禽多杀性巴氏杆菌C48-3株ompH基因敲除突变株的构建及其生物学功能

[目的]探讨ompH基因在禽多杀性巴氏杆菌致病过程中的作用.[方法]利用同源重组原理构建中间为四环素抗性基因,两侧为ompH基因上下游同源序列同源的敲除载体pWSK29△ompH,将敲除载体电击转入C48-3株感受态细胞中,通过四环素抗性和菌落PCR筛选ompH基因的敲除突变株,并通过组合PCR、逆转录PCR和DNA测序对突变株进行验证.用生物学功能实验比较野生株、互补株和突变株在生长速率、荚膜结构、粘着能力和致病性等方面的差异.[结果]组合PCR、逆转录PCR和DNA测序结果证实ompH基因的敲除突变株C48-3△ompH构建成功,电镜观察结果证实ompH基因的缺陷影响细菌的荚膜合成能力,粘附实验结果显示与野生株C48-3和互补株C48-3C相比突变株C48-3△ompH对CEF细胞的粘附能力显著降低(P＜0.01),而小鼠毒力实验结果表明突变株C48-3△ompH的致病性相对减弱.[结论]本实验构建的突变株C48-3△ompH,为进一步研究多杀性巴氏杆菌的致病机理奠定基础.     

A bicistronic baculovirus vector for transient and stable protein expression in mammalian cells

Baculoviruses are widely used for protein production in insect cells, and their potential for gene transfer to mammalian cells is increasingly being recognized. Here we describe a baculovirus vector with a bicistronic mammalian expression cassette and demonstrate its suitability for efficient transient and stable protein expression in human glioblastoma cells. Bicistronic baculovirus vectors are safe, cost efficient, and easy to produce; thus, they represent an excellent gene transfer system for mammalian cells.     

The design and synthesis of a new tumor-selective fluoropyrimidine carbamate, capecitabine

To identify an orally available fluoropyrimidine having efficacy and safety profiles greatly improved over those of parenteral 5-fluorouracil (5-FU: 1), we designed a 5-FU prodrug that would pass intact through the intestinal mucisa and be sequentially converted to 5-FU by enzymes that are highly expressed in the human liver and then in tumors. Among various N4-substituted 5'-deoxy-5-fluorocytidine derivatives, a series of N4-alkoxycarbonyl derivatives were hydrolyzed to 5'-deoxy-5-fluorocytidine (5'-DFCR: 8) specifically by carboxylesterase, which exists preferentially in the liver in humans and monkeys. Particularly, derivatives having an N4-alkoxylcarbonyl moiety with a C4-C6 alkyl chain were the most susceptible to the human carboxylesterase. Those were then converted to 5'-deoxy-5-fluorouridine (5'-DFUR: 4) by cytidine deaminase highly expressed in the liver and solid tumors and finally to 5-FU by thymidine phosphorylase (dThdPase) preferentially located in tumors. When administered orally to monkeys, a derivative having the N4-alkoxylcarbonyl moiety with a C5 alkyl chain (capecitabine: 6) The highest AUC and Cmax for plasma 5'-DFUR. In tests with various human cancer xenograft models, capecitabine was more efficacious at wider dose ranges than either 5-FU or 5'-DFUR and was significantly less toxic to the intestinal tract than the others in monkeys.     

Ribose 1,5-bisphosphate is a putative regulator of fructose 6-phosphate/fructose 1,6-bisphosphate cycle in liver

6-Phosphofructo-1-kinase and fructose-1,6-bisphosphatase are rate-limiting enzymes for glycolysis and gluconeogenesis respectively, in the fructose 6-phosphate/fructose 1,6-bisphosphate cycle in the liver. The effect of ribose 1,5-bisphosphate on the enzymes was investigated. Ribose 1,5-bisphosphate synergistically relieved the ATP inhibition and increased the affinity of liver 6-phosphofructo-1-kinase for fructose 6-phosphate in the presence of AMP. Ribose 1,5-bisphosphate synergistically inhibited fructose-1,6-bisphosphatase in the presence of AMP. The activating effect on 6-phosphofructo-1-kinase and the inhibitory effect on fructose-1,6-bisphosphatase suggest ribose 1,5-bisphosphate is a potent regulator of the fructose 6-phosphate/fructose 1,6-bisphosphate cycle in the liver.     

Immunoelectronmicroscopic localization of extracellular matrix components produced by bovine corneal endothelial cells in vitro

Bovine corneal endothelial cells deposit an extracellular matrix in short-term cultures, which contains various morphologically distinct structures when analysed by electron microscopy after negative staining. Amongst these were long-spacing fibers with a 150 nm periodicity, which appeared also to be assembled into more complex hexagonal lattices. Another structure was fine filaments, 10-40 nm in diameter, which occasionally exhibited 67 nm periodic cross-striation. Non-striated 10-20 nm filaments sometimes formed radially oriented bundles arranged in networks and fuzzy granular material was associated with the filaments in the bundles. Often, these bundles extended into solitary filaments, 10-20 nm in diameter, with a smooth surface. In addition, amorphous patches were seen, which contained dense aggregates of fibrillar and granular material. In longer-term cultures, some of the structures coalesced to form large fibrillar bundles. By using specific antibodies to various extracellular matrix components and immunolabeling with gold some of these structures could be identified as to their protein composition. Whereas fibronectin antibodies labeled a variety of structures--fine filaments with granular materials, radially oriented bundles, patchy amorphous aggregates and small granular material scattered throughout the background--type III collagen antibody predominantly labeled filaments with periodic banding (10-40 nm in diameter). A small amount of type III specific labeling was also observed over the networks of radially oriented fibrils and fine filaments associated with granular material. Type IV collagen and laminin antibodies localized in areas of the patchy amorphous aggregates. Type VI collagen antibodies, on the other hand, labeled fine filaments and the gold particles showed a pattern of 100 nm periodicity. Many of the fine 10-20 nm filaments exhibited a tubular appearance on cross-section, but they were not reactive with any of the antibodies used. Also negative were the long-spacing fibers and assemblies--including hexagonal lattices--containing this structural element.     

The action of sphingomyelinase from Bacillus cereus on ATP-depleted bovine erythrocyte membranes and different lipid composition of liposomes

The presence of cholesterol or phosphatidylethanolamine in sphingomyelin liposomes enhanced 2- to 10-fold the breakdown of sphingomyelin by sphingomyelinase from Bacillus cereus. On the other hand, the presence of phosphatidylcholine was either without effect or slightly stimulative at a higher molar ratio of phosphatidylcholine to sphingomyelin (3/1). In the bovine erythrocytes and their ghosts, the increase by 40-50% or the decrease by 10-23% in membranous cholesterol brought about acceleration or deceleration of enzymatic degradation of sphingomyelin by 50 or 40-50%, respectively. The depletion of ATP (less than 0.9 mg ATP/100 ml packed erythrocytes) enhanced K+ leakage from, and hot hemolysis (lysis without cold shock) of, bovine erythrocytes but decelerated the breakdown of sphingomyelin and hot-cold hemolysis (lysis induced by ice-cold shock to sphingomyelinase-treated erythrocytes), either in the presence of 1 mM MgCl2 alone or in the presence of 1 mM MgCl2 and 1 mM CaCl2. Also, ATP depletion enhanced the adsorption of sphingomyelinase onto bovine erythrocyte membranes in the presence of 1 mM CaCl2 up to 81% of total activity, without appreciable K+ leakage and hot or hot-cold hemolysis. These results suggest that the presence of cholesterol or phosphatidylethanolamine in biomembranes makes the membranes more susceptible to the attack of sphingomyelinase from B. cereus and that the segregation of lipids and proteins in the erythrocyte membranes by ATP depletion causes the deceleration of sphingomyelin hydrolysis despite the enhanced enzyme adsorption onto the erythrocyte membranes.     

Occurrence of ommochrome-containing pigment granules in the central nervous system of the silkworm, Bombyx mori

Dark-red pigment granules were found in the brain and ganglion of the normal strain of the silkworm, Bombyx mori, by light microscopy. No other pigmentation was seen in the brain or ganglia. Electron microscopy showed that the granules were electron-dense. The granules were similar to the ommochrome-containing pigment granules that are present in the epidermal cells of the quail mutant, as previously reported. The pigment in the larval central nervous system (CNS) of the normal silkworm was identical to the ommin standard with respect to the absorption spectrum, the infrared spectrum, and the Rf value in thin-layer chromatography (TLC). After acid hydrolysis of the pigment, 3-hydroxykynurenine was detected by TLC. The pigment granules in the CNS contained mainly ommin. An ommochrome-binding protein was also detected in the CNS by in vitro binding studies and Western blotting. The ommochrome granules may have an important function in the CNS of the silkworm.     

Missing piece of top predator-based conservation: Demographic analysis of an owl population on a remote subtropical island

Top predators are frequently the target of conservation programs. Owls are such predators. However, previous studies of owls are biased to species occurring in temperate regions, whereas most owl species occur in tropical or subtropical regions and are understudied. Furthermore, owls are often endemic to islands and of unknown conservation status. Demographic data for such species are especially scarce although they are essential for initiating and promoting their conservation. As a case study of demographic analysis of owls in a tropical or subtropical area and on islands, we applied an integrated population model to 7-year monitoring data (2012–2018) of the Ryukyu Scops Owl population on Minami-daito Island, Japan. We used survival history data from 903 individuals, reproduction and sex ratio data from 213 broods, and count data of 2,526 individuals in total. Long-term averages of annual survival rates were 0.73 for adult females and 0.74 for adult males, although the sexual difference was not significant. Sex ratio estimates fluctuated annually and long-term averages were slightly skewed to males: 0.51 among fledglings, 0.54 among yearlings, and 0.52 among adults. Long-term averages of population size were estimated to be 273.4 females and 296.8 males. The long-term average of population growth rate was 0.98, suggesting a slightly declining trend. It was fortunate to recognize the declining trend during the early phase. Considering the general lack of fundamental ecological data on owls of tropical or subtropical areas and on islands, it seems likely that many endangered owl populations await conservation efforts.