Assessment of cell engineering strategies for improved therapeutic protein production in CHO cells

Recombinant glycoprotein therapeutics have proven to be invaluable pharmaceuticals for the treatment of various diseases. Chinese hamster ovary (CHO) cells are widely used in industry for the production of these proteins. Several strategies for engineering CHO cells for improved protein production have been tried with considerable results. The focus has mainly been to increase the specific productivity and to extend the culture longevity by preventing programmed cell death. These CHO cell engineering strategies, particularly those developed in Korea, are reviewed here.     

Effects of Temperature on Photosynthetic Rates in Korean Fir （Abies koreana） between Healthy and Dieback Population

The present study was carried out on natural Korean fir forests (Abies koreana) growing In Mount Halla in Jeju Island, Korea (33°13-36′ N and 126° 12-57′ E). Mount Halla is the highest mountain (1950 m a.s.l.) in South Korea. On the Korean fir forests near the top of Mount Halla in Korea, we established permanent plots between dieback and healthy population. Each permanent plot includes both dieback and relatively healthy Korean fir individuals. Three sites in this study showed similar altitude, topographic position, aspects, slope, diameter at breast height, average height and ages. Net photosynthetic rates (PN) on different temperature regimes were evaluated to explain the forest dieback phenomenon on Korean fir populations. Light response curves were determined on three different temperature regimes: 15℃, 20℃and 25℃. The Irradiance response curve showed higher values in lower air temperatures. Generally, Irradiance response curves of healthy Korean fir populations were higher than the dieback population at all sites.     

Leaf traits and their interrelationship of 23 plant species in southeast of Keerqin Sandy Lands, China

Six leaf traits, i.e., fresh mass (FM), dry mass (DM), leaf dry matter content (DMC), area (AR), specific leaf area (SLA) and thickness (TH) from 23 plant species in the southeastern Keerqin Sandy Lands, China were measured. The results show that leaf traits of herbs were more diversified than those of shrubs and trees and average SLA tended towards a decreasing trend from herbs to shrubs to trees. On the contrary, DMC and DM show an upward trend from herbs to shrubs to trees. No apparent difference was found in TH. Except forDMand TH, there were significant variations in SLA and DMC among three different growth forms. Moreover, a significant correlation was found between SLA and DMC. It is concluded that SLA and DMC could be used to predict species position along a resource use gradient. __________ Translated from Chinese Journal of Ecology, 2006, 25: 921–925 [译自: 生态学杂志]     

Genes Encoding the N-Acyl Homoserine Lactone-Degrading Enzyme Are Widespread in Many Subspecies of Bacillus thuringiensis

Gram-negative bacteria can communicate with each other by N-acyl homoserine lactones (AHLs), which are quorum-sensing autoinducers. Recently, the aiiA gene (encoding an enzyme catalyzing the degradation of AHL) has been cloned from Bacillus sp. strain 240B1. During investigations in the course of the ongoing Bacillus thuringiensis subsp. morrisoni genome project, an aiiA homologue gene in the genome sequence was found. These results led to consideration of the possibility of the widespread existence of the gene in B. thuringiensis. aiiA homologue genes were found in 16 subspecies of B. thuringiensis, and their sequences were determined. Comparison of the Bacillus sp. strain 240B1 aiiA gene with the B. thuringiensis aiiA homologue genes showed high homologies of 89 to 95% and 90 to 96% in the nucleotide sequence and deduced amino acid sequence, respectively. Among the subspecies of B. thuringiensis having an aiiA gene, the subspecies aizawai, galleriae, kurstaki, kyushuensis, ostriniae, and subtoxicus were shown to degrade AHL. It was observed that recombinant Escherichia coli producing AiiA proteins also had AHL-degrading activity and could also attenuate the plant pathogenicity of Erwinia carotovora. These results indicate that insecticidal B. thuringiensis strains might have potential to compete with gram-negative bacteria in natural ecosystems by autoinducer-degrading activity.     

Continuous cultivation ofLactobacillus rhamnosus with cell recycling using an acoustic cell settler

Continuous production of lactic acid from glucose byLactobacillus rhamnosus with cell recycling using an acoustic cell settler was carried out. The performance of the system, such as the concentration of cell and product were compared with the control experiment without recycling. The acoustic settler showed cell separation efficiency of 67% during the continuous operation and the cell concentration in the fermentor with recycle exceeded that of the control by 29%. Compared with the control, lactic acid production was increased by 40%, while glucose consumption was only increased by 8%. The higher value of lactic acid production to substrate consumption (Y _P/S, product yield coefficient) achieved by cell recycling is interpreted to indicate that the recycled cell mass consumes less substrate to produce the same amount of product than the control. Within system environmental changes due to the longer mean cell residence time induced the cells maintaining the metabolic pathways to produce less by-product but more product, lactic acid.     

Ischemia-related changes in galanin expression in the dentate hilar region after transient forebrain ischemia in gerbils.

Although galanin (GAL) protects hippocampal neurons from ischemic damage, no study has examined ischemia-related changes in endogenous GAL in the hippocampal dentate gyrus. We investigated the chronological changes of GAL, well-known as the potassium channel opener, expression in the dentate gyrus at various times after 5 min of transient forebrain ischemia in gerbils. A few GAL-immunoreactive (IR) neurons were found in the polymorphic layer of the sham-operated group. Three hours after ischemia-reperfusion, the pattern of GAL immunoreactivity was similar to that of the sham-operated group and the number of GAL-IR neurons and immunoreactivity were highest 12 h after ischemic insult. At this time, GAL-IR neurons in the polymorphic layer showed strong GAL immunoreactivity. Thereafter, GAL-IR neurons and immunoreactivity significantly decreased in the dentate hilar region. Four days after ischemic insult, GAL-IR neurons were not detectable. In addition, the results of a Western blot study showed a pattern of GAL expression similar to the immunohistochemical changes. GAL protein content also was highest 12 h after ischemia. In conclusion, the increased expression of endogenous GAL in the dentate gyrus after ischemia is related to response to the ischemic damage.     

Generation of cloned transgenic cats expressing red fluorescence protein

A method for engineering and producing genetically modified cats is important for generating biomedical models of human diseases. Here we describe the use of somatic cell nuclear transfer to produce cloned transgenic cats that systemically express red fluorescent protein. Immature oocytes were collected from superovulating cat ovaries. Donor fibroblasts were obtained from an ear skin biopsy of a white male Turkish Angora cat, cultured for one to two passages, and subjected to transduction with a retrovirus vector designed to transfer and express the red fluorescent protein (RFP) gene. A total of 176 RFP cloned embryos were transferred into 11 surrogate mothers (mean = 16 +/- 7.5 per recipient). Three surrogate mothers were successfully impregnated (27.3%) and delivered two liveborn and one stillborn kitten at 65 to 66 days of gestation. Analysis of nine feline-specific microsatellite loci confirmed that the cloned cats were genetically identical to the donor cat. Presence of the RFP gene in the transgenic cat genome was confirmed by PCR and Southern blot analyses. Whole-body red fluorescence was detected 60 days after birth in the liveborn transgenic (TG) cat but not in the surrogate mother cat. Red fluorescence was detected in tissue samples, including hair, muscle, brain, heart, liver, kidney, spleen, bronchus, lung, stomach, intestine, tongue, and even excrement of the stillborn TG cat. These results suggest that this nuclear transfer procedure using genetically modified somatic cells could be useful for the efficient production of transgenic cats.     

Lipase-catalyzed synthesis of fatty acid sugar ester using extremely supersaturated sugar solution in ionic liquids

Artificial nanotransport systems inspired by intracellular transport processes have been investigated for over a decade using the motor protein kinesin and microtubules. However, only unidirectional cargo transport has been achieved for the purpose of nanotransport in a microfluidic system. Here, we demonstrate bidirectional nanotransport by integrating kinesin and dynein motor proteins. Our molecular system allows microtubule orientation of either polarity in a microfluidic channel to construct a transport track. Each motor protein acts as a nanoactuators that transports microspheres in opposite directions determined by the polarity of the oriented microtubules: kinesin-coated microspheres move toward the plus end of microtubules, whereas dynein-coated microspheres move toward the minus end. We demonstrate both unidirectional and bidirectional transport using kinesin- and dynein-coated microspheres on microtubules oriented and glutaraldehyde-immobilized in a microfluidic channel. Tracking and statistical analysis of microsphere movement demonstrate that 87-98% of microspheres move in the designated direction at a mean velocity of 0.22-0.28 microm/s for kinesin-coated microspheres and 0.34-0.39 microm/s for dynein-coated microspheres. This bidirectional nanotransport goes beyond conventional unidirectional transport to achieve more complex artificial nanotransport in vitro.     

Size-dependent scattering efficiency in dye-sensitized solar cell

Effect of scattering particle size on light scattering efficiency in dye-sensitized solar cell has been investigated with a FTO/semitransparent nano-TiO₂ layer (main-layer)/scattering layer (overlayer) structure, where two different rutile TiO₂ particles of 0.3 μm (G1) and 0.5 μm (G2) were used for a scattering overlayer and 20-nm anatase particle for nano-TiO₂ main-layer. The conversion efficiency of 7.55% for the 7 μm-thick main-layer film is improved to 8.94% and 8.78% when G1 and G2 particulate overlayers are introduced, respectively, corresponding to 18.4% and 16.3% increments. While the conversion efficiency of the 14 μm-thick main-layer is slightly improved from 8.60% to 9.09% and 9.15% upon depositing G1 and G2 particulate overlayers, respectively. Significant improvement and strong size-dependence upon deposition of scattering overlayer on the relatively thinner TiO₂ main-layer film are associated with the quantity and wavelength of transmitted light and the difference in reflectivity of G1 and G2 scattering particles.