槐种子发育中胚乳细胞半乳甘露聚糖积累的研究

槐 ( Sophora japonica L.)开花约 60 d至种子成熟 ,为胚乳半乳甘露聚糖积累期。用组织化学方法 ,对储藏于胚乳细胞壁上的半乳甘露聚糖的形成积累进行了观察 ,结果表明 ,半乳甘露聚糖最先在邻近胚的胚乳细胞的粗面内质网的囊泡腔内形成 ,并通过细胞质膜分泌至细胞壁周围。此后 ,半乳甘露聚糖的积累逐渐向种皮方向扩展 ,及至种子成熟时 ,除糊粉层外 ,所有胚乳细胞几乎全由多糖所填充。此外 ,对半乳甘露聚糖发生部位及其积累过程的消长变化进行了讨论     

Saponin-like substances inhibit α-galactosidase production in the endosperm of fenugreek seeds

When fenugreek (Trigonella foenum-graecum L.) endosperms plus testa (endosperms), which had been isolated from 5-h-imbibed seeds, were incubated for at least 2 h under germination conditions, they leaked substances which, like exogenous abscisic acid (ABA), inhibited the production of fenugreek endosperm -galactosidase. However, unlike ABA, 8 h treatment with these inhibitors had no effect on fenugreek endosperms which had been isolated from 15-h-imbibed seeds and leached for 2 h. This indicated that either their inhibitory action was on processes which were related to the production of -galactosidase and had been completed by this time, or that there might be factors present which inactivate these inhibitors. It was also concluded that the action of the endosperm leachate could not be attributed to the presence of ABA. The activity of the leachate decreased when it originated from endosperms imbibed for periods longer than 25 h and thin-layer chromatography (TLC) of extracts from these endosperms showed decreased contents of the leachable inhibitors as imbibition proceeded. From the seed leachate, which had a TLC pattern and inhibitory action similar to that of the endosperm, were isolated three substances which, when applied to endosperms, inhibited the production of -galactosidase activity. According to their chromatographic behaviour and their reaction with specific reagents, there are strong indications that these substances are saponins. These diffusible saponin-like substances were located in both endosperm and perisperm and their physiological role is discussed.Abbreviations ABA abscisic acid - PEG polyethylenglycol - TLC thin-layer chromatography We wish to thank the Alexander S. Onasis Public Benefit Foundation for a grant to K.Z. and Dr. J.S.G. Reid (University of Stirling, Scotland) for a kind gift of fenugreek seeds.     

Studies on the galactomannan-degrading enzymes produced bySporotrichum cellulophilum

Summary Extracellular mannanase activity produced bySporotrichum cellulophilum was purified into two components using acetone precipitation, SP-Sephadex C50 ion exchange chromatography and preparative polyacrylamide gel electrophoresis. The purified mannanse components, M1 and M2, had molecular weights of 108 000–112 000 and 32 200–36 000 respectively. Component M1 was shown to contain 2 subunits having molecular weights of 62 000 and 50 000. M1 and M2 had similar pH-activity profiles with pH optima of 5.5 and 6.0 respectively. M1 was more thermostable than M2: half lives of the enzymes at 70°C were 30 and 9 min for M1 and M2 respectively.     

曲霉菌半乳甘露聚糖单克隆抗体的制备及初步应用

目的:制备抗曲霉菌半乳甘露聚糖的单克隆抗体,并基于获得的抗体建立用于快速准确检测曲霉菌感染的双抗体夹心酶联免疫吸附法(ELISA),以期可用于侵袭性曲霉菌病的临床诊断。方法:提取曲霉菌半乳甘露聚糖后免疫BALB/c小鼠,筛选与制备抗曲霉菌半乳甘露聚糖的单克隆抗体,通过间接ELISA法与Western Blot方法开展单克隆抗体检测性能分析,使用获得的单克隆抗体建立双抗体夹心ELISA方法,并初步应用于曲霉菌感染血清检测。结果:获得抗曲霉菌半乳甘露聚糖单克隆抗体5株,均可特异性识别曲霉菌半乳甘露聚糖,以其中性能最佳的3C9抗体和辣根过氧化物酶标记的3C9抗体配对为基础,建立了双抗体夹心ELISA方法,通过初步评价确定该方法可应用于临床侵袭性曲霉菌病血清检测,并且该方法与现有商品化试剂盒相比检测背景值较低,可更有效区分曲霉菌感染阴阳性血清。结论:本研究筛选获得针对曲霉菌半乳甘露聚糖的特异性单克隆抗体,以该抗体为基础建立双抗体夹心ELISA方法具有潜在转化应用前景,可为侵袭性曲霉菌病的临床诊断提供支持。     

侵袭性真菌深部感染的早期实验室诊断

目的探讨假丝酵母菌甘露聚糖抗原和假丝酵母菌IgG/IgM抗体、曲霉半乳甘露聚糖抗原和烟曲霉IgG抗体在侵袭性真菌病早期临床诊断中的应用价值。方法收集已确诊侵袭性假丝酵母菌病患者18例,侵袭性烟曲霉病患者6例,单纯细菌感染患者20例,浅部真菌感染患者20例,健康体检者(正常对照组)20例,通过酶联免疫吸附法(ELISA)检测患者血清甘露聚糖和假丝酵母菌IgG/IgM抗体以及曲霉半乳甘露聚糖抗原和烟曲霉IgG抗体浓度,计算各指标的灵敏度、特异度、阳性预测值、阴性预测值和受试者工作特征(ROC)曲线下面积。结果甘露聚糖抗原和假丝酵母菌IgG/IgM抗体联合测定的敏感度为66.7%,特异度为83.3%,阴性预测值为100.0%,阳性预测值为85.7%,ROC曲线下面积为0.992(95%CI:0.974~1.000);半乳甘露聚糖抗原和烟曲霉IgG抗体联合测定的敏感度为66.7%,特异度为95.0%,阴性预测值为98.2%,阳性预测值为100.0%,ROC曲线下面积为0.978(95%CI:0.934~1.000)。结论甘露聚糖抗原和假丝酵母菌IgG/IgM抗体、半乳甘露聚糖抗原和烟曲霉IgG抗体联合检测对深部真菌感染的早期诊断具有重要意义。     

A multi-tolerant low molecular weight mannanase from Bacillus sp. CSB39 and its compatibility as an industrial biocatalyst

Bacillus sp. CSB39, isolated from popular traditional Korean food (Kimchi), produced a low molecular weight, thermostable mannanase (MnCSB39); 571.14 U/mL using locust bean gum galactomannan as a major substrate. It was purified to homogeneity using a simple and effective two-step purification strategy, Sepharose CL-6B and DEAE Sepharose Fast Flow, which resulted in 25.47% yield and 19.32-fold purity. The surfactant-, NaCl-, urea-, and protease-tolerant monomeric protein had a mass of ∼30 kDa as analyzed by SDS-PAGE and galactomannan zymography. MnCSB39 was found to have optimal activity at pH 7.5 and temperature of 70 °C. The enzyme showed ˃55% activity at 5.0–15% (w/v) NaCl, and ˃93% of the initial activity after incubation at 37 °C for 60 min. Trypsin and proteinase K had no effect on MnCBS39. The enzyme showed ˃80% activity in up to 3 M urea. The N-terminal amino acid sequence, ALKGDGX, did not show identity with reported mannanases, which suggests the novelty of our enzyme. Activation energy for galactomannan hydrolysis was 26.85 kJmol⁻¹ with a K_cat of 142.58 × 10⁴ s⁻¹. MnCSB39 had K_m and V_max values of 0.082 mg/mL and 1099 ± 1.0 Umg⁻¹, respectively. Thermodynamic parameters such as ΔH, ΔG, ΔS, Q₁₀, ΔG_E-S, and ΔG_E-T supported the spontaneous formation of products and the high hydrolytic efficiency and feasibility of the enzymatic reaction, which strengthen its novelty. MnCSB39 activity was affected by metal ions, modulators, chelators, and detergents. Mannobiose was the principal end-product of hydrolysis. Bacillus subtilis CSB39 produced a maximum of 1524.44 U mannanase from solid state fermentation of 1 g wheat bran. MnCSB39 was simple to purify, was active at a wide pH and temperature range, multi-stress tolerant and catalyzes a thermodynamically possible reaction, characteristics that suggests its suitability for application as an industrial biocatalyst.     

NMR spectroscopy and chemical studies of an arabinan-rich system from the endosperm of the seed of Gleditsia triacanthos

Exhaustive extraction of the endosperm from the seed of Gleditsia triacanthos using water at room temperature and 50 degrees C left a residue, which was further extracted at 95 degrees C. Precipitation of this extract with 2-propanol yielded major amounts of galactomannan components, while the supernatant was mainly composed of arabinose-rich constituents. Two fractions were obtained by anion-exchange chromatography. The fraction that eluted with water is an arabinan with (1-->5) alpha-L linkages and branching mainly on C-2, accompanied with equal amounts of a low-galactose galactomannan oligosaccharide, and a small proportion of a beta-(1-->4)-galactan. The fraction eluted with an increased ionic strength consists mainly of a similar arabinan, and lower proportions of a high-galactose galactomannan, galactan, and protein. The arabinan moiety in both fractions was characterized by chemical analysis and 1D and 2D NMR spectroscopic techniques.     

Effect of abscisic acid on galactomannan degradation and endo-β-mannanase activity in seeds of Sesbania virgata (Cav.) Pers. (Leguminosae)

Seeds of Sesbania virgata (Cav.) Pers. (Leguminosae) have an endosperm which accumulates galactomannan as a storage polysaccharide in the cell walls. After germination, it is hydrolysed by three enzymes: α-galactosidase (EC 3.2.1.22), endo-β-mannanase (EC 3.2.1.78) and β-mannosidase (EC 3.2.1.25). This work aimed at studying the effect of abscisic acid (ABA) on galactomannan degradation during and after germination. Seeds were imbibed in water or in 10⁻⁴ M ABA, and used to evaluate the effect of exogenous and endogenous ABA. Tissue printing was used to follow biochemical events by detecting and localising endo-β-mannanase in different tissues of the seed. The presence of exogenous ABA provoked a delay in the cellular disassembly of the endosperm and disappearance of endo-β-mannanase in the tissue. This led to a delay in galactomannan degradation. The testa (seed coat) of S. virgata contains endogenous ABA, which decreases ca. fourfold during storage mobilisation after germination, permitting the galactomannan degradation in the endosperm. Furthermore, endo-β-mannanase was immunolocalised in the testa, which has a living cell layer. The ABA appears to modulate storage mobilisation in the legume seed of S. virgata, and a cause–effect relationship between ABA (probably through testa) and activities of hydrolases is proposed.     

Diagnosis of invasive fungal disease in hospitalized patients with chronic obstructive pulmonary disease

Background

The role of culture-independent techniques (galactomannan, (1-3)-β-d-glucan) in the early diagnosis of invasive fungal diseases (IFD) is well assessed in hematological patients, but there are no clear conclusions in patients with chronic obstructive pulmonary disease (COPD).

Structural elucidation, modification and characterization of seed gum from Cassia javahikai seeds: A non-traditional source of industrial gums

A water-soluble seed gum was isolated from seed endosperm of Cassia javahikai. The acid-catalyzed fragmentation, methylation, selective enzymatic degradation and periodate oxidation suggested a heteropolymeric structure for the polysaccharide. The polysaccharide was shown to have a linear chain of β(1 → 4) linked d-mannopyranosyls units with side chains of α(1 → 6) d-galactopyranosyl units. Grafting of polyacrylamide onto the gum was performed using K₂S₂O₈/ascorbic acid redox system in presence of Ag⁺ as catalyst at 35 ± 2 °C. The viscosity of the gum solution increased on grafting and the grafted gum was observed to resist biodegradation for more than 256 h. Thermogravimetric analysis revealed that grafted gum was more thermally stable than native gum.