Metabolic engineering of isoflavone genistein in Brassica napus with soybean isoflavone synthase

Genistein, 4′,5,7-trihydroxyisoflavone, is an isoflavonoid compound predominantly restricted to legumes and known to possess phyto-oestrogenic and antioxidative activities. The key enzyme that redirects phenylpropanoid pathway intermediates from flavonoids to isoflavonoids is the isoflavone synthase (IFS). Brassica napus is a non-legume oilseed crop with vegetative tissues producing phenylpropanoids and flavonoids, but does not naturally accumulate isoflavones due to the absence of IFS. To demonstrate whether exogenous IFS is able to use endogenous substrate to produce isoflavone genistein in oilseed crop, the soybean IFS gene (GmIFS2) was incorporated into B. napus plants. The presence of GmIFS2 in B. napus was shown to direct the synthesis and accumulation of genistein derivatives in leaves up to 0.72 mg g⁻¹ DW. In addition, expression levels for most B. napus genes in the phenylpropanoid pathway were altered. These results suggest that the heterologous GmIFS2 enzyme is functionally active at using the B. napus naringenin as a substrate to produce genistein in oilseed rape.     

Acetone-soluble cellulose acetates prepared by one-step homogeneous acetylation of cornhusk cellulose in an ionic liquid 1-allyl-3-methylimidazolium chloride (AmimCl)

Cellulose samples extracted from cornhusk have been successfully acetylated in an ionic liquid 1-allyl-3-methylimidazolium chloride (AmimCl). Without using any catalyst, cornhusk cellulose acetates (CCAs) with the degree of substitution (DS) in a range from 2.16 to 2.63 were prepared in one-step. Under the homogeneous state, the DS value of CCAs was easily controlled by the acetylation time. The obtained CCAs were characterized by means of FT-IR, ¹³C NMR, DSC, TGA, and a mechanical test. The NMR results showed that the distribution of the acetyl moiety among the three OH groups of the anhydroglucose unit shows a preference at the C₆ position. The CCAs exhibited good solubility in some organic solvents, such as acetone and DMSO. The cast CCA films from their acetone solutions had good mechanical properties. At the end of each acetylation of cornhusk cellulose, the ionic liquid AmimCl could be effectively recovered. Therefore, this study presents a promising approach and “green process” to make use of crop by-products.     

Site-specific transfer of an intact beta-globin gene cluster through a new targeting vector

The ideal gene-therapy vector for treating genetic disorders should deliver intact therapeutic genes and their essential regulatory elements into the specific "safe genomic site" and realize long-term, self-regulatory expression. For beta-thalassemia gene therapy, viral vectors have been broadly used, but the accompanying insertional mutation and immunogenicity remain problematic. Hence, we aimed to develop new non-viral vectors that are efficient and safe in treating diseases. As previous studies have demonstrated that physiological expression of beta-globin genes requires both a 5' locus control region and 3' specific elements, we constructed a new human chromosome-derived targeting vector to transfer the intact beta-globin gene cluster into K562 cells. The whole beta-globin gene cluster was precisely integrated into the target site and expressed in a self-regulatory pattern. The results proved that the human chromosome-derived vector was specifically targeted to the human genome and this could provide a novel platform for further gene therapy research.     

Effect of Electroacupuncture on Neurotrophin Expression in Cat Spinal Cord after Partial Dorsal Rhizotomy

Neuroplasticity of the spinal cord following electroacupuncture (EA) has been demonstrated although little is known about the possible underlying mechanism. This study evaluated the effect of EA on expression of neurotrophins in the lamina II of the spinal cord, in cats subjected to dorsal rhizotomy. Cats received bilateral removal of L1–L5 and L7–S2 dorsal root ganglia (DRG, L6 DRG spared) and unilateral EA. They were sacrificed 7 days after surgery, and the L6 spinal segment removed and processed by immunohistochemistry and in situ hybridization histochemistry, to demonstrate the expression of neurotrophins. Significantly greater numbers of nerve growth factor (NGF) and neurotrophin-3 (NT-3) positive neurons, brain-derived neurotrophic factor (BDNF) immunoreactive varicosities and NT-3 positive neurons and glial cells were observed in lamina II on the acupunctured (left) side, compared to the non-acupunctured, contralateral side. Greater number of neurons expressing NGF mRNA was also observed on the acupunctured side. No signal for mRNA to BDNF and NT-3 was detected. The above findings demonstrate that EA can increase the expression of endogenous NGF at both the mRNA and protein level, and BDNF and NT-3 at the protein level. It is postulated that EA may promote the plasticity of the spinal cord by inducing increased expression of neurotrophins.     

A protocol for direct somatic embryogenesis of Protea cynaroides L. using zygotic embryos and cotyledon tissues

We describe a protocol for somatic embryogenesis of Protea cynaroides, with potential for high frequency production of this important horticultural species. Somatic embryos formed directly on both P. cynaroides mature zygotic embryos and excised cotyledons cultured on MS medium without growth regulators. The addition of growth regulators such as naphthalene acetic acid (NAA) (5; 13 and 27 μM) and 2,4-dichlorophenoxyacetic acid (2,4-D) (5; 11 and 23 μM), in combination with thidiazuron (TDZ) (1 μM), benzylaminopurine (BAP) (1 μM) or kinetin (1 μM) suppressed the formation of somatic embryos. After eight weeks in culture, formation of somatic embryos was observed. Zygotic explants formed the most embryos when cultured in a 12-h photoperiod in comparison to explants cultured in the dark. Up to 83% of these embryos germinated after transferal to the germination medium containing 0.3 μM GA₃. Significantly fewer embryos germinated in MS medium with no growth regulators, or supplemented with higher concentrations of GA₃, while low germination percentages were also observed in MS media containing casein hydrolysate and coconut water. The germination of normal somatic embryos (two separate cotyledons and a single radicle) was observed only in media containing either no growth regulators, 0.3 μM GA₃ or 1 μM GA₃. All embryos that germinated in high concentrations of GA₃ were malformed.     

A new role for cathelicidin in ulcerative colitis in mice

Cathelicidin, an antimicrobial peptide of the innate immune system, modulates microbial growth, wound healing, and inflammation. However, its association with inflammatory bowel diseases (IBDs) is unknown. Our objective was to determine whether cathelicidin would exert a modulatory effect on the progression of IBD and, if so, investigate the mechanism of action through which this effect occurred. We evaluated the potential for a synthetic cathelicidin, the mouse cathelin-related antimicrobial peptide (mCRAMP), to prevent the initiation and promote the healing of lesions from inflammatory colitis that was experimentally induced in mice with dextran sulfate sodium (DSS). During the experiment, mCRAMP was given: (i) as a parallel treatment starting together with 3% DSS feeding, and (ii) as a posttreatment starting 7 days after 3% DSS feeding. The body weight, fecal microflora populations, clinical symptoms, and histologic findings of colonic tissues were measured. Relative gene expression of mucins (MUC1, MUC2, MUC3, and MUC4) in colonic tissues was determined by real-time polymerase chain reaction. Intrarectal administration of mCRAMP ameliorated DSS-induced colitis with negligible effects on mucosal healing. The peptide also significantly reduced the increased number of fecal microflora in colitis animals. It reversed the decline of colonic mucus thickness during colitis through upregulation of the expression of mucin genes. Treatment with mCRAMP also prevented colitis development by suppressing the induction of apoptosis by DSS. The current study demonstrates for the first time that intrarectal administration of cathelicidin may be a novel therapeutic option for IBDs.     

Association of MT-ND5 gene variation with mitochondrial respiratory control ratio and NADH dehydrogenase activity in Tibet chicken embryos

NADH dehydrogenase (complex I) couples the oxidation of NADH for the reduction of ubiquinone with the generation of a proton gradient that can be used for the synthesis of ATP. We have found a missense mutation in the MT-ND5 subunit of NADH dehydrogenase in the Tibet chicken breed. In the present study, the mitochondrial respiratory control ratio (RCR) and NADH dehydrogenase activity in Tibet chicken embryonic brain with different genotypes were measured. Significant differences between animals carrying mitochondria with the EF493865.1:m.1627A vs. EF493865.1:m.1627C alleles were observed for RCR and enzyme activity.     

Binding of the environmental pollutant naphthol to bovine serum albumin

The interactions between naphthol and bovine serum albumin (BSA) were investigated by spectroscopy. Our results prove the formation of complex between naphthol and BSA. Hydrophobic interaction dominates in the association reaction. The isomers stack with the aromatic residues in their binding sites with different geometries. Effects of BSA on the excited-state proton transfer and fluorescence spectra of the isomers indicate the different characters of their binding sites. 1-Naphthol inserts deeply into a hydrophobic cavity whereas 2-naphthol is in a basic environment on the surface of BSA. Naphthol statically quenches the fluorescence of BSA in a concentration-dependent manner positively deviating from the linear Stern-Volmer equation. Naphthol binds near Trp-134 in the subdomain IA of the native BSA and is accessible to Trp-212 when BSA is unfolded by naphthol. The folding pattern of the main chain is altered at high naphthol concentration as revealed by the change in the secondary structure. The binding of 1-naphthol is more cooperative than that of 2-naphthol. The extent of cooperativity was estimated by the Hill equation.     

Endocytosis optimizes the dynamic localization of membrane proteins that regulate cortical polarity

Diverse cell types require the ability to maintain dynamically polarized membrane-protein distributions through balancing transport and diffusion. However, design principles underlying dynamically maintained cortical polarity are not well understood. Here we constructed a mathematical model for characterizing the morphology of dynamically polarized protein distributions. We developed analytical approaches for measuring all model parameters from single-cell experiments. We applied our methods to a well-characterized system for studying polarized membrane proteins: budding yeast cells expressing activated Cdc42. We found that a balance of diffusion, directed transport, and endocytosis was sufficient for accurately describing polarization morphologies. Surprisingly, the model predicts that polarized regions are defined with a precision that is nearly optimal for measured endocytosis rates and that polarity can be dynamically stabilized through positive feedback with directed transport. Our approach provides a step toward understanding how biological systems shape spatially precise, unambiguous cortical polarity domains using dynamic processes.     

Microarray-based analysis of changes in diversity of microbial genes involved in organic carbon decomposition following land use/cover changes

To increase our understanding of the impact of land use/cover changes on soil microbial decomposition genes involved in organic carbon decomposition, we analyzed soil samples in four sites with different land cover/use histories in a subalpine region of western Sichuan. One site was in a primitive Abies faxoniana forest, the second and the third sites were spruce plantations established in 1960's and 1980's, respectively, and the fourth site was in a cropland dating back to 1960's. The genomic DNA from the microbial community was isolated and hybridized against a functional gene microarray containing 1,961 probes. There were 39, 62, 41, and 28 gene probes with statistically significant positive signals and the gene diversity index (H') values were 3.59, 4.04, 3.70 and 3.16 in primitive forest, spruce plantations established in 1960s and 1980s and cropland, respectively. The results suggested that the number of functional genes and the gene diversity index were correlated with increasing amounts of soil organic carbon, except in the primitive Abies faxoniana forest site. cluster analysis demonstrated that primitive forest soil was clustered more closely to soil from the spruce plantation established in 1960s.