Establishment of a micropropagation supporting technology for the Fraxinus mandshurica × Fraxinus sogdiana

In Vitro Cellular & Developmental Biology - Plant - The interspecific hybridization can take advantage of heterosis and combine the double parental traits most extensively, and is an effective...     

Evidence for a synergistic effect of post‐translational modifications and genomic composition of eEF‐1α on the adaptation of Phytophthora infestans

Genetic variation plays a fundamental role in pathogen''s adaptation to environmental stresses. Pathogens with low genetic variation tend to survive and proliferate more poorly due to their lack of genotypic/phenotypic polymorphisms in responding to fluctuating environments. Evolutionary theory hypothesizes that the adaptive disadvantage of genes with low genomic variation can be compensated for structural diversity of proteins through post‐translation modification (PTM) but this theory is rarely tested experimentally and its implication to sustainable disease management is hardly discussed. In this study, we analyzed nucleotide characteristics of eukaryotic translation elongation factor‐1α (eEF‐lα) gene from 165 Phytophthora infestans isolates and the physical and chemical properties of its derived proteins. We found a low sequence variation of eEF‐lα protein, possibly attributable to purifying selection and a lack of intra‐genic recombination rather than reduced mutation. In the only two isoforms detected by the study, the major one accounted for >95% of the pathogen collection and displayed a significantly higher fitness than the minor one. High lysine representation enhances the opportunity of the eEF‐1α protein to be methylated and the absence of disulfide bonds is consistent with the structural prediction showing that many disordered regions are existed in the protein. Methylation, structural disordering, and possibly other PTMs ensure the ability of the protein to modify its functions during biological, cellular and biochemical processes, and compensate for its adaptive disadvantage caused by sequence conservation. Our results indicate that PTMs may function synergistically with nucleotide codes to regulate the adaptive landscape of eEF‐1α, possibly as well as other housekeeping genes, in P. infestans. Compensatory evolution between pre‐ and post‐translational phase in eEF‐1α could enable pathogens quickly adapting to disease management strategies while efficiently maintaining critical roles of the protein playing in biological, cellular, and biochemical activities. Implications of these results to sustainable plant disease management are discussed.     

肠出血性大肠杆菌O157∶H7 EspA和EspB蛋白的重组表达及其免疫效果

目的：通过DNA重组技术表达肠出血性大肠杆菌（EHEC）0157：H7的EspA和EspB蛋白,并分析它们的免疫保护性。方法：采用PCR技术从EHEC0157：H7基因组中扩增espA和espB基因,连接至pET-22b（4-）载体上,转化至宿主细胞大肠杆菌BL21（DE3）,经IPTG诱导表达,用亲和层析纯化目的蛋白,SDS-PAGE测定其相对分子质量,免疫小鼠分析其免疫保护性。结果：重组espA和espB基因片段的测序结果与GenBank中的相应基因序列完全一致,一致性均为100％;得到了纯度为95％以上的重组EspA和EspB蛋白,免疫小鼠所得到的抗体效价均为10^6。结论：重组EspA和EspB蛋白获得了可溶性表达,表达的蛋白具有良好的免疫保护性,为进一步制备疫苗奠定了基础。     

Bone morphogenetic protein 9 overexpression reduces osteosarcoma cell migration and invasion

Transforming growth factor-β (TGF-β) is known to promote tumor migration and invasion. Bone morphogenetic proteins (BMPs) are members of the TGF-β family expressed in a variety of human carcinoma cell lines. The role of bone morphogenetic protein 9 (BMP9), the most powerful osteogenic factor, in osteosarcoma (OS) progression has not been fully clarified. The expression of BMP9 and its receptors in OS cell lines was analyzed by RT-PCR. We found that BMP9 and its receptors were expressed in OS cell lines. We further investigated the influence of BMP9 on the biological behaviors of OS cells. BMP9 overexpression in the OS cell lines 143B and MG63 inhibited in vitro cell migration and invasion. We further investigated the expression of a panel of cancer-related genes and found that BMP9 overexpression increased the phosphorylation of Smad1/5/8 proteins, increased the expression of ID1, and reduced the expression and activity of matrix metalloproteinase 9 (MMP9) in OS cells. BMP9 silencing induced the opposite effects. We also found that BMP9 may not affect the chemokine (C-X-C motif) ligand 12 (CXCL12)/C-X-C chemokine receptor type 4 (CXCR4) axis to regulate the invasiveness and metastatic capacity of OS cells. Interestingly, CXCR4 was expressed in both 143B and MG63 cells, while CXCL12 was only detected in MG63 cells. Taken together, we hypothesize that BMP9 inhibits the migration and invasiveness of OS cells through a Smad-dependent pathway by downregulating the expression and activity of MMP9.     

Directional Modifications of Resibufogenin by Mucor subtilissimus and Pseudomonas aeruginosa

Directional modifications of resibufogenin 1 by Mucor subtilissimus and Pseudomonas aeruginosa were carried out. The substrate was hydroxylated at C-12 by M. subtilissimus AS 3.2454, from which a major product 12-hydroxyresibufogenin 2 was obtained. Then product 2 was dehydrogenated by P. aeruginosa AS 1.860, which resulted in a new compound 12β-hydroxy-3-keto-resibufogenin 3.     

Effects of High Dose of Anisodamine on the Respiratory Function of Patients with Traumatic Acute Lung Injury

The objective of this study is to observe the effects of high dose of anisodamine in the respiratory function of patients with acute lung injury (ALI) induced by trauma. Fifty cases of patients with complicated ALI after trauma were randomly divided into the anisodamine treatment group (n = 26) and the conventional treatment group (n = 24). Intravenous injection of high dose of anisodamine was administrated in the anisodamine treatment group on the basis of conventional treatment. Acute lung injury scores, oxygenation index, PaO₂, respiratory mechanic index, mechanical ventilation time, and the occurrence of adverse reactions in the two groups were observed. Acute lung injury scores and respiratory parameters were all significantly improved in the two groups of patients after the treatment (P < 0.05); compared with the conventional treatment group, the improvements of respiratory function in the anisodamine treatment group were more obvious (P < 0.05), the mechanical ventilation time was shorter (P < 0.05), and there was no significant adverse reaction. In conclusion, high dose of anisodamine contributed to improve the respiratory function of the patients with traumatic ALI.     

Laccases Involved in 1,8-Dihydroxynaphthalene Melanin Biosynthesis in Aspergillus fumigatus Are Regulated by Developmental Factors and Copper Homeostasis

Aspergillus fumigatus produces heavily melanized infectious conidia. The conidial melanin is associated with fungal virulence and resistance to various environmental stresses. This 1,8-dihydroxynaphthalene (DHN) melanin is synthesized by enzymes encoded in a gene cluster in A. fumigatus, including two laccases, Abr1 and Abr2. Although this gene cluster is not conserved in all aspergilli, laccases are critical for melanization in all species examined. Here we show that the expression of A. fumigatus laccases Abr1/2 is upregulated upon hyphal competency and drastically increased during conidiation. The Abr1 protein is localized at the surface of stalks and conidiophores, but not in young hyphae, consistent with the gene expression pattern and its predicted role. The induction of Abr1/2 upon hyphal competency is controlled by BrlA, the master regulator of conidiophore development, and is responsive to the copper level in the medium. We identified a developmentally regulated putative copper transporter, CtpA, and found that CtpA is critical for conidial melanization under copper-limiting conditions. Accordingly, disruption of CtpA enhanced the induction of abr1 and abr2, a response similar to that induced by copper starvation. Furthermore, nonpigmented ctpAΔ conidia elicited much stronger immune responses from the infected invertebrate host Galleria mellonella than the pigmented ctpAΔ or wild-type conidia. Such enhancement in eliciting Galleria immune responses was independent of the ctpAΔ conidial viability, as previously observed for the DHN melanin mutants. Taken together, our findings indicate that both copper homeostasis and developmental regulators control melanin biosynthesis, which affects conidial surface properties that shape the interaction between this pathogen and its host.     

Cyclin B1 is an efficacy-predicting biomarker for Chk1 inhibitors

Chk1 is the major mediator of cell-cycle checkpoints in response to various forms of genotoxic stress. Although it was previously speculated that checkpoint abrogation due to Chk1 inhibition may potentiate the efficacy of DNA-damaging agents through induction of mitotic catastrophe, there has not been direct evidence proving this process. Here, through both molecular marker and morphological analysis, we directly demonstrate that specific downregulation of Chk1 expression by Chk1 siRNA potentiates the cytotoxicities of topoisomerase inhibitors through the induction of premature chromosomal condensation and mitotic catastrophe. More importantly, we discovered that the cellular cyclin B1 level is the major determinant of the potentiation. We show that downregulation of cyclin B1 leads to impairment of the induction of mitotic catastrophe and correspondingly a reduction of the potentiation ability of either Chk1 siRNA or a small molecule Chk1 inhibitor. More significantly, we have extended the study by examining a panel of 10 cancer cell-lines with different tissue origins for their endogenous levels of cyclin B1 and the ability of a Chk1 inhibitor to sensitize the cells to DNA-damaging agents. The cellular levels of cyclin B1 positively correlate with the degrees of potentiation achieved. Of additional interest, we observed that the various colon cancer cell lines in general appear to express higher levels of cyclin B1 and also display higher sensitivity to Chk1 inhibitors, implying that Chk1 inhibitor may be more efficacious in treating colon cancers. In summary, we propose that cyclin B1 is a biomarker predictive of the efficacy of Chk1 inhibitors across different types of cancers. Unlike previously established efficacy-predictive biomarkers that are usually the direct targets of the therapeutic agents, cyclin B1 represents a non-drug-target biomarker that is based on the mechanism of action of the target inhibitor. This finding may be potentially very useful for the stratification of patients for Chk1 inhibitor clinical trials and hence, maximize its chance of success.     

An indigoidine biosynthetic gene cluster from Streptomyces chromofuscus ATCC 49982 contains an unusual IndB homologue

A putative indigoidine biosynthetic gene cluster was located in the genome of Streptomyces chromofuscus ATCC 49982. The silent 9.4-kb gene cluster consists of five open reading frames, named orf1, Sc-indC, Sc-indA, Sc-indB, and orf2, respectively. Sc-IndC was functionally characterized as an indigoidine synthase through heterologous expression of the enzyme in both Streptomyces coelicolor CH999 and Escherichia coli BAP1. The yield of indigoidine in E. coli BAP1 reached 2.78 g/l under the optimized conditions. The predicted protein product of Sc-indB is unusual and much larger than any other reported IndB-like protein. The N-terminal portion of this enzyme resembles IdgB and the C-terminal portion is a hypothetical protein. Sc-IndA and/or Sc-IndB were co-expressed with Sc-IndC in E. coli BAP1, which demonstrated the involvement of Sc-IndB, but not Sc-IndA, in the biosynthetic pathway of indigoidine. The yield of indigoidine was dramatically increased by 41.4 % (3.93 g/l) when Sc-IndB was co-expressed with Sc-IndC in E. coli BAP1. Indigoidine is more stable at low temperatures.