Biomedical subjects

Lili Wang

Publications and source records attributed to Lili Wang.

At least 73 records · Page 4Linked to original sources

PubMed2004

Production of transgenic blastocyst by nuclear transfer from different types of somatic cells in cattle.

The present study examined the effects of genetic manipulation to the donor cell and different types of transgenic donor cells on developmental potential of bovine nuclear transfer (NT) embryos. Four types of bovine somatic cells, including granulosa cells, fetal fibroblasts, fetal oviduct epithelial cells and fetal ovary epithelial cells, were transfected with a plasmid (pCE-EGFP-Ires-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes by electroporation. After 14 days selection with 800 microg/mL G418, transgenic cell lines from each type of somatic cells were obtained. Nontransgenic granulosa cells and all 4 types of transgenic somatic cells were used as nuclear donor to produce transgenic embryos by NT. There was no significant difference in development rates to the blastocyst stage for NT embryos from transgenic and nontransgenic granulosa cells (44.6% and 42.8%, respectively), and transfer of NT embryos derived from transgenic and nontransgenic granulosa cells to recipients resulted in similar pregnancy rates on day 90 (19% and 25%, respectively). The development rates to the blastocyst stage of NT embryos were significantly different among different types of transgenic donor cells (P<0.05). Blastocyst rates from fetal oviduct epithelial cell and granulosa cell (49.1% and 44.6%, respectively) were higher than those from fetal fibroblast (32.7%) and fetal ovary epithelial cell (22.5%). These results suggest that (i) genetic manipulation to donor cells has no negative effect on in vitro and early in vivo developmental competence of bovine NT embryos and (ii) granulosa and fetal oviduct epithelial cells can be used to produce transgenic bovine NT embryos more efficiently. In addition, GFP can be used to select transgenic NT embryos as a non-invasive selective marker.

Animals↗

PubMed2004

Generation of cloned calves from different types of somatic cells.

Six types of bovine somatic cell lines, including a granulosa cell line of Chinese red-breed yellow cattle (YGR), a granulosa cell line of Holstein cow (HGR), two skin fibroblast cell lines of two adult Holstein cows respectively (AFB1 and AFB2), a skin fibroblast cell line (FFB) and an oviduct epithelial cell line (FOV) of a Holstein fetus, were established. Somatic cell nuclear transfer (SCNT) was carried out using these cells as nuclei donor, and a total of 12 healthy calves were cloned. The effects of different types of donor cells on developmental potential of bovine SCNT embryos were investigated. (i) There was no significant difference in development rates to the blastocyst stage for SCNT embryos from YGR and HGR (33.2% and 35.1%, respectively). Pregnancy rates of them were 33.3% and 30.2%, respectively; and birth rates were 16.7% and 11.6%, respectively. (ii) Development rates to the blastocyst stage for SCNT embryos from different individuals (AFB1 and AFB2) differed significantly (27.9% and 39.4%, respectively, P < 0.05). Pregnancy rates of them were 36.2% and 36.4%, respectively; and birth rates were 14.9 % and 27.3%, respectively. (iii) There was significant difference in development rates to the blastocyst stage for SCNT embryos from FFB and FOV of the same fetus (37.9% and 41.5%, respectively, P < 0.05). Pregnancy rates of them were 45.7% and 24.1%, respectively; and birth rates were 22.9% and 10.3%, respectively. Finally, developmental potential of bovine SCNT embryos from all four types of somatic cells from Holstein cows (HGR, AFB, FFB and FOV) were compared. For in vitro development stage, development rates to the blastocyst stage for SCNT embryos from HGR, AFB, FFB and FOV were 35.1%A, 29.4%B, 37.9%A and 41.5%c, respectively (pABC < 0.05); for in vivo development stage, pregnancy rates of them were 30.2%, 36.2%, 45.7% and 24.1%, respectively; and birth rates of them were 11.6%, 17.2%, 22.9% and 10.3% respectively.

Animals↗

PubMed2003

Robust adenoviral and adeno-associated viral gene transfer to the in vivo murine heart: application to study of phospholamban physiology.

BACKGROUND: Viral gene transfer to the whole heart in vivo has been achieved in several mammalian species but remained difficult to accomplish in murine hearts. We postulated that a key impediment derives from the use of proximal aortic occlusion during virus injection, because this eliminates coronary perfusion gradients in mice as aortic root and left ventricle pressures equalize. METHODS AND RESULTS: Pressure-volume analysis confirmed these mechanics. In contrast, descending aortic occlusion with whole-body cooling (20 degrees C) preserved transmyocardial perfusion gradients and allowed for sustained (>10-minute) dwell times in an upper-body perfusion circuit. This approach yielded robust cardiac transfection with adenovirus (AdV) and adeno-associated virus (AAV) injected into the left ventricle cavity or more simply via a central vein. Cardio-specific expression was achieved with a myocyte-specific promotor. Optimal AdV transfection required 9-minute aortic occlusion, versus 5-minute occlusion for AAV. Using this method, we examined the in vivo function of phospholamban (PLB) by stably transfecting PLB-null mice with AAV encoding PLB (AAV(PLB)). AAV(PLB) restored PLB protein to near control levels that colocalized with SERCA2A in cardiomyocytes. At baseline, PLB-null hearts exhibited enhanced systolic and diastolic function, but frequency-dependent reserve was blunted versus wild-type controls. These properties, particularly the frequency response, returned toward control 3 months after AAV(PLB) transfection. CONCLUSIONS: The new simplified approach for murine whole-heart viral transfection should assist molecular physiology studies.

Adenoviridae↗

PubMed2003

Total correction of hemophilia A mice with canine FVIII using an AAV 8 serotype.

Despite the popularity of adeno-associated virus 2 (AAV2) as a vehicle for gene transfer, its efficacy for liver-directed gene therapy in hemophilia A or B has been suboptimal. Here we evaluated AAV serotypes 2, 5, 7, and 8 in gene therapy of factor VIII (FVIII) deficiency in a hemophilia A mouse model and found that AAV8 was superior to the other 3 serotypes. We expressed canine B domain-deleted FVIII cDNA either in a single vector or in 2 separate AAV vectors containing the heavy- and light-chain cDNAs. We also evaluated AAV8 against AAV2 in intraportal and tail vein injections. AAV8 gave 100% correction of plasma FVIII activity irrespective of the vector type or route of administration.

Adenoviridae↗

PubMed2003

Increase of TCR V beta accessibility within E beta regulatory region influences its recombination frequency but not allelic exclusion.

Seventy percent of the murine TCRbeta locus (475 kb) was deleted to generate a large deleted TCRbeta (beta(LD)) allele to investigate a possible linkage between germline transcription, recombination frequency, and allelic exclusion of the TCR Vbeta genes. In these beta(LD/LD) mice, the TCRbeta gene locus contained only four Vbeta genes at the 5' side of the locus, and consequently, the Vbeta10 gene was located in the original Dbeta1-Jbeta1cluster within the Ebeta regulatory region. We showed that the frequency of recombination and expression of the Vbeta genes are strongly biased to Vbeta10 in these mutant mice even though the proximity of the other three 5'Vbeta genes was also greatly shortened toward the Dbeta-Jbeta cluster and the Ebeta enhancer. Accordingly, the germline transcription of the Vbeta10 gene in beta(LD/LD) mice was exceptionally enhanced in immature double negative thymocytes compared with that in wild-type mice. During double negative-to-double positive transition of thymocytes, the level of Vbeta10 germline transcription was prominently increased in beta(LD/LD) recombination activating gene 2-deficient mice receiving anti-CD3epsilon Ab in vivo. Interestingly, however, despite the increased accessibility of the Vbeta10 gene in terms of transcription, allelic exclusion of this Vbeta gene was strictly maintained in beta(LD/LD) mice. These results provide strong evidence that increase of Vbeta accessibility influences frequency but not allelic exclusion of the TCR Vbeta rearrangement if the Vbeta gene is located in the Ebeta regulatory region.

Alleles↗

PubMed2003

Presenilin redistribution associated with aberrant cholesterol transport enhances beta-amyloid production in vivo.

Epidemiology, in vitro, and in vivo studies strongly implicate a role for cholesterol in the pathogenesis of Alzheimer's disease (AD). We have examined the impact of aberrant intracellular cholesterol transport on the processing of the amyloid precursor protein (APP) in a mouse model of Niemann-Pick type C (NPC) disease. In the NPC mouse brain, cholesterol accumulates in late endosomes/lysosomes. This was associated with the accumulation of beta-C-terminal fragments (CTFs) of APP, but the level of beta-secretase and its activity were not affected. Alpha-secretase activity and secreted APPalpha generation were also not affected, suggesting CTFs increased because of decreased clearance. The level of presenilin-1 (PS-1) was unchanged, but gamma-secretase activity was greatly enhanced, which correlated with an increase in Abeta40 and Abeta42 levels. These events were associated with abnormal distribution of PS-1 in the endosomal system. Our results show that aberrant cholesterol trafficking is associated with the potentiation of APP processing components in vivo, leading to an overall increase in Abeta levels.

Amyloid Precursor Protein Secretases↗

PubMed2003

Molecular characterization of the recurrent unbalanced translocation der(1;7)(q10;p10).

An unbalanced translocation der(1;7)(q10; p10) is a nonrandom chromosomal aberration commonly observed in myelodysplastic syndrome and acute myeloid leukemia. We molecularly analyzed the breakpoints of der(1;7)(q10;p10) by quantitative fluorescent in situ hybridization (FISH) analyses using centromeric satellite DNAs mapped to chromosomes 1 and 7 as probes. We found that the signal intensities of 2 centromere alphoid probes, D1Z7 on chromosome 1 and D7Z1 on chromosome 7, were almost invariably reduced on the derivative chromosome compared with those on their normal counterparts. These results suggest that this translocation results from the recombination between the 2 alphoids, which was further confirmed by fiber FISH experiments. Because the relative reduction in the intensities of D1Z7 and D7Z1 signals on the derivative chromosomes was highly variable among patients, it was estimated that the breakpoints in these patients were randomly distributed over several megabase pairs within each alphoid cluster except for its extreme end to the short arm. Our results provide a novel insight into the structural basis for generation of this translocation as well as its leukemogenic roles.

Acute Disease↗

PubMed2003

Cdk5 is a key factor in tau aggregation and tangle formation in vivo.

Tau aggregation is a common feature of neurodegenerative diseases such as Alzheimer's disease, and hyperphosphorylation of tau has been implicated as a fundamental pathogenic mechanism in this process. To examine the impact of cdk5 in tau aggregation and tangle formation, we crossed transgenic mice overexpressing the cdk5 activator p25, with transgenic mice overexpressing mutant (P301L) human tau. Tau was hyperphosphorylated at several sites in the double transgenics, and there was a highly significant accumulation of aggregated tau in brainstem and cortex. This was accompanied by increased numbers of silver-stained neurofibrillary tangles (NFTs). Insoluble tau was also associated with active GSK. Thus, cdk5 can initiate a major impact on tau pathology progression that probably involves several kinases. Kinase inhibitors may thus be beneficial therapeutically.

Animals↗

PubMed2003

Co-localization of cholesterol, apolipoprotein E and fibrillar Abeta in amyloid plaques.

Recent evidence strongly suggests a role for cholesterol and apolipoprotein E in the etiology of Alzheimer's disease. We have demonstrated the co-localization of cholesterol and apolipoprotein E with beta-amyloid immunoreactivity and thioflavin S immunofluorescence in AD type plaques of a transgenic mouse model. Cholesterol and apolipoprotein E co-localized to the core of thioflavin S-positive (fibrillar) plaques, but not thioflavin S-negative (diffuse) plaques from an early age. By 18 months of age, there was extensive coverage of fibrillar plaques immunopositive for apolipoprotein E and cholesterol oxidase. These findings support evidence that cholesterol and apolipoprotein E are involved in fibrillar plaque formation or maintenance, and suggest that cholesterol may impact amyloid formation extracellularly, as well as through an intracellular effect.

Alzheimer Disease↗

PubMed2003

Novel therapeutic approach for the treatment of Alzheimer's disease by peripheral administration of agents with an affinity to beta-amyloid.

Plaques containing beta-amyloid (Abeta) peptides are one of the pathological features of Alzheimer's disease, and the reduction of Abeta is considered a primary therapeutic target. Amyloid clearance by anti-Abeta antibodies has been reported after immunization, and recent data have shown that the antibodies may act as a peripheral sink for Abeta, thus altering the periphery/brain dynamics. Here we show that peripheral treatment with an agent that has high affinity for Abeta (gelsolin or GM1) but that is unrelated to an antibody or immune modulator reduced the level of Abeta in the brain, most likely because of a peripherally acting effect. We propose that in general, compounds that sequester plasma Abeta could reduce or prevent brain amyloidosis, which would enable the development of new therapeutic agents that are not limited by the need to penetrate the brain or evoke an immune response.

Alzheimer Disease↗

PubMed2003

Molecular cytogenetic analyses of HIG, a novel human cell line carrying t(1;3)(p36.3;q25.3) established from a patient with chronic myelogenous leukemia in blastic crisis.

Chromosomal abnormalities involving 1p36, 3q21, and/or 3q26 have been reported in a subset of myeloid neoplasms having characteristic dysmegakaryopoiesis, and the overexpression of EVI1 on 3q26 or of MEL1 on 1p36 has been implicated in their pathogenesis. We describe molecular cytogenetic analyses of a novel human cell line, HIG, established from a unique case in which a novel translocation t(1;3)(p36;q26) appeared as the sole additional chromosomal abnormality at the time of blastic transformation of chronic myelogenous leukemia. The patient displayed clinical features resembling those of the 3q21q26 syndrome. The HIG cell line retained der(1)t(1;3)(p36;q26) but lost t(9;22)(q34;q11). To identify the relevant gene that would be deregulated by this translocation, we molecularly cloned the translocation's breakpoints. They were distant from the breakpoint cluster regions of the 3q21q26 syndrome or t(1;3)(p36;q21), and neither the EVI1 nor the MEL1 transcript was detected in the HIG cell line. None of the genes located within 150 kilobase pairs of the breakpoints were aberrantly expressed, suggesting that in this case other gene(s) more distant from the breakpoints are deregulated by possible remote effects. Further analyses of the deregulated genes in the HIG cell line should provide important insight into the mechanisms involved in these types of leukemias.

Blast Crisis↗

PubMed2003

The R protein of SARS-CoV: analyses of structure and function based on four complete genome sequences of isolates BJ01-BJ04.

The R (replicase) protein is the uniquely defined non-structural protein (NSP) responsible for RNA replication, mutation rate or fidelity, regulation of transcription in coronaviruses and many other ssRNA viruses. Based on our complete genome sequences of four isolates (BJ01-BJ04) of SARS-CoV from Beijing, China, we analyzed the structure and predicted functions of the R protein in comparison with 13 other isolates of SARS-CoV and 6 other coronaviruses. The entire ORF (open-reading frame) encodes for two major enzyme activities, RNA-dependent RNA polymerase (RdRp) and proteinase activities. The R polyprotein undergoes a complex proteolytic process to produce 15 function-related peptides. A hydrophobic domain (HOD) and a hydrophilic domain (HID) are newly identified within NSP1. The substitution rate of the R protein is close to the average of the SARS-CoV genome. The functional domains in all NSPs of the R protein give different phylogenetic results that suggest their different mutation rate under selective pressure. Eleven highly conserved regions in RdRp and twelve cleavage sites by 3CLP (chymotrypsin-like protein) have been identified as potential drug targets. Findings suggest that it is possible to obtain information about the phylogeny of SARS-CoV, as well as potential tools for drug design, genotyping and diagnostics of SARS.

Amino Acid Sequence↗

PubMed2003

Adenovirus-mediated overexpression of follistatin enlarges intact liver of adult rats.

Under normal physiologic conditions, liver size is under strict regulatory control. Activin, a member of the transforming growth factor beta (TGF-beta) superfamily, is expressed in the intact adult liver and is an inhibitor of hepatocyte growth. However, the exact role played by endogenous activin in maintaining the size of a normal adult liver has yet to be completely examined in vivo. Here, we report the development of an adenoviral vector (AdexCAFS288) that expressed human follistatin-288, which binds to activin and neutralizes its biologic activities. AdexCAGFP, a control virus, expressed green fluorescent protein. AdexCAFS288 effectively expressed follistatin-288, as measured both in HepG2 cell lysate and conditioned medium and blocked activin signaling and its biologic functions in vitro. Intraperitoneal injection of AdexCAFS288 in vivo resulted in significant liver growth (146% of control) in intact liver of adult male rats 12 days following treatment without significant dysfunctions. The increase in liver size was attributed to increased hepatocyte proliferation, as monitored by the mitotic index. Furthermore, there was a significant correlation between serum follistatin levels and liver weight. In conclusion, our results suggest that activin plays a critical role in maintaining optimal liver size and implicates the endogenous activin system as a therapeutic target in the treatment of liver disease.

Activins↗

PubMed2003

Ex vivo evaluation of a novel polyiodinated compound for early detection of atherosclerosis.

Atherosclerosis is a primary cause of heart disease and stroke; it is the underlying cause of about 50% of all deaths in Western countries. It is known that early detection of atherosclerotic lesions would significantly reduce the risk of mortality. The objective of this study was to develop a radioimaging method for early detection of atherosclerotic plaques. A novel polyiodinated cholesterol analog, cholesteryl 1,3-diiopanoate glyceryl ether (C2I, patent pending), was synthesized and radiolabeled with 125I. 125I-C2I was incorporated into acetylated low-density lipoprotein (AcLDL), which is considered to be an atherosclerotic plaque-seeking carrier. 125I-C2I was also prepared as a chylomicron-like emulsion. Transgenic mice deficient in apoE and low-density lipoprotein receptors (LDLR), known as apoE/LDLR double knockout, were used as an animal model of early atherosclerosis. 125I-C2I/AcLDL or 125I-C2I emulsion was injected into the apoE/LDLR knockout mice via the tail vein, and the mice were killed humanely 24 h after injection. Various tissues including aorta were removed and radioactivity was determined. The aorta samples were also imaged to determine the accumulation of radioactivity from C2I. The images were compared to the atherosclerotic lesions revealed by histological studies. It was found that both 125I-C2I/AcLDL and 125I-C2I emulsion resulted in accumulation of radioactivity at the site of early atherosclerotic lesions, and they therefore may be useful for early detection of atherosclerosis.

Animals↗

PubMed2003

In vivo somatic cell gene transfer of an engineered Noggin mutein prevents BMP4-induced heterotopic ossification.

BACKGROUND: The formation of the skeleton requires inductive signals that are balanced with their antagonists in a highly regulated negative feedback system. Inappropriate or excessive expression of BMPs (bone morphogenetic proteins) or their antagonists results in genetic disorders affecting the skeleton, such as fibrodysplasia ossificans progressiva. BMP signaling mediated through binding to its receptors is a critical step in the induction of abnormal ossification. Therefore, we hypothesized that engineering more effective inhibitors of this BMP-signaling process may lead to the development of therapies for such conditions. METHODS: BMP4-induced heterotopic ossification was used as a model for testing the ability of the BMP antagonist Noggin to block de novo bone formation, either by local or systemic delivery. Since Noggin naturally acts locally, a Noggin mutein, hNOGDeltaB2, was engineered and was shown to circulate systemically, and its ability to block heterotopic ossification was tested in a mouse model with use of adenovirus-mediated somatic cell gene transfer. RESULTS: A mouse model of BMP4-induced heterotopic ossification was developed. Local delivery of wild-type NOG inhibited heterotopic ossification, but systemic administration was ineffective. In contrast, systemic delivery of the adenovirus encoding hNOGDeltaB2 resulted in systemic levels that persisted for more than two weeks and were sufficient to block BMP4-induced heterotopic ossification. CONCLUSIONS: BMP4-induced heterotopic ossification can be prevented in vivo either by local delivery of wild-type Noggin or after somatic cell gene transfer of a Noggin mutein, hNOGDeltaB2. Furthermore, the data in the present study provide proof of concept that a naturally occurring factor can be engineered for systemic delivery toward a desirable pharmacological outcome. CLINICAL RELEVANCE: Blocking bone formation is clinically relevant to disorders of heterotopic ossification in humans, such as fibrodysplasia ossificans progressiva. Furthermore, development of BMP antagonists as therapeutic agents may provide modalities for the treatment of other pathologic conditions that arise from aberrant expression of BMPs and/or from a lack of their antagonists.

Adenoviridae↗

PubMed2003

Acetylated low-density lipoprotein-encapsulated cholesteryl 1,3-diiopanoate glyceryl ether for the detection of atherosclerosis in rabbits.

UNLABELLED: A cholesteryl ester analog, cholesteryl 1,3-diiopanoate glyceryl ether (C2I), was synthesized and investigated for its potential use for the detection of atherosclerotic lesions in rabbits. METHODS: (125)I-labeled C2I was incorporated into acetylated low-density lipoprotein (AcLDL). The resultant complex, (125)I-C2I-AcLDL, was injected intravenously into 2 groups of rabbits, fed cholesterol and normal chow, at a dose of 555 kBq/kg. Tissue samples were taken 24 h after injection for the biodistribution study. Atherosclerotic lesions and C2I deposition in aortic samples were examined by Sudan IV staining and autoradiography, respectively. RESULTS: The levels of C2I in blood and aortic samples in cholesterol-fed animals were 2- to 3-fold higher than those in the control group (P < 0.05). The autoradiography results correlated well with the Sudan IV staining results, indicating sites of C2I deposition superimposed on lesion sites. CONCLUSION: C2I was preferentially taken up and retained at atherosclerotic lesion sites, suggesting its potential use for the detection of early atherosclerosis.

Animals↗

PubMed2003

Study on retinal ganglion cell apoptosis after explosive injury of eyeballs in rabbits.

PURPOSE: To explore the pathological changes of retinal ganglion cell apoptosis and its relation to the value of glutamate concentration in vitreous body after explosive injury of eyeballs in the rabbits. METHOD: Similar explosive injury models of eyeballs in 10 adult grey rabbits were made. The rabbits were killed on scheduled time. The retinal tissues of studied eyes and control eyes were obtained for the pathological examination with TUNEL method respectively. The value of glutamate in vitreum of injured eyes was measured and was compared with that of contralateral eyes. Statistical comparison analysis on the experiment data was performed. RESULT: The value of glutamate in vitreum of injured eyes was significantly higher than that of contralateral eyes in all rabbits in the study. A lot of TUNEL positive cells were observed in the injured eyes. It suggested that apoptosis of the retinal ganglion cells took place. CONCLUSION: We speculate that apoptosis of the retinal ganglion cells is perhaps among the causes resulting in damage of visual function after explosive injury of eyeballs and that the increasing of the value of glutamate in vitreum possibly associated with apoptosis of the retinal ganglion cells.

Animals↗

PubMed2002

Differential regulation between gene expression and histone H3 acetylation in the variable regions of the TCRbeta locus.

Histone acetylation is suggested to regulate gene expression in the TCR loci. Using R2CD3 mouse model, we previously showed that germline transcription of the TCRbeta chain gene is discrete between 5' Vbeta regions and Dbeta-Jbeta-Cbeta clusters plus Vbeta14 region. In this study, we investigated a role of histone H3 acetylation in transcriptional regulation of the TCRbeta locus. Our results showed that DN-DP transition is accompanied by significant promotion of histone H3 acetylation in both Dbeta-Jbeta-Cbeta cluster and Vbeta14 region, correlating with up-regulation of germline transcription. Surprisingly, termination of germline transcription of the 5' Vbeta regions was inversely correlated with histone H3 hyperacetylation. Moreover, histone H3 acetylation showed equivalent level in both functionally rearranged Vbeta region with active transcription and unrearranged Vbeta region without transcription in mature T cells. These results suggest that histone acetylation is not a sole limiting factor in both terminating germline Vbeta transcription during DN-DP transition and maintaining functionally rearranged Vbeta gene expression.

Acetylation↗