It is well known that the bona pellucidae of mouse oocytes become 'hardened' when they are allowed to mature in vitro in the absence of serum components. To see if oocytes already undergone meiotic resumption in vivo exhibit similar zona hardening, hardening of ZP of cumulus-enclosed oocytes(CEOs) was examined after culture in vitro since their release from follicles various hours after hCG injection. When CEOs matured in vivo for 3h or longer were subjected to culture in vitro for 14h with BSA alone, zona hardening was significantly reduced compared to those cultured in vitro from the begining of maturation. However, when CEOs matured in vivo for 5h were freed from cumulus cells and then cultured in vitro with BSA alone, little reduction of zona hardening was observed. Preincubation of CEOs for 5h with fetuin, one of the well known inhibitor of in vitro zone hardening, did not prevent bona hardening during its subsequent culture of CEOs for 14h without fetuin. However, when CEOs precultured with both fetuin and PMSG for 5h and then further cultured with BSA alone for 14h, zona hardening was dramatically reduced. Under these conditions, the expansion of cumulus cell was observed. In addition, CEOs cultured with both BSA and dbcAMP to prevent their meiotic resumption showed a significant increase of zona hardening. Whether the observed zona hardening was correlated with the conversion of ZP2 to ZP2f was examined. Zona pellucida, isolated from CEOs matured for 5h in vivo and then further cultured with BSA alone was subjected to SDS-PAGE. Most of ZP2 molecules from these CEOs did not undergo conversion from ZP2 to ZP2f. From these results, it is concluded that CEOs undergone meiotic resumption in vivo do not exhibit bona hardening when they were subsequently cultured in vitro without serum components. It appears that cumulus cells play an important role in this phenomenon.
Animals ; Bucladesine ; Cumulus Cells ; Electrophoresis, Polyacrylamide Gel ; Fetuins ; Herpes Zoster* ; Mice* ; Oocytes* ; Zona Pellucida

<b>OBJECTIVEb>To observe the targeted therapeutic effects of plasmid AF-pGL3-hTERT-TK on HepG2 cells.

<b>METHODSb>HepG2 cells were cultured and pGL3-hTERT-TK and AF-liposome were constructed. HepG2 and L02 cells were transfected with AF-pGL3-hTERT-TK. The growth, apoptosis of the cells and the bystander effects were studied using liquid scintillation analysis and tunnel and flow cytometry.

<b>RESULTSb>After the suicide gene was inserted into the downstream of hTERT, TK was effectively driven by the hTERT promoter, making the TK highly expressed in the HepG2 cells. The AF made the therapeutic gene enter the HepG2 cells more easily by recognizing and combining the ASGPR receptor protein on the HepG2 cell surfaces and induced their apoptosis and suicide with bystander effect. The apoptosis rate was 85%+/-3% in the HepG2 cells whereas in the normal L02 hepatic cells it was 16%+/-2%.

<b>CONCLUSIONb>AF-pGL3-hTERT-TK can target and attack HepG2 cells and has almost no influence on normal L02 hepatic cells. AF-pGL3-hTERT-TK has a potential in the treatment of hepatocellular carcinomas.

Apoptosis ; Asialoglycoproteins ; Bystander Effect ; Fetuins ; Ganciclovir ; metabolism ; Genes, Transgenic, Suicide ; Genetic Therapy ; Hep G2 Cells ; Humans ; Telomerase ; metabolism ; Thymidine Kinase ; metabolism ; Transfection ; alpha-Fetoproteins

<b>AIMb>To target for hepatocytic cell, liposomes was modified by special ligand.

<b>METHODSb>Sterically stabilized liposomes (SSL) was conjugated with asialofeticin (AF), the ligand of asialoglycoprotein receptor (ASGP-R) of hepatocyte. ASGP-R-BLM is the ASGP-R reconstructed on bilayer lipid membrane (BLM). The recognition reaction between AF-SSL and ASGP-R-BLM can be monitored by the varieties of membrane electrical parameters. The targetability of AF-SSL mediated to hepatocyte was detected by radioisotopic labeled in vitro and in vivo. The therapeutic effect of antihepatocarcinoma was observed also.

<b>RESULTSb>The lifetime of ASGP-R-BLM decreased with the added amount of AF-SSL. It was demonstrated that there was recognition reaction between AF-SSL and ASGP-R-BLM. The combination of AF-SSL with hepatocyte was significantly higher than that of SSL without AF-modified in vitro and in vivo. The survival time of rat for AF-SSL carriered ADM (adriamycin) group was much longer and the toxicities on heart, kidney and lung were lower than those SSL carried ADM group.

<b>CONCLUSIONb>It is possible to actively target the cell with specific receptor by ligand modified liposomes. The result prvide scientific basis of hepatocyte targeted liposomes.

Animals ; Antineoplastic Agents ; administration & dosage ; therapeutic use ; Asialoglycoprotein Receptor ; Asialoglycoproteins ; chemistry ; Doxorubicin ; administration & dosage ; therapeutic use ; Drug Carriers ; Drug Delivery Systems ; Fetuins ; Hepatocytes ; metabolism ; Ligands ; Lipid Bilayers ; Liposomes ; chemistry ; metabolism ; Liver ; metabolism ; Liver Neoplasms, Experimental ; drug therapy ; Male ; Mice ; Random Allocation ; Rats ; alpha-Fetoproteins ; chemistry

<b>AIMb>To study the transfection and anti-hepatitis B virus (HBV) effect of the co-modified hepatocytes-targeting cationic liposomes encapsulating anti-HBV antisense oligonucleotides (asON) , and to investigate the transfection mechanisms of the liposomes.

<b>METHODSb>Dipalmitoylphosphatidylcholine (DPPC) and 3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) were used as the lipids, beta-sitosterol-beta-D-glucoside (sito-G) and Brij 35 were used to modify the liposomes. Flow cytometry (FCM), fluorescence microscopy and enzyme-linked immunosorbent assay (ELISA) were utilized to evaluate the transfection improvement of the asON encapsulated in the liposomes in primary rat hepatocytes and the antigens inhibition activity in HepG 2.2.15 cells. The transfection mechanisms were evaluated based on the influence of wortmannin, nigericin, and asialofetuin on the antigens inhibition in HepG 2.2.15 cells by ELISA.

<b>RESULTSb>The co-modification with sito-G and Brij 35 significantly improved the transfection of the liposomes in primary rat hepatocytes and antigens inhibition effect in HepG 2.2.15 cells. Both transfection efficiency and antigens inhibition effect showed to be concentration-dependent with the asON-encapsulating liposomes. In fluorescence microscopy, the transfected cells showed strong fluorescence in primary rat hepatocytes, especially in the nuclei. Wortmannin, nigericin and asialofetuin decreased the antigens inhibition of the asON-encapsulating liposomes to different levels. Cationic liposomes modification with sito-G and Brij 35 could improve the transfection and antigens inhibition effect of the asON. The transfection mechanisms of the co-modified liposomes included endocytosis and membrane fusion. The ligand sito-G was confirmed to be able to enhance asialoglycoprotein receptor (ASGPR)-mediated endocytosis.

<b>CONCLUSIONb>Co-modified hepatocytes-targeting cationic liposomes would be a specific and effective carrier to transfer asON into hepatocytes.

Androstadienes ; pharmacology ; Animals ; Asialoglycoproteins ; pharmacology ; Cell Line, Tumor ; Cell Nucleus ; metabolism ; Cell Survival ; Cells, Cultured ; Endocytosis ; drug effects ; Female ; Fetuins ; Flow Cytometry ; Hepatitis B Antigens ; metabolism ; Hepatitis B virus ; genetics ; immunology ; Hepatocytes ; cytology ; drug effects ; metabolism ; Humans ; Liposomes ; Microscopy, Fluorescence ; Nigericin ; pharmacology ; Oligonucleotides, Antisense ; chemistry ; genetics ; Polyethylene Glycols ; chemistry ; Rats ; Rats, Wistar ; Sitosterols ; chemistry ; Transfection ; methods ; alpha-Fetoproteins ; pharmacology

Non-alcoholic fatty liver disease, which is considered a hepatic manifestation of metabolic syndrome, independently increases the risks of developing cardiovascular disease (CVD) and type 2 diabetes mellitus. Recent emerging evidence suggests that a group of predominantly liver-derived proteins called hepatokines directly affect the progression of atherosclerosis by modulating endothelial dysfunction and infiltration of inflammatory cells into vessel walls. Here, we summarize the role of the representative hepatokines fibroblast growth factor 21, fetuin-A, and selenoprotein P in the progression of CVD.
alpha-2-HS-Glycoprotein ; Atherosclerosis ; Cardiovascular Diseases* ; Diabetes Mellitus, Type 2 ; Fatty Liver ; Fibroblast Growth Factors ; Obesity* ; Selenoprotein P

Type 2 diabetes (T2D) is rapidly spreading throughout the world. It's an insidious disease and still treated in an indirect manner without having specific drug target. In majority cases T2D is treated with drugs that address type 1 diabetes, majority of drugs aim to increase insulin release although the root cause for T2D is not the dearth of insulin release, it occurs in the later stage of disease development. T2D silently progressed in the patient; it begins with insulin resistance that takes place due to the loss of insulin sensitivity. Though insulin resistance is the centre of pathogenesis, our treatment of the disease has not yet addressed it. It is now a fact that insulin resistance is manifested by lipid and fatty acids (FAs) play a critical role in blunting insulin sensitivity. Our understanding is still poor in deciphering how lipid impose insulin insensitivity, majority of workers suggest it is because of insulin signaling defects which implements insulin function in inhibiting glucose to the cell from circulation. Number of long chain saturated FA has been shown to produce insulin signaling defects. However, we really need further investigation to find specific target(s) for FA induced damage. In addition to these information, a new dimension of T2D is getting attractive is fetuin-A/alpha2-Heremans-Schmid Glycoprotein, a secretary protein from liver. Its gene locus has been identified as T2D susceptible. Fetuin-A's excess expression occurs by FA and it disrupts adipocyte function. It has been shown to be associated with T2D especially in obesity. In this review, we briefly discuss the present status on the mechanistic understanding of lipid induced insulin resistance that leads to T2D. More we understand the mechanism; opportunity to fight the battle with T2D will be increasing. Since, this field is now vast; we covered a few major events.
Adipocytes ; alpha-2-HS-Glycoprotein ; Fatty Acids ; Glucose ; Glycoproteins ; Hypogonadism ; Insulin ; Insulin Resistance ; Liver ; Mitochondrial Diseases ; Obesity ; Ophthalmoplegia

BACKGROUND: Coronary artery calcification is frequently seen in hemodialysis patients. The development and progression of coronary artery calcification is similar to osteogenesis, and a naturally occurring serum inhibitor of calcification may be involved. The aim of this study was to evaluate the relationship between coronary artery calcification, bone remodeling related factor, serum osteoprotegerin (OPG), calcification inhibitor and the serum fetuin-A levels in hemodialysis patients. METHODS: A total of 51 hemodialysis patient were assessed for their coronary artery calcium (CAC) scores with using multirow spiral computed tomography and measuring the serum OPG level, the serum fetuin-A level, the biochemical markers of inflammation, the lipid profile and the mineral metabolism. RESULTS: The mean serum OPG level was 3,561+/-1,160 pg/mL and the mean serum fetuin-A level was 28.5+/-4.1 mg/dL. The CAC scores were significantly correlated with the duration of dialysis (p=0.0225), hs-CRP (p=0.0392), serum phosphate (p=0.0341), Ca x P (p=0.0434), the serum OPG level (p=0.0026) and LDL-cholesterol (p=0.0438), after adjusting for age, gender, BMI, smoking history and the presence of diabetes. Multiple regression analysis showed that the CAC scores were significantly associated with the serum OPG level (p<0.0001) and the serum phosphate level (p=0.0003). The subgroup of the patients with a CAC score greater than 400 (the severe CAC group) had significantly higher OPG levels and lower fetuin-A levels than the groups of the patients with lower CAC scores. CONCLUSIONS: Our data suggest that the CAC scores in the patients undergoing hemodialysis were related with higher serum OPG and higher serum phosphate levels. The serum fetuin-A level was significantly lower in the patients with severe coronary artery calcification.
alpha-2-HS-Glycoprotein* ; Biomarkers ; Bone Remodeling ; Calcium ; Coronary Disease ; Coronary Vessels* ; Dialysis ; Humans ; Inflammation ; Kidney Failure, Chronic* ; Metabolism ; Osteogenesis ; Osteoprotegerin* ; Renal Dialysis ; Smoke ; Smoking ; Tomography, Spiral Computed

Coronary artery calcification is associated with the increased cardiovascular mortality and the extent of atheromatous plaque, especially in the hemodialysis patients. Vascular calcification was in the past considered a passive process, a degenerative consequence of aging or the result of disrupted mineral balance in the patients with chronic renal failure. It is now understood that calcium deposition in the vasculature is an active and regulated process similar to bone formation. In this issue of JKMS, Kim et al. investigate the clinical association between osteoprotegerin, an osteoclast inhibitory factor which was reported to be associated with coronary artery calcification, and fetuin-A, a systemic ectopic calcification inhibitory factor, with coronary artery calcification (CAC) score obtained from multi-slice CT. They showed that the high serum osteoprotegerin level is associated with increased CAC score and serum fetuin-A level is significantly lowered in the severe CAC score group. These results show that serum osteoprotegerin or fetuin-A level might be used as serum markers for the determination of the severity of coronary artery calcification. However, the role of serum osteoprotegerin in the formation of vascular calcification is uncertain and the pathophysiologic mechanism is not uncovered yet. Some studies suggested that the osteoprotegerin level is associated with vascular stiffness. Lower fetuin-A level is known to be a prognostic factor of cardiovascular disease mortality from several epidemiologic studies and a confirmed anti-calcifying agents in vitro experiments. In interpreting this issue of Kim et al., it is important that increased serum osteoprotegerin level might be associated with not vascular calcification but other vascular malfunction such as arterial stiffness. In conclusion, more sophisticated study needed for the clarification of the role of calcification-associated serum markers in the process of vascular calcification.
Aging ; alpha-2-HS-Glycoprotein ; Biomarkers* ; Calcium ; Cardiovascular Diseases ; Coronary Vessels* ; Humans ; Kidney Failure, Chronic ; Mortality ; Osteoclasts ; Osteogenesis ; Osteoprotegerin ; Renal Dialysis ; Vascular Calcification ; Vascular Stiffness

<b>OBJECTIVEb>To identify the single nucleotide polymorphisms (SNPs) of the alpha2-Heremans-Schmid glycoprotein (AHSG) gene and assess their association with the AHSG serum level.

<b>METHODSb>The SNPs of the AHSG gene were identified from 30 unrelated Han individuals from Guangzhou area by resequencing. Linkage disequilibrium(LD) was performed to observe the linkage disequilibrium pattern. Then tagSNPs were genotyped in 192 Han individuals from Beijing and 424 Han individuals from Guangzhou area. Finally, luciferase reporter gene assay was performed to determine whether the SNPs affected the promoter activity. Serum AHSG concentrations were measured in the 192 subjects from Beijing using ELISA.

<b>RESULTSb>Eight SNPs were detected in total. The linkage disequilibrium profile in the Guangzhou Han population was different from that in the Beijing Han population. However, the allele and genotype frequencies of tagSNPs between the two Han populations were not significantly different. The reporter gene assay showed that the -799A allele had significantly higher promoter activity than the -799T allele. Multiple regression analysis revealed that only the rs2248690 SNP was an independent contributor to serum AHAG concentration.

<b>CONCLUSIONb>The rs2248690 SNP in the promoter region of the AHSG gene might affect the AHSG gene transcription.

Blood Proteins ; genetics ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Genotype ; Linkage Disequilibrium ; Polymorphism, Single Nucleotide ; genetics ; Serum ; chemistry ; alpha-2-HS-Glycoprotein

<b>OBJECTIVEb>To explore the dynamic changes of fetuin-A expression and the influences of the changes on liver damage, hepatocyte apoptosis and inflammation in a mouse fulminant hepatic failure (FHF) model.

<b>METHODSb>The changes of fetuin-A expression were investigated by semi-quantitative RT-PCR and Western blot. Immunohistochemical staining was used in TNFa and fetuin-A detection. Hepatocyte apoptosis was detected by TUNEL.

<b>RESULTSb>Fetuin-A mRNA expression decreased after the FHF model was established for 3 hours (compared with the normal group, P less than 0.01), while the protein expression decreased after nine hours (compared with the normal group, P less than 0.01). Fetuin-A expressions were negatively correlated with the liver pathological scores and TNFa levels.

<b>CONCLUSIONb>In our mouse FHF model, fetuin-A is a possible protective factor for liver damage.

Animals ; Blood Proteins ; metabolism ; Female ; Liver ; metabolism ; pathology ; Liver Failure ; metabolism ; pathology ; Mice ; Mice, Inbred BALB C ; alpha-2-HS-Glycoprotein