The study on using dispase and trypsine 0.25% to detach cells from fetal monkey kidneys of Macaca mulatta showed that: the detach ability of dispase was much higher than trypsin. Some characteristics of dispase were suitable for detaching fetal cell in cold condition or serum condition. However, dispase has not been used as commonly as trypsin. Dispase can not be a good solution to detach cells without the effect of trypsin
Endopeptidase ; cells ; Kidney ; Haplorhini ; Fetus

In several human tumors, signal transducer and activator of transcription 3 (STAT3) and nuclear factor κB (NFκB) are activated and interact; how these STAT3–NFκB complexes are transported to the nucleus is not fully understood. In this study, we found that Rac1 was activated in starved cancer cells and that activated Rac1 coexisted with STAT3 and NFκB. Rac1 knockdown and overexpression of the dominant-negative mutant Rac1N19 inhibited the degradation of IκBα, an inhibitor of NFκB. MG132, an inhibitor of the ubiquitin proteasome pathway, increased the amount of non-phosphorylated IκBα, but not serine-phosphorylated IκBα, indicating that IκBα degradation by Rac1 in starved cancer cells is independent of IκBα serine phosphorylation by IKK. Rac1 knockdown also inhibited the nuclear translocation of STAT3–NFκB complexes, indicating that this translocation requires activated Rac1. We also demonstrated that the mutant STAT3 Y705F could form complexes with NFκB, and these unphosphorylated STAT3–NFκB complexes translocated into the nucleus and upregulated the activity of NFκB in starved cancer cells, suggesting that phosphorylation of STAT3 is not essential for its translocation. To our knowledge, this is the first study demonstrating the crucial role of Rac1 in the function of STAT3–NFκB complexes in starved cancer cells and implies that targeting Rac1 may have future therapeutic significance in cancer therapy.
Humans ; Phosphorylation ; Proteasome Endopeptidase Complex ; Serine ; STAT3 Transcription Factor ; Ubiquitin

It has been suggested that proteasome system has a role in initiation, progression, and complication stages of atherosclerosis. Although there is still controversy, there has been no research that compares the expression of proteasome in tissue and serum at each of these stages. This study aimed to investigated the expression of proteasome at different stages of atherosclerosis using rat model. We measured the expression of aortic proteasome by immunohistochemical analyses and were then analyzed using ImageJ software for percentage of area and integrated density. We used Photoshop version 3.0 to analyze aortic proteasome expression as a comparison. We measured serum proteasome expression by enzyme linked immunosorbents assays. Kruskal-Wallis test was used to compare mean value of percentage of area and serum proteasome. Analysis of variance test was used to compare mean value of integrated density. Correlation test between vascular proteasome expression and serum proteasome expression was made using Spearman test. A P-value of 0.05 was considered statistically significant. Compared with normal, percentage of area was higher in initiation, progression, and complication. Compared with normal, integrated density was higher in initiation and further higher in progression and complication. Data from Image J is similar with data from Photoshop. Serum proteasome expression was higher in initiation compared with normal, and further higher in progression and complication. It was concluded that there were different vascular proteasome expression and serum proteasome expression at the stages of atherosclerosis. These results may be used in research into new marker and therapeutic target in atherosclerosis.
Animals ; Atherosclerosis* ; Immunosorbents ; Models, Animal ; Proteasome Endopeptidase Complex* ; Rats*

The essence of endogenous turbidity in Chinese medicine (CM) is different from cream, fat, phlegm, retention, damp, toxicity, and stasis. Along with the development of modern scientific technologies and biology, researches on the essence of endogenous turbidity should keep pace with the time. Its material bases should be defined and new connotation endowed at the microscopic level. The essence of turbidity lies in abnormal functions of zang-fu organs. Sugar, fat, protein, and other nutrient substances cannot be properly decomposed, but into semi-finished products or intermediate metabolites. They are inactive and cannot participate in normal material syntheses and decomposition. They cannot be transformed to energy metabolism, but also cannot be synthesized as executive functioning of active proteins. If they cannot be degraded by autophagy-lysosome or ubiquitin-prosome into glucose, fatty acids, amino acids, and other basic nutrients to be used again, they will accumulate inside the human body and become endogenous turbidity. Therefore, endogenous turbidity is different from final metabolites such as urea, carbon dioxide, etc., which can transform vital qi. How to improve the function of zang-fu organs, enhance its degradation by autophagy-lysosome or ubiquitin-prosome is of great significance in normal operating of zang-fu organs and preventing the emergence and progress of related diseases.
Autophagy ; Humans ; Medicine, Chinese Traditional ; Proteasome Endopeptidase Complex

Small RNAs, especially microRNAs (miRNAs),widely exist in eukaryotic cells, with their main functions being regulating gene expression and function of target molecules through the degradation of cellular target RNAs by the ribonuclease-based system. Ubiquitins and ubiquitin-like proteins are polypeptides that exist in most eukaryotic cells, and their main function is almost to regulate protein level through the degradation of cellular proteins by ubiquitin proteasome system. Small RNAs, including miRNAs,and ubiquitins or ubiquitin-like proteins have similarities in many aspects although small RNAs and ubiquitin or ubiquitin-like proteins interact different substrates respectively. Therefore, miRNAs can be defined as ubiquitra (ubiquitous ribonucleic acid, ubiquitra or uRNA), and the other small RNAs can be defined as ubiquitra-like RNA or uRNA-like RNA. The concept of ubiquitra may be applied for explaining the biological essence of small RNAs diversity.
Gene Expression ; MicroRNAs ; Proteasome Endopeptidase Complex ; Proteins ; Ubiquitination

In order to study the physicochemical characteristics of cytosolic PrP (CytoPrP) and evaluate its possible influence on cell viability, a recombinant plasmid expressing human CytoPrP eukaryoticly was constructed and transfected into human neuroblastoma cell line SH-SY5Y transiently. Proteinase-resistant activities of CytoPrP were evaluated by a proteinase K (PK) digestion and cytotoxic effects of CytoPrP were tested by MTT assay and Trypan Blue cell-counting. The presence of CytoPrP in cytoplasm after transfection was controlled by the presence of protease inhibitor. Compared with wild-type PrP, CytoPrP possessed relatively stronger PK-resistant activities. Obvious cytotoxic effects were observed in the cells after inducement of CytoPrP in cytoplasm by protease inhibitor, showing a dose-dependent manner. The results provide useful scientific evidences for further studies of potential role of CytoPrP in pathological mechanism of prion disease.
Cell Line, Tumor ; Cell Survival ; Cytosol ; chemistry ; Endopeptidase K ; pharmacology ; Humans ; Prions ; genetics ; physiology ; Transfection

Human CRBN (cereblon) gene is located on chromosome 3 at 3p26 and its encoding protein is a member of E3 ubiquitin ligase complex (composed of CRBN, DDB1, CUL4A and ROC1). The E3 ubiquitin ligase complex functions in the ubiquitin-proteasome protein degradation pathway and attaches polyubiquitin chains to substrate proteins for degradation via the protease complex. Currently, there are no standardized assays for CRBN gene and protein measurement although quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunohistochemistry and Western blot are widely used. CRBN has been identified as a direct target for immunomodulatory drugs (IMiD) and plays a significant role in anti-proliferation, pro-apoptotic effects, anti-angiogenic activities, immunomodulatory activities and intervention of cell surface adhesion molecules between myeloma cells and bone marrow stromal cells. Recently, clinical data show that majority of the multiple myeloma patients treated with IMiD develop drug-resistance over time by unknown mechanisms. Fortunately, various in vivo and in vitro studies have revealed that the decreased CRBN expression or CRBN deletion is associated with resistance to IMiD in treating multiple myeloma, and CRBN expression levels may have a prognostic significance. Furthermore, the most recently discovered protein IKZF1, IKZF3, IRF4, C/EBPβ and Wnt/catenin signaling pathways may also be closely related to IMiD resistance in myeloma.
Drug Resistance, Neoplasm ; Humans ; Immunosuppressive Agents ; Multiple Myeloma ; Peptide Hydrolases ; Prognosis ; Proteasome Endopeptidase Complex ; Transcription Factors

Efforts to find etiologies and understand pathogenesis of Parkinson's disease (PD) have progressed over decades from epidemiological studies or biochemical studies on brain tissues of PD to discovering genes responsible for familial Parkinson's diseases and dissecting their roles and mechanisms in dopaminergic neuronal death at the molecular level. Eleven loci have been identified in familial PD and five genes have cloned. Although diverse genetic and environmental factors were identified, mechanisms of neurodegeneration by these etiological factors converge into four basic mechanisms; oxidative stress, mitochondrial complex I dysfunction, impairment of ubiquitin-proteasome pathway, and accumulation and aggregation of misfolded/unfolded proteins. These mechanisms appear to be closely interconnected with each other and lead to complex downstream cell death machineries. Understanding more sophisticated pathogenesis of dopaminergic neuronal death will help elucidating critical steps in dopaminergic cell death and developing therapeutic agents in the future.
Brain ; Cell Death ; Clone Cells ; Dopaminergic Neurons ; Oxidative Stress ; Parkinson Disease* ; Proteasome Endopeptidase Complex

Acute promyelocytic leukemia (APL) is characterized by PML-RARa expression. Ubiquitin proteasome-pathway (UPP) plays a key role in all-trans retinoid acid (ATRA) and arsenic trioxide (ATO)-induced degradation. In addition, the regulations of cell cycle and transcription are also related to this pathway. Deeply studying the role of ubiquitin-proteasome pathway in APL contributes to elucidate the mechanisms of some drugs and explode the clinical therapeutical insight for APL. In this article, the constitution of UPP, the role of UPP-mediated protein modification in APL, the application of ubiquitination-associated drugs in APL are reviewed.
Humans ; Leukemia, Promyelocytic, Acute ; Metabolic Networks and Pathways ; Proteasome Endopeptidase Complex ; Ubiquitin

As the major pathway mediating specific protein degradation in eukaryotes, ubiquitin-proteasome system (UPS) is involved in various physiological and pathological processes such as cell cycle regulation, immune response, signal transduction and DNA-repair. Deubiquitinases (DUB) maintain the balance of UPS and related physiological processes via reversibly removing ubiquitin from the covalently modified protein substrates, which have been implicated in various disease processes in case of their imbalance expression. Because DUB plays critical regulating roles in the UPS pathway, they may be also the ideal drug targets for severe and intractable human diseases, such as cancer and neurodegenerative disease. With the rapid development of proteomic technology, systematical investigation of specific substrates and interacting proteins of varied DUB via mass spectrometry approach may shed light on these DUB's biological function and regulating roles in the physiological and pathogenic states. In this review, we briefly introduce the characteristics of DUB and summarize the recent application and progresses of proteomics in DUB research.
Humans ; Mass Spectrometry ; Proteasome Endopeptidase Complex ; metabolism ; Proteomics ; Signal Transduction ; Ubiquitin ; metabolism ; Ubiquitin-Specific Proteases ; metabolism