Targeted Delivery Prodigiosin to Choriocarcinoma by Peptide-Guided Dendrigraft Poly-l-lysines Nanoparticles.

The available and effective therapeutic means to treat choriocarcinoma is seriously lacking, mainly due to the toxic effects caused by chemotherapy and radiotherapy. Accordingly, we developed a method for targeting delivery of chemotherapeutical drugs only to cancer cells, not normal cells, in vivo, by using a synthetic placental chondroitin sulfate (CSA)-binding peptide (plCSA-BP) derived from malarial protein VAR2CSA. A 28 amino acids placental CSA-binding peptide (plCSA-BP) from the VAR2CSA was synthesized as a guiding peptide for tumor-targeting delivery, dendrigraft poly-L-lysines (DGL) was modified with plCSA-BP and served as a novel targeted delivery carrier. Choriocarcinoma was selected to test the effect of targeted delivery carrier, and prodigiosin isolated from Serratia marcescens subsp. lawsoniana was selected as a chemotherapeutical drug and encapsulated in the DGL modified by the plCSA-BP nanoparticles (DGL/CSA-PNPs). DGL/CSA-PNPs had a sustained slow-release feature at pH 7.4, which could specifically bind to the JEG3 cells and exhibited better anticancer activity than that of the controls. The DGL/CSA-PNPs induced the apoptosis of JEG3 cells through caspase-3 and the P53 signaling pathway. DGL/CSA-PNPs can be used as an excellent targeted delivery carrier for anticancer drugs, and the prodigiosin could be an alternative chemotherapeutical drug for choriocarcinoma.

Biological Potential and Mechanism of Prodigiosin from Serratia marcescens Subsp. lawsoniana in Human Choriocarcinoma and Prostate Cancer Cell Lines.

Tripyrrole molecules have received renewed attention due to reports of numerous biological activities, including antifungal, antibacterial, antiprotozoal, antimalarial, immunosuppressive, and anticancer activities. In a screen of bacterial strains with known toxicities to termites, a red pigment-producing strain, HDZK-BYSB107, was isolated from Chamaecyparis lawsoniana, which grows in Oregon, USA. Strain HDZK-BYSB107 was identified as Serratia marcescens subsp. lawsoniana. The red pigment was identified as prodigiosin using ultraviolet absorption, LC-MS, and 1H-NMR spectroscopy. The bacterial prodigiosin had an inhibitory effect on both Gram-negative and Gram-positive bacteria. The main objective of this study was to explore the anticancer activities and mechanism of strain HDZK-BYSB107 prodigiosin by using human choriocarcinoma (JEG3) and prostate cancer cell lines (PC3) in vitro and JEG3 and PC3 tumor-bearing nude mice in vivo. In vitro anticancer activities showed that the bacterial prodigiosin induced apoptosis in JEG3 cells. In vivo anticancer activities indicated that the prodigiosin significantly inhibited the growth of JEG3 and PC3 cells, and the inhibitory activity was dose and time dependent. The anticancer efficacy of the bacterial prodigiosin on JEG3 and PC3 cells, JEG3 and PC3 tumor exhibited a correlation with the down regulation of the inhibitor of IAP family, including XIAP, cIAP-1 and cIAP-2, and the activation of caspase-9 and caspase-3 accompanied by proteolytic degradation of poly (ADP-ribose)-polymerase. The expressions of P53 and Bax/Bcl-2 in JEG3 and PC3 cells were significantly higher than in untreated groups. Our results indicated that the bacterial prodigiosin extracted from C. lawsoniana is a promising molecule due to its potential for therapeutic applications.

Bacillus subtilis subspecies virginiana, a new subspecies of antitermitic compound-producing endophytic bacteria isolated from Juniperus virginiana.

Termites are worldwide pests causing considerable damage to agriculture, forestry and buildings. Although physical and chemical methods have been tried to eliminate termite populations, they have the limitations such as low effectiveness, high-toxicity residue, environmentally harmful and high cost. Therefore, it has attracted much attention to develop highly effective, low-toxic, long residual period, environmentally friendly and low-cost termiticidals. Here, we report the characterization and antitermitic activities of a new antitermitic compound-producing endophytic bacterium HUB-I-47 isolated from eastern red-cedar, Juniperus virginiana L. The morphological, physiochemical characteristics of strain HUB-I-47 and its 16S rDNA sequences, and the antitermitic compound were analyzed by gas chromatography-mass spectrometry were studied. We found that the morphology of HUB-I-47 was very similar to that of Bacillus subtilis but presented some differences in shape and cell size. Growth evaluation showed that the lowest, highest, and optimum growth temperatures of HUB-I-47 were 12, 47, and 31 degrees C, respectively, which were different from those of reference strains. The 16S rDNA sequence analysis revealed a high similarity of 99% to those of B. subtilis. Based on these analyses, we named strain HUB-I-47 as B. subtilis subsp. virginiana D. P. Zhou, K. Zhao, J. Liu et W. X. Ping, subsp. nov. This is the first report on the analysis of antitermitic compounds from endophytic bacteria. Our study identified a new resource of antitermitic compounds through endophytic bacteria fermentation.

[Screening of high taxol producing fungi by mutagenesis and construction of subtracted cDNA library by suppression subtracted hybridization for differentially expressed genes].

OBJECTIVE: To screen mutants with high yield of taxol, and construct cDNA subtractive library of obtained mutant and primary strain HD(1-3). METHODS: The spores of taxol-producing fungus HD(1-3) were treated by diethyl sulphate (DES), ultraviolet radiation and diethyl sulphate (UV + DES). cDNA subtractive library of taxol producing fungi from the mRNA of obtained mutant with high yield of taxol tester and HD(1-3) driver was constructed by using suppression subtracted hybridization (SSH). RESULTS: The optimal conditions for mutagenesis of strain HD(1-3) were as follows: the spore suspension was treated with 8% DES for 15 min, followed by UV irradiation (30 w, 30 cm distance) for 45 sec under magnetic stirring, a mutant UD(14-1) which was able to produce taxol with high yield and could be stably passed on genetics was found. Its ability to produce taxol was improved from 232.73 +/- 4.61 microg/L (strain HD(1-3)) to 312.81 +/- 7.51 microg/L (strain UD(14-1)). The tilter of the constructed cDNA library was 1.2 x 10(7) cfu/mL, the recombinant rate reached to 75.3% and the length of the inserted fragments was mostly 300 bp-1.0 kb. CONCLUSION: A mutant UD(14-11) with high yield was obtained, and cDNA subtractive library of the mutant UD(14-11) and strain HD(1-3) was constructed. The study laid solid foundation for isolation of taxol biosynthesis related genes and construction of engineering strains with high yield of taxol by genetic techniques.

[Screening and breeding of high lysine-producing strains by genome shuffling].

OBJECTIVE: To screen and breed high lysine-producing strains by genome shuffling. METHODS: Corynebacterium pekinense 1 was used as the starting strain in this study. High lysine-producing strains were screened by genome shuffling. RESULTS: Five hereditarily stable strains with high lysine production were obtained by four cycles of genome shuffling. A high lysine producing strain, F4-36, was obtained, which produced 16.95 g/dL lysine, 37.1% higher than that of the starting strain. CONCLUSION: Genome shuffling can be an efficient tool to screen and breed high lysine production strains.

Screening and breeding of high taxol producing fungi by genome shuffling.

To apply the fundamental principles of genome shuffling in breeding of taxol-producing fungi, Nodulisporium sylviform was used as starting strain in this work. The procedures of protoplast fusion and genome shuffling were studied. Three hereditarily stable strains with high taxol production were obtained by four cycles of genome shuffling. The qualitative and quantitative analysis of taxol produced was confirmed using thin-layer chromatography (TLC), high performance liquid chromatography (HPLC) and LC-MS. A high taxol producing fungus, Nodulisporium sylviform F4-26, was obtained, which produced 516.37 microg/L taxol. This value is 64.41% higher than that of the starting strain NCEU-1 and 31.52%-44.72% higher than that of the parent strains.

[Recent advance and prospect on taxol production by endophytic fungus fermentation--a review].

Taxol has become a widely used clinical anti-cancer drug. Due to the scaricity of Taxus trees, current taxol output cannot meet the requirement of the market. Taxol produced by endophytic fungus fermentation has high prospective. We reviewed advantages of taxol production by fungus fermentation, research advances of isolation, biodiversity of taxol-producing fungi and methods of improved taxol output by endophytic fungus fermentation.

[Effects and mechanism of decorin on the proliferation of A549 lung adenocarcinoma cells in vitro].

BACKGROUND: Decorin is a member of the small proteglycans in extracellular matrix of tumor microenvironment, which is known to relate to the initiation, progression and growth of the tumor. The aim of this study is to investigate the effects and mechanism of decorin on the proliferation of A549 lung adenocarcinoma cell line in vitro. METHODS: Lung adenocarcinoma cell line A549 was cultured with decorin in a wide range of concentration for different time. Cell activities were studied by MTT. The changes of cell cycle and apoptosis were analyzed by FCM. Decorin mRNA expression was detected by RT-PCR. P21 expression was determined by Western blot. TGF-ß concentration in the culture supernatants was determined by ELISA. RESULTS: The proliferation of A549 cell could be inhibited by decorin in vitro and the inhibition effect was the time- and dose-dependent relationship. Apoptosis of adenocarcinoma cell could be efficiently induced by decorin in a time/dose-dependent manner. Decorin could upregulate the intrinsic decorin mRNA and P21 protein expression, downregulate the TGF-ß, and block cell cycle at G1 phase. CONCLUSIONS: Decorin can inhibit adenocarcinoma cell proliferation and induce apoptosis of adenocarcinoma cells in vitro. The proliferation of A549 cell could be inhibited in vitro by decorin through the mechanism of increasing decorin mRNA, decreasing TGF-ß, increasing P21 protein expression, inhibiting cell cycle and inducing cell apoptosis.

Isolation, Purification, and Identification of Taxol and Related Taxanes from Taxol-Producing Fungus Aspergillus niger subsp. taxi.

The content of taxol in the bark of yews is very low, and this is not affordable from the environmental point of view. Thus, it is a necessity to look for alternative sources of taxol production to solve its supply. Currently, a large portion of the taxol in the market comes from chemical semi-synthesis, but the semi-synthetic precursors such as baccatin III and 10-deacetyl-baccatin III are extracted from needles and twigs of yew trees. Taxol-producing fungi as a renewable resource is a very promising way to increase the scale of taxol production. Our group has obtained a taxol-producing endophytic fungus, Aspergillus niger subsp. taxi HD86-9, to examine if A. niger can produce the taxanes. Six compounds from the fermentation broth of strain HD86-9 were isolated and identified by 1H NMR, 13C NMR, and ESI-MS. The results showed that the six compounds included four taxane diterpenoids (taxol, cephalomannine, baccatin III, and 10-deacetyl-baccatin III) and two non-taxane compounds (ß-sitosterol and flavonoid isovitexin). The study verified that the taxanes can be produced by the A. niger, which is very important to taxol production via chemical semi-synthesis. Additionally, the finding is potentially very significant to solve the taxol semi-synthetic precursors extracted from needles and twigs of yew trees, and the precursor production can be easily increased through the culture condition optimization, genetic breeding, and metabolic engineering of the A. niger.

[Differential display technique and its progress].

Differential display technique is an important method to isolate differentially expressed gene. Comparing to other methods like representational difference analysis, suppression subtractive hybridization and serial analysis of gene expression, differential display technique is used in higher frequency. Since it was established in 1992, it has overcome many disadvantages and widened its practical fields through improvements and enhancements by global researchers. In this paper the principle and the main advantages and disadvantages of differential display technique were briefly introduced. Meanwhile, the four progressed aspects in designing primer, reducing false positives, identifying differentially expressed gene and techniques derived from DD were introduced in detail.

[Study on effect of Lactobacillus acidophilus MG2-1 on serum lipid metabolism in rats].

Wistar rats were fed with a high lipid diet supplemented with living or thermal death bacteria of Lactobacillus acidophilus MG2-1 which was isolated from koumiss in Mongolia and was of good ability of acid tolerance and decreasing the level of cholesterol in vitro. The effect of Lb. acidophilus MG2-1 on the metabolism of serum cholesterol was discussed. It was showed that it was on the 14th day of experiment that the inhibiting effects of the increase of serum cholesterol level of rat groups fed with living bacteria and heat-killed bacteria was significantly (p > 0.05) and very significantly (p < 0.01) higher than that of the high lipid diet group respectively; at the same time, the level of serum HDL-C of the thermal death bacteria group was significantly higher than that of the high lipid diet group (p < 0.05), also arteriosclerosis index of wistar rats in experimental group is significantly lower than that of the high lipid diet group (p < 0.01). The total bile acid level of the thermal death bacteria group in fecal is significantly higher than that of the high lipid diet group (p < 0.05). It is suggested that the increase of serum cholesterol level in rats can be inhibited and arteriosclerosis can also be prevented by this strain. During the period of tests, the effect of the strain on serum lipid in rats weaken with the time going, while the dose of bacteria fed was not changed.

[Breeding of high-yield strain of taxol by mutagensis of protoplast and primary discussion of genetic differences between mutants and their parent strain].

The breeding of high-yield strain of taxol was performed by protoplast mutagenesis of strain NCEU-1 using ultraviolet radiation and combined treatment of UV and LiCl. The mutants UV40-19 and UL50-6 were obtained, which raised the taxol yield from 314.07 microg/L to 376.38 microg/L and 392.63 microg/L respectively. Genetic differences between the mutants UV40-19, UL50-6 and their parent strain were primarily compared through random amplified polymorphic DNA (RAPD) and isozyme technique. The results showed that the genetic differences were very obviously between the parent strain and its mutants and between the two mutants, which laid foundation of molecular mechanism for the study of genes related to the taxol biosynthesis and mutants for raising the taxol yield.

[Study on breeding up high-yield strain of taxol by protoplast mutagensis].

In order to obtain resistant mutants to nystatin, ultraviolet radiation and LiCl were used to mutagenize the protoplasts of taxol-producing fungi NCEU-1, and four positive mutants with high yield of taxol were screened out on nystatin flat. After further screening experiments on fermentation, a mutant strain--UL04-5 which was able to produce taxol with high yield and could be stably passed on in genetics was eventually found, it's ability to produce taxol was improved from 314.07 microg/L (strain NCEU-1) to 418.24 microg/L (strain U04-5).