IGFBPrP1 accelerates autophagy and activation of hepatic stellate cells via mutual regulation between H19 and PI3K/AKT/mTOR pathway.

BACKGROUND: Our previous study found that insulin-like growth factor binding protein-associated protein (IGFBPrP1) drives hepatic stellate cells (HSCs) activation, and IGFBPrP1 and transforming growth factor ß1 (TGFß1) likely interact with each other to promote HSCs activation. TGFß1 reportedly promotes autophagy and contributes to HSCs activation; however, the mechanism between IGFBPrP1 and autophagy in liver fibrogenesis is yet unknown. Moreover, long noncoding RNA (lncRNA) H19 participates in autophagy regulation and plays a crucial function in liver fibrosis. AIMS: To define the relationship between IGFBPrP1 and autophagy and the role of H19 in IGFBPrP1-induced hepatic fibrosis. METHODS: IGFBPrP1 and autophagy were detected in bile duct ligation (BDL)-induced hepatic fibrosis. Adenovirus-mediated IGFBPrP1 was transfected into mouse liver and JS-1 cells with or without LY294002 or rapamycin to examine the effects of IGFBPrP1 on HSCs activation and autophagy as well as the PI3K/AKT/mTOR pathway. lncRNA H19 in liver fibrosis tissues and JS-1 cells induced by IGFBPrP1 were detected, then autophagy and HSCs activation level were detected in JS-1 cells by IGFBPrP1 with H19 overexpression or knowdown. RESULTS: IGFBPrP1 expression and autophagy level were concomitantly increased in liver tissue with BDL-induced hepatic fibrosis. Furthermore, we found that IGFBPrP1 stimulated autophagy and HSCs activation in vivo and in vitro, and PI3K/AKT/mTOR signaling pathway was involved in the regulation of autophagy by IGFBPrP1. In addition, H19 promoted autophagy by interacting with the PI3K/AKT/mTOR pathway in IGFBPrP1-induced HSCs activation. CONCLUSIONS: IGFBPrP1 promoted autophagy and contributed to HSCs activation via mutual regulation between H19 and the PI3K/AKT/mTOR pathway.

Insulin-like growth factor binding protein related protein 1 knockdown attenuates hepatic fibrosis via the regulation of MMPs/TIMPs in mice.

BACKGROUND: Previous research suggested that insulin-like growth factor binding protein related protein 1 (IGFBPrP1), as a novel mediator, contributes to hepatic fibrogenesis. Matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) play an essential role in hepatic fibrogenesis by regulating homeostasis and remodeling of the extracellular matrix (ECM). However, the interaction between IGFBPrP1 and MMP/TIMP is not clear. The present study was to knockdown IGFBPrP1 to investigate the correlation between IGFBPrP1 and MMP/TIMP in hepatic fibrosis. METHODS: Hepatic fibrosis was induced by thioacetamide (TAA) in mice. Knockdown of IGFBPrP1 expression by ultrasound-targeted microbubble destruction-mediated CMB-shRNA-IGFBPrP1 delivery, or inhibition of the Hedgehog (Hh) pathway by cyclopamine treatment, was performed in TAA-induced liver fibrosis mice. Hepatic fibrosis was determined by hematoxylin and eosin and Sirius red staining. Hepatic expression of IGFBPrP1, α-smooth muscle actin (α-SMA), transforming growth factor ß 1 (TGFß1), collagen I, MMPs/TIMPs, Sonic Hedgehog (Shh), and glioblastoma family transcription factors (Gli1) were investigated by immunohistochemical staining and Western blotting analysis. RESULTS: We found that hepatic expression of IGFBPrP1, TGFß1, α-SMA, and collagen I were increased longitudinally in mice with TAA-induced hepatic fibrosis, concomitant with MMP2/TIMP2 and MMP9/TIMP1 imbalance and Hh pathway activation. Knockdown of IGFBPrP1 expression, or inhibition of the Hh pathway, reduced the hepatic expression of IGFBPrP1, TGFß1, α-SMA, and collagen I and re-established MMP2/TIMP2 and MMP9/TIMP1 balance. CONCLUSIONS: Our findings suggest that IGFBPrP1 knockdown attenuates liver fibrosis by re-establishing MMP2/TIMP2 and MMP9/TIMP1 balance, concomitant with the inhibition of hepatic stellate cell activation, down-regulation of TGFß1 expression, and degradation of the ECM. Furthermore, the Hh pathway mediates IGFBPrP1 knockdown-induced attenuation of hepatic fibrosis through the regulation of MMPs/TIMPs balance.

Interaction between insulin-like growth factor binding protein-related protein 1 and transforming growth factor beta 1 in primary hepatic stellate cells.

BACKGROUND: We previously showed that insulin-like growth factor binding protein-related protein 1 (IGFBPrP1) is a novel mediator in liver fibrosis. Transforming growth factor beta 1 (TGFß1) is known as the strongest effector of liver fibrosis. Therefore, we aimed to investigate the detailed interaction between IGFBPrP1 and TGFß1 in primary hepatic stellate cells (HSCs). METHODS: We overexpressed TGFß1 or IGFBPrP1 and inhibited TGFß1 expression in primary HSCs for 6, 12, 24, 48, 72, and 96 hours to investigate their interaction and observe the accompanying expressions of α-smooth muscle actin (α-SMA), collagen I, fibronectin, and phosphorylated-mothers against decapentaplegic homolog 2/3 (p-Smad2/3). RESULTS: We found that the adenovirus vector encoding the TGFß1 gene (AdTGFß1) induced IGFBPrP1 expression while that of α-SMA, collagen I, fibronectin, and TGFß1 increased gradually. Concomitantly, AdIGFBPrP1 upregulated TGFß1, α-SMA, collagen I, fibronectin, and p-Smad2/3 in a time-dependent manner while IGFBPrP1 expression was decreased at 96 hours. Inhibition of TGFß1 expression reduced the IGFBPrP1-stimulated expression of α-SMA, collagen I, fibronectin, and p-Smad2/3. CONCLUSIONS: These findings for the first time suggest the existence of a possible mutually regulation between IGFBPrP1 and TGFß1, which likely accelerates liver fibrosis progression. Furthermore, IGFBPrP1 likely participates in liver fibrosis in a TGFß1-depedent manner, and may act as an upstream regulatory factor of TGFß1 in the Smad pathway.

A screening strategy for phenotypic detection of carbapenemase in the clinical laboratory.

Nosocomial infections caused by carbapenemase-producing Enterobacteriaceae have emerged as an important challenge worldwide and represent a great limitation for antimicrobial therapy. Detection of carbapenemase in Enterobacteriaceae species also remains challenging. Although the modified Hodge test is recommended, it lacks specificity and is unable to distinguish between carbapenemase types. Here, we demonstrated a screening strategy for the phenotypic detection of carbapenemases among Enterobacteriaceae isolates in the clinical laboratory by using ethylenediaminetetraacetic acid and phenylboronic acid. This strategy displayed an overall 100% sensitivity and 98.6% specificity for carbapenemase detection in Enterobacteriaceae, which was superior to that of the modified Hodge test (98.0% sensitivity and 84.3% specificity), and it also discriminated the carbapenemase phenotypes of KPC-2, VIM-1, and OXA-48.

PCR-based accurate synthesis of long DNA sequences.

Here we describe a simple and rapid method for assembly and PCR-based accurate synthesis (PAS) of long DNA sequences. The PAS protocol involves the following five steps: (i) design of the DNA sequence to be synthesized and of 60-bp overlapping oligonucleotides to cover the entire DNA sequence; (ii) purification of the oligonucleotides by PAGE; (iii) first PCR, to synthesize DNA fragments of 400-500 bp in length using 10 inner (template) and two outer (primer) oligonucleotides; (iv) second PCR, to assemble the products of the first PCR into the full-length DNA sequence; and (v) cloning and verification of the synthetic DNA by sequencing and, if needed, error correction using an overlap-extension PCR technique. This method, which takes approximately 1 wk, is suitable for synthesizing diverse types of long DNA molecule. We have successfully synthesized DNA fragments from 0.5 to 12.0 kb, with high G+C content, repetitive sequences or complex secondary structures. The PAS protocol therefore provides a simple, rapid, reliable and relatively inexpensive method for synthesizing long, accurate DNA sequences.

Vitreoscilla hemoglobin overexpression increases submergence tolerance in cabbage.

Agrobacterium tumefaciens was used to deliver the vhb gene into cabbage (Brassica oleracea var. Cabitata) cv. Xiaguang's parent line, 103. Using hypocotyls and cotyledon petioles as explants for infection, we obtained a transformation efficiency of 3-5% based on the number of transgenic shoots produced from the number of explants used for infection. Molecular analysis indicated that the vhb gene was stably integrated into the cabbage genome and that the vhb gene was expressed at the RNA level. Characterization of the vhb over-expressing transgenic plants revealed that transgenic seeds germinated faster than the wildtype controls. More importantly, the transgenic plants showed tolerance to a prolonged submergence treatment, suggesting that the vhb gene may provide an excellent tool for creation of submergence/flooding-tolerant cultivars of agriculturally important crops.

Different effects on ACC oxidase gene silencing triggered by RNA interference in transgenic tomato.

RNA interference (RNAi) is a potent trigger for specific gene silencing of expression in a number of organisms and is an efficient way of shutting down gene expression. 1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the oxidation of ACC to ethylene, a plant growth regulator that plays an important role in the tomato ripening process. In this research, to produce double-stranded (ds)RNA of tomato ACC oxidase, we linked the sense and antisense configurations of DNA fragments with 1,002-bp or 7-nt artificially synthesized fragments, respectively, and then placed these under the control of a modified cauliflower mosaic virus 35S promoter. The dsRNA expression unit was successfully introduced into tomato cultivar Hezuo 906 by Agrobacterium tumefaciens-mediated transformation. Molecular analysis of 183 transgenic plants revealed that the dsRNA unit was integrated into the tomato genome. With respect to the construct with the 1,002-bp linker, the severity of phenotypes indicated that 72.3% of the transformed plants had non-RNA interference, about 18.1% had semi-RNA interference, and only 9.6% had full-RNA interference. However when the construct with the 7-nt linker was used for transformation, the results were 13.0%, 18.0%, and 69.0%, respectively, indicating that the short linker was more efficient in RNAi of transgenic tomato plants. When we applied this fast way of shutting down the ACC oxidase gene, transgenic tomato plants were produced that had fruit which released traces of ethylene and had a prolonged shelf life of more than 120 days. The RNA and protein analyses indicated that there was non-RNA interference, semi-RNA interference and full-RNA interference of ACC oxidase in the transgenic tomato plants.

Isolation, characterization, and molecular cloning of the cDNA encoding a novel phytase from Aspergillus niger 113 and high expression in Pichia pastoris.

Phytases catalyze the release of phosphate from phytic acid. Phytase-producing microorganisms were selected by culturing the soil extracts on agar plates containing phytic acid. Two hundred colonies that exhibited potential phytase activity were selected for further study. The colony showing the highest phytase activity was identified as Aspergillus niger and designated strain 113. The phytase gene from A. niger 113 (phyI1) was isolated, cloned, and characterized. The nucleotide and deduced amino acid sequence identity between phyI1 and phyA from NRRL3135 were 90% and 98%, respectively. The identity between phyI1 and phyA from SK-57 was 89% and 96%. A synthetic phytase gene, phyI1s, was synthesized by successive PCR and transformed into the yeast expression vector carrying a signal peptide that was designed and synthesized using P. pastoris biased codon. For the phytase expression and secretion, the construct was integrated into the genome of P. pastoris by homologous recombination. Over-expressing strains were selected and fermented. It was discovered that ~4.2 g phytase could be purified from one liter of culture fluid. The activity of the resulting phytase was 9.5 U/mg. Due to the heavy glycosylation, the expressed phytase varied in size (120, 95, 85, and 64 kDa), but could be deglycosylated to a homogeneous 64 kDa species. An enzymatic kinetics analysis showed that the phytase had two pH optima (pH 2.0 and pH 5.0) and an optimum temperature of 60 degrees C.

A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences.

Chemical synthesis of DNA sequences provides a powerful tool for modifying genes and for studying gene function, structure and expression. Here, we report a simple, high-fidelity and cost-effective PCR-based two-step DNA synthesis (PTDS) method for synthesis of long segments of DNA. The method involves two steps. (i) Synthesis of individual fragments of the DNA of interest: ten to twelve 60mer oligonucleotides with 20 bp overlap are mixed and a PCR reaction is carried out with high-fidelity DNA polymerase Pfu to produce DNA fragments that are approximately 500 bp in length. (ii) Synthesis of the entire sequence of the DNA of interest: five to ten PCR products from the first step are combined and used as the template for a second PCR reaction using high-fidelity DNA polymerase pyrobest, with the two outermost oligonucleotides as primers. Compared with the previously published methods, the PTDS method is rapid (5-7 days) and suitable for synthesizing long segments of DNA (5-6 kb) with high G + C contents, repetitive sequences or complex secondary structures. Thus, the PTDS method provides an alternative tool for synthesizing and assembling long genes with complex structures. Using the newly developed PTDS method, we have successfully obtained several genes of interest with sizes ranging from 1.0 to 5.4 kb.

[Influence of signal peptide sequences on the expression of heterogeneous proteins in Pichia pastoris].

Pichia pastoris has been developed to be a very efficient expression host for the heterogeneous proteins since its alcohol promoter was isolated and cloned, and its transformation technique was established. For further improving the secretion expression of heterogeneous proteins, in this research, the signal sequences were studied. At first, the Saccharomyces cerevisiae mating factor alpha prepro-leader sequence was synthesized using successive PCR and designated as MF4I. Then, ten different signal sequences were constructed by adding the N-terminal residues of Pichia pastoris Aox1 protein to the N-terminal of the MF4I. These ten signal sequences were used for directing phytase gene secretion in Pichia pastoris, the secretion of phytase were increased in Pichia pastoris strains containing new signal sequence. Among these strains, the phytase secretion was highest in strain contain signal sequence added with A, I, P three Aox1 N-terminal residues; the phytase secretion of Pichia pastoris was 90 mg/L in flake. The secretion was six-fold of that with original Saccharomyces cerevisiae mating factor alpha prepro-leader sequence. In addition, insert of ten residues E E A E A E A E P K can further increase the phytase secretion by 35%, the secretion reach 120 mg/L.

[Double-antisense ACC oxidase and ACC synthase fusion gene introduced into tomato by Agrobacterium-mediated transformation and analysis the ethylene production of transgenic plants].

The tomato fruit-specific promoter 2A11 was amplified from tomato genomic DNA using PCR techniques. Total RNA was isolated from ripen fruit of tomato, then ACC oxidase gene and ACC synthase gene were obtained using reverse-transcription polymerase chain reaction. The fusion encoding ACC oxidase and ACC synthase gene was obtained through ACC oxidase gene and ACC synthase gene ligation. The fusion gene was then inserted into a plant binary vector pYPX145 in an inverted orientation. Finally, the binary plant expression vector pOSACC was constructed in which the double-antisense fusion gene was controlled by fruit-specific 2A11 promoter. By using hypocotyls and cotyledon petioles as explants, the unit of double-antisense fusion gene was successfully introduced into tomato (Lycopersicon esculentum Mill) cultivar "Hezuo 903" by Agrobacterium tumefaciens-mediated transformation. 105 transgenic plants were obtained through 200 mg/L kanamycin selection and GUS assay. Two lines of DR-1 and DR-2 were obtained through selecting the characteristics of prolonged shelf life and agriculture. The transgenic plants showed the characteristics of prolonged shelf life over 50 d. The amount of ethylene released from DR-1 and DR-2 fruits were reduced significantly to about 9.5% of that released by non-transformed controls.

[High expression of a heat-stable phytase in Pichia pastoris].

It is difficult to obtain naturally occurring phytase having the required thermostability for application in animal feeding. The 1.3 kb thermal stable phytase gene (fphy) of Aspergillus fumigatus was synthesized by using a successive PCR method for the optimal expression in Pichia pastoris. Though the nucleotides of synthetic fphy share only 74% homology with the natural gene, the amino acid sequences coded by both genes were identical. After being cloned into the yeast expression vector (pPIC9) and inserted into the chromosome of Pichia pastoris by homologous recombination, phytase was expressed in the yeast and secreted from the cell. The strains for phytase over-expression were selected out by SDS-PAGE and enzyme analysis. After fermentation in 5 L fermention tank and induced by 0.5% methanol for 60 h, about 5.6 g purified phytase was obtained per liter culture fluid. The activity of phytase was 130 000 u per microlitre fluid. The thermostable phytase remained 40% active after exposure at 90 degrees for 80 min.

[Molecular evolution of beta-glucuronidase in vitro: obtaining thermotolerant GUS gene].

The Escherichia coli beta-glucuronidase gene (gus) has been developed as a reporter gene for plants, and has been widely used for over a decade. Both chromogenic and fluorogenic GUS substrates have been synthesized, allowing rapid nonradioactive assays. The use of the Escherichia coli enzyme beta-glucuronidase (GUS) as a reporter in gene expression studies is limited by some plants and plant-associated bacteria express endogenous glucuronidase activities. The use of the enzyme as a reporter in transgenic plants is limited by high false positive. Laboratory evolution methods were used to enhance the thermostability and activity of the beta-glucuronidase. Using plasmid pBI121 as template, a 1.8 kb specific product was amplified and cloned into the vector pBluescript SK. The result of nucleotide sequence analysis was the same as reported. In vitro recombination (DNA shuffling), which involves DNase I digestion, primerless PCR, and primer PCR was used to generate mutant libraries. The mutant GUS3-3 gene was isolated after three rounds of mutation, DNA shuffling, and screening. The GUS3-3 enzyme can resistant high temperature up to 80 degrees C for 30 min. The nucleotide sequence analysis showed 99.2% homology between the GUS-ck gene from pBI121 and GUS3-3 gene. The deduced amino acid sequence demonstrated that 11 amino acid was changed. The Tm value of GUS3-3 is 80 degrees C and increased by 25 degrees C above GUS-ck (55 degrees C). The researches indicated the feasibility of the molecular evolution of beta-glucuronidase in vitro to improve enzymatic thermostability.

PCR-aided Synthesis and Stable Expression in E.coli of the cryIA (c)Bt Gene.

Insecticidal crystal proteins, known as delta-endotoxins, from a gram-positive bacterium Bacillus thuringiensis have been used as bio-pesticides for over 3 decades. By using a successive PCR method, the 1.8 kb cryIA (c)Bt gene coding for the fragment of protoxin was synthesized. Different from B. thuringiensis subsp. kurstaki Hd 73 cry gene, the synthesized gene has the codon usage pattern of an average Pseudomonas spp gene. 614 nucleotides were changed in the synthesized cryIA (c)Bt gene and the G C content was increased from 37.2% to 64%. The synthesized cryIA (c)Bt gene was cloned into pUT56 vector under the tac promoter and T1-T2 terminator. SDS-PAGE analysis revealed that 66 kD protein of the modified cryIA (c)Bt gene was expressed in E.coli and accounted for about 30% of total protein in the bacterial cells. Bioassays using crude expression products from host strains indicated that they had high toxicity to third instar larvae of the cabbage butterfly (Pieris brassicae). The LD(50) was calculated to be 0.024 &mgr;g/cm(2).