Dysregulation of Claudin family genes in colorectal cancer in a Chinese population

Claudins play an important role in tumor metastasis and in invasiveness of colorectal cancer (CRC). We have evaluated the relationship between CRC and expression of the claudin genes in Chinese patients with CRC. We measured CLDN1 and CLDN7 mRNA using quantitative PCR, and protein levels with immunohistochemistry in cancer tissues and adjacent normal tissue. Cancer tissues had significantly higher levels of CLDN1, and significantly lower levels of CLDN3, CLDN4, and CLDN7 than did normal tissue. CLDN3, CLDN4, and CLDN7 expression levels were higher in CRC of the protruded type than in CRC of the infiltrative type. Expression of CLDN7 correlated with lymph node metastasis. Stage N0 cancer tissues had higher levels of CLDN7 than did stages N1 and N2, suggesting that CLDN7 expression was closely related to the extent of lymph node metastasis. CLDN1 protein was upregulated, but CLDN7 protein was downregulated in cancer tissues when compared with expression in adjacent normal tissues. In conclusion, CLDN3, CLDN4, and CLDN7 were significantly downregulated, whereas CLDN1 was significantly upregulated in CRC. The altered expression of claudin genes may play a role in the initiation and development of CRC.     

Highly-sensitive organophosphorous pesticide biosensors based on nanostructured films of acetylcholinesterase and CdTe quantum dots

The optical transducer of CdTe semiconductor quantum dots (QDs) has been integrated with acetylcholinesterase enzyme (AChE) by the layer-by-layer (LbL) assembly technique, resulting in a highly sensitive biosensor for detection of organophosphorus pesticides (OPs) in vegetables and fruits based on enzyme inhibition mechanism. The detection limits of the proposed biosensors are as low as 1.05 × 10(-11) M for paraoxon and 4.47 × 10(-12) M for parathion, which are significantly better than those of the conventional GC/MS methods or amperometric biosensors (0.5 nM). These biosensors are used for quick determination of low concentrations of OPs in real vegetable and fruit samples and exhibit satisfactory reproducibility and accuracy. Moreover, the stock stability of the biosensors are very good due to the stabilizing environment for the enzyme in the nanostructures made by LbL technique. Many advantages provided by these biosensors, like fluorescent change recognized by naked eyes and mass production with low cost, will facilitate future development of rapid and high-throughput screening of OPs.     

Enhanced γ-aminobutyric acid-forming activity of recombinant glutamate decarboxylase (gadA) from Escherichia coli

γ-aminobutyric acid (GABA) is an important bioactive regulator, and its biosynthesis is primarily through the α-decarboxylation of glutamate by glutamate decarboxylase (GAD). The procedures to obtain GABA by bioconvertion with high activity recombinant Escherichia coli GAD have been seldom understood. In this study, Escherichia coli GAD (gadA) was highly expressed (about 70–75% of total protein) as soluble protein in Escherichia coli BL21(DE3) containing pET28a-gadA, which was induced by 0.4 mM IPTG in LB medium, and maximal GABA-forming activity of the recombinant GAD was 40 U/mL at a concentration (0.15 mM) of pyridoxal phosphate (PLP) and a concentration (0.6 mM) of Ca²⁺ at optimal pH of 3.8. The optimal concentration (7.5 mM) of Mn²⁺ can also improve the activity of recombinant enzyme, but the co-effect of Ca²⁺ and Mn²⁺ exhibited antagonism effect when added simultaneously. LB and 0.1% (w/v) lactose were selected as culture medium and inducer, respectively. The relative activity was markedly higher activated by Ca²⁺ (174%), Mn²⁺ (164%) than that by other seven bivalent cations. Finally, the yield of GABA was high of 94 g/L detected by paper chromatography or HPLC in 1 L reaction system with 30 mL crude GAD (12 U/mL). By entrapping Escherichia coli glutamate decarboxylase into sodium alginate and carrageenan gel beads, the activity of immobilized GAD (IGAD) remained 85% during the initial five batches and the activity still remained 50% at the tenth batch, these results indicated that the recombinant Escherichia coli GAD was feasible for the future industrial production of GABA.     

Three-dimensional reconstruction using an adaptive simultaneous algebraic reconstruction technique in electron tomography

Three-dimensional (3D) reconstruction of electron tomography (ET) has emerged as an important technique in analyzing structures of complex biological samples. However most of existing reconstruction methods are not suitable for extremely noisy and incomplete data conditions. We present an adaptive simultaneous algebraic reconstruction technique (ASART) in which a modified multilevel access scheme and an adaptive relaxation parameter adjustment method are developed to improve the quality of the reconstructed 3D structure. The reconstruction process is facilitated by using a column-sum substitution approach. This modified multilevel access scheme is adopted to arrange the order of projections so as to minimize the correlations between consecutive views within a limited angle range. In the adaptive relaxation parameter adjustment method, not only the weight matrix (as in the existing methods) but the gray levels of the pixels are employed to adjust the relaxation parameters so that the quality of the reconstruction is improved while the convergence process of the reconstruction is accelerated. In the column-sum substitution approach, the computation to obtain the reciprocal of the sum for the columns in each view is avoided so that the needed computations for each iteration can be reduced. Experimental results show that the proposed technique ASART is better based on objective quality measures than other methods, especially when data is noisy and limited in tilt angles. At the same time, the reconstruction by ASART outperforms that of simultaneous algebraic reconstruction technique (SART) in speed.     

Genome sequencing and comparison of two nonhuman primate animal models, the cynomolgus and Chinese rhesus macaques

The nonhuman primates most commonly used in medical research are from the genus Macaca. To better understand the genetic differences between these animal models, we present high-quality draft genome sequences from two macaque species, the cynomolgus/crab-eating macaque and the Chinese rhesus macaque. Comparison with the previously sequenced Indian rhesus macaque reveals that all three macaques maintain abundant genetic heterogeneity, including millions of single-nucleotide substitutions and many insertions, deletions and gross chromosomal rearrangements. By assessing genetic regions with reduced variability, we identify genes in each macaque species that may have experienced positive selection. Genetic divergence patterns suggest that the cynomolgus macaque genome has been shaped by introgression after hybridization with the Chinese rhesus macaque. Macaque genes display a high degree of sequence similarity with human disease gene orthologs and drug targets. However, we identify several putatively dysfunctional genetic differences between the three macaque species, which may explain functional differences between them previously observed in clinical studies.     

Identification and molecular mapping of a resistance gene to powdery mildew from the synthetic wheat line M53

Powdery mildew disease caused by Blumeria graminis f. sp. tritici (Bgt) is an economically important disease in wheat worldwide. The identification of germplasms resistant to the disease can not only facilitate the breeding of resistant cultivars, but can also broaden the diversity of resistance genes. The Mexican M53 is a synthetic hexaploid wheat line developed at the International Maize and Wheat Improvement Center (CIMMYT) from the cross between Triticum durum and Aegilops tauschii249. Infection of M53 with 15 different pathogen races revealed that the resistance in M53 was race-dependent and effective against the majority of the tested Bgt races, including the race 15 predominant in the Beijing wheat growing area. Inoculation of the parents of M53 with the race 15 demonstrated that M53 and Ae. tauschii249 were resistant, whereas T. durum was susceptible. The inoculation of three segregating F₂ populations developed from the crosses between M53 and three susceptible Chinese wheat cultivars with the race 15 showed that the resistant gene in M53 segregated in a single dominant manner. Amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers were used to map the gene in a segregating F₂ population consisting of 213 lines developed from the cross Wan7107 × M53. Two closely linked AFLP markers, Apm109 and Apm161, were identified to flank the gene with genetic distances of 1.0 cM and 3.0 cM, respectively. The recognized gene was assigned to the long arm of chromosome 5D as determined by three linked SSR markers, Xwmc289b, Xgwm583, and Xgwm292, and by the physical mapping of Apm109 using Chinese Spring nullisomic–tetrasomic and ditelosomic stocks. The resistance gene identified in M53, temporarily designated as Pm-M53, could be used in local wheat-breeding programs to improve powdery mildew resistance.     

A physical map for the Amborella trichopoda genome sheds light on the evolution of angiosperm genome structure

Background

Recent phylogenetic analyses have identified Amborella trichopoda, an understory tree species endemic to the forests of New Caledonia, as sister to a clade including all other known flowering plant species. The Amborella genome is a unique reference for understanding the evolution of angiosperm genomes because it can serve as an outgroup to root comparative analyses. A physical map, BAC end sequences and sample shotgun sequences provide a first view of the 870 Mbp Amborella genome.

Results

Analysis of Amborella BAC ends sequenced from each contig suggests that the density of long terminal repeat retrotransposons is negatively correlated with that of protein coding genes. Syntenic, presumably ancestral, gene blocks were identified in comparisons of the Amborella BAC contigs and the sequenced Arabidopsis thaliana, Populus trichocarpa, Vitis vinifera and Oryza sativa genomes. Parsimony mapping of the loss of synteny corroborates previous analyses suggesting that the rate of structural change has been more rapid on lineages leading to Arabidopsis and Oryza compared with lineages leading to Populus and Vitis. The gamma paleohexiploidy event identified in the Arabidopsis, Populus and Vitis genomes is shown to have occurred after the divergence of all other known angiosperms from the lineage leading to Amborella.

Conclusions

When placed in the context of a physical map, BAC end sequences representing just 5.4% of the Amborella genome have facilitated reconstruction of gene blocks that existed in the last common ancestor of all flowering plants. The Amborella genome is an invaluable reference for inferences concerning the ancestral angiosperm and subsequent genome evolution.     

Expression of a radish defensin in transgenic wheat confers increased resistance to Fusarium graminearum and Rhizoctonia cerealis

Fusarium head blight (scab), primarily caused by Fusarium graminearum, is a devastating disease of wheat (Triticum aestivum L.) worldwide. Wheat sharp eyespot, mainly caused by Rhizoctonia cerealis, is one of the major diseases of wheat in China. The defensin RsAFP2, a small cyteine-rich antifungal protein from radish (Raphanus sativus), was shown to inhibit growth in vitro of agronomically important fungal pathogens, such as F. graminearum and R. cerealis. The RsAFP2 gene was transformed into Chinese wheat variety Yangmai 12 via biolistic bombardment to assess the effectiveness of the defensin in protecting wheat from the fungal pathogens in multiple locations and years. The genomic PCR and Southern blot analyses indicated that RsAFP2 was integrated into the genomes of the transgenic wheat lines and heritable. RT-PCR and Western blot proved that the RsAFP2 was expressed in these transgenic wheat lines. Disease tests showed that four RsAFP2 transgenic lines (RA1–RA4) displayed enhanced resistance to F. graminearum compared to the untransformed Yangmai 12 and the null-segregated plants. Assays on Q-RT-PCR and disease severity showed that the express level of RsAFP2 was associated with the enhanced resistance degree. Two of these transgenic lines (RA1 and RA2) also exhibited enhanced resistance to R. cerealis. These results indicated that the expression of RsAFP2 conferred increased resistance to F. graminearum and R. cerealis in transgenic wheat.     

蝎毒对人肝癌细胞(SMMC7721)和宫颈癌(Hela)细胞抑制作用的研究

目的:探讨蝎毒粗粉及经初步纯化的蝎毒对人肝癌细胞(SMMC7721)和Hela细胞株的抑制作用。方法:以四甲基偶氮唑盐法(MTT),Western Blotting,免疫细胞化学以及荧光标记的方法,对人肝癌细胞(SMMC7721)和Hela细胞株的凋亡相关蛋白进行检测。结果:蝎毒粗毒及经初步纯化的蝎毒具有诱导SMMC7721和Hela凋亡的作用。结论:蝎毒粗毒及经初步纯化的蝎毒对人肝癌细胞(SMMC 7721)及Hela细胞的生长具有明显的抑制作用。