A Novel Approach to Cloning and Expression of Human Thymidylate Synthase

Lv, Ying-Tao,Du, Pei-Juan,Wang, Qiao-Yan,Tan, Yuan,Sun, Zong-Bin,Su, Zhong-Liang,Kang, Cong-Min Asian Pacific Journal of Cancer Prevention 2013 Asian Pacific journal of cancer prevention Vol.14 No.12

Thymidylate synthase (TS) catalyzes the transfer of a methyl group from methylenetetrahydrofolate to dUMP to form dTMP. It is a primary target in the chemotherapy of colorectal cancers and some other neoplasms. In order to obtain pure protein for analysis of structure and biological function, an expression vector TS-pET28b (+) was constructed by inserting wild-type human thymidylate synthase (hTS) cDNA into pET28b (+). Then an expression strain was selected after transformation of the recombined plasmid into Rosetta (DE3). Fusion protein with His-tag was efficiently expressed in the form of inclusion bodies after IPTG induction and the content was approximately 40.0% of total bacteria proteins after optimizing expression conditions. When inclusion bodies were washed, dissolved and purified by Ni-NTA under denatured conditions, the purity was up to 90%. On SDS-PAGE and West-blotting, the protein band was found to match well with the predicted relative molecular mass-36kDa. Bioactivity was 0.1 U/mg. The results indicated that high-level expression of wild-type hTS cDNA can be achieved in prokaryotes with our novel method, facilitating research into related chemotherapy.

<i>Yersinia pseudotuberculosis</i> Exploits CD209 Receptors for Promoting Host Dissemination and Infection

He, Ying-Xia,Ye, Cheng-Lin,Zhang, Pei,Li, Qiao,Park, Chae Gyu,Yang, Kun,Jiang, Ling-Yu,Lv, Yin,Ying, Xiao-Ling,Ding, Hong-Hui,Huang, Hong-Ping,Mambwe Tembo, John,Li, An-Yi,Cheng, Bing,Zhang, Shu-Sheng American Society for Microbiology 2019 Infection and immunity Vol.87 No.1

<P><I>Yersinia pseudotuberculosis</I> is a Gram-negative enteropathogen and causes gastrointestinal infections. It disseminates from gut to mesenteric lymph nodes (MLNs), spleen, and liver of infected humans and animals.</P><P><I>Yersinia pseudotuberculosis</I> is a Gram-negative enteropathogen and causes gastrointestinal infections. It disseminates from gut to mesenteric lymph nodes (MLNs), spleen, and liver of infected humans and animals. Although the molecular mechanisms for dissemination and infection are unclear, many Gram-negative enteropathogens presumably invade the small intestine via Peyer’s patches to initiate dissemination. In this study, we demonstrate that <I>Y. pseudotuberculosis</I> utilizes its lipopolysaccharide (LPS) core to interact with CD209 receptors, leading to invasion of human dendritic cells (DCs) and murine macrophages. These <I>Y. pseudotuberculosis</I>-CD209 interactions result in bacterial dissemination to MLNs, spleens, and livers of both wild-type and Peyer’s patch-deficient mice. The blocking of the <I>Y. pseudotuberculosis</I>-CD209 interactions by expression of O-antigen and with oligosaccharides reduces infectivity. Based on the well-documented studies in which HIV-CD209 interaction leads to viral dissemination, we therefore propose an infection route for <I>Y. pseudotuberculosis</I> where this pathogen, after penetrating the intestinal mucosal membrane, hijacks the <I>Y. pseudotuberculosis</I>-CD209 interaction antigen-presenting cells to reach their target destinations, MLNs, spleens, and livers.</P>

<i>Salmonella enterica</i> Serovar Typhimurium Interacts with CD209 Receptors To Promote Host Dissemination and Infection

Ye, Chenglin,Li, Qiao,Li, Xinyi,Park, Chae Gyu,He, Yingxia,Zhang, Yingmiao,Wu, Bicong,Xue, Ying,Yang, Kun,Lv, Yin,Ying, Xiao-Ling,Ding, Hong-Hui,Cai, Huahua,Alkraiem, Ayman Ahmad,Njiri, Olivia,Tembo, American Society for Microbiology 2019 Infection and immunity Vol.87 No.8

<P><I>Salmonella enterica</I> serovar Typhimurium, a Gram-negative bacterium, can cause infectious diseases ranging from gastroenteritis to systemic dissemination and infection. However, the molecular mechanisms underlying this bacterial dissemination have yet to be elucidated. A study indicated that using the lipopolysaccharide (LPS) core as a ligand, <I>S</I>.</P><P><I>Salmonella enterica</I> serovar Typhimurium, a Gram-negative bacterium, can cause infectious diseases ranging from gastroenteritis to systemic dissemination and infection. However, the molecular mechanisms underlying this bacterial dissemination have yet to be elucidated. A study indicated that using the lipopolysaccharide (LPS) core as a ligand, <I>S</I>. Typhimurium was able to bind human dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (hCD209a), an HIV receptor that promotes viral dissemination by hijacking antigen-presenting cells (APCs). In this study, we showed that <I>S</I>. Typhimurium interacted with CD209s, leading to the invasion of APCs and potentially the dissemination to regional lymph nodes, spleen, and liver in mice. Shielding of the exposed LPS core through the expression of O-antigen reduces dissemination and infection. Thus, we propose that similar to HIV, <I>S</I>. Typhimurium may also utilize APCs via interactions with CD209s as a way to disseminate to the lymph nodes, spleen, and liver to initiate host infection.</P>

Synthesis of (5R,8R)-2-(3,8-Dimethyl-2-oxo-1,2,4,5,6,7,8,8α-octahydroazulen-5-yl) Acrylic Acid (Rupestonic Acid) Amide Derivatives and in vitro Inhibitive Activities against Influenza A₃,B and Herpes Simplex Type 1 and 2 Virus

Yong, Jian-Ping,Lv, Qiao-Ying,Aisa, Haji Akber Korean Chemical Society 2009 Bulletin of the Korean Chemical Society Vol.30 No.2

19 Aromatic ring and L-amino acid ester contained rupestonic acid amide derivatives 2a~2l, 3a~3g were synthesized and preliminarily evaluated in vitro against influenza virus $A_3$,B and herpes simplex virus type 1 (HSV-1), 2(HSV-2) by the national center for drug screening of China. The rusults showed that 2i possessed the highest inhibition against both influenza virus $A_3\;(TC_{50}\;=\;120.6\;{\mu}mol/L,\;IC_{50}=\;19.2\;{\mu}$mol/L, SI = 6.3) and B (T$C_{50}\;=\;120.6\;{\mu}mol/L,\;IC_{50}=\;29.9\;{\mu}$mol/L, SI = 4.0); 2g was more active against influenza $A_3$ virus at very low cytotoxicity ($TC_{50}\;>\;2092.1\;{\mu}mol/L,\;IC_{50}=\;143.7\;{\mu}mol/L,$ SI > 14.6) than the parent compound; Compounds 2b, 2c, 2f showed higher activities both against HSV-1 and HSV-2 than that of the parent compound, and 2f was the most potent inhibitor of HSV-1 ($TC_{50}\;=\;200.0\;{\mu}mol/L,\;IC_{50}\;=\;11.3\;{\mu}mol$/L, SI = 17.7 ) and HSV-2 ($TC_{50}\;=\;200.0\;{\mu}mol/L,\;IC_{50}\;=\;20.7\;{\mu}mol$/L , SI = 9.7).

Synthesis of (5R,8R)-2-(3,8-Dimethyl-2-oxo-1,2,4,5,6,7,8,8α-octahydroazulen-5-yl) Acrylic Acid (Rupestonic Acid) Amide Derivatives and in vitro Inhibitive Activities against Influenza A3,B and Herpes Simplex Type 1 and 2 Virus

Jian-ping Yong,Qiao-ying Lv,Haji Akber Aisa 대한화학회 2009 Bulletin of the Korean Chemical Society Vol.30 No.2

19 Aromatic ring and L-amino acid ester contained rupestonic acid amide derivatives 2a~2l, 3a~3g were synthesized and preliminarily evaluated in vitro against influenza virus A3,B and herpes simplex virus type 1 (HSV-1), 2(HSV-2) by the national center for drug screening of China. The rusults showed that 2i possessed the highest inhibition against both influenza virus A3 (TC50 = 120.6 μmol/L, IC50 = 19.2 μmol/L, SI = 6.3) and B (TC50 = 120.6 μmol/L, IC50 = 29.9 μmol/L, SI = 4.0); 2g was more active against influenza A3 virus at very low cytotoxicity (TC50 > 2092.1 μmol/L, IC50 = 143.7 μmol/L, SI > 14.6) than the parent compound; Compounds 2b, 2c, 2f showed higher activities both against HSV-1 and HSV-2 than that of the parent compound, and 2f was the most potent inhibitor of HSV-1 (TC50 = 200.0 μmol/L, IC50 = 11.3 μmol/L, SI = 17.7 ) and HSV-2 (TC50 = 200.0 μmol/L, IC50 = 20.7 μmol/L , SI = 9.7).

Expression of RUNX2/LAPTM5 in the Induction of MC3T3-e1 Mineralization and Its Possible Relationship with Autophagy

Xing Lei,Li Yanqin,Li Wenhao,Liu Rong,Geng Yuanming,Ma Weiqun,Qiao Yu,Li Jianwen,Lv Yingtao,Fang Ying,Xu Pingping 한국조직공학과 재생의학회 2022 조직공학과 재생의학 Vol.19 No.6

BACKGROUND: The study aims to correlate osteogenesis with autophagy during the mineralization induction of MC3T3-e1 through exploring the expression of runt-related transcription factor 2 (RUNX2)/lysosomal-associated transmembrane protein 5 (LAMPT5). METHODS: The induction of mineralization in MC3T3-e1 was followed by detecting the expressions of osteogenesisrelated indexes such as RUNX2, alkaline phosphatase (ALP), osteocalcin (OCN), and LAPTM5 using RT-qPCR and Western blot from 0 to 14 days. Transmission electron microscope was utilised in visualizing the alterations of autophagosomes, which was followed by immunofluorescence detecting the subcellular localization of autophagy-related index sequestosome 1 (P62) and microtubule-associated protein 1 light 3 (LC3) protein and scrutinising the expression of P62 mRNA and P62 and LC3 proteins. RESULTS: Induction of MC3T3-e1 mineralization demonstrated an increased expression of osteogenesis-related indicators such as RUNX2, ALP, OCN, and LAPTM5 (p\0.05), as evident from the results of RT-qPCR and Western blot. Meanwhile, the expression of autophagosomes increased one day after mineralization induction and then experienced a gradual decline, and enhanced expression of LC3 protein was noted on days 1–2 of mineralization induction but was then followed by a corresponding reduce. In contrast, a continuous increase was reported in the expression of P62 mRNA and protein, respectively (p\0.05). Up- and down-regulating RUNX2/LAPTM5 expression alone confirmed the aforementioned results. CONCLUSION: It was therefore proposed that RUNX2 may be responsible for an early increase and then a gradual decrease in LAPTM5-mediated autophagy through the regulation of its high expression. Meanwhile, increased LAPTM5 expression in osteogenic mineralization presumed that RUNX2/LAPTM5 promoted autophagy and osteogenic expression, which may play a bridging role in the regulation of autophagy and osteogenesis.