A practical synthesis of beta-D-GlcA-(1-->3)-beta-D-Gal-(1-->3)-beta-D-Gal-(1-->4)-D-Xyl,a part of the common linkage region of a glycosaminoglycan

A practical synthesis of beta-D-GlcA-(1-->3)-beta-D-Gal-(1-->3)-beta-D-Gal-(1-->4)-beta-D-Xyl-(1-->OMe) was achieved by coupling of methyl 2,3,4-tri-O-acetyl-alpha-D-glucopyranosyluronate trichloroacetimidate with a trisaccharide acceptor. The trisaccharide acceptor was obtained by condensation of 3-O-allyl-2,4,6-tri-O-benzoyl-beta-D-galactopyranosyl-(1-->3)-2,4,6-tri-O-benzoyl-alpha-D-galactopyranosyl trichloroacetimidate with methyl 2,3-di-O-benzoyl-beta-D-xylopyranoside, followed by deallylation. The beta-(1-->3)-linked disaccharide was prepared readily with p-methoxyphenyl 3-O-allyl-2,4,6-tri-O-benzoyl-beta-D-galactopyranoside as the key synthon. The alpha-(1-->3)-linkage was formed in considerable amount with galactose mono- and disaccharide trichloroacetimidate donors with C-2 neighboring group participation.     

Cytopathological evaluations combined RNA and protein analyses on defined cell regions using single frozen tissue block

The co-existence of multiple cell components in tissue samples is the main obstacle for precise molecular evaluation on defined cell types. Based on morphological examination, we developed an efficient approach for paralleled RNA and protein isolations from an identical histological region in frozen tissue section. The RNA and protein samples prepared were sufficient for RT-PCR and Western blot analyses, and the results obtained were well coincident each other as well as with the corresponding parameters revealed from immunohistochemical examinations. By this way, the sampling problem caused by cell-cross contamination can be largely avoided, committing the experimental data more specific to a denned cell type. These novel methods thus allow us to use single tissue block for a comprehensive study by integration of conventional cytological evaluations with nucleic acid and protein analyses.     

Influence of inoculum preparation on the growth of Penicillium chrysogenum

AIMS: The influence of the spore preparation on subsequent fungal growth of Penicillium chrysogenum was assessed. METHODS AND RESULTS: The influence of four factors [the nature of the diluting solution (physiological water and physiological water added with Tween-80), the age of the sporulating culture (4, 8 and 12 days), the strain (737, 738 and 740) and the inoculum size (102, 103, 104 and 105 spores ml(-1)] on two responses (i.e. the radial growth rate, mu, and the lag time, lambda) was studied using an experimental screening methodology. CONCLUSIONS: The main conclusion was the strong effect of the inoculum size on lambda. In contrast, the diluting solution had no effect on both the experimental responses. In order to obtain the highest growth rates, it is recommended to use 4-day-old sporulating cultures with an inoculum size of 102 spores ml(-1). SIGNIFICANCE AND IMPACT OF THE STUDY: There is a need for standardizing spore preparation in predictive mycology. The screening methodology is a powerful tool to determine the influence of qualitative and quantitative factors on various biological responses and can be applied widely in microbiology.     

Localization of sucrose synthase and callose in freeze-substituted secondary-wall-stage cotton fibers

Summary.  Methods for cryogenic fixation, freeze substitution, and embedding were developed to preserve the cellular structure and protein localization of secondary-wall-stage cotton (Gossypium hirsutum L.) fibers accurately for the first time. Perturbation by specimen handling was minimized by freezing fibers still attached to a seed fragment within 2 min after removal of seeds from a boll still attached to the plant. These methods revealed native ultrastructure, including numerous active Golgi bodies, multivesicular bodies, and proplastids. Immunolocalization in the context of accurate structure was accomplished after freeze substitution in acetone only. Quantitation of immunolabeling identified sucrose synthase both near the cortical microtubules and plasma membrane and in a proximal exoplasmic zone about 0.2 μm thick. Immunolabeling also showed that callose (β-1,3-glucan) was codistributed with sucrose synthase within this exoplasmic zone. Similar results were obtained from cultured cotton fibers. The distribution of sucrose synthase is consistent with its having a dual role in cellulose and callose synthesis in secondary-wall-stage cotton fibers. Received August 19, 2002; accepted November 12, 2002; published online June 13, 2003 RID="*" ID="*" Correspondence and reprints: Department of Biological Sciences, Texas Tech University, Lubbock, TX 79409-3131, U.S.A. E-mail: candace.haigler@ttu.edu     

Change in substrate metabolism in isolated mouse hearts following ischemia-reperfusion

Several genetic and transgenic mouse models are currently being used for studying the regulation of myocardial contractility under normal conditions and in disease states. Little information has been provided, however, about myocardial energy metabolism in mouse hearts. We measured glycolysis, glucose oxidation and palmitate oxidation (using ³H-glucose, ¹⁴C-glucose and ³H-palmitate) in isolated working mouse hearts during normoxic conditions (control group) and following a 15 min global no-flow ischemic period (reperfusion group). Fifty min following reperfusion (10 min Langendorff perfusion + 40 min working heart perfusion) aortic flow, coronary flow, cardiac output, peak systolic pressure and heart rate were 44 ± 4, 88 ± 4, 57 ± 4, 94 ± 2 and 81 ± 4% of pre-ischemic values. Rates of glycolysis and glucose oxidation in the reperfusion group (13.6 ± 0.8 and 2.8 ± 0.2 mol/min/g dry wt) were not different from the control group (12.3 ± 0.6 and 2.5 ± 0.2 mol/min/g dry wt). Palmitate oxidation, however, was markedly elevated in the reperfusion group as compared to the control group (576 ± 37 vs. 357 ± 21 nmol/min/g dry wt, p < 0.05). This change in myocardial substrate utilization was accompanied by a marked fall in cardiac efficiency measured as cardiac output/oxidative ATP production (136 ± 10 vs. 54 ± 5 ml/mol ATP, p < 0.05, control and reperfusion group, respectively). We conclude that ischemia-reperfusion in isolated working mouse hearts is associated with a shift in myocardial substrate utilization in favour of fatty acids, in line with previous observations in rat.     

大熊猫气味标记DNA的制备和序列分析

大熊猫气味标记在其个体间的通讯中具有重要意义。用不同方法收集了７只大熊猫个体的９个气味标记样品,运用Ｉｎｓｔａｇｅｎｅ Ｋｉｔ制备出了ＤＮＡ。采用ＰＣＲ扩增线粒体Ｄ－环区和细胞色素ｂ基因、Ｔｈｒ－ｔＲＮＡ基因片段并作序列分析。结果提示,不同收集方式所得气味标记样品均有ＤＮＡ,但用干棉花收集样品的方法最佳。该方法为大熊猫的遗传多样性研究提供了新的简捷有效的ＤＮＡ来源。     

A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high quality plant chromosome spreads is challenging due to the rigid cell wall. One of the approved methods for the preparation of plant chromosomes is a so-called drop preparation, also known as drop-spreading or air-drying technique. Here, we present a protocol for the fast preparation of mitotic chromosome spreads suitable for the FISH detection of single and high copy DNA probes. This method is an improved variant of the air-dry drop method performed under a relative humidity of 50%-55%. This protocol comprises a reduced number of washing steps making its application easy, efficient and reproducible. Obvious benefits of this approach are well-spread, undamaged and numerous metaphase chromosomes serving as a perfect prerequisite for successful FISH analysis. Using this protocol we obtained high-quality chromosome spreads and reproducible FISH results for Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum and Secale cereale.     

蛋白质胶内酶切技术研究进展

胶内酶切是蛋白质组研究中衔接电泳分离和质谱鉴定的重要环节,对最终的蛋白质定性和定量分析结果有显著的影响。该技术自1992年初步建立以来,一直处于不断完善中,出现了种类繁多的改进方案。为了更有效地利用胶内酶切技术,从凝胶脱色、杂质去除、蛋白酶切、肽段提取4个方面归纳整理了近年来蛋白质胶内酶切技术的主要研究进展。     

深海白色侧齿霉遗传转化体系建立及渗透压调控相关的EaSHO1基因功能

海洋中具有丰富的动植物及微生物资源,海洋真菌是其重要组成之一.我们前期的研究发现一株深海真菌白色侧齿霉Engyodontium album能产生具有抑菌活性的次级代谢产物engyodontiuminA,该化合物能抑制黑曲霉、金黄色葡萄球菌及创伤弧菌等病原菌的生长,是一种潜在的海洋源抗菌药物.目前,该菌遗传转化体系尚未建...     

2‐Picoline‐borane: A non‐toxic reducing agent for oligosaccharide labeling by reductive amination

Analysis of N‐glycans is often performed by LC coupled to fluorescence detection. The N‐glycans are usually labeled by reductive amination with a fluorophore containing a primary amine to allow fluorescence detection. Moreover, many of the commonly applied labels also allow improved mass spectrometric detection of oligosaccharides. For reductive amination, the amine group of the label reacts with the reducing‐end aldehyde group of the oligosaccharide to form a Schiff base, which is reduced to a secondary amine. Here, we propose the use of 2‐picoline‐borane as the reducing agent, as a non‐toxic alternative to the extensively used, but toxic sodium cyanoborohydride. Using dextran oligosaccharides and plasma N‐glycans, we demonstrate similar labeling efficacies for 2‐picoline‐borane and sodium cyanoborohydride. Therefore, 2‐picoline‐borane is a non‐toxic alternative to sodium cyanoborohydride for the labeling of oligosaccharides.