The heat shock protein 70 inhibitor VER155008 suppresses the expression of HSP27, HOP and HSP90β and the androgen receptor,induces apoptosis,and attenuates prostate cancer cell growth

Heat shock proteins (HSPs) are molecular chaperones that play a pivotal role in correct folding, stabilization and intracellular transport of many client proteins including those involved in oncogenesis. HSP70, which is frequently overexpressed in prostate cancer (PCa), has been shown to critically contribute to tumor cell survival, and might therefore represent a potential therapeutic target. We treated both the androgen receptor (AR)-positive LNCaP and the AR-negative PC-3 cell lines with the pharmacologic HSP70 inhibitor VER155008. Although we observed antiproliferative effects and induction of apoptosis upon HSP70 inhibition, the apoptotic effect was more pronounced in AR-positive LNCaP cells. In addition, VER155008 treatment induced G1 cell cycle arrest in LNCaP cells and decreased AR expression. Further analysis of the HSP system by Western blot analysis revealed that expression of HSP27, HOP and HSP90β was significantly inhibited by VER155008 treatment, whereas the HSP40, HSP60, and HSP90α expression remained unchanged. Taken together, VER155008 might serve as a novel therapeutic option in PCa patients independent of the AR expression status.     

Development of a pronuclear DNA microinjection technique for production of green fluorescent protein-expressing bubaline (Bubalus bubalis) embryos

Oocytes from abattoir-derived bubaline (Bubalus bubalis) ovaries were subjected to IVM and IVF; the objective was to develop a pronuclear DNA microinjection technique to produce embryos expressing green fluorescent protein (GFP). The largest proportion (61.2%) of zygotes in which one (1 PN) or two pronuclei (2 PN) were visible was when centrifugation (14,000 x g for 15 min) was done 16 h after insemination. Centrifugation had no adverse effects on cleavage rate, development to morulae/blastocysts, and total cell number of embryos. Piercing the pronuclear but not the plasma membrane reduced (P<0.05) cleavage rate (44.0% vs. 51.0%), without affecting subsequent development. Following microinjection of a GFP-DNA construct, cleavage rate (55.9, 38.9, and 30.9%) and proportion of cleaved embryos that developed to morulae (39.9, 25.6, and 15.5%) and blastocyst stages (22.4, 13.4, and 2.8%) were higher (P<0.05) for non-injected controls than for those injected with buffer alone, which, in turn, were higher (P<0.05) than for those injected with buffer containing 5 microg/mL DNA. The cleavage rate (39.2% vs. 34.8%) and proportion of cleaved embryos that developed to morulae/blastocysts (37.5% vs. 10.9%) were higher (P<0.05) for microinjected zygotes with 2 PN than for those with 1 PN. The cleavage rate and the proportion of cleaved embryos that developed to morulae and blastocysts were higher (P<0.05) following culture of microinjected zygotes in mCR2aa medium (40.7, 32.7, and 9.1%, respectively) compared to those for mSOFaa (33.3, 26.0, and 6.5%, respectively) or after culture in TCM-199+co-culture with buffalo oviductal epithelial cells (31.2, 25.0, and 4.5%, respectively). The proportion of embryos expressing GFP was higher (P<0.01) for 2 PN than for 1 PN zygotes (15.9% vs. 13.7%). Thirty-five embryos expressed GFP; the proportion of mosaic embryos (62.8%) was higher (P<0.01) than of embryos in which all blastomeres expressed GFP (37.2%); eight and two of those embryos developed to the morula and blastocyst stages, respectively.     

Vitrification of buffalo (Bubalus bubalis) oocytes

The objective of the present study was to develop a method for the cryopreservation of buffalo oocytes by vitrification. Cumulus-oocyte complexes (COCs) were obtained from slaughterhouse ovaries. Prior to vitrification of COCs in the vitrification solution (VS) consisting of 4.5 M ethylene glycol, 3.4 M dimethyl sulfoxide, 5.56 mM glucose, 0.33 mM sodium pyruvate and 0.4% w/v bovine serum albumin in Dulbecco's phosphate buffered saline (DPBS), the COCs were exposed to the equilibration solution (50% VS v/v in DPBS) for 1 or 3 min at room temperature (25 to 30 degrees C). The COCs were then placed in 15-microL of VS and immediately loaded into 0.25-mL French straws, each containing 150 microL of 0.5 M sucrose in DPBS. The straws were placed in liquid nitrogen (LN2) vapor for 2 min, plunged and stored in LN2 for at least 7 d. The straws were thawed in warm water at 28 degrees C for 20 sec. For dilution, the COCs were equilibrated in 0.5 M sucrose in DPBS for 5 min and then washed 4 to 5 times in the washing medium (TCM-199+10% estrus buffalo serum). The proportion of oocytes recovered in a morphologically normal form was significantly higher (98 and 88%, respectively; P<0.05), and the proportion of oocytes recovered in a damaged form was significantly lower (2 and 12%, respectively; P<0.05) for the 3-min equilibration than for 1 min. For examining the in vitro developmental potential of vitrified-warmed oocytes, the oocytes were placed in 50-microL droplets (10 to 15 oocytes per droplet) of maturation medium (TCM-199+15% FBS+5 microg/mL FSH-P), covered with paraffin oil in a 35-mm Petri dish and cultured for 26 h in a CO2 incubator (5% CO2 in air) at 38.5 degrees C. Although the nuclear maturation rate did not differ between the 1- and 3-min equilibration periods (21.5+/-10.7 and 31.5+/-1.5%, respectively), the between-trial variation was very high for the 1-min period. This method of vitrification is simple and rapid, and can be useful for cryopreservation of buffalo oocytes.     

降解烤烟秸秆和烟碱菌株的筛选及其产酶特性

摘要:【目的】为获得能够降解烤烟秸秆和烟碱的菌株,并探索其降解烤烟秸秆的利用途径。【方法】以烤烟秸秆为唯一碳氮源,从烟田土壤中进行了菌株的筛选。采用形态学观察、生理生化特性鉴定、16S rRNA基因序列鉴定等方法对该菌株进行了鉴定,并对其以烤烟秸秆为底物进行液态发酵的产酶活性和木质纤维素降解效果进行了测定。【结果】结果表明:该菌株为巨大芽孢杆菌(Bacillus megaterium)。在以烤烟秸秆为主要营养物质液态发酵条件下该菌株具有较强的木质素降解能力,最大漆酶活力达到418.52 U/L,而木质素过氧化物酶和锰过氧化物酶的最大酶活分别为19.71 U/L 和64.71 U/L。此外,发酵20 d后该菌能够完全降解发酵液中的烟碱。【结论】本研究筛选到了1株能够较好降解烤烟秸秆和完全降解烟碱的巨大芽孢杆菌(Bacillus megaterium),且该菌株具有利用烤烟秸秆生产漆酶的应用价值。     

Human kallikrein 13 involvement in extracellular matrix degradation

The human kallikrein family is a group of 15 serine protease genes clustered on chromosome 19q13.4 and shares a high degree of homology. These proteolytic enzymes have diverse physiological functions in many different tissues. Growing evidence suggests that many kallikreins are differentially expressed in cancer and may play a role in metastasis. Human kallikrein gene 13 (KLK13) is a member of this family and codes for a trypsin-like, secreted serine protease (hK13) that is overexpressed in ovarian cancer patients. The aim of this study was to determine if hK13 can degrade extracellular matrix components. Recombinant hK13 was produced in yeast and purified using cation exchange and reverse-phase chromatography. The protein was used as an immunogen to generate mouse monoclonal antibodies. Enzymatic activity of hK13 was verified by using synthetic tri-peptide fluorogenic substrates and gelatin zymography. Active hK13 was incubated with biotinylated extracellular matrix (ECM) proteins and degradation was evaluated by Western blot analysis. hK13-secreting cancer cell lines were treated in a chemotaxis invasion chamber that was coated with various ECM proteins, to determine if hK13 plays a role in tumor cell migration and invasion. Assay with the synthetic substrates and zymography have shown that recombinant hK13 was enzymatically active. The Western blot results showed that hK13 was able to cleave the major components of the extracellular matrix. In the chemotaxis invasion chamber experiment, it was found that ovarian cancer cell lines that secreted hK13 and were treated with an hK13 neutralizing antibody migrated less than untreated cells. Human kallikrein13 may play a role in tissue remodeling and/or tumor invasion and metastasis. Targeting hK13 activity with neutralizing antibodies may have therapeutic applications.     

Pregnancies established from water buffalo (Bubalus bubalis) blastocysts derived from in vitro matured, in vitro fertilized oocytes and co-cultured with cumulus and oviductal cells

Buffalo ovaries were collected immediately after slaughter and were transported to laboratory in sterile saline at 37 degrees C. Follicular oocytes with the cumulus mass aspirated from 2 to 6 mm in diameter follicles were cultured in TCM-199 medium supplemented with 10% buffalo estrus serum (BES) in 5% CO(2) at 38.5 degrees C. After 20 to 24 h of incubation, the oocytes were inseminated with precapacitated frozen thawed spermatozoa for 6 h. The fertilization rate was 78.15% of the matured oocytes. Over an in vitro culture period of 3 to 9 d, 4.02% of the inseminated oocytes developed to the morula stage when cultured with cumulus cells alone and 17.83% when cumulus cells plus oviductal epithelial cells were used. The percentage of developed blastocysts was very low (0.57%) when the oocytes were co-cultured with cumulus cells from the original oocytes. However, 8% of the inseminated oocytes that were denuded 3 d after insemination developed to the blastocyst stage when they were co-cultured with cumulus and oviductal epithelial cells. Sixteen early/expanded blastocysts were transferred non-surgically to 16 recipients. Four of the 16 recipients became pregnant, of which 2 delivered normal buffalo male calves.