Biomedical subjects

J Xiang

Publications and source records attributed to J Xiang.

At least 73 records · Page 4Linked to original sources

PubMed1999

The JNKK2-JNK1 fusion protein acts as a constitutively active c-Jun kinase that stimulates c-Jun transcription activity.

c-Jun N-terminal protein kinase (JNK), a member of the mitogen-activated protein (MAP) kinase family, regulates gene expression in response to various extracellular stimuli. JNK is activated by JNK-activating kinase (JNKK1 and JNKK2), a subfamily of the dual specificity MAP kinase kinase (MEK) family, through phosphorylation on threonine (Thr) 183 and tyrosine (Tyr) 185 residues. The physiological functions of the JNK pathway, however, are not completely understood. A major obstacle is the lack of specific and activated kinase components that can stimulate the JNK pathway in the absence of any stimulus. Here we show that fusion of JNK1 to its upstream activator JNKK2 resulted in its constitutive activation. In HeLa cells, the JNKK2-JNK1 fusion protein showed significant JNK activity, which was comparable with that of JNK1 activated by many stimuli and activators, including EGF, TNF-alpha, anisomycin, UV irradiation, MEKK1, and small GTP binding proteins Rac1 and Cdc42Hs. Immunoblotting analysis indicated that JNK1 was phosphorylated by JNKK2 in the fusion protein on both Thr(183) and Tyr(185) residues. Like JNKK2, the JNKK2-JNK1 fusion protein was highly specific for the JNK pathway and did not activate either p38 or ERK2. Transient transfection assays demonstrated that the JNKK2-JNK1 fusion protein was sufficient to stimulate c-Jun transcriptional activity in the absence of any stimulus. Immunofluorescence analysis revealed that the JNKK2-JNK1 fusion protein was predominantly located in the nucleus of transfected HeLa cells. These results indicate that the JNKK2-JNK1 fusion protein is a constitutively active Jun kinase, which will facilitate the investigation of the physiological roles of the JNK pathway.

Fluorescent Antibody Technique↗

PubMed1999

Enzyme-linked immunosorbent assay-based selection and optimization of elution buffer for TAG72-affinity chromatography.

An enzyme-linked immunosorbent assay (ELISA)-elution assay was developed to screen a large variety of elution buffers for selection of a suitable one for purification of the fusion protein FV/TNF-alpha by affinity chromatography. Various commonly used buffer systems utilizing widely differing conditions such as extreme pH, denaturants, chaotropic ions and polarity reducing reagents were investigated. Ammonia solution (1 M, pH 11.5) proved to exert the most suitable influence on dissociation of the FV/TNF-alpha/TAG72 complex while having a minimal protein denaturing effect on FV/TNF-alpha. The total yield of purified FV/TNF-alpha using the TAG72-affinity column with this elution system was 300-fold higher than that using the common elution buffer, 0.1 M glycine, 0.5 M NaCl, pH 2.7. Our study indicates that the ELISA-elution assay will be most useful in the selection of suitable elution buffers for affinity chromatography.

Adenocarcinoma↗

PubMed1999

Blood-brain barrier mechanisms involved in brain calcium and potassium homeostasis.

This study examined the potential roles of the plasma membrane Ca2+-ATPase (PMCA) at the blood-CSF and blood-brain barriers in brain Ca2+ homeostasis and blood-brain barrier Na+/K+-ATPase subunits in brain K+ homeostasis. During dietary-induced hypo- and hypercalcemia (0.59+/-0.06 and 1.58+/-0.12 mM [Ca2+]) there was no significant change in choroid plexus PMCA (Western Blots) compared to normocalcemic rats (plasma [Ca2+]: 1.06+/-0.11 mM). In contrast, PMCA in cerebral microvessels isolated from hypocalcemic rats was 150% greater than that in controls (p<0.001). Comparison of the alpha3 subunit of Na+/K+-ATPase from cerebral microvessels isolated from hypo-, normo- and hyperkalemic rats (2.3+/-0.1, 3.9+/-0.1 and 7. 2+/-0.6 mM [K+]) showed a 75% reduction in the amount of this isoform during hyperkalemia. None of the other Na+/K+-ATPase isoforms varied with plasma [K+]. These results suggest that both PMCA and the alpha3 subunit of Na+/K+-ATPase at the blood-brain barrier play a role in maintaining a constant brain microenvironment during fluctuations in plasma composition.

Animals↗

PubMed1999

The 6svarsigma, 6dvarsigma, and 7dvarsigma Rydberg (1)Sigma(+)(g) States and Two Doubly Excited (1)Sigma(+)(g) States of Na(2).

Five highly excited (1)Sigma(+)(g) states of Na(2) were observed for the first time by pulsed optical-optical double resonance (OODR) fluorescence excitation spectroscopy. Three of the five states are assigned to the 6svarsigma (1)Sigma(+)(g), 6dvarsigma (1)Sigma(+)(g), and 7dvarsigma (1)Sigma(+)(g) Rydberg states. The other two states are assigned to doubly excited (1)Sigma(+)(g) states. Copyright 1999 Academic Press.

Journal Article↗

PubMed1999

8-10svarsigma, 8-10dvarsigma (1)Sigma(+)(g), and 10-15dpi (1)Pi(g) Rydberg States of Na(2).

With a sensitive space-charge-limited diode ionization detector, we studied 8-10svarsigma, 8-10dvarsigma (1)Sigma(+)(g), and 10-15dpi (1)Pi(g) Rydberg states of Na(2) and obtained their molecular constants for the first time. It is the first successful example of a diode ionization detector that is used in a pulsed OODR molecular spectroscopy experiment. Copyright 1999 Academic Press.

Journal Article↗

PubMed1999

Clonal growth of human acute leukemia cells in serum-free methylcellulose medium.

The clonal growth of human acute leukemia cell line (K562) and acute myeloid leukemia cells in the serum-free culture (SFC) was studied in order to establish a SFC system which could replace the effects of serum by using semi-solid methylcellulose culture technique. Our results showed that the clonal growth of K562 cells in semi-solid culture was dependent on exogenous serum. The K562 could be grown in SFC supplemented with 4 major replacing substances. The multifactor and multilevel orthogonal experiment demonstrated that the colony formation was statistically influenced by the 4 replacing substances at various concentrations (P < 0.01). Among them, bovine serum albumin had greatest effect on clonal growth of K562 cells with the optimal concentration being 15 mg/L, followed by transferring, cholesterol and insulin with their optimal concentrations being of 150 mg/L, 7.8 mg/L and 7.0 mg/L respectively. SFC system was formed with the 4 substances at their optimal concentrations. Colony formation of the blast cells in 10 patients with acute myeloid leukemia was observed in this SFC system. There was a heterogeneity of acute myeloid leukemia cells among the 10 patients in response to the growth substances. In SFC system, there was a linear relationship between the number of the clonal formation and the count of the added cells, indicating the colony growth of the cells. Primary acute leukemia cells maintained in SFC system in 10 cases could completely form clones. The colony formation number in some cases in SFC system was more than that of the serum-containing culture. The SFC system could partially replace the serum for study of the clonal formation of human leukemia cells.

Cell Division↗

PubMed1999

Visualization and characterization of GB virus-C particles: evidence for a nucleocapsid.

GB virus type C (GBV-C) is a member of the hepacivirus genus within the Flaviviradae. Persistent GBV-C infection is common in humans, yet it remains unclear if GBV-C causes any disease. Although GBV-C infection has been associated with acute non-A to non-E post-transfusion hepatitis, it does not appear to cause chronic hepatitis. GBV-C is closely related to hepatitis C virus (HCV), but indirect evidence suggests that it does not encode a core protein at the amino terminus of the open reading frame (ORF). This has led to speculation that GBV-C does not have a nucleocapsid. We evaluated the buoyant density of GBV-C, and found very low density particles consistent with virions, and intermediate density particles consistent with nucleocapsids in GBV-C-infected people. In addition, electron microscopy demonstrated an apparent nucleocapsid within an enveloped particle. Although these biophysical data strongly suggest that GBV-C utilizes a nucleocapsid, they do not indicate the origin of the protein content of this particle. To assess this, we evaluated patient plasma for reactivity with a synthetic oligopeptide representing a conserved region near the amino terminus of the predicted ORF. Specific antibody was detected in some individuals, similar to data of Feucht et al. who identified antibody against a recombinant core protein in GBV-C-infected people. These data indicate that GBV-C particles contain nucleocapsids. At least in some patients, the region upstream of the GBV-C E1 protein coding region appears to be expressed, and this region may represent the structural protein of the nucleocapsid.

Amino Acid Sequence↗

PubMed1999

Substitution of surface-exposed framework residues alters secretion of recombinant fusion protein Fv/tumor necrosis factor in Escherichia coli.

Substitution of hydrophobic residues with hydrophilic ones at surface-exposed positions may influence the yield of antibody fragment expression in Escherichia coli by reducing its aggregation potential. We introduced such substitutions at 8 surface-exposed framework region residues of a fusion protein Fv/Tumor Necrosis Factor, which resulted in the expression of 10 mutant fusion proteins (Mut 1-10) in E. coli. Our results showed that expression levels of Mut 1-3, with mutations of A9S, T18K, and G41D, respectively, decreased by 4- to 8-fold, whereas expression levels of Mut 4, 9, and 10, with mutations of S60D/S70D, T28D, and G65D, respectively, were not affected. In contrast, mutation of F83A at light-chain residue 83, which is usually buried at the variable/constant domain interface of antibody molecules but becomes surface-exposed in recombinant Fv molecules, increased the expression level of Mut 5 by 4-fold. Our results suggest that this important substitution of a hydrophobic residue with a hydrophilic one may also be applied to increase the secretion of other recombinant Fv molecules in E. coli periplasm.

Escherichia coli↗

PubMed1999

Expression of co-stimulatory 4-1BB ligand induces significant tumor regression and protective immunity.

Co-stimulatory molecules play an important role in initiating antitumor immune responses. Engineered tumor cells expressing co-stimulatory molecules have been used as cancer vaccines in both experimental tumor models and clinical trials. In this study, we cloned a cDNA gene coding for the mouse co-stimulatory molecule 4-1BBL by RTPCR. The expression vector pCI-4-1BBL was constructed by DNA recombinant technology and further transfected into a moderately immunogenic EL4 and a poorly immunogenic BL6-10 tumor cell line. Expression of the co-stimulatory molecule 4-1BBL is able to induce tumor regression of EL4/4-1BBL but not BL6-10/4-1BBL tumor cell line in syngeneic BALB/c mice. The tumor regression which is mainly mediated by CD8+ T cells further leads to protective immunity against the parental EL4 tumor. Our results thus indicate the potential utility of engineered tumor cells expressing co-stimulatory molecule 4-1BBL, especially in combination with other co-stimulatory molecules such as B7-1 in cancer vaccine.

4-1BB Ligand↗

PubMed1999

Adenovirus-mediated TNF-alpha gene transfer induces significant tumor regression in mice.

Recombinant adenovirus vectors are highly efficient at in vitro and in vivo gene delivery. The in vitro infection of a mouse colon adenocarcinoma cell line MCA-26 with the adenovirus AdV-LacZ can reach a maximal 75% of infectivity at an MOI of 1000. Intratumoral injection of AdV-LacZ (2X10(9) pfu) resulted in substantial gene transfer in nearly 70% of MCA-26 tumors. After the in vitro infection of AdV-TNF-alpha, infected MCA-26 cells showed significant secretion of TNF-alpha (45 ng/ml/10(6) cells) in tissue culture. The secretion peaks at day 2 and is diminished at day 4 following the viral infection. Infected MCA-26 tumor cells secreting TNF-alpha significantly reduced their tumorigenicity in syngeneic BALB/c mice. In mice bearing small tumors, intratumoral injection of 2X10(9) pfu of AdV-TNF-alpha virus with a repeated booster treatment resulted in complete regression of three tumors and significant diminution of the other two with a mean tumor-weight of 0.16 g; this is in contrast to 0.85 and 1.62 g for tumors injected with the control AdV-pLpA and PBS respectively (p < 0.01). Mice with complete tumor regression further developed protective immunity against the second challenge of MCA-26 inoculation. In mice bearing large tumors, this treatment also caused significant inhibition of tumor growth with a mean tumor weight of 0.65 g vis-a-vis 3.05 g for tumors injected with the control AdV-pLpA. On the contrary, in mice bearing large tumors, the treatment of tumors with pCI-TNF-alpha delivered by the gene gun did not induce significant tumor inhibition. These results indicate that the adenoviral delivery of TNF-alpha gene is more efficient than the particle-mediated gene gun device, and that adenovirus-mediated cytokine gene therapy may be a useful approach in the clinical management of human solid tumors.

Adenocarcinoma↗

PubMed1999

Light-chain framework region residue Tyr71 of chimeric B72.3 antibody plays an important role in influencing the TAG72 antigen binding.

The crystallographic study of chimeric B72.3 antibody illustrated that there are three FR side-chain interactions with either CDR residue's side chain or main chain. For example, hydrogen bonds are formed between the hydroxyl group of threonine at L5 in FR1 and the guanidinal nitrogen group of arginine at L24 in CDR1, between the hydroxyl group of tyrosine at L36 in FR2 and the amide nitrogen group of glutamine at L89 in CDR3 and between the hydroxyl group of tyrosine at L71 in FR3 and the carbonyl group of isoleucine at L29 as well as the amide nitrogen group of serine at L31 in CDR1. Elimination of these hydrogen bonds at these FR positions may affect CDR loop conformations. To confirm these assumptions, we altered these FR residues by site-directed mutagenesis and determined binding affinities of these mutant chimeric antibodies for the TAG72 antigen. We found that the substitution of tyrosine by phenylalanine at L71, altering main-chain hydrogen bonds, significantly reduced the binding affinity for the TAG72 antigen by 23-fold, whereas the substitution of threonine and tyrosine by alanine and phenylalanine at L5 and L36, eliminating hydrogen bonds to side-chain atoms, did not affect the binding affinity for the TAG72 antigen. Our results indicate that the light-chain FR residue tyrosine at L71 of chimeric B72.3 antibody plays an important role in influencing the TAG72 antigen binding. Our results will thus be of importance when the humanized B72.3 antibody is constructed, since this important mouse FR residue tyrosine at L71 must be maintained.

Amino Acid Sequence↗

PubMed1999

Attenuation of thrombin-induced brain edema by cerebral thrombin preconditioning.

BACKGROUND AND PURPOSE: Edema formation after intracerebral hemorrhage has been linked to thrombin toxicity induced by the clot. However, thrombin at low concentrations actually protects neurons and astrocytes in culture from hypoglycemic and ischemic cell death. It is also known that a brief episode of brain ischemia increases neuronal tolerance to a subsequent severe ischemic episode. The objective of this study was to investigate whether pretreatment of the brain with low-dose thrombin induces tolerance to a subsequent large dose of thrombin injected into brain parenchyma. METHODS: The rat brain was preconditioned with 1 U thrombin by direct infusion into the right caudate nucleus. After thrombin pretreatment, the effects of a large dose (5 U) of thrombin on brain edema formation were studied at different intervals. We examined whether heat-shock protein (HSP) 27, HSP32, and HSP70 were induced by Western blot analysis, immunocytochemistry, and immunofluorescent double staining. RESULTS: Thrombin pretreatment significantly attenuated the brain edema that normally follows the infusion of a large dose of thrombin (79.2+/-0.4 versus 84.0+/-0.3; P<0.01). This effect was abolished by the thrombin inhibitor hirudin. Time course studies showed that the maximal effect of thrombin preconditioning (TPC) on brain edema formation was 7 days after pretreatment. This time course corresponded to marked upregulation of HSP27 in the ipsilateral brain. TPC also induced HSP32, but this effect occurred earlier than the effect on edema formation. TPC had no effect on HSP70. Immunocytochemistry and immunofluorescent double labeling showed that HSP27 and HSP32 were expressed in astrocytes after TPC. CONCLUSIONS: OFF phenomenon of thrombin-induced tolerance of the brain to edema formation may be related to HSP27 induction.

Animals↗

PubMed1999

Effect of life styles on the risk of subsite-specific gastric cancer in those with and without family history.

To evaluate a combined effect of gastric cancer family history (GCFH) and selected living habits on the subsite-specific of gastric cancer, a hospital-based case-referent study was conducted in Tokai area of Japan. The study subjects were 850 newly diagnosed gastric cancer (GC) patients and 28,619 cancer-free first-visit outpatients. Odds ratios (ORs) of all subsites of GC in subjects with both GCFH and habitual smoking were significantly higher (OR = 4.22) compared with those with merely GCFH (OR = 1.81) or habitual smoking (OR = 2.83). When positive GCFH subjects frequently consumed raw vegetable, the risk of GC decreased in cardia (OR = 0.68), antrum (OR = 0.43) and all subsites (OR = 0.74). Our findings provided evidence that GCFH and habitual smoking increased the risk of GC with family history, while frequent intake of raw vegetable decreased the risk and it was modified by other environmental factors.

Adult↗

PubMed1999

Targeting cytokines to tumors to induce active antitumor immune responses by recombinant fusion proteins.

Cytokines such as interleukin-2(IL-2), gamma interferon (IFN-gamma) and alpha tumor necrosis factor (TNF-alpha) are important mediators in immune responses against tumors. However, their therapeutic efficacy and clinical utilities in treatment of human malignancies are in large part limited due to the low concentrations of cytokine in tumors and the severe toxic side-effects derived from high-dose administration of cytokines. One critical issue to improve therapeutic efficacy is how to increase the local concentration of cytokine in tumors without causing severe side-effects. A series of recent reports demonstrated that the introduction of cytokine genes into tumor cells and subsequent local secretion can circumvent the limitations associated with the systemic cytokine administration. An alternative means of cytokine delivery is to target cytokines to tumor cells with tumor specific antibodies. Thereby, effective local cytokine concentrations can be achieved at the tumor sites without resorting to patient-specific therapy. With the advance in biotechnology, two structurally disparate domains of immunoglobulin and cytokine can be brought together into one fusion protein molecule by protein engineering These engineered antibody-cytokine fusion proteins combine the unique targeting ability of tumor-specific antibodies with the multifunctional activity of cytokines. In general, there are two commonly engineered fusion proteins, the F(ab')2/cytokine expressed in mammalian cells and the single-chain FV/cytokine expressed in Escherichia coli. Both the tumor-binding reactivity and the functional cytokine activity are maintained in most of fusion proteins. Therefore, these fusion proteins may be useful in targeting cytokine to tumors to stimulate immune destruction of tumors, while limiting severe toxic side-effects by the high dose of cytokine administration. Recent preclinical studies have shown that these fusion proteins are able to target cytokines to tumors expressing the tumor-associated antigen in vivo, and to inhibit both the primary and metastatic tumors in an immune competent animal model. Therefore, these recombinant fusion proteins may represent a new generation of novel immunotherapeutic reagents for the treatment of human malignant diseases.

Animals↗

PubMed1999

An animal and clinical study on the change of neuropeptide Y release evoked by electrical stimulation and myocardial ischemia.

OBJECTIVE: To investigate the change characteristics of neuropeptide Y (NPY) release during acute myocardial ischemia period. METHODS: The animal test was carried out in in situ perfused guinea pig hearts with intact sympathetic innervation. Electrical stimulation-evoked exocytotic release of NPY during ischemia and reperfusion was tested by radioimmunoassay (RIA). The plasma NPY concentrations were measured in patients with acute myocardial infarction (AMI) and angina pectoris (AP) in different times. RESULTS: Electric stimulation of the left ganglion in guinea pig heart evoked an exocytic release of neuropeptide Y. Stimulation after 20 minutes of global ischemia (S2), compared with control period stimulation (S1) produced the inhibition of NPY to a certain extent (S2/S1: 0.72, P < 0.05), whereas the inhibition of NPY release disappeared after 5 minutes reperfusion (with S2/S1 of 1.01, P > 0.05). Ischemia alone, without the electric stimulation, did not apparently induce NPY release. The clinical test found that the plasma NPY level was increased significantly during the acute ischemia attack period of coronary heart disease (CHD). The plasma NPY level reached peak (136.3 +/- 66.5 pg/ml) in patients during the first day after AMI. It began to decrease from the third day and came to normal level in the end of the first week. The plasma NPY level was 159.3 +/- 98.5 pg/ml in AP patients during angina attack. After two weeks treatment, the plasma NPY level was decreased to 118.9 +/- 54.3 pg/ml (P < 0.05). CONCLUSIONS: The NPY release of global ischemia have some relation with sympathetic nerve activity. At the early stage of ischemia, NPY release is inhibited to some degree and the inhibition factors will fade away on reperfusion. NPY interferes with the pathogenesis and the pathophysiolgy.

Aged↗

PubMed1999

[Studies on chemical constituents of Forsythia suspensa (Thunb) Vahl].

OBJECTIVE: To study the chemical constituents of Forsythia suspensa. METHOD: Basedon silicacolumn chromatography, of which six compounds were obtained from the alcohol extract of F. suspensa, three identified by physicochemical and spectroscopic analyses. RESULT: The three compounds were identified as stearic acid, palmitic acid and beta-sitosterol. CONCLUSION: The three compounds were separated from F. suspensa fruits for the first time.

Forsythia↗

PubMed1999

[Chemical composition of Fordia cauliflora Hemsl].

OBJECTIVE: To study chemical constituents of the root of Fordia cauliflora. METHOD: Three compounds were obtained from the ethanolic extract of the roots of F. cauliflora by silica column chromatography, and identified by physico-chemical and spectroscopic analysis. RESULT: The compounds were identified as stearic acid, palmitic acid and beta-sitosterol. CONCLUSION: These compounds were obtained from this plant for the first time.

Fabaceae↗

PubMed1999

[The rebound of nasal resistance after decongestant used in nose].

OBJECTIVE: To investigate the effect of decongestant on nasal resistance so as to provide theories for studing rhinitis medicamentosa. METHOD: The changes of nasal blockage in 92 cases of acute rhinitis and chronic rhinitis after using decongestants were recorded, and the nasal resistance in 39 cases were measured by active anterior rhinomanometry. RESULT: According to the diseases and decongestants used, the percentage of nasal resistance rebound were 63.5%, 31.2%, 32.2%, 8.6% in 4 groups of investigated patients and 50%, 20%, 75% in 3 groups of measured patients respectively. CONCLUSION: Decongestants can cause the rebound of nasal resistance, which related to the diseases and the decongestants used. The mechanism may be that the factors dilating the vasa in nasal mucosa are activated.

Adolescent↗