Secretive Expression of Insect Antifungal Peptide-Encoded Genes in Pichia pastoris and Activity Assay of the Products

The antifungal peptides, drosomycin （Drs） and its isoform drosomycin-like C （Drs-lC） from Drosophila melanogaster and Thanatin from Podisus maculiventris, have potent activity with broad spectrum against filamentous fungi. Secretive expression of these genes in yeasts makes it possible to utilize the supernatants of yeast culture as protective reagents on fruit, vegetable, food and other agricultural products. So the study of effective secretion by yeast expression system is of great importance. Three genes, Drs, Drs-lC, and Thanatin, were cloned into pPICZαA and the recombinant vectors, pPICZαA-Drs, pPICZαA-Drs-lC, and pPICZαA-Thanatin were transformed into Pichia pastoris by the electric transfer method. The recombinant P. pastoris, which was screened by phenotype selection and PCR amplification, was induced to express antifungal peptide by methanol. The expressive products of the three recombinants showed antifungal activity against 5 out of 6 test fungi strains, and the products of Thanatin also had strong activity against the tested bacteria. The three antifungal peptide genes, Drs, Drs-lC, and Thanatin, were constructed into yeast P. pastoris. The expressed peptides were successfully secreted into the culture medium and exhibited potent activities against the test strains.

Improved Activity Assay Method for Arginine Kinase Based on a Ternary Heteropolyacid System

This paper presents a new system for the activity assay of arginine kinase (AK), based on the spectrophotometric determination of an ascorbic acid reduced blue ternary heteropolyacid composed of bismuth, molybdate and the released phosphate from N phospho L arginine (PArg) formed in the forward catalysis reaction. The assay conditions, including the formulation of the phosphate determination reagent (PDR), the assay timing, and the linear activity range of the enzyme concentration, have been tested and optimized. For these conditions, the ternary heteropolyacid color is completely developed within 1 min and is stable for at least 15 min, with an absorbance maximum at 700 nm and a molar extinction coefficient of 15.97 (mmol/L) -1 ·cm -1 for the phosphate. Standard curves for phosphate show a good linearity of 0.999. Compared with previous activity assay methods for AK, this system exhibits superior sensitivity, reproducibility, and adaptability to various conditions in enzymological studies. This method also reduces the assay time and avoids the use of some expensive instruments and reagents.

Gene Cloning and Expression of PKA Catalytic Subunit and Its Activity in Arabidopsis thaliana

Protein phosphorylation and dephosphorylation are the general means of regulation metabolism within a cell. A PKA catalytic subunit was found in Arabidopsis genome using Blast software. The cDNA was cloned by RT-PCR and sequencing result indicated a high degree of homology at protein level. The cDNA was subcloned into pET30a (+) and expressed in E. coli at different temperatures. The target protein was insoluble when induced at 37degreesC, while dissolvable if induced at 22degreesC with 0.01 mmol/L IPTG. Ni2+-NTA affinity chromatography was used to purify the target protein, which was shown to have cAMP-dependent protein kinase activity. Western blotting analysis indicated that stress treatments affected the expression of PKA catalytic subunit at protein level just to a small extent.

Potential bone-inducing activity in vitro of recombinant human bone morphogenetic protein-7 from a CHO expression system

Objective: To express the recombinant human bone morphogenetic protein-7(rhBMP-7) in Chinese hamster ovary(CHO) cells, and to establish the in vitro biological activity assay of rhBMP-7.Methods: Human BMP-7 cDNA was subcloned into p114 mammalian expression vector and transfected to CHO cells by using the Lipofectamine 2000 transfection method. CHO cell supernatants were harvested and analyzed to identify the molecule mass of secreted rhBMP-7 and examine its biological activity in vitro to stimulate the synthesis of alkaline phophatase(ALP), a characteristic of osteoblast phenotypes. Results: rhBMP-7 was produced stably in CHO cells, as a processed mature disulfide-linked homodimer, with an apparent molecular mass of 36 000. Examination of the rhBMP-7 biological activity showed that rhBMP-7 specifically stimulated the production of ALP(4-fold increase at 100 ng of rhBMP-7/ml). Conclusion: The rhBMP-7 from CHO expression system has significant biological activity in induction of osteoblast phenotype, which demonstrates rhBMP-7 has the potential bone regeneration activity.

Isolation and molecular identification of cellulolytic bacteria from Dig Rostam hot spring and study of their cellulase activity

Cellulose is the main structural component of lignocellulosic wastes that can be converted to sugars and biofuels by cellulase.Due to wide applications of this enzyme in various industries around the world,cellulase is considered as the third industrial enzyme.The ability of thermophilic bacteria in the production of heat-stable cellulases has made them valuable tools in biotechnology.The aim of this study was isolation and molecular identification of cellulolytic thermophile bacteria from Dig Rostam hot spring and investigating their cellulase activity.Samples were taken from water and sediments of this hot spring,and cellulolytic bacteria were enriched in media containing cellulose as the only carbon source.The bacteria were incubated at 60℃,and single colonies were then isolated on solid media.Congo red assay was used as a quick test for the qualitative screening of cellulase activity.According to these qualitative results,four colonies named CDB1,CDB2,CDB3,and CDB4 were isolated,and their growth curve and some other characteristics were determined by biochemical assays.Moreover,endoglucanase,exoglucanase,and FPase activities of the isolates were investigated quantitatively.Results indicated that CDB1 exhibited the highest endoglucanase(0.096 U/mL)and exoglucanase(0.156 U/mL)activities among other isolates.16S rDNA partial sequencing indicated that CDB1 had 99%similarity to the genus Anoxybacillus,and the other isolates showed the highest similarity to the genus Geobacillus.The cellulase gene of CDB1 isolate with the highest cellulase activity was also cloned,and its sequence is reported for the first time.Further studies on this thermophilic enzyme might be useful for industrial applications.

Synthesis of TP3 FragmentviaOne Pot Strategy and Its Immune Regulatory Activity

We have modified the previously described one-pot peptide synthesis method. The modified method has been successfully applied to the synthesis of TP3. Furthermore, the immune regulatory activity of TP3 has been characterized. The results show that the modified one-pot method can be used to synthesize the biological active peptide with the advantages of low cost and high productivity. Moreover, TP3 has a higher immune regulatory activity than TP5.

Optimization of reporter gene assay:several factors influencing detection of promoter activity

Background Promoter analysis is currently applied to detect the expression of the targeted gene in studies of signal transduction and transcriptional regulation. As a reporter gene, luciferase plays an important role and has been used widely in the promoter assay. Methods Human embryonic lung fibroblast cells （2BS）, HeLa cells and MCF-7 cells were transfected with various genes embedded by lipofectamine. This study determined vadous factors that affect promoter activity determination, such as the selection of the reporter genes and internal references, the dose and the type of the vectors carrying the transcription factors, the host cells and the instruments. Results The sensitivity of the luciferase assay was much higher than that of enhanced green fluorescence protein （EGFP）. Moreover, promoter activity is increased in a dose-related manner only in certain ranges outside of which the results may be reversed and the promoter activity is related to the expression vector which is carrying the cDNA. Otherwise, the length of the promoter, internal references and the host cell can also influence the promoter activity. Conclusions To detect the promoter activity accurately, a few factors including dose, vector, length and host cell which influence reporter gene assay aforementioned should be considered.

Sphingomyelin synthase activity measurement by the fluorescent product in cell culture medium or animal plasma

Sphingolipids, a new class of lipid mediators, are involved in a variety of important physiological and pathological processes. Sphingomyelin synthase （SMS） is an enzyme to convert the ceramide （Cer.） and phosphatidylcholine into sphingomyelin （SM） and diacylglycerol, which plays a key role in sphingolipid biosynthesis. Two SMS isoforms, SMS1 and SMS2, have been identified with different subceUular localizations and expression level in tissues. Previous studies have shown that SMS may serve as a potential therapeutic target for the treatment of various diseases, such as cardiovascular and metabolic diseases. Thus, there is an urgent need for a rapid and sensitive method for SMS activity analysis. In our study, we developed a novel method for SMS activity by monitoring the appearance of the product, NBD-SM, in the tissue culture medium or blood and applied this method in cells and mice. In Huh7 cells, the interassay coefficient of variation of the SMS activity assay was （3.60±0.07）% . In wild type （WT） mice, we observed accumulation of NBD-SM in blood in a time dependent fashion. In SMS2 KO mice, NBD-SM in plasma collected at 5- （0%, P〈0.01）, 30- （16%, P〈0.01）, and 60 min （21%, P〈0.01） after injection of fluorescence liposome solution was significantly decreased compared with WT mice. However, in SMS1 KO mice, NBD-SM in plasma collected 5- and 30 min is similar to that in WT mice. Our results suggest that this method could be used for SMS activity measurement in vitro and in vivo.

A Method for Determination of Hexokinase Activity by RP-HPLC

A method was established to determine the activity of hexokinase by reverse-phase high-performance liquid chromatography （RP-HPLC）. The activity of hexokinase was determined by detecting the amount of ADP in the reaction that converts glucose into glucose-6-phosphate. The separation degree of ATP and ADP was as good as 3.46 （separation degree^l.5）. In the range of 0.01-0.40 mmol/L, there was a good linear relationship between concentration of ADP and its peak areas （the correlation coefficient was 0.999）. The relative standard deviation（RSD） of intraday was 1.43%-1.46%, that of interday was 2.12%-2.15%. At 30℃ （optimal temperature） and pH7.0 （optimal pH）, the recovery rate of hexokinase was 95.3%-97.6%. The results showed that the method had good precision and high recovery rate, and the enzyme activity was determined through only one-step reaction. To some extent, this method can reduce the cost.

Synthesis and antitumor activity of some new pyrazolo[1,5-α]pyrimidines

New series of pyrazolo[1,5-α]pyrimidine derivatives 7a-i,11a-c and Schiff bases 13a-c were synthesized and screened for their in vitro antitumor activity against three human carcinoma cell lines,namely colorectal carcinoma（HCT116）,prostate adenocarcinoma（PC-3） and liver carcinoma（HepG-2） using MTT cytotoxicity assay at 100 μg/mL.Some of the tested compounds displayed good anticancer activities against HCT-116 and PC-3 cells.Whereas,compounds 7d and 11 a showed better antitumor activity than the rest of the compounds against both cell lines.A structure-activity relationship（SAR） has been discussed and structures of the newly synthesized compounds were confirmed by different spectral data（MS,IR,^1H NMR and ^13C NMR） and elemental analysis.