Hsi-Tien Wu - Academia.edu (original) (raw)

Papers by Hsi-Tien Wu

Research in Veterinary Science, Jun 1, 2019

Myostatin (MSTN) was identified as a negative regulator of skeletal muscle growth. MSTN inhibitio... more Myostatin (MSTN) was identified as a negative regulator of skeletal muscle growth. MSTN inhibition by myostatin propeptide (MSPP) increased skeletal muscle mass, myofiber growth and muscle force. Thus, this study was designed to produce wild-type porcine MSPP (WT-MSPP) and its mutated form (D75A-MSPP) in yeast Pichia pastoris and to investigate its potential enhancement of myoblast growth and differentiation. In an in vitro study, C2C12 myoblasts were treated with the purified WT-MSPP or D75A-MSPP (10 μg/mL) in either a regular culture medium or in a differentiation medium for 72 h. In an animal trial, post-weaning C57BL/6 mice fed with a high-fat diet (HFD) were administered WT-MSPP or D75A-MSPP for 6 weeks. The results showed that C2C12 myoblasts treated with the purified WT-MSPP or D75A-MSPP could dramatically promote cell proliferation. Both myoD and myogenin were significantly increased (p < .05) after WT-MSPP or D75A-MSPP treatment. D75A-MSPP was particularly more effective than WT-MSPP in promoting myotube formation (p < .05). The postweaning mice treated with D75A-MSPP significantly increased both body and muscle weights compared with the mock and WT-MSPP groups (p < .05). Furthermore, the mice treatment with D75A-MSPP could prevent increased glucose injection from inducing glucose elevation. Our data indicated that a mutant-type MSPP (D75A-MSPP) was superior to WT-MSPP in effectively enhancing myofiber growth due to the highly resistant to proteolytic cleavage by the bone morphogenetic protein-1/tolloid (BMP-1/TLD) and thus has potential applications for clinical muscle wasting diseases or for increasing muscle mass in meat-producing animals. increases the mass, size and strength of muscle and significantly attenuates the severity of muscular dystrophy (Wagner et al., 2002). Recently, the reduced level of the MSTN protein resulted in gross muscle hypertrophy and the development of unusual strength in humans (Schuelke et al., 2004). In addition, the loss of MSTN signaling has beneficial effects on muscle degeneration and metabolic diseases in mouse models (Zhu et al., 2000; Zimmers et al., 2002). However, treatment with MSTN in adult mice induced severe loss of muscle and

Asian-australasian Journal of Animal Sciences, 2004

Construction of recombinant vectors Goat β-casein 5' regulatory region was cloned and tested to i... more Construction of recombinant vectors Goat β-casein 5' regulatory region was cloned and tested to identify its expression capability. Briefly, a 6.2 kb fragment from pGB5-1 (Huang and Lin, 1996) was cloned into the promoterless GFP expression vector pEGFP-1 (Clontech, Palo Alto, CA, USA). The new clone named pGB562/GFP, and encompassed the sequences from-4,044 to +2,123 bp of the β-casein gene 5' flanking region, exon 1 and 2 including intron 1 to drive the GFP open reading

Reproduction in Domestic Animals, Dec 1, 2003

Contents BALB/c mice are widely used in genetic, tumour and immunological studies. However, the m... more Contents BALB/c mice are widely used in genetic, tumour and immunological studies. However, the mice demonstrate a lower reproduction rate, low fertility and small litters, because of their highly genetic homozygoisty. Based on in vitro fertilization (IVF), a routine technique for biomedical studies, it is worth to evaluate the effects to BALB/c mice on IVF efficiency. In order to test the genetic factor affecting the IVF efficiency of BALB/c, four reciprocal IVF tests of BALB/cByJ and FVB/NCrl mice were performed. The results showed that the average fertility of IVF sponsored by FVB/NCrl spermatozoa was 69.6%, but only 12.1% was obtained from BALB/cByJ strain. Effect of glucose contained in the culture medium to the IVF efficiency of BALB/ cByJ was also evaluated. The results showed that the fertility of BALB/cByJ spermatozoa incubated with 0, 2.7, 5.5, 11.1 and 22.2 mM of glucose in the TYH medium were 6.8, 9.9, 13.9, 32.7 and 22.2%, respectively. It is showed that IVF efficiency of BALB/cByJ spermatozoa could be improved depending on the concentration of glucose in the IVF medium. According to the results, it is beleived that lower IVF of BALB/cByJ mice might be due to the genetic defect in spermatozoa and increasing glucose in the IVF medium which significantly affect the IVF efficiency of BALB/cByl via activating the spermatozoa.

Journal of Animal Breeding and Genetics, Jan 12, 1998

SummarySequence polymorphism of the displacement loop (D‐loop) region in mitochondrial DNA (mtDNA... more SummarySequence polymorphism of the displacement loop (D‐loop) region in mitochondrial DNA (mtDNA) is a cytoplasmic DNA marker. Since the analysis of single‐strand conformation polymorphism (SSCP) has been used for detection of substitutions in nucleotide sequence, this technique was employed to investigate the porcine sequence polymorphism in the D‐loop region. The primers were prepared to amplify the most polymorphic site with 392‐bp in the D‐loop region from whole blood of pigs. The PCR‐SSCP patterns were identical in European breeds Landrace and Duroc, but were different in Yorkshire and in Taiwanese breeds Lanyu and Taoyuan. Nucleotide sequence analysis in the 392‐bp PCR products were completely in accordance with the PCR‐SSCP patterns. There was only a single nucleotide substitution between Yorkshire and Landrace/Duroc pig and 2–3 nucleotide substitutions (dependent on the breed and individual) between Taiwanese Lanyu and Taoyuan. As many as 10–11 nucleotides were different between European and Taiwanese breeds in which most differences clustered within a certain region in the D‐loop. The results suggest that PCR‐SSCP analysis may be used for detecting single nucleotide substitution in the porcine mtDNA D‐loop region and show the diversified sequence in this region between European and Asian origins.ZusammenfassungSSCP Analyse in der D‐Schleifen‐Region porciner mitochondrialer DNA bestätigt durch Sequenz DiversitätDa Einzelstrang Konformationspolymorphismus (SSCP) zur Entdeckung von Substitutionen in der Nukleotidesequenz benutzt wird, haben wir diese Technik zur Untersuchung der D‐Schleifen Region angewendet. Es wurden Primers zur Amplifikation der am stärksten polymorphen 392 bp konstruiert. Die PCR‐SSCP Muster waren zwischen Landrasse und Duroc identisch, unterschieden sich aber bei Yorkshire und den Taiwanesichen Rassen Lanyu und Taoyuan. Die Analyse der Nukleotidsequenzen im 392 bp PCR Produkt ergaben ein identisches Bild. Yorkshire unterschied sich von Landrasse/Duroc nur in einer Substitution und die Taiwanesichen Rassen voneinander um 2 bis 3. Von den europäischen unterschieden sich diese aber in 10 bis 11 Nukleotiden, wobei die Unterschiede in einer bestimmten D‐Schleifen Region gehäuft sind. Die Ergebnisse zeigen, daß PCR‐SSCP Analyse zu Entdeckung einzelner Nukleotid Substitutionen geeignet ist und diesbezüglishe Unterschiede zwischen Rassen europäischer und asiatischer Herkunft aufzeigt.

Asian-Australasian Journal of Animal Sciences, 1995

Reproduction in Domestic Animals, 2009

The aim of this study was to generate a transgenic mouse that ubiquitously expressed enhanced gre... more The aim of this study was to generate a transgenic mouse that ubiquitously expressed enhanced green fluorescent protein (EGFP) under the control of the murine phosphoglycerate kinase 1 promoter by allotransplantation of transgenic mouse ovaries. The EGFP transgenic mice expressed green fluorescence in many organs, and the fluorescence was detected as early as the embryonic stage. Ovaries from the EGFP transgenic mice were allotransplanted into recipients and these mice were mated with normal male mice. Histological sections of EGFP-allotransplanted ovaries from the recipient mice showed the well development and formation at follicles and corpora lutea. The green fluorescence was clearly detectable at the allotransplanted section of the ovaries, which had fused with the normal ovary. The average size of the first litter from these mice was 6.8 ± 1.2 pups per recipient, and 17.8% of the pups expressed EGFP. These results demonstrated that allotransplantation of transgenic ovaries can restore a normal reproductive lifespan and can be used to generate a ubiquitously expressing EGFP animal model.

Journal of the Agricultural Association of China, Dec 1, 1997

以本實驗室所選殖之山羊β-酪蛋白基因pGB5-1為模版，構築高表現能力之啟動子（promoter）序列，設計DNA引子，以β-酪蛋白5’端調節序列至exon 2為目標，利用PCR增殖出含exon... more 以本實驗室所選殖之山羊β-酪蛋白基因pGB5-1為模版，構築高表現能力之啟動子（promoter）序列，設計DNA引子，以β-酪蛋白5’端調節序列至exon 2為目標，利用PCR增殖出含exon 2之5’端β-酪蛋白基因調節序列。將其所增殖出之片段選殖入載體內，形成新質體pGB528（2.8 kb），pGB538（3.8 kb），pGB552（5.2 kb）及pGB565（6.5 kb）。經限制酶切割與南方吸漬分析，證實其序列之正確性，做為後續基因轉殖研究所需。酪蛋白基因之表現具有組織與階段專一性，可在泌乳相關內泌素及細胞外基質（extracellular matrix）之刺激下，在乳腺上皮細胞中表現。選殖5’端調節序列並包含exon 1、intron 1及exon 2的山羊β-酪蛋白基因（-4044至 +2123），與綠色螢光蛋白（green fluorescent protein, GFP）報導基因構築成新載體pGB562/GFP。將之以脂質體（liposome）轉染至小鼠乳腺上皮細胞株NMuMG中，觀察融合基因表現能力。pGB562/GFP轉染之NMuMG細胞經過24，48及72 h培養後，其GFP之表現明顯高出控制組分別有25，55及42倍之多。另以電穿孔法（electroporation）轉形至離體培養之泌乳母鼠乳腺組織，觀察融合基因表現能力，以螢光顯微鏡或雷射共軛焦顯微鏡（laser scanning confocal microscope）觀察，結果顯示離體培養24 h之乳腺組織中出現明顯綠色螢光。由此證明所選殖之6.2 kb山羊β-酪蛋白基因5’端調節序列GB562具有優越的啟動基因表現能力，可供後續連結其他外源蛋白基因之用。另一研究主題則是嘗試以體外受精（in vitro fertilization; IVF）技術與改變體外受精培養液成分，提高受精率，作為產製基因轉殖鼠所需受精卵之來源。試驗探討影響BALB/c小鼠IVF之因素，及改變培養液葡萄糖濃度，以提高其受精率之可行性。取BALB/cByJ 與FVB/NCrl 公小鼠精子，與母小鼠經超數排卵處理之卵子，進行體外受精，評估其受精率。實驗結果顯示，以FVB/NCrl公鼠為精子供應者，其受精率平均為69.6%；而BALB/cByJ公鼠為精子供應者，其受精率平均為12.1%。再者，培養液組成...

Chinese Journal of Physiology, 2022

Cell Biology International, 2020

Adult stem cells, such as bone marrow mesenchymal stem cells (BMSCs), are postdevelopmental cells... more Adult stem cells, such as bone marrow mesenchymal stem cells (BMSCs), are postdevelopmental cells found in many bone tissues. They are capable of multipotent differentiation and have low immune‐rejection characteristics. Hepatocytes may become inflamed and produce a large number of free radicals when affected by drugs, poisoning, or a viral infection. The excessive accumulation of free radicals in the extracellular matrix (ECM) eventually leads to liver fibrosis. This study aims to investigate the restorative effects of mouse bone marrow mesenchymal stem cells (mBMSCs) on thioacetamide (TAA)‐induced damage in hepatocytes. An in vitro transwell co‐culture system of HepG2 cells were co‐cultured with mBMSCs. The effects of damage done to TAA‐treated HepG2 cells were reflected in the overall cell survival, the expression of antioxidants (SOD1, GPX1, and CAT), the ECM (COL1A1 and MMP9), antiapoptosis characteristics (BCL2), and inflammation (TNF) genes. The majority of the damage done to HepG2 by TAA was significantly reduced when cells were co‐cultured with mBMSCs. The signal transducer and activator of transcription 3 (STAT3) and its phosphorylated STAT3 (p‐STAT3), as related to cell growth and survival, were detected in this study. The results show that STAT3 was significantly decreased in the TAA‐treated HepG2 cells, but the STAT3 and p‐STAT3 of HepG2 cells were significantly activated when the TAA‐treated HepG2 co‐cultured with mBMSCs. Strong expression of interleukin (Il6) messenger RNA in co‐cultured mBMSCs/HepG2 indicated mBMSCs secret the cytokines IL‐6, which promotes cell survival through downstream STAT3 activation and aid in the recovery of HepG2 cells damaged by TAA.

Bioresource technology, Jan 19, 2017

An alkali-tolerant Chlorella sp. AT1 mutant strain was screened by NTG mutagenesis. The strain gr... more An alkali-tolerant Chlorella sp. AT1 mutant strain was screened by NTG mutagenesis. The strain grew well in pH 6-11 media, and the optimal pH for growth was 10. The CO2 utilization efficiencies of Chlorella sp. AT1 cultured with intermittent 10% CO2 aeration for 10, 20 and 30min at 3-h intervals were approximately 80, 42 and 30%, respectively. In alkaline medium (pH=11) with intermittent 10% CO2 aeration for 30min at 3-, 6- and 12-h intervals, the medium pH gradually changed to 10, and the biomass productivities of Chlorella sp. AT1 were 0.987, 0.848 and 0.710gL(-1)d(-1), respectively. When Chlorella sp. AT1 was aerated with 10% CO2 intermittently for 30min at 3-h intervals in semi-continuous cultivation for 21days, the biomass concentration and biomass productivity were 4.35gL(-1) and 0.726gL(-1)d(-1), respectively. Our results show that CO2 utilization efficiency can be markedly increased by intermittent CO2 aeration and alkaline media as a CO2-capturing strategy for alkali-tolera...

Reproduction, Fertility and Development, 2003

The 5′-regulative sequence and intron 1 of the goat β-casein gene from −4044 to +2123 bp was clon... more The 5′-regulative sequence and intron 1 of the goat β-casein gene from −4044 to +2123 bp was cloned and fused with the reporter gene of green fluorescent protein (GFP) to create a plasmid termed pGB562/GFP. To detect GFP expression, pGB562/GFP was transfected in vitro via liposomes into the mammary epithelial cell line NMuMG. Cells could not express GFP unless the transfected NMuMG cells lined up to create functional alveoli. These functional cells were cultured with lactogenic hormones, including insulin, dexamethasone and prolactin, and were grown on a layer of the extracellular matrix Matrigel. Green fluorescent protein expression levels in NMuMG cells were 25-, 55- and 42-fold those in the control group at 24, 48, and 72 h after pGB562/GFP transfection respectively. In addition, pGB562/GFP was transfected ex vivo by electroporation into mammary gland fragments and cells were then cultured in vitro with a supplement of lactogenic hormones. Strong GFP expression localized in fragm...

Biochemical and Biophysical Research Communications, 2009

Granulocyte colony-stimulating factor (G-CSF) demonstrates neuroprotective effects through differ... more Granulocyte colony-stimulating factor (G-CSF) demonstrates neuroprotective effects through different mechanisms, including mobilization of bone marrow cells. However, the influence of G-CSF-mediated mobilization of bone marrow-derived cells on injured sciatic nerves remains to be elucidated. The administration of G-CSF promoted a short-term functional recovery 7 days after crush injury in sciatic nerves. A double-immunofluorescence study using green fluorescent protein-chimeric mice revealed that bone marrow-derived CD34+ cells were predominantly mobilized and migrated into injured nerves after G-CSF treatment. G-CSF-mediated beneficial effects against sciatic nerve injury were associated with increased CD34+ cell deposition, vascular endothelial growth factor (VEGF) expression, and vascularization/angiogenesis as well as decreased CD68+ cell accumulation. However, cell differentiation and VEGF expression were not demonstrated in deposited cells. The results suggest that the promotion of short-term functional recovery in sciatic nerve crush injury by G-CSF involves a paracrine modulatory effect and a bone marrow-derived CD34+ cell mobilizing effect.

Applied Biochemistry and Biotechnology, 2011

Expression of exogenous DNA in vitro is significantly affected by the particular transfection met... more Expression of exogenous DNA in vitro is significantly affected by the particular transfection method utilized. In this study, we evaluated the efficiency of two transfection methods, chemically mediated polyethyleneimine (PEI) treatment and physically mediated electroporation, on a rat heart myoblast cell line, H9c2(2-1). After PEI transfection of pPgk-1/ EGFP into H9c2(2-1) cells, EGFP expression could be easily detected by fluorospectrometer after 48 h (210±12 RFU) and continued to increase after 72 h (243±14 RFU). However, when H9c2(2-1) cells were transfected by electroporation (200 V, 500 μF, and one pulse), low level EGFP expression was observed after 48 h (49±4 RFU) or 72 h (21±14 RFU). In contrast, the easily transfectable control CHO-K1 cell line displayed a stronger EGFP expression than the H9c2(2-1) cells either by PEI or electroporation transfection. When transfection efficiencies were assayed by flow cytometry after 72 h, 13.6±2.2% of PEI and 10.1±1.5% of electroporation (250 V, 500 μF, and two pulses) transfected cells of H9c2(2-1) expressed EGFP, and PEI-transfected cells appeared to be less damaged (viability 93.6%) as compared to electroporation-transfected cells (39.5%). However, both PEI and electroporation (580 V, 50Ω, and 50 μF) were effective for transfection of CHO-K1 with a higher efficiency, cell viability, and EGFP expression than H9c2(2-1). Our results indicate that the transfection efficiency of different methods varies among cell lines and that PEI is more efficient than electropolation for transfection of H9c2(2-1) whereas both PEI and electroporation are suitable for CHO-K1 transfection.

Plant Molecular Biology, 2014

Expression of α-amylase genes in rice is induced not only by sugar starvation and gibberellin (GA... more Expression of α-amylase genes in rice is induced not only by sugar starvation and gibberellin (GA) but also by O2 deficiency. Promoters of two rice α-amylase genes, αAmy3 and αAmy8, have been shown to direct high-level production of recombinant proteins in rice suspension cells and germinated seeds. In the present study, we modified the cis-acting DNA elements within the sugar/GA response complex (SRC/GARC) of αAmy8 promoter. We found that addition of a G box and duplicated TA box leads to high-level expression of αAmy8 SRC/GARC and significantly enhances αAmy8 promoter activity in transformed rice cells and germinated transgenic rice seeds. We also show that these modifications have drastically increased the activity of αAmy8 promoter in rice seedlings under hypoxia. Our results reveal that the G box and duplicated TA box may play important roles in stimulating promoter activity in response to hypoxia in rice. The modified αAmy8 promoter was used to produce the recombinant human epidermal growth factor (hEGF) in rice cells and hypoxic seedlings. We found that the bioactive recombinant hEGF are stably produced and yields are up to 1.8% of total soluble protein (TSP) in transformed rice cells. The expression level of synthetic hEGF containing preferred rice codon usage comprises up to 7.8% of TSP in hypoxic transgenic seedlings. Our studies reveal that the modified αAmy8 promoter can be applicable in establishing a novel expression system for the high-level production of foreign proteins in transgenic rice cells and seedlings under hypoxia.

Applied Biochemistry and Biotechnology, 2013

Matrix metalloproteinase 9 (MMP-9), a member of MMP family, is involved in many physiological pro... more Matrix metalloproteinase 9 (MMP-9), a member of MMP family, is involved in many physiological processes, including cardiovascular disease (CVD). Tumor necrosis factor-α (TNF-α) is considered a cytokine with pleiotropic biological capabilities and leads to the process of CVD when TNF-α is abnormally released and stimulates MMP-9 expression and activation. In this study, we investigated the molecular mechanism of TNF-αregulated MMP-9 expression. The experimental results confirm that TNF-α could upregulate MMP-9 expression in heart myoblast H9c2 cells of rat. To evaluate the MMP-9 regulation at transcriptional level, a DNA fragment of 2.2 kb (−2168/+18) of human mmp-9 promoter region was cloned and constructed in a vector of luciferase reporter gene. The 2.2-kb sequences were identified as having three candidate nuclear factor-κ B (NF-κB) binding sites: NF-κB I (−1418/−1409), NF-κB II (−626/−617), and NF-κB III (−353/−345). A series of reporter vectors with the mutated NF-κB sites of mmp-9 promoter sequences were constructed and transfected into H9c2 cells. The results show that the NF-κB II binding site (−626/−617) within the promoter region of mmp-9 plays a key role in upregulation of mmp-9 expression by TNF-α induction. In addition, we also first identified that the NF-κB I, similar to c-Rel, might be one of the NF-κB families to regulate mmp-9 expression.

Sustainability

Microalgae-based carbon dioxide (CO2) biofixation and biorefinery are the most efficient methods ... more Microalgae-based carbon dioxide (CO2) biofixation and biorefinery are the most efficient methods of biological CO2 reduction and reutilization. The diversification and high-value byproducts of microalgal biomass, known as microalgae-based biorefinery, are considered the most promising platforms for the sustainable development of energy and the environment, in addition to the improvement and integration of microalgal cultivation, scale-up, harvest, and extraction technologies. In this review, the factors influencing CO2 biofixation by microalgae, including microalgal strains, flue gas, wastewater, light, pH, temperature, and microalgae cultivation systems are summarized. Moreover, the biorefinery of Chlorella biomass for producing biofuels and its byproducts, such as fine chemicals, feed additives, and high-value products, are also discussed. The technical and economic assessments (TEAs) and life cycle assessments (LCAs) are introduced to evaluate the sustainability of microalgae CO2...

Experimental Animals, 2018

Angiotensin converting enzyme II (ACE2), an angiotensin converting enzyme (ACE) homologue that di... more Angiotensin converting enzyme II (ACE2), an angiotensin converting enzyme (ACE) homologue that displays antagonist effects on ACE/angiotensin II (Ang II) axis in renin-angiotensin system (RAS), could play a protective role against liver damages. The purpose of this study is to investigate whether inflammation-mediated liver injury could be affected by ACE2 derived pathways in the RAS. Eight-weeks-old wild-type (WT; C57BL/6) and Ace2 KO (hemizygous Ace2-/y) male mice were used to induce liver fibrosis by thioacetamide (TAA) administration (0, 100, and 200 mg/kg BW). The mice administrated with TAA could be successfully induced liver fibrosis in a TAA-dose dependent manner. Compared to WT mice, the results show that Ace2 KO mice have high sensitive, and developed more serious reaction of hepatic inflammation and fibrosis by TAA administration. The physiological and pathological examinations demonstrated higher serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) levels, infiltration of white blood cells and fibrotic lesions within liver in the Ace2 KO mice. The severe liver damage of Ace2 KO mice were also confirmed by the evidence of higher expression of hepatic inflammation-related genes (IL-6 and Tnf) and fibrosis-related genes (Col1a1, Timp1 and Mmp9). Ace2 gene deficiency could lead to a severe inflammation and collagen remodeling in the liver administrated by TAA, and the responses lead the pathogenesis of liver fibrosis. Our studies provided the main messages and favorable study directions of relationship of Ace2 and liver disease.