赖氨酸修饰对梯棱羊肚菌黑色素结构和理化特性的影响

为了进一步提高梯棱羊肚菌黑色素的提取率及溶解性,本试验采用单因素、Plackett-Burman试验、响应面试验对纤维素酶-超声波协同提取梯棱羊肚菌黑色素的提取工艺进行优化研究。通过赖氨酸修饰,并对修饰前后的梯棱羊肚菌黑色素进行结构表征、理化性质及稳定性研究。结果表明,在NaOH浓度为1.54 mol/L,纤维素酶添加量为20 mg/g,纤维素酶酶解时间为78.6 min,料液比为1:30,酶解温度为40 ℃,超声时间为80 min条件下提取的梯棱羊肚菌黑色素最优。未修饰的梯棱羊肚菌黑色素不溶于水,色价值为480.24,修饰后的黑色素溶解度为1 016 g/L,色价值为1 771.18,比未修饰的黑色素在溶解性、色价值方面均有所提高。此外,在不同的温度、光照、pH条件下,修饰前后的梯棱羊肚菌黑色素均比较稳定。以上研究结果为梯棱羊肚菌黑色素的高效提取及其产品的开发利用奠定了理论基础。     

Acquired resistance triggered by elicitins in tobacco and other plants

Elicitins are a family of proteins excreted byPhytophthora spp. They exhibit high sequence homology but large net charge differences. They induce necrosis in tobacco plants which then become resistant to the tobacco pathogenPhytophthora parasitica var.nicotianae. In stem-treated plants, resistance was not restricted to the site of elicitin application, but could be demonstrated by petiole inoculation at all levels on the stem. Resistance was already maximum after two days and lasted for at least two weeks. It was effective not only towardsP. p. var.nicotianae infection, but also against the unrelated pathogenSclerotinia sclerotiorum. In contrast to dichloroisonicotinic acid, an artificial inducer of systemic acquired resistance, which was increasingly effective with doses ranging from 0.25 to 5mole per plant, the basic elicitin cryptogein exhibited a threshold effect, inducing near total resistance and extensive leaf necrosis above 0.1 nmole per plant. Between 1 and 5 nmole, acidic elicitins (capsicein and parasiticein) protected tobacco plants with hardly any necrotic symptom. Elicitins exhibited similar effects in various tobacco cultivars andNicotiana species, although with quantitative differences, but induced neither necrosis nor protection in other SolanaceÆ (tomato, petunia and pepper). Among 24 additional species tested belonging to 18 botanical families, only some BrassicaceÆ, noticeably rape, exhibited symptoms in response to elicitins, in a cultivar-specific manner. Elicitins appear to be natural specific triggers for systemic acquired resistance and provide a tool for unraveling the mechanisms leading to its establishment.Abbreviations AR acquired resistance - HR hypersensitive response - INA 2,6-dichloroisonicotinic acid - Ppn Phytophthora parasitica var.nicotianae - SAR systemic acquired resistance     

In vitro expression and inhibition of procoagulant activity produced by bovine alveolar macrophages and peripheral blood cells

Local and systemic activation of coagulation is frequently associated with bacterial sepsis. The coagulopathy is due, at least in part, to expression of tissue factor (TF) by monocytes and macrophages. The purpose of this study was to evaluate the expression of procoagulant activity by bovine alveolar macrophages, leukocytes and platelets, and to determine the relative potency of three chemical inhibitors of TF expression (pentoxifylline, retinoic acid, and cyclosporin A). Bovine alveolar macrophages were stimulated with lipopolysaccharide (LPS) derived from Pasteurella haemolytica or recombinant bovine tumour nervous factor (TNF) and dose- and time-dependent effects on TF expression were studied. LPS and TNF induced TF expression in alveolar macrophages and LPS treatment of whole blood induced TF expression in mononuclear cells. Neutrophils and platelets also expressed procoagulant activity, but this activity was not inhibited by anti-bovine TF monoclonal antibody. Pentoxifylline (40 mol/L), retinoic acid (0.01 mmol/L) and cyclosporin A (0.08 mol/L) inhibited TF expression when added concurrently with LPS or TNF, but not when added 4 h after stimulation. TF mRNA was not detected in unstimulated alveolar macrophages by Northern blot analysis. In contrast, exposure to LPS or TNF for 6 h induced marked expression of TF mRNA, which was inhibited by treatment with pentoxifylline, retinoic acid and cyclosporin A. Expression of TNF by alveolar macrophages stimulated with LPS was also inhibited by these compounds. Our results indicate that procoagulant activity expressed by alveolar macrophages and monocytes is associated with expression of TF, whereas procoagulant activity expressed by neutrophils and platelets is not. The concentrations of pentoxifylline and retinoic acid necessary for inhibition of TF expression in vitro may not be achievable in vivo owing to their toxic effects. However, the in vitro concentration of cyclosporin A that inhibited TF expression did not exceed the plasma concentration observed in humans, and therefore may be useful for inhibition of TF expression in vivo.Abbreviations BAL bronchoalveolar lavage - LPS lipopolysaccharide - cDNA cloned deoxyribonucleic acid - cAMP cyclic adenosine monophosphate - GAPDH glyceraldehyde phosphate dehydrogenase - mRNA messenger ribonucleic acid - TF tissue factor - TNF tumour necrosis factor - DPBS Dulbecco's phosphate-buffered saline     

Correlation between hydrolytic enzyme activities measured in bean seedlings afterTrichoderma koningii treatment combined with pregermination and the protective effect againstPythium splendens

An experimental protocol consisting in the colonisation of pregerminated bean seeds dressed withTrichoderma sp. was used in order to study the mechanisms correlated with the protective effect againstPythium splendens. Seed dressed with TH-11 (T. koningii) for 24 h presented a higher protective effect and a higher level of seed colonisation as compared to those dressed with TH-13 (T. longibranchiatum). The levels of seed coat colonisation by TH-11 and TH-13 was shown to be correlated with the carboxymethylcellulase activity, as measured in the seed coats retreived from germinating dressed bean seeds. The seed coat colonisation was also associated with an increased activities of endo-1,3--glucanase and endochitinase measured in seed extracts, and an inhibitory effect of seed extracts onPythium sporangia germination. Pretreatment of TH-13-dressed seeds with a commercial cellulase improved all parameters mentioned above, thus suggesting a role of cellulase activity in the colonisation process and the linked protective effect. The possible role of hydrolytic enzymes in the protective effects is discussed.     

Epidemiology of beet necrotic yellow vein virus in sugar beet at different initial inoculum levels in the presence or absence of irrigation

A field experiment was set up in 1988 to study the development of rhizomania disease of sugar beet at different inoculum levels of beet necrotic yellow vein virus (BNYVV) in soil. Five, tenfold different, inoculum levels were created by addition of the approximate amounts of 0, 0.5, 5, 50 and 500 kg infested soil per ha (the latter corresponding to 0.01% v/v calculated to the tillage layer). A drip irrigation treatment was applied to study the influence of soil moisture on disease. Susceptible sugar beet, cv. Regina, was grown for three consecutive years.In the first year, root symptoms were not observed, but BNYVV-infected plants were detected by ELISA in low numbers at all inoculum levels at harvest. After late drilling in 1989, high numbers of infected plants, up to 90–100% in plots with the highest inoculum level, were detected already in June. Root symptoms were also observed from June onwards. In both these years disease incidence increased in time and was significantly influenced by the initial inoculum level. In the third year, the whole field was heavily diseased, and only for the non-irrigated plots incidence differed for different initial inoculum levels. The expression of symptoms by BNYVV-infected plants was influenced by initial inoculum level, thus by the amount and timing of primary infection.Root weight at harvest was not affected, but sugar content decreased with increasing inoculum level already in 1988, leading to a reduction in sugar yield of 10% at the highest inoculum level. In 1989, both root weight and sugar content decreased progressively with increasing inoculum level, resulting in sugar yield reductions of 11–66% (down to approximately 3000 kg ha^–1) for low to high inoculum levels compared to the control. As the control plots became contaminated, all yields were low in 1990, still showing a decrease with increasing inoculum level in the non-irrigated plots, but an overall mean sugar yield of 3323 kg ha^–1 for the irrigated ones.Sodium and -amino nitrogen content of the root, additional quality parameters determining extractability of sucrose, showed an increase and decrease, respectively, with increasing initial inoculum level already in the first year. The relative differences in contents compared to those from the control were largest for Na content. A significant negative correlation was found between Na (mmol kg^–1 root) and sugar content (% of fresh weight); linear for 1988, exponential for 1989 and 1990.In spring 1989, the infestation of individual plots was assessed using a quantitative bioassay estimating most probable numbers (MPNs) of infective units of BNYVV per 100 g dry soil. The relationship between the MPns determined and root weight, sugar content and sugar yield at harvest could be described by Gompertz curves. The increase in disease incidence with increasing MPN in 1989 was adequately fitted with a logistic equation.     

加热光纤法同步测定土壤水热参数误差分析

土壤水热参数是研究土壤水热传输的基本物理参数。当前热脉冲探针法（HPP）可同步测定土壤水热参数,但该方法仅限于在点尺度下测定。与其具有相同理论基础的加热光纤法（SPHP-DTS）,可将测定尺度增大至田间千米尺度,但其测定精度尚未得到有效验证。为了探知SPHP-DTS法的误差,本研究进行了SPHP-DTS法与HPP法测定土壤水热参数的对比试验。结果表明,以HPP为标准,加热光纤法测定热导率的精度RMSE为0.13 W?m-1?℃-1。SPHP-DTS法测定的热导率显著高于HPP法,主要原因在于加热光纤时产生的温度效应。通过热导率法测定土壤含水率时,在热导率测定误差的影响下,SPHP-DTS法的测定精度明显低于HPP法。SPHP-DTS法测定土壤水热参数的其他误差来源包括光纤与土壤之间多个界面的接触热阻、光纤的温度敏感性、噪音干扰以及温度梯度驱动下的水分迁移。本研究可为SPHP-DTS法提升土壤水热参数测定精度提供理论参考。     

Characterisation of energy-dependent efflux of imazalil and fenarimol in isolates of Penicillium italicum with a low,medium and high degree of resistance to DMI-fungicides

Differential accumulation of [¹⁴C]imazalil and [¹⁴C]fenarimol by germlings of wild-type and DMI-resistant isolates ofPenicillium italicum was studied at various pH values. At pH 7 and 8 the low-resistant isolate E_300–3 accumulated 22% and 35%, respectively, less imazalil than the wild-type isolate W₅. Imazalil accumulation at pH 5 and 6 was similar. Isolate E_300–3 also accumulated less fenarimol as compared with the wild-type isolate. This difference was much more obvious than for imazalil and was observed at all pH values tested. Differences in accumulation of both imazalil and fenarimol between low (E_300–3), medium (H₁₇) and high resistant (I₃₃) isolates were not observed. These results suggest that decreased accumulation of DMIs is responsible for a low level of resistance only and that additional mechanisms of resistance might operate in isolates with a medium and high degree of resistance. With all isolates fenarimol accumulation was energy-dependent. This was not obvious for imazalil.The wild-type and DMI-resistant isolates had a similar plasma membrane potential as determined with the probe [¹⁴C]tetraphenylphosphonium bromide ([¹⁴C]TPP⁺). Various test compounds, among which ATPase inhibitors, ionophoric antibiotics and calmodulin antagonists, affected the accumulation of [¹⁴C]TPP⁺, [¹⁴C]imazalil and [¹⁴C]fenarimol. No obvious correlation between the effects of the test compounds on accumulation levels of the fungicides and [¹⁴C]TPP⁺ could be observed. These results indicate that the plasma membrane potential does not mediate the efflux of DMI fungicides byP. italicum.     

Determination of uric acid in canine serum and urine by high performance liquid chromatography

A reproducible high performance liquid chromatography (HPLC) method was developed for analysis of uric acid in canine serum and urine. The method consists of precipitating serum proteins with phosphotungstic acid prior to HPLC analysis. Urine is analyzed after dilution with buffer. Chromatography is performed on a reversed-phase C-18 column with UV detection at 292 nm. Sensitivity of the method will allow reproducible measurement of uric acid at concentrations of 0.05 mg/dl in serum and 0.1 mg/dl in urine. The HPLC method has been used to quantify hundreds of canine serum and urine samples. The method is superior to UV absorption or colorimetric methods because its lower limit of detection allows measurement of uric acid at concentrations found in canine serum and urine.