified euglycemic clamp to quantitate the amount of glucose required to maintain euglycemia over 60 min after administration of insulin (50 mU/kg). With normal innervation, intraportal infusions of acetylcholine (ACh, 2.5 micrograms.kg-1.min-1) did not alter insulin sensitivity, but intraportal venous atropine (3 mg/kg) reduced insulin sensitivity (49.4 +/- 5.8%, P < 0.001, n = 7). Liver denervation resulted in reduction of insulin responsiveness by 70.8 +/- 5.8% (P < 0.001, n = 8), which was fully (96.8 +/- 12.5%) reversed by ACh. ACh into the portal vein reversed insulin resistance produced by denervation, but intravenous ACh was without effect, thus showing that the liver was the site of ACh action. Regression analysis suggests that some component of insulin response is not dependent on hepatic cholinergic nerve effects but that virtually all of the variability in response to insulin in normal fed rats tested under these conditions could be accounted for by variability in the hepatic parasympathetic-dependent insulin sensitivity. These data suggest a major role for hepatic parasympathetic nerves in regulation of whole body clearance of glucose in response to insulin.
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