Response of under-ground bud bank to degradation in an alpine meadows on the Qinghai-Tibet Plateau, China.

Exploring the diversity and formation mechanism of under-ground bud banks is essential for understanding the renewal of plant populations and community succession. However, there are few studies on the response of bud bank size and composition to different degradation gradients in alpine meadows. In view of this, we investigated the size and composition of bud bank under four degradation gradients (non-degraded:ND, lightly degraded:LD, moderately degraded:MD, and heavily degraded:HD) caused by overgrazing in a typical alpine meadow in Tibet, China, using a unit area excavation sampling method, and analyzed the correlation between above-ground plant community composition and bud bank density. Our results showed that: (i) in the ND alpine meadow, rhizome buds were dominant, in the LD, tiller buds were dominant, and in the MD, root-sprouting buds were dominant; (ii) total bud bank and cyperaceae bud density decreased with increasing degradation gradient, the density of leguminosae was insignificant in each degradation gradient, and the density of gramineae and forb were dominant in LD and MD meadows, respectively; (iii) total bud bank density was significantly and positively correlated with total above-ground biomass in the LD gradient, tiller bud density was significantly positively correlated with the species diversity index of above-ground vegetation under the ND gradient, rhizome bud density was significantly and positively correlated with total above-ground biomass in the LD gradient, and root-sprouting density was significantly negatively correlated with total above-ground biomass in ND meadows, but was significantly positively correlated with the species diversity index of the LD gradient. Therefore, our research shows that rhizome buds are more important in ND meadow habitats, tiller buds are more important in LD meadow habitats, and root-sprouting buds are more important in MD meadows. The response of bud banks to degradation gradient varies with different types of bud banks and different functional groups of plants, and the survival strategy of bud banks is of great value for community restoration and regeneration, which should be paid more attention to in subsequent alpine meadow research.

[Effect of restoration types on the community structure of microbes harboring nifH and chiA genes in alpine meadow]. / 不同恢复方式对退化高寒草甸土壤nifH和chiA微生物群落结构的影响.

Biological nitrogen (N) fixation and organic N degradation are the main sources of soil available N, while microorganisms driving such processes play an important role in soil N supply and the maintenance of soil fertility. In this study, real-time quantitative PCR and amplicon sequencing technology were used to examine the effects of restoration types on the community structure of N2-fixing and chitin-degrading bacteria harboring nifH and chiA genes, respectively, and the gene abundance under four meadows (undisturbed, grazing, fencing, and fencing + reseeding mea-dows) in Qinghai-Tibet Plateau. The results showed that the abundance of nifH and chiA in the four meadows followed the order of undisturbed meadow > grazed meadow > fencing meadow > fencing + reseeding meadow. The abundance of nifH and chiA in the undisturbed meadow was 3.4-6.3 times and 3.3-8.3 times of that in the other three meadows. The α diversity of N2-fixing bacteria in gra-zing, fencing, and fencing + reseeding meadows was significantly higher than that in the undisturbed meadow, while the α diversity of chitin-degrading bacteria was higher in the undisturbed and grazing meadows. Grazing significantly increased the relative abundance of Proteobacteria, but decreased the relative abundance of Cyanobacteria and Actinobacteria. The abundance of nifH and chiA was significantly affected by soil moisture, nutrients, and vegetation characteristics, while the community structure of nifH and chiA was affected by soil moisture, soil organic carbon content, and soil pH. Compared with undisturbed meadow, grazing reduced the potential of N fixation and organic N degradation.The improvement of 10 years grazing prohibition with fencing and reseeding measures on the function of N fixation and organic N degradation was not obvious. The characteristics of functional microbes and their influencing factors should be comprehensively considered during meadow restoration, which might take longer time or take reasonable management measures to restore grazing meadow to undisturbed level.

Sensitive Tumor Cell Line for Annonaceous Acetogenins and High Therapeutic Efficacy at a Low Dose for Choriocarcinoma Therapy.

Annonaceous acetogenins (ACGs) have attracted much attention because of excellent antitumor activity. However, the lack of selectivity and the accompanying serious toxicity have eventually prevented ACGs from entering clinical application. To decrease the side effects of ACGs, the cytotoxicity of ACGs on 10 types of tumor cell lines was investigated by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) test to identify one that was very sensitive to ACGs. Meanwhile, ACGs nanoparticles (ACGs-NPs) were prepared using poloxamer 188 (P188) as an excipient so as to solve the problem of poor solubility and the in vivo delivery of ACGs. ACG-NPs were 163.9±2.5 nm in diameter, negatively charged, and spherical with a high drug loading content (DLC) of 44.9±1.2%. MTS assays demonstrated that ACGs had strong cytotoxicity against JEG-3, HeLa, SiHa, MCF-7, A375, A2058, A875, U-118MG, LN- 229, and A431 cells, among which JEG-3 cell line was extremely sensitive to ACGs with a 50% inhibitory concentration (IC50) value of 0.26 ng/mL, a very encouraging discovery. ACGs-NPs demonstrated very good dose-dependent antitumor efficacy in a broad range of 45?1200 µg/kg on JEG-3 tumor-bearing mice. At a very low dose (1200 µg/kg), ACGs-NPs achieved a high tumor inhibition rate (TIR) of 77.6% through oral administration, displaying a significant advantage over paclitaxel (PTX) injections that are currently used as first-line anti-choriocarcinoma drugs. In the acute toxicity study, the half lethal dose (LD50) of ACGs-NPs was 135.5 mg/kg, which was over 100 times as of the effective antitumor dose, indicating good safety of ACGs-NPs. ACGs-NPs show promise as a new type of and potent anti-choriocarcinoma drug in the future.

[Plant community and soil nutrient of alpine meadow in different degradation stages on the Tibetan Plateau, China.] / é’è—é«˜åŽŸé«˜å±±åµ©è‰é«˜å¯’è‰ç”¸ä¸åŒé€€åŒ–é˜¶æ®µæ¤ç‰©ç¾¤è½ä¸ŽåœŸå£¤å »åˆ†.

We examined soil physical and chemical properties and plant community characteristics of four Kobresia alpine meadows at different degradation stages in Damxung County, Lhasa City, Tibet Autonomous Region, China, with the method of spatial sequence instead of time succession. The results showed that soil organic carbon, total nitrogen, available phosphorus, available potassium, ammonium, nitrate, and water content showed a decreasing trend as the degree of soil degradation increased, while pH showed an increasing trend. Plant community height, richness, diversity index and evenness index of the moderately degraded meadow were the highest. Community coverage and total biomass were the largest in undegraded meadow and the smallest in the severely degraded meadow. As the degree of meadow degradation intensifies, the biomass and proportion of Cyperaceae decreased, the biomass and proportion of legumes and weeds increased, and the biomass and proportion of Gramineae first increased and then decreased. The aboveground biomass of meadow was significantly positively correlated with soil organic carbon content, total nitrogen content, total phosphorus content, and soil water content, and significantly negatively correlated with soil pH. With the vegetation degradation in meadow, soil degradation had worsened, which ultimately manifested as a significant decline in grassland productivity.

Combined administration on You-Gui Yin and low-dose Raloxifene partially attenuates the bone loss in ovariectomized mice through the proliferation and osteogenic differentiation of bone marrow stromal cells.

BACKGROUND: Osteoporosis is a systemic skeletal disease of fragility fractures due to the loss of mass and deterioration of the microarchitecture of bone. PURPOSE: The aim of the study was to assess the osteogenic effects and the underlying mechanisms of the combined administration of You-Gui Yin (YGY) and Raloxifene hydrochloride (RLX) in ovariectomized (OVX) mice. METHODS: First, a classic animal model was used to mimic postmenopausal osteoporosis through the removal of the ovary of mice. Second, the OVX mice were administered YGY, RLX, and YGY + RLX for 12 weeks. Next, the bone microtomographic histomorphometry and bone mineral density (BMD) were assessed by micro-CT, and the biochemical markers of procollagen type I N-terminal propeptide (P1NP) and beta-isomerized C-telopeptide (ß-CTX) in serum were assessed. Finally, primary bone marrow stromal cells (BMSCs) were isolated from the tibia and cultured to evaluate cell proliferation and osteogenic differentiation. RESULTS: The results showed that BMD on the YGY + RLX group was higher than that on the RLX group (p < 0.05) and did not have a significant difference when compared with the sham group. Notably, the YGY + RLX group had a dramatically increased trabecular number (Tb.N) compared with that of the YGY group (p < 0.05). Moreover, the BV/TV (bone volume/total volume) and Tb.N in the YGY + RLX group were higher than that in the RLX group (p < 0.05), and the Tb.Sp (trabecular separation) was lower than that in the RLX group (p < 0.05). Moreover, the serum level of P1NP from the YGY + RLX group dramatically increased when compared with that from the YGY and RLX groups (YGY group: p < 0.05; RLX groups: p < 0.01). Notably, there was no significant difference between the YGY and YGY + RLX groups. In addition, cell proliferation from the co-administration of YGY and RLX was clearly higher than a single use of YGY and RLX (p < 0.01, respectively). The ALP/BCA (alkaline phosphatase/bicinchoninic acid) in the YGY + RLX group was higher than that in the RLX group (p < 0.01). CONCLUSION: Overall, co-administered YGY and RLX could partially attenuate bone loss and were more effective than individually using either one; this outcome might be associated with the proliferation and osteogenic differentiation of BMSCs.

[Preliminary study on synthesis of novel amphiphilic molecules and their application as drug vectors].

OBJECTIVE: To synthesize three amphiphilic molecules (TEG-R1, TEG-R2, TEG-R3), with branched oligo polyethylene glycol as hydrophilic fractions and aliphatic chains (containing six, eight and twelve carbon atoms respectively) as hydrophobic fractions, and study them as insoluble drug vectors. METHOD: Three compounds were successfully through acylation, substitution reaction, reduction reaction and esterification. Their structures were verified by NMR analysis; and the critical micelle concentrations (CMC) of TEG-R1, TEG-R2, TEG-R3 were determined by pyrene fluorescence probe spectrometry. Transmission electronic microscopy (TEM) photos displayed the state of the aqueous solution. The self-assembly solution evaporation method was adopted to prepare drug loading podophyllotoxin micelles, and characterize their grain size, in order to detect the hemolysis of the three amphiphilic molecules. RESULT: Nuclear magnetism showed the successful synthesis of three amphiphilic molecules, with critical micelle concentrations of TEG-R1, TEG-R2, TEG-R3 of 50, 50, 10 mg x L(1), respectively. Transmission electronic microscopy (TEM) photos displayed a spherical-like state, with diameter of 20-50 nm. All of the three amphiphilic molecules could be prepared into drug-loading micelles, with the range of grain sizes between 100-200 nm. Hemdytic experiment showed that, among the amphiphilic molecules of the graft six-carbon aliphatic chain, TEG-R1 could not cause hemolysis. CONCLUSION: All of the three amphiphilic molecules are micellized in water solution, and can be used as insoluble drug vectors. Among them, TEG-R1 could not cause hemolysis, and is expected to become a new-type drug vector.

Lectin-directed enzyme activated prodrug therapy (LEAPT): Synthesis and evaluation of rhamnose-capped prodrugs.

The lectin-directed enzyme activated prodrug therapy (LEAPT) bipartite drug delivery system utilizes glycosylated enzyme, localized according to its sugar pattern, and capped prodrugs released by that enzyme. In this way, the sugar coat of a synthetic enzyme determines the site of release of a given drug. Here, prodrugs of doxorubicin and 5-fluorouracil capped by the nonmammalian l-rhamnosyl sugar unit have been efficiently synthesized and evaluated for use in the LEAPT system. Both are stable in blood, released by synthetically d-galactosylated rhamnosidase enzyme, and do not inhibit the uptake of the synthetic enzyme to its liver target. These results are consistent with their proposed mode of action and efficacy in models of liver cancer, and confirm modular flexibility in the drugs that may be used in LEAPT.

[Synthesis of G3.5 dendrimer-2-aminoethyl ß-D-galactopyranoside conjugates and its specificity on hepatocyte targeting in vitro].

OBJECTIVE: To prepare a valuable nanodevice targeting for hepatic parenchymal cells to improve the effects of drugs for treatment of hepatitis B and liver cancer. METHODS: Generation 3.5 dendrimer (G3.5 PAMAM) was conjugated with fluoresceine isothiocyanate (FITC) to obtain G3.5 PAMAM-FITC, which was then conjugated with 2-aminoethyl ß-D-galactopyranoside (Dgal) in different molar ratios (1:5, 1:10, 1:15, 1:20) to obtain Dgal(n)-PAMAM-FITC. Flow cytometry was applied to examine which of the molar ratios was the best. RESULTS: Through active ester method, we got conjugates in different molar ratios of G3.5 PAMAM-FITC to Dgal (1:5, 1:10, 1:15, 1:20). Cellular entry of FITC-labeled PAMAM conjugated with different numbers of Dgal was evaluated in vitro by using rat hepatocytes with flow cytometry, and it was found that one molecule of PAMAM conjugated with 20 molecules of Dgal was the best ratio. CONCLUSION: Dgal(20)-G3.5 PAMAM-FITC can be used for liver-targeting drug delivery system.

Blood compatibility of polyamidoamine dendrimers and erythrocyte protection.

Dendrimers are global macromolecules with promising biomedical application. In order to investigate their blood compatibility, different generations of polyamidoamine (PAMAM) dendrimers terminated with different types of surface functional groups were incubated with red blood cells (RBC) for time course of haemolysis assay and RBC morphology observation. Cationic PAMAM dendrimers demonstrated obvious generation, concentration and time dependent haemolysis while anionic and neutral PAMAM dendrimers were much less hemolytic. The surface amino groups and the polymeric structure of cationic PAMAM dendrimers played pivotal roles in haemolysis induction; meanwhile the basic pH of cationic PAMAM solutions was an inneglectable factor. Due to the presence of plasma proteins in abundance, whole blood was found tolerable to haemolysis induced by dendrimers, possibly through complex formation of plasma proteins with cationic PAMAM dendrimers. It was deduced that poly-carboxylic acids may have the similar effect as that of plasma proteins. So by means of pH adjustment to the physical 7.4, reduction of administration dose and the combined use of plasma protein (or other protective additives such as negatively charged molecules), the blood compatibility and even biocompatibility of cationic dendrimers may be enhanced to "safe" level.

LEAPT: lectin-directed enzyme-activated prodrug therapy.

Targeted drug delivery to selected sites allows reduced toxicity, enhanced efficiency and interchangeable target potential [Langer, R. (2001) Science 293, 58-59 and Molema, G. & Meijer, D. K. F., eds. (2001) Drug Targeting (Wiley-VCH, Weinheim, Germany)]. We describe a bipartite drug-delivery system that exploits (I) endogenous carbohydrate-to-lectin binding to localize glycosylated enzyme conjugates to specific, predetermined cell types followed by (II) administration of a prodrug activated by that predelivered enzyme at the desired site. The carbohydrate structure of an alpha-L-rhamnopyranosidase enzyme was specifically engineered through enzymatic deglycosylation and chemical reglycosylation. Combined in vivo and in vitro techniques (gamma scintigraphy, microautoradiography and confocal microscopy) determined organ and cellular localization and demonstrated successful activation of alpha-L-rhamnopyranoside prodrug. Ligand competition experiments revealed enhanced, specific localization by endocytosis and a strongly carbohydrate-dependent, 60-fold increase in selectivity toward target cell hepatocytes that generated a >30-fold increase (from 0.02 to 0.66 mg) in protein delivered. Furthermore, glycosylation engineering enhanced the serum-uptake rate and enzyme stability. This created enzyme activity (0.2 units in hepatocytes) for prodrug therapy, the target of which was switched simply by sugar-type alteration. The therapeutic effectiveness of lectin-directed enzyme-activated prodrug therapy was shown through the construction of the prodrug of doxorubicin, Rha-DOX, and its application to reduce tumor burden in a hepatocellular carcinoma (HepG2) disease model.

[Preparation of thrombus-targeted urokinase liposomes and its thrombolytic effect in model rats].

AIM: To prepare thrombus-targeted urokinase liposomes and observe its improved thrombolytic efficacy on thrombus model rats. METHODS: The ligand H-Arg-Gly-Asp-Ser-OH (RGDS) which has specific affinity to thrombus was synthesized by liquid phase method and anchored on the surface of liposomes by incorporating its conjugate with DSPE-PEG3,500-COOH into liposomal lipid bilayers, thus thrombus-targeted liposomes were produced. Urokinase (UK) liposomes were prepared at room temperature through method modification using hydrogenated soy phosphatidylcholine (HSPC); the in vivo thrombolysis of the obtained thrombus-targeted UK liposomes and its comparison with TBS (Tris-HCl buffered solution) control, free UK and UK liposomes were assessed on common carotid artery model rats. RESULTS: The obtained liposomes were characteristic of high UK entrapment efficiency, small mean diameter and good storage stability. At the same dose (60,000 U.kg-1), compared to the wet thrombi weights of TBS control group, those of free UK group and UK liposome group showed no statistical difference, while those of targeted UK liposomes group were significantly decreased (P < 0.001); when evaluated in term of dry thrombi weights the result was slightly different. Compared to UK liposomes of the same dose, the targeted UK liposomes showed significantly improved thrombolytic efficacy (P < 0.01 in wet weights decrease and P < 0.05 in dry weights decrease respectively). CONCLUSION: The targeted UK liposomes displayed good targeted thrombolytic effect.

[Study on the hepatocytic cell targetability of liposomes].

AIM: To target for hepatocytic cell, liposomes was modified by special ligand. METHODS: Sterically stabilized liposomes (SSL) was conjugated with asialofeticin (AF), the ligand of asialoglycoprotein receptor (ASGP-R) of hepatocyte. ASGP-R-BLM is the ASGP-R reconstructed on bilayer lipid membrane (BLM). The recognition reaction between AF-SSL and ASGP-R-BLM can be monitored by the varieties of membrane electrical parameters. The targetability of AF-SSL mediated to hepatocyte was detected by radioisotopic labeled in vitro and in vivo. The therapeutic effect of antihepatocarcinoma was observed also. RESULTS: The lifetime of ASGP-R-BLM decreased with the added amount of AF-SSL. It was demonstrated that there was recognition reaction between AF-SSL and ASGP-R-BLM. The combination of AF-SSL with hepatocyte was significantly higher than that of SSL without AF-modified in vitro and in vivo. The survival time of rat for AF-SSL carriered ADM (adriamycin) group was much longer and the toxicities on heart, kidney and lung were lower than those SSL carried ADM group. CONCLUSION: It is possible to actively target the cell with specific receptor by ligand modified liposomes. The result prvide scientific basis of hepatocyte targeted liposomes.

Studies on alginate-chitosan microcapsules and renal arterial embolization in rabbits.

Spherical and well-dispersed alginate-chitosan microcapsules, with a mean diameter of 77.28+/-0.93 microm (n=3), were prepared by the emulsification-gelation method. Adriamycin hydrochloride (ADM) was used as a model drug to investigate the drug loading capacity and release characteristics of the microcapsules. The drug/carrier ratio and chitosan concentration influenced the encapsulation efficiency of adriamycin. The adriamycin release from microcapsules was obviously different in 0.1 mol/l HCl from that in phosphate-buffered saline (PBS, pH 7.4). The drug was completely and rapidly released in 0.1 mol/l HCl, while it showed a sustained release after a burst release in PBS. The increase in chitosan concentration had no effect on adriamycin release in PBS. Using sulforhodamin B (SRB)-staining survival assay, the inhibition of adriamycin alginate-chitosan microcapsules (ADM-ACM) to different cancer cell lines (human BGC-823 cells, Bel-7402 cells and Hela cells) in vitro was determined. The inhibitory rate of ADM-ACM suspension to the three cell lines significantly outran that of ADM solution, no matter at high or low concentration. The effects of blank alginate-chitosan microcapsules (BACM) on renal arterial embolization were examined with transcatheter arterial embolization in rabbits. The angiogram and histopathological results indicated the blank microcapsules had excellent short- and long-term effects on renal arterial embolization.

Phospholipid deformable vesicles for buccal delivery of insulin.

To investigate the possibility of the enhancing effect of deformable vesicles on buccal delivery of insulin, two kinds of vesicles with and without the presence of sodium deoxycholate (deformable vesicles and conventional vesicles) were prepared by reverse phase evaporation methods. The liposomal entrapment efficiency was determined by column chromatography. The particle size and morphology of the vesicles were also evaluated. The hypoglycemic effects, insulin concentrations, and residual amounts of insulin deposited in the buccal membrane after buccal administration of insulin vesicles to rabbits were investigated. Compared with subcutaneous administration of insulin solution, the relative pharmacological bioavailability and the relative bioavailability of buccal administration of insulin vesicles were determined. The results showed that the entrapment efficiencies of the deformable and conventional vesicles were 18.87+/-1.78% (n=3) and 22.07+/-2.16% (n=3), respectively. The particle sizes of the deformable and conventional vesicles were 42.5+/-20.5 nm and 59.7+/-33.8 nm, respectively. There were no significant differences in appearance between the two types of vesicle. Compared with subcutaneous administration of insulin solution, the relative pharmacological bioavailability and the relative bioavailability in the insulin-deformable vesicles group were 15.59% and 19.78%, respectively, which were higher than in the conventional insulin vesicles (p<0.05), blank deformable vesicles and insulin mixture groups (p<0.05). Deformable vesicles have an enhancing effect on buccal delivery of insulin and may be a better carrier than conventional vesicles for buccal delivery of protein drugs.