Chronic activation of neutral ceramidase protects beta-cells against cytokine-induced apoptosis.

AIM: To investigate the activity and expression of neutral ceramidase (N-CDase) in the insulin-secreting cell line INS-1 and its role in the cellular response to cytokines. METHODS: HPLC, Western blotting, and quantitative real-time PCR were performed to detect the activity and expression of N-CDase in INS-1 cells treated with a cytokine mixture (5 ng/mL interleukin-1beta, 10 ng/mL TNF-alpha, and 50 ng/mL interferon-gamma). The expression and activity of N-CDase in the INS-1 cells were specifically inhibited using N-CDase-siRNA transfection. Annexin V-fluorescein- isothiocyanate/propidium iodide flow cytometry was used to assess apoptosis in the INS-1 cells. RESULTS: The INS-1 cells exhibited some basal N-CDase activity, and cytokines induced a time-dependent delay in the activation of NCDase. As a result, the activation of N-CDase was first detectable at 8 h after stimulation. It peaked at 16 h and remained elevated at 24 h. Cytokines also upregulated the mRNA and protein expression of N-CDase in the INS-1 cells. Furthermore, when N-CDase activity was inhibited by RNA interference, cytokine-induced apoptosis in the INS-1 cells was markedly increased. CONCLUSION: The N-CDase pathway is active in INS-1 cells, and the chronic activation of N-CDase is involved in the pathological response of beta-cells to cytokines, potentially providing protection against cytokine toxicity.

Early Application of Auxiliary Partial Orthotopic Liver Transplantation in Murine Model of Wilson Disease.

BACKGROUND: Liver transplantation (LT) is the only option of treatment for Wilson disease (WD) when chelation therapy fails, but it is limited due to the shortage of donor. Auxiliary partial orthotopic LT (APOLT) has been performed successfully in end-stage WD patients, which expands the donor pool. METHODS: Atp7bmice were used as experimental model of WD. Eight- and 20-week-old mice were used as different timepoints to perform APOLT. Serum copper, tissue copper, serum ceruloplasmin (CP), and liver histological examination were observed after operation. RESULTS: Hepatic and serum copper levels in Atp7b mice decreased after APOLT, and copper metabolism disorder of WD mice was relieved at both early and late stages. The progression of pathology in the native liver was delayed only when transplantation was performed at an early stage. CONCLUSIONS: Auxiliary partial orthotopic LT can significantly improve copper metabolism disorder in the Atp7b mice, and early transplantation may prevent the disease progression.

Preparation and characterization of polyclonal antibodies against ARL-1 protein.

AIM: To prepare and characterize polyclonal antibodies against aldose reductase-like (ARL-1) protein. METHODS: ARL-1 gene was inserted into the E. coli expression vector pGEX-4T-1(His)(6)C and vector pQE-30. Recombinant ARL-1 proteins named ARL-(His)(6) and ARL-GST were expressed. They were purified by affinity chromatography. Sera from domestic rabbits immunized with ARL-(His) (6) were purified by CNBr-activated sepharose 4B coupled ARL-GST. Polyclonal antibodies were detected by Western blotting. RESULTS: Recombinant proteins of ARL-(His)(6) with molecular weight of 35.7 KD and ARL-GST with molecular weight of 60.8 KD were highly expressed. The expression levels of ARL-GST and ARL-(His)(6) were 15.1 % and 27.7 % among total bacteria proteins, respectively. They were soluble, predominantly in supernatant. After purification by non-denatured way, SDS-PAGE showed one band. In the course of polyclonal antibodies purification, only one elution peak could be seen. Western blotting showed positive signals in the two purified proteins and the bacteria transformed with pGEX-4T-1(His) (6) C-ARL and pQE-30-ARL individually. CONCLUSION: Polyclonal antibodies are purified and highly specific against ARL-1 protein. ARL-GST and ARL-(His) (6) are highly expressed and purified.

[Expression analysis of mouse homologous proteins with human aldose reductase like-1].

OBJECTIVE: To detect expression of mouse ARL-1 homologous proteins in mouse tissues, and analyze homology, genetic distance and phylogenetic relationship between human aldose reductase like-1 (ARL-1) and mouse homologous proteins. METHODS: Homology of mouse ARL-1 homologous proteins with human ARL-1 was analyzed by software Clustal X 1.8 using GenBank and Swiss-Prot database; genetic distance and phylogenetic relationship between mouse ARL-1 homologous proteins and human ARL-1 were analyzed by software Mega 2.0; mouse tissues were detected by Western blotting using polyclonal antibodies against ARL-1 protein from domestic rabbits. RESULTS: The amino acid sequence of human ARL-1 was 83%, 82%, 81%, 79%, 70%, 51%, 50% and 45% identical to that of the Chinese hamster ovary reductase (CHO-Red), the mouse fibroblast growth factor-regulated protein (FR-1), rat aldose reductase-like protein (rARLP), the mouse vas deferens protein (MVDP), rat lens aldose reductase (LeAR), delta4-3-ketosteroid-5beta-reductase (5beta-Red), rat aldo-keto reductase protein c (RaK-c) and 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD). Among all the mouse ARL-1 homologous proteins, the genetic distance between CHO-Red and human ARL was the shortest (18.0%, P = 0.023), next was FR-1 (19.1%, P=0.023) and rARLP (19.9%, P = 0.025). From the phylogenetic tree, the protein whose relationship with human ARL-1 was the closest with CHO-Red, next was mouse FR-1, rARLP, MVDP and LeAR. Homologous proteins were found in mouse tissues including vas deferens, testis, bladder and uterus by Western blotting using polyclonal antibodies against ARL-1 protein from domestic rabbits. CONCLUSIONS: CHO-Red has the highest homology, the shortest genetic distance and the closest relationship with human ARL-1, next is FR-1, rARLP, MVDP. The major distribution of mouse ARL-1 homologous proteins is in vas deferens, testis, bladder and uterus, deducing they might be CHO-Red, FR-1, rARLP or MVDP

Involvement of c-Jun N-terminal kinase in reversal of multidrug resistance of human leukemia cells in hypoxia by 5-bromotetrandrine.

5-Bromotetrandrine (BrTet), a candidate multidrug resistance (MDR) modulator, is a potential compound for use in cancer therapy when combined with anticancer agents such as daunorubicin (DNR) and paclitaxel. The purposeof this study was to investigate the mechanism of reversal of P-glycoprotein (P-gp)-mediated MDR by BrTet and the involvement of the c-Jun N-terminal kinase (JNK)/c-Jun signaling pathway in both adriamycin-sensitive K562 and adriamycin-resistant K562 (KA) leukemia cells in hypoxia. The combination of BrTet and DNR decreased both phosphorylated JNK1/2 and MDR1/P-gp levels under hypoxic conditions. Furthermore, a pharmacological inhibitor of JNK, SP600125, or small interfering RNA (siRNA) oligonucleotides to both JNK1 and JNK2 reversed BrTet- or DNR-induced JNK phosphorylation and MDR1/P-gp levels. We further demonstrated that the decreased JNK phosphorylation and MDR1/P-gp levels were associated with a significant increase in intracellular accumulation of DNR, which dramatically enhanced the sensitivity of drug-resistant KA cells to DNR, and led to cellular apoptosis through activation of the caspase-3 pathway. It is concluded that using BrTet in combination with other chemotherapeutic agents and pharmacological inhibitors of JNK can abrogate the P-gp-induced MDR in adriamycin-resistant K562 cells, which has potential clinical relevance in cancer therapy for chemotherapeutic-resistant human leukemia.

[Preparation and characterization of monoclonal antibodies against AR protein].

AIM: To prepare monoclonal antibodies (mAbs) against aldose reductase (AR) and compare with anti-aldose reductase-like protein (ARL-1) mAb. METHODS: The AR gene was gained by RT-PCR and inserted into pGEX-4T-1 (His)(6)C. Recombinant protein GST-AR was used to immunize BALB/c mouse. MAb was prepared by hybridoma technique and detected by ELISA and Western blot. Simultaneously, according to the analysis of AR by software Clustalx and Antheprot, GST-dAR(80-142 aa), GST-dA1(1-79 aa), GST-dA2(80-99 aa), GST-dA3(111-142 aa) and GST-dA4(143-316 aa) were expressed in E. coli Rosetta. All the proteins were used to analyze the binding sites of the mAb and AR protein by Western blot. RESULTS: Three clones secreting anti-AR mAb were obtained. They were all of IgG1. And the titer of mAb in ascites was 1:400,000 while in cell culture was 1:10,000. All of the three anti-AR mAbs reacted to GST-AR and proteins of placenta tissues and had no cross-reaction to GST-ARL-1 and GST protein. And the three anti-AR mAb could recognize GST-dA1, GST-dA3 and GST-dA4, respectively. CONCLUSION: Three specific mAbs against AR are obtained and recognize the 1-79, 111-142, 143-316 amino acid sites of AR, respectively. The anti-AR mAb, together with the anti-ARL-1 mAb, may be a useful tool for further study of the function of AR and ARL-1 and the relationship between the two proteins and relevant diseases as well as for the epidemiological investigation.