Biomedical subjects

Liming Yu

Publications and source records attributed to Liming Yu.

9 recordsLinked to original sources

PubMed2006

Receptor activation alters inner surface potential during phagocytosis.

The surface potential of biological membranes varies according to their lipid composition. We devised genetically encoded probes to assess surface potential in intact cells. These probes revealed marked, localized alterations in the charge of the inner surface of the plasma membrane of macrophages during the course of phagocytosis. Hydrolysis of phosphoinositides and displacement of phosphatidylserine accounted for the change in surface potential at the phagosomal cup. Signaling molecules such as K-Ras, Rac1, and c-Src that are targeted to the membrane by electrostatic interactions were rapidly released from membrane subdomains where the surface charge was altered by lipid remodeling during phagocytosis.

Animals↗

PubMed2005

High-throughput phagocytosis assay utilizing a pH-sensitive fluorescent dye.

We describe a development of a novel high-throughput phagocytosis assay based on a pH-sensitive cyanine dye, CypHer5E, which is maximally fluorescent in an acidic environment. This dye is ideally suited for the study of phagocytosis because of the acidic conditions generated in the intracellular phagocytic vesicles after particle uptake. Use of CypHer5E-labeled particles results in greatly reduced background from noninternalized particles and makes the assay more robust. Additionally, CypHer5E-labeled particles are resistant to fluorescence quenching observed in the aggressive and acidic environment of the phagosome with traditional dyes. The CypHer5E-based assay has been shown to work reliably in a variety of cell types, including primary human monocytes, primary human dendritic cells, primary human endothelial cells, human monocytic THP-1 cell line, and human/mouse hybrid macrophage cell line WBC264-9C. Inhibition of CypHer5E bead uptake by cytochalasin D was studied, and the 50% inhibition concentration (IC50) was determined. The assay was performed in 96- and 384-well formats, and it is appropriate for high-throughput cellular screening of processes and compounds affecting phagocytosis. The CypHer5E phagocytosis assay is superior to existing protocols because it allows easy distinction of true phagocytosis from particle adherence and can be used in microscopy-based measurement of phagocytosis.

Animals↗

PubMed2004

WGA-coated yttrium oxide beads enable an imaging-based adenosine 2a receptor binding scintillation proximity assay suitable for high throughput screening.

Adenosine receptors belong to the superfamily of G protein-coupled receptors and are involved in a variety of physiologic functions. Traditionally, binding assays to detect adenosine 2a (A2a) antagonists and agonists have used filtration methods that are cumbersome to run and not amenable to HTS. We developed scintillation proximity assays (SPA trade mark ) utilizing HEK293 RBHA2AM cell membranes, either wheat germ agglutinin (WGA)-coated yttrium silicate (YSi) or red-shifted yttrium oxide (YO) beads and the A2a-selective radioligand [(3)H]SCH 58261. Both beads gave windows (total binding/nonspecific binding) of >5 and K(d) values of 2-3 nM for the radioligand, in agreement with results obtained by filtration. In contrast, WGA-polyvinyltoluene as well as other bead types had windows of <3 and significant radioligand binding to the uncoated beads. A 384-well WGA-YO bead SPA was optimized utilizing a LEADseeker imaging system and an automated trituration process for dispensing the dense yttrium-based beads. Signals were stable after 4 h, and Z' values were 0.7-0.8. The LEADseeker imaging assay tolerated 2% dimethyl sulfoxide and generated IC(50) values of 3-5 nM for the A2a antagonist CGS 15943, comparable to that obtained by the filtration method. A number of adenosine and xanthine analogues were identified as hits in the Library of Pharmacologically Active Compounds (LOPAC). This imaging-based A2a SPA enables HTS and is a major improvement over the filtration method.

Adenosine A2 Receptor Agonists↗

PubMed2004

The bHLH protein MyoR inhibits the differentiation of early embryonic endoderm.

MyoR is a bHLH protein whose expression was reported to be almost exclusively restricted to the precursors of the skeletal muscle lineage where it was postulated to function as a transcriptional repressor of myogenesis. However, previous studies in our laboratory suggested a much broader role for MyoR in embryonic cell differentiation. We demonstrated that, besides being expressed in several adult tissues of non-muscle lineage, MyoR was expressed at a much earlier stage in mammalian development than had previously been reported, that is, as early as the blastocyst stage, well before skeletal muscle specification. We also found that, as in skeletal muscle precursor cells, MyoR expression is inversely correlated with the cellular differentiative state of ectodermal, non-muscle embryonal carcinoma (EC) cells. Retinoic acid (RA) treatment of ectodermal EC or embryonal stem (ES) cells promotes their differentiation into primitive endoderm. However, in the present study, we show that the RA-induced expression of endodermal markers such as EndoA, collagen IV, and t-PA are inhibited by exogenous MyoR expression and that the level of inhibition of these markers correlates with the level of MyoR expressed. Conversely, knock-down of MyoR expression via RNA interference enhances RA-induced differentiation of EC cells, promoting earlier and much higher expression of the above-mentioned endodermal markers following RA treatment. Finally, we have narrowed the period of exogenous MyoR-induced embryonic lethality to between 3.5 and 5.5 days post-coitum (dpc), the period during which embryonic endoderm differentiates from the embryonic ectoderm. Our results suggest, therefore, that inhibition of endodermal differentiation between 3.5 and 5.5 dpc contributes to the embryonic death of mouse embryos overexpressing exogenous MyoR and consequently that MyoR may serve as a repressor of embryonal endoderm differentiation.

Animals↗

PubMed2004

[Effect of long-term suboptimal temperature and short-term low temperature under low light density on cucumber growth and its photosynthesis].

The study on the effects of long-term suboptimal temperature (T1) and short-term low temperature (T2) under low light density on cucumber seedling's growth and its photosynthesis showed that the growth speed, photosynthetic rate (Pn), carboxylation efficiency (CE), apparent quantum yield (AQY) and actual quantum yield of P(S II) electron transport (phi(PS II)) all declined remarkably, and the photochemical efficiency of PS II (Fnu/Fm) also decreased to some extent. The decreasing extents of Pn, CE and AQY of T1 were less than those of T2, while the recovery rate of T1 was slower than that of T2. The Fnu/Fm and phi(PS II) of T1 declined to less extent and resiled quickly, while those of T2 decreased to larger extent and recovered slowly. The contents of chlorophyll a, chlorophyll b and carotenoid of T1 increased remarkably after being treated, while those of T2 decreased evidently. The Chl a/b of both T1 and T2 declined remarkably. With recovery time going, the pigment of T1 declined under most conditions, while that of T2 increased gradually. Three days later, the pigment content of T1 and T2 were both higher than control. The Chl a/b had no significant change during the recovery period, and was still obviously lower than control.

Cucumis sativus↗

PubMed2003

MyoR is expressed in nonmyogenic cells and can inhibit their differentiation.

The development of skeletal muscle in mammals is promoted by the muscle-specific basic helix-loop-helix transcription factors of the MyoD family. Evidence also suggests that there are basic helix-loop-helix proteins that specifically inhibit skeletal myogenesis, including Mtwist, Mist1, and the most recently described, MyoR. It has been suggested that MyoR expression is limited to the precursors of the skeletal muscle lineage and acts as a transcriptional repressor of the muscle differentiation program. However, our results demonstrate that MyoR is expressed in several different, nonmuscle adult tissues. Furthermore, MyoR is expressed in the embryonic ectoderm of blastocyst stage mouse embryos, well before skeletal muscle specification and even before delineation of the mesodermal germ layer. Using embryonic ectoderm analogous stem cells, we demonstrate that in these nonmuscle cells, as in skeletal muscle precursor cells, expression of MyoR is inversely correlated with the extent of cellular differentiation as induced by retinoic acid. Our preliminary results indicate that overexpression of exogenous MyoR inhibits retinoic-acid-induced differentiation in EC cells and is lethal to early mouse embryos. Our results suggest a much broader role for MyoR in the repression and/or determination of embryonic cell differentiation.

Animals↗

PubMed2002

Fluorescence-based, high-throughput DNA polymerase assay.

The commonly used DNA polymerase assay is based on the detection of incorporated radiolabeled nucleotides in a DNA elongation reaction. It is laborious, radioactive, and can be highly variable. Here we report a nonradioactive fluorescence-based assay. The method consists of Cydye-labeled nucleotides, biotinylated primer, and a streptavidin-coated microplate. The assay is found to have sensitivity and dynamic range comparable to the classical radioactive method. Moreover, it has the advantages of being simple, stable, nonradioactive, and suitable for high-throughput applications. We have also found that, to ensure efficient measurement of the enzyme activity, the template DNA used in this method should have a sequence that avoids the incorporation of the fluorescence-labeled nucleotide in a consecutive way.

Base Sequence↗

PubMed2002

[A report on a large family with X-linked recessive nonsyndromic hereditary low frequency neuropathic hearing impairment].

OBJECTIVE: To analyze the genetic causes of low frequency neuropathic hearing impairment. METHODS: Using the network established by our institute, the proband of the low frequency neuropathic hearing loss pedigree was found. Then, investigation was carried out in the family from the proband. Cyrillic 2.1 software was set up to draw the pedigree and genetic characterization and phenotypes were analyzed in this family. RESULTS: One-hundred and one alive family members were investigated and the clinic audiologic examinations were performed in 43 of 101 individuals. Six of forty-three individuals appeared to be low frequency neuropathic hearing loss and all patients were males without systemic disorders except hearing loss. The clinic phenotypes were mild, middle, severe and profound hearing loss with disappearing of the auditory brainstem response(ABR) and partial normal results of distortion product otoacoustic emission (DPOAE) in the affected individuals. The onset of hearing loss was at 10-16 years old and the age of all patients was arranged from 18 to 26 years old. CONCLUSION: A large five generations family with hereditary low frequency neuropathic hearing impairment was found in our study. The genetic pattern in this family is male dominant X-linked recessive (XR) nonsyndromic hearing loss. Our findings suggest that some low frequency neuropathic disorders might be attributed to genetic factors.

Adolescent↗

PubMed2002

[KCNQ4 gene mutations affected a pedigree with autosomal dominant hereditary hearing loss].

OBJECTIVE: To investigate if the KCNQ4 gene contributes to a Chinese non-syndromic hearing loss pedigree and to detect the gene mutations in the pedigree using candidate approach. METHODS: PCR-SSCP and clone sequencing were performed to identify the mutations and polymorphism in PCR products of KCNQ4 coding sequence in the six-generations pedigree of autosomal dominant hereditary hearing loss. RESULTS: Mutations and polymorphism detection were performed on the KCNQ4 coding sequence in 36 family members of the pedigree. A molecular polymorphism marker located in the exon2 and exon3 intron sequence, which resulted from a copy variation of 47 base pairs insertion or deletion, was found in KCNQ4 sequence. CONCLUSION: A new molecular polymorphism marker with different genotypes was proved to locate at the intron sequence between at exon2 and exon3. The correlation between genotype and phenotype was analyzed. Deaf individuals were accompanied by the increase of the intron copies in the family. These findings suggest that the changes of the copies of intron between exon2 and exon3 of KCNQ4 might be a specific marker for the hearing loss of the pedigree.

Exons↗