Biomedical subjects

Maokai Gong

Publications and source records attributed to Maokai Gong.

5 recordsLinked to original sources

PubMed2006

Ethanol destabilizes liver Gal beta l, 4GlcNAc alpha2,6-sialyltransferase, mRNA by depleting a 3'-untranslated region-specific binding protein.

Asialoconjugates are viable biomarkers for alcohol abuse. We previously showed that chronic ethanol feeding down-regulated liver Gal beta l, 4GlcNAc alpha2,6-sialyltransferase (ST6Gal l) mRNA by destabilizing it. Since RNA-binding proteins are known to stabilize many eukaryotic mRNAs by interacting with the 3'-untranslated region (UTR), we have delineated the possible mechanism by which ethanol destabilizes ST6Gal l mRNA. Using (32)P-labeled RNA probes generated from a 2.7-kb 3'-UTR of ST6Gal l mRNA, we identified a liver cytosolic 41-kDa specific binding protein that interacts with its 3'-UTR domain and protects it from degradation in normal rat liver but disappears after chronic ethanol treatment. Mapping of the binding region revealed that four RNA probes of 80-base pair (bp) length spanning the 304 bp of the 3'-UTR of ST6Gal l mRNA showed equal binding intensity. The corresponding cDNA sequences for the four 80-bp RNA probes share the 13-bp consensus sequence. Mutagenesis analysis identified that four nucleotides, AG and TC, among the consensus sequences were critical for the RNA-protein interaction. Therefore, 5'-CAGCCTCCTCCCT-3' serves as a cis-element critically involved in this interaction. The RNA-protein complex formation progressively decreased with increasing dietary ethanol, resulting in its virtual disappearance with 36% of the dietary calories as ethanol. Concomitantly, the same ethanol diet decreased sialic acid index of plasma apolipoprotein J by 45% (p < 0.05). Thus, depletion of a binding protein that specifically interacts with its 3'-UTR region of ST6Gal l mRNA may account for its destabilization and consequent appearance of asialoconjugates as alcohol biomarkers.

3' Untranslated Regions↗

PubMed2005

Ultrasound-accelerated tissue fixation/processing achieves superior morphology and macromolecule integrity with storage stability.

We demonstrate that high-frequency and high-intensity ultrasound (US) can be applied to both tissue fixation and tissue processing to complete the conventional overnight formalin-fixation and paraffin-embedding (FFPE) procedures within 1 hr. US-facilitated FFPE retains superior tissue morphology and long-term room temperature storage stability than conventional FFPE. There is less alteration of protein antigenicity after US-FFPE preservation so that rapid immunohistochemical reactions occur with higher sensitivity and intensity, reducing the need for antigen retrieval pretreatment. US-FFPE tissues present storage stability so that room temperature storage up to 7 years does not significantly affect tissue morphology, protein antigenic properties, RNA distribution, localization, and quantitation. In addition, during fixation, tissue displays physical changes that can be monitored and reflected as changes in transmission US signals. As far as we know, this is the first effort to monitor tissue physical changes during fixation. Further study of this phenomenon may provide a method to control and to monitor the level of fixation for quality controls. The mechanism of less alteration of protein antigenicity by US-FFPE was discussed.

Autopsy↗

PubMed2005

Mechanism of action of ethanol in the down-regulation of Gal(beta)1, 4GlcNAc alpha2,6-sialyltransferase messenger RNA in human liver cell lines.

Gal beta l, 4GlcNAc alpha 2,6-sialyltransferase (2,6-ST) mediates the addition of alpha 2,6-linked sialic acid to glycoproteins in the Golgi compartment. Down-regulation of its gene and consequent impaired activity of 2,6-ST seems to be the major cause for the appearance of asialoconjugates in the blood of long-term alcoholics. Therefore, mechanism(s) involved in the regulation of 2,6-ST gene is important and clinically relevant. Our previous work showed that long-term ethanol feeding in rats caused a marked 59% decrease of 2,6-ST activity as well as 2,6-ST messenger RNA (mRNA) level in liver that were due to the decreased stability of its mRNA. We now mimic these actions of ethanol using ( a ) human liver HepG2 cells stably transfected with ethanol-inducible human cytochrome P4502E1 (CYP2E1 cells), or ( b ) with high alcohol dehydrogenase (HAD cells) but not in wild-type HepG2 cells lacking either of the above 2 enzymes as models. Incubation of these cells for 72 hours with 100 mmol/L ethanol caused decreases (up to 76%, P < .05) of 2,6-ST mRNA levels in CYP2E1 and HAD cells but not in the wild type. However, incubation of wild-type cells with acetaldehyde at concentrations of 50 and 100 micro mol/L showed a dramatic decrease (up to 69%, P < .02) in the 2,6-ST mRNA levels. Furthermore, exposure of CYP2E1 cells to 4-hydroxy-2-nonenal, an endogenous lipid peroxidation product of reactive oxygen species, strongly decreased 2,6-ST mRNA level by 61% ( P < .02). These results demonstrate that 2,6-ST gene is highly sensitive to ethanol action in human liver cells either via its oxidation product, acetaldehyde, or via reactive oxygen species leading to the generation of a more reactive aldehyde such as 4-hydroxy-2-nonenal. Thus, this study assumes major importance and clinical relevance because 2,6-ST gene regulation in a human liver cell model is demonstrated within a few days of ethanol exposure, whereas its in vivo regulation in liver generally takes prolonged period of ethanol exposure.

Alcohol Dehydrogenase↗

PubMed2005

A nondestructive molecule extraction method allowing morphological and molecular analyses using a single tissue section.

In clinical practice, molecular analysis of tumor specimens is often restricted by available technology for sample preparation. Virtually all current methods require homogenization of tissues for molecule extraction. We have developed a simple, rapid, nondestructive molecule extraction (NDME) method to extract proteins and nucleic acids directly from a single fixed or frozen tissue section without destroying the tissue morphology. The NDME method is based upon exposure of micron-thick tissue section to extraction buffer with the help of heating and/or intact physical forces (ultrasound and microwave) to facilitate release of macromolecules into the buffer. The extracted proteins and nucleic acids can be used directly without further purification for downstream SDS-PAGE analysis, immunoblotting, protein array, mass spectra protein profiling, PCR, and RT-PCR reactions. Most importantly, the NDME procedure also serves as an antigen retrieval treatment, so that after NDME, the same tissue section can be used for histopathological analyses, such as H&E staining, immunohistochemistry, and in situ hybridization. Thus, the NDME method allows, for the first time, both histological diagnosis and molecular analysis on a single tissue section, whether it is from frozen or fixed tissue specimens.

Animals↗

PubMed2002

The multidrug resistance transporter ABCG2 (breast cancer resistance protein 1) effluxes Hoechst 33342 and is overexpressed in hematopoietic stem cells.

The human ATP-binding cassette superfamily G (White) member 2 (ABCG2) gene and its murine homologue breast cancer resistance protein 1 (Bcrp1) are recently described ATP-binding cassette transporters associated with drug resistance in tumor cell lines, including the MCF-7 cell line, selected for its resistance to mitoxantrone (MCF-7/MitoR). Infection of MCF-7 cells with the retroviral vector containing ABCG2 cDNA (G1-ABCG2) resulted in cells (MCF-7/ABCG2) that were resistant to mitoxantrone at levels similar to those observed in MCF-7/MitoR cells. Previous studies have shown that pluripotent hematopoietic stem cells overexpress the multidrug-resistant transport (MDR1) gene and efflux rhodamine, a substrate for the MDR1 transporter. Other studies have identified a primitive hematopoietic stem cell population, or side population (SP) cells, which are identified by their efflux of the fluorescent dye, Hoechst 33342. In an attempt to identify the transport genes responsible for this phenotype, we examined the uptake of Hoechst 33342 into MCF-7, MCF-7/MitoR, and MCF-7 cells infected with a retroviral vector expressing the ABCG2 gene (MCF-7/ABCG2). MCF-7/MitoR cells as well as MCF-7/ABCG2 cells demonstrated lower levels of Hoechst 33342 uptake compared with the parental MCF-7 cells. We also examined the level of the mouse Bcrp1 RNA in SP cells and non-SP cells isolated from mouse hematopoietic cells. Mouse SP cells expressed relatively high levels of Bcrp1 mRNA relative to non-SP cells. These results suggest that Hoechst 33342 is a substrate for the ABCG2 transporter and that ABCG2/Bcrp1 expression may serve as a marker for hematopoietic stem cells in hematopoietic cells.

ATP Binding Cassette Transporter, Subfamily G, Mem↗