Enhanced production of (+)-terrein by Aspergillus terreus strain PF26 with epigenetic modifier suberoylanilide hydroxamic acid

New strategies for improving the fermentation yield of (+)-terrein which is a fungal metabolite with multiple bioactivities are very urgent. In this study, the effect of suberoylanilide hydroxamic acid, one kind of epigenetic modifier, on the biosynthesis of (+)-terrein by Aspergillus terreus strain PF26 isolated from the marine sponge Phakellia fusca was investigated. It was found that suberoylanilide hydroxamic acid exhibited a positive impact on (+)-terrein production, resulting from promoting the biosynthesis of 6-hydroxymellein, the precursor of (+)-terrein. Through optimization of feeding concentration and time of suberoylanilide hydroxamic acid, 5.58 g/L (+)-terrein could be obtained in shake flask cultivation, 29.5% higher than the control. Correspondingly, the fermentation of A. terreus strain PF26 in 7.5-L stirred bioreactor with feeding suberoylanilide hydroxamic acid (900 μM, day 4) yielded 9.07 g/L (+)-terrein, 77.1% higher than the control. These results showed that the epigenetic modifier-suberoylanilide hydroxamic acid could be utilized to enhance the production of (+)-terrein, which laid the foundation of massive production of (+)-terrein by fermentation.     

Influences of sex,group size,and spatial position on vigilance behavior of Przewalski’s gazelles

Group-living animals may need to spend less time being vigilant, consequently, having more time for other important activities such as foraging (i.e., group size effect). Przewalski’s gazelle (Procapra przewalskii) is a group-living social animal, and a study was conducted in Qinghai Province of China during June–August 2006 by using a continuous focal sampling method to investigate the influences of group size, sex, within-group spatial position, and nearest-neighbor distance on individual vigilance level (defined as scanning frequency per minute). Male gazelles were more vigilant than females. The gazelle’s vigilance level decreased with group size (group size effect), but only for females. The individuals at the central positions within a group were less vigilant than those at the peripheral positions, but the nearest-neighbor distance did not have any significant influence on the individual vigilance level. Our results support the hypotheses of group size effect and edge effects, but the sexual difference in vigilance level and in the response to group size effect on vigilance suggests that there may be sexual difference in the function and targets of vigilance behavior of Przewalski’s gazelles, which warrants more investigation, with incorporation of within-group spatial position, to better understand the mechanism underlying the group size effect and edge effect.     

绵羊胞内单精子注射技术

In this study, the possibility of sheep transgenesis by intracytoplasmic sperm injection (ICSI) was assessed. In experiment 1, activation of ovine oocytes matured in vitro in preparation for ICSI has been investigated with 3.42 mmol/L Ca2+ treatment, ionomycin alone and ionomycin followed by 6-dimethylaminopurine (DMAP) after 3-h delay (group 1, 2 and 3, respectively). After activation, the oocytes were then cultured in SOFaaBSA medium. Cleavage rates were significantly (P<0.05) different among three groups (18.4%, 91.8% and 71.7%, respectively). In additional culture, no parthenotes in group 1, whereas 11% and 17.4% in group 2 and 3 developed to the blastocyst stage. Therefore we used the third activation method in the following ICSI tests. In experiment 2, development of ovine oocytes after ICSI was investigated. Thawed semen from two rams was separated by Percoll centrifugation and was used for ICSI or in vitro fertilization (IVF) trails. A total of 71.8% of oocytes reached the 2-cell stage following living sperm injection, which was significantly (p>0.05) different from those following IVF (41.4%) and sham-ICSI (30.2%). After seven days' culture, no sham-injected oocytes developed into the blastocyst stage, although 7% in ICSI and 16.1% in IVF-oocytes developed into the blastocyst stage, but there was no significant difference in ICSI and IVF groups (p>0.05). In the further study, the possibility of sheep transgenesis by ICSI was assessed. After coinjection of ovine oocytes matured in vitro with dead sperm cold to -20 degrees C and exogenous DNA encoding green fluorescent protein (GFP), seventy-three percent of coinjected oocytes developed to 2-cell stage (33/45) and two of them were transgene-expressing embryos. Among ten embryos at the 16-cell stage, all embryonic cells in one transgenic embryo still expressed GFP. Four coinjected blastocysts were thawed and transferred to the uterine of the two progesterone-synchronized recipient ewe. No pregnancies were detected on the 60th day. These results suggested sheep transgenic embryos could be produced by ICSI and further studies should be performed.     

Linear correlation between thermal stability and folding kinetics of lysozyme

We have studied the refolding and thermal denaturation of hen egg white lysozyme in a wide range of pH values (from 1.5 to 9.4) using stopped-flow circular dichroism (CD) and differential scanning calorimetry (DSC). A linear correlation was found between the thermal denaturation temperature (T(m)) and the logarithm of the refolding rate of the slow folding phase of hen egg white lysozyme (lnk(2)).     

Design and synthesis of new piperidone grafted acetylcholinesterase inhibitors

Alzheimer’s disease (AD) is a neurodegenerative disorder affecting 35 million people worldwide. A common strategy to improve the well-being of AD patients consists on the inhibition of acetylcholinesterase with the concomitant increase of the neurotransmitter acetylcholine at cholinergic synapses. Two series of unreported N-benzylpiperidines 5(a–h) and thiazolopyrimidines 9(a–q) molecules were synthesized and evaluated in vitro for their acetylcholinesterase (AChE) inhibitory activities. Among the newly synthesized compounds, 5h, 9h, 9j, and 9p displayed higher AChE enzyme inhibitory activities than the standard drug, galantamine, with IC₅₀ values of 0.83, 0.98, and 0.73 μM, respectively. Cytotoxicity studies of 5h, 9h, 9j, 9n and 9p on human neuroblastoma cells SH-SY5Y, showed no toxicity up to 40 μM concentration. Molecular docking simulations of the active compounds 5h and 9p disclosed the crucial role of π-π-stacking in their binding interaction to the active site AChE enzyme. The presented compounds have potential as AChE inhibitors and potential AD drugs.     

Differential antibacterial activity of nitric oxide from the immunological isozyme of nitric oxide synthase transduced into endothelial cells.

Primary cultures of endothelial cells, grown on the three-dimensional matrix Gelfoam where they take on the morphology of these cells in vivo, were found to phagocytose Staphylococcus aureus and two strains of Escherichia coli. The phagocytosis was independent of opsonization, although once opsonized, these bacteria were phagocytosed by endothelial cells. As cytochalsin D inhibited the internationalization of S. aureus and E. coli, the phagocytosis by endothelial cells appears to be actin-dependent. Transducing the gene for nitric oxide synthase (NOS) II into endothelial cells allowed us to determine the importance of NO(*) in host immunity against these bacteria. While the growth of S. aureus was impeded by NOS II endothelial cells, two strains of E. coli were killed by an NO(*)-dependent pathway. We conclude that endothelial cells have microbicidal mechanisms that are selective for the type of pathogen encountered.