Objective To develop a new method to screen the potential microcystin-producing cyanobacteria in natural waters. Methods Cyanobacteria were cultured and waters samples were analyzed by polymerase chain reaction with special primers for gene of the operon mcyD which encodes a microcystin synthetase combining conserved gene 16S rRNA of alga, subsequently, whose microcystins were detected by ELISA. Results There was a unique amplified product of approximately 870 bp in microcystin-producing blue-green alga and water samples, but no target band in non-toxic strains and samples. Conclusion It is feasible to use the molecular biological method used mcyD as the microcystin molecular biomarker to screen microcystin-producing cyanobacteria in natural water.

[Objective]To evaluate the clinical effect for the treatment of severe thoracolumbar vertebral burst fracture by the method of anterior vertebral decompression and fusion combining anterior or posterior fixation,and investigate its indications in different conditions.[Method]At the standard of Denis,twenty-eight cases were treated by anterior vertebral decompression and fusion combining anterior or posterior fixation.In a total of 28 cases,25cases were single vertebral fractures,2 cases were two adjacent vertebral fractures and 1 case was two vertebral fractures comparted by one unfractured centrum,the level of vertebral destruction was ruled by LSC scale of McCormack.[Result]26 cases were reviewed,all patients' pain were relieved obviously after the operation,(VAS scale improved),the height and shape of the fractured centrums recovered obviously(Cobb's angle improved 14.5? in average).After an average of 21.5 months follow-up,the pain all disappeared,the intervertebral grafted bone reached bony union,no Cobb's angle increased and no apparent implication occurred in all cases.[Conclusion]Application of anterior vertebral decompression and fusion combining anterior or posterior fixation can increase the stability of the anterior column of the spine,decompress completely,the intervertebral bone graft with kinds of interbody fixation enables the patients to move as early as possible,it is an effective method in treatment of severe thoracolumbar vertebral burst fracture.

[Objective]To investigate the effects of the expansive Z-open-door laminoplasty for the ossification of posterior longitudinal ligaments(OPLL).[Methods]Thirty-six cases of OPLL were operated by the expansive Z-open-door laminoplasty.According to JOA score(17 Points) the proximate effects of all patients were analyzed.[Results]All cases were followed-up for average 18.6 months.JOA improved from 8.3 preoperatively to 13.8 postoperatively.Improved rate averaged 58.6%.Image show:3 of 31cases of anterior protruding type were changed to straight type,5 cases of straight type still kept initial type.None of all cases changed into posterior protruding type after operation.The flexion and extention range of the neck were less 1/3 and the axis-ward pain happened commonly.[Conclusion]Improved expensive Z-open door laminoplasty for DPLL can decompression clearly.It has advantages of adeguate canal expansion,preventing re-dosing the door,and being safe and reliable.

Aim To explore the effects of lipoteichoic acid (LTA) induced delayed preconditioning (PC) on hypoxia-reoxygenation (H/R) injury of cultured human coronary artery endothelial cells (HCAECs), and to investigate the potential role of endogenous nitric oxide (NO) participated in the protective mechanism. Methods HCAECs were incubated for 2 h in a hypoxic atmosphere and reoxygenated for 4 h in a normoxic atmosphere. The delayed PC was induced by pretreatment with LTA assessed by the percentage of cellular injury with Trypan blue exclusion and by the amount of lactate dehydrogenase (LDH) in culture media. The NO level of the culture media was measured detect the expression of eNOS mRNA by RT-PCR method after cells were recovered from different points.Results LTA pretreatment significantly decreased the percentage of the killed cell and the concentration of LDH in media. Also, LTA pretreatment obviously raised the concentrations of NO in culture media. The protective effects of LTA were abrogated by pretreatment with N-monomethyl-L-arginine (L-NMMA).Moreover, the expression of eNOS mRNA was significantly upregulated after HCAECs exposure to LTA for 4 h following 2 h or 4 h recovery. Conclusion LTA could induce the delayed protection against H/R induced endothelial injury and dysfunction of cultured HCAECs. NO produced by eNOS acts initially as a trigger and subsequently as a mediator of delayed PC.

Recombinant HLA-Ⅰ molecules/antigenic peptide complexes (pHLA complexes) are applied in the research of human T cell-specific immune responses. The preparation of pHLA complex is based on genetic engineering and protein in vitro dilution and folding-refolding technology. In an in vitro refolding system, recombinant HLA-Ⅰ molecules correctly fold and bind with antigenic peptides to form complexes. In this study, ultrafiltration-high performance liquid chromatography (ultrafiltration-HPLC) was used for quantitative determination of the antigenic peptides in recombinant pHLA complexes, especially for those in a small amount of prepared products. By adding the recombinant HLA-Ⅰ molecules and antigenic peptides into the refolding buffer, the heavy chain (HC) and light chain (β2m) of recombinant HLA-Ⅰ molecules were refolded and bond with the VYF antigenic peptide containing anchor residues to form a pHLA complex. The unbound free antigenic peptide VYF was removed by ultrafiltration to retain the complex. Finally, the pHLA complex was treated by acid to destroy its interaction, thus releasing the antigenic peptide. The results showed that the prepared recombinant pHLA complex was recognized by HLA-Ⅰ molecule specific antibody W6/32, which indicated that the recombinant HLA-Ⅰ class molecule had correct folding and was identified as pHLA complex. The antigen peptide VYF contained in the pHLA complex was also detected by ultrafiltration-HPLC, so it is feasible to apply ultrafiltration-HPLC for determination of pHLA complex. Compared with Western blotting, the concentration of antigenic peptides detected by ultrafiltration-HPLC was 0-9 μg/mL. The binding conditions can be optimized according to the amount of antigenic peptides bound in the complex in order to improve the folding efficiency of HLA-Ⅰ molecules and promote the binding of HLA-Ⅰ molecules to antigenic peptides. The production rate of pHLA complexes in the refolding system can also be calculated according to the content of antigenic peptides bound by pHLA complexes. Therefore, ultrafiltration-HPLC in this study can be used for the quality control of the preparation process of pHLA complexes, and may facilitate the research of T cell-specific immunity, artificial antigen-presenting cells, and development of specific tetramer probe applications.
Amino Acid Sequence ; Antigens ; Chromatography, High Pressure Liquid ; Humans ; Peptides/chemistry* ; Ultrafiltration

To explore the potential of lipoteichoic acid (LTA) induced cardioprotection against ischemia-reperfusion (I/R) injury in isolated rat hearts and whether endogenous nitric oxide (NO) participates in the protection, the rats were pretreated with LTA (1 mg/kg, i.p.) 24 h before the experiment, and the isolated hearts were subjected to 30 min no-flow normothermic global ischemia and 60 min reperfusion after a 20-min stabilization period by the langendorff method. Cardiac functions were evaluated at the end of stabilization, and at 30 min, 60 min of reperfusion. The amounts of MB isoenzyme of creatine kinase (CK-MB), lactate dehydrogenase(LDH) and total NO oxidation products in the coronary effluent were measured spectrophotometrically at the end of reperfusion. It was revealed that pretreatment with LTA could significantly improve the recovery of cardiac function, reduce the release of CK-MB and LDH, and increase the concentrations of NO in coronary effluent. The protective effects were abrogated by pretreatment of the rats with L-NAME. It was concluded that LTA could induce the delayed cardioprotection against I/R injury, and endogenous NO may be involved in the mechanisms.
Animals ; Cardiotonic Agents ; pharmacology ; Creatine Kinase ; metabolism ; Creatine Kinase, MB Form ; In Vitro Techniques ; Ischemic Preconditioning, Myocardial ; Isoenzymes ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Lipopolysaccharides ; pharmacology ; Male ; Myocardial Reperfusion Injury ; prevention & control ; Nitric Oxide ; metabolism ; Rats ; Rats, Wistar ; Teichoic Acids ; pharmacology

Ethylene regulates sex expression in cucumber plants (Cucumis sativus L.). ACC synthase is a key factor during the ethylene biosynthesis. Pairs of PCR primers were synthesized corresponding to the conserved sequences of ACC synthase gene family. The 1 188 bp DNA fragment of ACC synthase gene (CS-ACS2) was amplified from genomic DNA of 8 different sexual phenotypes of cucumber respectively (GenBank accession number is DQ115884～DQ115886 and DQ115875～DQ115879). 8 SNPs have been identified by sequences analysis between 3 monoecious lines and 5 subgynoecious lines and gynoecious lines, which including 4 A←→G and 4 T←→C transition. Of these 8 SNPs, one locus is in intron and 7 loci in exons. Of the 7 SNPs located in exons, 3 SNPs are non-coding SNPs and 4 SNPs are coding SNPs (cSNPs) of which 3 induced changes of encoding amino acid of ACC synthase. The results of SNPs from subgynoecious lines and gynoecious lines suggest that single nucleotide mutation events of CS-ACS2 might be correlated with the development of subgynoecious lines and gynoecious lines in cucumber. Furthermore, CAPS marker C-MT705 was developed for identifying elite subgynoecious cultivar MT-705, which could be valuable in cucumber breeding. Besides, the SNPs and CAPS markers obtained in the study enriched molecular markers of cucumber.

Pituitary adenoma is one of the most common intracranial tumors, and most of them can be well removed by endoscopic transsphenoidal surgery. Pituitary adenoma is located in the sella, which can compress the pituitary and optic nerve, invade the sphenoid sinus and cavernous sinus, invade and grow in all directions, and affect the endocrine function at the same time. Endoscopic transsphenoidal pituitary adenoma resection (EETS) has the advantages of minimally invasive, close observation, panoramic view, high tumor resection rate and high preservation rate of normal pituitary function, but it can cause pituitary endocrine disorder, diabetes insipidus, electrolyte disorder, cerebrospinal fluid leakage, intracranial infection, cerebral nerve paralysis, tumor cavity, intracranial hemorrhage, rhinitis and other related complications. It affects the prognosis and quality of life of patients. The postoperative complications of EETS are not only related to the pathophysiological characteristics of pituitary adenoma, but also closely related to the depth of understanding of pathology and anatomy of pituitary adenoma, surgical skills, proficiency and clinical experience. Familiarity with endoscopic anatomy and fine surgical techniques, rich surgical experience, strict preoperative endocrine function evaluation, hormone replacement therapy, etc. can reduce the incidence of postoperative complications of EETS.

An efficient genetic transformation system and suitable promoters are essential prerequisites for gene expression studies and genetic engineering in streptomycetes. In this study, firstly, a genetic transformation system based on intergeneric conjugation was developed in Streptomyces rimosus M527, a bacterial strain which exhibits strong antagonistic activity against a broad range of plant-pathogenic fungi. Some experimental parameters involved in this procedure were optimized, including the conjugative media, ratio of donor to recipient, heat shock temperature, and incubation time of mixed culture. Under the optimal conditions, a maximal conjugation frequency of 3.05×10^-5 per recipient was obtained. Subsequently, based on the above developed and optimized transformation system, the synthetic promoters SPL-21 and SPL-57, a native promoter potrB, and a constitutive promoter permE^* commonly used for gene expression in streptomycetes were selected and their activity was analyzed using gusA as a reporter gene in S. rimosus M527. Among the four tested promoters, SPL-21 exhibited the strongest expression activity and gave rise to a 2.2-fold increase in β-glucuronidase (GUS) activity compared with the control promoter permE^*. Promoter SPL-57 showed activity comparable to that of permE^*. Promoter potrB, which showed the lowest activity, showed a 50% decrease in GUS activity compared with the control permE^*. The transformation system developed in this study and the tested promotors provide a basis for the further modification of S. rimosus M527.
Conjugation, Genetic ; Glucuronidase/genetics* ; Promoter Regions, Genetic ; Streptomyces rimosus/genetics*

OBJECTIVETo investigate the effect of TiO₂nanotube arrays covalently modified by recombinant human bone morphogenetic protein- 2(rhBMP- 2) on the early bioactivity of mesenchymal stem cells (MSC) in vitro and to provide experimental evidence for the biochemical modification of titanium implants.

METHODSIn the experiment group, double titanium nanotube arrays were prepared by anodization, and were chemically grafted with rhBMP- 2. Mechanically polished pure titanium was used as blank control group, and titanium dioxide nanotubes was used as negative control A group, and titanium dioxide nanotubes + carbonyldiimidazole as negative control B group. Field emission scanning electron microscope (FE- SEM) and X- ray photoelectron spectroscopy (XPS) were used to detect the morphology and physicochemical properties of the experiment group, blank control group and the negative control group. Cell adhesion on the specimen surface of the experiment group, blank control group and negative control group on the 1st day was tested. Cell proliferation on the 1st, 3rd and 5th day and alkaline phosphatase activity on the 5th, 7th and 11th day was also tested.

RESULTSFE- SEM showed that the surface of titanium nanotubes loaded with rhBMP- 2 possessed visible miliary particulate matter. XPS showed that nitrogen peak in the group of titanium nanotubes loaded with rhBMP-2 was significantly greater that those in the other groups. FE- SEM showed that the cells on the surface of the experimental group on the 1st day spread well, better than those in the control group and negative control group. Cell proliferation activity on the 1st day in different groups was not obvious (P>0.05), the A value of the experimental group on the 3rd and 5th day (3.295 ± 0.153, 3.823 ± 0.059) were significantly higher than those in the control group (2.479 ± 0.064, 3.131 ± 0.096) and negative control A group (2.715 ± 0.075, 3.371 ± 0.047) and negative control B group (2.756 ± 0.132, 3.637 ± 0.047) (P<0.05). Alkaline phosphatase activity on the 5th, 7th and 11th day in the experimental group (0.0477 ± 0.0287, 0.0615 ± 0.0016, 0.0667 ± 0.0018) were better than those in the control group, negative control A group and negative control B group (P<0.05).

CONCLUSIONSTitanium nanotube arrays can be loaded with rhBMP-2 by biochemical methods and have good biocompatibility.

Alkaline Phosphatase ; metabolism ; Bone Morphogenetic Protein 2 ; pharmacology ; Cell Adhesion ; Cell Proliferation ; Humans ; Materials Testing ; Mesenchymal Stromal Cells ; Microscopy, Electron, Scanning ; Nanotubes ; chemistry ; ultrastructure ; Photoelectron Spectroscopy ; Recombinant Proteins ; pharmacology ; Surface Properties ; Titanium ; chemistry ; Transforming Growth Factor beta ; pharmacology