Calcium has been implicated as a primary pathogenetic mediator of cellular injury under conditions of oxygen and substrate deprivation in the kidney aswell as other tissues. It has been proposed that calcium entry from the external medium increases intracellular free calcium to toxic levels there-by converting cellular injury from a potentially reversible to an irreversible state. Cellular calcium homeostasis is a fundamental prerequisite for many coordinate cellular functions. The regulation of cellular Ca++ -metabolism is maintained mainly by Ca++ -ATPase. The purpose of the present study was to determine the effects of ischemia on the subcellular specific activity of Ca++ -ATPase of renal tissues in the rabbits. 1. In both normal cortical and medullary tissues, the specific activities Ca++ -ATPase were higher in mitochondria than in cytosol. 2. In cortical tissue, the specific activities of mitochondrial Ca++ -ATPase were increased with duration of ischemic time under normothermia. But the specific activities were decreased comparing to those of control under hypothermia, and the activities were not different with those of control in Verapamil treated group. 3. The specific activities of Ca++ -ATPase of cortical cytosol were increased as in mitochondria under normothermia. And the activities were similar with those of control in hypothermic and Verapamil treated groups. 4. In medullary tissues, the specific activities of mitochondrial Ca++ -ATPase were increased with duration of ischemic time under normothermia. In Verapamil treated group, the activities were decreased comparing to those of control. And there was no difference of activities between hypothermia and control groups. 5. As in cortical tissues, the specific activities of Ca++ -ATPase of medullary, cytosol were increased in normothermic group. And the activities were similar with those of control in hypothermic and Verapamil treated groups. Above results suggested that calcium contents of mitochondria and cytosol were increased with duration of ischemic time under normothermia. Hypothermia and Verapamil treatment seemed to be beneficial effects in prevention of calcium accumulation in the renal tissues.
Adenosine Triphosphatases* ; Calcium ; Cytosol ; Homeostasis ; Hypothermia ; Ischemia* ; Kidney ; Mitochondria ; Oxygen ; Rabbits* ; Subcellular Fractions* ; Verapamil

PURPOSE: The possible mechanisms of increased thiol me thyltransferase (TMT) activity in cholestatic rat livers and serum were studied. METHODS: Rats were divided into seven groups: rats receiv ing a sham operation, rats with a bile duct obstruction (BDO) alone (BDO group), rats with BDO plus taurocholic acid (TCA) injection (BDO plus TCA group), rats with BDO plus tauroursodeoxycholic acid (TUDCA) injection (BDO plus TUDCA group), rats receiving a choledoco-caval shunt (CCS) operation (CCS groups), rats receiving a CCS operation plus TCA Injection (CCS plus TCA group), and rats receiving a CCS operation plus TUDCA injection (CCS plus TUDCA group). The TMT activities in the serum and in the hepatic subcellular fractions isolated from these experimental rats were determined. The values of Km and Vmax in this he patic enzyme were measured. RESULTS: The activities of liver mitochondrial and microsomal TMTs as well as the Vmax values of TMT were found to be increased significantly in both the CCS plus TCA and the BDO plus TCA groups, compared with the CCS and BDO groups. On the other hand, the Km values of hepatic subcellular TMT were the same in all experimental groups. The serum TMT activity increased significantly in both the CCS plus TCA and the BDO plus TCA groups, compared with the control, CCS and BDO groups. However, these serum and hepatic enzyme activities were the same in the CCS plus TUDCA and the BDO plus TUDCA groups. CONCLUSION: The above results suggest that TCA stimulates the biosynthesis of TMT in the liver. Also, the elevated TMT activity in the serum is thought to be caused by an increase in membrane permeability of hepatocytes from liver cell necrosis caused by TCA.
Administration, Intravenous* ; Animals ; Cholestasis ; Hand ; Hepatocytes ; Liver ; Membranes ; Necrosis ; Permeability ; Rats* ; Subcellular Fractions ; Taurocholic Acid*

PURPOSE: The possible mechanisms of increased arylamine N-methyl- transferase (AMT) activity in cholestatic rat livers and serum were studied. METHODS: Rats were divided into eight groups: rats receiving a sham operation, rats with a bile duct obstruction (BDO) alone (BDO group), rats with a BDO plus taurocholic acid (TCA) injection (BDO plus TCA group), rats with a BDO plus tauroursode oxycholic acid (TUDCA) injection (BDO plus TUDCA group), rats receiving a choledocho-caval shunt (CCS) operation (CCS groups), rats receiving a CCS operation plus TCA injection (CCS plus TCA group), and rats receiving a CCS operation plus TUDCA injection (CCS plus TUDCA group). The AMT activities in the serum and in the hepatic subcellular fractions isolated from the above experimental rats were determined. The values of Km and Vmax in this hepatic enzyme were measured. RESULTS: The activities of liver mitochondrial and microsomal AMTs as well as the Vmax values of AMT, were found to be increased significantly in both the CCS plus TCA group and the BDO plus TCA group compared with the CCS and BDO groups. On the other hand, the values of Km of hepatic subcellular AMT was the same in all experimental groups. The serum AMT activity increased significantly in both the CCS plus TCA group and the BDO plus TCA group compared with control the CCS and BDO group. However, these serum and hepatic enzyme activities were the same in both the CCS plus TUDCA group and the BDO plus TUDCA group. CONCLUSION: The above results suggest that TCA stimulates the biosynthesis of AMT in the liver. Also, the elevated AMT activity in the serum is thought to be caused by an increase in the membrane permeability of hepatocytes from liver cell necrosis caused by TCA.
Animals ; Cholestasis ; Cholestasis, Extrahepatic ; Hand ; Hepatocytes ; Liver ; Membranes ; Necrosis ; Permeability ; Rats* ; Subcellular Fractions ; Taurocholic Acid* ; Transferases

OBJECTIVETo develop a method for extracting cytoskeletons and cytoskeleton-associated proteins for proteomic analysis.

METHODSA subcellular sequential proteome extraction method was exploited. The extraction procedure was optimized and controlled according to observed cell morphology changes and one- and two-dimensional electrophoresis images. The extraction efficiency and selectivity were evaluated by Western blotting and mass spectrometry.

RESULTSFour extracted fractions clearly displayed distinct patterns. Western blotting detected the fraction-marker proteins FAK, integrin-β1, histone H1 and cytokeratin 19 only in their expected fractions. About 90% of the protein spots in the cytoskeleton fraction were identified by mass spectrometry as cytoskeleton and/or its associated proteins.

CONCLUSIONThe subcellular proteome sequential fractionation method facilitates the detection of proteins of low abundance and shows a high reproducibility and selectivity, and thus can serve as an ideal pre-fractionation method prior to two-dimensional electrophoresis.

Cytoskeleton ; chemistry ; Electrophoresis, Gel, Two-Dimensional ; methods ; Humans ; Proteome ; analysis ; Proteomics ; methods ; Subcellular Fractions

In order to provide a theoretical basis for better understanding the function and properties of proteins, we proposed a simple and effective feature extraction method for protein sequences to determine the subcellular localization of proteins. First, we introduced sparse coding combined with the information of amino acid composition to extract the feature values of protein sequences. Then the multilayer pooling integration was performed according to different sizes of dictionaries. Finally, the extracted feature values were sent into the support vector machine to test the effectiveness of our model. The success rates in data set ZD98, CH317 and Gram1253 were 95.9%, 93.4% and 94.7%, respectively as verified by the Jackknife test. Experiments showed that our method based on multilayer sparse coding can remarkably improve the accuracy of the prediction of protein subcellular localization.
Algorithms ; Amino Acid Sequence ; Computational Biology ; Protein Transport ; Proteins ; Subcellular Fractions ; Support Vector Machine

PURPOSE: To Study the possible mechanisms of change of thiosulfate sulfurtransferase (TST) activity in cholestatic rat liver and serum. METHODS: Rats were divided into seven groups: those receiving a sham operation (Sham group), with a bile duct obstruction (BDO) alone (BDO group), with a BDO plus taurocholic acid (TCA) injection (BDO plus TCA group), with a BDO plus tauroursodeoxycholic acid (TUDCA) injection (BDO plus TUDCA group), a choledocho-caval shunt (CCS) operation (CCS groups), a CCS operation plus TCA injection (CCS plus TCA group) and a CCS operation plus TUDCA injection (CCS plus TUDCA group). The TST activities in the serum and in the hepatic subcellular fractions isolated from above experimental rats were determined. The Km and Vmax values of this hepatic enzyme were measured. RESULTS: The liver cytosolic, mitochondrial and microsomal TSTs activities, as well as the TST Vmax values were found to be significantly decreased in the BDO plus TCA and BDO groups compared to the control group. The activity and Vmax value of the liver cytosolic TST were also found to be significantly decreased in the CCS plus TCA group. Conversely, there was no variation in the Km values of the hepatic enzymes in any of the above experimental groups. The serum TST activities in the CCS plus TCA and BDO plus TCA groups, were significantly increased compared with the control, CCS and BDO groups. However, the serum and hepatic enzyme activities were unchanged in both the CCS plus TUDCA and BDO plus TUDCA groups. CONCLUSION: The above results indicate that TCA represses the biosynthesis of TST in the liver. Also, the elevated TST activity in the serum is most likely due to an increase in the permeability of hepatocytes membrane upon TCA mediated liver cell necrosis.
Administration, Intravenous* ; Animals ; Cholestasis ; Cytosol ; Hepatocytes ; Liver* ; Membranes ; Necrosis ; Permeability ; Rats* ; Subcellular Fractions ; Taurocholic Acid* ; Thiosulfate Sulfurtransferase*

PURPOSE: To Study the possible mechanisms of change of thiosulfate sulfurtransferase (TST) activity in cholestatic rat liver and serum. METHODS: Rats were divided into seven groups: those receiving a sham operation (Sham group), with a bile duct obstruction (BDO) alone (BDO group), with a BDO plus taurocholic acid (TCA) injection (BDO plus TCA group), with a BDO plus tauroursodeoxycholic acid (TUDCA) injection (BDO plus TUDCA group), a choledocho-caval shunt (CCS) operation (CCS groups), a CCS operation plus TCA injection (CCS plus TCA group) and a CCS operation plus TUDCA injection (CCS plus TUDCA group). The TST activities in the serum and in the hepatic subcellular fractions isolated from above experimental rats were determined. The Km and Vmax values of this hepatic enzyme were measured. RESULTS: The liver cytosolic, mitochondrial and microsomal TSTs activities, as well as the TST Vmax values were found to be significantly decreased in the BDO plus TCA and BDO groups compared to the control group. The activity and Vmax value of the liver cytosolic TST were also found to be significantly decreased in the CCS plus TCA group. Conversely, there was no variation in the Km values of the hepatic enzymes in any of the above experimental groups. The serum TST activities in the CCS plus TCA and BDO plus TCA groups, were significantly increased compared with the control, CCS and BDO groups. However, the serum and hepatic enzyme activities were unchanged in both the CCS plus TUDCA and BDO plus TUDCA groups. CONCLUSION: The above results indicate that TCA represses the biosynthesis of TST in the liver. Also, the elevated TST activity in the serum is most likely due to an increase in the permeability of hepatocytes membrane upon TCA mediated liver cell necrosis.
Administration, Intravenous* ; Animals ; Cholestasis ; Cytosol ; Hepatocytes ; Liver* ; Membranes ; Necrosis ; Permeability ; Rats* ; Subcellular Fractions ; Taurocholic Acid* ; Thiosulfate Sulfurtransferase*

OBJECTIVETo investigate the effects of Hedan Tablet () on serum lipid profile, proprotein convertase subtilisin/kexin type 9 (PSCK9) and high-density lipoprotein (HDL) subfractions in patients with hyperlipidemia.

METHODSThirty-seven patients with hyperlipidemia were randomized to treatment with Hedan Tablet 4.38 g/day as Hedan group (18 cases) or placebo (19 cases) as control group for 8 weeks. The lipid profile, PCSK9 and HDL subfractions were determined at day 0 and week 8 in both groups respectively.

RESULTSHedan treatment for 8 weeks mildly decreased serum low-density lipoprotein cholesterol (LDL-C) levels, while no changes were found in total cholesterol (TC), triglycerides (TG) and PCSK9 concentrations. Furthermore, Hedan treatment increased the concentration of large high-density lipoprotein cholesterol (HDL-C) and the percentage of large HDL subfraction, while decreased the concentration of small HDL-C and the percentage of small HDL subfraction without changing serum HDL-C levels in patients with hyperlipidemia.

CONCLUSIONHedan treatment of 4.38 g per day for 8 weeks could confer a favorable effects on serum LDL-C concentration as well as HDL subfractions.

Drugs, Chinese Herbal ; therapeutic use ; Female ; Humans ; Hyperlipidemias ; blood ; drug therapy ; enzymology ; Lipoproteins, HDL ; blood ; Male ; Middle Aged ; Proprotein Convertase 9 ; metabolism ; Subcellular Fractions ; metabolism

Arginine methylation has been implicated in the signal transduction pathway leading to cell growth. Here we show that a regenerating rat liver following partial hepatectomy exhibited elevated methyltransferase activity as shown by increased methylation of a subset of endogenous proteins in vitro. The 20-kDa protein was shown to be a major cytosolic protein undergoing methylation in regenerating hepatocytes. Methylation of the 20-kDa protein peaked at 1 d following partial hepatectomy, which gradually declined to a basal level within the next 14 d. Likewise, methylation of exogenously added bulk histones followed the similar time kinetics as the 20-kDa protein, reflecting time-dependent changes in methyltransferase activity in regenerating hepatocytes. Presence of exogenously added bulk histone in the in vitro methylation assay resulted in dose-dependent inhibition of methylation of the 20-kDa protein. All the histone subtypes tested, histone 1, 2A, 2B, 3 or 4, were able to inhibit methylation of the 20-kDa protein while addition of cytochrome C, a-lactalbumin, carbonic anhydrase, bovine serum albumin, and g globulin minimally affected methylation of the 20-kDa protein. Since methylation of the 20-kDa protein preceded proliferation of hepatocytes upon partial hepatectomy, it is tempting to speculate that the methylated 20-kDa protein by activated histone-specific methyltransferase may be involved in an early signal critical for liver regeneration.
Animals ; Cytoplasm/*chemistry ; *Hepatectomy ; Histones/metabolism ; Humans ; Liver Regeneration/*physiology ; Methylation ; Methyltransferases/metabolism ; Protein Isoforms/metabolism ; Proteins/*metabolism ; Rats ; Rats, Sprague-Dawley ; Research Support, Non-U.S. Gov't ; Signal Transduction/physiology ; Subcellular Fractions/chemistry/metabolism

The molecular pathogenesis of leukemia is poorly understood. Earlier studies have shown both Wilms' tumor 1 suppressor gene (WT1) and CML28 abnormally expressed in malignant diseases of the hematopoietic system and WT1 played an important role in leukemogenesis. However, the relationship between molecular CML28 and WT1 has not been reported. Here we described the use of small interfering RNA (siRNA) against WT1 and CML28 in leukemic cell line K562 to examine the interaction between CML28 and WT1. WT1 and CML28 gene expression in transfected K562 cells was detected by using RQ-PCR and Western blotting. K562 cells transfected with WT1-siRNA could greatly decrease both mRNA and protein expression levels of WT1 and CML28. In contrast, CML28-siRNA did not exert effect on WT1. Further, subcellular co-localization assay showed that the two proteins could co-localize in the cytoplasm of K562 cells, but WT1/CML28 complexes were not detected by using immunoprecipitation. It was suggested that there exists the relationship between CML28 and WT1. CML28 may be a downstream target molecule of WT1 and regulated by WT1, which will provide important clues for further study on the role of CML28 and WT1 in leukemic cells.
Antigens, Neoplasm ; metabolism ; Cell Line, Tumor ; Exosome Multienzyme Ribonuclease Complex ; metabolism ; Humans ; K562 Cells ; Leukemia, Erythroblastic, Acute ; metabolism ; Neoplasm Proteins ; metabolism ; Protein Interaction Mapping ; RNA-Binding Proteins ; metabolism ; Subcellular Fractions ; metabolism ; WT1 Proteins ; metabolism