Preparation and in vitro evaluation of redox-sensitive polypeptide vector for co-delivery of gene and chemotherapeutic drugs / 第二军医大学学报

Objective To prepare a lipoic acid (LA) modified intrinsically disordered protein-cytosol-localizing internalization peptide 6 (CL) nanocomplex (LA-CL) entering cells by non-endocytosis mechanism for co-delivery of gene and chemotherapeutic drugs, and to investigate its transfection efficiency and cellular uptake on human embryonic kidney cell line HEK293 cells and its release behavior in vitro. Methods We synthesized four disulfide cross-linked lipoic acid modified LA-CLss(1-4) at different cross-linked degrees using different mass fractions (2.5%, 5%, 10% and 20%) of cysteine as cross-linking agent. The construction of LA-CLss was characterized by1H nuclear magnetic resonance (1HNMR) and gel permeation chromatography. The LA-CLss/plasmid enhanced green fluorescent protein (pEGFP) nanocomplexes were self-assembled with LA-CLss and pEGFP at different nitrogen/phosphorus (N/P) ratios (2.5, 5, 10, 20, 40 and 80). The size and zeta potential of LA-CLss/pEGFP nanocomplexes were determined by particle size analyzer, and the pEGFP enrichment capacity of LA-CLss was determined by agarose gel electrophoresis. The docetaxel (DTX)-loaded micelles were prepared by ultrasonic emulsification, and the critical micelle concentration of LA-CLss3 was determined by pyrene fluorescence probe spectroscopy. The LA-CLss/pEGFP nanocomplexes were co-cultured with HEK293 cells, and the transfection efficiencies of LA-CLss/pEGFP nanocomplexes at different cross-linked degrees were investigated. Results1 HNMR results showed the LA-CLss was successfully synthesized. When N/P ratio was 40, the transfection efficiency of LA-CLss3/pEGFP nanocomplex by HEK293 cells was significantly higher than that of LA-CL/pEGFP, LA-CLss1/pEGFP, LA-CLss2/pEGFP and LA-CLss4 nanocomplexes. The encapsulation efficiency and drug loading of docetaxel-loaded micelles prepared by ultrasonic emulsification were (85.25±0.04)% and (8.81±0.02)%, respectively. Cellular uptake test showed that the gene could be effectively delivered into the HEK293 cells by the LA-CLss micelles. In vitro release experiments showed that the LA-CLss micelles had redox-responsive drug release behavior. Conclusion The prepared LA-CLss/DTX/pEGFP nanocomplex is expected to become an efficient vector for co-delivery of gene and chemotherapeutic drugs.

Research on relationship between cytosol phospholipase A2α and chronic obstructive pulmonary disease / 中国中西医结合急救杂志

Objective To approach the changes of cytosol phospholipase A2α(cPLA2α)and nitric oxide (NO)in patients with chronic obstructive pulmonary disease(COPD)and its significance. Methods One hundred patients with COPD admitted into Department of Critical Care Medicine of Affiliated Wuqing Traditional Chinese Medicine(TCM)Hospital of Tianjin University of TCM were enrolled,and according to the COPD severity grading standards,they were divided into mild group(25 cases),moderate group(25 cases),severe group(26 cases) and extremely severe group(24 cases);simultaneously,90 cases with normal pulmonary function who had taken health examination were chosen and assigned to the healthy control group. The cPLA2α level was detected by enzyme linked immunosorbent assay(ELISA),the level of uric acid(UA),total cholesterol(TC),triacylglycerol (TG)were detected by enzymatic method,and serum NO metabolites(NOx)level was detected by nitrate reductase method. Results Compared with the healthy control group,the serum levels of cPLA2α and UA in patients with different severity of COPD were significantly increased;along with the increase of patient's COPD grade of severity,the cPLA2α,UA levels were gradually increased,while NOx level was gradually decreased in mild, moderate, severe, extremely severe groups〔cPLA2α(ng/L):125.60±8.17, 155.20±6.42, 190.20±9.32, 255.80±11.28 vs. 88.50±7.99;UA(μmol/L):381.23±32.22,434.95±87.71,464.81±52.65,487.45±82.61 vs. 241.95±52.33;NOx(μmol/L):59.90±17.52,45.60±6.17,38.20±4.08,25.70±3.04 vs. 74.90±18.31,all P0.05). The cPLA2αand NOx levels presented significant negative correlation(rs=-0.798,P=0.013). Conclusion The combined examination of blood cPLA2αand serum NOx levels can evaluate the severity degree of COPD patients,and cPLA2αcan be used as a new target index for COPD grading.

Cytosolic estrogen and progesterone receptor content in the endometriotic tissues and endometrium in women with and without endometriosis.

In order to have a profile of cytosolic estrogen and progesterone receptors in either endometriotic tissue or endometrium in patients with and without endometriosis a cross-sectional study was performed involving 43 infertile women. They consisted of 31 (72.09%) endometriosis and 12 (27.91%) non-endometriosis cases; their average age was 32 ± 4 years and 32 ± 3 years respectively, with the average length of menstrual cycle 31 ± 8 days and 29 ± 1 days respectively. The endometriotic tissue was obtained by excision during operative laparoscopy procedure, while the endometrium was obtained by biopsy following hysteroscopy procedure. These procedures were conducted within the periovulatory period (on Day 13-18 of the cycle). The sex steroid receptor content in the cytosol was measured quantitatively using enzyme-immunoassay method, and calculated as sex steroid receptor/cytosol protein (fmol/ml cytosol). It was found that the average cytosolic estrogen receptor concentration in the respective tissues were 512.99 fmol/ml in the endometriotic ovary compared with 2369.17 in normal ovary and 632.18 fmol/ml in the endometriotic peritoneum compared with 9607.61 fmol/ml in normal peritoneum; while 99.28 fmol/ml and 608.33 fmol/ml in the endometrium of women with endometriosis and those without endometriosis respectively. The average cytosolic progesterone receptor concentration found in the respective tissues were 50.64 fmol/ml in the endometriotic ovary compared with 6469.42 fmol/ml in normal ovary and 1631.40 fmol/ml in endometriotic peritoneum compared with 12466.99 in normal peritoneum, while 21.26 fmol/ml and 599.61fmol/ml in the endometrium of women with endometriosis and those without endometriosis respectively. There is no significant difference in the receptor concentration between each tissue according to its topographic origin. However, this result may assume that the responsivity on hormonal treatment in endometriosis cases will depend on the cytosolic sex steroid receptor content in the sick tissues, and the peritoneal lesions will possibly give better response than those in other sites. A further clinical trial is necessary.

Analysis of the Cell Lysate Proteome of a Korean Mycobacterium tuberculosis Isolate K01 with H37Rv and H37Ra Strains

Despite recent economic prosperity, Korea still has high prevalence of tuberculosis. Molecular biologic characterization of Korean Mycobacterium tuberculosis strains might provide a deeper understanding of the forces contributing to the spread of tuberculosis in Korea. Therefore, we analyzed the cell lysate proteome of a representative Korean Mycobacterium tuberculosis isolate (K01) in comparison with laboratory reference strains H37Rv and H37Ra. Seven spots were strongly expressed only in K01 strain compared with M. tuberculosis H37Rv and H37Ra. Through continuous MALDI-MS analysis, these spots were identified as hypothetical protein Rv3849, secreted immunogenic protein Mpt64, Acetyl/propionyl-CoA Carbpxylase (AccD1), alkyl hydroperoxide reductase C (AhpC), N-acetylmuramyl-L-alanine amidase, a putative UDP glucose epimerase, and a transposase. A deeper study of these proteins may provide a clue in the development of effective new anti-tuberculosis vaccines against Korean M. tuberculosis isolates.

The Evaluation of Liver Cytosol Extracts Affecting Liver Regeneration after Partial Hepatectomy in Rat

It was known that the regenerating liver cytosol accelerated liver regeneration, whereas normal liver cytosol inhibited it during first 28 hours, but promoted it later by the thymidine incorporation test. In experiments, Sprague-Dawley male rats were divided into 3 experimental groups (A, B and C) which had 10 rats in each. Also, each group was equally subdivided into 2 subgroups such as A-8 and A-28 in the A group, B-8 and B-28 in the B group and C-8 and C-28 in the C group. All groups were partially hepatectomized equally. The remnant livers were procured at 8 hours in the 3 subgroups A-8, B-8 and C-8 and at 28 hours in the other 3 subgroups A-28, B-28 and C-28. Another experiment was performed in 10 rats under the same partial hepatectomy to get the average weight of the remnant liver. These data could be taken as the initial weight of the remnant liver in the above experimental animals. The normal saline, normal liver cytosol extract and regenerating liver cytosol extract were injected intraperitoneally after the partial hepatectomies, 4 ml of in the A, B, and C groups, respectively. Since specific structural changes might precede demonstrable functional alterations, morphometric measurements were done in the histological specimens of the 3 subgroups A-8, B-8, and C-8. The remnant livers, obtained in all six subgroups, were weighed to calculate the regeneration rates. The overall results are as follows : 1) The liver regeneration rates in the six subgroups were 4.1% in A-8, 3.8% in B-8, 4.3% in C-8, 28.4% in A-28, 16.7% in B-28, and 32.8% in C-28. 2) The morphometric study showed that the nucleus/cytoplasm ratios in the three subgroups were 0.1389 in A-8, 0.2048 in B-8, and 0.3705 in C-8. These results mean that regenerating liver cytosol extract promotes liver regeneration and that normal liver cytosol extract inhibits it during first 28 hours after a partial hepatectomy, but promotes cell division for subsequent regeneration.

Characterization of cytosolic phosphoglucoisomerase from immature wheat (Triticum aestivum L.) endosperm.

Phosphoglucoisomerase from cytosol of immature wheat endosperm was purified 650-fold by ammonium sulphate fractionation, isopropyl alcohol precipitation, DEAE-cellulose chromatography and gel filtration through Sepharose CL-6B. The enzyme, with a molecular weight of about 130,000, exhibited maximum activity at pH 8·1. It showed typical hyperbolic kinetics with both fructose 6-P and glucose 6-P with Km of 0·18 mM and 0·44mM respectively. On either side of the optimum pH, the enzyme had lower affinity for the substrates. Using glucose 6-P as the substrate, the equilibrium was reached at 27% fructose 6-P and 73% glucose 6-P with an equilibrium constant of 2·7. The ΔF' calculated from the apparent equilibrium constant was +597 cal mol–1 . The activation energy calculated from the Arrhenius plot was 5500 cal mol–1. The enzyme was completely inhibited by ribose 5-P, ribulose 5-P and 6-phosphogluconate, with Ki values of 0·17, 0·25 and 0·14 mM respectively. The probable role of the enzyme in starch biosynthesis is discussed.

Effects of human fetal hepatic stimulator substance on D-galac-tosamine-induced liver necrosis in rats / 第三军医大学学报

The effects of human fetal hepatic stimulator substance (h-HSS) and liver cytosol (h-FLC) on the survival rate,the intensity of liver damage,and liver regeneration in rats with liver necrosis induced with D-galactosamine were observed.The physico-chemical properties of h-HSS were preliminarily determined.It was found that both h-HSS and H-FLC could markedly increase the.survival rate of the rats with toxic liver necrosis,significantly decrease the serum level of ornithine carbamoyl transferase and mitochondria aspartic trans-aminase,improve the hepaplastin activity,and elevate the incorporation rate of 3H-thymidine with liver DNA.h-HSS was stable after heated to 95℃ for 30 minutes,and sensitive to trypsin treatment.It demonstrated one major band at 15 000 daltons and 3 minor ones at 24 000,34 000,and 40 000 daltons respectively on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The relative content of the major band was 47.57%.

Effects of dog regenerating liver cytosol on MPS function of rats with experimental liver injury / 中国病理生理杂志

Using the technique for measuring clearance rate of ~(125)I-microaggregated albumin(~(125)I-MAA), we investigated the effects of dog regenerating liver cytosol(DRLC) on the function of mononuclear phagocytic system (MPS) of rats with liver injury induced by D-galactosamine(D-GalN). The results showed that the clearance rate of ~(125)I-MAA from plasma in rats with liver injury was markedly inhibited, the half life(T_(1/2)) of ~(125)I-MAA was significantly prolonged(P

Enhancements of Mouse Hepatic Cytosol Enzyme Activities Involved in UDP-Glucuronic Acid Synthesis, Glutathione Reduction and Conjugation with Butylated Hydroxyanisole (BHA) and Its Structural Analogs

Activities of hepatic cytosol enzymes involved in UDP-g1ucuronic acid synthesis as well as in glutathione reduction and conjugation systems were determined following administrations of butylated hydroxyanisole (approximately 5 mmol/kg body weight/day) and of equimolar intake doses of its structural anglogs. These compounds included the multi-functional group side chain compounds (t-butyl hydroquinone, 4-hydroxy- anisole, hydroquinone, benzoquinone) and the mono-functional side chain compounds (t-butyl benzene, anisole, phenol). They were administered to mice for 10 days either by mixing them in the diet or by oral intubations. Results showed that glutathione Stransferase activities were markedly increased by all tested compounds except for the t-butyl benzene. Activities of glutathione reductase and glucose 6-phosphate dehydrogenase were increased together on1y by BHA and t-butyl hydroguinone. UDP-glucose dehydrogenase and NADH:quinone reductase activities were significantly elevated by the multi-functional side chain compounds, but not by the mono-functional analogs. The relations between chemical structures of tested BHA analogs and elevations of the measured hepatic cytosol conjugation (detoxification) system enzyme activities for the metabolism and excretion of BHA analogs are discussed.

Analysis of the Components of Cell-free Preparationsof Human Fetal Liver and Its Effect on Survivalof Experimental Hepatic Failure / 第三军医大学学报

Effect of cell-free preparation of human fetal liver (cytosol and its dialysa-te) , human fetal muscle cytosol(FMC) and human adult liver cytosol (ALC) on survival rate of rats with acute hepatic failure induced by D-galactosamine was studied. The components of the 4 preparations were analysed preliminarily. Results: 1 ,fetal liver cytosol(FLC) could increase the survival rate(SR) of rats with hepatic failure(HF) comparing with normal saline(P0.05) . The SR of rats with HF was reduced by ALC markedly) 2,IgG,IgA,IgM and C3 could not be detected in FLC, but a few kinds of hormones, trace elements, glucose and lipids, and biological large molecule substances (BLMS) such as nucleic acids and proteins were found. As to free amino acid,content of branch chain amino acids in FLC was significantly higher than that in ALC, but the content of aromatic amino acids and ammonia was markedly lower than that in ALC.The components in FLD were almost the same as in FLC except BLMS.These data suggest that the soluble BLMS in FLC may play a key role in treating HF. ALC could reduce SR and contained a large amount of poisonous substances such as aromatic amino acids and ammonia.

Relationship between Cytosol Estrogen Receptor Level in Uterine Leiomyoma and Sex Steroid Level in Plasma, Leiomyoma and Myometrium / 第三军医大学学报

This study was designed to evaluate the effect of estrogen to the growth of leiomyoma and the mechanism of testosterone propionate action on leiomy-oma.The levels of estrogen receptor in uterine tissud and those of estradiol and progesterone in uterine tissue and plasma were determined in 47 women with leiomyoma.The levels of estrogen receptor and estradiol in leiomyomatous tissues were 37.6?4.0 fmol/mg protein and 401.7?92.6 pg/g tissue,respectively.Both of them were higher than the corresponding levels in normal uterine tissues (P

THE EFFECTS OF CHRONIC ADMINISTRATION OF HYDROCORT- ISONE ON THE GLUCOCORTICOID RECEPTOR OF HEPATIC CYTSOL IN RATS / 第二军医大学学报

The daily intramuscular injections of hydrocortisone acetate for about 20 days (2 mg/100g body weight per day), resulted in a marked decrease of specific binding capacity (Ro) and an increase of the equilibrium dissociation constant (Kd) of the hepatic cytosol with [3H] dexamethasone in rats. There were no changes of Ro and Kd in the control groups after saline injection for about 20 days or a single injection of hydro-cortisone acetate 5 hours or 24 hours prior to sacrification of the animals. The results indicated the exsistence of down-regulation of glucocorticoid hormone to its homologous receptor after chronic admnistration of glucocrticoid hormone.

Hexokinase isoenzymes in diabetes.

Hexokinase is present in the tissues in four isoenzymic forms. Cerebral tissue contains predominantly Type I hexokinase which is believed to be insulin-insensitive. In cerebral tissue about 60 to 70% of the hexokinase is bound to the particulate fraction. The changes in the distribution of hexokinase Type I and Type II together with the bound and free hexokinase have been studied in control, diabetic and diabetic animals treated with insulin. The results indicate that the presence of insulin is essential for the normal binding of the hexokinase to the particulate fraction. In heart tissue, Type II hexokinase bound to the pellet shows a significant decrease in diabetes, which is reversed on insulin administration.