Generation and analyses of the transgenic potatoes expressing heterologous thermostable β-amylase

β-Amylase hydrolyzes the -1,4-glycosidic linkages of starch resulting in the release of maltose. This reaction is of industrial importance for maltose production and for the preparation process of fermented foods and alcoholic beverages. A demand for an acceleration of the rate of enzymatic cleavage of the starch macro-molecule is a prerequisite for large-scale and highly efficient production. Increasing the temperature up to the optimum of approximately 60 °C can significantly speed up the reaction. However, at higher temperatures, the effect on protein denaturation becomes dominant, and the conversion rate decreases. The primary objective of this study was to generate transgenic plants of the “Kennebec” potato variety for production of thermostable β-amylase using Agrobacterium-mediated transformation. Four chimeric genes encoding the β-amylase with or without signal peptide sequences for targeting expression in cytoplasm, amyloplasts, or vacuoles were constructed and driven by high tuber expression promoter from Sucrose synthetase gene Sus4. Forty-two transgenic lines were selected for this study. Transgenic lines with various β-amylase constructs were verified for the existence and expression of the transgenes by PCR approaches. The expression level of the introduced β-amylase protein was estimated by immunoblot analyses using polyclonal antibodies. Recombinant β-amylase was successfully expressed in Escherichia coli B21 (DE3), and temperature ranges of these inducible recombinant proteins were found to be between 40 and 90 °C. This enzymatic complex produced in the in vitro cultured microtubers and field-grown tubers from transgenic potatoes were proved to be stable and active at 60 °C. The relative activities of β-amylase in tubers of field-grown potatoes were compared, and the maximum increase was found with transgenic line #6A of the pSUS4-AMY construct which has an 11-fold greater increase than the untransformed “Kennebec”. Variations of the chemical compositions were found in the selected transgenic lines. Results of this study suggest the feasibility of utilizing thermostable β-amylase in transgenic potatoes for the starch-processing industries.     

Surface α-Enolase Promotes Extracellular Matrix Degradation and Tumor Metastasis and Represents a New Therapeutic Target

In previous research, we found α-enolase to be inversely correlated with progression-free and overall survival in lung cancer patients and detected α-enolase on the surface of lung cancer cells. Based on these findings, we hypothesized that surface α-enolase has a significant role in cancer metastasis and tested this hypothesis in the current study. We found that α-enolase was co-immunoprecipitated with urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor, and plasminogen in lung cancer cells and interacted with these proteins in a cell-free dot blotting assay, which can be interrupted by α-enolase-specific antibody. α-Enolase in lung cancer cells co-localized with these proteins and was present at the site of pericellular degradation of extracellular matrix components. Treatment with antibody against α-enolase in vitro suppressed cell-associated plasminogen and matrix metalloproteinase activation, collagen and gelatin degradation, and cell invasion. Examination of the effect of treatment with shRNA plasmids revealed that down regulation of α-enolase decreases extracellular matrix degradation by and the invasion capacity of lung cancer cells. Adoptive transfer of α-enolase-specific antibody to mice resulted in accumulation of antibody in subcutaneous tumor and inhibited the formation of tumor metastasis in lung and bone. This study demonstrated that surface α-enolase promotes extracellular matrix degradation and invasion of cancer cells and that targeting surface α-enolase is a promising approach to suppress tumor metastasis.     

Colchicine modulates calcium homeostasis and electrical property of HL‐1 cells

Colchicine is a microtubule disruptor that reduces the occurrence of atrial fibrillation (AF) after an operation or ablation. However, knowledge of the effects of colchicine on atrial myocytes is limited. The aim of this study was to determine if colchicine can regulate calcium (Ca²⁺) homeostasis and attenuate the electrical effects of the extracellular matrix on atrial myocytes. Whole‐cell clamp, confocal microscopy with fluorescence, and western blotting were used to evaluate the action potential and ionic currents of HL‐1 cells treated with and without (control) colchicine (3 nM) for 24 hrs. Compared with control cells, colchicine‐treated HL‐1 cells had a longer action potential duration with smaller intracellular Ca²⁺ transients and sarcoplasmic reticulum (SR) Ca²⁺ content by 10% and 47%, respectively. Colchicine‐treated HL‐1 cells showed a smaller L‐type Ca²⁺ current, reverse mode sodium–calcium exchanger (NCX) current and transient outward potassium current than control cells, but had a similar ultra‐rapid activating outward potassium current and apamin‐sensitive small‐conductance Ca²⁺‐activated potassium current compared with control cells. Colchicine‐treated HL‐1 cells expressed less SERCA2a, total, Thr17‐phosphorylated phospholamban, Cav1.2, CaMKII, NCX, Kv1.4 and Kv1.5, but they expressed similar levels of the ryanodine receptor, Ser16‐phosphorylated phospholamban and Kv4.2. Colchicine attenuated the shortening of the collagen‐induced action potential duration in HL‐1 cells. These findings suggest that colchicine modulates the atrial electrical activity and Ca²⁺ regulation and attenuates the electrical effects of collagen, which may contribute to its anti‐AF activity.     

Characterization of Genomic Inheritance of Intergeneric Hybrids between Ascocenda and Phalaenopsis Cultivars by GISH,PCR-RFLP and RFLP

BackgroundThe intergeneric hybrids between Ascocenda John De Biase ‘Blue’ and Phalaenopsis Chih Shang''s Stripes have been generated to introduce the blue color into the Phalaenopsis germplasm in prior study. In order to confirm the inheritance in hybrid progenies, genomic in situ hybridization (GISH) and restriction fragment length polymorphism (RFLP) analysis were conducted to confirm the intergeneric hybridization status.Methods/ResultsGISH analysis showed the presence of both maternal and paternal chromosomes in the cells of the putative hybrids indicating that the putative hybrid seedlings were intergeneric hybrids of the two parents. Furthermore, twenty-seven putative hybrids were randomly selected for DNA analysis, and the external transcribed spacer (ETS) regions of nrDNA were analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and RFLP analyses to identify the putative hybrids. RFLP analysis showed that the examined seedlings were intergeneric hybrids of the two parents. However, PCR-RFLP analysis showed bias to maternal genotype.ConclusionsBoth GISH and RFLP analyses are effective detection technology to identify the intergeneric hybridization status of putative hybrids. Furthermore, the use of PCR-RFLP analysis to identify the inheritance of putative hybrids should be carefully evaluated.     

Ankrd17, an ubiquitously expressed ankyrin factor, is essential for the vascular integrity during embryogenesis

Ankyrin repeat domain 17 (Ankrd17) encodes an ubiquitously expressed protein with two clusters of ankyrin repeats. We have used gene targeting strategy to ablate the Ankrd17 gene in mouse. The Ankrd17-deficient mice died between embryonic day (E) 10.5 and E11.5 due to cardiovascular defects. Serious hemorrhages were detected and the vascular smooth muscle cells (vSMCs) surrounding the vessels were drastically reduced in the Ankrd17-deficient embryos, suggesting that the vascular maturation was not completed. Interestingly, vSMC differentiation marker genes were up-regulated in the mutant embryos. Our data have demonstrated the indispensability of Ankrd17 functioning for vascular maturation during early development. The Ankrd17-deficient mice also provide a new animal model for the analysis of the regulatory pathways of the differentiation of vSMC precursor cells.     

Molecular delineation of the Y-borne Sry gene in the Formosan pangolin (Manis pentadactyla pentadactyla) and its phylogenetic implications for Pholidota in extant mammals

The systematic status of Pholidota has been a matter of debate, particularly regarding the apparent inconsistency between morphological and molecular studies. The Sry gene, a master regulator of male sex determination in eutherian mammals, has not yet been used for phylogenetic analyses of extant mammals. The objective of the present study was to clone and characterize the complete gene (1300 base pairs; bp) and amino acid sequences (229 residues) of Sry from the Formosan pangolin (Manis pentadactyla pentadactyla), a member of Pholidota. The Sry amino acid identity between pangolin and other reported species ranged from 42.5% (mouse, Mus musculus) to 84.1% (European hare, Lepus europaeus). Sequence conservation was primarily in the high motility group (HMG) box (234 bp), whereas homology outside the HMG box was low. The cloned Sry was mapped to the pangolin Y chromosome by fluorescence in situ hybridization (FISH); this was confirmed to be the first Y-borne molecular marker identified in Pholidota. Based on Bayesian phylogenetic analysis for Sry HMG sequences from 36 representative taxa, including the Formosan pangolin, Pholidota was more closely related to Carnivora than to Xenarthra, consistent with the emerging molecular tree inferred from markers not located on the Y chromosome. In conclusion, this study characterized the gene structure of Sry of the Formosan pangolin and provided insights into the phylogenetic position of Pholidota.     

Adaptive introgression of anticoagulant rodent poison resistance by hybridization between old world mice

Polymorphisms in the vitamin K 2,3-epoxide reductase subcomponent 1 (vkorc1) of house mice (Mus musculus domesticus) can cause resistance to anticoagulant rodenticides such as warfarin [1-3]. Here we show that resistant house mice can also originate from selection on vkorc1 polymorphisms acquired from the Algerian mouse (M. spretus) through introgressive hybridization. We report on a polymorphic introgressed genomic region in European M. m. domesticus that stems from M. spretus, spans >10 Mb on chromosome 7, and includes the molecular target of anticoagulants vkorc1 [1-4]. We show that in the laboratory, the homozygous complete vkorc1 allele of M. spretus confers resistance when introgressed into M. m. domesticus. Consistent with selection on the introgressed allele after the introduction of rodenticides in the 1950s, we found signatures of selection in patterns of variation in M. m. domesticus. Furthermore, we detected adaptive protein evolution of vkorc1 in M. spretus (Ka/Ks = 1.54-1.93) resulting in radical amino acid substitutions that apparently cause anticoagulant tolerance in M. spretus as a pleiotropic effect. Thus, positive selection produced an adaptive, divergent, and pleiotropic vkorc1 allele in the donor species, M. spretus, which crossed a species barrier and produced an adaptive polymorphic trait in the recipient species, M. m. domesticus.