Digital image analysis of cytoskeletal F-actin disintegration in renal microvascular endothelium following ischemia/reperfusion.

BACKGROUND: Damaged and/or dysfunctional microvascular endothelium has been implicated in the pathogenesis of ischemic acute renal failure (ARF). Rapidly occurring changes in the endothelial F-actin cytoskeleton as observed in vitro might be responsible, but have been proven difficult to measure accurately in situ. Therefore, the purpose of this study was to examine several methods of digital image analysis in order to quantify the alterations of endothelial F-actin after renal ischemia and reperfusion (I/R), and to relate these to deterioration of renal function. METHODS: Frozen sections of Sham and I/R rat kidneys were fixed in 4% formaldehyde and stained with rhodamine-phallo?din. Microvascular structures were captured using a 3i Marianastrade mark digital imaging fluorescence microscope workstation. Images were analyzed using 3i SlideBooktrade mark software, employing several masking techniques and line-scans. RESULTS: Digital image analysis demonstrated a decrease in the mean intensity of rhodamine-phallo?din fluorescence after I/R from 1030 +/- 187 to 735 +/- 121 a.u. (arbitrary units, mean +/- SD, n = 7). The number of F-actin fragments per pixel increased from (15.8 +/- 4.9) x 10(-5) to (20.7 +/- 3.5) x 10(-5) (n = 7), indicating cytoskeletal fragmentation. In addition, line-scan analysis demonstrated a disturbed spatial orientation of the F-actin cytoskeleton after I/R. Finally, the loss of F-actin correlated with a rise in plasma creatinine. CONCLUSIONS: The methods of digital image analysis described in the present study demonstrate that renal I/R induces profound changes in the F-actin cytoskeletal structure of microvascular endothelial cells, implicating an injured and dysfunctional microvascular endothelium, which may contribute to acute renal failure (ARF).     

刺桐胰蛋白酶抑制剂亲和填料的制备及纯化瑞替普酶

刺桐属胰蛋白酶抑制剂(ETI)是一种丝氨酸蛋白酶抑制剂,能特异性的抑制胰蛋白酶、组织纤溶酶原激活剂(tPA)、瑞替普酶(rPA)等丝氨酸蛋白酶。实验利用ETI工程菌,经诱导表达、体外复性及纯化获得ETI蛋白,并将该蛋白键合到CNBr活化的琼脂糖凝胶,制备ETI亲和填料,纯化瑞替普酶 。     

Genetic Analysis of a Rat Model of Aerobic Capacity and Metabolic Fitness

Aerobic capacity is a strong predictor of all-cause mortality and can influence many complex traits. To explore the biological basis underlying this connection, we developed via artificial selection two rat lines that diverge for intrinsic (i.e. inborn) aerobic capacity and differ in risk for complex disease traits. Here we conduct the first in-depth pedigree and molecular genetic analysis of these lines, the high capacity runners (HCR) and low capacity runners (LCR). Our results show that both HCR and LCR lines maintain considerable narrow-sense heritability (h²) for the running capacity phenotype over 28 generations (h² = 0.47 ± 0.02 and 0.43 ± 0.02, respectively). To minimize inbreeding, the lines were maintained by rotational mating. Pedigree records predict that the inbreeding coefficient increases at a rate of <1% per generation, ~37-38% slower than expected for random mating. Genome-wide 10K SNP genotype data for generations 5, 14, and 26 demonstrate substantial genomic evolution: between-line differentiation increased progressively, while within-line diversity deceased. Genome-wide average heterozygosity decreased at a rate of <1% per generation, consistent with pedigree-based predictions and confirming the effectiveness of rotational breeding. Linkage disequilibrium index r² decreases to 0.3 at ~3 Mb, suggesting that the resolution for mapping quantitative trait loci (QTL) can be as high as 2-3 cM. To establish a test population for QTL mapping, we conducted an HCR-LCR intercross. Running capacity of the F1 population (n=176) was intermediate of the HCR and LCR parentals (28 pairs); and the F2 population (n=645) showed a wider range of phenotypic distribution. Importantly, heritability in the F0-F2 pedigree remained high (h²~0.6). These results suggest that the HCR-LCR lines can serve as a valuable system for studying genomic evolution, and a powerful resource for mapping QTL for a host of characters relevant to human health.     

Characterization of novel linuron-mineralizing bacterial consortia enriched from long-term linuron-treated agricultural soils

Linuron-mineralizing cultures were enriched from two linuron-treated agricultural soils in the presence and absence of a solid support. The cultures contained linuron-degrading bacteria, which coexisted with bacteria degrading either 3,4-dichloroaniline (3,4-DCA) or N,O-dimethylhydroxylamine (N,O-DMHA), two common metabolites in the linuron degradation pathway. For one soil, the presence of a solid support enriched for linuron-degrading strains phylogenetically related to but different from those enriched without support. Most linuron-degrading consortium members were identified as Variovorax, but a Hydrogenophaga and an Achromobacter strain capable of linuron degradation were also obtained. Several of the linuron-degrading isolates also degraded 3,4-DCA. Isolates that degraded 3,4-DCA but not linuron belonged to the genera Variovorax, Cupriavidus and Afipia. Hyphomicrobium spp. were involved in the metabolism of N,O-DMHA. Whereas several isolates degraded linuron independently, more efficient degradation was achieved by combining linuron and 3,4-DCA-degraders or by adding casamino acids. These data suggest that (1) linuron degradation is performed by a group of metabolically interacting bacteria rather than by individual strains, (2) there are other genera in addition to Variovorax that degrade linuron beyond 3,4-DCA, (3) linuron-degrading consortia of different origins have a similar composition, and (4) interactions between consortium members can be complex and can involve exchange of both metabolites and other nutrients.     

Microbial community of granules in expanded granular sludge bed reactor for simultaneous biological removal of sulfate, nitrate and lactate

This study studied the cultivation of granules from an expanded granular sludge bed reactor that simultaneously transforms sulfates, nitrates, and oxygen to elementary sulfur, nitrogen gas, and carbon dioxides, respectively. The living cells accumulate at the granule outer layers, as revealed by the multicolor staining and confocal laser scanning microscope technique. The microbial community comprises sulfate-reducing bacteria (SRB, Desulfomicrobium sp.), heterotrophic (Pseudomonas aeruginosa and Sulfurospirillum sp.), and autotrophic denitrifiers (Sulfurovum sp. and Paracoccus denitrificans) whose population dynamics at different sulfate and nitrate loading rates are monitored with the single-strand conformation polymorphism and denaturing gradient gel electrophoresis technique. The Desulfomicrobium sp. presents one of the dominating strains following reactor startup. At high sulfate and nitrate loading rates, the heterotrophic denitrifiers overcompete autotrophic denitrifiers to reduce SRB activities. Conversely, suddenly reducing nitrate loading rates completely removes the heterotrophic denitrifier Sulfurospirillum sp. from the granules and activates the autotrophic denitrifiers. The physical fixation of different groups of functional strains in granules fine-tunes the strains' activities, and hence the reactor performance.     

Diversity and virulence of soil-dwelling fungi Isaria spp. and Paecilomyces spp. against Solenopsis invicta (Hymenoptera: Formicidae)

To comprehend the diversity and potential control of soil-dwelling fungi, Isaria and Paecilomyces, against the red imported fire ant Solenopsis invicta (Hymenoptera, Formicidae), an investigation was carried out between 2004 and 2008. From 258 soil samples collected from 16 central and southern provinces and cities in China, a total of 171 isolates of the genra Isaria and Paecilomyce were isolated, and the species I. javanicus, P. marquandii and I. fumosoroseus were found more abundant than I. cateniobliquus, P. carneus, P. inflatus and P. lilacinus. Geographic differences of isolating rates were observed as well. Samples from the southern areas had higher fungal isolating rates than those from the central areas. Subsequently, 47 isolates were further tested for pathogencity against the red imported fire ant. All isolates except P115 showed certain pathogenic potential (the mean is 52.3% at 4000 conidiospores/mL) to the ant. I. javanicus was the most effective species with a mean pathogenicity of 80.6%, while pathogenicities of P. marquandii, P. gunni and I. fumosoroseus were 44, 21 and 49%, respectively. Furthermore, the more effective isolates P028 of I. javanicus and P003 of I. fumosoroseus were tested in a virulence experiment. The LD₅₀ values of P028 and P003 against major and miner workers were determined as 412,280 and 854,451 conidiospores/cm², respectively. Meanwhile, the LT₅₀ values at 1000 conidiospores/cm² were 7.1 and 6.6 d in isolate P003 and 6.8 and 6.6 d in isolate P028.     

硒对培养人胚肝细胞Ⅲ型前胶原,羟脯氨酸合成的影响

原代培养人胚肝细胞经１．１５６×１０￣(－７）ｍｏｌ／Ｌ硒预处理４ｈ,加入２０ｍｍｏｌ／Ｌ四氟化碳作用２０ｈ,观察硒对其Ⅲ型前胶原（ＰＣⅢ）和羟脯氨酸（Ｈｙｐ）生成的影响。结果培养液中ＰＣⅢ水平、细胞内Ｈｙｐ含量及细胞内外丙二醛（ＭＤＡ）水平均降低,与未加硒对照组比较差别有显著性（Ｐ＜０．０１）。而硒谷腕甘肽过氧化物酶（Ｓｅ－ＧＳＨ－ＰＸ）活性则较对照组显著增高（Ｐ＜０．００１）,且ＰＣⅢ水平与Ｓｅ－ＧＳＨ－Ｐ＿Ｘ／ＭＤＡ比值呈负相关（ｒ＝－０．９１５６,Ｐ＜０．０１）。提示硒可提高Ｓｅ－ＧＳＨ－Ｐ＿Ｘ／ＭＤＡ比值,抑制脂质过氧化激发的肝细胞胶原合成。     

表达O型口蹄疫病毒VPl基因的重组病毒BHV-1的构建与鉴定

[目的]为了构建表达口蹄疫病毒(O/china/99)VP1基因的牛疱疹病毒1型,将人工合成的口蹄疫病毒VP1基因插入到巨细胞病毒(CMV)启动子之下构建gE基因缺失转移载体.[方法]利用磷酸钙介导转染法将该转移载体与亲本病毒BHV-1/gE-/LacZ+的基因组DNA共转染牛鼻甲细胞后收获增殖的病毒.通过筛选白色病毒蚀斑,得到重组病毒BHV-1/gE-/VP1.[结果]PCR检测结果表明VP1基因已经插入到了重组病毒BHV-1/gE-的基因组中,间接免疫荧光试验和Westem blot证实了BHV-1/gE-/VP1中的VP1基因在感染的细胞中获得了表达.[结论]本研究成功地构建了表达口蹄疫病毒VP1基因的重组病毒BHV-1/gE-/VP1,为研制口蹄疫及其他重要牛传染病的BHV-1病毒载体疫苗奠定了基础.     

Difference in antifungal activity of morphotypes of clavicipitaceous endophytes within and between species

In a previous study, a total of 484 endophytic fungi were isolated and purified from seven populations of Achnatherum sibiricum (L.) Keng collected at six geographical locations in Inner Mongolia, China. Based on growth rates as well as morphological characteristics, the isolates were classified into five morphotypes. Among them, morphotypes A, B and C were ascribed to the same species, Neotyphodium chisosum, based on ITS sequences. Morphotype E was identified as Epichloë amarillans. In the present study, four morphotypes, A, B, C and E, belonging to two species, were chosen for an in vitro pathogen trial. The results showed that both endophyte colonies and endophyte filtrate of all morphotypes could inhibit the mycelia growth and spore germination of the pathogen fungi tested. The magnitude of inhibition varied not only between species, but also among morphotypes of the same species. Overall, the antifungal ability of E. amarillans (morphotype E) was higher than that of N. chisosum. Within N. chisosum, the antifungal ability was highest in morphotype C, followed by morphotype A, and lowest in morphotype B. This variability suggests that different morphotypes might represent different genotypes of endophyte. The effect of endophyte infection on the host grass should be examined not only on the species level but also on the morphotype level to determine the possible interactions.