Evaluation of different culture conditions for high-level soluble expression of human cyclin A(2) with pET vector in BL21 (DE3) and spectroscopic characterization of its inclusion body structure

In this paper, we evaluated various parameters of culture condition affecting high-level soluble expression of human cyclin A, in Escherichia coli BL21(DE3), and demonstrated that the highest protein yield was obtained using TB(no glycerol) + 0.5% glucose medium at 25 degrees C. By single immobilized metal ion affinity chromatography, we got highly purified human cyclin A(2) with a yield ranged from 20 to 30 mg/L. By amyloid-diagnostic dye ThT binding and Fourier transform infrared spectroscopy, we observed a significant decrease in alpha-helix content and an increase in beta-sheet structure in cyclin A(2) inclusion body in comparison to its native protein, and confirmed the resemblance of the internal organization of cyclin A(2) inclusion body and amyloid fibrils.

Cultivation of the brown alga Sargassum horneri: sexual reproduction and seedling production in tank culture under reduced solar irradiance in ambient temperature

As a large conspicuous intertidal brown alga, individuals of Sargassum horneri can reach a length of more than 7 m with a fresh weight of 3 kg along the coasts of the Eastern China Sea. The biomass of this alga as a vital component in coastal water ecology has been well documented. In recent years, a steady disappearance of the algal biomass along the once densely populated coastal areas of the Eastern China Sea has drawn attention in China. Efforts have been made to reconstruct the subtidal algal flora or even to grow the alga by use of long-lines. As part of the efforts to establish an efficient technique for producing seedlings of S. horneri, in this investigation a series of culture experiments were carried out in indoor raceway and rectangular tanks under reduced solar irradiance at ambient temperature in 2007-2008. The investigation demonstrated that: (1) sexual reproduction of S. horneri could be accelerated in elevated temperature and light climates, at least 3 months earlier than in the wild; (2) eggs of S. horneri had the potential to be fertilized up to 48 h, much longer than that of known related species; (3) suspension and fixed culture methods were both effective in growing the seedlings to the long-line cultivation stage; and (4) the life cycle of S. horneri in culture could be shortened to 4.5 months, thus establishing this alga as an appropriate model for investigating sexual reproduction in dieocious species of this genus.

Suspension culture of gametophytes of transgenic kelp in a photobioreactor

Transgenic Laminaria japonica gametophytes producing a recombinant tissue-type plasminogen activator (rtPA) protein, which is an effective third-generation thrombolytic agent for acute myocardial infarction (AMI), were cultured in an illuminated bubble column bioreactor. A maximum final dry cell weight of 1120 mg l(-1) was obtained in batch culture with an initial dry cell weight of 126 mg l(-1) and with aeration rate of 1.2 l air min(-1) l(-1) culture, nitrate at 1.5 mM and phosphate at 0.17 mM. The yield of rtPA was 56 mu g g(-1) dry cell wt. This is the first report regarding cultivation of a transgenic macroalga in a bioreactor.

Cultivation of the brown alga Hizikia fusiformis (Harvey) Okamura: enhanced seedling production in tumbled culture

The reuse of holdfasts for regeneration of young seedlings or using wild juvenile plants as the seedling source has played the major role in commercial cultivation of the brown alga Hizikia fusiformis in East Asia over the past 20 years. The possibility of employing zygote-derived germlings for producing seedlings has been discussed in the literature, but has not yet become a reality. Three main obstacles have limited the use of zygotes as a main source of seedlings, (1) the dioecious nature of the algal life cycle which may lead to asynchronous male and female receptacle development and thus different timing of egg and spermatozoa expulsion, (2) the low attachment rate when using zygote-derived germlings with developed rhizoids from wild parental plants for seeding production, and (3) the problem of culturing young germlings in regions where water temperature is high in summer. In this investigation, shifting the timing of receptacle formation earlier than in nature was performed by tumbling the algae in a long-day tank (16-h light per day). Synchronization of egg and spermatozoa expulsion and thereafter fertilization were conducted in indoor tanks. Receptacle formation in constant long days could be shifted by 20 days earlier than in plants cultured on long lines in the open sea, or I month earlier than in plants growing on intertidal rocks. Synchronized expulsion of eggs and spermatozoon led to a high rate of fertilization. This was achieved by tumbling the male and female receptacle-bearing branchlets in the same tank at low density in high irradiance. In two independent trials, a total of 1,400,000 zygote-derived germlings were obtained from 620 g (fresh weight) female sporophytes. The germlings shed from the receptacles were at an identical developmental stage indicating high synchronization of expulsion of eggs and spermatozoon followed by fertilization. Approximately 63% ( +/-9.6%) of the germlings were shed from the receptacle between 16 and 24 It after fertilization and 20% ( +/-11.9%) remained on the receptacle for 3 days after fertilization. Germlings were seeded on string collectors before rhizoids started to elongate and the attachment efficiency was enhanced. Young seedlings reached 800 ( +/-50) mum in length in 25 days at 25 degreesC before they were transferred to open sea cultivation. These results provide the basis of a practical way of seedling production by use of zygote-derived germlings in the commercial cultivation of Hizikia fusiformis. (C) 2004 Elsevier B.V All rights reserved.

New life cycles of Porphyra katadae var. hemiphylla in culture

Porphyra katadae Miura var. hemiphylla Tseng et T. J. Chang, a species distributed around the Liaodong and Shandong Peninsulas of China, produces gametophytes from late winter to early spring. These are monoecious with male and female reproductive tissues in distinct halves or sectors. Vegetative tissues from sectors expected to differentiate into sexual tissue were cultured in the laboratory. Male and female reproductive organs, as well as conchocelis and blades, were differentiated from these tissues. The male and female reproductive tissues were in patches and mixed on the cultured tissue pieces. This was quite different from the wild-type sectored individuals. The F-1 conchospore germlings also produced monospores, carposporangia, spermatangia and conchocelis. These carposporangia and spermatangia were in patches and were mixed on the F-1 fronds. The results imply that P. katadae var. hemiphylla is possibly sex-differentiated rather than sex-determined. This is the first report of such a dimorphic life history in the genus Porphyra.

Mass culture of Undaria gametophyte clones and their use in sporeling culture

Undaria pinnatifida (Harv.) Sur. is one of the three main seaweed species under commercial cultivation in China. In the mid-1990s the annual production was about 20000 tons dry. The supply of healthy sporelings is key to the success of commercial cultivation of Undaria. Previous studies demonstrated that instead of the zoospore collection method, sporelings can be cultured through the use of gametophyte clones. This paper reports the experimental results on mass culture of clones and sporeling raising in commercial scale. Light had an obvious effect on growth of gametophyte clones. Under an irradiance of 80 mumol m(-2) s(-1) and favorable temperature of 22-25degreesC, mean daily growth rate may reach as high as 37%. Several celled gametophyte fragments were sprayed onto the palm rope frame. Gametogenesis occurred after 4-6 days. Juvenile sporeling growth experiments showed that nitrate and phosphate concentrations of 2.9 10(-4) mol 1(-1) and 1.7 10(-5) mol 1(-1) were sufficient to enable the sporelings to maintain a high daily growth rate. Sporelings can reach a length of 1 cm in a month. Since 1997, extension of the clone technique has been carried out in Shandong Province. Large-scale production of sporelings for commercial cultivation of 14 and 31 hectares in 1997 and 1998 had been conducted successfully.

Primary culture and characteristic morphologies of medulla terminalis neurons in the eyestalks of Chinese shrimp, Fenneropenaeus chinensis

A method for culturing medulla terminalis (MT) neurons in the eyestalk of Chinese shrimp, Fenneropenaeus chinensis, was first established. The neurons showed immediate outgrowth in the culture medium supplemented with glutamine, glucose and antibiotics. The cells grew for about 2-7 days and then sustained for a week or more. At least six types of neurons were distinguished on the basis of size and form of soma and outgrowth pattern of cells. (C) 2003 Elsevier Science B.V. All rights reserved.