Animals ; Cell Differentiation ; Hepatocyte Growth Factor ; genetics ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Mice ; Mouse Embryonic Stem Cells ; cytology ; metabolism ; Protein Precursors ; genetics ; metabolism ; Serine Endopeptidases ; genetics ; metabolism

Increasing the production and secretion of endogenous opioid peptide by immune cell can significantly induce myocardial protective effects against ischemia-reperfusion injury. Gene therapy is promising to increase endogenous enkephalin (ENK). However, classical viral and plasmid vectors for gene delivery are hampered by immunogenicity, gene recombination, oncogene activation, the production of antibacterial antibody and changes in physiological gene expression. Minimalistic immunologically defined gene expression (MIDGE) can overcome all the deficients of viral and plasmid vectors. The exon of rat's preproenkephalin (PPENK) gene was amplified by PCR and the fragments were cloned into pEGFP-N1 plasmids. The recombined plasmids were digested with enzymes to obtain a linear vector contained promoter, preproenkephalin gene, RNA stable sequences and oligodesoxy nucleotides (ODNs) added to both ends of the gene vector to protect gene vector from exonuclease degradation. A nuclear localization sequence (NLS) was attached to an ODN to ensure the effective transport to the nucleus and transgene expression. Flow cytometry, laser confocal microscopy and Western blotting demonstrated that PPENK-MIDGE-NLS can transfect leukocyte of rat in vivo, increase the expression of proenkephalin (PENK) in tissue, and the transfection efficiency depends on gene vector's dosage. These results indicate that PPENK-MIDGE-NLS could be an innovative method to protect and treatment of myocardial ischemia-reperfusion injury.
Animals ; Cloning, Molecular ; Enkephalins ; genetics ; Gene Expression ; Genetic Therapy ; Genetic Vectors ; Leukocytes ; Plasmids ; Promoter Regions, Genetic ; Protein Precursors ; genetics ; Rats ; Transfection ; Transgenes

OBJECTIVETo observe the intervention of electroacupuncture (EA) with different current frequencies and treatment frequencies on pain thresholt in rats with bone-cancer pain, so as to optimize treatment parameters of EA against bone cancer pain; and by measuring gene expression of opioid receptor and precursor in different tissues to preliminarily explore the possible mechanism of EA against bone cancer pain.

METHODSNinety healthy female SD rats were randomly divided into a control group, a model group, EA groups (6 subgroups according to different frequencies) and a sham EA group, ten rats in each one. Rats in the control group were injected with 10 µL of amicrobic phosphate buffer solution (PBS) into tibial cavity; rats in the remaining groups were injected with Walker 256 cancer cells to establish model of bone-cancer pain. No treatment was given to rats in the control group and model group; rats in the EA groups were treated with EA at bilateral "Housanli" (ST 36) and "Genduan" with 3 different current frequencies (2 Hz, 100 Hz and 2 Hz/100 Hz), once a day and once every other day, 30 min per treatment (1mA for 15 min, 2 mA for 15 min); rats in the sham EA group were treated with identical acupoints as the EA group, but the acupoints were needled subcutaneously and EA was connected with power off. All the treatment was given for 14 days. Dynamic plantar aesthesiometer was applied to measure the paw withdrawal thresholds (PWTs) of the affected side before the model establishment, 6d, 8d, 10d, 12d, 14d, 16d, 18d, and 20d after model establishment. The mRNA expressions of µ-opioid receptor (MOR), κ-opioid receptor (KOR), δ-opioid receptor (DOR), proopiomelanocortin (POMC) and prodynorphin (PDYN) in dorsal root ganglion (DRG) and lumbar spinal cord dorsal horn (SCDH) of L4-L6 of the affected side were detected by PCR method.

RESULTSThere were no differences in PWTs among all groups before model establishment (P>0. 05). Each time point after model establishment, PWTs in model group were obviously lower than those in the control group (all P<0. 01). Compared with the model group, PWTs in each EA subgroup were all increased (all P<0.05), but the differences at different time points were not significant among EA subgroups (P>0.05). The mRNA expressions of MOR, KOR, POMC, and PDYN in L4-L6 DRG in the 2 Hz/100 Hz II group were significantly higher than those in model group (P<0. 05, P<0. 01), while the mRNA expressions of MOR, KOR, DOR, POMC and PDYN in SCDH were not different compared with the model group (P>0. 05).

CONCLUSIONEA treatment has obvious analgesic effect on bone-cancer pain, however, its effect is not related with current frequency and treating frequency. EA against bone-cancer pain may be related with increasing the mRNA expression of some peripheral opioid receptors and precursor.

Acupuncture Analgesia ; instrumentation ; methods ; Acupuncture Points ; Animals ; Bone Neoplasms ; complications ; Electroacupuncture ; instrumentation ; methods ; Enkephalins ; metabolism ; Female ; Ganglia, Spinal ; metabolism ; Humans ; Pain ; etiology ; genetics ; metabolism ; Pain Management ; instrumentation ; methods ; Protein Precursors ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Opioid ; genetics ; metabolism

Previous studies have reported the association of prodynorphin (PDYN) promoter polymorphism with temporal lobe epilepsy (TLE) susceptibility, but the results remain inconclusive. To further precisely evaluate this association, we performed a meta-analysis. Published studies of TLE and PDYN polymorphism up to February 2015 were identified. Subgroup analysis by TLE subtype was performed. Moreover, sensitivity, heterogeneity, and publication bias were also analyzed. Seven case-control studies were finally included in this meta-analysis with 875 TLE cases and 1426 controls. We did not find synthetic evidence of association between PDYN promoter polymorphism and TLE susceptibility (OR=1.184, 95% CI: 0.873-1.606, P=0.277). Similar results were also obtained in non-familial-risk TLE subgroup. However, in the familial-risk TLE subgroup analysis, a significant association was observed (OR=1.739, 95% CI: 1.154-2.619, P=0.008). In summary, this meta-analysis suggests that PDYN gene promoter polymorphism might contribute to familial-risk TLE.
Case-Control Studies ; Enkephalins ; genetics ; Epilepsy, Temporal Lobe ; diagnosis ; genetics ; pathology ; Family ; Gene Expression ; Genetic Association Studies ; Genetic Predisposition to Disease ; Humans ; Inheritance Patterns ; Odds Ratio ; Polymorphism, Genetic ; Prognosis ; Promoter Regions, Genetic ; Protein Precursors ; genetics

Hepatitis B surface antigen (HBsAg) carrying preS sequences could be an ideal candidate for a new hepatitis B virus (HBV) vaccine with higher efficacy. Here we report the success in achieving efficient and stable expression of hepatitis B virus S antigen and preS1 epitope fusion protein (S/preS1) in CHO cells. The HMRCHEF53u/Neo-S/preS1 expression vector carrying S/preS1 gene was constructed and transfected into CHO-S cells. A stable and high-expression CHO cell line, named 10G6, was selected by ELISA and limiting dilution analysis. Western blotting analysis showed S/preS1 expressed from 10G6 cells possessed both S and preS1 antigenicity. 10G6 cells displayed characters of favorable growth and stable S/preS1 expression in repeated batch cultures as evaluated by viable cell density, viability and S/preS1 concentration. And cultivation of 10G6 cells in fed-batch mode resulted in S/preS1 production at 17-20 mg/L with viable cell density at 7 x 10(6)-10 x 10(6) cells/mL.
Animals ; CHO Cells ; Cricetulus ; Epitopes ; biosynthesis ; genetics ; Hepatitis B Surface Antigens ; biosynthesis ; genetics ; immunology ; Hepatitis B Vaccines ; biosynthesis ; genetics ; Hepatitis B virus ; Protein Precursors ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection

OBJECTIVETo explore the central mechanism of moxibustion on analgesic effect.

METHODSMale Wistar rats were screened by pain threshold value before making model, and 48 rats whose pain threshold was (250 +/- 25) g were selected. Twelve male Wistar rats were randomly selected as a normal group. For the rest rats the rheumatoid arthritis (RA) model was duplicated by raising in a windy, cold and wet environment combined with injection of Freund's complete adjuvant (FCA), and then they were randomly divided into a model group, a moxibustion group and a moxa volatile oil group, 12 rats in each group. The moxibustion and the moxa volatile oil igroup were treated with moxibustion and moxa volatile oil at "Shenshu"(BL 23) and "Zusanli"(ST 36), respectively, for 15 days. No interventions were added on the model group and the normal group. The pain threshold in Iinjured foot and the expression of hypothalamic POMC mRNA and PDYN mRNA in rats were observed.

RESULTSCompared with the normal group, the pain threshold and the expression of hypothalamic POMC mRNA and PDYN mRNA in the model group were increased (all P < 0.01). Compared with the model group, the pain threshold and the expression of hypothalamic POMC mRNA and PDYN mRNA in the moxibustion group were increased significantly (all P < 0.01), but no statistically significance in the moxa volatile oil group (P > 0.05). Compared with the moxa volatile oil group, the above-mentioned observative indices in moxibustion group were all increased significantly (all P < 0.01).

CONCLUSIONMoxibustion has obvious analgesic effect and its mechanism may be related to the increasing expression of hypothalamic POMC and PDYN mRNA through the warming effect of moxibustion.

Animals ; Arthritis, Rheumatoid ; genetics ; metabolism ; therapy ; Enkephalins ; genetics ; metabolism ; Humans ; Hypothalamus ; metabolism ; Male ; Moxibustion ; Pro-Opiomelanocortin ; genetics ; metabolism ; Protein Precursors ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar

OBJECTIVETo establish a quantitative method to detect circulating tumor cells (CTC) in patients with small cell lung cancer, and analyze its sensitivity and stability.

METHODSA specific primer and probe for prepro-gastrin-releasing peptide (preproGRP) was designed and a quantitative RT-PCR method was established to detect preproGRP mRNA. Cell incorporation method was used to evaluate the sensitivity. Magnetic cell sorting (MACS) was used to isolate and purify CTC from peripheral blood, and the MACS in combination with morphological diagnosis were used for cell counting.

RESULTSThe isolation rate of CTC by MACS was 30% and the lower detection limit was 5 cells per ml blood. The sensitivity of quantitative RT-PCR in detection of preproGRP mRNA in CTC was 0.64 cells per reaction, and the lower detection limit was 50 cells per ml blood, which was lower than that of MACS. However, the cell numbers calculated by Ct value was in greater accordance (about 80%) with actual cell numbers than that obtained by MACS.

CONCLUSIONSPreproGRP quantitative RT-PCR and MACS have both advantages and disadvantages in detecting CTC of SCLC patients. MACS has a higher sensitivity, and is more favorable when CTC count is below 50 per ml blood. Meanwhile, preproGRP mRNA quantitative RT-PCR is more reliable in calculating actual cell numbers.

Humans ; Immunomagnetic Separation ; Lung Neoplasms ; blood ; metabolism ; pathology ; Neoplastic Cells, Circulating ; Peptides ; genetics ; metabolism ; Protein Precursors ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Small Cell Lung Carcinoma ; blood ; metabolism ; pathology

The purpose of this study was to measure the thickness of canine epidermis at various anatomical sites according to localization of cornified envelopes (involucrin and filaggrin), keratins (keratin 10, 5), and their mRNA expression. This was done in the skin of five breeds of dogs including seven poodles, six golden retrievers, six Shih Tzus, four pugs, and four Labrador retrievers. Epidermal thickness of the stratum corneum and nucleated epidermal layer was significantly different. The greatest thickness was observed in the digital web area and the thinnest epidermis was in the axilla. Epidermal thickness was also significantly different between the breeds (p < 0.05). Immunohistochemical staining scores revealed significant decreases of involucrin, filaggrin, and keratin 10 in the ventral and weight-bearing sites, and a relative increase of keratin 5 (p < 0.05). q-PCR analysis showed that their the levels of mRNA were positively correlated with expression of the corresponding proteins in skin samples (p < 0.05). The present study is the first to report the relationship between epidermal gene expression and histologic morphology of the skin in normal dogs. Further studies will be essential to fully understand the pathogenesis of skin barrier dysfunctions in canines.
Animals ; DNA, Complementary/genetics/metabolism ; Dogs/anatomy & histology/genetics/*metabolism ; Gene Expression Regulation/*physiology ; Intermediate Filament Proteins/genetics/*metabolism ; Keratin-10/genetics/*metabolism ; Keratin-5/genetics/*metabolism ; Polymerase Chain Reaction/methods/veterinary ; Protein Precursors/genetics/*metabolism ; RNA/genetics/metabolism ; Skin/anatomy & histology/metabolism

Rhizopus oryzae lipase (ROL) is not only a biocatalyst used in a broad range of biotechnological fields, but also a model to investigate the function of intramolecular chaperone in the post-translational processing of lipase. In this study, we cloned and expressed the mature lipase gene (m-ROL) containing the pre-sequence (pro-ROL) of R. oryzae HU3005 in Pichia pastoris GS115 and characterized their enzymatic activities. m-ROL exhibited higher hydrolysis activity towards middle-chain substrates (C10 and C12) at pH 9.0, whereas pro-ROL preferred short-chain substrates (C4) and displayed maximal activity at pH 8.0. Moreover, pro-ROL possessed better thermal stability than m-ROL. This enzymatic discrepancy between m-ROL and p-ROL may be due to the pre-sequence that affects the folding and conformation of the mature lipase domain. To improve the expression level of m-ROL in P. pastoris, overlap extension PCR was conducted to substitute eight less-frequently used codons of m-ROL with frequently used codons. After methanol-induced expression for 72 h, the activity and protein content of the codon optimized m-ROL reached 132.7 U/mL and 50.4 mg/L, while the activity of the parental m-ROL and pro-ROL are 28.7 U/mL and 14.4 mg/L, 29.6 U/mL and 14.1 mg/L, respectively.
Codon ; Enzyme Precursors ; biosynthesis ; chemistry ; genetics ; Enzyme Stability ; Lipase ; biosynthesis ; genetics ; metabolism ; Pichia ; enzymology ; genetics ; Protein Engineering ; methods ; Protein Folding ; Recombinant Proteins ; biosynthesis ; genetics ; Rhizopus ; enzymology ; genetics ; Substrate Specificity

Hepatitis B virus X protein (HBx) has various functions and plays a crucial role in the development of hepatocellular carcinoma (HCC). However, due to different transfection efficiency levels and experimental approaches, it is difficult to correlate the exact functions of HBx to HBV-associated HCC. In this study, we constructed two prokaryotic expression vectors, pGEX-HBx-EGFP-TLM and pGEX-EGFP-TLM, which expressed HBx-EGFP-TLM and EGFP-TLM fusion proteins respectively. Both vectors contained a coding sequence of TLM transduction motif derived from the PreS2-domain of Hepatitis B Virus surface antigens. In addition, EGFP was expressed as a reporter reflecting the transduction efficiency of TLM. The fusion protein HBx-EGFP-TLM or EGFP-TLM purified from Escherichia coli BL21(DE3) by AKTA Purifier system was incubated with AML12 and SMMC-7721 cells. Both Western blotting and laser confocal results indicated that the translocation motif TLM could lead HBx-EGFP and EGFP into the cytoplasm. Dual-Luciferase Reporter Assay revealed that the activity of mEZH2 promoter could be up-regulated by the recombinant HBx. In conclusion, we expressed a cell-permeable HBx, which could provide a new method to study the functions of HBx.
Amino Acid Motifs ; genetics ; Cell-Penetrating Peptides ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Hepatitis B Surface Antigens ; genetics ; Protein Precursors ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Trans-Activators ; biosynthesis ; genetics