Biomedical subjects
Publications and source records attributed to F Shen.
A novel non-invasive method for diabetes diagnosis based on an electronic nose is proposed in this paper. The principles of the method and results of clinical experiments are presented. These results show the convenience, painlessness and non-invasiveness of the method.
Antitumor immune responses are mediated primarily by T cells. Downregulation of the major histocompatibility complex (MHC) and the molecules that costimulate the immune response is associated with defective signaling by tumor cells for T-cell activation. In vitro treatment with a combination of cytokines significantly increased the expression of MHC class I and adhesion molecules on tumor cell surfaces. When tumor cells were first incubated with a bispecific monoclonal antibody that binds antigen on tumor cells to CD28 on T cells, the modified tumor cells become immunogenic and are able to stimulate naive T cells, generating tumor-specific cytotoxic T cells in vitro. Immunization with the modified tumor cells elicits an immune response mediated by CD8+ T cells. This response protected against a challenge with parental tumor cells and cured established tumors. The approach was effective in both low immunogenic and nonimmunogenic tumor model systems. Modification of tumor cells with this two-step procedure may provide a strategy for development of tumor vaccines that is effective for cancer immunotherapy.
With the same forage to the mice, three experiments were done. The control group took water and experimental group took 1% taurine. 1. After the mice were vaccinated with S180 sarcoma, every group was further divided into two groups, one treated with cytoxan (CTX), another one treated with nothing. The survival period, weight of the tumor and the tumor inhibition rate were investigated. The result shows: The survival period of the group fed with taurine was (35.75 +/- 23.7) days longer than that of the control group (18.7 +/- 5.6) days, and the tumor inhibition rate was 42.26%. The mice fed with taurine and treated with CTX survived to 61 days with no death, and no tumor could be seen grossly, so the tumor inhibition rate is 100%. The survival period and weight of the tumor were not different between the CTX treated and control groups. 2. Tumor cell membrane fluidity was observed after the mice were vaccinated with Erlish ascitic tumor. The result shows: in the taurine group, the fluorescence polarization (p) is 0.108 +/- 0.020, the viscosity (eta) is 0.618 +/- 0.140 and in the control group p is 0.139 +/- 0.20, eta is 0.874 +/- 0.150, respectively. Thus the cell membrane fluidity of the taurine group is also obviously better than that of the control group (P < 0.01). 3. The immune functions of the two groups were determined by carbon clearance test and capacity of serum hemolysin. The immune function of the taurine group is also obviously higher than that of control group (P < 0.01). All the results described above show:taurine is functioning for tumor inhibition, and has obvious synergic effect while treated with CTX as chemotherophy. It is considered that the tumor inhibit function of taurine may be related to its effects of immune enhancing and membrane profecting.
To investigate a new kind of anti-tumor immunological cells and improve surgical results of hepatocellular carcinoma by anti-recurrence application, we activated T cells isolated from tumor infiltrating lymphocytes by double stimulating signals: the one was autologus HCC cells which were treated with IFN-gamma and TNF-alpha for enhancing expression of MHC class I and presented tumor antigen, and the other was costimulation signals which was from ICAM-1 and B7 molecules expressed on treated HCC cells as well as CD28 mAbs. Activated T cells which bound to HCC cells were expanded selectively as tumor specific cytotoxic T lymphocytes (TS-CTLs), their cytotoxic activity in vitro and anti-tumor effects in vivo were observed. Our results suggested that TS-CTLs expressed high cytotoxicity against autologous HCC cells with MHC class I restriction manner. Adoptive TS-CTLs treatment could decrease serum AFP level, inhibit ascites formation and prolong survival in SCID mice bearing human HCC. In clinical trail of 12 cases of HCC, TS-CTLs treatment was able to delay tumor recurrence after HCC resection. Our data demonstrated that TS-CTLs as new immunological treatment modality, are of great value in further application of tumor comprehensive management.
OBJECTIVE: To study the changes of synthesis and secretion of transferrin (Tf) in human hepatocellular carcinoma (HCC) cells and search the causes of reduced membrane-bond transferrin in HCC tissue. METHODS: By radioisotope incorporation and immunoprecipitation to determine the synthesis and secretion rate of transferrin in human HCC cells and peritumor hepatocytes. RESULTS: When compared with peritumor hepatocytes the quantity of transferrin synthesized was not changed in HCC cells, but its secretion rate was accelerated significantly while the transferrin storage was significantly decreased. CONCLUSION: The results suggest that HCC growth might be enhanced by increased Tf secretion and help explain the decreased membrane-bond form of Tf in HCC tissue.
Ovarian carcinoma is the fourth most common cause of cancer death in women and there has been a steady increase in the age-adjusted cancer death rates in the past 25 years in the US. However, patients who become cisplatin resistant respond poorly to available cytotoxic agents; therefore, discovering novel targets for ovarian carcinoma is vital. Quercetin, an anticancer agent, arrests the cell cycle at G1 and S phase boundary. Genistein, a plant flavonoid, attacks the cell cycle at G2 and/or early M phases in most carcinoma cells. Quercetin and genistein block the phosphatidylinositol conversion to IP3 signal transduction pathway mainly by inhibiting 1-phosphatidylinositol 4-kinase (PI kinase, EC 2.7.1.67) and 1-phosphatidylinositol 4-phosphate 5-kinase (PIP kinase, EC 2.7.1.68), respectively. Because each drug attacks a different phase of the cell cycle and reduces IP3 concentration by attacking different signal transduction enzymes, we tested the hypothesis that the two drugs might be synergistic in human carcinoma cells. In human ovarian carcinoma OVCAR-5 cells in growth inhibition assay, the IC50S for quercetin and genistein were (mean +/- SE) 66 +/- 3.0 and 32 +/- 2.5 microM; in clonogenic assays they were 15 +/- 1.2 and 5 +/- 0.5 microM, respectively. When quercetin was added to the cultures of OVCAR-5 cells followed 8 h later by genistein, synergism was observed in growth inhibition and clonogenic assays. The synergistic action of quercetin and genistein may be of interest in clinical treatment of human ovarian carcinoma.
Eukaryotic transcriptional regulatory proteins typically comprise both a DNA binding domain and a regulatory domain. Although the structures of many DNA binding domains have been elucidated, no detailed structures are yet available for transcriptional activation domains. The activation domain of the herpesvirus protein VP16 has been an important model in mutational and biochemical studies. Here, we characterize the VP16 activation domain using time-resolved and steady-state fluorescence. Unique intrinsic fluorescent probes were obtained by replacing phenylalanine residues with tryptophan at position 442 or 473 of the activation domain of VP16 (residues 413-490, or subdomains thereof), linked to the DNA binding domain of the yeast protein GAL4. Emission spectra and quenching properties of Trp at either position were characteristic of fully exposed Trp. Time-resolved anisotropy decay measurements suggested that both Trp residues were associated with substantial segmental motion. The Trp residues at either position showed nearly identical fluorescence properties in either the full-length activation domain or relevant subdomains, suggesting that the two subdomains are similarly unstructured and have little effect on each other. As this domain may directly interact with several target proteins, it is likely that a significant structural transition accompanies these interactions.
The transcriptional activation domain of the herpesvirus protein VP16 resides in the carboxyl-terminal 78 amino acids (residues 413-490). Fluorescence analyses of this domain indicated that critical amino acids are solvent-exposed in highly mobile segments. To examine interactions between VP16 and components of the basal transcriptional machinery, we incorporated (at position 442 or 473 of VP16) tryptophan analogs that can be selectively excited in complexes with other Trp-containing proteins. TATA-box binding protein (TBP) (but not transcription factor B (TFIIB)) caused concentration-dependent changes in the steady-state anisotropy of VP16, from which equilibrium binding constants were calculated. Quenching of the fluorescence from either position (442 or 473) was significantly affected by TBP, whereas TFIIB affected quenching only at position 473. 7-aza-Trp residues at either position showed a emission spectral shift in the presence of TBP (but not TFIIB), indicating a change to a more hydrophobic environment. In anisotropy decay experiments, TBP reduced the segmental motion at either position; in contrast, TFIIB induced a slight change only at position 473. Our results support models of TBP as a target protein for transcriptional activators and suggest that ordered structure in the VP16 activation domain is induced upon interaction with target proteins.
Rab proteins are Ras-related small GTPases that are digeranylgeranylated at carboxyl-terminal cysteines, a modification essential for their action as molecular switches regulating intracellular vesicular transport. Geranylgeranylation of Rabs is a complex reaction that requires a catalytic Rab geranylgeranyl transferase (GGTase) and a Rab escort protein (REP). REP binds unprenylated Rab and presents it to Rab GGTase. After GG transfer, REP remains associated with diGG-Rab, which leads to insertion of the Rab into a specific membrane. We used recombinant Rab1a single cysteine mutants that accept only one GG group to study the mechanism of the digeranylgeranylation reaction. Using the prenylation assay, gel filtration chromatography, and density ultracentrifugation, we show that REP, but not Rab GGTase, forms a stable complex with unprenylated, monoGG- and diGG-Rab1a. The REP.monoGG-Rab1a complex is stable in the presence of detergents or phospholipids, whereas the REP.diGG-Rab1a complex partially dissociates under these conditions. The stoichiometry of the REP.Rab complex appears to be 1:1 before prenylation. Prenylation induces a change in complex stoichiometry, with the formation of a 2:2 or 2:1 REP.Rab complex. A possible mechanism by which Rab proteins are digeranylgeranylated is suggested by the current studies. We propose that each geranylgeranyl addition is an independent reaction that leads to the production of monoGG-Rab and diGG-Rab, respectively. The stability of the REP.monoGG-Rab complex prevents monoGG-Rab from dissociating from REP prior to the second geranylgeranylation reaction, ensuring efficient digeranylgeranylation of Rab substrates.
Although several pathophysiological sequences, such as protease activation, free radical generation, and inflammatory mediator release, have been described in acute pancreatitis, the precise mechanism by which acute pancreatitis is initiated is unknown. Cellular calcium, a key physiological signaling element in cell function and also a crucial pathological intracellular messenger in cell injury, appears to be involved in the initiation and development of acute pancreatitis. The present study provides several lines of evidence supporting this suggestion. First, verapamil (a calcium channel blocker) administration was associated with a significant protection of rats from acute pancreatitis induced by high doses of cerulein (50 micrograms/kg/hr, subcutaneously), as evidenced both histologically and biochemically. Second, verapamil was found to minimize the increased tissue levels of calcium, platelet-activating factor, and thromboxane B2 detected during acute pancreatitis. Third, acute pancreatitis could be observed in rats with elevated serum calcium levels at low doses of cerulein (5 micrograms/kg/hr, subcutaneously), but could not be observed in rats with normal serum calcium levels treated with low doses of cerulein. It is proposed that cellular calcium, which is a critical signaling component in the synthesis and release of inflammatory mediators and several other events, may be an important factor in the pathogenesis of cerulein-induced acute pancreatitis.
Hormone-stimulated cellular Ca2+ mobilization in the isolated perfused rat pancreas was investigated by analyzing the efflux profiles of 45Ca2+ from 45Ca(2+)-loaded pancreata following agonist stimulation. The increased 45Ca2+ efflux reflects the enhanced exchange of Ca2+ across the plasma membrane as a result of increased [Ca2+]i. Both high and low concentrations of the cholecystokinin analog, cerulein, applied to the isolated perfused pancreas gave rise to an increased release of 45Ca3+. The patterns of the increase in 45Ca2+ release were consistently different for high and low concentrations of the agonist. Cerulein infused at a concentration of 10(-11) M induced a release of a small but significant amount of 45Ca2+ which could be abolished by 8-(N,N-diethylamine)octyl-3,4,5-trimethoxy-benzoate (TMB-8), but was not affected by ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). Cerulein stimulation at 10(-9) M elicited a marked increase in 45Ca2+ release which was minimized by EGTA, but not by TMB-8. Also, infusion of cerulein stimulated a concentration-dependent amylase secretion response which displayed the same TMB-8- and EGTA-sensitivity pattern as the 45Ca2+ release response. The present study suggests (i) that cellular Ca2+ influx is a prominent feature of the increased 45Ca2+ efflux (i.e., increased [Ca2+]i) induced by pharmacological concentrations of cerulein while physiological concentrations of cerulein cause an increase in [Ca2+]i which is due predominantly to a release of internal Ca2+; and (ii) [Ca2+]i changes are essential for pancreatic enzyme secretion. Although isolated pancreatic acini or cells may lose their sensitivity and physiological responses to various agonists during isolation and preparation, the isolated perfused pancreas is a suitable and very sensitive model in which to study the physiology of Ca2+ mobilization and enzyme secretion.
(1) In all examined rat and human tissues and cells, PIP kinase activity was rate-limiting and PLC activity was present in great excess. (2) The steady-state activities of the signal transduction enzymes, PI kinase, PIP kinase and PLC, and the concentration of the end product, IP3, were determined in rat liver and hepatomas of different malignancies. The activities of all three enzymes were elevated in the hepatomas in a non-random fashion. A generalization emerged that the enzyme with the lowest activity in liver, PIP kinase, increased to the highest extent and the enzyme with the highest activity in liver, PLC, increased to the smallest extent in rapidly growing hepatomas. The IP3 concentration in the hepatomas was elevated in a progression-linked fashion. (3) The three signal transduction enzyme activities were elevated in human ovarian carcinoma samples and in human breast carcinoma cells. (4) When human breast carcinoma MDA-MB-435 cells were allowed to go through lag, log and plateau phases, the IP3 concentration reached a 20-fold peak at 12 hr after plating. The elevation in IP3 concentration preceded the rise in PI and PIP kinase activities which increased 11-fold in the log phase. The IP3 concentration and PI and PIP kinase activities returned to their baseline levels when the plateau phase was reached. The PLC activity did not change significantly during the whole period. (5) Administration of cycloheximide i.p. in rats revealed short half-lives in the bone marrow for the two kinases (8 min) and a long half-life for PLC (> 6 hr). In a group of 10 enzymes, the half-lives of the kinases were the shortest. In cycloheximide-injected rats, the bone marrow IP3 concentration was reduced to about 50% in 30 min. The reduction of IP3 concentration is attributed to the decline to 15 and 12%, respectively, in PI and PIP kinase activities since PLC activity did not change. (6) In 3-day starved rats, the bone marrow PI and PIP kinase were reduced to activities (13%) that were markedly lower than the decrease in the protein concentration (to 55%). By contrast, the PLC activity was preferentially maintained (to 78%) over the protein level. Under starvation, the IP3 concentration decreased (to 24%), indicating that starvation can markedly disrupt IP3 homeostasis. Refeeding returned the enzymic activities and the IP3 concentration to the normal level in bone marrow in 24 hr. (7) Comparison of the absolute activities of PI and PIP kinases and PLC showed that PLC is present in an excess; therefore, it does not appear to have a rate-limiting action in cycloheximide treated rats or in starvation. (8) Whereas PI and PIP kinases have short half-lives and apparently rapid synthetic rates, PLC has high activity, a long half-life and responds to starvation with only a small decrease. (9) The gain in function manifested in the over-expressed capacity for signal transduction confers growth advantages to cancer cells. These increased activities, particularly those of PI and PIP kinases, should be sensitive targets for chemotherapy.
Analysis of segregation and heritability were performed, with the aid of Penrose's method, maximum likelihood estimation and Falconer's method, in 215 cases with systemic lupus erythematosus (SLE) and their pedigrees to explore its possible model of inheritance. Results showed that prevalence of SLE in first-degree relatives of the proband with SLE was 0.85% and higher than that in general population (0.03%). A ratio of s/q approached 1/square root q with Penrose's method, segregation proportion was equal to 0.029 with simple segregation analysis, with a chi-square for goodness-of-fit of 0.0001 (P > 0.05), and the heritability of SLE was 56.7% (P < 0.05). It suggests that SLE follows a pattern of multifactorial inheritance rather than single-gene one.
As a part of a pilot clinical study, a high-performance reversed-phase liquid chromatography analysis was developed to quantify temozolomide in plasma and urine of patients undergoing a chemotherapy cycle with temozolomide. All samples were immediately stabilized with 1 M HCl (1 + 10 of biological sample), frozen and stored at -20 degrees C prior to analysis. The clean-up procedure involved a solid-phase extraction (SPE) of clinical sample (100 microliters) on a 100-mg C18-endcapped cartridge. Matrix components were eliminated with 750 microliters of 0.5% acetic acid (AcOH). Temozolomide was subsequently eluted with 1250 microliters of methanol (MeOH). The resulting eluate was evaporated under nitrogen at RT and reconstituted in 200 microliters of 0.5% AcOH and subjected to HPLC analysis on an ODS-column (MeOH-0.5% AcOH, 10:90) with UV detection at 330 nm. The calibration curves were linear over the concentration range 0.4-20 micrograms/ml and 2-150 micrograms/ml for plasma and urine, respectively. The extraction recovery of temozolomide was 86-90% from plasma and 103-105% from urine over the range of concentrations considered. The stability of temozolomide was studied in vitro in buffered solutions at RT, and in plasma and urine at 37 degrees C. An acidic pH (< 5-6) should be maintained throughout the collection, the processing and the analysis of the sample to preserve the integrity of the drug. The method reported here was validated for use in a clinical study of temozolomide for the treatment of metastatic melanoma and high grade glioma.
A novel isoform of the human folate receptor (FR, type gamma) was recently identified in hematopoietic tissues [Shen et al. (1994) Biochemistry 33, 1209-1215]. In that report, Cos-1 cells, transiently transfected with the cDNA for FR-gamma, produced relatively poor expression of the receptor on the cell surface. In this study, several recombinant Chinese hamster ovary (CHO) cell lines were produced by stable transfection with the cDNA for FR-gamma followed by amplification. Similar recombinant CHO cell lines were produced that expressed the glycosylphosphatidylinositol-(GPI-) anchored FR type beta and a truncated form of FR type beta (FR-beta delta), in which the normal carboxyl-terminal signal for GPI anchor attachment was deleted. Both FR-gamma-and FR-beta delta-expressing CHO cells produced a [3H]folic acid binding protein in the medium with a similar time course over a 24-h period; in contrast to intact FR-beta, relatively insignificant amounts of either FR-gamma or FR-beta delta were associated with the CHO cell surface and this was unaltered by the absence of serum in the medium. The FR-gamma- and FR-beta delta-producing CHO cells did not differ significantly in intracellular FR levels. Furthermore, the mRNA level for FR-gamma did not exceed that for FR-beta delta. When deglycosylated with hydrogen fluoride, both FR-gamma and FR-beta delta showed similar apparent molecular weights on Western blots as predicted for the intact polypeptides.(ABSTRACT TRUNCATED AT 250 WORDS)
Fraction A of the armadillo submandibular glycoprotein (ASG-A) is one of the simplest glycoproteins among mammalian salivary mucins. The carbohydrate side chains of this mucous glycoprotein have one-third of the NeuAc alpha 2-->6GalNAc (sialyl-Tn) sequence and two thirds of Tn (GalNAc alpha-->Ser/Thr) residues. Those of the desialylated product (ASG-Tn) are almost exclusively unsubstituted GalNAc residues (Tn determinant). When the binding properties of these glycoproteins were tested by a precipitin assay with Gal, GalNAc and GlcNAc specific lectins, it was found that ASG-Tn reacted strongly with all of the Tn-active lectins and completely precipitated Vicia villosa (VVL both B4 and mixture of A and B), Maclura pomifera (MPA), and Artocarpus integrifolia (jacalin) lectins. However, it precipitated poorly or negligibly with Ricinus communis (RCA1); Dolichos biflorus (DBA); Viscum album, ML-I; Arachis hypogaea (PNA), and Triticum vulgaris (WGA). The reactivity of ASG-A (sialyl-Tn) was as active as that of ASG-Tn with MPA and less or slightly less active than that of ASG-Tn with VVL-A+B, VVL-B4, HPA, WFA, and jacalin, as one-third of its Tn was sialylated. These findings indicate that ASG-A and its desialylated product (ASG-Tn) are highly useful reagents for the differentiation of Tn, T (Gal beta 1-->3GalNAc), A (GalNAc alpha 1-->3Gal) or Gal specific lectins and monoclonal antibodies against such epitopes.
The in vitro demonstration of the ability of NK cells to secrete cytokines prompted in vivo studies that illustrated the importance of NK cell-derived cytokines in regulating immune responses. Cross-linking of CD16 on NK cells can stimulate cytokine production. CD16-independent interactions capable of stimulating cytokine production have also been described, but molecules mediating such stimulations remain to be biochemically defined. We report here that cross-linking of CD45 specifically stimulates IFN-gamma production in human NK cells. The NK cells used were IL-2-activated adherent NK cells and from the NK3.3 cell line. The ability of CD45 mAbs to stimulate NK cells appears not to be dependent on CD16, as CD45 mAbs of both IgG1 and IgG2a isotypes were equally stimulatory, as were F(ab')2 compared with whole anti-CD45 mAbs. Resting NK cells, like T cells, express predominantly CD45RA, whereas IL-2 activated adherent NK cells acquire expression of CD45RO. Abs specific for CD45RO, but not CD45RA, were able to stimulate IFN-gamma production in NK cells. It has been reported that one ligand for CD45RO is CD22 beta. We tested the ability of CD22-expressing transfectants to bind to and stimulate NK cells. Whereas NK cells bound to CD22 alpha and CD22 beta transfectants, this interaction was not inhibited by CD45RO Abs. In addition, neither of the CD22-transfectants were able to stimulate NK3.3 cells to secrete IFN-gamma. These observations collectively suggest that binding of NK3.3 cells to CD22 may be independent of CD45RO on NK3.3 cells.
To identify specific environmental, viral, and genetic risk factors for hepatocellular carcinoma (HCC) and the interaction of such factors, we are conducting a prospective study in a high-incidence area of China. Questionnaires were completed and biosamples collected by 60,984 men ages 30-64 years, at study entry. Within 2.5 years, 183 deaths from HCC had occurred. Each HCC case was matched with 5 controls and compared for items on the questionnaire. In addition to chronic hepatitis B virus (HBV) infection, the significant risk factors were: occupation (peasant), corn consumption (in the 1970s), family history of HCC, and history of an episode of acute hepatitis as an adult. HBV, consumption of aflatoxins, a genetic factor, and possibly a second hepatitis virus infection contribute to the risk of HCC.