Effect of LuxS/AI-2-mediated quorum sensing system on bacteriocin production of Lactobacillus plantarum NMD-17.

Lactobacillus plantarum NMD-17 separated from koumiss could produce a bacteriocin named plantaricin MX against Gram-positive bacteria and Gram-negative bacteria. The bacteriocin synthesis of L. plantarum NMD-17 was remarkably induced in co-cultivation with Lactobacillus reuteri NMD-86 as the increase of cell numbers and AI-2 activity, and the expressions of luxS encoding signal AI-2 synthetase, plnB encoding histidine protein kinase, plnD encoding response regulator, and plnE and plnF encoding structural genes of bacteriocin were significantly upregulated in co-cultivation, showing that the bacteriocin synthesis of L. plantarum NMD-17 in co-cultivation may be regulated by LuxS/AI-2-mediated quorum sensing system. In order to further demonstrate the role of LuxS/AI-2-mediated quorum sensing system in the bacteriocin synthesis of L. plantarum NMD-17, plasmids pUC18 and pMD18-T simple were used as the skeleton to construct the suicide plasmids pUC18-UF-tet-DF and pMD18-T simple-plnB-tet-plnD for luxS and plnB-plnD gene deletion, respectively. luxS and plnB-plnD gene knockout mutants were successfully obtained by homologous recombination. luxS gene knockout mutant lost its AI-2 synthesis ability, suggesting that LuxS protein encoded by luxS gene is key enzyme for AI-2 synthesis. plnB-plnD gene knockout mutant lost the ability to synthesize bacteriocin against Salmonella typhimurium ATCC14028, indicating that plnB-plnD gene was a necessary gene for bacteriocin synthesis of L. plantarum NMD-17. Bacteriocin synthesis, cell numbers, and AI-2 activity of luxS or plnB-plnD gene knockout mutants in co-cultivation with L. reuteri NMD-86 were obviously lower than those of wild-type strain in co-cultivation at 6-9 h (P < 0.01). The results showed that LuxS/AI-2-mediated quorum sensing system played an important role in the bacteriocin synthesis of L. plantarum NMD-17 in co-cultivation.

LuxS-mediated quorum sensing system in Lactobacillus plantarum NMD-17 from koumiss: induction of plantaricin MX in co-cultivation with certain lactic acid bacteria.

A bacteriocin termed plantaricin MX with a broad antimicrobial spectrum was produced by Lactobacillus plantarum NMD-17, which was isolated from Inner Mongolia traditional koumiss of china. Among 300 strains of lactic acid bacteria (LAB) belonging to the genera Lactococcus, Lactobacillus, Streptococcus, Leuconostoc, and Enterococcus, five strains including Lactobacillus reuteri NMD-86, Lactobacillus helveticus NMD-137, Lactococcus lactis NMD-152, Enterococcus faecalis NMD-178, and Enterococcus faecium NMD-219 were revealed to significantly induce the bacteriocin synthesis and greatly increase the cell numbers of Lactobacillus plantarum NMD-17 and activity of AI-2 signaling molecule. Bacteriocin synthesis was not increased by cell-free supernatants and autoclaved cultures of inducing strains, demonstrating that intact cells of inducing strains were essential to the induction of bacteriocin synthesis. The existence of bacteriocin structural plnEF genes and the plnD and luxS genes involved in quorum sensing was confirmed by PCR, and the presence of plnB gene encoding histidine protein kinase was determined by single oligonucleotide nested PCR (Son-PCR). Quantitative real-time PCR demonstrated that plnB, plnD, luxS, plnE, and plnF genes of L. plantarum NMD-17 were upregulated significantly (P < 0.01) in co-cultivation with L. reuteri NMD-86. The results showed that the bacteriocin synthesis of L. plantarum NMD-17 in co-cultivation might have a close relationship with LuxS-mediated quorum sensing system.

Heterologous expression of an Agropyron cristatum SnRK2 protein kinase gene (AcSnRK2.11) increases freezing tolerance in transgenic yeast and tobacco.

The sucrose non-fermenting-1 related protein kinase 2 (SnRK2) family plays an important role in the response to abiotic stress. To characterize the function of the SnRK2 gene from Agropyron cristatum in stress protection, we cloned the complete coding sequence of the AcSnRK2.11 gene from A. cristatum and generated AcSnRK2.11-overexpressing tobacco lines. The open reading frame of AcSnRK2.11 was 1083 bp in length and encoded a polypeptide of 360 amino acid residues. The sequence analysis results showed that AcSnRK2.11 contained conserved domains typified in SnRK2 protein kinases. Subcellular localization analysis showed that AcSnRK2.11 was located in the nucleus. AcSnRK2.11 was constitutively expressed in all of the examined tissues, and its transcription was induced by cold, dehydration, and salt stress, but not by abscisic acid treatment. Overexpression of the AcSnRK2.11 gene conferred freezing tolerance in yeast. AcSnRK2.11-overexpressing tobacco lines showed higher tolerance to freezing stress than did wild-type (WT) based on higher survival rates, lower malondialdehyde content and increased relative water content retention, chlorophyll yields, superoxide dismutase activities, reactive oxygen species content, peroxidase levels, and soluble carbohydrates under low-temperature conditions. The transcripts of NtDREB1, NtDREB2, NtERD10A, NtERD10B, NtERD10C, NtERD10D, NtMnSOD, NtCDPK15, and NtMPK9 in AcSnRK2.11-overexpressing tobacco lines were more abundant than in WT plants under low-temperature stress. These results suggest that AcSnRK2.11 may function as a regulatory factor associated with a cold-response pathway and could be used in plant breeding for cold resistance.

Characterization of a broad spectrum bacteriocin produced by Lactobacillus plantarum MXG-68 from Inner Mongolia traditional fermented koumiss.

An agar well diffusion assay (AWDA) was used to isolate a high bacteriocin-producing strain with a broad spectrum of antibacterial activity, strain MXG-68, from Inner Mongolia traditional fermented koumiss. Lactobacillus plantarum MXG-68 was identified by morphological, biochemical, and physiological characteristics and 16S rDNA analysis. The production of antibacterial substance followed a growth-interrelated model, starting at the late lag phase of 4 h and arriving at a maximum value in the middle of the stationary phase at 24 h. Antibacterial activity was abolished or decreased in the presence of pepsin, chymotrypsin, trypsin, proteinase, and papain K. The results showed that antibacterial substances produced by L. plantarum MXG-68 were proteinaceous and could thus be classified as the bacteriocin, named plantaricin MXG-68. The molar mass of plantaricin MXG-68 was estimated to be 6.5 kDa, and the amino acid sequence of its N-terminal was determined to be VYGPAGIFNT. The mode of plantaricin MXG-68 action was determined to be bactericidal. Bacteriocin in cell-free supernatant (CFS) at pH 7 was stable at different temperatures (60 °C, 80 °C, 100 °C, 121 °C for 30 min; 4 °C and - 20 °C for 30 days), as well as at pH 2.0-10.0. Antibacterial activity maintained stable after treatment with organic solvents, surfactants, and detergents but increased in response to EDTA. Response surface methodology (RSM) revealed the optimum conditions of bacteriocin production in L. plantarum MXG-68, and the bacteriocin production in medium optimized by RSM was 26.10% higher than that in the basal MRS medium.

Strain Screening from Traditional Fermented Soybean Foods and Induction of Nattokinase Production in Bacillus subtilis MX-6.

The plasminogen-free fibrin plate assay method was used to isolate Bacillus subtilis MX-6, a strain with high production of nattokinase from Chinese douchi. The presence of aprN, a gene-encoding nattokinase, was verified with PCR method. The predicted amino acid sequence was aligned with homologous sequences, and a phylogenetic tree was constructed. Nattokinase was sublimated with ammonium sulfate, using a DEAE-Sepharose Fast Flow column, a CM-Sepharose Fast Flow column and a Sephadex G-75 gel filtration column. SDS-PAGE analysis indicated that the molecular weight of the purified nattokinase from Bacillus subtilis MX-6 was about 28 kDa. Fermentation of Bacillus subtilis MX-6 nattokinase showed that nattokinase production was maximized after 72 h; the diameter of clear zone reached 21.60 mm on the plasminogen-free fibrin plate. Nattokinase production by Bacillus subtilis MX-6 increased significantly after supplementation with supernatant I, supernatant II and soy peptone but decreased substantially after the addition of amino acids. This result indicated that the nattokinase production by B. subtilis MX-6 might be induced by soybean polypeptides. The addition of MgSO4 and CaCl2 increased B. subtilis MX-6 nattokinase production.

A new Em-like protein from Lactuca sativa, LsEm1, enhances drought and salt stress tolerance in Escherichia coli and rice.

Late embryogenesis abundant (LEA) proteins are closely related to abiotic stress tolerance of plants. In the present study, we identified a novel Em-like gene from lettuce, termed LsEm1, which could be classified into group 1 LEA proteins, and shared high homology with Cynara cardunculus Em protein. The LsEm1 protein contained three different 20-mer conserved elements (C-element, N-element, and M-element) in the C-termini, N-termini, and middle-region, respectively. The LsEm1 mRNAs were accumulated in all examined tissues during the flowering and mature stages, with a little accumulation in the roots and leaves during the seedling stage. Furthermore, the LsEm1 gene was also expressed in response to salt, dehydration, abscisic acid (ABA), and cold stresses in young seedlings. The LsEm1 protein could effectively reduce damage to the lactate dehydrogenase (LDH) and protect LDH activity under desiccation and salt treatments. The Escherichia coli cells overexpressing the LsEm1 gene showed a growth advantage over the control under drought and salt stresses. Moreover, LsEm1-overexpressing rice seeds were relatively sensitive to exogenously applied ABA, suggesting that the LsEm1 gene might depend on an ABA signaling pathway in response to environmental stresses. The transgenic rice plants overexpressing the LsEm1 gene showed higher tolerance to drought and salt stresses than did wild-type (WT) plants on the basis of the germination performances, higher survival rates, higher chlorophyll content, more accumulation of soluble sugar, lower relative electrolyte leakage, and higher superoxide dismutase activity under stress conditions. The LsEm1-overexpressing rice lines also showed less yield loss compared with WT rice under stress conditions. Furthermore, the LsEm1 gene had a positive effect on the expression of the OsCDPK9, OsCDPK13, OsCDPK15, OsCDPK25, and rab21 (rab16a) genes in transgenic rice under drought and salt stress conditions, implying that overexpression of these genes may be involved in the enhanced drought and salt tolerance of transgenic rice. Thus, this work paves the way for improvement in tolerance of crops by genetic engineering breeding.

Stress-responsive gene RsICE1 from Raphanus sativus increases cold tolerance in rice.

The ICE1 transcription factor plays a critical role in plant cold tolerance via triggering CBF/DREB1 cold-regulated signal networks. In this work, a novel MYC-type ICE1-like gene, RsICE1, was isolated from radish (Raphanus sativus L.), and its function in cold tolerance was characterized in rice. The RsICE1 gene was expressed constitutively with higher transcriptional levels in the roots and stems of radish seedlings. The NaCl, cold, and ABA treatments could significantly upregulate RsICE1 expression levels, but dehydration stress had a weak effect on its expression. Ectopic expression of the RsICE1 gene in rice conferred enhanced tolerance to low-temperature stress grounded on a higher survival rate, higher accumulation of soluble sugars and free proline content, a decline in electrolyte leakage and MDA levels, and higher chlorophyll levels relative to control plants. OsDREBL and OsTPP1, downstream cold-regulated genes, were remarkably upregulated at transcription levels in rice overexpressing RsICE1 under low-temperature stress, which indicated that RsICE1 was involved in CBF/DREB1 cold-regulated signal networks. Overall, the above data showed that RsICE1 played an active role in improving rice cold tolerance, most likely resulting from the upregulation of OsDREBL and OsTPP1 expression levels by interacting with the RsICE1 gene under low-temperature stress.

The Interactions Between Aligned Poly(L-Lactic Acid) Nanofibers and SH-SY5Y Cells In Vitro.

Aligned nanofibers have been regarded as promising nanomaterials in facilitating nerve regeneration. Investigating the interactions between aligned nanofibers and neuronal cells will be critically important for the design and application of aligned nanofibers in nerve tissue engineering. In this study, we explored the effects of electrospun Poly(L-Lactic Acid) (PLLA) aligned nanofibers on SH-SY5Y cells (a type of human neuroblastoma cell line) and specifically focused on the role of integrin in the PLLA aligned nanofiber-SH-SY5Y cell interaction. We found that PLLA aligned nanofibers could significantly guide the neurite outgrowth of SH-SY5Y cell, and promote the viability, proliferation, glucose and lactic acid metabolism of SH-SY5Y cell. This promotion effect could be alleviated when the functions of integrins on the SH-SY5Y cell membrane were hampered by pentapeptide GRGDS. Moreover, we found that PLLA aligned nanofibers could enhance the expression of phosphorylated-ERK1/2 (p-ERK1/2) in the SH-SY5Y cells and blocking the integrins would decrease p-ERK1/2 expression. These results suggested that PLLA aligned nanofibers might affect many cellular behaviors of SH-SY5Y cells via integrin mediated ERK pathway. Our findings provided more insights for understanding the interaction between aligned nanofibers and neuronal cells.

Reactive oxygen species generated from skeletal muscles are required for gecko tail regeneration.

Reactive oxygen species (ROS) participate in various physiological and pathological functions following generation from different types of cells. Here we explore ROS functions on spontaneous tail regeneration using gecko model. ROS were mainly produced in the skeletal muscle after tail amputation, showing a temporal increase as the regeneration proceeded. Inhibition of the ROS production influenced the formation of autophagy in the skeletal muscles, and as a consequence, the length of the regenerating tail. Transcriptome analysis has shown that NADPH oxidase (NOX2) and the subunits (p40(phox) and p47(phox)) are involved in the ROS production. ROS promoted the formation of autophagy through regulation of both ULK and MAPK activities. Our results suggest that ROS produced by skeletal muscles are required for the successful gecko tail regeneration.

The HMGB1 signaling pathway activates the inflammatory response in Schwann cells.

Schwann cells are not only myelinating cells, but also function as immune cells and express numerous innate pattern recognition receptors, including the Toll-like receptors. Injury to peripheral nerves activates an inflammatory response in Schwann cells. However, it is unclear whether specific endogenous damage-associated molecular pattern molecules are involved in the inflammatory response following nerve injury. In the present study, we demonstrate that a key damage-associated molecular pattern molecule, high mobility group box 1 (HMGB1), is upregulated following rat sciatic nerve axotomy, and we show colocalization of the protein with Schw-ann cells. HMGB1 alone could not enhance expression of Toll-like receptors or the receptor for advanced glycation end products (RAGE), but was able to facilitate migration of Schwann cells. When Schwann cells were treated with HMGB1 together with lipopolysaccharide, the expression levels of Toll-like receptors and RAGE, as well as inflammatory cytokines were upregulated. Our novel findings demonstrate that the HMGB1 pathway activates the inflammatory response in Schwann cells following peripheral nerve injury.

CD59 mediates cartilage patterning during spontaneous tail regeneration.

The regeneration-competent adult animals have ability to regenerate their lost complex appendages with a near-perfect replica, owing to the positional identity acquired by the progenitor cells in the blastema, i.e. the blastemal cells. CD59, a CD59/Ly6 family member, has been identified as a regulator of positional identity in the tail blastemal cells of Gekko japonicus. To determine whether this function of CD59 is unique to the regenerative amniote(s) and how CD59 mediates PD axis patterning during tail regeneration, we examined its protective role on the complement-mediated cell lysis and intervened CD59 expression in the tail blastemal cells using an in vivo model of adenovirus transfection. Our data revealed that gecko CD59 was able to inhibit complement-mediated cell lysis. Meanwhile, CD59 functioned on positional identity through expression in cartilage precursor cells. Intervening positional identity by overexpression or siRNA knockdown of CD59 resulted in abnormal cartilaginous cone patterning due to the decreased differentiation of blastemal cells to cartilage precursor cells. The cartilage formation-related genes were found to be under the regulation of CD59. These results indicate that CD59, an evolutionarily transitional molecule linking immune and regenerative regulation, affects tail regeneration by mediating cartilage patterning.

The regenerating spinal cord of gecko maintains unaltered expression of ß-catenin following tail amputation.

The Wingless/Integrated (Wnt) signaling pathway plays important roles in central nervous system (CNS) development and regeneration, and ß-catenin, the central component, has been considered in association with adult neurogenesis. To decipher its roles on spontaneous spinal cord regeneration, we cloned ß-catenin from Gekko japonicus and examined its function in regenerating spinal cord. The protein was localized in the neurons and oligodendrocytes and maintained a stable expression levels during the spinal cord regeneration. The temporal pattern of expression has been found to be completely distinct with those of glycogen synthase kinase 3ß (GSK3ß). Experiments of gain-of-function by overexpression of full length ß-catenin or stabilized ΔN90-ß-catenin revealed that the accumulated protein attenuates the elongation of neurites and oligodendrocyte process. Knockdown of endogenous ß-catenin, however, decreased proliferation of oligodendrocytes by affecting expression of downstream lef1 and c-jun. The upregulated extracellular matrix fibronectin in injured cord was found to be inefficient in regulation of ß-catenin expression. Our results suggest that a tightly regulated stable expression of ß-catenin is required for the spontaneous spinal cord regeneration.

Mesenspheres of neural crest-derived cells enriched from bone marrow stromal cell subpopulation.

Neural crest-derived cells (NCCs) can be used for cell replacement therapy of neurodegenerative diseases and nerve injury, and it is of significance to open readily accessible tissue sources for NCCs due to their insufficient supply. In this study, we aimed to examine the possibility of enriching NCCs from bone marrow stromal cell (BMSC) subpopulation. The epidermal growth factor/fibroblast growth factor-2 (EGF/FGF2)-responsive BMSC subpopulation (BMSC-C2) was isolated from rat bone marrow by repetitive two-step condition culture. The BMSC-C2 subpopulation showed a long-term proliferative capacity and high cell growth rate, and possessed a significant sphere-forming ability. The mesenspheres derived from BMSC-C2 subpopulation were self-renewable and could express NCC markers, such as CD29, CD44, nestin, CD133 and p75(NTR). In particular, the mesenspheres could be induced to differentiate into neuron- and glia-like cells in vitro. Collectively, our results might provide a basis for in-depth studies of recruiting postmigratory NCCs from bone marrow and various neural crest-derived tissues.

Comparison in the biological characteristics between primary cultured sensory and motor Schwann cells.

Schwann cells (SCs) express distinct sensory and motor phenotypes, which are associated with modality-specific promotion of axon growth. Here we compared cell proliferation and migration of primary cultured sensory and motor SCs and determined the mRNA expression of several genes, nap1l1, dok4, lpp, mmp-9 and l1cam, in two phenotypes of SCs. The results showed that the rate of cell proliferation or migration was higher in sensory SCs than in motor SCs, and the five proliferation or migration-related genes also had higher expression in sensory SCs than in motor SCs. These findings may provide a basis for deeply studying the biological differences between sensory and motor SCs.

Comparative proteomic analysis of differentially expressed proteins between peripheral sensory and motor nerves.

Peripheral sensory and motor nerves have different functions and different approaches to regeneration, especially their distinct ability to accurately reinervate terminal nerve pathways. To understand the molecular aspects underlying these differences, the proteomics technique by coupling isobaric tags for relative and absolute quantitation (iTRAQ) with online two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) was used to investigate the protein profile of sensory and motor nerve samples from rats. A total of 1472 proteins were identified in either sensory or motor nerve. Of them, 100 proteins showed differential expressions between both nerves, and some of them were validated by quantitative real time RT-PCR, Western blot analysis, and immunohistochemistry. In the light of functional categorization, the differentially expressed proteins in sensory and motor nerves, belonging to a broad range of classes, were related to a diverse array of biological functions, which included cell adhesion, cytoskeleton, neuronal plasticity, neurotrophic activity, calcium-binding, signal transduction, transport, enzyme catalysis, lipid metabolism, DNA-binding, synaptosome function, actin-binding, ATP-binding, extracellular matrix, and commitment to other lineages. The relatively higher expressed proteins in either sensory or motor nerve were tentatively discussed in combination with their specific molecular characteristics. It is anticipated that the database generated in this study will provide a solid foundation for further comprehensive investigation of functional differences between sensory and motor nerves, including the specificity of their regeneration.