Biomedical subjects

Shyh-Jye Lee

Publications and source records attributed to Shyh-Jye Lee.

6 recordsLinked to original sources

PubMed2007

Molecular cloning, expression and phylogenetic analyses of parvalbumin in tilapia, Oreochromis mossambicus.

The gene expression of parvalbumin (Pvalb), a high-affinity calcium-binding protein and the major fish allergen, was significantly increased in the tilapia fry treated with methyltestosterone (MT) as examined using a subtractive hybridization assay. Using the real-time quantitative PCR, we further confirmed the increased Pvalb expression in the MT-treated tilapia fry. The 568 base pairs (bp) tilapia Pvalb (tPvalb) cDNA clone was fully sequenced and found to contain a coding region of 330 bp, which encodes a 108 amino acids protein with a molecular weight of 11,370.5 and an calculated isoelectric point of 4.56. The predicted secondary structure of tPvalb is comprised of seven alpha helices. It contains two characteristic EF-hand calcium-binding motifs, one PKC and five casein kinase II consensus phosphorylation sites. The tPvalb is highly homologous to the selected fish Pvalbs at a similarity ranging from 53% to 80%. The phylogenetic tree analysis showed that the tPvalb is closest to the Scomber japonicus Pvalb. The tPvalb was found to express in the heart, muscle, gill, kidney, brain and ovary of adult fish by RT-PCR analysis. In situ hybridization also revealed that the tPvalb was highly expressed in the hypothalamus and sarcoplasmic reticulum. A tPvalb glutathione S-transferase (GST) fusion protein was generated and digested by thrombin to remove the GST moiety. Further Western analysis showed that the tPvalb protein was cross-reacted to an anti-rat Pvalb antibody. Those results suggest that Pvalb is evolutionally conserved in tilapia.

Amino Acid Sequence↗

PubMed2006

Potential of a simple solid-phase extraction method coupled to analytical and bioanalytical methods for an improved determination of microcystins in algal samples.

Artemia assays and protein phosphatase assays are commonly used for the screening of microcystins (MCs) in algal samples instead of the standard mouse toxicity assay. However, it has been shown that their results are often biased because of the matrix effects. To eliminate the possible interferences in the algal matrices, a new solid-phase extraction (SPE) method using silica gel as a sorbent was developed and evaluated. Results show that this SPE method could not only reduce the toxicity of the Microcystis samples towards brine shrimp by 50-80% but also eliminate 90-100% of the endogenous phosphatase activity from Spirulina and Chlorella samples, thus improving the determination of microcystins in algal samples using either of the two bioanalytical methods. The application of this SPE method as an off-line cleanup for high-performance liquid chromatography (HPLC) with UV detection is also described in this study. After SPE, the HPLC chromatograms of Microcystis samples have clear baselines that have no interferences with the analyte peaks.

Animals↗

PubMed2006

Comparison of protein phosphatase inhibition activities and mouse toxicities of microcystins.

Eight naturally purified microcystins (MCs), including MC-LR, -FR, -WR, -RR, [d-Asp(3)]MC-FR, -WR, -RR, and [Dha(7)]MC-RR were utilized to determine the effects of amino acid substitutions and modifications on MC-induced protein phosphatase inhibition activity and mouse toxicity. Catalytic subunits of protein phosphatase 1 (PP-1) and 2A (PP-2A) were purified and subjected to the inhibition assays, and intraperitoneal injection was used to administer MCs into mice for the toxicity assay. It is found that the replacement of the non-polar amino acid l-leucine at the second position of these heptacyclic peptide toxins by a polar l-arginine reduces their mouse toxicities and inhibitory activities against PP-1 and PP-2A to different extends. Demethylation of methyldehydroalanine (Mdha) at the seventh amino acid of MC-RR exhibits the least mouse toxicity and phosphatase inhibition. The loss of a methyl group on the common methylaspartic acid (MeAsp) at the third position of MC-FR, -WR, and -RR does not alter their toxicity levels, but dominantly reduces their activities in PP-1 inhibition compared to other substitutions or modifications. This suggests that the methyl group on MeAsp is also important for MCs inhibition. However, such a tendency is not observed for PP-2A. By comparing the LD(50) values of the mouse toxicity assay and IC(50) values of the PP-1 and PP-2A inhibition assay of eight MCs using linear regression, it is evident that the MC-induced toxicity is much more related to the inhibition of PP-2A than PP-1, which suggests that PP-2A inhibition may play a major role in the MC-induced mouse toxicity.

Animals↗

PubMed2005

Probing lectin and sperm with carbohydrate-modified quantum dots.

We report the encapsulation of quantum dots with biologically important beta-N-acetylglucosamine (GlcNAc) in different ratios, together with studies of their specific/sensitive multivalent interactions with lectins and sperm by fluorimetry, transmission electron microscopy, dynamic light scattering microscopy, confocal imaging techniques, and flow cytometry. These GlcNAc-encapsulated quantum dots (QDGLNs) specifically bind to wheat germ agglutinin, and cause fluorescence quenching and aggregation. Further studies of QDGLNs and the mannose-encapsulated QDs (QDMANs) with sperm revealed site-specific interactions, in which QDGLNs bind to the head of the sperm, while QDMANs spread over the whole sperm body.

Acetylglucosamine↗

PubMed2005

Rho mediates cytokinesis and epiboly via ROCK in zebrafish.

To study the regulation of embryonic development by Rho, we microinjected Clostridium botulinum C3-exoenzyme (C3) into zebrafish embryos. We found that C3 inhibited cytokinesis during early cleavages. C3 inhibition appeared to be specific on RhoA, since the constitutively active RhoA could partially rescued the C3-induced defects. Distributions of actin and the cleavage furrow associated beta-catenin were disrupted by C3. Belbbistatin, a myosin II inhibitor, also caused blastomeres disintegration. It suggested that Rho mediates cytokinesis via cleavage furrow protein assembly and actomyosin ring constriction. Furthermore, C3 blocked cellular movements during epiboly and gastrulation as evident by the impairment on no tail and goosecoid expression in blastoderm front runner cells and the dorsal lip of blastopore, respectively. Y-27632, an antagonist of Rho-associated kinase (ROK/ROCK), had the similar inhibitory effects on zebrafish development as the C3 treatments. Taken together, these results suggest that Rho mediates cleavage furrow protein assembly during cytokinesis and cellular migration during epiboly and gastrulation via a ROK/ROCK-dependent pathway.

ADP Ribose Transferases↗

PubMed2004

Inhibition of embryonic development by microcystin-LR in zebrafish, Danio rerio.

Microcystin-LR (MC-LR), a cyanobacterial toxin, is a potent inhibitor of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A). PP1 and PP2A are critical regulators in embryonic development. However, the effects of MC-LR in embryonic development have been controversial. MC-LR has been demonstrated to be highly toxic in medaka, but not in zebrafish or rabbit embryos. The causes of difference may be due to membrane impermeability that impaired the delivery of MC-LR into cytoplasm of zebrafish and rabbit embryos. Therefore, we microinjected MC-LR directly into developing zebrafish embryos and investigated the effects of MC-LR on embryonic development. We demonstrated that MC-LR induced the lethality of zebrafish embryos in a dose- and time-dependent manner. MC-LR also induced the loss of blastomere coherence via the interference of beta-catenin and cadherins distributions. Furthermore, the MC-LR treated fry revealed various developmental defects. These results suggested that MC-LR might affect the phosphorylation equilibrium of signaling molecules, including beta-catenin and cadherins, required early in zebrafish embryonic development.

Animals↗