Overexpression of the Gossypium barbadense Actin-Depolymerizing Factor 1 Gene Mediates Biological Changes in Transgenic Tobacco

The function of a member of the actin-depolymerizing factor family from Gossypium barbadense, GbADF1, was investigated. Tobacco (Nicotiana tabacum) lines expressing GbADF1 were produced by Agrobacterium-mediated transformation. Southern and northern blot analyses showed that GbADF1 was successfully incorporated as a single copy into the tobacco genome and stably expressed in three lines of T₁ transgenic tobacco plants. Biological changes were detected in these transgenic lines, wherein GbADF1 transgenic seedlings exhibited shorter hypocotyls along with fewer root hairs than those of control plants. Moreover, guard cells of leaves of the transgenic plants were induced to close stomata, while flowering was delayed 5 days in T₁ lines compared to those of empty vector transgenic control plants. Segregation of GbADF1 in the T₂ generation fits the expected 3:1 ratio corresponding to a single dominant gene. Subsequently, GbADF1 was fused to the green fluorescent protein gene to generate a fusion expression vector. Transient expression analysis indicated that this fusion protein was localized in the nucleus and cytoskeleton of epidermal cells of onion. These results suggest that actin-depolymerizing factor 1 gene from G. barbadense plays an important role in the process of plant cell morphogenesis.     

Theoretical insight into the structural mechanism for the binding of vinblastine with tubulin

Vinblastine (VLB) is one of vinca alkaloids with high cytotoxicity toward cancer cells approved for clinical use. However, because of drug resistance, toxicity, and other side effects caused from the use of VLB, new vinca alkaloids with higher cytotoxicity toward cancer cells and other good qualities need to develop. One strategy is to further study and better understand the essence why VLB possesses the high cytotoxicity toward cancer cells. In present work, by using molecular simulation, molecular docking, density functional calculation, and the crystal structure of α,β-tubulin complex, we find two modes labeled in catharanthine moiety (CM) and vindoline moiety (VM) modes of VLB bound with the interface of α,β-tubulin to probe the essence why VLB has the high cytotoxicity toward cancer cells. In the CM mode, nine key residues B-Ser178, B-Asp179, B-Glu183, B-Tyr210, B-Asp226, C-Lys326, C-Asp327, C-Lys336, and C-Lys352 from the α,β-tubulin complex are determined as the active sites for the interaction of VLB with α,β-tubulin. Some of them such as B-Ser178, B-Glu183, B-Tyr210, B-Asp226, C-Lys326, C-Asp327, and C-Lys336 are newly identified as the active sites in present work. The affinity between VLB and the active pocket within the interface of α,β-tubulin is ?60.8 kJ mol^?1 in the CM mode. In the VM mode, that is a new mode established in present paper, nine similar key residues B-Lys176, B-Ser178, B-Asp179, B-Glu183, B-Tyr210, B-Asp226, C-Lys326, C-Asp327, and C-Lys336 from the α,β-tubulin complex are found as the active sites for the interaction with VLB. The difference is from one key residue C-Lys352 in the CM mode changed to the key residue B-Lys176 in the VM mode. The affinity between VLB and the active pocket within the interface of α,β-tubulin is ?96.3 kJ mol^?1 in the VM mode. Based on the results obtained in present work, and because VLB looks like two faces, composed of CM and VM both to have similar polar active groups, to interact with the active sites, we suggest double-faces sticking mechanism for the binding of VLB to the interface of α,β-tubulin. The double-faces sticking mechanism can be used to qualitatively explain high cytotoxicity toward cancer cells of vinca alkaloids including vinblastine, vincristine, vindestine, and vinorelbine approved for clinical use and vinflunine still in a phase III clinical trial. Furthermore, this mechanism will be applied to develop novel vinca alkaloids with much higher cytotoxicity toward cancer cells.     

Zinc Transporter 7 Induced by High Glucose Attenuates Epithelial-to-Mesenchymal Transition of Peritoneal Mesothelial Cells

Zinc (Zn) is an essential micronutrient and cytoprotectant involved in preventing many types of epithelial-to-mesenchymal transition (EMT)-driven fibrosis in vivo. The zinc-transporter family SLC30A (ZnT) is a pivotal factor in the regulation of Zn homeostasis. However, its function in EMT in peritoneal mesothelial cells (PMCs) remains unknown. This study explored the regulation of zinc transporters and the role they play in cell EMT, particularly in rat peritoneal mesothelial cells (RPMCs), surrounding glucose concentrations and the molecular mechanism involved. The effects of high glucose (HG) on zinc transporter gene expression were measured in RPMCs by real-time PCR. We explored ZnT7 (Slc30A7): the effect of ZnT7 over-expression and siRNA-mediated knock-down on HG-induced EMT was investigated as well as the underlying molecular mechanisms. Over-expression of ZnT7 resulted in significantly inhibited HG-induced EMT in RPMCs, while inhibition of ZnT7 expression using a considerable siRNA-mediated knock-down of RPMCs increased the levels of EMT. Furthermore, over-expression of ZnT7 is accompanied by down-regulation of TGF-β/Smad pathway, phospho-Smad3,4 expression levels. The finding suggests that the zinc-transporting system in RPMCs is influenced by the exposure to HG. The ZnT7 may account for the inhibition of HG-induced EMT in RPMCs, likely through targeting TGF-β/Smad signaling.     

Dongia rigui sp. nov., isolated from freshwater of a large wetland in Korea

A motile, curved to twisted rod-shaped aerobic bacterium, designated strain 04SU4-P^T, was isolated from freshwater collected from the Woopo wetland (Republic of Korea). Cells were observed to be Gram-stain negative, catalase negative and oxidase positive. The major fatty acids (>10 % of the total) were identified as C_19:0 ω8c cyclo (24.6 %), C_16:0 (24.3 %) and C_18:1 ω7c (13.1 %). The DNA G+C content was determined to be 71.5 mol%. The major polar lipids were identified as phosphatidylglycerol, phosphatidylethanolamine, one unknown phospholipid and one unknown aminolipid. The major ubiquinone was determined to be Q-10. A phylogenetic tree based on 16S rRNA gene sequences showed that strain 04SU4-P^T forms an evolutionary lineage within the genus Dongia and its nearest neighbour is Dongia mobilis LM22^T (98.0 % sequence similarity). Genomic DNA–DNA hybridization of stain 04SU4-P^T with D. mobilis LM22^T showed relatedness of only 34.2 %. The phenotypic characteristics indicate the strain 04SU4-P^T can be distinguished from the sole member of the genus Dongia. On the basis of the data presented in this study, strain 04SU4-P^T represents a novel species, for which the name Dongia rigui is proposed. The type strain is 04SU4-P^T (KCTC 23341^T = JCM 17521^T).     

Activation of a mechanosensitive BK channel by membrane stress created with amphipaths

Some BK channels are activated in response to membrane stretch. However, it remains largely unknown which membrane component transmits forces to the channel and which part of the channel senses the force. Recently, we have shown that a BK channel cloned from chick heart (named SAKCa channel) is a stretch activated channel, while deletion of a 59 amino acids splice insert (STREX) located in the cytoplasmic side, abolishes its stretch-sensitivity. This finding raised a question whether stress in the bilayer is crucial for the mechanical activation of the channel. To address this question we examined the effects of membrane perturbing amphipaths on the stretch activation of the SAKCa channel and its STREX-deletion mutant. We found that both anionic amphipath trinitrophenol (TNP) and cationic amphipath chlorpromazine (CPZ) could dose-dependently activate the channel by leftward shifting the voltage activation curve when applied alone. In contrast, TNP and CPZ compensated each other's effect when applied sequentially. These results can be understood in the framework of the bilayer couple hypothesis, suggesting that stress in the plasma membrane can activate the SAKCa channel. Interestingly, the STREX-deletion mutant channel has much less sensitivity to the amphipaths, suggesting that STREX acts as an intermediate structure that can indirectly convey stress in the membrane to the gate of the SAKCa channel via an unidentified membrane associated protein(s) that can detect or transmit stress in the membrane.     

In vitro antimicrobial and anticancer potential of hinokitiol against oral pathogens and oral cancer cell lines

Hinokitiol is a natural component isolated from Chamacyparis taiwanensis. It has anti-microbial activity, and has been used in oral care products. The minimal inhibitory concentration (MIC) and minimal microbicidal concentration (MMC) of hinokitiol against MRSA, Aggregatibacter actinomycetemcomitans, Streptococcus mutans, and Candida albicans were determined by the agar and broth dilution method (MIC: 40–110 μM; MMC: 50–130 μM); the paradoxical inhibition phenomenon (PIP) was observed in A. actinomycetemcomitans and S. mutans. The PIP can be described as microbial growth occurring in the presence of both high and low concentrations of a compound, between which microbial growth is inhibited. The PIP was confirmed using a kinetic microplate and inhibition zone methods. The PIP was also observed in MRSA. The low autolysin activity somehow correlated to the PIP positive. The cell diameter was increased in all the pathogens, and the transition was inhibited in C. albicans following hinokitiol treatment. Hinokitiol is also a potential anticancer drug. The 200 μM of hinokitiol has significant antimicrobial and cytotoxic activities against oral pathogens and oral squamous cell carcinoma cell lines, respectively, and lower cytotoxic effects for normal human oral keratinocytes, indicating that hinokitiol displays a high potential for safe and effective applications in oral health care.     

Characterization of saponins and phenolic compounds: antioxidant activity and inhibitory effects on α-glucosidase in different varieties of colored quinoa (Chenopodium quinoa Willd)

ABSTRACT

This study investigated the contents of saponins and phenolic compounds in relation to their antioxidant activity and α-glucosidase inhibition activity of 7 colored quinoa varieties. The total saponin content was significantly different among 7 varieties and ranged from 7.51 to 12.12 mg OAE/g DW. Darker quinoa had a higher content of phenolic compounds, as well as higher flavonoids and antioxidant activity than that of light varieties. Nine individual phenolic compounds were detected in free and bound form, with gallic acid and ferulic acid representing the major compounds. The free and bound phenolic compounds (gallic acid and ferulic acid in particular) exhibited high linear correlation with their corresponding antioxidant values. In addition, the free phenolic extracts from colored quinoa exhibited higher inhibitory activity against α-glucosidase than the bound phenolic extracts. These findings imply that colored quinoa with abundant bioactive phytochemicals could be an important natural source for preparing functional food.     

The current state of the art of quantitative phosphoproteomics and its applications to diabetes research

Protein phosphorylation is a fundamental regulatory mechanism in many cellular processes and aberrant perturbation of phosphorylation has been implicated in various human diseases. Kinases and their cognate inhibitors have been considered as hotspots for drug development. Therefore, the emerging tools, which enable a system-wide quantitative profiling of phosphoproteome, would offer a powerful impetus in unveiling novel signaling pathways, drug targets and/or biomarkers for diseases of interest. This review highlights recent advances in phosphoproteomics, the current state of the art of the technologies and the challenges and future perspectives of this research area. Finally, some exemplary applications of phosphoproteomics in diabetes research are underscored.     

High glucose concentration induces endothelial cell proliferation by regulating cyclin‐D2‐related miR‐98

Cyclin D2 is involved in the pathology of vascular complications of type 2 diabetes mellitus (T2DM). This study investigated the role of cyclin‐D2‐regulated miRNAs in endothelial cell proliferation of T2DM. Results showed that higher glucose concentration (4.5 g/l) significantly promoted the proliferation of rat aortic endothelial cells (RAOECs), and significantly increased the expression of cyclin D2 and phosphorylation of retinoblastoma 1 (p‐RB1) in RAOECs compared with those under low glucose concentration. The cyclin D2‐3′ untranslated region is targeted by miR‐98, as demonstrated by miRNA analysis software. Western blot also confirmed that cyclin D2 and p‐RB1 expression was regulated by miR‐98. The results indicated that miR‐98 treatment can induce RAOEC apoptosis. The suppression of RAOEC growth by miR‐98 might be related to regulation of Bcl‐2, Bax and Caspase 9 expression. Furthermore, the expression levels of miR‐98 decreased in 4.5 g/l glucose‐treated cells compared with those treated by low glucose concentration. Similarly, the expression of miR‐98 significantly decreased in aortas of established streptozotocin (STZ)‐induced diabetic rat model compared with that in control rats; but cyclin D2 and p‐RB1 levels remarkably increased in aortas of STZ‐induced diabetic rats compared with those in healthy control rats. In conclusion, this study demonstrated that high glucose concentration induces cyclin D2 up‐regulation and miR‐98 down‐regulation in the RAOECs. By regulating cyclin D2, miR‐98 can inhibit human endothelial cell growth, thereby providing novel therapeutic targets for vascular complication of T2DM.