[Telomere, telomerase and cancers].
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Animals↗
Biomedical subjects
S Wu
Publications and source records attributed to S Wu.
Semi-quantitative study of calcitonin gene methylation in myelodysplastic syndrome.
OBJECTIVE: To evaluate whether hypermethylation of calcitonin (CT) gene could serve as a transforming signal of myelodysplastic syndrome (MDS) to leukemia. METHODS: Bone marrow aspirates from 35 MDS patients, including 25 refractory anemia (RA), 10 refractory anemia with excess of blasts (RAEB) or refractory anemia with excess of blasts in transformation (RAEBt) and 7 cases of acute myeloid leukemia (AML) transformed from MDS, were studied on methylation rate in 5' end of CT gene by polymerase chain reaction (PCR) technique using methylation-sensitive endonuclease Hpa II with external references of undigested DNA and Msp I digested DNA and internal reference of 112 bp fragment containing codon 61 of N-ras oncogene. The results were expressed as calcitonin gene methylation rate (CTMR) calculated from the densitometer-analyzed integral calculus of PCR products of 566 bp CT(a1), 112 bp N-ras(b1) by using Hpa II-digested DNA and PCR products of 566 bp CT (a0), 112 bp N-ras(b0) by using undigested DNA according to the formula, CTMR = (a1/b1)/(a0/b0) x 100%. RESULTS: The CTMRs in total 35 MDS, 25 RA, 10 RAEBt and 7 cases of AML transformed from MDS were 36.87% +/- 25.10%, 28.12% +/- 24.01%, 58.74% +/- 16.49%, and 54.03% +/- 7.06% respectively, significantly higher than that in control group (P < 0.001, P < 0.05, P < 0.001 and P < 0.001, respectively). CONCLUSION: The results suggest that hypermethylation of CT gene occurs in early stage of leukemic transformation and CTMR might be a useful marker in predicting the transformation of MDS to AML.
Adolescent↗
[A clinical study of six cases of Pneumocystis carinii pneumonia].
OBJECTIVE: To heighten the awareness of pneumocystis carinii pneumonia. METHOD: Six cases of pneumocystis carinii pneumonia were retrospectively analyzed. RESULT: The underlying diseases were leukemia, lymphoma, renal transplantation and autoimmune hemolytic anemia. The clinical features were dyspnea, cough and fever. X rays typically showed a bilateral diffuse or patchy interstitial infiltrate. Three cases complicated with acute respiratory failure showed hypoxemia, intrapulmonary shunt and reduced compliance resembling ARDS. CPAP/PEEP was required. High-level PEEP (> 1.47 kPa) can be applied to the refractory hypoxemia. SMZco and dapsone were the initial choice of treatment. CONCLUSION: The prognosis of pneumocystis carinii pneumonia with acute respiratory failure was poor. Aggressive early diagnosis and treatment were critical to improve survival.
Acute Disease↗
[An experimental study on antitumor activity of psoralen on mammary cancer cell line EMT6 in vitro and in vivo].
To investigate the antitumor activity of psoralen on mammary cancer cells of EMT6 line, the cytotoxity of the drug against EMT6 cells was tested by MTT assay; an experimental therapy was carried out in 15 nude mice that were subcutaneously injected with EMT6 cells; the morphological changes of tumor tissues were observed under electron microscope; and the DNA content was measured and analysed with a computerized MPV-microspectrophotometer. The result shows that psoralen has a strong antitumor effect on mammary cancer EMT6 cells both in vivo and in vitro, with an IC50 value of 2.23 micrograms/ml. The antitumor effect might be related to the relative decrease of cell DNA content and the degeneration and cavitation of cellular mitochondriums. Therefore psoralen may be an effective agent for the treatment of mammary cancer.
Animals↗
[Compositional analysis of two metal crowns and its cements unearthed from a Ming Dynasty grave in China by SEM].
OBJECTIVE: To investigate the prosthodontic level of our country in Ming Dynasty (from A. D. 1368 to A. D. 1644) and provide our courtry's history of stomatological development with some details. METHODS: By means of range estimation and SEM, the structure and shape of the metal crowns on the two human teeth which were unearthed from a Ming Dynasty grave, numbered 0754/0046 and stored in Wujin museum of Jiangsu province, as well as the compositions of the crowns and its cements were examined and analysed preliminarily. RESULTS: The two teeth looked like upper anterior teeth and no marks of preparations were observed. The golden-yellow metal crowns might have been hammered and welded into shape. The spectrum analysis of SEM showed the crowns were made from gold-copper alloy and the cements contains Zinc. CONCLUSION: The defects of teeth could have been restored with extra-hard gold-copper alloy and cement luting materials during A. D. 1500-1600 in China.
China↗
[Generation of anti-rhEPO ScFv by using phage display technology].
In this paper, the recombinant phage antibody techniques was used to construct, clone, screen, and express anti-rhEPO single chain antibody ScFv. The variable region genes of antibody were amplified by PCR from a hybridoma cell line D3 which secreted monoclonal antibody to rhEPO. The ScFv gene fragments were successfully cloned into phagemid vector pCANTAB5E. The recombinant phages were panned by rhEPO which was coated on a microtiter plate. After three rounds of panning, 8 clones were determined specifically binding to rhEPO antigen. The positive recombinant phagemids were extracted and transformed into non-suppressed E. coli HB2151. The soluble single chain antibody was expressed, and the specificity of the expressed ScFv was determined by ELISA. Western blot and Dot-blot. The result of SDS-PAGE indicated that the apparent molecular weight of the target peptide which is mainly in culture supernatant is 32 kD. The DNA sequence data showed that the ScFv gene included 783 bp, encoding 261 amino acids.
Amino Acid Sequence↗
[The study on in vivo erythropoietin gene transfer for treatment of renal anemia in animal model].
To explore the possibility of gene therapy to renal anemia, the receptor mediated erythropoietin (EPO) gene transfer in vivo had been tested. EPO gene was inserted to an EBV replicon expression vector to form the plasmid pEPO, Renal anemia rat had been induced by feeding with adenine, and 450 micrograms of the pEPO was delivered by perfusion into the rat peripheral circulation in the form of soluble DNA/galactosylate histone complex. 13 days after injection, the number of peripheral red cells was counted and the concentration of hemoglobin was measured. The results demonstrated that symptom of anemia had been significantly improved(P < 0.01), when compared with that of the control test. (the red cell number was 492 vs 409, and the hemoglobin was 11.4 vs 9.1).
Anemia↗
[Microscopic identification of Herba Dianthi grown in Shandong].
The medicinal materials of four species and two varieties of Herba Dianthi grown in Shandong were identified. The result shows that they are identified easily and accurately according to the outer properties, and they are apparently divided into the Shizhu group, the Qumai group and the Dianthus shandongensis on the basis of their morphological and structural characteristics of the stem and leaf, but they have not obvious distinction among the species of every group.
Dianthus↗
[Determination of zinc, copper, iron and manganese contents in hair for MPA patients and healthy men].
In this paper the contents of zinc, copper, iron and manganese in hair for MPA patients and 38 hair-healthy men have been determined by FAAS. The results show that the hair zinc and manganese levels in MPA patients are significantly lower than that in hair-healthy men. The hair copper level in MPA patients is significantly higher than that in hair-healthy men. There are statistic difference (P < 0.05) in hair zinc, copper and menganese levels among two groups. There is no statistic difference (P > 0.05) in the iron level among the two gopups. This paper provides useful material for the research of the cause and treatment of the MPA.
Alopecia↗
Antibody to caspase-cleaved actin detects apoptosis in differentiated neuroblastoma and plaque-associated neurons and microglia in Alzheimer's disease.
During apoptosis, activation of a family of cysteine proteases related to interleukin-1beta-converting enzyme (ICE)-related proteases or "caspases" results in endoproteolytic cleavage of multiple substrates at specific aspartate residues. We have sought to develop new antibody probes for the neoepitopes in protein fragments produced by ICE-related proteolytic cleavage as specific markers of events tightly linked to apoptotic mechanisms. Here, we demonstrate that an antibody probe specific for the C terminus of a 32-kd actin fragment produced by ICE-like activity specifically labels apoptotic but not necrotic, differentiated human neuroblastoma cells in culture. Unlike probes for nonspecific DNA strand breaks confined to the nucleus or cell body, this method allows the detection of cytoskeletal fragments in cell processes as well as the perikaryon long before DNA fragmentation and cell death and therefore serves as a novel marker of apoptosis-related events in distal parts of cells such as axons and dendrites. To illustrate this new tool, we show that the antibody detects the processes and cell bodies of degenerating neurons and plaque-associated microglia in Alzheimer's disease. In situ detection of caspase-cleaved actin provides a new means to evaluate the role of caspase activation in pathological and physiological processes.
Actins↗
T cell immunobiology of alloimmune thrombocytopenias.
Allorecognition and generation of alloantisera depend on T cell help. Here we summarize our recent work on identifying the T cells that recognize a specific platelet alloantigen generated by a Pro to Leu polymorphism in beta-chain of the platelet integrin alphaIIb beta3. The ability to generate alloantibodies is restricted by HLA-DRB3*0101. By measuring peptide binding to HLA-DRB3*0101, we have shown that the polymorphism controls the generation of the T cell epitope. This is not a result of the polymorphism changing a T cell contact residue, but rather by its generating a peptide anchor. The result is a directional antibody response in which only the beta-chain that is antigenic is the one that produces a peptide that binds to the HLA-DR. This mechanism of generating alloantibodies may be a paradigm for a whole class of responses.
Animals↗
Reduction of both RAR and RXR levels is required to maximally alter sensitivity of CA-OV3 ovarian tumor cells to growth suppression by all-trans-retinoic acid.
We wished to determine the effect of altering the levels or functional activity of retinoid receptors, in particular retinoic acid receptor-alpha (RAR-alpha) and retinoid X receptor-alpha (RXR-alpha) on the growth sensitivity of ovarian tumor cells to all-trans-retinoic acid (all-trans-RA). We found that CA-OV3 cells could be made resistant to all-trans-RA growth inhibition by overexpressing RAR-beta(R269Q), an efficient dominant negative mutant which inhibits the function of all RAR subtypes. Antisense technology was then used to prepare stable transfectants of the retinoid-sensitive ovarian carcinoma cell line CA-OV3 in which expression of RAR-alpha, RXR-alpha, or both RAR-alpha and RXR-alpha was reduced. The effect of all-trans-RA on ovarian tumor cell growth was determined by MTT assay, autoradiographic analysis of DNA synthesis, and anchorage-independent colony formation in soft agar. Our results show that cell lines expressing reduced levels of either RAR-alpha alone or RXR-alpha alone exhibited a small decrease in sensitivity to growth inhibition by all-trans-RA. However, maximum RA resistance was obtained in cell lines in which the levels of both RAR-alpha and RXR-alpha were reduced. These results demonstrate the importance of both retinoid nuclear receptors and retinoid-X receptors in general, and RAR-alpha and RXR-alpha in particular, as mediators of ovarian carcinoma cell growth inhibition by retinoids.
Animals↗
Mitochondrial dysfunction in lymphocytes from old mice: enhanced activation of the permeability transition.
Aging is associated with mitochondrial dysfunction in excitable tissues such as nerve and muscle. However, it is not known if immunosenescence is similarly associated with mitochondrial dysfunction in lymphocytes. We have found that spleen lymphocytes from old mice have lower respiration rates than lymphocytes from young mice. Cyclosporin, an inhibitor of the mitochondrial Permeability Transition, PT, restored normal respiration rates to lymphocytes from old mice, suggesting enhanced susceptibility to PT activation. Lymphocytes from old mice also had a lower mitochondrial membrane potential (delta psi m) than lymphocytes from young mice, which was also restored by cyclosporin. Oxidized FAD fluorescence was higher in lymphocytes from old mice suggesting a more oxidized state, which may be the cause of the enhanced activation of PT. Incubation of lymphocytes from old mice with the lipophilic cationic dye DiOC6(3), which inhibits electron transport, induced the appearance of apoptotic cells. These findings suggest that the mitochondrial PT is more susceptible to activation in lymphocytes from old mice. This activation may inhibit energy metabolism and enhance apoptosis, and may therefore contribute to immunosenescence.
Aging↗
Induction of mucosal and systemic responses against human immunodeficiency virus type 1 glycoprotein 120 in mice after oral immunization with a single dose of a Salmonella-HIV vector.
Previous studies from our group showed that a Salmonella-HIV vector vaccine that expressed recombinant HIV-1 envelope protein gp120 stably in the vector cytoplasm elicited type 1 helper T cell (Th1) responses to gp120. Despite the promise of such vaccines, a major limitation in their use was that multiple immunizations were required to elicit even small responses. For this reason, we sought a modified vector configuration that would induce more potent gp120-specific T cell responses exhibiting a broader spectrum of effector functions after a single inoculation. In this article we describe the construction and immunogenicity of a Salmonella-HIV vector that displays a truncated derivative of HIV-1(IIIB) envelope in the periplasm of the vector. A single oral dose of this Salmonella vector, called H683(pW58-asd+), generated a gp120-specific proliferation response in the spleen 14 days after immunization. In agreement with our previous findings, the gp120-specific splenic CD4+ T cells elicited by H683(pW58-asd+) displayed a Th1 phenotype; however, gp120-specific splenic CD4+ Th2 cells were also evident. In addition, this strain induced strong gp120-specific IgA antibody-secreting cell (ASC) responses in the intestinal lamina propria and mesenteric lymph nodes. As many as 2% of the total lamina propria and mesenteric lymph node IgA ASCs were found to be specific for gp120 28 days after a single oral dose of H683(pW57-asd+). Because the proliferative response following a single dose of H683(pW58-asd+) was comparable to that seen previously after three doses of an analogous construct expressing recombinant gp120 in the cytoplasm, these observations suggest that Salmonella-vectored secreted HIV-1 antigens elicit higher T cell responses than their cytoplasmically bound analogs.
Administration, Oral↗
Identification of a human protein that recognizes the 3' splice site during the second step of pre-mRNA splicing.
Accurate splicing of precursor mRNAs (pre-mRNAs) requires recognition of the 5' and 3' splice sites at the intron boundaries. Interactions between several splicing factors and the 5' splice site, which occur prior to the first step of splicing, have been well described. In contrast, recognition of the 3' splice site, which is cleaved during the second catalytic step, is poorly understood, particularly in higher eukaryotes. Here, using site-specific photo-crosslinking, we find that the conserved AG dinucleotide at the 3' splice site is contacted specifically by a 70 kDa polypeptide (p70). The p70-3' splice site crosslink has kinetics and biochemical requirements similar to those of splicing, was detected only in the mature spliceosome and occurs subsequent to the first step. Thus, p70 has all the properties expected of a factor that functionally interacts with the 3' splice site during the second step of splicing. Using antisera to various known splicing factors, we find that p70 corresponds to a previously reported 69 kDa protein of unknown function associated with the Sm core domain of spliceosomal small nuclear ribonucleoproteins.
Cross-Linking Reagents↗
Viral infection. II. Hemin induces overexpression of p67 as it partially prevents appearance of an active p67-deglycosylase in baculovirus-infected insect cells.
The roles of p67-deglycosylase (p67-DG) in the regulation of protein synthesis in baculovirus-infected insect cells were studied. Like vaccinia viral infection, baculovirus infection of insect cells also induced the appearance of a p67-DG. However, p67-DG activity could not be detected because these cells do not contain a detectable level of p67. The baculovirus expression vector system (BEVS), however, promotes significant expression of cloned p67-cDNA. The expression of p67 was significantly enhanced by the addition of hemin to the growth medium. Maximum enhancement was observed at 5 microM hemin. Data suggest that hemin prevents the activation of latent p67-DG inside the cell and does not have any effect on p67 gene transcription. To gain a better understanding of the mechanism of p67-DG activation and hemin stimulation of p67 synthesis, we have now purified p67-DG from baculovirus-infected insect cells. We prepared antibodies against this protein. These antibodies reacted with a 105-kDa protein in cell extracts from the uninfected insect cells (Sf9), KRC-7, and L929 (animal cells). In addition, these antibodies reacted with an additional 60-kDa protein in the cell extracts of baculovirus-infected Sf9 cells and vaccinia virus-infected KRC-7 and L929 cells. Data are also presented to show that the antibodies against p67-DG reacted more efficiently (40%) with the 60-kDa protein in both hemin-deficient reticulocyte lysate and hemin-deficient baculovirus-infected cells. We suggest that hemin prevents the conversion of an inactive p67-DG into an active form possibly by covalent modification such as protein phosphorylation or protein glycosylation. The active form is more efficiently recognized by the p67-DG antibodies since these antibodies were prepared against the active form of p67-DG.
Acetylglucosaminidase↗
Molecular cloning, expression, and functional significance of a cytochrome P450 highly expressed in rat heart myocytes.
A cDNA encoding a P450 monooxygenase was amplified from reverse transcribed rat heart and liver total RNA by polymerase chain reaction using primers based on the 5'- and 3'-end sequences of two rat pseudogenes, CYP2J3P1 and CYP2J3P2. Sequence analysis revealed that this 1,778-base pair cDNA contained an open reading frame and encoded a new 502 amino acid protein designated CYP2J3. Based on the deduced amino acid sequence, CYP2J3 was approximately 70% homologous to both human CYP2J2 and rabbit CYP2J1. Recombinant CYP2J3 protein was co-expressed with NADPH-cytochrome P450 oxidoreductase in Sf9 insect cells using a baculovirus expression system. Microsomal fractions of CYP2J3/NADPH-cytochrome P450 oxidoreductase-transfected cells metabolized arachidonic acid to 14,15-, 11,12-, and 8, 9-epoxyeicosatrienoic acids and 19-hydroxyeicosatetraenoic acid as the principal reaction products (catalytic turnover, 0.2 nmol of product/nmol of cytochrome P450/min at 37 degrees C). Immunoblotting of microsomal fractions prepared from rat tissues using a polyclonal antibody raised against recombinant CYP2J2 that cross-reacted with CYP2J3 but not with other known rat P450s demonstrated abundant expression of CYP2J3 protein in heart and liver. Immunohistochemical staining of formalin-fixed paraffin-embedded rat heart tissue sections using the anti-CYP2J2 IgG and avidin-biotin-peroxidase detection localized expression of CYP2J3 primarily to atrial and ventricular myocytes. In an isolated-perfused rat heart model, 20 min of global ischemia followed by 40 min of reflow resulted in recovery of only 44 +/- 6% of base-line contractile function. The addition of 5 microM 11, 12-epoxyeicosatrienoic acid to the perfusate prior to global ischemia resulted in a significant 1.6-fold improvement in recovery of cardiac contractility (69 +/- 5% of base line, p = 0.01 versus vehicle alone). Importantly, neither 14,15-epoxyeicosatrienoic acid nor 19-hydroxyeicosatetraenoic acid significantly improved functional recovery following global ischemia, demonstrating the specificity of the biological effect for the 11, 12-epoxyeicosatrienoic acid regioisomer. Based on these data, we conclude that (a) CYP2J3 is one of the predominant enzymes responsible for the oxidation of endogenous arachidonic acid pools in rat heart myocytes and (b) 11,12-epoxyeicosatrienoic acid may play an important functional role in the response of the heart to ischemia.
8,11,14-Eicosatrienoic Acid↗
p67 transcription regulates translation in serum-starved and mitogen-activated KRC-7 cells.
The regulation of protein synthesis was studied in KRC-7 cells (rat hepatoma) grown in complete medium, during serum starvation, and mitogen activation. Upon serum starvation, the cells lost almost completely p67 mRNA, p67 protein, and protein synthesis activity. After phorbol 12-myristate 13-acetate addition, the same serum-starved cells regained p67 mRNA, p67 protein, and protein synthesis activity. Also, the extracts from the serum-starved cells phosphorylated the eukaryotic initiation factor-2 (eIF-2) alpha-subunit. This eIF-2 alpha-subunit phosphorylation was not observed when the extracts from either the cells grown in complete medium or mitogen-activated cells were used (Gupta, S., Wu, S., Chatterjee, N., Ilan, J., Ilan, J., Osterman, J. C., and Gupta, N. K. (1995) Gene Expr. 5, 113-122). We now report the following. 1) The eIF-2 kinase activity was the same in the cells grown in complete medium, after serum starvation, and subsequent mitogen stimulation. However, the eIF-2 kinase in the cells grown in complete medium and also after mitogen activation of the serum-starved cells cannot phosphorylate eIF-2 alpha-subunit as these cells contain p67. After removal of endogenous p67 by p67 antibodies, the extracts from all these cells similarly phosphorylated exogenously added eIF-2. 2) None of the cell extracts showed p67 deglycosylase activity. 3) The p67 mRNA was synthesized in serum-starved cells by expression of a p67 cDNA. The appearance of p67 mRNA in the serum-starved cells was accompanied by the appearance of p67 protein. Also, the rates of protein synthesis in the serum-starved cells were restored nearly to the level observed in the confluent cells. The expression of p67 cDNA also significantly increased protein synthesis rates in the cells grown in complete medium and in mitogen-activated cells. These results show that the loss of protein synthesis activity in serum-starved cells was due to loss of p67 mRNA. The expressed p67 mRNA was stable in serum-starved cells. These results, therefore, suggest that the loss of p67 mRNA in serum-starved cells is due to loss of p67 transcription. The p67 transcription regulates translation.
Aminopeptidases↗