~（60）Co－γ射线诱变柠檬酸产生菌黑曲霉Co9－6的研究

本试验采用￣（６０）Ｃｏ－γ射线对柠檬酸产生菌黑曲霉Ｃｏ９－６进行辐射,经两次处理后选育出Ｌ１２１７和Ｌ８０１两株优良柠檬酸产生菌。中试结果表明菌株Ｌ１２１７较Ｌ８０１更优：产酸率较菌株Ｃｏ９－６提高１７．６％、发酵周期缩短１３．４％、对糖转化率提高１３．３％。     

Cloning, expression and characterization of a UDP-galactose 4-epimerase from Escherichia coli

The gene galE encoding UDP-galactose 4-epimerase was cloned into E. coli BL21(DE3) from the chromosomal DNA of E. coli strain K-12. High expression of the soluble recombinant epimerase was achieved in the cell lysate. In order to evaluate the use of this epimerase in enzymatic synthesis of important -Gal epitopes (oligosaccharides with a terminal Gal1,3Gal sequence), a new radioactivity assay (1,3-galactosyltransferase coupled assay) was established to characterize its activity in producing UDP-galactose from UDP-glucose. Approximately 2700 units (100 mg) enzyme with a specific activity of 27 U mg^–1 protein could be obtained from one liter of bacterial culture. The epimerase was active in a wide pH range with an optimum at pH 7.0. This expression system established a viable route to the enzymatic production of -Gal oligosaccharides to support xenotransplantation research.     

Optimal conditions for hepatitis B core antigen production in shaked flask fermentation

The effects of various environmental factors such as pH (5, 6, 7, 8 and 9), temperature (30, 37 and 40°C) and rotational speed (150, 200 and 250 rpm) on the growth and the hepatitis B core antigen (HBcAg) production ofEscherichia coli W3110IQ were examined in the present study. The highest growth rate is achieved at PH 7, 37°C and at a rotational speed of 250 rpm which is 0.927 h⁻¹. The effect of pH on cell growth is more substantial compared to other parameters; it recorded a 123% different between the highest growth rate (0.927 h⁻¹) at pH 7 and lowest growth at pH 5. The highest protein yield is achieved at pH 9, rotational speed of 250 rpm and 40°C. The yield of protein at pH 7 is 154% higher compared to the lowest yield achieved at pH 5. There is about 28% different of the protein yield for theE. coli cultivated at 250 rpm compared to that at 150 rpm which has the lowest HBcAg yield. The yield of protein at 40°C is 38% higher compared to the lowest yield achieved, at 30°C.     

Fine-Scale Population Genetic Structure of Zhikong Scallop (Chlamys farreri): Do Local Marine Currents Drive Geographical Differentiation?

Marine scallops, with extended planktonic larval stages which can potentially disperse over large distances when advected by marine currents, are expected to possess low geographical differentiation. However, the sessile lifestyle as adult tends to form discrete "sea beds" with unique population dynamics and structure. The narrow distribution of Zhikong scallop (Chlamys farreri), its long planktonic larval stage, and the extremely hydrographic complexity in its distribution range provide an interesting case to elucidate the impact of marine currents on geographical differentiation for marine bivalves at a fine geographical scale. In this study, we analyzed genetic variation at nine microsatellite DNA loci in six locations throughout the distribution of Zhikong scallop in the Northern China. Very high genetic diversity was present in all six populations. Two populations sampled from the same marine gyre had no detectable genetic differentiation (F (ST) = 0.0013); however, the remaining four populations collected from different marine gyres or separated by strong marine currents showed low but significant genetic differentiation (F (ST) range 0.0184-0.0602). Genetic differentiation was further analyzed using the Monmonier algorithm to identify genetic barriers and using the assignment test conducted by software GeneClass2 to ascertain population membership of individuals. The genetic barriers fitting the orientation of marine gyres/currents were clearly identified, and the individual assignment analysis indicated that 95.6% of specimens were correctly allocated to one of the six populations sampled. The results support the hypothesis that significant population structure is present in Zhikong scallop at a fine geographical scale, and marine currents can be responsible for the genetic differentiation.     

Constitutive expression of human angiostatin in <Emphasis Type="Italic">Pichia pastoris</Emphasis> by high-density cell culture

A high-density cell culture method to produce human angiostatin has been successfully established by constitutive expression of the protein in Pichia pastoris. The fermentation was carried out in a 20 l bioreactor with a 10 l working volume, using a high-density cell culture method by continuously feeding with 50% glycerol−0.8% PTM4 to the growing culture for 60 h at 30°C. Dissolved oxygen level was maintained at 25–30% and pH was controlled at 5 by the addition of 7 M NH₄OH. Angiostatin was constitutively expressed during the fermentation by linking its expression to the P. pastoris constitutive GAP promoter (pGAP). But after 36 h of fermentation, the peak biomass growth was 305 as measured by absorption of 600 nm, while the peak angiostatin expression was 176 mg/l. Similar to the product expressed from inducible system [24], angiostatin produced from constitutive system also inhibited the angiogenesis on the CAM and suppressed the growth of B16 melanoma in C57BL/6J mouse. The above results suggest that GAP promoter is more efficient than AOX1 promoter for the expression of angiostatin in P. pastoris by shake flask culture or high-density cell fermentation and is likely to be an alternative to AOX1 promoter in large-scale expression of angiostatin and other heterologous proteins. Supported by the Natural Science Foundation of China (39670013) and “225” Science and Technology Program of Guangzhou Municipal Government of China (99-Z-004-001).     

Micropropagation of <Emphasis Type="Italic">Penthorum chinense</Emphasis> through axillary bud

This study reports a protocol for successful micropropagation of Penthorum chinense using nodal explants on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA) or kinetin (Kn). The presence of BA promoted a higher rate of shoot multiplication than Kn. Maximum multiple shoot formation was observed in 59.2% of nodal explants cultured on MS medium supplemented with 2.0 mg l⁻¹ BA after 6 wk. After subculture for 4 wk, the maximum number of shoots (6.4) was obtained on a medium with 2.0 mg l⁻¹ BA, but shoots were too short and not suitable for micropropagation. The taller shoots that regenerated in the presence of lower BA concentration (1.0 mg l⁻¹) were selected for root induction study. Most shoots (98.8%) rooted in the presence of 0.5 mg l⁻¹ indole-3-acetic acid after 3 wk, with each shoot forming an average of 10.0 roots. Plantlets were transferred to soil and successfully acclimatized.     

A study on floral biology of seedlings in vitro in Orychophragmus violaceus: Induction of flowers in seedlings of O. violaceus cultured in vitro

Seedlings of O. violaceus were cultured on MS media and treated at low temperature. Cold treatment at 5–7 °C for more than 7 days was needed for flower induction of seedlings in vitro originated from germinated seeds. When cultured on MS media supplemented with 2.5 mg l⁻¹ zeatin and 2 mg l⁻¹ gibberellin (GA₃), seedlings in vitro did initiate flowers without cold treatment. When MS media was used with a reduced amount of NH₄NO₃, flower induction of seedlings in vitro could be accelerated. This revised version was published online in June 2006 with corrections to the Cover Date.     

The WD40 Domain Is Required for LRRK2 Neurotoxicity

Background

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson disease (PD). LRRK2 contains an “enzymatic core” composed of GTPase and kinase domains that is flanked by leucine-rich repeat (LRR) and WD40 protein-protein interaction domains. While kinase activity and GTP-binding have both been implicated in LRRK2 neurotoxicity, the potential role of other LRRK2 domains has not been as extensively explored.

Principal Findings

We demonstrate that LRRK2 normally exists in a dimeric complex, and that removing the WD40 domain prevents complex formation and autophosphorylation. Moreover, loss of the WD40 domain completely blocks the neurotoxicity of multiple LRRK2 PD mutations.

Conclusion

These findings suggest that LRRK2 dimerization and autophosphorylation may be required for the neurotoxicity of LRRK2 PD mutations and highlight a potential role for the WD40 domain in the mechanism of LRRK2-mediated cell death.