Multiplication of the Recombinant Strain Re-7 of Avian Influenza Virus Subtype H5 in MDCK Cells

This study was conducted to explore the multiplication pattern of the recombinant strain Re-7 of avian influenza virus subtype H5 in Madin Darby Canine Kidney （MDCK） cells and to determine the optimal multiplicity of infection （MOI） and the optimal time for virus harvest. The recombinant strain Re-7 was inoculated at different MOIs into MDCK cells grown in serum-free medium in 100 L bioreactors for replication. Then, the hemagglutination（HA） titer, 50% tissue culture infectious dose （TCID50） and 50% embryo infectious dose （EID50） of culture medium were measured once every 12 h from 24 h after virus inoculation to determine the optimal MOI. After that, virus was inoculated at the optimal MOI determined above into MDCK cells for large-scale virus replication to determine the optimal time for virus harvest. The results showed that the optimal MOI was 10 2, and the optimal time for virus harvest was 60 h after inoculation. Under these conditions, the HA titer, TCIDso per 1 mL and EIDso per 0.1 mL were increased to 1：102 4, 10^7.33 and 10^6.83, respectively. This study provides relatively stable parameters for large-scale production of the recombinant strain Re-7 of avian influenza virus subtype H5.

Effect of Different Culture Media on the Proliferation of Avian Influenza Virus H9 Subtypes in MDCK Cells

[Objective] To screen the best culture media for the proliferation of avian influenza virus （AIV） H9 subtypes in MDCK cells. [Method] The DMEM containing 10% （V/V） newborn calf serum, low-serum containing medium （ MEM-MD-611 ） and serum-free medium （SFE4Mega） were used to culture the MDCK monolayer ceils, which were then inoculated with different dilutions of AIV H9 subtypes, and the 3 kinds of media were al- so used as the maintenance solution to culture the virus. The cytopathic changes were observed at every 24 h, and the HA titers of the culture su- pernatants were also determined. [ Result] After culturing for 72 -96 h, the HA titers of the serum-free media were higher than that of low-serum culture media, while the HA titers were higher in the low-serum media than in the serum containing media. [ Conclusion] The 3 kinds of media can all used for the proliferation of AIV_ but the low-serum culture medium （MEM-MD-611 ） and serum-free medium （SFE4Meaa3 are preferred.

FITC labeling of human insulin and transport of FITC-insulin conjugates through MDCK cell monolayer

Fluorescein isothiocyanate-labeled insulin(FITC-insulin)has been widely used for bioanalytical applications.Due to the high cost of commercial FITC-insulin and tedious labeling procedures described in the literature,there is still a need to develop a cost effective,reliable and quick labeling method for insulin.The purpose of the present work was to develop a quick and affordable method for FITC labeling of human insulin and to determine the effect of different conjugations of FITC to human insulin on its permeability through the MDCK cell monolayer.FITC labeling of insulin gives mono-,di-or tri-conjugates depending on the reaction time and the molar ratio of FITC:insulin.Mono-conjugate with unlabeled insulin,mixture of di-and tri-conjugate,and tri-conjugate with very little amount of di-conjugate were synthesized in less than 4 h.Degree of conjugation had an effect on the permeability of insulin through the MDCK cell monolayer.Mono-conjugate had higher permeability than the unlabeled insulin due to increase in partition coefficient.However,tri-conjugate showed lower permeability than the unlabeled insulin due to the increase in molecular weight.

Mitochondria as the main source of vanadyl acetylacetonate-induced reactive oxygen species generation in renal epithelial cell lines

In this study, we aimed to clarify the source of the reactive oxygen species （ROS） generation induced by vanadium compounds. We used vanadyl acetylacetonate （VO（acac）2）, a highly effective agent in controlling hyperglycemia, to determine the source of ROS generation in two renal cell lines LLC-PK1 and MDCK. Four commonly fluorescent dyes were used to assess VO（acac）2-induced H202 and ＂02 production and their location. It demonstrated that VO（acac）2 can induce significant ROS generation in both LLC-PKI and MDCK cells, which were primarily derived from mitochondria. The results obtained in this study raised the possibility to reduce ROS level induced by vanadium compounds locally and thus avoid affecting its activity.