Meta analysis of aerobic exercise improving intelligence and cognitive function in patients with Alzheimer's disease.

OBJECTIVE: Alzheimer's disease (AD) is a neurodegenerative disease. This study aims to explore the intervention and treatment effects of aerobic exercise and different exercise modes on AD through meta-analysis. METHODS: Using the set inclusion and exclusion criteria, retrieve the China national knowledge infrastructure (CNKI), Wanfang Data Knowledge Service Platform, China Science and Technology Journal Database, Cochrane Library, and PubMed were searched from January 1, 2012, to December 31, 2021. Cochrane risk bias assessment tool was used to evaluate the quality of the included articles, and ReMan5.4.1 was used for forest plot analysis of mini-mental state exam (MMSE) score indicators included in the included articles. RESULTS: Twelve randomized controlled trials and 795 samples were included. Meta analysis of all articles: I2 = 91%, P ≤ .00001, (MD = 2.95, 95%CI [2.49, 3.40], P ≤ .00001). Meta analysis of 5 fit aerobics groups: I2 = 4%, P = .38, (MD = 1.53, 95%CI [0.72, 2.33], P = .0002); meta-analysis of three spinning groups: I2 = 3%, P = .36, (MD = 1.79, 95%CI [0.29, 3.29], P = .02). CONCLUSION: Aerobic exercise can effectively improve intellectual and cognitive impairment in AD patients, and for different forms of aerobic exercise, the therapeutic effect of spinning aerobic exercise is better than that of fit aerobics.

Discovery of potent and highly selective thienopyridine Janus kinase 2 inhibitors.

Developing Janus kinase 2 (Jak2) inhibitors has become a significant focus for small molecule drug discovery programs in recent years due to the identification of a Jak2 gain-of-function mutation in the majority of patients with myeloproliferative disorders (MPD). Here, we describe the discovery of a thienopyridine series of Jak2 inhibitors that culminates with compounds showing 100- to >500-fold selectivity over the related Jak family kinases in enzyme assays. Selectivity for Jak2 was also observed in TEL-Jak cellular assays, as well as in cytokine-stimulated peripheral blood mononuclear cell (PBMC) and whole blood assays. X-ray cocrystal structures of 8 and 19 bound to the Jak2 kinase domain aided structure-activity relationship efforts and, along with a previously reported small molecule X-ray cocrystal structure of the Jak1 kinase domain, provided structural rationale for the observed high levels of Jak2 selectivity.

Utility of lyophilized PMA and ionomycin to stimulate lymphocytes in whole blood for immunological assays.

BACKGROUND: The need to implement robust biomarkers in clinical trials has never been greater, and such efforts can be easily compromised by reagent instability or simple human error during assay set-up. Many biotechnology and pharmaceutical companies are introducing efforts to conduct biomarker studies under more rigorous settings, and the use of plates or tubes pre-loaded with stimulation or staining reagents could be of value for studies that involve flow cytometry. METHODS: Five reagents lyophilized from ethanol or CHAPS buffer stock solution of phorbol 12-myristate 13-acetate (PMA) and ionomycin were benchmarked against standard DMSO liquid formulation for their stimulation equivalency. The median fluorescence intensity of phosphorylated ribosomal protein S6 in lymphocytes was assessed on a BD FACSCalibur. RESULTS: We demonstrate here that tubes pre-loaded with lyophilized versions of the liquid reagents can provide equivalent stimulation in healthy volunteer specimens. CONCLUSIONS: The value of this approach is that it safeguards against omission or erroneous addition of bulk liquid formulations of PMA and ionomycin to the reaction vessel (i.e., plate or tube) and also lends itself to extended stability/shelf-life of these reagents. On the basis of this initial success, we plan to expand our evaluation of lyophilized reagents so that they can be incorporated into our clinical biomarker campaigns as appropriate.

[Effect of heat stress on expression of gp96 in K562 cell line of the chronic myeloid leukemia and its significance].

This study was purposed to investigate the effect of different heat stress conditions on expression level of heat shock protein gp96 in K562 cell line of chronic myeloid leukemia in order to provide experiment basis for seeking optimal heat stress condition increasing extraction amount of gp96 from K562 cells. The expression changes of gp96 in K562 cell line was detected by immunocytochemistry under 38, 40, 42, 44, 46 and 48 degrees C for 30 minutes in water, by flow cytometry under 40, 44, 48 and 52 degrees C for 30 minutes in water, by Western blot under 40, 44, 48 and 52 degrees C for 30 minutes in water. Immunocytochemistry assay showed that gp96 existed mainly in cytoplasm. The peak of gp96 expression was at 30 minutes after 48 degrees C in water. The result of flow cytometry was consistent to immunocytochemistry detection results under temperatures 40, 44, 48 and 52 degrees C (P < 0.01). Western blot showed that detection result was the same as the immunocytochemistry and flow cytometry detections. In conclusion, the expression of gp96 in K562 cell line reached peak at 30 minutes after 48 degrees C in water. This condition may be an effective preparative condition for extraction of gp96 from K562 cells.

Identification and expression of a new type II transmembrane protein in human mast cells.

A cDNA encoding a new type II transmembrane protein has been isolated from human mast cells by subtraction cloning. This cDNA contains an open reading frame of 186 amino acids. RT-PCR analysis showed that this gene is differentially expressed in mast cells. Therefore, the peptide encoded by this gene was termed mast cell-expressed membrane protein 1 (MCEMP1). The MCEMP1 gene contains seven exons and was mapped to human chromosome 19p13.3. The epitope-tagged MCEMP1 has been expressed in mammalian cells and found to be localized to the cellular membrane with its C-terminus extending to the outside of the membrane and N-terminus into the cytoplasmic compartment. Monoclonal antibodies against MCEMP1 were generated and characterized by immunoprecipitation and FACS. The results showed that the native MCEMP1 is expressed in cord blood-derived mast cells and HMC-1 and THP-1 cell lines, but not in other cell types that we have tested. Immunochemical staining of human lung sections showed that MCEMP1 staining is specifically associated with lung mast cells.

Amphiregulin expression in human mast cells and its effect on the primary human lung fibroblasts.

BACKGROUND: Amphiregulin is a member of the epidermal growth factor family and has been shown to stimulate the proliferation of human keratinocytes in an autocrine manner. OBJECTIVE: The aim of the present study was to examine the expression change of growth factors, especially amphiregulin, in human mast cells induced by IgE cross-linking. METHODS: Microarray analysis and RT-PCR were used to analyze the gene expression profile of human cord blood-derived mast cells (CBMCs) stimulated with IgE cross-linking. Protein secretion in the supernatants of CBMCs was measured by means of ELISA. Double-immunofluorescence staining was used to analyze the expression in the lung mast cells. RESULTS: Of the 64 different growth factor genes analyzed, 5 were found to be substantially upregulated. Among them, amphiregulin mRNA was induced by 44-fold in CBMCs on activation through IgE cross-linking. Secretion of amphiregulin protein was evident in CBMCs 8 hours after stimulation. Amphiregulin was also expressed in human lung mast cells from patients with asthma, as demonstrated by means of double-immunofluorescence staining. Amphiregulin promoted the proliferation of the primary human lung fibroblasts, and amphiregulin-treated primary human lung fibroblasts showed a marked increase in the expression of c-fos , a proto-oncogene that facilitates or is required for the proliferation of a wide variety of cells. CONCLUSION: Human CBMCs secreted amphiregulin on IgE cross-linking, and the amphiregulin induced proliferation of primary human lung fibroblasts. These data suggest that local release of amphiregulin by human mast cells could play an important role in lung fibrosis by promoting the proliferation of primary human lung fibroblasts.

[Differentiation and function of human dendritic cells influenced by heat shock protein gp96 purified from K562 cells].

This study was to establish the method of purifying heat shock protein GP96 from K562 cells and explore the differentiation and function of human DC influenced by heat shock prolein (HSP). Using ammonium sulfate precipitation, conA-sepharose affinity chromatography and DEAE-sephacel anion exchange chromatography GP96 from K562 cells lysate was isolated and purified. The identification of the purified protein was controlled by Western blot with anti-human GP96 IgG. Human dendritic cell derived from peripheral blood mononuclear cell were cultured with purified GP96. The phenotype changes of DC were analyzed by flow cytometry and MLR was detected by MTT. The results showed that 60-80 microg GP96 was purified successfully from 1 x 10(10) K562 cells. DC stimulated with HSP-GP96 had higher expression rates of CD83, CD86, HLA-DR and lower expression rates of CD1a and had stronger ability to induce T cells proliferation. It is concluded that heat shock protein GP96 can be isolated and purified from K562 cells and could induce maturation of dendritic cell. HSP-DC vaccine show stronger ability to induce T cell proliferation than DC.

[Effect of transforming growth factor-beta1 on proliferation and apoptosis of mononuclear cells of cord blood].

To explore the mechanism of transforming growth factor-beta1 (TGF-beta1) effect on umbilical cord blood mononuclear cells proliferation and apoptosis, 5-bromo-2'-deoxyurine (BrdU) incorporation assay was adopted to detect effect of TGF-beta1 on synthesis of DNA in cells. Western blot method was used to examine effect of TGF-beta1 on expression of cyclin A, Cyclin D1, CDK2 and CDK4 in G1 phase of cell cycle. Giemsa staining and flow cytometry (FCM) were performed to detected effect of TGF-beta1 on cell apoptosis. The results showed that (1) after culture of cells with IMDM containing 10% FBS, 10% FBS + 1 ng/ml TGF-beta1, 10% FBS + 2 ng/ml TGF-beta1 or 10% FBS + 5 ng/ml TGF-beta1 for 12 hours the OD values of TGF-beta1 group were significantly lower than control group (P <0.01); after culture for 24 hours the OD values of 1 ng/ml TGF-beta1 group had no significant difference compared with control group (P >0.05), but the OD values of 2 ng/ml and 5 ng/ml TGF-beta1 groups were significantly lower than control group (P <0.05). (2) 2 ng/ml TGF-beta1 could significantly inhibit the production of cyclin A, cyclin D1, CDK2 and CDK4, the protein levels were significantly lower than control group. (3) when the cells were co-cultured with 2 ng/ml TGF-beta1 for 12 and 24 hours, Giemsa staining and FCM detection could display typical apoptosis, the apoptosis rates were 14.42% and 31.98%, while apoptosis rate in control were 4.71% and 5.76%. It is concluded that TGF-beta1 can inhibit production of G1 cyclins and CDKs of umbilical cord blood mononuclear cells, arrest cells in the G1 phase of cell cycle and induce cell apoptosis. Thus, TGF-beta1 may be an important negative modulator in hematopoiesis.

[The heat shock protein 70 antisense oligomers enhance the sensitivity of bladder cancer cell EJ to mitomycin C].

OBJECTIVE: To investigate whether the heat shock protein (HSP) 70 antisense oligomers can enhance the sensitivity of bladder cancer cell EJ to mitomycin C. METHODS: The HSP70 mRNA of EJ cells was blocked by the 10 micromol/L HSP70 antisense oligomers, while its effect on cell growth was evaluated by methyl thiazolyl tetrazolium (MTT) and colony forming ability test. RESULTS: The HSP70 expressions in HSP70 antisense treated group were lower than the corresponding sense and nonsense treated groups (P < 0.01). While, the increased sensitivity of EJ to mitomycin C was found in antisense treated group, compared with the corresponding sense and nonsense treated groups (P < 0.01). CONCLUSION: The sensitivity of bladder cancer cell EJ to mitomycin C was enhanced by the blockage of the HSP70 expression.

[Apoptin induces G2-M arrest in cancer cells].

OBJECTIVE: To further study the effect of apoptin in inducing cancer cell specific apoptosis and the possible applications in cancer therapy. METHODS: Apoptin gene was amplified by PCR and inserted into pcDNA3.1(+) with a FLAG tag in front of the multi-cloning-site. Apoptin gene with the FLAG tag was sub-cloned into an adenovirus vector. Several cancer cell lines were transfected with pcDNA3.1/FLAG/apoptin or infected with apoptin containing recombinant adenoviruses to study the morphologic changes. Ad/apoptin infected cells were also analyzed by flowcytometry after staining with PI. RESULTS: Expressed apoptin was localized in the nucleus of cancer cells. Chromatin condensation occurred 2 or 3 days after Ad/apoptin(+) infection. Cell number in G(2)-M phase increased dramatically after Ad/apoptin(+) infection. CONCLUSION: Apoptin can induce cell cycle G(2)-M arrest and chromatin condensation in cancer cells.

A common IL-13 Arg130Gln single nucleotide polymorphism among Chinese atopy patients with allergic rhinitis.

Allergic rhinitis is a major public health problem and has seen its prevalence increase during the past few decades. Interleukin 13 (IL-13) has been implicated in the pathogenesis and in the regulation of immunoglobulin E (IgE) production. Single nucleotide polymorphisms (SNPs) have been found in both the coding sequence and the promoter region of IL-13, and such SNPs have been associated with allergic asthma. We have investigated whether IL-13 SNPs are associated with allergic rhinitis. Among 188 Chinese adult patients with allergic rhinitis and 87 normal controls, no significant difference was found in either allele or haplotype frequency of the SNPs between the two groups. Within patients, there was a significant association of the IL-13 Arg130Gln SNP, but not of the IL-13 promoter -1112(C/T) SNP, with serum total IgE levels. Patients with a Gln/Gln genotype showed much higher serum total IgE than those with an Arg/Arg genotype. When tested for serum-specific IgE, patients allergic to Derp 1, but not those allergic to Artemisia pollen, showed a significant association with the IL-13 promoter SNP. Thus, our results suggest a possible involvement of IL-13 SNPs in the regulation of IgE production in response to allergens in this Chinese population.

[Immortalization of human osteoblast by transferring hTERT gene].

OBJECTIVE: To establish an immortalized osteoblast cell line used for basal and clinical research of orthopaedics. METHODS: Human marrow stromal cells (hMSCs) had been inducted into osteoblasts directionally, and human telomerase reverse transcriptase (hTERT) cDNA was transferred into the osteoblasts by retroviral vector pLPChTERT. The resultant stable clones reproduced successively and the expression of hTERT as well as the osteogenesis characteristics of different eras were identified. RESULT: hTERT gene has been transferred into human osteoblasts successfully. The transformed cells expressed telomerase activity and divided vigorously. p62 has been obtained so far. The expression of bone specific Alkaline phosphatase, Collagen type I and Osteopontin of p25, p55 were checked and it is proved that the immortalized cell line preserved typical osteogenesis characteristics. CONCLUSION: Osteoblasts can be immortalized by transferring exogenous hTERT gene to reconstitute telomerase activity, and the immortalized cell reserved osteogenesis characteristics.

Regulation of activin A expression in mast cells and asthma: its effect on the proliferation of human airway smooth muscle cells.

Activin A, a homodimeric protein (betaAbetaA) and a member of the TGF-beta superfamily, is involved in the inflammatory repair process. Using cDNA microarray analysis, we discovered strong induction of the activin betaA gene in human mast cells (MC) on stimulation with PMA and calcium ionophore (A23187). Activin betaA mRNA was also highly induced in primary cultured murine bone marrow MC (BMMC) after stimulation by IgE receptor cross-linking. Secretion of activin A was evident in human mast cell-1 line cells 3 h after stimulation and progressively increased over time. Activin A was present in the cytoplasm of activated but not unstimulated murine bone marrow MC as demonstrated by immunofluorescence studies, suggesting that secretion of activin A by MC was due to de novo synthesis rather than secretion of preformed protein. Activin A also colocalized with human lung MC from patients with asthma by double-immunofluorescence staining. Furthermore, secretion of activin A was significantly increased in the airway of wild-type mice after OVA sensitization followed by intranasal challenge. Secretion of activin A, however, was greatly reduced in MC-deficient WBB6F(1)-W/W(v) mice as compared with wild-type mice, indicating that MC are an important contributor of activin A in the airways of a murine asthma model. Additionally, activin A promoted the proliferation of human airway smooth muscle cells. Taken together, these data suggest that MC-derived activin A may play an important role in the process of airway remodeling by promoting the proliferation of airway smooth muscle.

Calcineurin/nuclear factors of activated T cells (NFAT)-activating and immunoreceptor tyrosine-based activation motif (ITAM)-containing protein (CNAIP), a novel ITAM-containing protein that activates the calcineurin/NFAT-signaling pathway.

We report in this study the identification and characterization of a novel protein that we designated as calcineurin/NFAT-activating and immunoreceptor tyrosine-based activation motif (ITAM)-containing protein (CNAIP). The predicted 270-amino acid sequence contains an N-terminal signal peptide, an immunoglobin domain in the extracellular region, a transmembrane domain and an ITAM in the cytoplasmic tail. Quantitative reverse transcription-PCR showed that CNAIP was preferentially expressed in neutrophils, monocytes, mast cells, and other immune-related cells. Co-transfection of CNAIP expression constructs with luciferase reporter plasmids in HMC-1 cells resulted in activation of interleukin-13 and tumor necrosis factor-alpha promoters, which was mediated through the calcineurin/NFAT-signaling pathway. Mutation of either or both tyrosines in the ITAM abolished transcriptional activation induced by CNAIP, indicating that the ITAM is indispensable for CNAIP function in activating cytokine gene promoters. Thus, it is concluded that CNAIP is a novel ITAM-containing protein that activates the calcineurin/NFAT-signaling pathway and the downstream cytokine gene promoters.

[A quantitative assay for telomerase activity in peripheral blood mononuclear cells from patients with acute leukemia].

To establish a quantitative assay for telomerase activity and analyze the telomerase activity in peripheral blood mononuclear cells (PBMNC) from patients with acute leukemia, a fluorescent dye, PicoGreen, was added to the products after telomere repeat amplification protocol. The samples were excited at 480 nm and the fluorescence emission intensity was measured at 520 nm using a spectrofluorometer. Telomerase activity was detected in PBMNCs from 20 cases of normal individuals and 25 patients with acute leukemia. The results showed that the fluorescence of PicoGreen binding to double-stranded DNA specifically was enhanced with increase of DNA quantities. In conclusion, the met hod is rapid, simple and quantitative, the telomerase activities of PBMNCs from acute leukemia patients are significantly higher than that of the normal controls.

[Dendritic cells cultured with human umbilical cord serum instead of fetal calf serum].

To investigate whether the dendritic cells (DC) could grow up in cultural system with umbilical cord serum (UCS), the UCS was used in the culture instead of fetal calf serum. The phenotype of dendritic cells was detected by flow cytometry and the antigen presenting ability of DC in allo-MLR was measured by MTT assay. The results showed that DC grown in UCS (UCS-DC) had higher expression rate of CD86, CD83 and HLA-DR than that in grown in FCS (FCS-DC). (P < 0.05), and their expression of CD1a was lower than that of FCS-DC. The ability to induce T cell proliferation had no difference between UCS-DC and FCS-DC. It is suggested that dendritic cells with more mature phenotype had been produced in the medium containing UCS than those in the medium containing FCS, and UCS-DC possessed potent ability to stimulate proliferation of allogeneic T cells.

[p21(WAF1) transfection decreases sensitivity of K562 cells to VP-16].

The objective of this study was to explore the effect of p21(WAF1) on the sensitivity to chemotherapeutic agent VP-16, etoposide, in leukemia cell line K562. A p21(WAF1) retroviral expression vector was constructed, and mediated by FuGENE(TM)6, it was transfected into K562 cells, which without p21(WAF1) expression. The ecotropic expression of p21(WAF1) in K562 cells was identified by RT-PCR and Western blot, and named K562-p21(WAF1) cell. The K562-p21(WAF1) cells were exposed to VP-16 at different concentrations and different times, then, the sensitivity to VP-16 was examined by cell viability and MTT assay, the apoptosis induced by VP-16 was examined by DNA fragments electrophoresis and flow cytometric Annexin V-PI dual labeling technique. The results showed that the ecotropic expression of p21(WAF1) decreased the sensitivity of K562 cells to VP-16. After treatment of VP-16, the DNA ladder was examined in control K562-neo cells at 20 microgram/ml, but in K562-p21(WAF1) cells at 80 microgram/ml. With FCM, the number of apoptotic K562-neo cells was 14.9% and 25.4% respectively after treated with 20 microgram/ml VP-16 for 12 and 24 hours, and it was 6.94% and 10.96% in K562-p21(WAF1) cells. Our results suggest that the expression of p21(WAF1) could reduce the sensitivity of K562 cells to VP-16 and inhibit the apoptosis of K562 cells

[p21(WAF1) Gene Transfection Inhibits Proliferation of Leukemia Cell Line K562]

To explore the functional role of p21(WAF1) gene on the proliferation of leukemia cell line K562, a p21(WAF1) retroviral expression vector was constructed. Mediated by FuGENE trade mark 6, p21(WAF1) was transfected into leukemia cell line K562, which was without p21(WAF1) expression. After selected in G418, K562 cell clones that expressed p21(WAF1) stabaly were isolated and named K562-p21(WAF1). The ectopic expression of p21(WAF1) mRNA and protein in K562 cells was identified by RT-PCR and Western Blot. The cell proliferaton was tested in liquid and soft agar culture after transfection. The cell cycle was tested by FCM. The expression of p21(WAF1) protein and mRNA could be detected in K562-p21(WAF1) cells. A strong inhibition of cell proliferation was observed in K562-p21(WAF1) cell clones cultured in liquid media as well as soft agar (P < 0.01). The cell number in G(0)/G(1) phase was remarkably increased. The findings showed that p21(WAF1) can inhibit the proliferation of leukemia cells, and it could be a potential target gene for leukemia gene therapy.

[Transfer and Expression of Recombinant Human Thrombopoietin Gene in COS-7 Cells and Mice In Vivo]

In order to investigate the expression of recombinant TPO gene in COS-7 cells and in vivo of the mouse model, eukaryotic expressing plasmid pcd2/TPO with human TPO cDNA was constructed with DNA recombinant techniques. The plasmid pcd2/TPO was transiently transfected into the COS-7 cells by means of lipofection, the naked pcd2/TPO plasmid was injected into the skeletal muscle of mice with electric pulses. RT-PCR and ELISA methods were used to detect the TPO expression of the transfected COS-7 cells, both showed high level expression. The MTT test showed the expressed TPO had proliferative activity to TPO-dependent cell line. High efficiency of gene transfer in transgenic mice was also observed by RT-PCR and immunohistochemical methods. The serum TPO level [(1 185 +/- 264) ng/L] in transgenic mice was quite different compared with the normal mice [(250 +/- 76) ng/L]. All these results provided solid foundations for the research of TPO gene therapy in the future.