Biomedical subjects

Chunlai Wu

Publications and source records attributed to Chunlai Wu.

5 recordsLinked to original sources

PubMed2026

Generation of full-length wild-type and mutated futsch transgenes in Drosophila-efficient Gibson Assembly of ultra-large cDNAs.

Drosophila Futsch is a key microtubule-associated protein (fly homolog of MAP1B) that regulates microtubule organization, synaptic terminal growth, and neuronal development. Functional analysis of futsch has long been limited by the inability to clone and express a full-length futsch transgene, owing to its exceptional size (~16.5 kb) and extensive repetitive sequences. Here, I present an efficient and reproducible method for cloning both wild-type and mutated full-length Drosophila futsch cDNA (16,488 bp) using Gibson Assembly. These resulting cDNAs were used to generate UAS-futsch transgenes. When expressed in neurons, the wild‑type transgenic Futsch associated with microtubule and rescued the synaptic morphological defects observed in futschK68 mutants. This approach substantially reduces the time and complexity compared with traditional cloning techniques. Furthermore, I highlight common pitfalls encountered during the cloning process and provide practical solutions to enhance cloning efficiency. This protocol offers a broadly applicable and cost-effective framework for cloning otherwise intractable large cDNAs from Drosophila and other organisms.

Animals↗

PubMed2006

Preparation of xylenol orange functionalized silica gel as a selective solid phase extractor and its application for preconcentration--separation of mercury from waters.

A new selective solid phase extractor was prepared from silica gel modified with xylenol orange (SGMXO). The solid phase extractor is stable in 6molL(-1) HCl, common organic solvents, and pH 1.0-9.0 buffer solutions. In the batch experiments, Hg(II) can be adsorbed on SGMXO at pH 1.0 with 90.0% retention, whereas the retention of other common coexisting metal ions such as Cd(II), Pb(II), Cu(II), Ni(II), Co(II), Mn(II), Zn(II), and Fe(III) is less than 4.1%.. The adsorption equilibration for Hg(II) was achieved within 3min. At optimum conditions, the adsorption capacity of the extractor is 18.26micromolg(-1) of dry modified silica gel, and the preconcentration factor is as high as 333. The recovery is still higher than 95% for the preconcentration of 10ngmL(-1) Hg(II). The new solid phase extractor has been used for the preconcentration of low level of Hg(II) in surface water, tap water in chemistry laboratory and student's dormitory and a simulated sea water samples, recoveries of 98.2-100.6% were obtained. It is showed that low level of Hg(II) can be effectively preconcentrated by this new selective solid phase extractor.

Adsorption↗

PubMed2006

Differential role of 14-3-3 family members in Xenopus development.

The 14-3-3 proteins are intracellular dimeric phosphoserine/threonine binding molecules that participate in signal transduction, checkpoint control, nutrient sensing, and cell survival pathways. Previous work established that 14-3-3 proteins are required in early Xenopus laevis development by modulating fibroblast growth factor signaling. Although this general requirement for 14-3-3 proteins in Xenopus early embryogenesis is established, there is no information about the specific role of individual 14-3-3 genes. Botanical studies previously demonstrated functional specificity among 14-3-3 genes during plant development. In this study, an antisense morpholino oligo microinjection approach was used to characterize the requirement for six specific 14-3-3 family members in Xenopus embryogenesis. Microinjection experiments followed by Western blot analysis showed that morpholinos reduced specific 14-3-3 protein levels. Embryos lacking specific 14-3-3 isoforms displayed unique phenotypic defects. In particular, reduction of 14-3-3 tau (tau) protein, and to a lesser extent, 14-3-3 epsilon (epsilon), resulted in embryos with prominent gastrulation and axial patterning defects and reduced mesodermal marker gene expression. In contrast, reduction of 14-3-3 zeta (zeta) protein caused no obvious phenotypic abnormalities. Reduction of 14-3-3 gamma (gamma) protein resulted in eye defects without gastrulation abnormalities. Therefore, individual 14-3-3 genes have separable functions in vertebrate embryonic development.

14-3-3 Proteins↗

PubMed2005

Highwire function at the Drosophila neuromuscular junction: spatial, structural, and temporal requirements.

Highwire is a huge, evolutionarily conserved protein that is required to restrain synaptic growth and promote synaptic transmission at the Drosophila neuromuscular junction. Current models of highwire function suggest that it may act as a ubiquitin ligase to regulate synaptic development. However, it is not known in which cells highwire functions, whether its putative ligase domain is required for function, or whether highwire regulates the synapse during development or alternatively sets cell fate in the embryo. We performed a series of transgenic rescue experiments to test the spatial, structural, and temporal requirements for highwire function. We find that presynaptic activity of highwire is both necessary and sufficient to regulate both synapse morphology and physiology. The Highwire RING domain, which is postulated to function as an E3 ubiquitin ligase, is required for highwire function. In addition, highwire acts throughout larval development to regulate synaptic morphology and function. Finally, we show that the morphological and physiological phenotypes of highwire mutants have different dosage and temporal requirements for highwire, demonstrating that highwire may independently regulate the molecular pathways controlling synaptic growth and function.

Animals↗

PubMed2002

Role of 14-3-3 proteins in early Xenopus development.

14-3-3 proteins are intracellular dimeric phosphoserine/threonine-binding molecules that participate in signal transduction and checkpoint control pathways. 14-3-3 proteins are required for normal eye development, brain function, and terminal patterning in Drosophila melanogaster, but the role of 14-3-3 proteins in vertebrate development is undefined. In this work an unphosphorylated peptide inhibitor of 14-3-3, R18, was used to determine the role of 14-3-3 proteins in Xenopus embryonic development. Biochemical analysis demonstrated that R18 was specific and efficient at attenuating global 14-3-3 activities in Xenopus embryos. Microinjection experiments showed a requirement for 14-3-3 function in mesodermal specification. Inhibition of 14-3-3 resulted in embryos with axial patterning defects and reduced expression of mesodermal marker genes. These phenotypic defects were caused by impaired fibroblast growth factor signaling in R18-injected embryos. These results establish the importance of 14-3-3 proteins in vertebrate embryonic development.

14-3-3 Proteins↗