Peptide cyclization, a pivotal approach to modifying linear precursors of proteins and pepticles, has been used to enhance their biological activities and serum stabilities. Recently, sortase A (SrtA) from Staphyloccus aureus becomes a promising new technology for efficiently incorporating site specific modifications into proteins, conjugating the cell surface and cyclizing the linear peptides. In this study, we constructed two recombinant expression systems, one with chitin binding domain and the other with six-histidine tag and chitin binding domain on the N-terminal of SrtA, separately. The results of enzymatic kinetics indicate that the two recombinant tags do not impair the transpeptidase activity of SrtA compared with the standard reaction reported under the same reaction condition. The two synthesized peptides with N-ternimal three glycines and C-terminal penta-amino acid motif, LPETG, were cyclized using immobilized and recycled SrtA. The SrtA-based cyclization promises to represent a simple method for easy and efficient enzymatic synthesis of large cyclic peptides.
Aminoacyltransferases ; metabolism ; Bacterial Proteins ; metabolism ; Cyclization ; Cysteine Endopeptidases ; metabolism ; Enzymes, Immobilized ; metabolism ; Kinetics ; Peptides ; metabolism ; Peptides, Cyclic ; biosynthesis ; Staphylococcus aureus ; enzymology

OBJECTIVETo study the effect of tea polyphenols (TP) on the apoptosis of germ cells in rats with experimental varicocele.

METHODSThirty-two adolescent male Wistar rats were randomly and equally divided into groups A (sham-operation), B (high-dose TP), C (low-dose TP), and D (experimental left varicocele). Experimental varicocele was induced by partial ligation of the left renal vein in the latter three groups of rats. The animals in groups A and D were fed with normal saline, while those in B and C with TP at 40 and 10 mg per kg per d, respectively, all for 4 weeks. Then, all the rats were sacrificed and the left testes harvested for determination of the expression of HIF-1, Bcl-2, Bax, CytC, and caspase-3 by immunohistochemistry and measurement of the apoptosis index (AI) of spermatogenic cells.

RESULTSThe expression of Bcl-2 was higher in groups B and C than in D but lower than in A (P < 0.05), and lower in C than in B (P < 0.05). However, the expressions of HIF-1, Bax, CytC, and caspase-3 were lower in groups B and C than in D but higher than in A (P < 0.05), and higher in C than in B (P < 0.05). The AI of spermatogenic cells was the lowest in group A, higher in D than in the other groups but lower in B than in C (P < 0.05).

CONCLUSIONTP can reduce the apoptosis of spermatogenic cells in a dose-dependent manner in varicocele rats.

Animals ; Apoptosis ; drug effects ; Caspase 3 ; Cytochromes c ; metabolism ; Dose-Response Relationship, Drug ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Ligation ; Male ; Polyphenols ; administration & dosage ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Renal Veins ; Spermatozoa ; drug effects ; Tea ; chemistry ; Testis ; metabolism ; Varicocele ; complications ; metabolism ; bcl-2-Associated X Protein ; metabolism

This paper was to study the angiogenic inhibitory effect and the potential antitumor effect of the constructed recombinant DNA vaccine CRT/HPV6bE7 in vivo. The C57BL/6 mice were vaccinated respectively with recombinant CRT/HPV6bE7 DNA plamids. The inhibitory effects on angiogenesis of generated vaccines in vivo were evaluated by a bFGF-induced angiogenesis assay using the Matrigel kit. To investigate the potential antitumor effect, the mean tumor weights, sizes and tumor appearing times were measured in C57BL/6 mice treated with HPV6bE7-expressing B16 cells. The results indicated that the recombinants CRT180/HPV6bE7 and CRT180 showed strong anti-angiogenic effects in bFGF-induced angiogenesis in vivo. Moreover, CRT180/HPV6bE7 and CRT180 DNA vaccines could significantly inhibit the tumor growth in tumor challenge experiment, and CRT180/HPV6bE7 was superior to other vaccines in delaying tumor formation time, limiting tumor size and weight in tumor protection experiment. In conclusion, recombinant CRT180/HPV6bE7 DNA could elicit a most efficient anti-angiogenic effect and inhibit tumor growth in mice inoculated with DNA vaccines. The antiangiogenic activity of CRT were suggested residing in a domain between CRT 120-180 aa.
Animals ; Calreticulin ; genetics ; Female ; Human papillomavirus 6 ; immunology ; Mice ; Mice, Inbred C57BL ; Neovascularization, Pathologic ; prevention & control ; Oncogene Proteins, Viral ; genetics ; Papillomavirus Infections ; therapy ; Papillomavirus Vaccines ; immunology ; Recombinant Fusion Proteins ; genetics ; Vaccines, DNA ; immunology

OBJECTIVETo determine the mechanism of pseudo-tears of the lateral meniscus caused by the transverse geniculate ligament (TL) and the miniscofemoral ligament(MFL) and to investigate a method to differentiate pseudo-tears from true tear of the lateral meniscus.

METHODSForm June 2012 to February 2014, MR examinations of 72 knees (44 left knees and 28 right knees) without tear of the lateral meniscus verified by arthroscopy were performed in the sagittal and coronal plane. There were 41 males and 31 females in the group, with an average age of 33.7 years old (ranged from 25 to 61). The MR appearance of the TL and the MFL was carefully observed.

RESULTSThere existed fatty tissue in the gap between the TL and the anterior horn of the lateral meniscus and its central tendinous attachment. On the sagittal images, the fatty tissue formed a linear high-signal cleft between the TL and the anterior horn of the lateral meniscus. This might be mistaken as an oblique tear within the anterior horn of the lateral meniscus. It was called as pseudo-tears of the anterior horn of the lateral meniscus. In sagittal plane, the MFL was identified as a circle-like or short stick-like area of low signal intensity anterior or posterior to the posterior cruciateligament. Nevertheless, a belt-shaped area of low signal intensity from the posterior horn of the lateral meniscus to lateral facet of the medial femoral condyle was identified in the coronal plane. A linear area of high signal intensity between the MFL and the lateral meniscus was found in sagittal plane, which might be mistaken as an oblique tear within the posterior horn of the lateral meniscus. It was called pseudo-tears of the posterior horn of the lateral meniscus. The occurrence rate of the TL was 34.7% (25/72). The prevaleribe of pseudo-tears of the anterior horn of the lateral meniscus was 18 cases. The shape of the anterior horn of the lateral meniscus was regular, and the course of the pseudo-tears cleft was oblique. The occurrence rate of the MFL was 73.6% (53/72), which included the anterior MFL 23.6% (17/72), the posterior MFL 70.8% (51/72) and the two ligaments coexisted 16.7% (12/72). The prevalence of pseudo-tears of the posterior horn of the lateral meniscus was 25 cases. All observed pseudo-tears had either in posteroinferiorly oblique direction (19/25) or in vertical direction (6/25).

CONCLUSIONBased on the location and direction of pseudo-tears and observation in the continuous sagittal plane and the coronal plane, pseudo-tears is easily differentiated from the true tear of the lateral meniscus

Adult ; Female ; Humans ; Knee Injuries ; diagnostic imaging ; surgery ; Knee Joint ; diagnostic imaging ; surgery ; Magnetic Resonance Imaging ; Male ; Menisci, Tibial ; diagnostic imaging ; surgery ; Middle Aged ; Radiography ; Tibial Meniscus Injuries

OBJECTIVETo compare the ability of osthole (OST) and genistein (GEN) in enhancing bone peak bone mass of rats to prevent osteoporosis.

METHODSThirty-six female one-month-old SD rats of (125 +/- 3) g body weight were randomly divided into three groups, 12 rats in each group, one group was orally administered osthole at 9 mg x kg(-1) d(-1), one group was given genistein at 10 mg x kg(-1) d(-1) and another was given equal quantity of distilled water as the control. The body weight was monitored weekly and the bone mineral density (BMD) of total body was measured every month. All rats were sacrificed after three months, the femoral bone mineral density, the serum levels of osteocalcin (OC) and anti-tartaric acid phosphatase 5b (TRACP 5b) were measured by Elisa. The bone microarchitectures were analyzed with micro-CT and the bone biomechanics properties were tested with universal material machine.

RESULTSNo significant differences were observed between O-treated or GEN group and the control for the food-intake and body weight during three months. However, the rats treated with OST had significant higher BMD for both total body and femur than the control and GEN group. The O-treated rats also had higher level of serum OC and lower level of TRACP 5b. Besides, they owned bigger bone volume/tissue volume, trabecular thickness, trabecular number but smaller trabecular spacing. In the three point bending tests of femurs,they were found to have larger maximum load, the young's modulus and structural model index (SMI).

CONCLUSIONOrally administered osthole could efficiently increase the peak bone mass of rats,which provide new ideas for preventing osteoporosis.

Acid Phosphatase ; blood ; Animals ; Body Weight ; drug effects ; Bone Density ; drug effects ; Coumarins ; pharmacology ; Female ; Femur ; diagnostic imaging ; drug effects ; pathology ; Genistein ; pharmacology ; Isoenzymes ; blood ; Osteocalcin ; blood ; Radiography ; Rats ; Rats, Sprague-Dawley ; Tartrate-Resistant Acid Phosphatase

Objective To study the protective effect of glucocorticoid preconditioning on the myocardial ischemic and reperfused hearts.Methods Totally 18 rabbits were randomly divided into three groups: myocardial ischemia-reperfusion model(model),high-dose glucocorticoid given by one time group(high-dose group) and low-dose glucocorticoid given by several times group(low-dose group),with six rabbits in each group.Myocardial ischemia was induced by left anterior descending coronary artery ligation.ST segments were recorded by the BL-420 biological signal acquisition system.Plasma malondial dehyde(MDA) was examined before ischemia,at 15 min after ischemia and 30 min after reperfusion;ischemic heart muscles were prepared with cryotomy and stained histochemically.Succinic dehydrogenase activity was observed in the ischemic region.Results There was shorter time of ST-segment recovery in the high-dose group and the low-dose group than that in the model group.Serum level of MDA in the high-dose group was lower than in the low-dose group(P

Gene transfer technology is being used to enhance agronomic performance or improve quality traits in a wide variety of crop species. However, it is sometimes severely handicapped by difficulty in obtaining material in which transgene expression is predictable and stable over many generations. Because integration seemed to occur randomly in the plant genome, it was thought that some transgenes would be integrated in a relatively uncondensed, transcriptionally active chromatin environment, while others in a condensed, transcriptionally inert chromatin structure. Nuclear matrix attachment regions (MARs) are defined as DNA sequences that bind preferentially to the proteins of the nuclear matrix. They typically are localized at the borders of gene domains, implicating them in the formation of individual loops of higher order chromatin structure and transcription regulation. When MARs are positioned on either side of a transgene their presence usually results in higher and more stable espression in transgenic plants, most likely by minimizing gene silencing. In this review, we focus mainly on novel findings and our observations concerning the function of MARs in transcription regulation. Our objective is not only to summarize the current data and present several possible models to explain MAR effects on the transcription regulation, but also to point out some open questions involving the utilization of MARs in constructing high efficient expression vectors.
Chromatin ; physiology ; DNA ; metabolism ; Gene Expression Regulation, Plant ; Models, Genetic ; Nuclear Matrix ; metabolism ; Nuclear Proteins ; metabolism ; Transcription, Genetic ; Transgenes ; genetics

OBJECTIVETo investigate the protective effect of phosphodiesterase type 5 inhibitors (tadalafil) on the testis following testicular ischemia-reperfusion injury in rats.

METHODSEighty-four healthy adult male SD rats were randomly and equally divided into groups A (sham operation), B (testicular torsion + low-dose tadalafil), C (testicular torsion + high-dose tadalafil), and D (testicular torsion + placebo). Models were established in the latter three groups by 7200 torsion of the right testis for 2 hours. The animals in groups A and B were treated by gavage with tadalafil at the dose of 0. 5 mg per kg per day, those in group C at 2 mg per kg per day, and those in group D with saline at the same dose. After 3, 7, and 14 days of treatment, the torsioned testes were harvested for evaluation of the superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in the testis tissue. The pathological changes in the testis were observed under the light microscope.

RESULTSAt 3, 7, and 14 days, the SOD activity was (254.46 +/- 7.43), (278.49 +/- 8.33), and (317.99 +/- 3.31) nU/mg prot in group B, and (277.12 +/- 8.80), (309.40 +/- 2.14), and (320.39 +/- 4.72) nU/mg prot in group C, all obviously higher than in D ([223.21 +/- 4.65], [231.45 +/- 4.16] and [248.28 +/- 5.74] nU/mg prot), while the MDA content was lower in the former two groups than in the latter. At 3 and 7 days, the SOD activity was significantly higher and the MDA level significantly lower in group C than in B (both P < 0.01) , while at 14 days, neither showed any remarkable differences between the two groups (P > 0.05). No obvious histopathological change was observed in the testis tissue of group A. At 3 and 7 days, pathological examination of the testis tissue revealed significant differences in the number of seminiferous epithelial layers, testicular histological score, and seminiferous tubule diameter in group B (P < 0.01), but the three indexes at 14 days in group B and at 7 days in group C exhibited no remarkable differences from those at 14 days in group A.

CONCLUSIONTadalafil can alleviate testicular ischemia-reperfusion injury following testis torsion/detorsion in a time- and dose-dependent manner.

Animals ; Biomarkers ; metabolism ; Carbolines ; administration & dosage ; pharmacology ; Dose-Response Relationship, Drug ; Male ; Malondialdehyde ; metabolism ; Phosphodiesterase 5 Inhibitors ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; prevention & control ; Seminiferous Tubules ; pathology ; Spermatic Cord Torsion ; complications ; Superoxide Dismutase ; metabolism ; Tadalafil ; Testis ; blood supply ; metabolism ; pathology ; Time Factors

OBJECTIVETo evaluate effect of amygdalin on expression of four biomarkers in the animal model of pulmonary fibrosis induced by bleomycin.

METHODSRats were given one dose (5 mg/kg) of bleomycin in bleomycin-treated groups, amygdalin-treated groups and saline in controls by intratracheal instillation exposed surgically. The amygdalin-treated groups rats were treated with intraperitoneal injection of amygdalin (15 mg x kg(-1) x day(-1)). The rats were sacrificed 7, 14 and 28 days after bleomycin administration. Polarized light microscopy and Image-Pro Plus detected I and III collagen expressed in Paraffin-embedded lung sections stained with Sirius red. Surface-enhanced laser desorption-ionization time-of-flight mass spectrometry (SELDI-TOF MS) with weak cationic proteinchip (CM10) detected differentially expressed proteins in the pooled serum samples of all groups.

RESULTSConsistent fibrotic responses were found in all bleomycin and amygdalin-tread groups. On the 7th, 14th and 28th day after bleomycin or saline instillation, four differentially expressed proteins were detected in the pooled serum of all groups rats, consisting of 4 proteins with mass/charge ratio of 3530.7, 7043.5, 8332.6 and 9068.0, respectively. Compared with control groups, protein peaks intensity ratio with mass/charge ratio of 3530.7 on 7, 28 d and 7043.5, 8332.6 and 9068.0 on 7, 14 and 28 d was > 2 in bleomycin-treated groups. Compared with amygdalin-treated groups, protein peaks intensity with mass/charge ratio of 3530.7 at 7, 14, 28 d had no change almost, but protein peaks intensity ratio with mass/charge ratio of 7043.5 at 7 d, 8332.6 on 28 d and 9068.0 on 14 d was > 2 in bleomycin-tread groups. All the four protein peaks intensity had no change almost at other point.

CONCLUSIONAmygdalin may reduce the bleomycin-induced increase of differentially expressed protein peak intensities in rat serum.

Amygdalin ; pharmacology ; Animals ; Biomarkers ; blood ; Bleomycin ; adverse effects ; Blood Proteins ; metabolism ; Male ; Pulmonary Fibrosis ; blood ; chemically induced ; Rats ; Rats, Wistar

This research is to study the relationship between HPLC fingerprints of Moutan Cortex, Paeoniae Radix Rubra and Paeoniae Radix Alba and their activity on lipopolysaccharide-induced acute lung injury. HPLC fingerprints of each extract of Moutan Cortex,Paeoniae Radix Rubra and Paeoniae Radix Alba were established by an optimized HPLC-MS method. The activities of all samples against protein and tumor necrosis a factor were tested by the model of lipopolysaccharide-induced acute lung injury. The possible relationship between HPLC-MS fingerprints and the activitieswere deduced by the Partial least squares regression analysis method. Samples were analyzed by HPLC-MS/MS to identify the major peaks. The results showed that each sample had some effect on acute lung injury. Four components with a lager contribution rate of efficacy were calculated by the research of spectrum-effect relationship. Moutan Cortex exhibited good activity on acute lung injury, and gallic acid, paeoniflorin, galloylpaeoniflorin and paeonol were the main effective components.
Acetophenones ; chemistry ; pharmacology ; Acute Lung Injury ; chemically induced ; drug therapy ; Animals ; Bridged Bicyclo Compounds, Heterocyclic ; chemistry ; pharmacology ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Gallic Acid ; chemistry ; pharmacology ; Glucosides ; chemistry ; pharmacology ; Lipopolysaccharides ; pharmacology ; Male ; Monoterpenes ; chemistry ; pharmacology ; Paeonia ; chemistry ; Plant Roots ; chemistry ; Rats ; Rats, Wistar ; Tandem Mass Spectrometry ; methods