Compound Danshen tablets downregulate amyloid protein precursor mRNA expression in a transgenic cell model of Alzheimer's disease Effects and a comparison with donepezil

After gene mutation, the pcDNA3.1/APP595/596 plasmid was transfected into HEK293 cells to establish a cell model of Alzheimer＇s disease. The cell model was treated with donepezil or compound Danshen tablets after culture for 72 hours. Reverse transcription-PCR showed that the mRNA expression of amyloid protein precursor decreased in all groups following culture for 24 hours, and that there was no significant difference in the amount of decrease between donepezil and compound Danshen tablets. Our results suggest that compound Danshen tablets can reduce expression of the mRNA for amyloid protein precursor in a transgenic cell model of Alzheimer＇s disease, with similar effects to donepezil.

Dominant-negative inhibition of glucose-dependent insulinotropic polypeptide impairs function of β cells in transgenic pigs

Dear Editor： Glucose-dependent insulinotropic polypeptide （GIP） and proglucagon product glucagon-like peptide-1 （GLP- 1） and their corresponding receptors promote secretion of glucose-dependent insulin and may be responsible for up to 70% of postprandial insulin secretions.

Expression of Hepatitis B Surface Antigen Gene in Ginseng Cells

The recombinant plasmid pBIBSa containing the HBsAg DNA fragment was transferred into Agrobacterium tumefaciens strain LBA4404 directly. Ginseng cells were transfected with A. tumefaciens carrying pBIBSa and the ginseng cell lines carrying HBsAg-S gene were obtained. The presence of target gene in the transfect cells was confirmed by PCR and RT-PCR. A clear band at the site of 700 bp was observed by agarose electrophoresis analysis of the samples containing the target gene. HBsAg expressed by the transgenic ginseng cells was detected by Western blot. Maximum expression levels of 184 ng HBsAg/g FW and 0. 009% of the total soluble proteins were observed by ELISA. HBsAg in ginseng cells was located both on the cell membrane and in the nuclei.

STABLE EXPRESSION OF HUMAN CYTOCHROME CYP2B6 AND CYP1A1 IN CHINESE HAMSTER CHL CELLS:THEIR USE IN MICRONUCLEUS ASSAYS

With specific designed prmers. CYP2B6 and CYP1A1 cDNA were generatecl by reverse transcrlI7tion-Polymerase chain reaction(RT-PCR )technlque Performed on total RNAs isolated frorn hum1ln liver and 3-rnethylch(,lanthrene(3-Mtt)induc human amnion FL, cells. Cell llnes (CHL, 2B6 and CtHL-1A1 ) capableof expressing hunlan cytochome P 15O (CYP ) 2B6 and 1A1 were establishecl after transfection of corre-sponding eukaryotic reconlbinant expression plasmid with human CYP2ll6 and 1A1 cDNA lnserts respectlvely. These cell lines stably expressed the mRNAs and the enzymatic activltles cc)rresI’onding to ttYP2B6and CYP1A1, respectively’ Compared with Chinese hamster lung (CHL) cells, the n1icr()nucleus frecluencyin CHl,-2B6 cells is markedly lncreased when exPosed to nitrosamines,aflatoxln B, (AFB1) and cyclophos-Phamide (CPA). Thls is also in CHL-1A1 cells,when exposed to carcinogenic polycycllc aromatic hydrocar-bons.

NON-DESTRUCTIVE ANALYSIS OF THE NUCLEI OF TRANSGENIC LIVING CELLS USING LASER TWEEZERS AND NEAR-INFRARED RAMAN SPECTROSCOPIC TECHNIQUE

Transgenic cell lines of loblolly pine （Pinus taeda L.） were analyzed by a compact laser-tweezers-Raman-spectroscopy （LTRS） system in this investigation. A low power diode laser at 785 nm was used for both laser optical trapping of single transgenic cells and excitation for near-infrared Raman spectroscopy of the nuclei of synchronized cells, which were treated as single organic particles, at the S-phase of the cell cycle. Transgenic living cells with gfp and uidA genes were used as biological samples to test this LTRS technique. As expected, different Raman spectra were observed from the tested biological samples. This technique provides a high sensitivity and enables real-time spectroscopic measurements of transgenic cell lines. It could be a valuable tool for the study of the fundamental cell and molecular biological process by trapping single nucleus and by providing a wealth of molecular information about the nuclei of cells.

Transgenic mice with overexpression of human scavenger receptor A on endothelial cells

Objectives To establish a new transgenic mouse model for determining the function and role of human scavenger receptor A (SR-A) in atherosclerosis in vivo.Methods Human scavenger receptor minigene-driven mouse tie-1 promoter was constructed and confirmed by endonuclease digestion and sequence analysis. Transgenic mice were generated via the microinjection method. PCR and Southern blot were used to screen the positive transgenic mice. RT-PCR and immunohistochemical analysis were used to detect the level and location of human SR-A Ⅰ expression in transgenic mice. The activity of human SR-A Ⅰ was determined by morphologic observation of aortic endothelial cells of transgenic mice under transmission electron microscopy.Results The electrophoresis assay showed the expected 4 fragments of 0.9 kb, 1.1 kb, 1.2 kb and 4.2 kb in the Sma Ⅰ digest and 2 fragments of 0.8 kb and 6.7 kb in Bgl Ⅱ digest of plasmids pTie-1/hSR-A. The fragment sequence of tie-1 promoter and human SR-A cDNA in plasmids pTie-1/hSR-A was correct and no ATG before the translation initiation sites of human SR-A was found by sequence analysis. 561 injected and surviving embryos with the purified human SR-A minigene were implanted into the oviducts of 19 ICR pseudopregnant mice. Among the 54 surviving pups from 13 foster mothers, 7 were identified by PCR and Southern blot analysis. The results of RT-PCR and imrnunohistochemical analysis showed human SR-A was specifically expressed on vascular endothelial cells of the aorta and renal artery, as well as hepatic sinusoidal endothelial cells in transgenic mice. Transmmion electron microscope (TEM) of aorta of transgenic mice showed that a large number of vesicles, multivesicle bodies and swollen mitochondria filled the plasma of endothelial cells.Conclusions A transgenic mouse model with overexpression of human SR-A in endothelial cells was successfully established. The transgene was integrated and transmitted into the chromosome of transgenic mice. Tie-1 promoter controlled the transgene to express in endothelial cells in mice. Pinocytic activity of aortic endothelial cells in transgenic mice was higher than that of C57BL/6J mice. Our studies will provide a new transcgenic model for investigation of atherosclerosis and functions of human SR-A.

Genome-wide pan-GPCR cell libraries accelerate drug discovery

G protein-coupled receptors(GPCRs)are pivotal in mediating diverse physiological and pathological processes,rendering them promising targets for drug discovery.GPCRs account for about 40%of FDA-approved drugs,representing the most successful drug targets.However,only approximately 15%of the 800 human GPCRs are targeted by market drugs,leaving numerous opportunities for drug discovery among the remaining receptors.Cell expression systems play crucial roles in the GPCR drug discovery field,including novel target identification,structural and functional characterization,potential ligand screening,signal pathway elucidation,and drug safety evaluation.Here,we discuss the principles,applications,and limitations of widely used cell expression systems in GPCR-targeted drug discovery,GPCR function investigation,signal pathway characterization,and pharmacological property studies.We also propose three strategies for constructing genome-wide pan-GPCR cell libraries,which will provide a powerful platform for GPCR ligand screening,and facilitate the study of GPCR mechanisms and drug safety evaluation,ultimately accelerating the process of GPCR-targeted drug discovery.

Post-transcriptional Gene Silencing Induced by Short Interfering RNAs in Cultured Transgenic Plant Cells

Short interfering RNA (siRNA) is widely used for studyingpost-transcriptional gene silencing and holds great promise as a tool for both identifying functionof novel genes and validating drug targets. Two siRNA fragments (siRNA-a and -b), which weredesigned against different specific areas of coding region of the same target green fluorescentprotein (GFP) gene, were used to silence GFP expression in cultured gfp transgenic cells of rice(Oryza sativa L.; OS), cotton (Gossypium hirsutum L.; GH), Eraser fir [Abies fraseri (Pursh) Poir;AF], and Virginia pine (Pinus virginiana Mill.; PV). Differential gene silencing was observed in thebombarded transgenic cells between two siRNAs, and these results were consistent with theinactivation of GFP confirmed by laser scanning microscopy, Northern blot, and siRNA analysis intested transgenic cell cultures. These data suggest that siRNA-mediated gene inactivation can be thesiRNA specific in different plant species. These results indicate that siRNA is a highly specifictool for targeted gene knockdown and for establishing siRNA-mediated gene silencing, which could bea reliable approach for large-scale screening of gene function and drug target validation.

Effect of Early Intervention with Extract of Huannao Yicong Decoction(还脑益聪方) on the Pathologic Picture of Hippocampus and Neurocyte Apoptosis in APP Transgenic Mice Model of Dementia

Objective：To observe the effect of early intervention using extract of Huannao Yicong Decoction （还脑益聪方,HYD） on the pathological picture of hippocampus,neurocyte apoptosis,and associated regulatory genes inβ-amyloid precursor protein（APP） transgenic mice model of dementia.Methods：Sixty APP695^（V7171） transgenic mice were randomly divided into four groups of 15.The model group was treated with distilled water, the positive control group was treated with donepezil（0.65 mg/kg）,and the two HYD groups were treated with high dose（2.8 g/kg） and low dose（1.4 g/kg） HYD,respectively.All testing drugs were administered through gastrogavage by dissolving in equal volume of distilled water,once a day for six successive months.In addition, a normal control group with 15 healthy C57BL/6J mice of the same age and genetic background was set up with distilled water treatment.The pathologic picture of brain tissue was observed by microscopy with HE stain;the amount of apoptosis cells in the hippocampal CA1 area was detected by TUNEL;and expressions of associated genes,Bcl-2,and Bax were determined by immunohistochemical method.Results：Pathologic pictures of hippocampus showed that in the model group,cells messily arranged,neurons markedly decreased, and the surrounding tissue of some cells was loosened with edema,necrosis,and widened gap with glia cells proliferation.Compared with those in the normal group,the amount of apoptosis cells in the CA1 area was increased,Bcl-2 expression decreased,and Bax expression increased significantly,with markedly reduced Bcl-2/Bax ratio in the model group.Compared to the model group,the pathological changes were significantly milder in the HYD-treated groups,showing rather regularly arranged cells,significantly increased neurons, only few denatured necrotic cells with milder edema,less proliferation of glia cells,and obviously reduced cell apoptosis in CA1 area（P0.05 or P0.01）.Besides,Bcl-2 expression was up-regulated and Bax expression down-regulated,and Bcl-2/Bax ratio significantly increased in the two HYD groups（P0.05 or P0.01）. Conclusion：Early intervention with HYD could improve the abnormal pathologic picture of hippocampus and regulate the expressions of associated genes to suppress cell apoptosis,which might be its mechanism of action in alleviating cognitive functional disorder.

Effect of oxLDL on the uptake and clearance rate of cholesterol in v-SMC originated from human apoA1 transgenic mice

To study the inhibition effect of oxLDL on the uptake and clearance of intra ce llular 3H cholesterol in v SMC from the human apoA1 transgenic m ice (C57BL/6) and the changes in human apoA1 mRNA expression in v SMC from hum an apoA1 transgenic mice after oxLDL stimulation Methods v SMC originally isolated from human apoA1 transgenic mice connected with a rec ombined mouse metallothionein Ⅰ (MT Ⅰ) promoter was used, and the effect of oxLDL on the uptake and clearance of intracellular 3H cholesterol was s tudied in v SMC of the transgenic and control mice respectively, the study of h apoAⅠ mRNA expression from v SMC of the transgenic mice were done by RT PCR and Northern blot Results oxLDL (30?μg/ml) strongly promoted the SMC proliferation No difference was f ound in 3H cholesterol uptake between nSMC and trSMC, and the uptak e rates of both kinds of SMC rose 100% after oxLDL stimulation The efflux rate s of 3H cholesterol in trSMC were much higher than those of nSMC (40% -50%) Afte r ox LDL stimulation, the clearance rates fell by 28% and 10%, respectively, for nSMC and trSMC The result of RT PCR and Northern blot showed that h apoA1 gene e xpression was markedly increased by the stimulation of oxLDL Conclusion Expression of the h apoA1 gene in C57BL/6 mice enables them to reduce the accum ulation of cholesterol in v SMC The trSMC can alleviate the harmful effect of oxLDL due to the increase of h apoA1 expression