TaqMan-MGB探针检测文森巴尔通体博格霍夫亚种实时荧光定量PCR方法的建立及应用

【目的】应用TaqMan-MGB探针技术,建立具有种水平特异性、高敏感性的荧光定量PCR方法,用于快速检测文森巴尔通体博格霍夫亚种。【方法】在序列特异性扩增区标记(Sequence characterized amplifiedregion,SCAR)技术基础上,依据文森巴尔通体博格霍夫亚种一段特有的基因序列设计探针和引物,分别优化扩增反应的退火温度、探针和引物的反应浓度;分析此方法的特异性、敏感性及重复性;绘制标准曲线,评估PCR反应的扩增效率和稳定性。【结果】本研究设计的TaqMan-MGB探针具有种水平特异性;最低检出限为每个PCR反应11个拷贝;组内和组间的变异系数CV值分别为0.12%-0.70%和0.14%-0.55%,在允许范围内;标准曲线线性关系良好(R2=1),扩增效率高(E=104.7%)。【结论】本研究建立的基于TaqMan-MGB探针技术的荧光定量PCR方法能够在种水平特异性、高灵敏度检出文森巴尔通体博格霍夫亚种,为这种巴尔通体所引起的一系列疾病的早期快速诊断、监测和流行病学调查等研究提供有效手段。     

Molecular characterization of avian-like H1N1 swine influenza a viruses isolated in Eastern China, 2011

Currently, three predominant subtypes of influenza virus are prevalent in pig populations worldwide: H1N1, H3N2, and H1N2. European avian-like H1N1 viruses, which were initially detected in European pig populations in 1979, have been circulating in pigs in eastern China since 2007. In this study, six influenza A viruses were isolated from 60 swine lung samples collected from January to April 2011 in eastern China. Based on whole genome sequencing, molecular characteristics of two isolates were determined. Phylogenetic analysis showed the eight genes of the two isolates were closely related to those of the avian-like H1N1 viruses circulating in pig populations, especially similar to those found in China. Four potential glycosylation sites were observed at positions 13, 26, 198, 277 in the HA1 proteins of the two isolates. Due to the presence of a stop codon at codon 12, the isolates contained truncated PB1-F2 proteins. In this study, the isolates contained 591Q, 627E and 701N in the polymerase subunit PB2, which had been shown to be determinants of virulence and host adaptation. The isolates also had a D rather than E at position 92 of the NS1, a marker of mammalian adaptation. Both isolates contained the GPKV motif at the PDZ ligand domain of the 3′ end of the NS1, a characteristic marker of the European avian-like swine viruses since about 1999, which is distinct from those of avian, human and classical swine viruses. The M2 proteins of the isolates have the mutation (S31N), a characteristic marker of the European avian-like swine viruses since about 1987, which may confer resistance to amantadine and rimantadine antivirals. Our findings further emphasize the importance of surveillance on the genetic diversity of influenza A viruses in pigs, and raise more concerns about the occurrence of cross-species transmission events.     

Molecular Cloning of a Novel Gene ZAhi-1 and its Expression Analysis during Zebrafish Gametogenesis

Proto-oncogen Ahi-1 is closely related to a lot of human and mouse diseases. Ahi-1 mutation will lead to leukemia in mice and Joubert syndrome in human beings. We have cloned the full cDNA sequence of Ahi-1 homologous in zebrafish, and RT-PCR results of ZAhi-1 in different tissues reveal that ZAhi-1 expressed highest in the mature gonad. In situ hybridization results of zebrafish gonad show that ZAhi-1 only expressed in the early stages’ gamete cells. RT-PCR analyses of mouse Ahi-1 in different stages of spermatogenesis have been done according to the published Ahi-1 sequence, and the findings reveal that Ahi-1 is expressed in gamete of pachytene stage. It can then be safely concluded that Ahi-1 might take place in the spermatocyte from the early pachytene stage to the late pachytene stage.     

Aquaporin water channels and lung physiology

Fluid transport across epithelial and endothelial barriers occurs in the neonatal and adult lungs. Biophysical measurements in the intact lung and cell isolates have indicated that osmotic water permeability is exceptionally high across alveolar epithelia and endothelia and moderately high across airway epithelia. This review is focused on the role of membrane water-transporting proteins, the aquaporins (AQPs), in high lung water permeability and lung physiology. The lung expresses several AQPs: AQP1 in microvascular endothelia, AQP3 in large airways, AQP4 in large- and small-airway epithelia, and AQP5 in type I alveolar epithelial cells. Lung phenotype analysis of transgenic mice lacking each of these AQPs has been informative. Osmotically driven water permeability between the air space and capillary compartments is reduced approximately 10-fold by deletion of AQP1 or AQP5 and reduced even more by deletion of AQP1 and AQP4 or AQP1 and AQP5 together. AQP1 deletion greatly reduces osmotically driven water transport across alveolar capillaries but has only a minor effect on hydrostatic lung filtration, which primarily involves paracellular water movement. However, despite the major role of AQPs in lung osmotic water permeabilities, AQP deletion has little or no effect on physiologically important lung functions, such as alveolar fluid clearance in adult and neonatal lung, and edema accumulation after lung injury. Although AQPs play a major role in renal and central nervous system physiology, the data to date on AQP knockout mice do not support an important role of high lung water permeabilities or AQPs in lung physiology. However, there remain unresolved questions about possible non-water-transporting roles of AQPs and about the role of AQPs in airway physiology, pleural fluid dynamics, and edema after lung infection.     

冬眠周期长短不同的蒙古黄鼠（Citellus dauricus）...

Adult Mongolian ground squirrels (Citellus dauricus) were kept at 5 degrees C in winter and divided into four experimental groups according to the bout length. The first group was not hibernating until decapitation. The bout length of the second group was between 4-10 days, the third group 11-17 days and the fourth group longer than 20 days. All pineals were sampled at the end of January. Morphometric analytical procedures were used to study the ultrastructure of the distal part of the pineal gland. The statistical results demonstrated that 1) the euthermic animals have larger cross areas of pinealocyte, longer and narrower Golgi apparatus and more number of saccules of each Golgi apparatus (P less than 0.01). But they also have smaller volume density of vaculoes, less lipid droplets and associated vesicles around Golgi apparatus (P less than 0.01). 2) the hibernating animals with variety of bout length had no significant differences in the number of mitochondria, lipid droplets, lysosomes, the size of Golgi apparatus and the cross areas of nucleus and cytoplasm (P greater than 0.05). However, the number and the cross areas of vacuoles were significantly increased with the bout length (P less than 0.01). This might suggest that the bout length was not related to the metabolic activity of pinealocytes in Citellus dauricus and vacuoles might play some important roles in maintenance of individual bout of hibernation in this species.     

Voltage-gated K+ channel KCNQ1 regulates insulin secretion in MIN6 β-cell line

Bone homeostasis is maintained by a dynamic balance between bone resorption by osteoclasts and bone formation by osteoblasts. Since excessive osteoclast activity is implicated in pathological bone resorption, understanding the mechanism underlying osteoclast differentiation, function and survival is of both scientific and clinical importance. Osteoclasts are monocyte/macrophage lineage cells with a short life span that undergo rapid apoptosis, the rate of which critically determines the level of bone resorption in vivo. However, the molecular basis of rapid osteoclast apoptosis remains obscure. Here we report the role of a BH3-only protein, Noxa (encoded by the Pmaip1 gene), in bone homeostasis using Noxa-deficient mice. Among the Bcl-2 family members, Noxa was selectively induced during osteoclastogenesis. Mice lacking Noxa exhibit a severe osteoporotic phenotype due to an increased number of osteoclasts. Noxa deficiency did not have any effect on the number of osteoclast precursor cells or the expression of osteoclast-specific genes, but led to a prolonged survival of osteoclasts. Furthermore, adenovirus-mediated Noxa overexpression remarkably reduced bone loss in a model of inflammation-induced bone destruction. This study reveals Noxa to be a crucial regulator of osteoclast apoptosis, and may provide a molecular basis for a new therapeutic approach to bone diseases.     

绿洲农业生态经济系统的结构与功能分析──以张掖绿洲为例

通过分析张掖绿洲农业生态经济系统的结构与功能,发现其种群结构单一、产业结构不甚合理、空间结构脆弱,决定了绿洲功能较差,产投比低下的特点,并据此从可持续发展的角度提出了改善绿洲农业生态经济系统结构、提高其功能的对策与建议.     

Design and application of highly responsive fluorescence resonance energy transfer biosensors for detection of sugar in living Saccharomyces cerevisiae cells

A protein sensor with a highly responsive fluorescence resonance energy transfer (FRET) signal for sensing sugars in living Saccharomyces cerevisiae cells was developed by combinatorial engineering of the domain linker and the binding protein moiety. Although FRET sensors based on microbial binding proteins have previously been created for visualizing various sugars in vivo, such sensors are limited due to a weak signal intensity and a narrow dynamic range. In the present study, the length and composition of the linker moiety of a FRET-based sensor consisting of CFP-linker(1)-maltose-binding protein-linker(2)-YFP were redesigned, which resulted in a 10-fold-higher signal intensity. Molecular modeling of the composite linker moieties, including the connecting peptide and terminal regions of the flanking proteins, suggested that an ordered helical structure was preferable for tighter coupling of the conformational change of the binding proteins to the FRET response. When the binding site residue Trp62 of the maltose-binding protein was diversified by saturation mutagenesis, the Leu mutant exhibited an increased binding constant (82 microM) accompanied by further improvement in the signal intensity. Finally, the maltose sensor with optimized linkers was redesigned to create a sugar sensor with a new specificity and a wide dynamic range. When the optimized maltose sensors were employed as in vivo sensors, highly responsive FRET images were generated from real-time analysis of maltose uptake of Saccharomyces cerevisiae (baker's yeast).