Nowadays,small peptides are always expressed in the form of fusion protein.The expression product contains many superfluous amino acids which can affect the biological functions of small peptides even expressed by GST fusion protein expression system.SUMO protease can cut SUMO fusion protein expressed by fusion expression system without any amino acid residues left on target protein thus become a hot topic in this field.Recombinant His-UlP1/pET3c/BL21(DE3)engineering strain was constructed by genetic engineering technology and the expression conditions were optimized in shake flaks.The process of high density fermentation was explored and different purification conditions were detected by chromatography.The results showed that SUMO protease could be expressed well after inducing the engineering strain by IPTG of 1.0mmol/L at 30℃ for 6 hours.The expression level of the strain in fermentation pots could reach 24.3% analyzed by SDS-PAGE.The purity of SUMO protease was more than 98% after further purification by cation exchange chromatography.The yield was 355mg SUMO protease per liter fermentation liquid.Western blot analysis demonstrated that there were immune reactions between IlP1 and 6?His antibodies,so it has established a good foundation for large-scale industralazation in the future.

Objective:To screen the ectodysplasin A (EDA)gene mutation in the patients with non-syndromic tooth agenesis and ectodermal dysplasia,and to analyze the phenotype of missing teeth pattern in these two groups of patients.Methods:In the study,174 patients with tooth agenesis (143:non-syn-dromic,31:ectodermal dysplasia)and 451 health control volunteers were enrolled from the clinic,and the genome DNA was extracted from either peripheral blood or oral mucosal swab.The coding region of EDA gene was then amplified by PCR,sequenced and blasted to online NCBI database.The missing teeth were recorded for all patients,and the missing teeth from patients with EDA mutation were com-pared among the different dentition sites.Results:33 patients were identified with EDA mutation.In the non-syndromic patients,13 /143(9.09%)were identified with EDA mutation,while in patients with ec-todermal dysplasia,20 /31 (64.52%)were found with EDA mutation.Ten novel EDA mutations were identified (c.769G >C[p.G257R],c.936C >G[p.I312M],c.223G >A[p.E75K],c.1166C >T[p. P389L],c.133G >C[p.G45R],c.1109G >A[p.E370K],c.914G >T[p.S305I],c.916C >T[p. Q306X],c.602G >T[p.G201V],c.88 -89insG[p.A30GfsX69]).For each dentition site there was no statistic difference in the number of missing teeth between the left and right sides,so the number from both sides were combined later in the analysis.In the patients with EDA mutation,the non-syndromic pa-tients had fewer missing teeth (15.9 ±6.4 missing teeth for each,207 /364 in total)than the patients with ectodermal dysplasia (23.9 ±4.3,478 /560).In the non-syndromic patients with EDA mutation, the maxillay central incisors and first molars were less affected,with the same missing rate as 19.2% (5 /26).While the mandibular central incisors (with a missing rate of 76.9%,20 /26),the maxillary late-ral incisors (the missing rate:88.5%,23 /26 ),the mandibular lateral incisors (the missing rate:80.8%,21 /26),and the maxillary first premolars (the missing rate:80.8%,21 /26)were more likely to be missing.In the ectodermal dysplasia patients with EDA mutation,only maxillary central incisors (the missing rate:60%,24 /40),maxillary canines (the missing rate:70%,28 /40),mandibular ca-nines (the missing rate:67.5%,27 /40),maxillary first molars (the missing rate:65%,26 /40)and mandibular first molars (the missing rate:72.5%,29 /40)had higher possibility of persistence.Teeth at other dentition sites were more likely to be affected (the minimum missing rate:87.5%,35 /40). Conclusion:The findings would help to reveal the EDA gene and its function in ectodermal organogene-sis.

HCV RNA positive serum was first selected by RT-PCR test kit from several anti-HCV positive sera obtained from Xi'an.HCV RNA extrac ted from the elected sera was converted to cDNA by reverse transcription with ra ndom primer.Half-nested PCR was performed.The amplified product was 852 bp.The purified PCR product was digested by restriction endonucleases and then ligated to epressio vector pET-22b\++.Its nucleotide sequence was determined by dideoxy chain termination method.A comparison of the sequence with several isolates rep orted previously showed that the sequence belonged to HCV type Ⅱ.

Objective To evaluate the efficacy of Lianhua Dingchuan Tablets in bronchial asthma.Methods Fifty BALB/C mice were randomly and equally divided into control (Con) group,ovalbumin (OVA) group,dexamethasone (DEX) group,high-dose Lianhua group,low-dose Lianhua group.The mice were sensitized and challenged with OVA plus aluminium hydroxide to establish asthmatic model and were pre-treated 30 minutes before challenge.Specific airway resistance (sRaw) was used to evaluate airway hyperresponsiveness,and airway inflammatory changes were measured.ELISA and Magnetic Luminex(R) were used to quantified the levels of IL-4,IL-13 and INF-γ.Results Airway resistance significantly decreased in DEX group and High-dose Lianhua group (P＜0.05).Levels of inflammatory cells and IL-13 in BALF evidently reduced in DEX group,high-dose Lianhua group and low-dose Lianhua group (P ＜ 0.05),while IL-13 level in serum only decreased in DEX group.There was no significant changes in the levels of IL 4 and INF γ among those groups.Conclusions Lianhua Dingchuan Tablets might relieve the symptoms of asthma by reducing IL-13 level and inhibiting the airway inflammation.

ObjectiveTo analyze the classifications of the abnormalities of cervical curvature and its treatments with manipulation. Methods209 patients with cervical lordosis were analyzed. The abnormalities of the cervical curvature were classified as over-curvature (>45°), hypo-curvature (0°～30°), the straightened cervix (0°), backward curvature(0°～―20°), multiple curvatures (the cervical curvature showed "S" shaped). The C_5 subluxation were restored with manipulation in patients with hypo-curvature and straightened cervix, or other vertebra subluxation in multiple curvatures. Results1~4 yeared follow-up, 127 cases were excellently well being, 59 were well being, 23 were improved.ConclusionManipulation is effective on abnormalities of cervical curvature.

This study investigated the protective effect of chrysin on hepatic fibrosis by regulating AMP-activated kinase (AMPK)-NOD-like receptor protein 3 (NLRP3) mediated pyroptosis pathway. The hepatic fibrosis model of mice was established by thioacetamide (TAA) in vivo. Except the control and chrysin alone groups, the mice were injected intraperitoneally with TAA at 100 mg·kg^-1, three times per week for the first week. From the 2^nd to 5^th week, mice were injected intraperitoneally with TAA at 200 mg·kg^-1, three times per week for the next 4 weeks. Chrysin groups were intragastrically administrated once per day to 5^th week. The histopathological changes were detected by HE and Masson staining. The levels of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were assessed by the kits. All animal experiments were approved by the Medical Ethics Committee of Affiliated Zhongshan Hospital of Dalian University (DWLL2019060). LX-2 cells were stimulated by (transforming growth factor-β, TGF-β) in vitro. The protein expressions of AMPKα, p-AMPKα, NLRP3, cysteinyl aspartate specific proteinase-1 (caspase-1), gasdermin D (GSDMD) were detected by Western blot, and the mRNA levels of collagen-Ι, α-smooth muscle actin (α-SMA), interleukin-1β (IL-1β), IL-18, caspase-1, GSDMD were analysis by reverse transcription-polymerase chain reaction (RT-PCR). Chrysin attenuated the increases in serum AST and ALT levels in the TAA group, while significantly improved the changes of liver morphology, reduced liver tissue inflammatory cell infiltration and inhibited collagens deposition. Compared with TAA group, chrysin effectively activated AMPKα phosphorylation and inhibited hepatic NLRP3 inflammasome activation. Additionally, the protein expressions and mRNA levels of IL-1β, IL-18, caspase-1 and GSDMD in chrysin groups were decreased. Chrysin inhibited the expressions of collagen-Ι and α-SMA, enhanced the phosphorylation of AMPKα, and decreased the expressions of NLRP3 and GSDMD. Therefore, chrysin may inhibit inflammatory injury and pyroptosis possibly by activating AMPK and inhibiting NLRP3 inflammasome to alleviate hepatic fibrosis.

OBJECTIVE: To study the percentages of polymorphonuclear leukocytes (PMN), mononuclear cells (MNC) and fibroblastic cells (FBC) in different post-traumatic intervals after skeletal muscle mechanical injury in rats. METHODS: The rat model of skeletal muscle mechanical injury was established. The rats were divided into injured groups (6 h, 12 h, 1 d, 3 d, 7 d, 10 d and 14 d after injury) and control group. The percentages of PMN, MNC and FBC in different post-traumatic intervals after skeletal muscle mechanical injury were assessed with HE staining and image analysis. RESULTS: At post-injury 6-12h, the percentages of PMN and MNC infiltration appeared in injured sites and that of PMN reached peak. At 1 d, the percentage of MNC infiltration appeared and reached peak, while that of PMN decreased. At 3-7 d, the percentage of FBC gradually increased, while that of PMN and MNC decreased. At 10-14d, the percentage of FBC reached peak. CONCLUSION The percentages of PMN, MNC and FBC in injured zones showed time-dependent changes, which might be used as reference index for determination of age of skeletal muscle injury.
Animals ; Fibroblasts ; Muscle, Skeletal/injuries* ; Neutrophils ; Rats ; Time Factors

To explore the growth and development and analyze the quality of the parthenocarpy fruit induced by exogenous hormones of Siraitia grosvenorii. the horizontal and vertical diameter, volume of the fruit were respectively measured by morphological and the content of endogenous hormones were determined by ELISA. The size and seed and content of mogrosides of mature fruit were determined. The results showed that the fruit of parthenocarpy was seedless and its growth and development is similar to the diploid fruit by hand pollination and triploid fruit by hand pollination or hormones. But the absolute value of horizontal and vertical diameter, volume of parthenocarpy fruit was less than those of fruit by hand pollination, while triploid was opposite. The content of IAA, ABA and ratio of ABA/GA was obviously wavy. At 0-30 d the content of IAA and ABA of parthenocarpy fruit first reduced then increased, content of IAA and GA parthenocarpy fruit was higher than that of fruit by hand pollination. Mogrosides of parthenocarpy fruit was close to pollination fruit. Hormones can induce S. grosvenorii parthenocarpy to get seedless fruit and the fruit shape and size and quality is close to normal diploid fruit by hand pollination and better than triploid fruit by hormone or hand pollination.
Cucurbitaceae ; chemistry ; drug effects ; genetics ; growth & development ; Diploidy ; Fruit ; chemistry ; genetics ; growth & development ; Plant Growth Regulators ; pharmacology

Aim To investigate the effect of hypercholesterolemia on L-type Ca2+(ICa-L) current and intracellular calcium concentration ([Ca2+]i) in single ventricular myocytes of hypercholesterolemic rats.Methods 12 male wistar rats were randomly divided into two dietary groups:a group fed a normal diet(n=6)and a group fed high-cholesterol diet(n=6) for 4 weeks,respectively. The level of serum lipid was examined.Zymolytic method was used to isolate single ventricular myocytes from hypercholesterolemic and normal rats,which were loaded with Ca2+-sensitive fluorescent indicator Fluo-3/AM.[Ca2+]i represented by fluorescent intensity(FI)was measured by laser scanning confocal microscope.Whole cell patch clamp technique was used to record ICa-L.Results There was no significant influence exhibited on TG level.However, the serum total cholesterol(TC)level of hypercholesterolemic rats was much higher than that of model control group; at the test potential of 0 mV, ICa-L decreased from(-8.56?1.29)pA/pF(Control)to(-5.24?0.90) pA/pF(HC)(n=6 in each group,P

OBJECTIVE: To research the relation between the time-dependent appearances of myotibroblasts during the repair of contused skeletal muscle in rat and wound age determination. METHODS: A total of 35 SD male rats were divided into the control and six injured groups according to wound age as follows: 12 h, 1 d, 5 d, 7 d, 10 d and 14 d after injury. The appearances of myofibroblasts were detected by HE staining, immunohistochemistry and confocal laser scanning microscopy. Masson's trichrome staining was utilized to examine collagen accumulation in the contused areas. RESULTS: Immunohistochemical staining showed that α-SMA+ myofibroblasts were initially observed at 5 d post-injury. The average ratio of myofibroblasts was highest at 14 d post-injury, with all samples, ratios more than 50%. In the other five groups, the average of α-SMA positive ratios were less than 50%. The collagen stained areas in the contused zones, concomitant with myofibroblast appearance, were increasingly augmented along with advances of posttraumatic interval. CONCLUSION The immunohistochemical detection of myofibroblasts can be applied to wound age determination. The myofibroblasts might be involved in collagen deposition during the repair of contused skeletal muscle in rat.
Animals ; Collagen/metabolism* ; Contusions/metabolism* ; Immunohistochemistry ; Male ; Microscopy, Confocal ; Muscle, Skeletal/metabolism* ; Myofibroblasts/metabolism* ; Rats ; Time Factors ; Wound Healing