Increased efficiency of cloning large DNA fragments using a lower copy number plasmid.
Explore the source record for details and available documents.
Cloning, Molecular↗
Biomedical subjects
L Zhou
Publications and source records attributed to L Zhou.
Fluorescence-based functional assay for Wnt/beta-catenin signaling activity.
Aberrant activation of beta-catenin signaling has been implicated in the development of human cancers. As a Wnt signal transducer, beta-catenin forms a complex with the lymphocyte enhancer-binding factor/T cell factor transcription factor and activates downstream targets that promote cell proliferation. Here we developed a Wnt-dependent beta-catenin-mediated heterologous transactivation system, which consisted of a chimeric transcription factor constructed by fusing the GAL4 DNA-binding domain with the full-length beta-catenin, and a GAL4-responsive reporter expressing GFP. The chimeric transcription factor was highly unstable and exerted no detectable transactivating effect on the GAL4-responsive reporter. However, lithium and Wnt1 significantly stabilized this chimeric transactivator, indicating that this transactivation system is regulated by beta-catenin in a Wnt-responsive fashion. Thus, this transactivation system could be used as a functional reporter to identify potential upstream factors that deregulate beta-catenin signaling during tumorigenesis, as well as to screen for potential anti-cancer agents that specifically inhibit beta-catenin signaling in human tumors.
Adenocarcinoma↗
A large carbon sink in the woody biomass of Northern forests.
The terrestrial carbon sink, as of yet unidentified, represents 15-30% of annual global emissions of carbon from fossil fuels and industrial activities. Some of the missing carbon is sequestered in vegetation biomass and, under the Kyoto Protocol of the United Nations Framework Convention on Climate Change, industrialized nations can use certain forest biomass sinks to meet their greenhouse gas emissions reduction commitments. Therefore, we analyzed 19 years of data from remote-sensing spacecraft and forest inventories to identify the size and location of such sinks. The results, which cover the years 1981-1999, reveal a picture of biomass carbon gains in Eurasian boreal and North American temperate forests and losses in some Canadian boreal forests. For the 1.42 billion hectares of Northern forests, roughly above the 30th parallel, we estimate the biomass sink to be 0.68 +/- 0.34 billion tons carbon per year, of which nearly 70% is in Eurasia, in proportion to its forest area and in disproportion to its biomass carbon pool. The relatively high spatial resolution of these estimates permits direct validation with ground data and contributes to a monitoring program of forest biomass sinks under the Kyoto protocol.
Biomass↗
Direct extraction of specific pharmacophoric flavonoids from gingko leaves using a molecularly imprinted polymer for quercetin.
A new straightforward approach to extract active components from Chinese traditional herb was reported by using a molecularly imprinted polymer (MIP) as the sorbent material. The MIP was prepared using quercetin, a typical active compound of the flavonoid family in gingko leaves as the template. Acrylamide was used as the functional monomer and two polar solvent tetrahydrofuran was used as the porogen. Afterwards, the polymer was evaluated by chromatographic analysis. It exhibited high selectivity for quercetin and good affinity to its structural analogues. Specific binding amount of quercetin on the MIP in the solvent methanol was 12 microg/g polymer by the cartridge test. The MIP cartridge could directly trap a specific class of compounds including quercetin and kaempferol from the hydrolyzate of gingko leaves. The result demonstrated the possibility of direct extraction of certain pharmacophoric constituents from herb by MIP technology. The compounds extracted were confirmed by time-of-flight mass spectrometry.
Chromatography, High Pressure Liquid↗
Neuralized functions as an E3 ubiquitin ligase during Drosophila development.
The Notch pathway is a widely studied means of intercellular signaling responsible for the determination of cell fate, cell differentiation, and boundary formation (reviewed in ). The main effectors of this pathway, Notch (N) and Delta (Dl), have been shown to function as a receptor and ligand, respectively. Genetic and phenotypic studies suggest that Neuralized (Neu), a RING finger protein, also plays a role within the N-Dl pathway, although its biochemical function is unknown. Here, we show that Neu is required at the plasma membrane for functional activity and that its RING finger domain acts as an E3 ubiquitin ligase. These data suggest that the role of Neu is to target components of the N-Dl pathway for ubiquitination, allowing for propagation and/or regulation of the signal.
Animals↗
Detection of chemically induced DNA damage in layered films by catalytic square wave voltammetry using Ru(bpy)3(2+).
A sensor constructed by alternate layer-by-layer adsorption of PDDA cations and double-stranded (ds)-DNA on oxidized pyrolytic graphite electrodes was evaluated for detection of chemical damage to ds-DNA from known damage agent styrene oxide. Films made with PDDA ions of structure (PDDA/DNA)2 were approximately 6 nm thick and contained 0.23 microg of ds-DNA. Catalytic oxidation using 50 microM Ru(bpy)3(2+) (bpy = 2,2'-bipyridine) and square wave voltammetry (SWV) provided more sensitive detection of DNA damage than direct SWV oxidation. The catalytic peaks increased linearly with time during incubations with styrene oxide, but only minor changes were detected during incubation with nonreactive toluene. For best sensitivity, the outer layer of the film must be ds-DNA, and analysis should be done at low salt concentration. Studies of DNA and polynucleotides in solutions and films suggested that oxidation of guanine and chemically damaged adenine in partly unraveled, damaged DNA were the most likely contributors to the catalytic peak.
2,2'-Dipyridyl↗
Tetracycline-regulated gene expression mediated by a novel chimeric repressor that recruits histone deacetylases in mammalian cells.
Regulated gene expression will provide important platforms from which gene functions can be investigated and safer means of gene therapy may be developed. Histone deacetylases have recently been shown to play an important role in regulating gene expression. Here we investigated whether a more tightly controlled expression could be achieved by using a novel chimeric repressor that recruits histone deacetylases to a tetracycline-responsive promoter. This chimeric repressor was engineered by fusing the tetracycline repressor (TetR) with an mSin3-interacting domain of human Mad1 and was shown to bind the tetO(2) element with high affinity, and its binding was efficiently abrogated by doxycycline. The chimeric repressor was shown to directly interact with mSin3 of the histone deacetylase complex. This inducible system was further simplified by using a single vector that contained both a chimeric repressor expression cassette and a tetracycline-responsive promoter. When transiently introduced into mammalian cells, the chimeric repressor system exhibited a significantly lower basal level of luciferase activity (up to 25-fold) than that of the TetR control. When stably transfected into HEK 293 cells, the chimeric repressor system was shown to exert a tight control of green fluorescent protein expression in a doxycycline dose- and time-dependent fashion. Therefore, this novel chimeric repressor provides an effective means for more tightly regulated gene expression, and the simplified inducible system may be used for a broad range of basic and clinical studies.
Base Sequence↗
AML1-ETO expression is directly involved in the development of acute myeloid leukemia in the presence of additional mutations.
The t(8;21) is one of the most frequent chromosomal abnormalities associated with acute myeloid leukemia (AML). The translocation, which involves the AML1 gene on chromosome 21 and the ETO gene on chromosome 8, generates an AML1-ETO fusion transcription factor. To examine the effect of the AML1-ETO fusion protein on leukemogenesis, we made transgenic mice in which expression of AML1-ETO is under the control of the human MRP8 promoter (hMRP8-AML1-ETO). AML1-ETO is specifically expressed in myeloid cells, including common myeloid progenitors of hMRP8-AML1-ETO transgenic mice. The transgenic mice were healthy during their life spans, suggesting that AML1-ETO alone is not sufficient for leukemogenesis. However, after treatment of newborn hMRP8-AML1-ETO transgenic mice and their wild-type littermates with a strong DNA-alkylating mutagen, N-ethyl-N-nitrosourea, 55% of transgenic mice developed AML and the other 45% of transgenic mice and all of the wild-type littermates developed acute T lymphoblastic leukemia. Our results provide direct evidence that AML1-ETO is critical for causing myeloid leukemia, but one or more additional mutations are required for leukemogenesis. The hMRP8-AML1-ETO-transgenic mice provide an excellent model that can be used to isolate additional genetic events and to further understand the molecular pathogenesis of AML1-ETO-related leukemia.
Animals↗
Nature of e(g) electron order in La(1-x)Sr(1+x)MnO(4).
X-ray scattering measurements of the low-temperature structure of La(1-x)Sr(1+x)MnO(4) ( 0.33< or =x< or =0.67) indicate the existence of three distinct regions: a disordered phase (x<0.4), a charge-ordered phase (x> or =0.5), and a mixed phase (0.4< or =x<0.5). For x>0.5, the modulation vector associated with the charge order is incommensurate with the lattice and depends linearly on the concentration of e(g) electrons. The primary superlattice reflections are strongly suppressed along the modulation direction and the higher harmonics are weak, implying the existence of a largely transverse and nearly sinusoidal structural distortion, consistent with a charge-density wave of the e(g) electrons.
Journal Article↗
SNARE-dependent signaling at the Drosophila wing margin.
The wing of Drosophila melanogaster has long been used as a model system to characterize intermolecular interactions important in development. Implicit in our understanding of developmental processes is the proper trafficking and sorting of signaling molecules, although the precise mechanisms that regulate membrane trafficking in a developmental context are not well studied. We have therefore chosen the Drosophila wing to assess the importance of SNARE-dependent membrane trafficking during development. N-Ethylmaleimide-sensitive fusion protein (NSF) is a key component of the membrane-trafficking machinery and we constructed a mutant form of NSF whose expression we directed to the developing wing margin. This resulted in a notched-wing phenotype, the severity of which was enhanced when combined with mutants of VAMP/Synaptobrevin or Syntaxin, indicating that it results from impaired membrane trafficking. Importantly, we find that the phenotype is also enhanced by mutations in genes for wingless and components of the Notch signaling pathway, suggesting that these signaling pathways were disrupted. Finally, we used this phenotype to conduct a screen for interacting genes, uncovering two Notch pathway components that had not previously been linked to wing development. We conclude that SNARE-mediated membrane trafficking is an important component of wing margin development and that dosage-sensitive developmental pathways will act as a sensitive reporter of partial membrane-trafficking disruption.
Animals↗
Conformational regulation of the fibronectin binding and alpha 3beta 1 integrin-mediated adhesive activities of thrombospondin-1.
The recognition of extracellular matrix components can be regulated by conformational changes that alter the activity of cell surface integrins. We now demonstrate that conformational regulation of the matrix glycoprotein thrombospondin-1 (TSP1) can also modulate its binding to an integrin receptor. F18 1G8 is a conformation-sensitive TSP1 antibody that binds weakly to soluble TSP1 in the presence of divalent cations. However, binding of the antibody to melanoma cells was strongly stimulated by adding exogenous TSP1 in the presence of calcium, suggesting that TSP1 undergoes a conformational change following its binding to the cell surface. This conformation was not induced by known cell surface TSP1 receptors, whereas binding of F18 was stimulated when TSP1 bound to fibronectin but not to heparin or fibrinogen. Conversely, binding of F18 to TSP1 enhanced TSP1 binding to fibronectin. Exogenous fibronectin also stimulated TSP1-dependent binding of F18 to melanoma cells. Binding of the fibronectin-TSP1 complex to melanoma cells was mediated by alpha4beta1 and alpha5beta1 integrins. Furthermore, binding to F18 or fibronectin strongly enhanced the adhesive activity of immobilized TSP1 for some cell types. This enhancement of adhesion was mediated by alpha3beta1 integrin and required that the alpha3beta1 integrin be in an active state. Fibronectin also enhanced TSP1 binding to purified alpha3beta1 integrin. Therefore, both fibronectin and the F18 antibody induce conformational changes in TSP1 that enhance the ability of TSP1 to be recognized by alpha3beta1 integrin. The conformational and functional regulation of TSP1 activity by fibronectin represents a novel mechanism for extracellular signal transduction.
Animals↗
Optimized analytical method for cyclosporin A by high-performance liquid chromatography-electrospray ionization mass spectrometry.
The Micromass Platform LCZ mass detector parameters were optimized for simultaneous recording of the protonated (CsA-H+), sodium adduct (CsA-Na+) and potassium adduct (CsA-K+) of cyclosporin A eluted from a Symmetry Shield RP8 column. The optimized procedure allows a precise analysis of CsA in whole blood or serum without removal of salts prior to analysis. The ratio of the three forms of CsA varied depending on the assay condition and the types of specimens being analyzed. The summation of three ionic forms of CsA detected by LC-ESI-MS is a reliable and simple method to assess CsA concentration in the blood.
Cations, Monovalent↗
Induction of hepatitis B virus-specific cytotoxic T lymphocytes response in vivo by filamentous phage display vaccine.
The ability of inducing MHC class I restricted cytotoxic T lymphocytes response in vivo via recombinant filamentous phage was investigated. The recombinant filamentous phage particles that displayed the Hepatitis B virus epitope S(28--39) were injected into BALB/c (H-2d) mice without adjuvants. A MHC class I restricted HBs specific CTL response was found 8 days after injection. The potentiality of using the recombinant filamentous phage as anti-virus vaccine was discussed.
Animals↗
N(1)-C(5')-linked dimer hydrates of 5-substituted uracils produced by anodic oxidation in aqueous solution.
Electrochemical dimerization reactivity has been studied for 5-substituted uracils (5XU) including thymine (1a: X = Me) and 5-halouracil derivatives (1b: X = F; 1c: X = Cl; 1d: X = Br; 1e: X = I). Upon galvanostatic electrolysis of Ar-saturated aqueous solution 1a underwent anodic oxidation to produce N(1)-C(5')- and N(1)-C(6')-linked dimer hydrates, 1-(6'-hydroxy-5',6'-dihydrothymin-5'-yl)thymine (5a) and 1-(5'-hydroxy-5',6'-dihydrothymin-6'-yl)thymine (6a), as the major products. These N-C-linked dimerizations were accompanied by the formation of novel stereoisomeric C(5)-C(5')-linked dimers (meso isomer: 13a[meso]; racemic isomer: 13a[rac]) with a condensed tetrahydrofuran ring skeleton. Similar electrolyses of 5-fluorouracil (1b) and 5-chlorouracil (1c) also afforded the corresponding N(1)-C(5')-linked dimer hydrates, 1-(5'-fluoro-6'-hydroxy-5',6'-dihydrouracil-5'-yl)-5-fluorouracil (5b) and 1-(5'-chloro-6'-hydroxy-5',6'-dihydrouracil-5'-yl)-5-chlorouracil (5c), respectively, while resulting in neither N(1)-C(6')-linked dimer analogues nor C(5)-C(5')-linked dimers, unlike the reactivity of 1a. In contrast to 1a-c, no dimeric products were obtained from 5-bromouracil (1d) and 5-iodouracil (1e). The present electrochemical method was applicable to the cross-dimerization into N(1)-C(5')-linked heterodimer hydrates composed of binary 5-substituted uracils that occurred in competition with the formation of homodimer hydrates. A mechanism of the N(1)-C(5')-linked dimerization of 1a-c has been proposed, by which allyl-type radical intermediates with limiting mesomeric forms of N(1)-centered and C(5)-centered pyrimidine radicals (2a-c [N(1)]/2a-c [C(5)]) are generated via anodic one-electron oxidation and subsequent deprotonation at N(1) and undergo a head-to-tail coupling.
Dimerization↗
The mouse lens fiber-cell intrinsic membrane protein MP19 gene (Lim2) and granule membrane protein GMP-17 gene (Nkg7): Isolation and sequence analysis of two neighboring genes.
PURPOSE: The lens fiber cell intrinsic membrane protein MP19 appears to play a key role in lens fiber cell structure or communication, and thus cataractogenesis. The goal of this study was to isolate and characterize the entire gene structure of the MP19 gene, termed Lim2, and to investigate gene sequences surrounding this lens-specific gene. METHODS: A 129/SvJ mouse genomic DNA library was screened using radioisotope labeled bovine MP19 cDNA. From this screening, an 11 kb genomic fragment was isolated which contained the entire Lim2 gene, and a neighboring gene, Nkg7, which codes for a 17 kDa granulocyte membrane protein termed GMP-17. The nucleotide sequence of this entire fragment was obtained using double strand automated sequencing techniques. Using CAT and green fluorescent protein reporter constructs, Lim2 5'-upstream promoter sequences were analyzed. RESULTS: An 11,182 base pair genomic clone containing the entire murine Lim2 gene and another downstream gene, Nkg7, was obtained and completely sequenced. These two genes are only 1,182 base pairs apart, from the poly(A) signal of the Lim2 gene to the published transcriptional start site of Nkg7. Interestingly, the protein coded for by Nkg7, GMP-17, is very similar to the product of the lens Lim2 gene, MP19, in many respects. Both proteins are transmembrane proteins, with each having 4 transmembrane loops. The amino acid sequence of the two proteins is 34% identical, and 49% with respect to similar amino acids. The size of mouse Lim2 is 5,896 base pairs from the transcriptional start site to the poly(A) signal, and contains five exons and four introns. Exons 2-5 of the Lim2 gene encode a polypeptide of 173 amino acids, having over 92% identity to human MP19. Using chloramphenicol acetyltransferase (CAT) and green fluorescent protein (GFP) reporter constructs, it was determined that about 160 bp of sequence upstream from the start of transcription is both necessary and sufficient for efficient expression levels as well as tissue specificity of expression. CONCLUSIONS: The mouse Lim2 gene is very similar to the human LIM2 gene, both having the same number of exons and introns. The coding nucleotide sequences from both species are 88% identical, and 92% identical at the amino acid level. In the immediate 5'-upstream region of these two genes, several highly conserved regions are observed. Due to the similarity of the MP19 and GMP-17 proteins, it is interesting to speculate that the lens MP19 and the lymphocyte-associated GMP-17 may have originated from one primordial gene which, through genetic drift, resulted in two separate proteins having similar functions in two widely separated tissue types.
Amino Acid Sequence↗
[Microsurgery of brainstem cavernous malformation].
OBJECTIVE: To evaluate the effectiveness and benefits of surgical treatment of cavernous malformations (CMs) in brainstem. METHODS: From 1992 to 1999, 7 cases of brainstem cavernous malformations (six females and one male, aged 44 on average) with 6 lesions in pons, one in mesencephalon, and one in medulla oblongata, were operated on with microsurgery. In the recent three years neuronavigation system has been applied. RESULTS: All of the eight lesions were removed without death toll. The functional disorders of nervous system were improved after operation in four cases. The neurological deficits worsened in two cases, however, their neurological functions improved gradually to the preoperative level in four months or three years. The neurological function in the case with lesion in mesencephalon worsened after operation. Pathology revealed a grade one oligodendroglioma in addition to the CM lesion. Neuronavigation helped facilitate the accurate location of the lesions and minimize the surgical injury. CONCLUSION: Microsurgery is effective in treating brainstem cavernous malformations with appropriate indications and minimal invasive technique.
Adult↗
Chromatographic identification of a biochemical alteration in the aqueous humour of megalophthalmic Black Moor goldfish.
PURPOSE: To observe if any biochemical abnormalities exist between the eye of megalophthalmic and non-megalophthalmic goldfish by high-performance liquid chromatography (HPLC). METHOD: Aqueous humour and sera from megalophthalmic and non-megalophthalmic goldfish were subjected to HPLC and monitored by photodiode array detection (Waters, MA, USA). RESULTS: An unusual accumulation of a compound with a UV absorption maximum at 290 nm was observed in the aqueous humour of megalophthalmic eye. This compound was also present in the sera of both normal goldfish and one of its megalophthalmic mutant. However, it was significantly elevated in the aqueous humour of the megalophthalmic eye only. This compound concentration was very high in the eye of small fish and its concentration increased only slightly with the expansion of the eye in larger fish. CONCLUSIONS: The presence of this compound in the serum and aqueous humour indicates a specific systemic metabolic variation in Black Moor goldfish not seen other animal species we had studied (humans, bovine, chick, rabbits and rats). The marked elevation of this compound in the megalophthalmic eye indicates a possible association of this compound with the metabolic variation accounting for the expansion of the eye in megalophthalmic goldfish.
Animals↗
In vivo evaluation of a novel antitumor prodrug, 1-(2'-oxopropyl)-5-fluorouracil (OFU001), which releases 5-fluorouracil upon hypoxic irradiation.
PURPOSE: We previously proposed that a prodrug of 5-fluorouracil (5-FU), OFU001, is activated through capturing of hydrated electrons produced by hypoxic irradiation. Because hydrated electrons are readily deactivated by oxygen, the 5-FU release occurs specifically upon hypoxic irradiation. In this study, we investigated the in vivo efficacy, pharmacokinetics, and toxicity of OFU001. METHODS AND MATERIALS: Female 10-week-old C3H/He mice bearing SCCVII tumors were used. To measure release of 5-FU from OFU001 in vivo, the mice were given 100 mg/kg of OFU001 intraperitoneally and irradiated. Thereafter, 5-FU levels in the tumor and serum were measured by high-performance liquid chromatography. To evaluate in vivo efficacy, OFU001 was administered 30 min before irradiation, and radiation-potentiating effects were investigated by means of a tumor growth delay assay and a 50% tumor control dose (TCD-50) assay. The lethal dose of OFU001 was evaluated in the same mice. RESULTS: Following administration of OFU001 and irradiation at 30 Gy, the average 5-FU levels in the tumor and serum were 179 ng/g and 83 ng/mL, respectively. Administration of OFU001 (100-200 mg/kg) to the tumor-bearing mice before a single dose of 15-Gy irradiation produced a mean tumor growth delay of 1-5 days as compared to radiation alone (although the delay was not significant). However, no additional growth delay was observed when OFU001 was combined with 5 radiation fractions of 4 Gy each. The enhancement ratio of OFU001 in the TCD-50 assay was 1.2. No mice died after administration of 0.6-1.2 g/kg of OFU001. CONCLUSIONS: OFU001 appears to work in vivo via the proposed mechanism of activation. Although the in vivo effect of this compound was not strong enough for clinical efficacy, these results should encourage further research on the development of prodrugs of more potent anticancer agents activated through the same mechanism.
Animals↗