Homocysteine-potentiated Kelch-like ECH-associated protein 1 promotes senescence of neuroblastoma 2a cells via inhibiting ubiquitination of ß-catenin.

Elevated serum homocysteine (Hcy) level is a risk factor for Alzheimer's disease (AD) and accelerates cell aging. However, the mechanism by which Hcy induces neuronal senescence remains largely unknown. In this study, we observed that Hcy significantly promoted senescence in neuroblastoma 2a (N2a) cells with elevated ß-catenin and Kelch-like ECH-associated protein 1 (KEAP1) levels. Intriguingly, Hcy promoted the interaction between KEAP1 and the Wilms tumor gene on the X chromosome (WTX) while hampering the ß-catenin-WTX interaction. Mechanistically, Hcy attenuated the methylation level of the KEAP1 promoter CpG island and activated KEAP1 transcription. However, a slow degradation rate rather than transcriptional activation contributed to the high level of ß-catenin. Hcy-upregulated KEAP1 competed with ß-catenin to bind to WTX. Knockdown of both ß-catenin and KEAP1 attenuated Hcy-induced senescence in N2a cells. Our data highlight a crucial role of the KEAP1-ß-catenin pathway in Hcy-induced neuronal-like senescence and uncover a promising target for AD treatment.

Acinetobacter faecalis Sp. Nov., Isolated from Elephant Faeces.

A Gram-negative coccobacillus, YIM 103518T, isolated from wild elephant feces in Xishuangbanna, Yunnan Province, West China, was characterized and identified using a polyphasic taxonomic approach. The strain was strictly aerobic, non-motile, catalase-positive and oxidase-negative, colonies were round, convex, smooth, and pale yellow. The strain growth at 4-40 ℃ (optimum, 28 ℃), pH 6.0-10.0 (optimum, pH 7.0) and 0-4% NaCl (optimum, 0%) in culture medium YIM 38. The major fatty acids of strain YIM 103518T were summed feature 3 (C16:1 ω6c/C16:1 ω7c), C16:0, and C18:1 ω9c. The predominant ubiquinone was Q-9. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine and phospholipids. The 16S rRNA gene sequence showed moderate level of similarity with Acinetobacter portensis AC 877T (98.7%), Acinetobacter sichuanensis CCTCC AB 2018118T (97.1%), and Acinetobacter cumulans CCTCC AB 2018119T (97.1%). The G+C content of the genomic DNA was 36.5 mol%. Strain YIM 103518T showed an average nucleotide identity value of 86.6%, 77.3% and 78.5%, a digital DNA-DNA hybridizations value of 31.2%, 21.9% and 23.0% with the type strain of A. portensis, A. sichuanensis and A. cumulans based on draft genome sequences, respectively. The results of the phenotypic, chemotaxonomic and phylogenetic analyses, showed that strain YIM 103518T represents a novel species of the genus Acinetobacter, for which the name Acinetobacter faecalis sp. nov. is proposed. The type strain is YIM 103518T (=CCTCC AB 2019201T = NBRC 114057T).

Nakamurella leprariae sp. nov., isolated from a lichen sample.

A novel actinobacterium, YIM 132084T, was isolated from Lepraria sp. lichen collected from Yunnan province, south-west PR China and identified by a polyphasic taxonomic approach. The strain was Gram-stain-positive, aerobic, catalase-positive, oxidase-negative, non-motile and coccus-shaped. Colonies were round, convex, smooth and light orange yellow in color. It grew at 10-40 °C (optimum 28 °C), at pH 6.0-11.0 (optimum pH 7.0) and in the presence of 0-4% NaCl (optimum 0%). Strain YIM 132084T comprised diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol as the major polar lipids, MK-8(H4) as the predominant menaquinone, and anteiso-C15:0, anteiso-C17:0, iso-C15:0 and iso-C16:0 as major fatty acids. Strain YIM 132084T had meso-diaminopimelic acid as the diagnostic diamino acid in the cell-wall peptidoglycan, and mannose, ribose, glucose and rhamnose as whole-cell sugars. The 16S rRNA gene sequence showed high level of similarity with Nakamurella flavida KCTC 19127T (97.7%) and Nakamurella flava CGMCC 4.7524T (97.7%). The G + C content of the genomic DNA was 72.4 mol%. Based on draft genome sequences, strain YIM 132084T showed an average nucleotide identity value of 76.1% and 74.9%, a digital DNA-DNA hybridization value of 20.9% and 20.6% with the reference strains Nakamurella flavida and Nakamurella flava, respectively. The results of the phenotypic, chemotaxonomic and phylogenetic analyses showed that strain YIM 132084T represents a novel species of the genus Nakamurella, for which the name Nakamurella leprariae sp. nov. is proposed. The type strain is YIM 132084T (= CGMCC 4.7667T = NBRC 114280T = KCTC 49367T).

Liraglutide Ameliorates Hyperhomocysteinemia-Induced Alzheimer-Like Pathology and Memory Deficits in Rats via Multi-molecular Targeting.

Hyperhomocysteinemia (Hhcy) is an independent risk factor for Alzheimer's disease (AD), and insulin-resistance is commonly seen in patients with Hhcy. Liraglutide (Lir), a glucagon-like peptide that increases the secretion and sensitivity of insulin, has a neurotrophic or neuroprotective effect. However, it is not known whether Lir ameliorates the AD-like pathology and memory deficit induced by Hhcy. By vena caudalis injection of homocysteine to produce the Hhcy model in rats, we found here that simultaneous administration of Lir for 2 weeks ameliorated the Hhcy-induced memory deficit, along with increased density of dendritic spines and up-regulation of synaptic proteins. Lir also attenuated the Hhcy-induced tau hyperphosphorylation and Aß overproduction, and the molecular mechanisms involved the restoration of protein phosphatase-2A activity and inhibition of ß- and γ-secretases. Phosphorylated insulin receptor substrate-1 also decreased after treatment with Lir. Our data reveal that Lir improves the Hhcy-induced AD-like spatial memory deficit and the mechanisms involve the modulation of insulin-resistance and the pathways generating abnormal tau and Aß.

[Reversal of mdrl gene-dependent multidrug resistance in multidrug resistance human leukemia cell line K562/ADM using short hairpin RNA expression vectors].

OBJECTIVE: To explore the role of reversal multidrug resistance (MDR) using short hairpin RNA (shRNA) expression vectors in multidrug resistance human leukemia cell line K562/ADM. METHODS: The oligonucleotides with 19-mer hairpin structure were synthesized. The shRNA expression vectors were constructed and introduced into K562/ADM cells. Expression of mdr1 mRNA was assessed by RT-PCR, and P-gp expression was determined by Western blot. The apoptosis and sensitivity of the K562/ADM cells to doxorubicin were quantified by flow cytometry and methyl thiazolyl tetrazolium (MTT) assays, respectively. Cellular daunorubicin accumulation was assayed by laser confocal scanning microscope (LCSM). RESULTS: In positive clones of K562/ADM cells stably transfected with pSilencer 3.1-HI neo mdr1-A and mdr1-B shRNA expression vectors, RT-PCR showed that mdr1 mRNA expression was significantly reduced to 35.9% (P < 0.05), 27.5% (P < 0.01), respectively. Western blot showed that P-gp expression was significantly and specifically inhibited. Resistance against doxorubicin was decreased from 79-fold to 38-fold (P < 0.05), 30-fold (P < 0.01) respectively. Furthermore, the fluorescence intensity of K562/ADM cells was increased significantly compared with the control. shRNA vectors significantly enhanced the cellular daunorubicin accumulation. The percent of the apoptosis cell was significantly enhanced to 18.1% (P < 0.05) , 54.4% (P < 0.01) respectively. CONCLUSIONS: shRNA expression vectors can effectively reverse MDR, and restore the sensitivity of drug-resistance K562/ADM cells to conventional chemotherapeutic agents.

Study on the bone marrow mesenchymal stem cells induced drug resistance in the U937 cells and its mechanism.

BACKGROUND: The hematopoietic microenvironment (HM) plays a critical role in malignant cell growth, patient survival, and response to chemotherapy in hematologic malignancies. However, mechanisms associated with this environmental influence remain unclear. In this study, we investigated the role of bone marrow derived mesenchymal stem cells (MSCs) in U937 cell line, to find out the relations between leukemia drug resistance and the MSCs. METHODS: U937 cells were cultured in suspension or grew adherently with MSCs. The cell growth curve was drawn and the cell cycle was measured by flow cytometry. Apoptosis and sensitivity of U937 to daunoblastina (DNR) were quantified by DNA ladder detection and trypan blue exclusion assays, respectively. The gene expression profile chip technology was used to determine and analyze the changes in apoptosis-related gene expression after adherent culture and the expression of MDR1 mRNA was assessed by reverse transcriptional polymerase chain reaction (RT-PCR) at the same time. RESULTS: In the adherent culture, the proliferation of the U937 cells was inhibited, the G0/G1 phase cells increased (F = 64.9726, P < 0.0001), G2/M phase cells were decreased (F = 98.1361, P < 0.0001) and the natural apoptosis rate was decreased (F = 24.0866, P < 0.0001) compared with those in the suspended culture. U937 cell viability was enhanced and cell apoptosis was blocked during DNR treatment in adherent culture with MSCs. Thirty-nine differently expressed genes were screened from the 487 apoptosis related genes in the adherent culture U937 cells. Among the 37 upregulated genes, Bcl-XL was upregulated most significantly. Two genes were downregulated. Adherent culture did not induce MDR1 mRNA expression in U937 cells. CONCLUSIONS: MSCs play a role in modulating the proliferation of U937 cells and response of U937 cells to DNR, and Bcl-XL apoptosis-inhibiting gene may be most important in determining the sensitivity of leukemic cells to treatment, which is not related to MDR1.

[Influence of human mesenchymal stem cells on cell proliferation and chemo-sensitivity of K562 cells].

This study was aimed to compare K562 cell proliferation, chemo-sensitivity and alteration of MDR1 before and after adhesive culture with MSC, so as to evaluate the relationship between chemodrug-resistance of leukemia cells and hemopoietic microenvironment. K562 cell cultivated in suspension and adhesively cultivated with MSC were collected respectively and cell proliferation curves were drawn; the cell cycle was determined by flow cytometry; the effect of chemotherapy on cellular viability and apoptosis of K562 cell was investigated, the MDR1 gene expression was determined by RT-PCR. The results showed that K562 cells adhesively cultivated with MSC were inhibited and cells in G0/G1 increased (P < 0.05), cells in S phase decreased (P < 0.05) and those in G0/G1 increased (P < 0.01), compared with that cultivated in suspension. In process of daunomycin-inducing apoptosis, K562 cell apoptosis in the adhesive culture with MSC was inhibited (P < 0.05). MDR1 gene expression in K562 cells was not induced or altered by adhesive co-cultivation. It is concluded that by co-culture of cell-cell contact with MSC, growth suppression and induction of chemo-resistance of K562 cells take place. The mechanism, however, seems not relevant with MDR1.

Reversal of MDR1 gene-dependent multidrug resistance using short hairpin RNA expression vectors.

BACKGROUND: RNA interference using short hairpin RNA (shRNA) can mediate sequence-specific inhibition of gene expression in mammalian cells. A vector-based approach for synthesizing shRNA has been developed recently. Overexpression of P-glycoprotein (P-gp), the MDR1 gene product, confers multidrug resistance (MDR) to cancer cells. In this study, we reversed MDR using shRNA expression vectors in a multidrug-resistant human breast cancer cell line (MCF-7/AdrR). METHODS: The two shRNA expression vectors were constructed and introduced into MCF-7/AdrR cells. Expression of MDR1 mRNA was assessed by RT-PCR, and P-gp expression was determined by Western Blot and immunocytochemistry. Apoptosis and sensitization of the breast cancer cells to doxorubicin were quantified by flow cytometry and methyl thiazolyl tetrazolium (MTT) assays, respectively. Cellular daunorubicin accumulation was assayed by laser confocal scanning microscopy (LCSM). Statistical significance of differences in mean values was evaluated by Student's t tests. P < 0.05 was considered statistically significant. RESULTS: In MCF-7/AdrA cells transfected with MDR1-A and MDR1-B shRNA expression vectors, RT-PCR showed that MDR1 mRNA expression was reduced by 40.9% (P < 0.05), 30.1% (P < 0.01) (transient transfection) and 37.6% (P < 0.05), 28.0% (P < 0.01) (stable transfection), respectively. Western Blot and immunocytochemistry showed that P-gp expression was significantly and specifically inhibited. Resistance against doxorubicin was decreased from 162-fold to 109-fold (P < 0.05), 54-fold (P < 0.01) (transient transfection) and to 108-fold (P < 0.05), 50-fold (P < 0.01) (stable transfection). Furthermore, shRNA vectors significantly enhanced the cellular daunorubicin accumulation. The combination of shRNA vectors and doxorubicin significantly induced apoptosis in MCF-7/AdrR cells. CONCLUSIONS: shRNA expression vectors effectively reduce MDR expression in a sustained fashion and can restore the sensitivity of drug-resistant cancer cells to conventional chemotherapeutic agents.

[Effects of donor-specific bone marrow cell infusion on chimerism and acute rejection in kidney transplantation].

OBJECTIVE: To evaluate the effect of donor-specific bone marrow cell infusion on the production of chimerism and acute rejection in kidney transplantation. METHODS: Sixty-one patients, 48 males and 13 females, aged 38.4 (23 - 45), underwent transplantation of cadaveric kidneys, 24 of which underwent kidney transplantation combined with donor-specific bone marrow cell infusion and 37 of which underwent pure transplantation of kidney. During the kidney transplantation combined with donor-specific bone marrow cell infusion the donor bone marrow cells (DBMC) were isolated from the thoracic vertebrae and iliac bones of the donors-cadavers and infused into the recipients of the kidneys of the same donors. After operation the peripheral blood was collected from the 61 recipients every 2 - 3 months for at least 1 year to undergo PCR to detect the presence of chimerism, and the rates of chimerism and acute rejection were compared between these 2 groups. RESULTS: During the follow-up the presence rate of chimerism was 87.5% (21/24) in the marrow recipients, significantly higher than that in the control group (40.5%, 15/37, P < 0.001). The prevalence of acute rejection in the chimerism positive patients was 19.4% (7/16), significantly lower than that in the chimerism negative patients (44%, 11/25, P < 0.05). CONCLUSION: Donor-specific bone marrow cell infusion in cadaver kidney transplantation induces the production of chimerism, and increases the immune tolerance to the donor organs, thus finally decreasing the incidence of acute rejection. Chimerism is correlated with immune tolerance.