ABSTRACT
Streptomyces Sp FJS31-2 is a strain isolated from special habitat soils in the early stage of our laboratory for producing a new type of halogenated type II polyketide antibiotic with good anti-MRSA activity. In this experiment, a variety of chromatographic and spectroscopic methods was used to isolate and identify a milbemycin compound VM48130 from the ethyl acetate extract of the fermentation products. To investigate its bioactivity, Cell Counting Kit-8 (CCK-8) assay was used to test the cytotoxic activity of the compound against a variety of cancer cells (human liver cancer cell line MHCC97H and SK-Hep1, human nasopharyngeal carcinoma cell line CNE1, mouse melanoma cell line B16, human colon cancer cell line LOVO, human lung adenocarcinoma cell line A549) and normal cells (human bronchial epithelial cell line 16HBE, human normal liver cell line L02, human nasopharyngeal epithelial cell line NP69). The results showed that the compound had significant cytotoxic activity against the above cancer cells, and the IC50 values were 21.96 ± 1.45, 22.18 ± 0.55, 19.42 ± 0.71, 18.61 ± 1.68, 18.62 ± 0.67, 18.52 ± 0.64 µM, respectively. Furthermore, the CCK-8 method was used to evaluate the compound's reversal of cisplatin resistance in multidrug resistant cisplatin-resistant human lung adenocarcinoma (A549/DDP) cells. The results indicated that when the compound concentration was 0.5 µM, the reversal fold (RF) reached 6.25 and showed a dose-dependent effect. At 5 µM, the RF reached 8.35, which was approximately equivalent to the reversal effect of the positive drug verapamil at the same concentration. The expression of MDR1, MRP1, LRP, MAST1 resistance genes and the corresponding proteins were analyzed by quantitative RT-PCR and Western blot assay, and found that the compound could significantly down-regulate the expression of these genes and proteins. These results indicated that VM48130 had the potential of being a lead compound for the treatment or adjuvant treatment of cancer.
Subject(s)
Adenocarcinoma of Lung/drug therapy , Antineoplastic Agents/pharmacology , Cisplatin/pharmacology , Drug Resistance, Neoplasm/drug effects , Lung Neoplasms/drug therapy , Macrolides/pharmacology , Streptomyces , A549 Cells , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B/metabolism , Adenocarcinoma of Lung/genetics , Adenocarcinoma of Lung/metabolism , Adenocarcinoma of Lung/pathology , Antineoplastic Agents/isolation & purification , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm/genetics , Gene Expression Regulation, Neoplastic , Humans , Inhibitory Concentration 50 , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Macrolides/isolation & purification , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/metabolism , Streptomyces/chemistry , Vault Ribonucleoprotein Particles/genetics , Vault Ribonucleoprotein Particles/metabolismABSTRACT
A novel antifreeze protein cDNA was cloned by RT-PCR from the larva of the yellow mealworm Tenebrio molitor. The coding fragment of 339 bp encodes a protein of 112 amino acid residues and was fused to the expression vectors pET32a and pTWIN1. The resulted expression plasmids were transformed into Escherischia coli strains BL21 (DE3), ER2566, and Origami B (DE3), respectively. Several strategies were used for expression of the highly disulfide-bonded beta-helix-contained protein with the activity of antifreeze in different expression systems. A protocol for production of refolded and active T. molitor antifreeze protein in bacteria was obtained.
Subject(s)
Antifreeze Proteins/metabolism , Escherichia coli/metabolism , Gene Expression Regulation , Tenebrio/metabolism , Amino Acid Sequence , Animals , Antifreeze Proteins/chemistry , Antifreeze Proteins/genetics , Base Sequence , Cloning, Molecular , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Escherichia coli/genetics , Molecular Sequence Data , Temperature , Tenebrio/chemistry , Tenebrio/geneticsABSTRACT
CD9 is a member of the tetraspanin family proteins and has recently been shown to be essential for sperm-oocyte fusion in mice. The giant panda (Ailuropoda melanoleuca) CD9 (gpCD9) cDNA was amplified for the first time by RT-PCR from ovary total RNA and cloned, sequenced and analyzed. The result revealed that the open reading frame (ORF) of gpCD9 was 681 bp, which has the same length as that of mouse. Sequence analysis and structure prediction displayed that the amino acid sequence of gpCD9 is over 80% identity to those of mammals with the conserved structures, including the four transmembrane domains (TM) and certain characteristic residues. The results of sperm-egg fusion experiments demonstrated that giant panda CD9 large extracellular loop (LEL) significantly inhibited (P < 0.05) the mouse gamete fusion when the recombinant protein was added. However, when three amino acid residues TVT (173-175) of the gpCD9 were mutated to AAA, the large extracellular loop (LELM) of mutated protein was rarely inhibiting the gamete fusion of mice. Our results may be useful in improving an insight into understanding the potential mechanism of gamete fusion and genetic characteristics of giant panda.
