Effects of sleep deprivation on liver circadian clock gene expression in rats / 军事医学

Objective To investigate the effects of 72 h sleep deprivation(SD)on circadian clock gene expression in the rat liver.Methods Twelve rats were randomly divided into control group and SD group.An SD instrument was used to deprive the rats′sleep for 72 h.Then the abdominal cavity was exposed to obtain liver,and the expression of clock genes was detected by RT-PCR and Western blotting analysis, respectively.Results Compared with the control group, the mRNA levels of clock,npas2 and rev-erbαstrikingly decreased in the livers of the SD group rats.However,per1,per2 and rorαmRNA levels obviously increased.bmal1 and cry1 mRNA expression hardly changed in the control and SD groups. Meanwhile,the protein levels of liver BMAL1,CLOCK,NPAS2,CRY1 and REV-ERBαwere significantly down-regulated and PER1,PER2 and RORαprotein levels were up-regulated in SD group compared with control group.Conclusion 72 h SD can result in abnormal expressions of several circadian clock genes in the rat liver at both transcriptional and translational levels.

Effects of sleep deprivation on spleen circadian clock gene expression in rats / 军事医学

Objective To investigate the effects of 72 h sleep deprivation (SD) on circadian clock gene expression in the rat spleen.Methods The rats were randomly divided into control group and SD group.An SD instrument was used to deprive the rats of sleep for 72 h.Then the lymphocytes from the spleen were obtained by Ficoll seperation medium before the expression of clock genes was detected by RT-PCR and Western blotting analysis respectively.Results Compared with the control group,the mRNA levels of bmal1,clock,per2 and rev-erbα strikingly decreased in the spleens of the SD group rats.However,npas2,per1,rorα and cry1 mRNA expression hardly changed in the control and SD group.Meanwhile,the protein levels of spleen BMAL1,NPAS2,CRY1 and RORα were significantly down-regulated and PER1 protein levels were up-regulated in SD group compared with control group.However REV-ERBα protein expression remained unchanged in the control and SD group.Conclusion 72 h SD can result in abnormal expressions of several circadian clock genes in lymphocytes of the spleen at both transcription and translation levels.

Immunoregulation effects in vitro of the xenoprotein in combination with recombinant human granulocyte-macrophage colony stimulating factor and bacillus Calmette-Guerin / 中国实验血液学杂志

This study was aimed to investigate the effects of xenogeneic antigen neu-Fc in combination with the recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) and Bacillus Calmette-Guerin (BCG) on the regulation of Th1 and Th2 immune response in vitro. The rat neu L2-S2 domain was engineered as a chimeric protein with human IgG Fc. The eukaryotic expression vector was constructed. The recombinant protein was stably expressed in CHO cells and purified by rProtein A Sepharose Fast Flow column. The recombinant protein was identified by SDS-PAGE and Western blot. Peripheral blood mononuclear cells (PBMNCs) were obtained by means of standard Ficoll separation from the blood of healthy donors. Neu-Fc-induced PBMNC proliferation was tested by MTT. The production of IL-12 and IL-10 was measured by ELISA. The results showed that the level of IL-12 decreased and IL-10 increased after PBMNCs were incubated with MCF-7 cultural supernatant. 10 nmol/L neu-Fc strongly induced the cell proliferation. Compared with neu-Fc or GM-CSF or BCG treatment alone, neu-Fc in combination with GM-CSF and BCG significantly stimulated IL-12 production and inhibited IL-10 production (p < 0.01). It is concluded that the neu-Fc can stimulate the proliferation activity of PBMNCs. neu-Fc, GM-CSF and BCG costimulation efficiently induces Th1 immune response.

Nano-ESI-MS/MS identification on differentiation-associated proteins in M1 mouse myeloid leukemia cells induced by IL-6 / 中国医学科学院学报

<p><b>OBJECTIVE</b>To identify two differentiation-associated proteins induced by rhIL-6 in M1 mouse myeloid leukemia cells.</p><p><b>METHODS</b>Protein spots were excised from 2-D gels and digested in-gel with trypsin. The trypsin lysis products were first analyzed by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) through peptide mass fingerprinting and then performed peptide sequencing by nano-electrospray ionization mass spectrometry/mass spectrometry (nano-ESI-MS/MS). The database search was finished with the Mascot search engine (http://www.matrixscience.co.uk) using the data processed through MaxEnt3 and MasSeq.</p><p><b>RESULTS</b>The two proteins were not revealed by peptide mass fingerprint using MALDI-TOF-MS, while they were respectively identified as Destrin and Putative protein after the sequence of their trypic peptides were obtained by the nano-ESI-MS/MS techniques.</p><p><b>CONCLUSION</b>Nano-ESI-MS/MS technique can successfully identify the two differentiation-associated proteins induced by rhIL-6 and has great advantage in protein analysis.</p>

The effect of eIF-5A on the G1-S in cell cycle regulation / 中国实验血液学杂志

Eukaryotic initiation factor 5A (eIF-5A) contains an unusual amino acid, hypusine, which is formed post-translationally. Although eIF-5A and its hypusine modification are essential for eukaryotic cell viability, the precise physiological function of it has remained elusive. The aim of the study is to investigate how hypusine formation modulate the proliferation, cell cycle and apoptosis in leukaemia cells. The effects of 1,7-diaminoheptane (DAH), a potent inhibitor of deoxyhypusine synthase, on proliferation and cell viability of leukemia cell lines (Mo7e, TF-1 and THP-1) and MCF-7 cells, were investigated. eIF-5A expression level was detected after cell synchronization. The results showed that inhibition of cell proliferation by DAH was in a concentration-dependent manner while apoptosis was also induced at the same time. Upon treatment of the cell lines with DAH, cell growth was inhibited. Cell cycle analysis showed that DAH induced cell growth arrest at the G(1)-S boundary of the cell cycle. In synchronized MCF-7 cells, the expression level of eIF-5A peaked at G(1) phase but very low at S and G(2)/M phases. It is concluded that hypusine formation of eIF-5A exits in the regulation of cell cycle and the results suggest that eIF-5A is involved in the expression of proteins regulating transition of G(1)-S phase of cell cycle.

Induction of Apoptosis in Leukemic Cells by Inhibiting the Ubiquitin-Proteasome Pathway and Its Possible Mechanism / 中国实验血液学杂志

The ubiquitin-proteasome pathway is the principal mechanism for the degradation of short-lived proteins in eukaryotic cells. Recently, proteasome inhibitors have been shown to induce apoptosis in many kinds of human malignant cells. In this study, the mechanism of apoptosis induced by proteasome inhibitor in leukemic cells was examined. Evaluated by MTT assay, treatment of leukemic cells with Z-LLL-CHO, a reversible proteasome inhibitor, induced cell death in a dose-dependent manner. Appearance of the sub G(0)/G(1) fraction of cell cycle observed in flow cytometry assay suggested the induction of apoptosis, which was further proved by typical DNA ladder and morphological study. Western blot displayed the cleavage of bcl-2 into a shortened 22 kD fragment and the decrease in the levels of caspase-3 precursor. A highly sensitive colorimetric assay was employed and the elevation of caspase-3 activity was detected in both cell lines after treatment with Z-LLL-CHO. By comparison, these results showed that the leukemic cell line M-07e and KG-1a, which both express bcl-2 at a relative high level, had different susceptibility to undergo apoptosis induced by Z-LLL-CHO, which possibly due to their different levels of expression and activation of caspase-3 precursor, as well as their different degree of bcl-2 cleavage after treated by Z-LLL-CHO.