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To evaluate the effects of nitrite on the oxidative damage of blood cells of Grass Carp Ctenopharyngodon idella, the isolated hemocytes were exposed to nitrite (0, 1, 10, or 100 mg/L) for up to 24 h. Hemoglobin (Hb) and methemoglobin (MetHb) concentrations, reactive oxygen species (ROS) and malondialdehyde (MDA) levels, mitochondrial membrane potential (∆Ψm), and antioxidant enzyme activity were assayed to assess hematological parameters and the antioxidant defense mechanism. Results showed a remarkable decrease in Hb concentration with increasing nitrite concentration after a 24-h exposure, while the MetHb concentration increased significantly in nitrite exposure groups. The levels of ROS, ∆Ψm, and MDA increased to varying degrees with increases in nitrite exposure concentration and time. The total antioxidant capacity, catalase (CAT) activity, glutathione peroxidase (GPx) activity, and glutathione content showed a trend of rising initially and then decreasing with prolonged exposure time. Superoxide dismutase (SOD) activity was higher in the 1-mg/L nitrite exposure group and lower in the 100-mg/L group than in the control. The relative messenger RNA expression ratios of cat, sod1, and gpx were up-regulated significantly in the 1- and 10-mg/L groups and then declined in the 100-mg/L group. Therefore, it can be concluded that nitrite exposure activates the antioxidant defense mechanism of Grass Carp hemocytes and that the balance of oxidant-antioxidant homeostasis will be undermined by higher nitrite doses or longer exposure periods.

BACKGROUND Growth arrest-specific gene 2 (GAS2) plays a role in modulating in reversible growth arrest cell cycle, apoptosis, and cell survival. GAS2 protein is universally expressed in most normal tissues, particularly in the liver, but is depleted in some tumor tissues. However, the functional mechanisms of GAS2 in hepatocellular carcinoma (HCC) are not fully defined. AIM To investigate the function and mechanism of GAS2 in HCC. METHODS GAS2 expression in clinic liver and HCC specimens was analyzed by real-time PCR and western blotting. Cell proliferation was analyzed by counting, MTS, and colony formation assays. Cell cycle analysis was performed by flow cytometry. Cell apoptosis was investigated by Annexin V apoptosis assay and western blotting. RESULTS GAS2 protein expression was lower in HCC than in normal tissues. Overexpression of GAS2 inhibited the proliferation of HCC cells with wide-type p53, while knockdown of GAS2 promoted the proliferation of hepatocytes (P < 0.05). Furthermore, GAS2 overexpression impeded the G1-to-S cell cycle transition and arrested more G1 cells, particularly the elevation of sub G1 (P < 0.01). Apoptosis induced by GAS2 was dependent on p53, which was increased by etoposide addition. The expression of p53 and apoptosis markers was further enhanced when GAS2 was upregulated, but became diminished upon downregulation of GAS2. In the clinic specimen, GAS2 was downregulated in more than 60% of HCCs. The average fold changes of GAS2 expression in tumor tissues were significantly lower than those in paired non-tumor tissues (P < 0.05). CONCLUSION GAS2 plays a vital role in HCC cell proliferation and apoptosis, possibly by regulating the cell cycle and p53-dependent apoptosis pathway.

The hot compressive deformation behavior of hot isostatically pressed Ti-47. 5Al-2Cr-2Nb-0. 2W-0. 2B alloy using gas atomization powders was systematically investigated and the processing map was obtained in the temperature range of 1323−1473 K and strain rate range of 0.001−0.5 s−1 . The calculated activation energy in the above variational ranges of temperature and strain rate possesses a low activation energy value of approximately 365.6 kJ/mol based on the constitutive relationship models developed with the Arrhenius-type constitutive model respectively considering the strain rate and deformation temperature. The hot working flow behavior during the deformation process was analyzed combined with the microstructural evolution. Meanwhile, the processing maps during the deformation process were established based on the dynamic material model and Prasad instability criterion under different deformation conditions. Finally, the optimal hot processing window of this alloy corresponding to the wide temperature range of 1353−1453 K and the low strain rate of 0.001−0.1 s−1 was obtained.

An analytical approach was developed to investigate nucleation, recalescence and growth of Ti-48Al (at.%) alloy droplets during their flight in a gas atomization process. Evolution of microstructure of the solidified powders was quantitatively investigated using scanning electron microscopy, transmission electron microscopy and electron back-scatter diffraction. Relationships among the initial number of nuclei, liquid/solid interface velocity and cooling rate have been established. Results showed that statistical nucleation events increased exponentially with increasing droplet size, and the powder microstructures were transformed from a twinned spherical segment into a concentric liquid/solid interface geometry. Variations of initial temperature, cooling rate and solid fraction of the droplets as a function of cooling durations for the droplets with various sizes were investigated.

The paper focuses on the phase transformation of undercooled Ti–48Al (at.%) droplets atomized by high pressure gas. The microstructural evolution was analyzed using the transient nucleation theory in combination with the microstructural examinations. The primary phase, final phase volume fraction and the hardness are related to the droplets size to provide fundamental understanding. The competitive formation of the primary phases α and β are strongly controlled by the droplets size, and a critical diameter of about 25 μm was identified corresponding to an undercooling of 102 K. The final phase volume fraction of γ increased with the increase of powder size from 12% to 85%. The highest hardness with the value of 652 HV was obtained for powder of 50 μm in diameter.

Tubificidae is often used in the wastewater treatment systems to minimize the sludge production. A two-compartment kinetic model was applied to simulate the uptake and release of C, N and P-bearing pollutants by Tubificidae in a sludge reduction system. The nonlinear fitting method was used to calculate the uptake rate constantK1and the release rate constantK2for TOC, TN and TP, which were 4.983, 25.764, 10.154 and 3.671, 23.683, 9.023, respectively. The model fitted quite well with the collected data, suggesting that the model was applicable. The results showed that Tubificidae activity would increase the C, N and P concentrations in the system, and the specific release rates of C, N and P-bearing pollutants were 4.31 mg COD/gL. hoffmeisteri· d, 0.722 mg TN/gL. hoffmeisteri· d, and 0.14 mg TP/gL. hoffmeisteri· d,respectively. Ammonia nitrogen contributed up to 70.42% of the released nitrogen.

Low-cycle fatigue (LCF) fracture of the spray formed GH738 alloy was investigated under total strain controlled mode at 650°C. Basic theory for evaluating fatigue lives by fatigue striations has been introduced. The crack length a and the crack propagation rate da/dN were measured and the curve of da/dN in crack propagation zone was obtained with Paris formula and tabulation trapezoidal formula, respectively. The size and symmetry of rapid fracture zone of fatigue were also studied. The results showed that the relationship of a and N was linear with tabulation trapezoidal formula, and the da/dN and a was conic with Paris formula. At last, quantitative analysis of fatigue fracture was also discussed.

Purpose: Osteonecrosis of the jaw (ONJ) is a growing concern in patients who receive bisphosphonates that target osteoclasts. As osteoclasts play multifunctional roles in the bone marrow, their suppression likely affects bone homeostasis and alters wound healing of the jaw. The objective was to delineate the impact of osteoclast suppression in the bone marrow and wound healing of the jaw. Experimental Design: Zoledronate was administered to senile rats for 14 weeks. A portion of the gingiva was removed to denude the palatal bone. Gene expression in the bone marrow was assessed and histologic sections were analyzed to determine the wound healing status. Results: Angiogenesis-related genes, CD31 and VEGF-A, were not altered by zoledronate. VEGF-C, which plays a role in lymphangiogenesis, was suppressed. There was a decrease in gene expression of Tcirg1 and MMP-13. Bone denudation caused extensive osteocyte death indicative of bone necrosis. In zoledronate-treated rats, the necrotic bone was retained in the wound while, in controls, osteoclastic resorption of the necrotic bone was prominent. Even though large necrotic bone areas existed in zoledronate-treated rats, overlaying soft tissue healed clinically. Immunohistochemical staining showed rich vascularity in the overlaying soft tissue. Conclusions: Zoledronate therapy impacts bone marrow by suppressing genes associated with lymphangiogenesis and tissue remodeling, such as VEGF-C and MMP-13. Zoledronate was associated with impaired osseous wound healing but had no effect on angiogenic markers in the bone marrow or soft tissue wound healing. Zoledronate selectively blunts healing in bone but does not affect soft tissue healing in the oral cavity. Clin Cancer Res; 17(6); 1405–14. ©2010 AACR.

Haloxylon ammodendron (C. A. Mey.) Bunge is a host for the holoparasitic plant Cistanche deserticola Y. C. Ma, the original source of medicinal material known as Herba Cistanchis. The inter-simple sequence repeat marker was used to assess the genetic variations and relationships among six accessions of H. ammodendron with a total of 120 individuals collected from three localities in the Alxa Desert, Inner Mongolia, China. At each locality, individuals both parasitized (PP) by C. deserticola and non-parasitized (NP) were sampled. The results showed that Nei's gene diversity and Shannon's index of PP accessions were higher, but were not significantly different, from those of NP accessions. An unweighted pair-group method arithmetic average dendrogram showed two clusters, one that included all PP accessions, and the other the NP accessions. Genetic differentiation therefore existed between PP and NP accessions, which might be attributed to low gene flow between the NP and PP groups (Nm

Tubificidae is now used in the wastewater treatment systems to successfully minimize the sludge production, which has been proved an effective, economical and sustainable technology. But the excess sludge inevitably contains a variety of heavy metals, especially the sludge from industrial wastewater treatment plant. In order to apply tubificidae to these systems, Chromium was selected as pollutant object and the physiological responses of tubificidae to Chromium were studied in this paper. Acute toxicity was analyzed and Median lethal concentrations (LC50) were determined over 96 h periods for Cr. Results indicated that 24 h LC50 and 96 h LC50 were 7.94 mg x L(-1) and 0.49 mg x L(-1), respectively. The duration f tubificidae in Cr solution decreased with increasing Cr concentration. Under the Cr stress, a highest respiration rate was obtained when the concentration of Cr(VI), temperature, pH and DO was 2.50 mg x L(-1), 26 degrees C, 6.0 and 6.0 mg x L(-1), respectively. The order of these factors was the concerntration of Cr(VI), temperature, DO and pH. The respiration experiments demonstrated that low concentration (2.50 mg x L(-1)) of Cr could promote the respiration rate of tubificidaes. On the other hand, when the concentration of Cr was 8.00 mg x L(-1), it could remarkably inhibit the respiratory rates of tubificidae.

AIM: To construct different conformations of a plasmid DNA/vector complex (pcDNA3.1/IFN-gamma-ASOR-PLL) and transfect cells of the hepatoma cell line BEL7402 to investigate the optimal conformation of the complex for improved expression efficiency in the target cell. METHODS: Double-distilled water and adjuvant were added to the naked pcDNA3.1/IFN-gamma, target vector ASOR-PLL and the ASOR-PLL-pcDNA3.1/IFN-gamma complex to create different conformations; molecules that were transfected into BEL7402 cells and the expression efficiency was determined by measuring the IFN-g concentration in the culture supernatant by ELISA. RESULTS: Naked pcDNA3.1/IFN-gamma DNA distributed linearly in double-distilled water and condensed into a mica configuration in adjuvant; ASOR-PLL had a net-like distribution without adjuvant and a spider-like form in the adjuvant-treated group; the ASOR-PLL-pcDNA3.1/IFN-g complex had a divaricate form without adjuvant, but a bead-like or granular conformation in 0.1 and 0.2 mol/L of adjuvant, a homogeneous bacilliform or chromatoid-shaped conformation in 0.3 mol/L adjuvant, and varied shapes in 0.4 and 0.5 mol/L adjuvant. The supernatant IFN-gamma expression in the bacilliform/chromatoid conformation complex group was the highest among the different conformation groups and controls. When chloroquine was added the supernatant IFN-gamma concentration increased in the liposome group and decreased in the bacilliform/chromatoid conformation group . CONCLUSIONS: The two structural molecules and their complex, ASOR-PLL-pcDNA3.1/IFN-gamma, were adjustable in the liquid mode. The specific bacilliform/chromatoid conformation of complex was lysosome enzyme-resistant and could play an active role in improving the efficiency of gene expression. The hypothesis that a chromosome-like conformation of the target gene molecule is involved in enhancing exogenous gene expression is proposed.

Micellar electrokinetic chromatography (MEKC) conditions were developed to analyze the constituents of Scutellariae Radix (SR) and Scutellaria baicalensis roots. Using the MEKC method, the major flavonoid constituents of baicalin, baicalein and wogonin of wild and cultivated S. baicalensis roots were compared. In a preliminary comparison of electropherogram, one special peak was found in a wild sample but not in a 2-year-cultivated one. The compound corresponding to the peak was isolated and identified as a phenylethanoid glycoside, acteoside, by comparing the 1H- and 13C-NMR spectral data with that of the authentic compound. This is the first time acteoside has been isolated from the Scutellaria genus. It could only be found in SR derived from wild S. baicalensis roots and 4-year-cultivated plants, but not in plant materials cultivated for 3 years. Applying the MEKC method established in this study, rapid and simultaneous determinations of acteoside together with 3 flavonoids in samples were achieved. The method can thus be used for the quality control of SR in a shorter analysis period than HPLC.

To construct the recombinant plasmid of pGPC3-EGFP containing human AFP(542-550) gene, EGFP gene and GPC3 gene to express fusion protein GPC3-hAFP(542-550)-EGFP and to discover its localization on cytoplasmic membrane.GPC3 gene was obtained from total RNA of human placental tissues by RT-PCR; After the enhanced green fluorescent protein (EGFP) gene was amplified from pEGFP-N1 plasmid and the gene segment of-KOZAK-GPCN + afp(542-550)-was chemically synthesized, the recombinant plasmid pcDNA3.1(+)/GPCN+afp(542-550)-EGFP-GPCC (pGPC3-EGFP) containing three chimeric genes of strong epitope hAFP(542-550), GPI-anchored protein GPC3 and EGFP was constructed. The fusion protein (GPC3-hAFP(542-550)-EGFP) was detected on RNA and protein levels at 24 h and 48 h after pGPC3-EGFP was transfected into HepG2 (GPC3(+)AFP(+)) via lipofectamine 2000. EGFP expression was observed by fluorescent microscopy after pGPC3-EGFP was transfected into HepG2 using pEGFP-N1 plasmid transfection as a positive control. The fusion protein in both membrane proteins and soluble proteins extracted from the transfected 293 cells (GPC3(-)AFP(-)) was detected by Western blot using GPC3 monoclonal antibody as primary antibody.The recombinant plasmid pGPC3-EGFP was successfully constructed through restriction endonuclease digestion and sequencing; pGPC3-EGFP expression in HepG2 cells was detected not only by RT-PCR using specific primers (GPCN-F and EGFP-r) but also by Western blot using GFP polyclonal antibody and GPC3 monoclonal antibody. Green fluoresce was mainly found around pGPC3-EGFP transfected HepG2 cell periphery beside sporadic distribution in cytoplasm, but that of pEGFP-N1 transfected HepG2 cell was evenly distributed in the whole cell. Moreover, the fusion protein was not detected in soluble proteins but membrane proteins extracted from transfected 293 cells.The recombinant plasmid of pGPC3-EGFP based on protein engineering theory can express fusion protein (GPC3-hAFP(542-550)-EGFP) in eukaryotic cells. Furthermore, the fusion protein is still located on cytoplasmic membrane, which is a characteristic of GPI-anchored membrane protein, and is a new GPI-reanchored protein.

Introduction: B-cell leukemia/lymphoma 2 (Bcl2) is a proto-oncogene best known for its ability to suppress cell death. However, the role of Bcl2 in the skeletal system is unknown. Bcl2 has been hypothesized to play an important anti-apoptotic role in osteoblasts during anabolic actions of PTH. Although rational, this has not been validated in vivo; hence, the impact of Bcl2 in bone remains unknown. Materials and Methods: The bone phenotype of Bcl2 homozygous mutant (Bcl2−/−) mice was analyzed with histomorphometry and μCT. Calvarial osteoblasts were isolated and evaluated for their cellular activity. Osteoclastogenesis was induced from bone marrow cells using RANKL and macrophage-colony stimulating factor (M-CSF), and their differentiation was analyzed. PTH(1–3;34) (50 μg/kg) or vehicle was administered daily to Bcl2+/+ and Bcl2−/− mice (4 days old) for 9 days to clarify the influence of Bcl2 ablation on PTH anabolic actions. Western blotting and real-time PCR were performed to detect Bcl2 expression in calvarial osteoblasts in response to PTH ex vivo. Results: There were reduced numbers of osteoclasts in Bcl2−/− mice, with a resultant increase in bone mass. Bcl2−/− bone marrow–derived osteoclasts ex vivo were significantly larger in size and short-lived compared with wildtype, suggesting a pro-apoptotic nature of Bcl2−/− osteoclasts. In contrast, osteoblasts were entirely normal in their proliferation, differentiation, and mineralization. Intermittent administration of PTH increased bone mass similarly in Bcl2+/+ and Bcl2−/− mice. Finally, Western blotting and real-time PCR showed that Bcl2 levels were not induced in response to PTH in calvarial osteoblasts. Conclusions: Bcl2 is critical in osteoclasts but not osteoblasts. Osteoclast suppression is at least in part responsible for increased bone mass of Bcl2−/− mice, and Bcl2 is dispensable in PTH anabolic actions during bone growth.

AIM: To describe the pattern of inheritance and confirm the diagnostic criteria of primary shunt hyperbilirubinaemia (PSH). METHODS: Forty members of a family pedigree across four generations were included in this study. All family members were interviewed and investigated by physical examination, hematology and liver function test and the pattern of inheritance was analyzed. RESULTS: Nine of the forty family members suffered primary shunt hyperbilirubinaemia. The mature erythrocytes of the propositus were irregular in shape and size. The pedigree showed transmission of the trait through four generations with equal distribution in male and female. No individual with a primary shunt hyperbilirubinaemia was born to unaffected parents. The penetrance was complete in adult. CONCLUSION: The pattern of inheritance is autosomal dominant. The abnormality of erythrocytes and decrease in white blood cell could be supplemented in the diagnosis of PSH. The PSH is a genetic disorder and could by renamed as hereditary shunt hyperbilirubinaemia.

Studying the mechanism of why the abnormal protein coded by mutant gene of hepatoma carcinoma cell possesses potential antigenicity, but can not form effective immunogen, searching for specific antigen of hepatoma, and developing DNA vaccine for hepatoma treatment will have active impact on the immunotherapy of hepatoma. This study was to construct fluorescent eukaryotic expression vector of mouse alpha fetoprotein (AFP) cDNA fragment with or without the secretory signal peptide (termed AFP1 cDNA and AFP2 cDNA), and observe its expression in mouse dendritic cells (DCs) and in vitro antitumor effect on hepatoma.AFP1 cDNA and AFP2 cDNA were amplified from mouse hepatoma cell line H22 by reverse transcription-polymerase chain reaction (RT-PCR), and cloned into eukaryotic expression vector PEGF-N3 to form the recombinant PEGF-N3/AFP1 and PEGF-N3/AFP2. The mouse bone marrow cells were induced with recombinant granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and recombinant interleukin-4 (rmIL-4) to form mature DCs. The recombinant PEGF-N3/AFP1 and PEGF-N3/AFP2 were transfected into DCs. The DCs vaccine were co-cultured with isogenous mouse spleen lymphocytes. The expression of gamma-interferon (IFN-gamma) in the spleen lymphocytes stimulated by AFP1/DCs or AFP2/DCs was measured by ELISA assay; the specific killing activity of cytotoxic T lymphocytes (CTLs) was evaluated by 51Cr release assay.Sequencing analysis verified that AFP1 cDNA and AFP2 cDNA were correctly amplified from H22 cells. The recombinant PEGF-N3/AFP1 and PEGF-N3/AFP2 were stably expressed in mouse DCs. AFP2/DCs obviously stimulated proliferation of T lymphocytes; the stimulation index (SI) was significantly higher in AFP2/DCs group than in AFP1/DCs group, empty vector group, and blank control group (5.12 +/- 1.46 vs. 1.41 +/- 0.65, 1.42 +/- 0.7, and 1.41 +/- 0.55, P0.01); the specific killing activity of CTLs was stronger in AFP2/DCs group than in empty vector group [(88.15 +/- 16.4)% vs. (12.72 +/- 5.45)%].The recombinant expression vectors PEGF-N3/AFP1 and PEGF-N3/AFP2 with or without signal peptide were constructed successfully. AFP2/DCs can specifically activate T lymphocytes in vitro and effectively induce antitumor immune response.

To identify Radix Scutellariae (Huangqin) of different growth years, to distinguish whether it's wild or cultivated and to provide useful information for the quality control of Huangqin crude drug.By using morphological and histological methods, we studied 87 individuals of 45 specimens from 12 habitats of 5 provinces of China, which grew wild or were cultivated in different growing years. Moreover, 22 commercial samples of Huangqin from 7 provinces were also investigated.The identification was performed base on morphological and histological characteristics, such as, the shape, color, cork, remaining stems, decayed central xylem, and vessels arrangement, xylem cork ring, growth rings, etc.We established an identification method for distinguishing Huangqin wild or cultivated in different growing years. Furthermore, the structure of annual rings in the transection of Radix Scutellariae has been discovered for the first time.

Rhei Rhizoma (Dahuang in Chinese) is widely known as a purgative and antiinflammatory agent. In the Japanese Pharmacopoeia, Rhei Rhizoma is prescribed for four Rheum species, Rheum palmatum, R. tanguticum, R. officinale, and R. coreanum, while the first three species are prescribed for Dahuang in the Chinese Pharmacopoeia. Due to the morphologic similarity of the aerial parts and frequent occurrence of intermediate forms, the taxonomy of this genus and the correct identification of Rheum species and their derivative drugs are very difficult. To resolve taxonomic problems of the genus Rheum and develop an ultimate identification method for plants and drugs, molecular analysis of the chloroplast matK gene and nuclear 18S ribosomal RNA gene were performed on nine species. The sequence comparison of the matK gene revealed that most species had variable sequences not only inter- but also intraspecies. However, the specimens of the same species belonged to the same subclade in the phylogenetic tree constructed based on matK gene sequences, except for R. palmatum, in which specimens belonged to three subclades related to their production areas. The nucleotide differences at positions 587, 707, and 838 distinguished official species from others, while specific nucleotides at positions 367 and 937 became identification markers for R. palmatum, R. tanguticum, and R. officinale (or R. coreanum). Moreover, three groups of R. palmatum, each belonging to three subclades, were characterized by the nucleotides at positions 619, 769, 883, and 1061. By detecting marker nucleotides, the botanical origins of Rhei Rhizoma were determined.

To construct the double-subunit co-expression plasmid P(+)/IL-12 to fulfill the gene therapy.The full length cDNAs of P40 and P35, two subunits of hIL12, were amplified from the reverse transcription production of human embryo kidney using polymerase chain reaction (PCR) and respectively cloned into eukaryotic expression vector pcDNA3.1(+/-) to construct the single-subunit plasmids--P(+)/P40 and P(-)/P35. Then we linked cDNAs of P40 and P35 tandem and cloned them into pcDNA3.1(+) to get P(+)/IL-12 plasmid. The hIL-12 protein in supernate was detected with enzyme-linked immunoabsorbent assay (ELISA) after P(+)/IL-12 was transfected into HepG2 through liposome.The plasmid of P(+)/IL-12 was testified using enzyme cutting and sequencing analysis. ELISA showed that hIL-12 protein could be expressed after transfecting into HepG2.The successful construction of P(+)/IL-12 plasmid can not only simplify the operation steps but also mimic hIL-12 physiological expression style in the gene therapy, thus laying the foundation for the use of hIL-12 in the gene therapy.

AIM: To examine the protein expression alterations in liver injury/repair network regulation as a response to gut-derived lipopolysaccharide (LPS) treatment, in order to anticipate the possible signal molecules or biomarkers in signaling LPS-related liver injury. METHODS: Male BALB/c mice were treated with intra-peritoneal (i.p.) LPS (4 mg/kg) and sacrificed at 0, 6, 24 and 30 h to obtain livers. The livers were stained with hematoxylin and eosin for histopathologic analyses. Total liver protein was separated by two-dimensional gel electrophoresis (2-DE). The peptide mass of liver injury or repair related proteins were drawn up and the protein database was searched to identify the proteins. RESULTS: Observations were as follows: (1) TRAIL-R2 was down regulated in livers of LPS-treated mice. TNFAIP1 was significantly up regulated at 6 h, then down-regulated at 24, 30 h with silent expression during senescent stage. (2) The amount of metaxin 2 and mitochondria import inner membrane translocase subunit TIM8a (TIMM8A) was increased upon treatment with LPS. (3) P34 cdc2 kinase was significantly up-regulated 30 h after LPS administration with silent expression during senescent, 6, 24 h treated stage. (4) The amount of proteasome activator 28 alpha subunit (PA28), magnesium dependent protein phosphatase (MDPP) and lysophospholipase 2 was decreased 6 h after LPS treatment but recovered or up-regulated 24 and 30 h after LPS treatment. CONCLUSION: LPS-treated mouse liver displaying a time-dependent liver injury can result in expression change of some liver injury or repair related proteins.

AIM: To increase exogenous gene expression level by modulating molecular conformations of targeting gene drugs. METHODS: The full length cDNAs of both P40 and P35 subunits of human interleukin 12 were amplified through polymerase chain reaction (PCR) and cloned into eukaryotic expressing vectors pcDNA3.1 (±) to construct plasmids of P (+)/IL-12, P (+)/P40 and P (-)/P35. These plasmids were combined with ASOR-PLL to form two targeting gene drugs [ASOR-PLL-P (+)/IL-12 and ASOR-PLL-P (+)/P40 + ASOR-PLL-P (-)/P35] in optimal ratios. The conformations of these two drugs at various concentrations adjuvant were examined under electron microscope (EM) and the drugs were transfected into HepG2 (ASGr+) cells. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed with total RNA extracted from the transfected cells to determine the hIL12 mRNA transcript level. The hIL12 protein in the cultured supernatant was measured with enzyme-linked immunosorbent assay (ELISA) 48 hours after transfection. RESULTS: Targeting gene drugs, whose structures were granular and circle-like and diameters ranged from 25 nm to 150 nm, had the highest hIL-12 expression level. The hIL-12 expression level in the group co-transfected with ASOR-PLL-P (+)/P40 and ASOR-PLL-P (-)/P35 was higher than that of ASOR-PLL-P (+)/IL-12 transfected group. CONCLUSION: The molecular conformations of targeting gene drugs play an important role in exogenous gene expression level, the best structures are granular and circle-like and their diameters range from 25 nm to 150 nm. The sizes and linking styles of exogenous genes also have some effects on their expression level.