Biomedical subjects

M Yu

Publications and source records attributed to M Yu.

At least 235 records · Page 13Linked to original sources

PubMed1997

[Cloning of the NS1 gene of dengue-2 virus and determination of its partial nucleotide sequence].

In this study, RT-PCR product of the NS1 gene of dengue-2 virus isolated in China was inserted into the DNA of T-vector plasmid. DNAs of the positive clones, which were identified by using the blue/white spot and PCR, were digested with enzymes, indicating that the inserted fragments were the same sizes as those of the amplified NS1 genes. The result of the nucleotide sequence determined by dideoxy chain-termination method indicated that 5'-terminal nucleotide sequence of the amplified NS1 fragment was not changed.

Cloning, Molecular↗

PubMed1997

[The anti-leukemia effects of IL-2 and IL-3 genes co-transfected leukemia vaccine].

OBJECTIVE: To investigate the anti-leukemia activity of IL-2 and IL-3 genes co-transfected leukemia vaccine. METHODS: The vaccine was prepared with FBL-3 erythroleukemia cells which were in vitro transfected with IL-2 and/or IL-3 genes by recombinant adenovirus and then inactivated by 60Co irradiation. The vaccine was injected peritoneally into FBL-3 leukemia mice, and the tumor growth, survival time of the mice, cytotoxicities of the peritoneal macrophages, splenic NK cells and CTL cells were observed. RESULTS: A fter treatment with the vaccine, the leukemic cell tumor in the mice showed significant growth inhibition and the survival time of the mice were prolonged when treated with IL-2 transfected vaccine, the cytotoxicities of splenic NK and CTL were increased significantly, while in the case of IL-3 transfected vaccine, the number and cytotoxicity of peritoneal macrophages were increased. The antileukemia effect was augmented when treated with IL-2 and IL-3 genes co-transfected vaccine. The antileukemia activity of the vaccine was also augmented by low dose cyclophosphamide administration. CONCLUSION: Leukemia vaccine gained more antitumor effect via induction of antitumor immunity.

Animals↗

PubMed1997

The effects of 50 Hz magnetic field exposure on dimethylbenz(alpha)anthracene induced thymic lymphoma/leukemia in mice.

Several epidemiological investigations have suggested an increased incidence of lymphoma, leukemia, and brain tumor in residents living near power transmission lines. However, some observers failed to confirm such a positive correlation. To evaluate the effects of extremely low frequency (ELF) magnetic fields on leukemogenesis, an experimental animal model was used, in which thymic lymphoma/leukemia was induced by dimethylbenz(alpha)anthracene (DMBA) injected subcutaneously into the interscapular region of newborn mice within 24 h after birth. Beginning at the second week of age, 165 mice were exposed to 50 Hz magnetic field at 1 mT, 3 h/day, 6 days/week for 16 weeks, and 155 animals exposed to sham conditions. All surviving animals were killed by cervical dislocation at the age of 32 weeks and were examined pathologically. The results showed that the incidences of advanced thymic lymphoma, complicated with lymphomatous leukemia, were 21.8 and 23.9% in the two groups, respectively, without statistically significant differences. But dense metastatic infiltration by lymphoma cells into liver in the field exposure group greater (50%) than that in the sham-exposure group (16.2%) was observed (chi 2 = 9.847, P < 0.01). To determine whether ELF acts as a tumor promoter, further experiments are required.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1997

Expression in Escherichia coli and purification of human thrombopoietin.

Human thrombopoietin (TPO) has been successfully overexpressed in Escherichia coli, with an expression level of about 12% of total cellular protein. The full-length TPO gene was subcloned into the prokaryotic expression vector pKK233-2 under the control of the inducible tac promoter. The recombinant protein was produced mainly in the form of inclusion body. By efficient renaturation and one-step purification, the recombinant protein was purified to homogeneity. The specific activity and yield of recombinant TPO can reach 2 x 10(4) units/mg and 2 mg/g of wet E. coli cells respectively.

Chromatography, Liquid↗

PubMed1997

An experimental study on application of PCR in detection of kala-azar.

A polymerase chain reaction (PCR) technique was developed to detect Kala-azar. Out of 25 patients, 24 were detected, a sensitivity of 96%. The specificity rate, false positive rate, false negative rate, coincidence rate and Youdent's index were 100% (60/60), 0, 4%, 99% and 0.96 respectively. It was shown that the PCR technique can directly detect Kala-azar in samples (1 microliter) from peripheral blood.

Animals↗

PubMed1997

[The relationship between autoimmune thyroiditis and TNF alpha: a preliminary study].

OBJECTIVE: To observe the action of tumor necrosis factor-alpha (TNF alpha) in the pathogenesis of autoimmune thyroiditis. METHODS: The activity of TNF alpha of peripheral blood mononuclear cells was detected in 25 cases of autoimmune thyroiditis, 9 cases of Grave's disease, and 18 normal controls by ELISA and bioassay. RESULTS: The activity of TNF alpha in patients with autoimmune thyrioditis was significantly higher than that in normal controls (P < 0.001), and in patients with Grave's diseases significantly lower than that in normal controls (P < 0.01). TNF alpha activity detected by two methods showed a good correlation (r = 0.972 P < 0.001). There were a negative correlation between TNF alpha and TT3 or FT3 (r = -0.437, -0.432 respectively, both P < 0.05) and a positive correlation between TNF alpha and TPO-Ab or TGA (r = 0.53, 0.51 respectively, both P < 0.01). TNF alpha activity was not associated with the period of disease and the size of thyriod. CONCLUSION: TNF alpha exerts an important role in the pathogenesis of autoimmune thyroiditis.

Adult↗

PubMed1997

[Transfer and expression of antisense genes of hepatitis B virus (HBV) and their anti-HBV effects].

OBJECTIVE: To observe the transfer and expression of HBV antisense genes and their anti-HBV effects in 2.2.15 cells mediated by retroviral vector-packaging cell line system. METHODS: The recombinant retroviral vectors mediating expression of HBV antisense RNA complementary to 1402-2906 fragment (preC/C) or 2839-1986 fragment (preS/S) of HBV DNA were used to transduce into NIH 3T3 cells and 2.2.15, respectively. Then, the total RNAs of tranduced NIH 3T3 cells were extracted and hybridized with HBV DNA probe. The HBsAg and HBeAg in the supernatants of cultured 2.2.15 cells were assayed with RIA method and the DNAs were hybridized with HBV DNA probe by dot blot. RESULTS: The results showed that the HBV antisense genes could be transfered into and expressed in the NIH 3T3 cells mediated by recombinant retroviral vector packaging cell line (PA317 cell). The inhibitory effects on the expression of HBV antigens by antisense RNAs appeared as early as on the day 3 after transduced, reached peak level on the day 5, and persisted at least for eleven days. The inhibitory rates of HBsAg and HBeAg were 71% and 23% by antisense preS/S, and 23% and 59% by antisense preC/C on the day 5 after transduction. In comparison to blank control or sense--gene expressing vectors, the inhibitory effects of HBV antisense vectors were highly significant (P < 0.01). HBV DNA level in the supernatant of the 2.2.15 cells transduced with either antisense preS/S or preC/C in comparison to the blank control vectors was also reduced on the day 5 after transduction as detected by DNA dot blot assays, but the viability of transduced 2.2.15 cells was not affected as detected by MTT assays. CONCLUSIONS: HBV antisense genes can be transfered and expressed in the eukaryotic cells and the expression of HBV antisense RNAs in 2.2.15 cells can inhibit the replication and expression of HBV.

3T3 Cells↗

PubMed1996

RIC, a calmodulin-binding Ras-like GTPase.

Neuronal activity dramatically increases the concentration of cytosolic Ca2+, which then serves as a second messenger to direct diverse cellular responses. Calmodulin is a primary mediator of Ca2+ signals in the nervous system. In a screen for calmodulin-binding proteins, we identified RIC, a protein related to the Ras subfamily of small GTPases. In addition to the ability to bind calmodulin, a number of unique features distinguished RIC from other Ras-like GTPases, including the absence of a signal for prenylation and a distinct effector (G2) domain. Furthermore, we describe two human proteins, RIN and RIT, which were 71% and 66% identical to RIC respectively, shared related G2 domains with RIC, and lacked prenylation signals, suggesting that the RIC family is conserved from flies to humans. While Ric and RIT were widely expressed, expression of RIN was confined to the neuron system.

Amino Acid Sequence↗

PubMed1996

Alternative RNA splicing of the MLL gene in normal and malignant cells.

The human MLL (mixed-lineage leukemia or myeloid-lymphoid leukemia) gene belongs to the trithorax gene family of which the Drosophila trithorax (trx) gene is known to regulate homeotic genes through alternative RNA splicing. To test if such a splicing mechanism also operates in MLL, we evaluated mRNA transcripts from a large number of normal and malignant human cells, making use of RT-PCR, PCR cloning, DNA sequencing and Northern blot analysis. Our findings indicate that different cell types transcribe MLL mRNA species lacking exons that generally encode putative regulatory domains such as AT hooks (exon 3), repression domain (exon 6), zinc finger motifs (exon 8) and activation domain (exon 18). Such findings suggest that posttranscriptional regulation by alternative RNA splicing may play an important role in MLL gene expression and provides the rationale for a mechanism by which this gene, with multiple functional domains, could produce discrete protein products that may prove critical in the regulation of human homeobox genes.

Alternative Splicing↗

PubMed1996

Artificial neural networks aided deconvolving overlapped peaks in chromatograms.

A novel method for deconvolving overlapped peaks in chromatograms is proposed. The basic idea of this method consists of finding a set of parameters which characterize the shape of the overlapped peaks and using a multi-layered perceptron network for quantitatively correlating the parameters with the percentage area of an individual peak. The proposed method performs very well with high accuracy and less computing time compared to other conventional methods.

Chromatography↗

PubMed1996

CD34+ cell dose predicts survival, posttransplant morbidity, and rate of hematologic recovery after allogeneic marrow transplants for hematologic malignancies.

After autologous or allogeneic transplants of peripheral blood stem cells (PBSC), an adequate dose of CD34+ cells is necessary to ensure early and sustained hematopoietic engraftment and favorable clinical outcome. There are no comparable data on the relationship between CD34+ cell dose and recovery after allogeneic bone marrow transplants (BMT). Twenty-eight patients with hematologic malignancies received a BMT from an HLA-identical sibling, using T-cell depletion and cyclosporin for graft-versus-host disease prophylaxis and delayed donor lymphocyte transfusions in an attempt to prevent leukemia relapse. The treatment-related mortality (TRM), primarily due to infections and cytopenias, was significantly higher for 13 patients receiving less than 1 x 10(6) CD34+ cells/kg (64.9% +/- 12.8% v 6.9% +/- 6.4%, P = .003). Survival at a median follow-up of 1 year was also lower in the group receiving less than 1 x 10(6) CD34+ cells/kg (30.8% +/- 12.8 v 74.3% +/- 13.7%, P = .005). The CD34+ cell dose was the only variable significantly associated with TRM. The dose of CD34+ cells also correlated with speed of hematopoistic recovery. Patients receiving more than 2 x 10(6) CD34+ cells/kg showed significantly earlier recovery of monocytes and a trend for earlier recovery of lymphocytes. They achieved platelet and red blood cell transfusion independence earlier, required less granulocyte colony-stimulating factor support during ganciclovir treatment, and spent fewer days in the hospital after transplantation. These results suggest that, for allogeneic T-cell-depleted BMT, the higher CD34+ cell doses may improve outcome in engrafting patients.

Adult↗

PubMed1996

Redox regulation of GA-binding protein-alpha DNA binding activity.

We have investigated the reduction/oxidation (redox) regulation of the heteromeric transcription factor GA-binding protein (GABP). GABP, also known as nuclear respiratory factor 2, regulates the expression of nuclear encoded mitochondrial proteins involved in oxidative phosphorylation, including cytochrome c oxidase subunits IV and Vb, as well as the expression of mitochondrial transcription factor 1. GABP is composed of two subunits, the Ets-related GABP-alpha, which mediates specific DNA binding, and GABP-beta, which forms heterodimers and heterotetramers on DNA sequences containing the PEA3/Ets motif ((C/A)GGA(A/T)(G/A)). We demonstrate here that GABP DNA binding activity and GABP-dependent gene expression in 3T3 cells are inhibited by pro-oxidant conditions. DNA binding of recombinant GABP-alpha was activated by chemical reduction (dithiothreitol) and by thioredoxin; however, GSSG inhibited GABP DNA binding activity. Treatment of GABP-alpha, but not GABP-beta1, with sulfhydryl-alkylating agents also inhibited GABP DNA binding activity. Our results suggest that GABP DNA binding activity is redox-regulated in vivo, possibly by thioredoxin-mediated reduction and by GSSG-mediated oxidation of the GABP-alpha subunit. The regulation of GABP (nuclear respiratory factor 2) DNA binding activity by cellular redox changes provides an important link between mitochondrial and nuclear gene expression and the redox state of the cell.

3T3 Cells↗

PubMed1996

Analysis of the expression of glycosylphosphatidylinositol anchored proteins on platelets from patients with paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH), an acquired clonal disorder is manifested by failure of hematopoietic cells to express phosphatidylinositol glycan-anchored proteins (PIG-AP). Since the PIG-A mutation is present at the stem cell level, all cell lines may be affected. Although the pathogenesis of hemolytic anemia in PNH is related to the absence of CD55 and CD59 molecules on the membrane of red cells, the mechanism responsible for the increased incidence of thrombotic events in PNH is not clear. In this study we measured two glycosylphosphatidylinositol (GPI)-linked molecules on platelets (CD55 and CD59) and two GPI-linked proteins on neutrophils (CD14 and CD16), comparing their expression on normal and PNH patients. Using two-color flow cytometric analysis with antibodies directed against CD42b and CD41a, we found that CD55 and CD59 were constitutively expressed by normal fresh platelets, but that the expression levels decreased during the five day storage of platelets. A substantial population of platelets lacking the GPI-linked proteins were detected in most cases. We demonstrated varying degrees of deficiency in the expression of GPI-anchored molecules with neutrophils, monocytes and platelets with the highest proportion of deficient cells found within monocytic lineage. Similar numbers of platelets with the PNH phenotype and normal platelets expressed activation markers before and after exposure to platelet agonists. Flow cytometry is more sensitive than Ham's test in monitoring expression of PNH in platelets. Differences in the numbers of circulating GPI-deficient platelets and myeloid cells suggest that either the survival of platelets and mature myeloid cells differs or megakaryocytopoeisis is abnormal within the GPI-deficient clone.

Anemia, Aplastic↗

PubMed1996

Generation of autonomously pathogenic neo-autoreactive Th1 cells during the development of the determinant spreading cascade in murine autoimmune encephalomyelitis.

Chronic progression of autoimmune disease is accompanied by the acquisition of autoreactivity to new self-determinants. Recent evidence indicates that this process, commonly referred to as determinant spreading, may be pathogenic for chronicity. Our studies on experimental autoimmune encephalomyelitis (EAE), a murine model widely used in multiple sclerosis (MS) studies, have shown that determinant spreading develops as a predictable sequential cascade of neo-autoimmunity during progression to chronic disease. By 7-8 weeks after immunization of (SWR x SJL)F1 mice with the immunodominant myelin proteolipid protein determinant (PLP 139-151), splenocytes consistently respond to the immunodominant myelin basic protein determinant (MBP 87-99). In the present study, we directly address the pathogenicity of neo-autoimmunity resulting from endogenous self-priming during the course of disease. Our results indicate that T cells responding to the spreading MBP 87-99 determinant produce a proinflammatory cytokine profile consistent with type 1 helper T cells (Th1) cells. In addition, splenocytes activated to the spreading MBP 87-99 determinant consistently transfer acute EAE in naive recipients even when T cells reactive to the priming PLP 139-151 immunogen are eliminated by bromodeoxyuridine (BUdR)-mediated photolysis. Our data indicate that endogenous neo-autoantigen priming during chronic autoimmune disease generates type 1 helper T cells (Th1) cells that are autonomously pathogenic. These results provide further evidence supporting the view that determinant spreading is a pathogenic process that leads to chronic progression of autoimmune disease.

Adoptive Transfer↗

PubMed1996

Fine mapping of a C-terminal linear epitope highly conserved among the major envelope glycoprotein E2 (gp51 to gp54) of different pestiviruses.

Envelope glycoprotein E2 (gp51 to gp54) is the major neutralizing antigen of pestiviruses, which include classical swine fever virus (CSFV), bovine viral diarrhoea virus (BVDV), and border disease virus (BVD). Previous studies carried out using a panel of monoclonal antibodies raised against CSFV strain Brescia have revealed the existence of four antigenic domains, A to D, of the E2 protein, all of which are located at the N-terminal half of the molecule. Here we report the detailed mapping, using three complementary techniques, of a novel linear epitope located at the C-terminal part of the molecule, which reacted with a monoclonal antibody (4-9D4) as well as polyclonal animal sera. This epitope is highly conserved in the three different members of pestiviruses and hence can be used as a genus-specific diagnosis tool. The observation that this epitope is not accessible on the native virus surface, together with its C-terminal location, supports a recently proposed structural model, indicating that the C-terminal part of E2 is membrane-bound while the N-terminal half of the molecule is exposed on the virus surface.

Amino Acid Sequence↗

PubMed1996

Prevention of islet allograft rejection with engineered myoblasts expressing FasL in mice.

Allogeneic transplantation of islets of Langerhans was facilitated by the cotransplantation of syngeneic myoblasts genetically engineered to express the Fas ligand (FasL). Composite grafting of allogeneic islets with syngeneic myoblasts expressing FasL protected the islet graft from immune rejection and maintained normoglycemia for more than 80 days in mice with streptozotocin-induced diabetes. Graft survival was not prolonged with composite grafts of unmodified myoblasts or Fas-expressing myoblasts. Islet allografts transplanted separately from FasL-expressing myoblasts into the contralateral kidney were rejected, as were similarly transplanted third-party thyroid allografts. Thus, the FasL signal provided site- and immune-specific protection of islet allografts.

Animals↗

PubMed1996

RIG-E, a human homolog of the murine Ly-6 family, is induced by retinoic acid during the differentiation of acute promyelocytic leukemia cell.

In vivo all-trans-retinoic acid (ATRA), a differentiation inducer, is capable of causing clinical remission in about 90% of patients with acute promyelocytic leukemia (APL). The molecular basis for the differentiation of APL cells after treatment with ATRA remains obscure and may involve genes other than the known retinoid nuclear transcription factors. We report here the ATRA-induced gene expression in a cell line (NB4) derived from a patient with APL. By differential display-PCR, we isolated and characterized a novel gene (RIG-E) whose expression is up-regulated by ATRA. The gene is 4.0 kb long, consisting of four exons and three introns, and is localized on human chromosome region 8q24. The deduced amino acid sequence predicts a cell surface protein containing 20 amino acids at the N-terminal end corresponding to a signal peptide and an extracellular sequence containing 111 amino acids. The RIG-E coded protein shares some homology with CD59 and with a number of growth factor receptors. It shares high sequence homology with the murine LY-6 multigene family, whose members are small cysteine-rich proteins differentially expressed in several hematopoietic cell lines and appear to function in signal transduction. It seems that so far RIG-E is the closest human homolog of the LY-6 family. Expression of RIG-E is not restricted to myeloid differentiation, because it is also present in thymocytes and in a number of other tissues at different levels.

Amino Acid Sequence↗

PubMed1996

Expression and function of voltage-dependent potassium channel genes in human airway smooth muscle.

Patch clamp and RNA-polymerase chain reaction methods were used to determine the expression of voltage-dependent potassium channel currents and mRNAs in human airway smooth muscle cells, and tension measurements were used to examine the functional role of specific potassium channel gene products in human bronchial smooth muscle. RNA from airway smooth muscle tissue revealed the presence of Kv1.2 (11 kilobases (kb)) and Kv1.5 (3.5 and 4.4 kb) transcripts, as well as Kv1.1 mRNA (9.5 kb), which has not previously been reported in smooth muscle; transcripts from other gene families were not detected. RNA-polymerase chain reaction from cultured human myocytes confirmed that the identified transcripts were expressed by smooth muscle cells. The available voltage-dependent potassium current in human airway myocytes was insensitive to charybdotoxin (200 nM) but blocked by 4-aminopyridine. Dendrotoxin (1-300 nM; inhibits Kv1.1 and Kv1.2 channels), charybdotoxin (10 nM to 1 microM; inhibits KCa and Kv1.2 channels), and glybenclamide (0.1-100 microM; inhibits KATP channels) had no effect on resting tone. Conversely, 4-aminopyridine increased resting tension with an EC50 (1.8 mM) equivalent to that observed for current inhibition (1.9 mM). Human airway myocytes express mRNA from several members of the Kv1 gene family; the channel that underlies the predominate voltage-dependent current and the regulation of basal tone appears to be Kv1.5.

Base Sequence↗