[Protective effect of Pinus massoniana bark extracts against cisplatin-induced nephrotoxicity in human embryonic kidney cells].

OBJECTIVE: To investigate the in vitro protective effect of Pinus massoniana bark extracts (PMBE) against cisplatin-induced nephrotoxicity in human embryonic kidney cells (HEK293), and preliminarily study its mechanism. METHOD: Human embryonic kidney cells (HEK293) were cultured in vitro. The MTT assay was adopted to test the effect of PMBE and cisplatin on growth of HEK293 cells, and the protective effect of PMBE on cisplatin-induced nephrotoxicity of HEK293, and then detect the intracellular reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH) content, catalase (CAT), superoxide dismutase (SOD) and activity of thioredoxin reductase (TrxR). RESULT: PMBE could promote growth of HEK293 cells at low concentrations, but generate slight nephrotoxicity at high concentration. Cisplatin could inhibit growth of HEK293 cells, increase ROS and MDA content, while reducing SOD, CAT and TrxR. The pre-protective PMBE was added to reduce cisplatin's injury to HEK293 cells, ROS, MDA and GSH content, SOD, CAT and TrxR within certain range. CONCLUSION: PMBE at specific concentration has the protective effect in cisplatin-induced nephrotoxicity in HEK293 cells. Its mechanism may be related to PMBE's antioxidant activity.

MpAsr encodes an intrinsically unstructured protein and enhances osmotic tolerance in transgenic Arabidopsis.

Abscisic acid-, stress- and ripening (ASR) -induced proteins are plant-specific proteins whose expression is up-regulated under abiotic stresses or during fruit ripening. In this study, we characterized an ASR protein from plantain to explore its physiological roles under osmotic stress. The expression pattern of MpAsr gene shows that MpAsr gene changed little at the mRNA level, while the MpASR protein accumulates under osmotic treatment. Through bioinformatic-based predictions, circular dichroism spectrometry, and proteolysis and heat-stability assays, we determined that the MpASR protein is an intrinsically unstructured protein in solution. We demonstrated that the hydrophilic MpASR protein could protect L: -lactate dehydrogenase (L: -LDH) from cold-induced aggregation. Furthermore, heterologous expression of MpAsr in Escherichia coli and Arabidopsis enhanced the tolerance of transformants to osmotic stress. Transgenic 35S::MpAsr Arabidopsis seeds had a higher germination frequency than wild-type seeds under unfavorable conditions. At the physiological level, 35S::MpAsr Arabidopsis showed increased soluble sugars and decreased cell membrane damage under osmotic stress. Thus, our results suggest that the MpASR protein may act as an osmoprotectant and water-retaining molecule to help cell adjustment to water deficit caused by osmotic stress.

Characterization of a novel plantain Asr gene, MpAsr, that is regulated in response to infection of Fusarium oxysporum f. sp. cubense and abiotic stresses.

Asr (abscisic acid, stress, ripening induced) genes are typically upregulated by a wide range of factors, including drought, cold, salt, abscisic acid (ABA) and injury; in addition to plant responses to developmental and environmental signals. We isolated an Asr gene, MpAsr, from a suppression subtractive hybridization (SSH) cDNA library of cold induced plantain (Musa paradisiaca) leaves. MpAsr expression was upregulated in Fusarium oxysporum f. sp. cubense infected plantain leaves, peels and roots, suggesting that MpAsr plays a role in plantain pathogen response. In addition, a 581-bp putative promoter region of MpAsr was isolated via genome walking and cis-elements involved in abiotic stress and pathogen-related responses were detected in this same region. Furthermore, the MpAsr promoter demonstrated positive activity and inducibility in tobacco under F. oxysporum f. sp. cubense infection and ABA, cold, dehydration and high salt concentration treatments. Interestingly, transgenic Arabidopsis plants overexpressing MpAsr exhibited higher drought tolerance, but showed no significant decreased sensitivity to F. oxysporum f. sp. cubense. These results suggest that MpAsr might be involved in plant responses to both abiotic stress and pathogen attack.

[Study on industrialized extraction technology and function of hyperlipidemic regulating of Laminaria japonica polysaccharides].

OBJECTIVE: To study the industrialized extraction technology and the function of hyperlipidemic regulating of Laminaria japonica polysaccharides. METHODS: With the orthogonal design L9 (3(4)), different influential elements of the industrialized extraction technology of Laminaria japonica polysaccharides were investigated. Mice treated with different doses of polysaccharides were applied to investigate its-function of hyperlipidemic regulating for 15 days. RESULTS: 5% Na2CO3, 3 hours and 80 degrees C was the best extraction condition, and the extraction rate was 2.13%. Different groups of polysaccharides with different levels of purity and dose lowered the TG, TC level of the mice in various degree. CONCLUSION: Laminaria japonica polysaccharides have obvious effect on hyperlipidemic regulating.

Carrot antifreeze protein does not exhibit the polygalacturonase-inhibiting activity of PGIP family.

The carrot (Daucus carota) antifreeze protein (DcAFP) has a strong antifreeze activity and identified as belonging to the plant polygalacturonase-inhibiting protein (PGIP) family based on its sequence similarities, including the presence of a leucine-rich repeat (LRR) motif. In this study, yeast two-hybrid technology was used to analyze whether the carrot AFP could act as a PGIP. The complete DcAFP and polygalacturonase (PGase; obtained from fungus Alternaria alternata by RT-PCR) coding sequences were cloned into the bait and capture vectors, respectively, and yeast two-hybrid assays were performed. The results revealed that there was no evidence of an interaction between DcAFP and PGase, which suggests that DcAFP probably lacks PGIP activity. An analysis of the electrostatic potential of DcAFP and other PGIPs revealed that a large number of nonconservative residues within the beta-helix of the DcAFP LRR motif had been substituted to basic amino acids, thus changing the surface from negative to positive. This will electrostatically prevent DcAFP from binding with the positively charged surface of PGase. This is the first report that showed the correlation between nonconservative amino acids within the LRR motif of the DcAFP and its loss of polygalacturonase inhibiting activity.

Significance of conservative asparagine residues in the thermal hysteresis activity of carrot antifreeze protein.

The approximately 24-amino-acid leucine-rich tandem repeat motif (PXXXXXLXXLXXLXLSXNXLXGXI) of carrot antifreeze protein comprises most of the processed protein and should contribute at least partly to the ice-binding site. Structural predictions using publicly available online sources indicated that the theoretical three-dimensional model of this plant protein includes a 10-loop beta-helix containing the approximately 24-amino-acid tandem repeat. This theoretical model indicated that conservative asparagine residues create putative ice-binding sites with surface complementarity to the 1010 prism plane of ice. We used site-specific mutagenesis to test the importance of these residues, and observed a distinct loss of thermal hysteresis activity when conservative asparagines were replaced with valine or glutamine, whereas a large increase in thermal hysteresis was observed when phenylalanine or threonine residues were replaced with asparagine, putatively resulting in the formation of an ice-binding site. These results confirmed that the ice-binding site of carrot antifreeze protein consists of conservative asparagine residues in each beta-loop. We also found that its thermal hysteresis activity is directly correlated with the length of its asparagine-rich binding site, and hence with the size of its ice-binding face.

Antitumor effects of Pinus massoniana bark extract in murine sarcoma S180 both in vitro and in vivo.

Pinus massoniana bark extract (PMBE) is a mixture of flavonoids, and showed a capability of inducing cell apoptosis; however, its properties have not yet been fully investigated. This paper evaluates the antitumor effects of PMBE in murine sarcoma S180 both in vitro and in vivo. In vitro, the growth inhibition of S180 cells was concentration dependent on PMBE as shown by the CCK-8 assay. The AO/EB staining and flow cytometry assay showed that PMBE induced S180 cell apoptosis. Cell cycle analysis revealed that the cells in the S phase were decreased by treatment with PMBE. In vivo, the treatment of 100, 200, and 300 mg/kg PMBE reduced the tumor weight and volume of S180-bearing NIH mice by 9%-67% and 13%-68%, respectively. Peripheral leukocyte count and lymphoproliferation were increased significantly after treatment with PMBE. Our results suggest that PMBE inhibits the tumor cell growth by inducing cell apoptosis and improving lymphoproliferation.

Expression, purification, and antifreeze activity of carrot antifreeze protein and its mutants.

Antifreeze proteins (AFPs) enable organisms to survive under freezing or sub-freezing conditions. AFPs have a great potential in the low temperature storage of cells, tissues, organs, and foods. This process will require a large number of recombinant AFPs. In the present study, the recombinant carrot AFP was highly expressed in Escherichia coli strain BL21 (DE3). The activity of the purified and refolded recombinant proteins was analyzed by measurement of thermal hysteresis (TH) activity and detection of in vitro antifreeze activity by measuring enhanced cold resistance of bacteria. Two carrot AFP mutants generated by site-directed mutagenesis were also expressed and purified under these conditions for use in parallel experiments. Recombinant DcAFP displayed a TH activity equivalent to that of native DcAFP, while mutants DcAFP-N130Q and rDcAFP-N130V showed 32 and 43% decreases in TH activity, respectively. Both the recombinant DcAFP and its mutants were able to enhance the cold resistance of bacteria, to degrees consistent with their respective TH activities.