Biomedical subjects

Fu-Chu He

Publications and source records attributed to Fu-Chu He.

8 recordsLinked to original sources

PubMed2003

[Subcellular localization of basic Krüppel-like factor].

To understand the function of basic Krüppel-like factor (BKLF), it was confirmed by direct fluorescence and indirect fluorescence observation that hBKLF was localized in nucleus, and distributed throughout nucleoplasm in a speckled pattern, except the nucleoli. This pattern is similar to many but not all transcription factors. To clarify the specific sequence responsible for its nuclear localization, a series of deletion mutants of GFP/hBKLF were constructed. By observing their subcellular localization, it was found that the three zinc fingers of hBKLF and the N-terminal aside from the fingers all served as nuclear localization signals (NLS); the sub-NLS of hBKLF was located in the N-terminus, including the CtBP-binding motif and the proline rich domain. These results provided a basis for further clarifying the function of BKLF.

Animals↗

PubMed2003

[Transcriptional regulation of gamma- and epsilon-globin genes by basic Krüppel-like factor].

To study the transcription regulatory function of basic Krüppel-like factor (BKLF)on gamma- and epsilon-globin genes, recombinant expression vectors containing the full-length human BKLF gene, and a deletion mutant that lost N-terminal 40 amino acids, were constructed and used, respectively, to transiently transfect COS7 cells in order to assay their reporter activities. Results showed that hBKLF was able to repress the activity of gamma- and epsilon-globin gene promoters, while the antisense nucleic acid specific for hBKLF activated the transcription of these promoters. Deleting 40 amino acids from N-terminus did not influence the transcriptional repression of hBKLF. The stimulatory function of FKLF on gamma- and epsilon-globin gene promoters was also significantly reduced by hBKLF. In addition, BKLF bound the CACCC element in the SHP1 (SH2-containing protein tyrosine phosphatase 1) gene promoter. These results suggest that gamma- and epsilon-globin genes may be transcriptional targets of BKLF, providing evidence for further studies on the role of BKLF in participating the transcriptional regulation of haemocyte-specific genes.

3T3 Cells↗

PubMed2003

[cDNA cloning, subcellular localization and tissue expression of a new human Krüppel-like transcription factor: human basic Krüppel-like factor (hBKLF)].

The FLD4585 clone from the cDNA library of human fetal liver may encode a hematopoietic related transcription factor. Here we tried to clone its full-length cDNA from the 22 weeks-gestation human fetal liver and study its functional domains, genomic structure, chromosomal localization, subcellular site and expression pattern. To obtain the full-length cDNA of FLD4585 clone, 5' RACE technique was used. Bioinformatics was used to analyze its genomic structure, chromosomal localization and potential functional domains. Its subcellular localization was shown by GFP fusion technique. The expression pattern was studied by Northern blot, RT-PCR and Western blot. The results show the full-length cDNA encoded by FLD4585 clone is 1810 bp long and encodes a 345 amino acids protein with high homology to mouse BKLF (basic Krüppel-like factor). Its characteristic C-terminal three contiguous C2H2 zinc fingers place it within the family of Krüppel-like factors. Bioinformatics studies show hBKLF gene spans over 33 kb on chromosome 4p15.2-4p16.1 and contains 6 exons and 5 introns. GFP-hBKLF fusion technique showed hBKLF was present in the nuclei of COS-7 cells in a punctate pattern, whereas it was absent in the nucleoli. By Northern blot, hBKLF has two transcripts, one between 4.4 kb-7.5 kb and the other between 1.35 kb-2.4 kb. The larger transcript exists widely in human tissues. However, the smaller transcript was more restricted in blood leukocytes, liver and bone marrow. RT-PCR showed erythrocytes and granulocytes could both express hBKLF and its level increased as they matured. The expression level in fetal liver decreased as it developed towards adult liver and its hematopoietic function gradually lost. Taken together, in this paper we have successfully cloned the full length cDNA of hBKLF. Expression study suggests it may have broad functions in vivo, especially the functions in hematopoietic tissues.

Amino Acid Sequence↗

PubMed2002

Identification and characterization of a novel isoform of hepatopoietin.

AIM: To isolate a novel isoform of human HPO (HPO-205) from human fetal liver Marathon-ready cDNA and characterize its primary biological function. METHODS: 5'-RACE (rapid amplification of cDNA 5' ends) was used to isolate a novel isoform of hHPO in this paper. The constructed pcDNA(HPO-205), pcDNA(HPO) and pcDNA eukaryotic expression vectors were respectively transfected by lipofectamine method and the stimulation of DNA synthesis was observed by (3)H-TdR incorporation assay. Proteins extracted from different cells were analyzed by Western blot. RESULTS: A novel isoform of hHPO (HPO-205) encoding a 205 amino acid ORF corresponding to a translated production of 23 kDa was isolated and distinguished from the previous HPO that lacked the N-terminal 80 amino acids. The dose-dependent stimulation of DNA synthesis of HepG2 hepatoma cells by HPO-205 demonstrated its similar biological activity with HPO in vitro. The level of MAPK (Mitogen-activated protein kinase) phosphorylation by Western blot analysis revealed that HPO-205 might have the stronger activity of stimulating hepatic cell proliferation than that of HPO. CONCLUSION: A novel isoform of hHPO (HPO-205) was isolated from hepatic-derived cells. The comparison of HPO-205 and HPO will lead to a new insight into the structure and function of hHPO, and provide the new way of thinking to deeply elucidate the biological roles of HPO/ALR.

Amino Acid Sequence↗

PubMed2002

[Isolation and characterization of a novel transcript of hepatopoietin].

A novel transcript of human HPO (HPO-205) encoding a 205 amino acid ORF corresponding to a translated product of 23 kD different from the previous HPO that lacks the N-terminal 80 amino acids, was isolated from human fetal liver cDNA library by 5'RACE methods. The dose-dependent stimulation of DNA synthesis of HepG2 hepatoma cells by HPO-205 demonstrated its similar biological activity with HPO in vitro. The level of MAPK (mitogen-activated protein kinase)phosphorylation was further examined by Western blot analysis and the result revealed that HPO-205 protein might have stronger activity on stimulating hepatic cell proliferation than that of HPO. The similar result was observed by FACS technique. Therefore, our results suggest that the HPO-205 has activity to stimulate proliferation of liver derived cells, and this activity may be stronger than HPO.

Amino Acid Sequence↗

PubMed2000

Development and Preliminary Application of a Peptide Mass Fingerprinting Technique in Proteome Research.

Proteome means the total proteins expressed by the genome in a cell or tissue. Two-dimensional electrophoresis (2-DE) is now the most powerful separating technique and the key separation method used in proteome. Peptide mass fingerprinting (PMF) is becoming a widely used and vastly developed technique for protein identification in 2-D gels. In this research, a systematic method to identify the proteins in polyacrylamide gels by PMF was developed. Proteins were digested in-gel by enzyme and the masses of generated peptides were measured by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The data obtained from PMF were used in protein database search and the protein identification. The proteins from human lung cancer cells isolated by 2-DE were subjected to identification by the PMF method developed in this work. Three spots of proteins in gel were identified as G3P2_HUMAN, UBL1_ HUMAN and TPIS_HUMAN.

Journal Article↗

PubMed1998

Partial isolation and identification of hepatic stimulator substance mRNA extracted from human fetal liver.

AIM:To partially isolate and identify hepatic stimulator substance mRNA from human fetal liver tissues.METHODS:The poly (A)mRNA was extracted from human fetal liver tissues of 4-5 month gestation, fractionated by size on sucrose gradient centrifugation, translated into protein from each fraction in vitro and then its products were tested for HSS activity.RESULTS:Twenty-two 500 total RNA was obtained from human fetal liver tissues and pooled.mRNA of 420 was yielded,processed by oligo(dT)-cellulose column chromatography, then was size-fractionated by ultracentrifution on a continuous sucrose density gradient (5%-25%), and separated into 18 fractions.Translated products of mRNA in fraction 8 and 9 could produce a two-fold increase in the incorporation of( 3)H-TdR into DNA of SMMC-7721 hepatoma cells and in a heat resistant and organ-specific way.CONCLUSION:The partially purified HSS mRNA was obtained and this would facilitate the cloning of HSS using expression vectors.

Journal Article↗

PubMed1997

Expression and Activity of Recombinant Human Augmenter of Liver Regeneration.

The cDNA of human augmenter of liver regeneration was subcloned onto the downstream of the P(R)P(L) promoter of the expression plasmid pBV220. The recombinant plasmid could stably express ALR with high efficiency (up to 20% of the total bacterial proteins) in E. coli through thermal induction. The expressed recombinant ALRs could produce a significant increase in the incorporation of (3)H-TdR into liver DNA of a 1/3 hepatectomized test animal and also had a potent antihepatitis effect. These results suggest that ALR appears to be an important regulator of liver regeneration and may be used in clinical trial for enhancing liver regeneration in the treatment of hepatic diseases.

Journal Article↗