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

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

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

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*

BACKGROUND: The extraction methods of DNA from clinical samples are the major obstacle to use the PCR(Polymerase Chain Reaction) in routine labortary for early detection of M. tuberculosis. We tried to improve the extraction method of DNA from sputum for establishment of the PCR in routine labortary by reducing the possibility of cross contamination and performing it easily and safely. METHODS: We used the InstaGene(TM) DNA extraction kit(BioRad Co.) using Chelex 100 ion exchange resin for preparation of DNA. We compared InstaGene method in 100 cases of sputum from proteinase K method which is known as the most commonly used method for DNA purification(Experiment 1). And we compared InstaGene method in 98 cases of sputum from Microwave method developed by a company in Korea(Experiment 2). In experiment 1, 245bps of IS6110 were amplified and then 188bps were amplified by nested PCR. In experiment 2, 536bps in primary PCR and 276bps in nested PCR were amplified and analysed by agarose gel electrophoresis and EtBr staining. RESULTS: When we chose AFB smear, culture, or AFB smear and culture as a standard test, PCR had low specificity and positive predictive value in both experiments. The InstaGene method has higher value in sensitivity and negative predictive value significantly than proteinase K method. The InstaGene method and the Microwave methods were similar in sensitivity, specificity, positive predictive value and negative predictive value.. CONCLUSION: Even though both methods had lower possibility of cross contamination, shorter time requrirement, simplicity, and economic advantages than Proteinase K method, the InstaGene method was a little simpler than the Microwave method. Therefore, in terms of usfulness in clinical application, the Instagene method seems to be the most useful method in DNA extraction for detection of M. tuberculosis using PCR. The reliability of this method will be clarified by further studies with enough clinical samples.
DNA* ; Electrophoresis, Agar Gel ; Endopeptidase K ; Ion Exchange* ; Microwaves ; Polymerase Chain Reaction* ; Sensitivity and Specificity ; Sputum ; Tuberculosis*

Multilocus sequence typing (MLST) of Salmonella is useful method for replacing serotyping using antisera but is limited by difficulties associated with in polymerase chain reaction (PCR). We optimized the PCR reaction, especially annealing temperature and extension time (94degrees C for 2 min; 40 cycles at 94degrees C for 30 sec, 56.8degrees C for 1 min, 72degrees C for 2 min; and 72degrees C for 10 min). The degradation of PCR product by thermostable nucleases was inhibited by using template DNAs treated proteinase K or purified by a commercialized preparation kit. The resulting modified MLST was used as accurate and fast typing method.
DNA ; Endopeptidase K ; Immune Sera ; Multilocus Sequence Typing* ; Polymerase Chain Reaction ; Salmonella* ; Serotyping*

The acid digestion method for extracting diatoms has been widely used to confirm death by drowning, but its reliability is still disputed because some diatoms can be destroyed during the extraction process due to treatment with strong acid and heat. There is a need to develop an efficient and reliable digestive method to overcome the limitation of the present analytical procedure. In this study, the reliability and efficacy of quantitative and qualitative diatom analysis from seawater by an enzymatic digestion method was evaluated. We confirmed the merit of the enzymatic method that used proteinase K instead of nitric acid in the conventional method. As a result, the enzymatic method showed a higher recovery ratio and better preservation of the diatom structure, which is essential for quantitative (diatom density) and qualitative (species) interpretation of diatom analysis. This result indicates that the enzymatic method can replace the conventional acid digestion method to confirm cases of death by drowning since it is more reliable and yields conclusive results.
Diatoms ; Digestion ; Drowning ; Endopeptidase K ; Hot Temperature ; Nitric Acid ; Plankton ; Seawater

BACKGROUND: Proteasomes are multi-subunit enzyme complexes present in the cytoplasm and nucleus of eukaryotic cells. Proteasomes are involved in the pathophysiological process resulting in development of many diseases. Release of proteasomes from lyzed erythrocytes has been suggested in recent reports. Accumulation of proteasomes in blood products could contribute to formation of storage lesions and have adverse effects on recipients; therefore, we conducted an analysis of changes in concentration of proteasomes in blood products during storage. METHODS: Concentrations of 20S proteasomes in supernatant of whole blood products obtained from eight healthy volunteers and in segments of 16 packed red blood cell (pRBC) units transfused to patients were measured by ELISA. Plasma samples containing several hemoglobin concentrations were prepared in order to assess the relationship between proteasome concentration and degree of hemolysis. RESULTS: Proteasome concentrations in whole blood products on day one of storage were significantly lower than those on day seven of storage and later (P<0.05). In segments of pRBC units, the proteasome concentration was 8.072+/-11.802 microg/mL (storage day: 13.8+/-4.7). Of the 32 pRBC units, two showed extremely high proteasome concentrations (36.662 and 62.798 microg/mL). Proteasome concentrations in plasma increased with increasing hemoglobin concentrations. CONCLUSION: During storage of whole blood products, except during the first seven storage days, levels of proteasome do not undergo significant change. However, hemolysis may be related to accumulation of proteasome. Further study to evaluate the effects of blood components containing high proteasome concentrations on recipients should be conducted.
Cytoplasm ; Enzyme-Linked Immunosorbent Assay ; Erythrocytes ; Eukaryotic Cells ; Hemoglobins ; Hemolysis ; Humans ; Plasma ; Proteasome Endopeptidase Complex

PURPOSE: Anti-p185HER2/neu monoclonal antibody (mAb) that can induce phenotypic reversion and monoclonal antibodies specific for the p185HER2/neu growth factor receptor that are able to diminish its kinase-signaling properties represent a specific advance in the therapy for p185HER2/neu-expressing human cancers. With mAb treatment, down-regulation of p185HER2/neu surface receptors and attenuation of the kinase-signaling properties have been observed and regarded as a basic phenomenon; however, the mechanisms for mAb-induced phenotypic reversion are not clear. METHODS: We used human tumor-cell lines of SK-BR-3, T6-17, and U373MG. With immunoprecipitation and Western blotting, we investigated the changes in p185HER2/neu receptor phosphorylation and the expression of signal-regulatory proteins (SIRPs) after mAb treatment. To identify the proteins interacting with Tat-binding protein-1 (TBP1), we used the Clonotech Gal4 matchmaker two-hybrid system. RESULTS: Minimal to moderate reduction in phosphotyrosine (pTyr) content was observed in SK-BR-3 and T6-17 cells with short-term (10-30 minutes) incubation after mAb treatment, but that did not alter total p185HER2/neu receptor density. SIRPs phosphorylation after peptide treatment was increased. With mAb treatment, three proteins were shown to interact with TBP1, and all of the interacting proteins are subunits of proteasome 26S. Collectively, anti- p185HER2/neu mAb or peptide down-regulates the surface receptors and attenuates the kinase signaling, which then both induces higher proteasome activity through increased TBP1 and increases SIRPs expres sion. CONCLUSION: Increased proteasomal activity may degrade abnormal proteins and increased SIRPs may regulate signal transduction toward the norm. Therefore, activation of a protein-degradation pathway and induction of signal-regulatory proteins may be possible mechanisms for the ultimate anti-tumor effects of the anti-p185HER2/neu mAb or peptide.
Antibodies, Monoclonal ; Blotting, Western ; Down-Regulation ; Humans ; Immunoprecipitation ; Phosphorylation ; Phosphotransferases ; Phosphotyrosine ; Proteasome Endopeptidase Complex ; Signal Transduction