Serial propagation of mammalian cells on gelatin-coated microcarriers

The ability to serially propagate mammalian cells in microcarrier cultures is essential for large-scale operation. The success of such serial propagation depends on viable dissociation of cells from microcarriers and the normal growth and product formation after subsequent reinoculation. The high pH treatment developed for dissociating cells from DEAE-derivatized microcarriers was not as effective for a number of cell strains cultivated on gelatin-coated microcarriers. By prewashing the cell-laden microcarriers with buffer containing a chelating agent, bovine kidney cells, BK, human embryonic foreskin fibroblasts, FS-4, and continuous human kidney cells, TCL-598 which produces prourokinase, were viably dissociated from commercially available gelatin-coated microcarriers, Cytodex-3. Cells dissociated from microcarriers reattached and grew on micro-carriers subsequent to inoculation into subcultures. However, after subculturing, cells may attach at different rates to newly added beads and to conditioned microcarriers which cells had previously grown. It resulted in an uneven cell distribution on microcarriers and inferior growth kinetics. This effect was more profound for BK and FS-4 cells which are propagated with a low multiplication ratio. Specifically, BK cells attach to conditioned beads at a faster rate than to new beads, while FS-4 cells attach to new beads faster than to conditioned beads. Thus, for these two cell strains, a separator was used to separate the microcarriers from the suspension of dissociated cells before subsequent inoculation. For TCL-598 cells, which are propagated at a high multiplication ratio, this dissociation technique can be applied directly without the separation of dissociated cells and conditioned microcarriers. All the three cell lines tested exhibit normal growth kinetics in serial propagation on microcarriers. Furthermore, the production of prourokinase by TCL598 cells serially propagated on microcarriers was comparable to that inoculated from roller bottles.     

Seasonal Variations in Rubber Biosynthesis, 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase,and Rubber Transferase Activities in Parthenium argentatum in the Chihuahuan Desert

The rubber content and the activities of enzymes in the polyisoprenoid pathway in Parthenium argentatum (guayule) were examined throughout the growing season in field plots in the Chihuahuan Desert. The rubber content of the plants was low in July and August and slowly increased until October. From October to December there was a rapid increase in rubber formation (per plant) from 589.0 mg to 4438.0 mg. The percentage of rubber in the plants increased from 0.7% (mg/g dry weight) in August and 1.27% in October to 5.5% in December. The rapid increase in rubber formation may result from exposing the plants to low temperatures of 5 to 7[deg]C. The activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) was 21.1 nmol mevalonic acid (MVA) h-1 g-1 fresh weight in the bark of the lower stems in June during seedling growth and decreased to 5.1 nmol MVA h-1g-1 fresh weight in July and 2.9 nmol MVA h-1 g-1 fresh weight in September. From October to December, the activity increased from 5.0 to 29.9 nmol MVA h-1 g-1 fresh weight. The activity of rubber transferase was 65.5 nmol isopentenyl pyrophosphate (IPP) h-1 g-1fresh weight in the bark in September and increased to 357.5 nmol IPP h-1 g-1 fresh weight in December. The rapid increase in the activities of HMGR and rubber transferase coincided with the rapid increase in rubber formation. The activities of MVA kinase and IPP isomerase did not significantly increase in the fall and winter. A tomato HMGR-1 cDNA probe containing a highly conserved C-terminal region of HMGR genes hybridized at low stringency with several bands on blots of HindIII-digested genomic DNA from guayule. In northern blots with the HMGR-1 cDNA probe at low stringency, HMGR mRNA was high in June and November, corresponding to periods of high HMGR activity during seedling growth and rapid increase in rubber formation. The seasonal variations in rubber formation and HMGR mRNA, HMGR activity, and rubber transferase activity may be due to low temperature stimulation in the fall and winter months.     

海洋药物的抗病毒研究

海洋由于其特殊的生态环境,包含着极丰富的生物来源的天然产物。自本世纪七十年代以来,已经从海藻类、海绵类、海鞘类、海星类、腹足动物、棘皮动物、腔肠动物、软体动物、珊瑚及海洋微生物等海洋生物中分离出一系列有抗病毒作用的天然化合物,其中有些结构类型已成为抗病毒药物研究的导向化合物。基本现代分离和分析技术的发展、新的实验模型的建立和在病毒学方面分子生物学研究的进展,从海洋生物中寻找新的抗病毒药物已步入一个     

The nuclear bodies formed by histone demethylase KDM7A

Dear Editor, In the nucleus of higher eukaryotes, chromatin occupies only a small proportion of the nuclear space, while many proteins and RNAs segregate into membrane-less nuclear bodies (NBs).These NBs follow a stochastic or ordered assembly model and constantly exchange components with the surrounding nucleoplasm (Jain et al., 2016).Typical NBs include nucleoli, nuclear speckles, paraspeckles, PML bodies, Cajal bodies, polycomb bodies and Sam68 bodies,which play critical roles in various biological processes such as ribosome assembly, RNA processing, and protein modification.The dysfunction of nuclear bodies may cause diseases, such as cancer (Li et al., 2019).     

Molecular deconvolution of the neutralizing antibodies induced by an inactivated SARS-CoV-2 virus vaccine

Dear Editor, The rapid emergence and persistence of the pandemic caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) has had enormous impacts on global health and the economy.Effective vaccines against SARS-CoV-2 are urgently needed to control the coronavirus disease 2019(COVID-19) pandemic,and multiple vaccines have been found to be efficacious in preventing symptomatic COVID-19(Polack et al.,2020;Wu et al.,2020;Jones and Roy,2021).We have developed a traditional beta-propiolactone-inacti-vated aluminum hydroxide-adjuvanted whole-virion SARS-CoV-2 vaccine (BBIBP-CorV),which elicited protective immune responses in clinical trials (Wang et al.,2020;Xia et al.,2021).The vaccine has been granted conditional approvals or emergency use authorizations (EUAs) in China and other countries.     

One-step electrochemically co-assembled redox-active [Ru(bpy)(2)(tatp)](2+)-BSA-SWCNTs hybrid film for non-redox protein biosensors

A redox-active [Ru(bpy)(2)(tatp)](2+)-BSA-SWCNTs (bpy=2,2'-bipyridine, tatp=1,4,8,9-tetra-aza-triphenylene, BSA=bovine serum albumin, SWCNTs=single-walled carbon nanotubes) hybrid film is fabricated on an indium-tin oxide (ITO) electrode via one-step electrochemical co-assembly approach. BSA is inherently dispersive and therefore served as the linking mediator of SWCNTs, which facilitate the redox reactions of [Ru(bpy)(2)(tatp)](2+) employed as a reporter of BSA. The evidences from differential pulse voltammetry, cyclic voltammetry, scanning electron microscope, emission spectroscopy and fluorescence microscope reveal that the [Ru(bpy)(2)(tatp)](2+)-BSA-SWCNTs hybrid can be electrochemically co-assembled on the ITO electrode, showing two pairs of well-defined Ru(II)-based redox waves. Furthermore, the electrochemical co-assembly of the [Ru(bpy)(2)(tatp)](2+)-BSA-SWCNTs hybrid is found to be strongly dependent on the simultaneous presence of BSA and SWCNTs, indicating a good linear response to BSA in the range from 6 to 50mgL(-1). The results from this study provide an electrochemical co-assembly method for the development of non-redox protein biosensors.     

Proliferation inhibition and apoptosis promotion by dual silencing of VEGF and Survivin in human osteosarcoma

Simultaneous silencing of multiple upregulated genes is an attractive and viable treatment strat-egy for many incurable diseases including cancer.Herein we used a dual gene-targeted siRNA conjugate composed of VEGF and Survivin siRNA sequences in the same backbone to inhibit proliferation and angiogenesis in two human osteosarcoma cell lines.We synthesized siRNA sequences targeting the VEGF and Survivin genes individually (VEGF siRNA and Survivin siRNA) or simultaneously (one-chain-double-target siRNA:dual siRNA).VEGF and Survivin mRNA and protein expression levels in human osteosarcoma MG-63 and Saos-2 cells were detected by qRT- PCR and western blot analysis.VEGF and Survivin protein location and expression were evaluated by immunohistochemistry and immunofluorescence staining.MG-63 and Saos-2 cell migration,proliferation,apoptosis,and angiogenesis were detected by scratch test,MTT assay,flow cytometry,and capillary tube assay respectively.The dual siRNA induced similar downregulation of VEGF and Survivin mRNA and protein levels,compared with VEGF siRNA or Survivin siRNA alone.The dual siRNA caused greater suppression of MG-63 and Saos-2 cell migration,proliferation and angiogenesis,and promoted more cell apoptosis than VEGF siRNA or Survivin siRNA alone,suggesting that the effects of the dual siRNA on inhibiting cell proliferation,migration,and angiogenesis and promoting apoptosis were superior to those of the single-target siRNAs.Simultaneous silencing of VEGF and Survivin using the dual siRNA may be an advantageous alternative for the development of therapeutic strategies against human osteosarcoma.     

Global,regional, and cladistic patterns of variation in climatic niche breadths in terrestrial elapid snakes

We obtai ned geo-refere need occurre nee and climatic data from in dividual localities for 59 species of terrestrial elapid snakes, used phylogenetic generalized least squares regression to investigate spatial and cladistic patter ns of variation in climatic niche breadths, and compared patter ns within and across regions and clades to see if they parallel or differ from each other. Specifically, we test (1) whether a species' climatic niche breadth on a given niche axis relates to its position along that axis, and to its climatic niche breadth on another niche axis, and (2) whether variation in niche breadths among species is explained by within-locality variation in climatic conditions or by among-locality variation. We found that:(1) there is an overall global patter n, and patter ns in individual regi ons or clades gen erally parallel each other and global patter ns;(2) species in wanner environments have narrower temperature niche breadths (TNBs);(3) precipitation niche breadth (PNB) and position are positively related;⑷ TNB and PNB are not related;and ⑸ within-locality variation in climatic conditions explains most variation in TNBs, whereas among-locality variation explains most variation in PNBs. Our results are consistent with those reported for lizards of the families Phrynosomatidae and Varanidae, con firm the importance of withi n-locality n iche breadth to species n iche breadth, and show a more im porta nt role of amon g-locality n iche breadth in affecting species niche breadth in terrestrial elapids than in lizards.     

Synthesis and antifungal properties of sulfanilamide derivatives of chitosan

Sulfanilamide derivatives of chitosan (2-(4-acetamido-2-sulfanimide)-chitosan (HSACS, LSACS), 2-(4-acetamido-2-sulfanimide)-6-sulfo-chitosan (HSACSS, LSACSS) and 2-(4-acetamido-2-sulfanimide)-6-carboxymethyl-chitosan (HSACMCS, LSACMCS)) were prepared using different molecular weights of chitosan (CS), carboxymethyl chitosan (CMCS) and chitosan sulfates (CSS) reacted with 4-acetamidobenzene sulfonyl chloride in dimethylsulfoxide solution. The structures of the derivatives were characterized by FT-IR spectroscopy and elemental analysis, which showed that the substitution degree of sulfanilamide group of HSACS, HSACSS, HSACMCS, LSACS, LSACSS and LSACMCS were 0.623, 0.492, 0.515, 0.576, 0.463 and 0.477, respectively. The solubility of the derivatives (pH<7.5) was higher than that of chitosan (pH<6.5). The antifungal activities of the derivatives against Aiternaria solani and Phomopsis asparagi were evaluated based on the method of Jasso et al. in the experiment. The results indicated that all the prepared sulfanilamide derivatives had a significant inhibiting effect on the investigated fungi in the polymer concentration range from 50 to 500 microg mL(-1). The antifungal activities of the derivatives increased with increasing the molecular weight, concentration or the substitution degree. The sulfanilamide derivatives of CS, CMCS and CSS show stronger antifungal activities than CS, CMCS and CSS.     

Existence of an endogenous glutamate and aspartate transporter in Chinese hamster ovary cells

Chinese hamster ovary cells show endogenous high-affinity Na^＋ -dependent glutamate transport activity. This transport activity is kinetically similar to a glutamate transporter family strategically expressed in the central nervous system and is pharmacologically unlike glutamate transporter- 1 or excitatory amino acid carrier 1. The cDNA of a glutamate/aspartate transporter （GLAST）-like transporter was obtained and analyzed. The deduced amino acid sequence showed high similarity to human, mouse, and rat GLAST. We concluded that a GLAST-like glutamate transporter exists in Chinese hamster ovary cells that might confer the endogenous high-affinity Na^＋ -dependent glutamate transport activity evident in these cells.