Production of aflatoxin on rice

A method has been developed for the production of aflatoxin by growing Aspergillus flavus strain NRRL 2999 on the solid substrate rice. Optimal yields, more than 1 mg of aflatoxin B₁ per g of starting material, were obtained in 5 days at 28 C. A crude product containing aflatoxins was isolated by chloroform extraction and precipitation with hexane from concentrated solutions. The crude product consisted of 50% aflatoxin in the following ratio: B₁-B₂-G₁-G₂, 100:0.15:0.22:0.02. Aflatoxin B₁ was separated from almost all the impurities and from the other aflatoxins by chromatography on silica gel with 1% ethyl alcohol in chloroform. Analytically pure aflatoxin B₁ was recrystallized from chloroform-hexane mixtures.     

鼠李糖乳杆菌LV108及其发酵乳免疫调节作用的比较

目的探讨鼠李糖乳杆菌LV108及其发酵乳对免疫抑制小鼠免疫功能的调节作用。方法将BALB/c小鼠随机分为5组,每组10只,即空白组(正常小鼠)、模型组(免疫抑制小鼠)、药物组(免疫抑制小鼠食物中添加左旋咪唑)、LV108菌悬液组(免疫抑制小鼠食物中添加LV108菌悬液)和LV108发酵乳组(免疫抑制小鼠食物中添加LV108发酵乳),除空白组外其余组构建免疫抑制小鼠模型。干预4周后,分别测定各组小鼠体质量和脏器指数,血清中白细胞介素2(IL2)、肿瘤坏死因子α(TNFα)和免疫球蛋白G(IgG)含量,血清溶血素含量、耳肿胀度和肝、脾巨噬细胞吞噬能力。结果相比模型组,LV108菌悬液组和LV108发酵乳组小鼠体质量增长速度、脏器指数、血清IL2与IgG水平、血清溶血值、耳肿胀度和巨噬细胞吞噬能力显著升高(均P<0.05);在脾脏指数、血清IL2与TNFα水平、血清溶血素含量和耳肿胀度免疫指标上,LV108菌悬液组与LV108发酵乳组之间比较差异具有统计学意义(均P<0.05)。结论LV108菌体及发酵乳对免疫抑制小鼠具备较全面的免疫调节作用,均可提高小鼠的自身免疫力;LV108发酵乳对小鼠的免疫调节作用强于LV108菌体。     

Phosphomannomutase activity in congenital disorders of glycosylation type Ia determined by direct analysis of the interconversion of mannose-1-phosphate to mannose-6-phosphate by high-pH anion-exchange chromatography with pulsed amperometric detection

Congenital disorders of glycosylation (CDG) are a group of multisystemic disorders resulting from defects in the synthesis and processing of N-linked oligosaccharides. The most common form, CDG type Ia (CDG-Ia), results from a deficiency of the enzyme phosphomannomutase (PMM). PMM converts mannose 6-phosphate (man-6-P) to mannose-1-phosphate (man-1-P), which is required for the synthesis of GDP-mannose, a substrate for dolichol-linked oligosaccharide synthesis. The traditional assay for PMM, a coupled enzyme system based on the reduction of NADP(+) to NADPH using man-1-P as a substrate, has limitations in accuracy and reproducibility. Therefore, a more sensitive, direct test for PMM activity, based on the detection of the conversion of man-1-P to man-6-P by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), was developed. Using this assay, the activity of PMM was markedly deficient in fibroblasts and lymphoblasts from 23 patients with CDG-Ia (range 0-15.3% of control, average 4.9+/-4.7%) and also decreased in seven obligate heterozygotes (range 33.0-72.0% of control, average 52.2+/-14.7%). Unlike the spectrophotometric method, there was no overlap in PMM activity among patients, obligate heterozygotes, or controls. Thus, the PMM assay based on HPAEC-PAD has increased utility in the clinical setting, and can be used, together with transferrin isoelectric focusing, to diagnose patients with CDG-Ia and to identify heterozygotes when clinically indicated.     

Reciprocal and nonreciprocal recombination at the glucocerebrosidase gene region: implications for complexity in Gaucher disease

Gaucher disease results from an autosomal recessive deficiency of the lysosomal enzyme glucocerebrosidase. The glucocerebrosidase gene is located in a gene-rich region of 1q21 that contains six genes and two pseudogenes within 75 kb. The presence of contiguous, highly homologous pseudogenes for both glucocerebrosidase and metaxin at the locus increases the likelihood of DNA rearrangements in this region. These recombinations can complicate genotyping in patients with Gaucher disease and contribute to the difficulty in interpreting genotype-phenotype correlations in this disorder. In the present study, DNA samples from 240 patients with Gaucher disease were examined using several complementary approaches to identify and characterize recombinant alleles, including direct sequencing, long-template polymerase chain reaction, polymorphic microsatellite repeats, and Southern blots. Among the 480 alleles studied, 59 recombinant alleles were identified, including 34 gene conversions, 18 fusions, and 7 downstream duplications. Twenty-two percent of the patients evaluated had at least one recombinant allele. Twenty-six recombinant alleles were found among 310 alleles from patients with type 1 disease, 18 among 74 alleles from patients with type 2 disease, and 15 among 96 alleles from patients with type 3 disease. Several patients carried two recombinations or mutations on the same allele. Generally, alleles resulting from nonreciprocal recombination (gene conversion) could be distinguished from those arising by reciprocal recombination (crossover and exchange), and the length of the converted sequence was determined. Homozygosity for a recombinant allele was associated with early lethality. Ten different sites of crossover and a shared pentamer motif sequence (CACCA) that could be a hotspot for recombination were identified. These findings contribute to a better understanding of genotype-phenotype relationships in Gaucher disease and may provide insights into the mechanisms of DNA rearrangement in other disorders.     

Comparison of Isoflurane and α-Chloralose in an Anesthetized Swine Model of Acute Pulmonary Embolism Producing Right Ventricular Dysfunction

Pulmonary embolism (PE) is a leading cause of sudden cardiac death, and a model is needed for testing potential treatments. In developing a model, we compared the hemodynamic effects of isoflurane and α-chloralose in an acute swine model of PE because the choice of anesthesia will likely affect the cardiovascular responses of an animal to PE. At baseline, swine that received α-chloralose (n = 6) had a lower heart rate and cardiac output and higher SpO_2, end-tidal CO_2, and mean arterial pressure than did those given isoflurane (n = 9). After PE induction, swine given α-chloralose compared with isoflurane exhibited a lower heart rate (63 ± 10 compared with 116 ± 15 bpm) and peripheral arterial pressure (52 ± 12 compared with 61 ± 12 mm Hg); higher SpO₂ (98% ± 3% compared with 95% ± 1%), end-tidal CO₂ (35 ± 4 compared with 32 ± 5), and systolic blood pressure (121 ± 8 compared with 104 ± 20 mm Hg); and equivalent right ventricular:left ventricular ratios (1.32 ± 0.50 compared with 1.23 ± 0.19) and troponin I mean values (0.09 ± 0.07 ng/mL compared with 0.09 ± 0.06 ng/mL). Isoflurane was associated with widely variable fibrinogen and activated partial thromboplastin time. Intraexperiment mortality was 0 of 6 animals for α-chloralose and 2 of 9 swine for isoflurane. All swine anesthetized with α-chloralose survived with sustained pulmonary hypertension, RV-dilation-associated cardiac injury without the confounding vasodilatory or coagulatory effects of isoflurane. These data demonstrate the physiologic advantages of α-chloralose over isoflurane for anesthesia in a swine model of severe submassive PE.Abbreviations: LV, left ventricle; PAP, pulmonary arterial pressure; PE, pulmonary embolism; RV, right ventriclePulmonary embolism (PE) is one of the leading causes of noncardiac sudden death in Western nations and is the third most common cause of cardiovascular morbidity.^4,6,7,18 In survivors, severe PE damages the right heart, leading to a clinical course complicated by hypotension and circulatory shock, suggesting acute right heart failure in about 10% of patients and followed by persistent pulmonary hypertension or right ventricular dysfunction and dyspnea in at least 15% of patients.^{9,15,16,23,29} To test treatments to reduce right heart failure, a standardized model that is repeatable, accurate, and precise and that mimics the gross pathologic, cardiovascular, pulmonary, autonomic, hematologic, biochemical, and cellular characteristics of PE in humans with disease is needed.⁸Three lines of rationale favor domestic pigs as a model for PE. Several publications, using different methods of anesthesia, have found that swine manifest hemodynamic responses similar to those of humans in the presence of autologous PE, including elevated heart rate, decreased cardiac output, and reduced oxygen saturation.^2,12,30 Swine have similar platelet concentrations, and their coagulation profile on thromboelastography has been shown to be similar to humans, with the exception of higher fibrin crosslinking but less fibrin, leading to resistance to plasmin.^5,11,19,34 Market swine, which would otherwise be destined for slaughter, are relatively cost effective compared with other large animals and are of sufficient size for placement of an adult pulmonary arterial catheter for measurement of pulmonary vascular resistance in a closed-chest preparation.In view of the differences in the hemodynamic effects of different anesthetic agents, the choice of anesthesia will likely affect the cardiovascular responses of an animal to PE. However, current literature lacks a methodologic publication that compares the cardiovascular, right ventricular, pulmonary, and hematologic responses to PE in closed-chest swine models incorporating different anesthetic regimens.Figure 1 presents features of an ideal animal model for the purpose of testing treatments for PE. To develop a swine model of PE that closely resembles this physiologic ideal model, we induced PE in swine maintained in a surgical plane of anesthesia with either isoflurane or α-chloralose. Each of these agents has potential advantages and disadvantages. Isoflurane can be titrated minute by minute but causes undesirable vasodilation, whereas α-chloralose is believed to preserve cardiovascular reflexes but requires heating to dissolve and continuous infusion or repeated boluses.^26,35 We hypothesized that, compared with isoflurane, α-chloralose would meet more of the features described in Figure 1.Open in a separate windowFigure 1.Desirable features of large animal model of severe submassive PE designed for translational research.     

中国环境管理分区:方法与方案

我国生态环境可持续性及其影响因素的区域差异显著,各地区环境管理面临的主要挑战和需要优先解决的生态环境问题不同。进行环境管理分区,根据各地区生态环境特征及其影响因素的差异性,制定有针对性的环境管理政策,将有效促进我国区域生态环境的整体优化。采取定性和定量分析相结合的方法进行我国环境管理分区。首先,在我国3大自然区的基础上,根据我国的自然地理格局和已有的相关区划成果,把我国划分为4个环境管理大区,包括:南部季风区、北部季风区、西北干旱区和青藏高寒区。其次,通过建立的包含13个指标的环境管理分区指标体系,采用一维化欧式距离法分析各环境管理大区下相邻省级行政区环境特征的相似性,把环境特征相似性大的相邻地区划分到同一分区,得到以省级行政区为基本单元的我国环境管理分区方案。然后,结合地区间历史渊源和区域未来发展趋势分析,对基于相似性分析的初步分区方案进行调整,把我国划分为8个以省级行政区为基本单元环境管理区。最后,根据相关调整原则和方法,对以省级行政区为基本单元的分区方案的边界线进行调整,得到以地级行政区为基本单元的分区方案,把我国划分为东北地区、华北平原区、华北山地与高原区、东南沿海地区、长江流域中游地区、西南地区、西北干旱区和青藏高寒区8个环境管理区。     

Interactions of the Human LIP5 Regulatory Protein with Endosomal Sorting Complexes Required for Transport

The endosomal sorting complex required for transport (ESCRT) pathway remodels membranes during multivesicular body biogenesis, the abscission stage of cytokinesis, and enveloped virus budding. The ESCRT-III and VPS4 ATPase complexes catalyze the membrane fission events associated with these processes, and the LIP5 protein helps regulate their interactions by binding directly to a subset of ESCRT-III proteins and to VPS4. We have investigated the biochemical and structural basis for different LIP5-ligand interactions and show that the first microtubule-interacting and trafficking (MIT) module of the tandem LIP5 MIT domain binds CHMP1B (and other ESCRT-III proteins) through canonical type 1 MIT-interacting motif (MIM1) interactions. In contrast, the second LIP5 MIT module binds with unusually high affinity to a novel MIM element within the ESCRT-III protein CHMP5. A solution structure of the relevant LIP5-CHMP5 complex reveals that CHMP5 helices 5 and 6 and adjacent linkers form an amphipathic “leucine collar” that wraps almost completely around the second LIP5 MIT module but makes only limited contacts with the first MIT module. LIP5 binds MIM1-containing ESCRT-III proteins and CHMP5 and VPS4 ligands independently in vitro, but these interactions are coupled within cells because formation of stable VPS4 complexes with both LIP5 and CHMP5 requires LIP5 to bind both a MIM1-containing ESCRT-III protein and CHMP5. Our studies thus reveal how the tandem MIT domain of LIP5 binds different types of ESCRT-III proteins, promoting assembly of active VPS4 enzymes on the polymeric ESCRT-III substrate.