Subchronic toxicity study in rats evaluating herbicide-tolerant soybean DAS-68416-4.

A subchronic toxicity study was conducted in Wistar rats to evaluate the potential health effects of genetically modified (GM) herbicide-tolerant soybean DAS-68416-4. Rats were fed with diets containing toasted meal produced from GM soybean engineered with aad-12 and pat genes or containing non-GM soybean at a dose of 30.0, 15.0, or 7.5%,w/w% and 0% (control group) for 90 consecutive days. Animals were evaluated for general behavior, body weight gain, food consumption, food use efficiency, etc. At the middle and end of the study, blood and serum samples were collected for routine and biochemical assays. Internal organs were taken for calculating relative weights and doing histopathological examination. The rats were active and healthy without any abnormal symptoms during the entire study period. No biological differences in hematological or biochemical indices were detected. No histopathological changes were observed. Under the conditions of this study, herbicide-tolerant soybean DAS-68416-4 did not cause any treatment-related effects in Wistar rats following 90 days of dietary administration.

Subchronic toxicity study in rats evaluating genetically modified DAS-81419-2 soybean.

A 90-day feeding study in rats was conducted to evaluate the subchronic oral toxicity of genetically modified (GM) DAS-81419-2 soybean. Wistar rats were fed with diets containing toasted soybean meal produced from DAS-81419-2 soybean grain that expresses the Cry1F, Cry1Ac, and Pat proteins or containing conventional soybean at doses of 30.0%, 15.0%, 7.5%, or 0% (control group) for 90 consecutive days. The general behavior, body weight and food consumption were observed. At the middle and end of the experiment, blood, serum, and urine samples were collected for biochemical assays. At the conclusion of the study, the internal organs were weighed and histopathological examination was completed. The rats exhibited free movement and shiny coats without any abnormal symptoms or abnormal secretions in their noses, eyes, or mouths. There were no adverse effects on body weight in GM soybean groups and conventional soybean groups. No biological differences in hematological, biochemical, or urine indices were observed. No significant differences in relative organ weights were detected between the experimental groups and the control group. No histopathological changes were observed. Under the conditions of this study, DAS-81419-2 soybean did not cause any treatment-related effects in Wistar rats following 90 days of dietary administration.

Effects of low concentrations of di-(2-ethylhexyl) and mono-(2-ethylhexyl) phthalate on steroidogenesis pathways and apoptosis in the murine leydig tumor cell line MLTC-1.

The aim of this study was to evaluate the effects of low concentrations of DEHP and MEHP on steroidogenesis in a murine Leydig tumor cell line (MLTC-1) in vitro. The result of flow cytometry analysis revealed that the proportion of apoptotic cells was significantly increased after the exposure to DEHP. All three genes (P450scc, P450c17, and 3ßHSD) under study showed an increased expression following exposure to DEHP or MEHP, although some insignificant inhibitory effects appeared in the 10 µmol/L treatment group as compared with the controls. It was also found that compared with the controls. It was also found that DEHP or MEHP stimulated INSL3 mRNA and protein especially in the 0.001 µmol/L treatment group. Testosterone secretions were stimulated after the exposure to DEHP or MEHP. Alternations of steroidogenic enzymes and INSL3 in MLTC-1 cells might be involved in the biphasic effects of DEHP/MEHP on androgen production.

[Effects of dibutyl phthalate and monobutyl phthalate on testosterone secretion and insulin-like factor 3 expression of Leydig tumor cells in mice].

OBJECTIVE: To observe the effects of dibutyl phthalate (DBP) and monobutyl phthalate (MBP) on the mRNA and protein expression of insulin-like factor 3 (INSL3) in the Leydig tumor cells (MA-10) of mice and the level of testosterone secreted from MA-10 cells. METHODS: The MA-10 cells of mice, used as a cellular model, were exposed to DBP and MBP. The content of testosterone in the supernatant medium was measured by enzyme-linked immunosorbent assay; the mRNA and protein expression levels of INSL3 in MA-10 cells were measured by quantitative PCR and Western Blot. RESULTS: Compared with the control group, MA-10 cells showed increased synthesis of testosterone when exposed to low concentrations of DBP and MBP (10(-9) ∼ 10(-6) mol/L) and inhibited synthesis of testosterone when exposed to high concentrations of DBP and MBP (10(-3) mol/L), and the typical two-way effects became more significant as the time went one and the concentrations increased (P < 0.05). Compared with the control group, MA-10 cells showed significantly lower mRNA and protein expression levels of INSL3 when exposed to 10(-6) and 10(-4) mol/L DBP (P < 0.05); MA-10 cells showed increased protein expression of INSL3 when exposed to 10(-7) mol/L MBP, and the mRNA and protein expression levels of INSL3 decreased as the concentration of MBP increased. CONCLUSION: DBP and MBP can inhibit the secretion of testosterone from MA-10 cells at high concentrations, but stimulate the secretion of testosterone at low concentrations. Both DBP and MBP have inhibitory effects on the mRNA and protein expression of INSL3 in MA-10 cells.

Antihepatoma peptide, scolopentide, derived from the centipede scolopendra subspinipes mutilans.

BACKGROUND: Centipedes have been used to treat tumors for hundreds of years in China. However, current studies focus on antimicrobial and anticoagulation agents rather than tumors. The molecular identities of antihepatoma bioactive components in centipedes have not yet been extensively investigated. It is a challenge to isolate and characterize the effective components of centipedes due to limited peptide purification technologies for animal-derived medicines. AIM: To purify, characterize, and synthesize the bioactive components with the strongest antihepatoma activity from centipedes and determine the antihepatoma mechanism. METHODS: An antihepatoma peptide (scolopentide) was isolated and identified from the centipede scolopendra subspinipes mutilans using a combination of enzymatic hydrolysis, a Sephadex G-25 column, and two steps of high-performance liquid chromatography (HPLC). Additionally, the CCK8 assay was used to select the extracted fraction with the strongest antihepatoma activity. The molecular weight of the extracted scolopentide was characterized by quadrupole time of flight mass spectrometry (QTOF MS), and the sequence was matched by using the Mascot search engine. Based on the sequence and molecular weight, scolopentide was synthesized using solid-phase peptide synthesis methods. The synthetic scolopentide was confirmed by MS and HPLC. The antineoplastic effect of extracted scolopentide was confirmed by CCK8 assay and morphological changes again in vitro. The antihepatoma effect of synthetic scolopentide was assessed by the CCK8 assay and Hoechst staining in vitro and tumor volume and tumor weight in vivo. In the tumor xenograft experiments, qualified model mice (male 5-week-old BALB/c nude mice) were randomly divided into 2 groups (n = 6): The scolopentide group (0.15 mL/d, via intraperitoneal injection of synthetic scolopentide, 500 mg/kg/d) and the vehicle group (0.15 mL/d, via intraperitoneal injection of normal saline). The mice were euthanized by cervical dislocation after 14 d of continuous treatment. Mechanistically, flow cytometry was conducted to evaluate the apoptosis rate of HepG2 cells after treatment with extracted scolopentide in vitro. A Hoechst staining assay was also used to observe apoptosis in HepG2 cells after treatment with synthetic scolopentide in vitro. CCK8 assays and morphological changes were used to compare the cytotoxicity of synthetic scolopentide to liver cancer cells and normal liver cells in vitro. Molecular docking was performed to clarify whether scolopentide tightly bound to death receptor 4 (DR4) and DR5. qRT-PCR was used to measure the mRNA expression of DR4, DR5, fas-associated death domain protein (FADD), Caspase-8, Caspase-3, cytochrome c (Cyto-C), B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), x-chromosome linked inhibitor-of-apoptosis protein and Cellular fas-associated death domain-like interleukin-1ß converting enzyme inhibitory protein in hepatocarcinoma subcutaneous xenograft tumors from mice. Western blot assays were used to measure the protein expression of DR4, DR5, FADD, Caspase-8, Caspase-3, and Cyto-C in the tumor tissues. The reactive oxygen species (ROS) of tumor tissues were tested. RESULTS: In the process of purification, characterization and synthesis of scolopentide, the optimal enzymatic hydrolysis conditions (extract ratio: 5.86%, IC50: 0.310 mg/mL) were as follows: Trypsin at 0.1 g (300 U/g, centipede-trypsin ratio of 20:1), enzymolysis temperature of 46 °C, and enzymolysis time of 4 h, which was superior to freeze-thawing with liquid nitrogen (IC50: 3.07 mg/mL). A peptide with the strongest antihepatoma activity (scolopentide) was further purified through a Sephadex G-25 column (obtained A2) and two steps of HPLC (obtained B5 and C3). The molecular weight of the extracted scolopentide was 1018.997 Da, and the peptide sequence was RAQNHYCK, as characterized by QTOF MS and Mascot. Scolopentide was synthesized in vitro with a qualified molecular weight (1018.8 Da) and purity (98.014%), which was characterized by MS and HPLC. Extracted scolopentide still had an antineoplastic effect in vitro, which inhibited the proliferation of Eca-109 (IC50: 76.27 µg/mL), HepG2 (IC50: 22.06 µg/mL), and A549 (IC50: 35.13 µg/mL) cells, especially HepG2 cells. Synthetic scolopentide inhibited the proliferation of HepG2 cells (treated 6, 12, and 24 h) in a concentration-dependent manner in vitro, and the inhibitory effects were the strongest at 12 h (IC50: 208.11 µg/mL). Synthetic scolopentide also inhibited the tumor volume (Vehicle vs Scolopentide, P = 0.0003) and weight (Vehicle vs Scolopentide, P = 0.0022) in the tumor xenograft experiment. Mechanistically, flow cytometry suggested that the apoptosis ratios of HepG2 cells after treatment with extracted scolopentide were 5.01% (0 µg/mL), 12.13% (10 µg/mL), 16.52% (20 µg/mL), and 23.20% (40 µg/mL). Hoechst staining revealed apoptosis in HepG2 cells after treatment with synthetic scolopentide in vitro. The CCK8 assay and morphological changes indicated that synthetic scolopentide was cytotoxic and was significantly stronger in HepG2 cells than in L02 cells. Molecular docking suggested that scolopentide tightly bound to DR4 and DR5, and the binding free energies were-10.4 kcal/mol and-7.1 kcal/mol, respectively. In subcutaneous xenograft tumors from mice, quantitative real-time polymerase chain reaction and western blotting suggested that scolopentide activated DR4 and DR5 and induced apoptosis in SMMC-7721 Liver cancer cells by promoting the expression of FADD, caspase-8 and caspase-3 through a mitochondria-independent pathway. CONCLUSION: Scolopentide, an antihepatoma peptide purified from centipedes, may inspire new antihepatoma agents. Scolopentide activates DR4 and DR5 and induces apoptosis in liver cancer cells through a mitochondria-independent pathway.

Molecular Characterization of Mg-Chelatase CHLI Subunit in Pea (Pisum sativum L.).

As a rate-limiting enzyme for chlorophyll biosynthesis, Mg-chelatase is a promising target for improving photosynthetic efficiency. It consists of CHLH, CHLD, and CHLI subunits. In pea (Pisum sativum L.), two putative CHLI genes (PsCHLI1 and PsCHLI2) were revealed recently by the whole genome sequencing, but their molecular features are not fully characterized. In this study, PsCHLI1 and PsCHLI2 cDNAs were identified by PCR-based cloning and sequencing. Phylogenetic analysis showed that PsCHLIs were derived from an ancient duplication in legumes. Both PsCHLIs were more highly expressed in leaves than in other organs and downregulated by abscisic acid and heat treatments, while PsCHLI1 was more highly expressed than PsCHLI2. PsCHLI1 and PsCHLI2 encode 422- and 417-amino acid proteins, respectively, which shared 82% amino acid identity and were located in chloroplasts. Plants with a silenced PsCHLI1 closely resembled PsCHLI1 and PsCHLI2 double-silenced plants, as both exhibited yellow leaves with barely detectable Mg-chelatase activity and chlorophyll content. Furthermore, plants with a silenced PsCHLI2 showed no obvious phenotype. In addition, the N-terminal fragment of PsCHLI1 (PsCHLI1N, Val63-Cys191) and the middle fragment of PsCHLI1 (PsCHLI1M, Gly192-Ser336) mediated the formation of homodimers and the interaction with CHLD, respectively, while active PsCHLI1 was only achieved by combining PsCHLI1N, PsCHLI1M, and the C-terminal fragment of PsCHLI1 (Ser337-Ser422). Taken together, PsCHLI1 is the key CHLI subunit, and its peptide fragments are essential for maintaining Mg-chelatase activity, which can be used to improve photosynthetic efficiency by manipulating Mg-chelatase in pea.

STAT3 influences the characteristics of stem cells in cervical carcinoma.

The purpose of the study was to investigate the role of the signal transducer and activator of transcription 3 (STAT3), signal transduction protein in regulating the biological characteristics of stem cells in cervical carcinoma. Overexpressed plasmid of STAT3 was constructed and used to transfect SiHa into cervical carcinoma cells. STAT3-targeted specific siRNA was designed and produced. The effects of STAT3 upregulation (or inhibition) on the expression of NANOG, OCT4 and SOX2 markers of stem cells were measured, using western blot analysis and RT-qPCR. In addition, the tumor sphere experiment was also conducted to detect the formation of tumor spheres after the intervention of expression of STAT3 and the expression of STAT3, NANOG, OCT4 and SOX2 was detected in 35 cases of cervical carcinoma tissues and 31 cases of normal cervical tissues using immunohistochemistry. We determined whether the STAT3 overexpression plasmid was successfully constructed using enzyme digestion, PCR for bacterium solution, western blot analysis and RT-qPCR and found that the plasmid met the requirements of subsequent procedures. Compared with the empty plasmid group and STAT3 low expression group, the mRNA and protein expression of markers of stem cells, OCT4, SOX2 and NANOG were significantly elevated in the STAT3 overexpression group with statistically significant differences (P<0.05), the formation ratio of tumor spheres in the STAT3 overexpression group was also significantly higher than those in the other two groups (P<0.05). The positive expression of STAT3, OCT4, NANOG and SOX2 in the cervical squamous carcinoma group was also markedly higher than that in the chronic cervicitis group (P<0.05). This study led us to a conclusion that STAT3 can regulate the characteristics of stem cells in cervical carcinoma, and STAT3, NANOG, OCT4 and SOX2 are highly expressed in cervical squamous carcinoma, thus able to promote the progression of cervical carcinoma.

[Experimental study of transplanted endothelial progenitor cells transfected with VEGF165 gene augment the survival volume of transplanted fat tissue].

OBJECTIVE: To investigate the feasibility of transplanting endothelial progenitor cells (EPCs) transfected with VEGF165 gene to free transplanted fat tissue for increasing neovascularization and the survival. METHODS: EPCs isolated from human cord blood were cultured in vitro and identified by immunocytochemistry. After transfection by VEGF165 gene, the expression of VEGF was assessed using ELISA. Then EPCs with (VEGF gene transfection group) and without VEGF165 gene transfection (EPCs group) were transplanted to free transplanted fat tissue at 18 nude mice's back, and nine nude mice transplanted with free fat tissue were injected with M199 (control group). CM-DiI was used to trace the transplanted cells. The capillary density of transplanted fat tissue was detected by CD34 immunohistochemistry. RESULTS: EPCs expressed cell markers CD34, KDR and CD133. After transfection, the expression of VEGF was positive. Transplanted EPCs survived and proliferated, and transplanted EPCs were incorporated into the capillary networks in the transplanted fat tissue. The percent of survival volume of transplanted fat tissue of VEGF gene transfection group was (96.2 +/- 8.6)%, significantly higher than that of the EPCs group [(75.3 +/- 6.8)%, P < 0.05) and M199 group [(40.2 +/- 2.5)%, P < 0.05). The capillary density of transplanted fat tissue of VEGF gene transfection group was significantly higher than those of the EPCs group and M199 group (P < 0.05). CONCLUSIONS: EPCs from human cord blood can increase free transplanted fat tissue neovascularization and the survival volume, and the ability of promoting neovascularization of EPCs transfected with VEGF165 gene is more potent than EPCs alone.

Effects of di(n-butyl) and monobutyl phthalate on steroidogenesis pathways in the murine Leydig tumor cell line MLTC-1.

Di(n-butyl) phthalate (DBP) and its active metabolite monobutyl phthalate (MBP) have been shown to disrupt reproductive organ growth. The objective of this study was to evaluate the effects of DBP/MBP on steroidogenesis in the murine Leydig tumor cell line MLTC-1 in vitro. MLTC-1 cells were incubated with various concentrations of DBP (100, 1, 0.01, and 0µmol/l in DMSO) and MBP (1000, 10, 0.1, and 0µmol/l in DMSO) for 24h. Testosterone secretion was stimulated at the lowest doses and inhibited at higher treatment doses of DBP and MBP. The mRNA levels of the side-chain cleavage enzyme (P450scc), cytochrome p450c17 (P450c17) and 3ß-hydroxy-steroid dehydrogenase (3ßHSD) were significantly reduced in the phthalate-exposed groups, whereas, the transcription and translation of insulin-like hormone 3 (INSL3) was affected by DBP and MBP. Alterations of the steroidogenic enzymes and INSL3 in MLTC-1 cells may be involved in the biphasic effects of DBP/MBP on androgen production.

[Experiment of augmenting the survival areas of ischemic flap by transplanting endothelial progenitor cells].

OBJECTIVE: To investigate the feasibility of transplanted endothelial progenitor cells to ischemic flap with increased neovascularization and augmented the survival areas. METHODS: EPCs were isolated from human cord blood, cultured in vitro, identified by immunohistochemistry. Then EPCs were transplanted to ischemic flaps of 9 nude mice's back (experimental group), and 9 nude mice's back flaps was injected with M199(control group). And pedicle division time was 4 days after operation. CM-DiI was used to trace the transplanted cells. The blood perfusion of flaps was monitored by the laser Doppler flowetry, and the capillary density of flaps was detected by CD34 immunohistochemistry. RESULTS: EPCs expressed cell markers CD34, KDR and CD133. Transplanted EPCs survived and was incorporated into the capillary networks in the ischemic flaps of nude mice. The percent of experimental group's flap survival area was (60.3 +/- 2.1)%, significantly higher than the control group[ (34.2 +/- 1.8)%, P < 0.05 ]. The blood perfusion, capillary density of flaps of experimental group was significantly higher than the control group (P < 0.05). CONCLUSIONS: EPCs from human cord blood can increase ischemic flaps neovascularization and augment the survival areas.