pKILLIN: a versatile positive-selection cloning vector based on the toxicity of Killin in Escherichia coli

The invention of DNA cloning over 40 years ago marked the advent of molecular biology. The technique has now become a routine practice in any modern biomedical laboratory. Although positive-selection of recombinants in DNA cloning seems to be superior to blue/white selection based on the disruption of the lacZ gene, it is rarely practiced due to its high background, lack of multiple cloning sites, and inability to express the genes of interest or purify the protein products. Here we report the creation of a new positive-selection cloning vector dubbed pKILLIN, which overcomes all of the above pitfalls. The essence behind its high cloning efficiency is the extreme toxicity and small size of the toxic domain of killin, a recently discovered p53 target gene. Insertion inactivation of killin within the multiple cloning site via either blunt- or sticky-end ligation not only serves as a highly efficient cloning trap, but also may allow any cloned genes to be expressed as His-tagged fusion proteins for subsequent purification. Thus, pKILLIN is a versatile positive-selection vector ideal for cloning PCR products, making DNA libraries, as well as routine cloning and bacterial expression of genes.     

广东省野生植物一新记录科——蜡梅科

报道了广东省野生植物一新记录科--蜡梅科(Calycanthaceae)、一新记录属--蜡梅属(Chimonanthus)及一新记录种--山蜡梅(Chimonanthus nitens Oliv.).标本存放于华南农业大学林学院树木标本室(CANT).     

Characterization and mapping of a novel mutant sms1 (senescence and male sterility 1) in rice

Plant senescence plays diverse important roles in development and environmental responses.However,the molecular basis of plant senescence is remained largely unknown.A rice spontaneous mutant with the character of early senescence and male sterility (sms) was found in the breeding line NT10-748.In order to identify the gene SMS1 and the underlying mechanism,we preliminarily analyzed physiological and biochemical phenotypes of the mutant.The mutant contained lower chlorophyll content compared with the wild t...     

大豆初生根凯氏带对铜离子的通透性

采用在根内生成有色铜沉淀的方法研究大豆(Glycine max)初生根凯氏带对铜离子的通透性。用真空泵抽取浓度为200 μmol·L^–1 的CuSO₄溶液进入根中, 然后在重力作用下从根基部灌注400 μmol·L^–1的K₄[Fe(CN)₆]溶液, 两种物质在根内相遇即可产生棕色的Cu₂[Fe(CN)₆]沉淀, 根据沉淀的位置来确定铜离子所经过的途径。结果表明: Cu²⁺可以穿过内皮层凯氏带, 在木质部导管壁以及凯氏带至木质部之间的细胞壁处产生棕色沉淀, 侧根发生的部位也产生了大量的沉淀; 当抽取K₄[Fe(CN)₆]溶液后再灌注CuSO₄溶液, 发现Cu²⁺仍然可以穿过凯氏带, 并在凯氏带外侧以及外皮层细胞的细胞壁处产生棕色沉淀。研究结果证明凯氏带并不是一个可以完全阻止离子进出的完美屏障。     

Variations of floral traits among different life forms illustrate the evolution of pollination systems in Potamogeton species from China

Four leaf morpha groups (floating-leaved, submerged broad-leaved, submerged linear-leaved and filiform-leaved) and three inflorescences type groups (erect, facultative floating and floating) can be found in the genus Potamogeton. Life forms in the genus correlate with pollination types (anemophily, epihydrophily and hydroautogamy). Floral traits vary greatly among different life forms and pollination types. Both floating and submerged broad-leaved species produce erect inflorescences that are suited for anemophilous pollination. These flowers also have the highest pollen/ovule (P/O) ratio (25,013) and the smallest pollen grain volume (6046 μm³). On the contrary, submerged filiform-leaved species produce only floating inflorescences and their flowers produce the largest pollen grains (17,567 μm³) and have the lowest P/O ratio (6752), which is typical of the syndromes of hydroautogamy and epihydrophily. The relatively prolific seed production (about 70%) and wide dispersal in the floating and submerged broad-leaved species and the submerged filliform-leaved species indicate that these two groups are well adapted to the aquatic environment. This is in contrast with the less well-adapted complex and unstable pollination system found in linear-leaved species. We propose that the pollination systems and life forms in the Potamogeton, have undergone modification in response to the demands imposed by the aquatic environment during the evolutionary process in the genus.     

Generation of mouse conditional and null alleles of the type III sodium‐dependent phosphate cotransporter PiT‐1

Accelerated vascular calcification occurs in several human diseases including diabetes and chronic kidney disease (CKD). In patients with CKD, vascular calcification is highly correlated with elevated serum phosphate levels. In vitro, elevated concentrations of phosphate induced vascular smooth muscle cell matrix mineralization, and the inorganic phosphate transporter‐1 (PiT‐1), was shown to be required. To determine the in vivo role of PiT‐1, mouse conditional and null alleles were generated. Here we show that the conditional allele, PiT‐1^flox, which has loxP sites flanking exons 3 and 4, is homozygous viable. Cre‐mediated recombination resulted in a null allele that is homozygous lethal. Examination of early embryonic development revealed that the PiT‐1^{Δe3,4/Δe3,4} embryos displayed anemia, a defect in yolk sac vasculature, and arrested growth. Thus, conditional and null PiT‐1 mouse alleles have been successfully generated and PiT‐1 has a necessary, nonredundant role in embryonic development. genesis 47:858–863, 2009. © 2009 Wiley‐Liss, Inc.     

Novel Cell-Free Strategy for Therapeutic Angiogenesis: In Vitro Generated Conditioned Medium Can Replace Progenitor Cell Transplantation

Background

Current evidence suggests that endothelial progenitor cells (EPC) contribute to ischemic tissue repair by both secretion of paracrine factors and incorporation into developing vessels. We tested the hypothesis that cell-free administration of paracrine factors secreted by cultured EPC may achieve an angiogenic effect equivalent to cell therapy.

Methodology/Principal Findings

EPC-derived conditioned medium (EPC-CM) was obtained from culture expanded EPC subjected to 72 hours of hypoxia. In vitro, EPC-CM significantly inhibited apoptosis of mature endothelial cells and promoted angiogenesis in a rat aortic ring assay. The therapeutic potential of EPC-CM as compared to EPC transplantation was evaluated in a rat model of chronic hindlimb ischemia. Serial intramuscular injections of EPC-CM and EPC both significantly increased hindlimb blood flow assessed by laser Doppler (81.2±2.9% and 83.7±3.0% vs. 53.5±2.4% of normal, P<0.01) and improved muscle performance. A significantly increased capillary density (1.62±0.03 and 1.68±0.05/muscle fiber, P<0.05), enhanced vascular maturation (8.6±0.3 and 8.1±0.4/HPF, P<0.05) and muscle viability corroborated the findings of improved hindlimb perfusion and muscle function. Furthermore, EPC-CM transplantation stimulated the mobilization of bone marrow (BM)-derived EPC compared to control (678.7±44.1 vs. 340.0±29.1 CD34⁺/CD45⁻ cells/1×10⁵ mononuclear cells, P<0.05) and their recruitment to the ischemic muscles (5.9±0.7 vs. 2.6±0.4 CD34⁺ cells/HPF, P<0.001) 3 days after the last injection.

Conclusions/Significance

Intramuscular injection of EPC-CM is as effective as cell transplantation for promoting tissue revascularization and functional recovery. Owing to the technical and practical limitations of cell therapy, cell free conditioned media may represent a potent alternative for therapeutic angiogenesis in ischemic cardiovascular diseases.     

Synthesis,initial SAR and biological evaluation of 1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-4-amine derived inhibitors of IκB kinase

A new series of tricyclic-based inhibitors of IKK have been derived from an earlier lead compound. The synthesis and structure–activity relationships (SAR) are described. Compound 4k inhibited TNF production in rats stimulated with LPS.     

Bifidobacteria-mediated immune system imprinting early in life

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