Human carotid lesion linoleic acid hydroperoxide inhibits paraoxonase 1 (PON1) activity via reaction with PON1 free sulfhydryl cysteine 284

Paraoxonase 1 (PON1) is an HDL-associated lactonase with antiatherogenic properties. These include dampening the oxidation properties of human carotid lesion lipid extract (LLE), which in turn inactivates the enzyme. The aims of this study were to identify the PON1 inhibitor in LLE and explore the mechanism of inhibition. LLE inhibited both recombinant PON1 and HDL-PON1 lactonase activity in a dose- and time-dependent manner. Addition of antioxidants or electrophiles to LLE did not prevent PON1 inhibition. LLE was unable to inhibit a PON1 mutant lacking Cys284, whereas it did inhibit all other PON1 mutants tested. The inhibitor in the LLE was identified as linoleic acid hydroperoxide (LA-OOH) and inhibition was specific to this hydroperoxide. During its inhibition, PON1 acted like a peroxidase enzyme, reducing LA-OOH to LA-hydroxide via its Cys284. A similar reaction occurred with external thiols, such as DDT or cysteine, which also prevented PON1 inhibition and restored enzyme activity after inhibition. Thus, the antiatherogenic properties of HDL could be, at least in part, related to the sulfhydryl-reducing characteristics of its associated PON1, which are further protected and recycled by the sulfhydryl amino acid cysteine.     

玉米弯孢叶斑病菌ATMT突变株构建及致病力分析

利用农杆菌介导的基因转化(Agrobacterium-mediated transformation,ATMT)技术,转化玉米弯孢叶斑病菌 (Curvularia lunata),共获得转化子1 454个。对其中菌落形态、生长速率等性状有显著变化的8个突变株进行分析。通过离体叶片接种进行筛选,发现4个突变株致病性降低,2个突变株致病性增强。通过测定生长速率、产孢量及细胞壁降解酶活性发现,其中致病性减弱的突变株,其产孢量均有所下降甚至不产孢,突变株的PG酶活性普遍增强,但Cx酶活性没有明显变化,而2株强致病性突变株的Cx酶活性明显增强。     

灰葡萄孢分生孢子产生相关基因的克隆及功能分析

[目的]克隆灰葡萄孢分生孢子产生相关基因,并研究其功能,为进一步研究灰葡萄孢分生孢子产生机理和灰葡萄孢侵染及致病机理奠定基础.[方法]通过筛选灰葡萄孢ATMT突变体库,获得一株不能产生分生孢子的突变菌株BCt78,采用PCR和Southern Blotting技术,对突变菌株BCt78进行分子鉴定.利用TAIL-PCR技术获得T-DNA插入位点的侧翼序列;将所获得侧翼序列与灰葡萄孢基因组数据库中的已知基因序列进行BLAST分析,推测出T-DNA的插入位点;通过PCR进一步验证T-DNA的插入位点,利用RT-PCR技术确定突变基因;最后对突变菌株的菌落形态、生长速度、胞壁降解酶活力、粗毒素的生物活性、对番茄叶片的致病能力及部分致病相关基因的表达情况进行研究.[结果]TAIL-PCR结果证实T-DNA插入到灰葡萄孢BCIG 12707.1基因的ATG起始密码子区;RT-PCR结果证实突变基因为BCIG_12707.1,该基因DNA全长为135 bp,编码一个44个氨基酸的假定蛋白(Hypothetical protein).突变菌株在PDA培养基上菌落呈灰白色,生长速度减慢,不能产生分生孢子及菌核;对番茄叶片的致病性增强,且胞壁降解酶(PG、PMG和Cx)活力增强;突变菌株中参与细胞壁降解的角质酶基因cutA和多聚半乳糖醛酸酶基因Bepg1,信号转导途径基因(PKA1、PKA2、Bac、Bmp3),产毒素基因BcBOT2(Sesquiterpene synthase),漆酶基因Lac1,跨膜蛋白基因Btp1表达都增强.[结论]BC1G_ 12707.1基因在灰葡萄孢分生孢子产生、菌核形成及致病力等方面起重要作用.     

Kinetic studies of the differential effect of detergents on the peptidase activities of the multicatalytic proteinase from rat liver

We present here a detailed study of the effect of detergents on the three peptidase activities (hydrolysis of the LLVY, ARR, and LLE peptides) of the purified multicatalytic proteinase from rat liver. At Triton X-100 and sodium dodecyl sulfate (SDS) concentrations of 0.1%, all three peptidase activities are inhibited. Lower concentrations of the two detergents (0.01%) do not affect the hydrolysis of the ARR peptide, whereas they behave differently on the hydrolysis of the LLVY and LLE peptides. Triton X-100 inhibits and SDS strongly activates LLVY peptide hydrolysis by decreasing and increasing Vmax, respectively. In the absence of detergents, the saturation curve for the LLE peptide can be analyzed as the result of two components, one showing cooperative (nH = 1.6) with higher affinity (S0.5 = 60 microM) and lower Vmax than a second, noncooperative component (Km = 320 microM). SDS (0.01%) activates LLE peptide hydrolysis by suppressing cooperativity, slightly increasing Vmax, and decreasing the half-saturation concentration (Km = 30 microM) of the enzyme. Triton X-100 (0.01%) also suppresses the cooperativity and decreases the half-saturation concentration (Km = 25 microM) for the LLE peptide; in contrast, it reduces Vmax by inhibition of the low affinity, high Vmax component observed in the absence of detergents. Based on these observations, it can be concluded that both detergents behave like allosteric activators of peptidylglutamyl-peptide hydrolyzing activity and that the multicatalytic proteinase has at least three different classes of active sites: two independent noncooperative sites that catalyze the hydrolysis of trypsin and chymotrypsin-like substrates and one class for peptidylglutamyl-peptide hydrolysis having two components: one cooperative (two or more sites) and one noncooperative.     

紫外线诱变蝉拟青霉对白粉虱致病性的影响(Ⅰ)

对蝉拟青霉进行UV诱变处理,比较诱变前菌株3L和诱变后M1、M2、M3及M4各个菌株对白粉虱的致病性和菌生理特性的变化情况,并进行相关性分析。结果表明,菌株的几丁质酶活性、孢子萌发率及生长势与致病性有密切的相关性;而产孢量、蛋白质酶活性则与致病性的关系不大。     

Alcohol oxidase is a novel pathogenicity factor for Cladosporium fulvum, but aldehyde dehydrogenase is dispensable

Cladosporiumfulvum is a mitosporic ascomycete pathogen of tomato. A study of fungal genes expressed during carbon starvation in vitro identified several genes that were up regulated during growth in planta. These included genes predicted to encode acetaldehyde dehydrogenase (Aldh1) and alcohol oxidase (Aox1). An Aldh1 deletion mutant was constructed. This mutant lacked all detectable ALDH activity, had lost the ability to grow with ethanol as a carbon source, but was unaffected in pathogenicity. Aox1 expression was induced by carbon starvation and during the later stages of infection. The alcohol oxidase enzyme activity has broadly similar properties (Km values, substrate specificity, pH, and heat stability) to yeast enzymes. Antibodies raised to Hansenula polymorpha alcohol oxidase (AOX) detected antigens in Western blots of starved C. fulvum mycelium and infected plant material. Antigen reacting with the antibodies was localized to organelles resembling peroxisomes in starved mycelium and infected plants. Disruption mutants of Aox1 lacked detectable AOX activity and had markedly reduced pathogenicity as assayed by two different measures of fungal growth. These results identify alcohol oxidase as a novel pathogenicity factor and are discussed in relation to peroxisomal metabolism of fungal pathogens during growth in planta.     

灰葡萄孢犬尿氨酸途径关键酶基因的鉴定

【背景】灰葡萄孢是一种重要的植物病原真菌,实验室前期明确了灰葡萄孢犬尿氨酸单加氧酶(kynurenine3-monooxygenase,KMO)基因BcKMO参与调控病菌的生长发育和致病力。犬尿氨酸单加氧酶(KMO)是犬尿氨酸途径的关键酶,但灰葡萄孢是否存在犬尿氨酸途径及其在病菌生长、发育和致病过程中的功能尚未见相关报道。【目的】鉴定灰葡萄孢犬尿氨酸途径中的关键酶基因,确定灰葡萄孢犬尿氨酸途径的存在,为阐明灰葡萄孢生长发育和致病力的分子机理奠定基础。【方法】利用生物信息学方法,对灰葡萄孢犬尿氨酸途径中犬尿氨酸酶(kynureninase,KYN)、吲哚-2,3-双加氧酶(indoleamine-2,3-dioxygenase,IDO)、犬尿氨酸氨基转移酶(kynurenine amino transferase,KAT)的编码基因进行分析;利用Real-time PCR技术,检测灰葡萄孢野生型BC22、BcKMO基因T-DNA插入突变体BCG183、恢复菌株BCG183/BcKMO中犬尿氨酸途径关键酶基因的表达水平;利用真菌犬尿氨酸酶KYN检测试剂盒,测定BcKMO突变体中犬尿氨酸酶(KYN)的含量。【结果】灰葡萄孢中含有2个犬尿氨酸氨基转移酶(KAT)的编码基因、3个吲哚-2,3-双加氧酶(IDO)的编码基因、10个犬尿氨酸氨基转移酶(KAT)的编码基因。灰葡萄孢KYN编码基因、IDO编码基因、KAT编码基因在突变体BCG183中的表达水平显著高于或低于在野生型和恢复菌株。突变体BCG183中犬尿氨酸酶(KYN)的含量显著低于野生型BC22和恢复菌株。【结论】灰葡萄孢中存在犬尿氨酸途径,灰葡萄孢BcKMO基因突变影响KYN、IDO和KAT编码基因的表达以及犬尿氨酸酶(KYN)的含量。     

Paraoxonase 1 protects macrophages from atherogenicity of a specific triglyceride isolated from human carotid lesion

Human atherosclerotic lesions contain oxidized lipids that facilitate further oxidation of macrophages, LDLs, and oxidative stress (OS)-sensitive markers and inhibit the antiatherogenic enzyme paraoxonase 1 (PON1). Our aim was to isolate and identify the oxidizing agent in a human atherosclerotic lesion lipid extract (LLE) and to explore the mechanisms of oxidation and of PON1's effect on the oxidizing agent. Of the five main fractions separated from the LLE, only fraction 2 (F2) promoted macrophage reactive oxygen species (ROS) production via a mechanism requiring mitochondrial involvement, whereas the NADPH oxidase system was not involved. Incubation of F2 with PON1 abridged the former's peroxide value and reduced its capacity to oxidize OS markers. The active agent was a triglyceride composed of palmitic, oleic, and linoleic acids, with 0.3% of its linoleic moiety in oxidized form. Incubation of either F2 or an identical synthetic triglyceride with PON1 reduced their ability to oxidize macrophages, without affecting cellular accumulation of triglycerides. We conclude that macrophage ROS production by LLE occurs in the presence of a specific triglyceride and requires mitochondrial involvement. Lipid peroxide in the triglyceride can also facilitate lipid autoxidation. Both atherogenic pathways are suppressed by PON1, which acts as an antiatherogenic element.     

Biotechnological approach to the synthesis of 9alpha-hydroxylated steroids

The steroid 9alpha-hydroxylase gene has been cloned from Mycobacterium smegmatis into Escherichia coli BL21. Progesterone added to bioreactors was subjected to in vivo transformation into 9alpha-hydroxyprogesterone. In 7 days, 43.6 mg 9alpha-hydroxyprogesterone was formed from 53.8 mg/L progesterone. The enzyme also has shown evidence of processing 4-androstene-3,17-dione in vivo. An extensive analytical method development, including LLE, HPLC-DAD, MS, and NMR was performed to verify the product and to enable a quantitative analysis. Protocols for analytical and preparative separation have been developed, using binaphtol as internal standard. Both the growth pattern and the bioconversion rate were unaffected by the presence of binaphtol in the bioreactor. The enzyme was purified by immobilised metal affinity and ion exchange chromatography, resulting in low in vitro activity.     

The Ustilago maydis ubc4 and ubc5 genes encode members of a MAP kinase cascade required for filamentous growth

Ustilago maydis, the causal agent of corn smut disease, displays dimorphic growth in which it alternates between a budding haploid saprophyte and a filamentous dikaryotic pathogen. We are interested in identifying the genetic determinants of filamentous growth and pathogenicity in U. maydis. To do this we have taken a forward genetic approach. Earlier, we showed that haploid adenylate cyclase (uac1) mutants display a constitutively filamentous phenotype. Mutagenesis of a uac1 disruption strain allowed the isolation of a large number of budding suppressor mutants. These mutants are named ubc, for Ustilago bypass of cyclase, as they no longer require the production of cyclic AMP (cAMP) to grow in the budding morphology. Complementation of a subset of these suppressor mutants led to the identification of the ubc4 and ubc5 genes, which are required for filamentous growth and encode a MAP (mitogen-activated protein) kinase kinase kinase and a MAP kinase kinase, respectively. Evidence suggests that they are important in the pheromone response pathway and in pathogenicity. These results further support an important interplay of the cAMP and MAP kinase signal transduction pathways in the control of morphogenesis and pathogenicity in U. maydis.     

Physiology and morphology of descending neurons in pheromone-processing olfactory pathways in the male moth Manduca sexta

1. We have characterized the responses and structure of olfactory descending neurons (DNs) that reside in the protocerebrum (PC) of the brain of male sphinx moths Manduca sexta and project toward thoracic ganglia. 2. Excitatory responses of DNs to pheromone blends were of two general types: (a) brief excitation (BE) that recovered to background in less than 1 s after the stimulus, and (b) long-lasting excitation (LLE) that outlasted the stimulus by greater than or equal to 1 s and, in many cases, as long as 30 s. Individual pheromone components were ineffective in eliciting LLE. 3. Some neurons showing LLE also exhibited state-dependent responses to pheromonal stimuli. When such neurons were in a state of low background firing, stimulation with pheromone blend elicited LLE. When they were in a state of LLE, an identical stimulus reduced firing for 5-10 s after which firing gradually increased to the initial higher level. 4. Thirteen stained DNs were reconstructed from serial sections for detailed analysis of their morphology in the brain. DNs exhibiting LLE had neurites concentrated in the lateral accessory lobes (LALs) in the protocerebrum and adjacent neuropil. Most DNs exhibiting only BE to pheromonal stimuli and other DNs showing responses only to visual or mechanosensory stimuli did not have branches in the LALs.     

Triterpenoid-rich loquat leaf extract induces growth inhibition and apoptosis of pancreatic cancer cells through altering key flux ratios of glucose metabolism

Introduction

Loquat leaf extract (LLE) is a mixture rich in terpenoids, and has broad biological activities including the inhibition of cancer cell growth. The exact metabolic mechanism of this growth inhibiting effect is not known.

Objectives

We investigated the cellular metabolic effect of LLE, and ursolic acid (UA) on pancreatic cancer cells using a ¹³C carbon tracing technology.

Methods

MIA PaCa-2 cells were cultured in medium containing [1, 2 ¹³C₂]-glucose in the presence of either LLE (50 µg/ml), UA (50 µM), or metformin (1 mM). The mass isotopomer distribution of glucose, lactate, ribose, glutamate and palmitate in medium was determined. Based on the mass isotopomer distribution in metabolites we were able to determine individual ¹³C enrichment (∑M?×?n) and the minimum fraction of new synthesis?(1-M0) in each metabolite. Several flux ratios of energy metabolic pathways were calculated from the mass isotopomer ratios of these metabolites.

Results

We found that tumor viability was suppressed by LLE and UA in a dose dependent manner, and the tumor-inhibiting effect was associated with the changes in oxidative/non-oxidative pentose (Ox/Non-ox) and pyruvate dehydrogenase/isocitrate dehydrogenase (PDH/ICDH) flux ratios resulting in decreased new syntheses of ribose and fatty acids.

Conclusion

Metabolic homeostasis (balance of fluxes) in cancer cells is maintained through the regulation of metabolic fluxes by oncogenes and tumor-suppressor genes. Treatment of MIA PaCa-2 cells by LLE, UA and metformin likely altered key metabolic flux ratios affecting metabolic homeostasis required for energy and macromolecular production in tumor growth.

     

Relationship between variations in pathogenicity and lag phase at 37°C of Listeria monocytogenes previously stored at 4°C

s. BUNCIC AND S.M. AVERY. 1996. Three haemolytic, pathogenic strains of Listeria monocytogenes (a reference strain, a food-derived strain and a human strain) were held at 4°C for 4 weeks in phosphate-buffered saline pH 5.5 or 7.0, with and without 0.2% potassium sorbate or 0.3% sodium acetate. The number of viable cells did not change significantly during this storage. Pathogenicity of non-growing L. monocytogenes cells for 14-d-old chick embryos was determined before and after storage. Storage at 4°C resulted in decreased pathogenicity, but effects were strain-, pH- and substrate-dependent. After 4 weeks storage at 4°C non-growing bacterial cells were transferred to Brain Heart Infusion broth and growth characteristics were determined during incubation at 37°C. Strains that showed decreased pathogenicity had significantly longer lag phases at 37°C than strains that maintained pathogenicity. It is concluded that decreased pathogenicity of L. monocytogenes stored without growth at 4°C for 4 weeks and subsequent long lag phase at 37°C are correlated.     

Nucleoside diphosphate kinase: role in bacterial growth, virulence, cell signalling and polysaccharide synthesis

Nucleoside diphosphate kinase (Ndk) is an important enzyme that generates nucleoside triphosphates (NTPs) or their deoxy derivatives by terminal phosphotransfer from an NTP such as ATP or GTP to any nucleoside diphosphate or its deoxy derivative. As NTPs, particularly GTP, are important for cellular macromolecular synthesis and signalling mechanisms, Ndk plays an important role in bacterial growth, signal transduction and pathogenicity. Specific examples of the role of Ndk in regulating growth, NTP formation and cell surface polysaccharide synthesis in two respiratory tract pathogens, Pseudomonas aeruginosa and Mycobacterium tuberculosis , are discussed.     

Peptidylglutamyl-peptide hydrolase activity of the multicatalytic proteinase complex: evidence for a new high-affinity site, analysis of cooperative kinetics, and the effect of manganese ions.

The multicatalytic proteinase (MCP) complex or proteasome is a major nonlysosomal proteinase of eukaryotic cells. The proteinase can cleave peptide bonds on the carboxyl side of hydrophobic, basic, or acidic amino acid residues. These activities have been referred to as "chymotrypsin-like", "trypsin-like", and "peptidylglutamyl-peptide hydrolase" activities, respectively, and have been shown to be catalyzed at distinct sites. The latter activity is often assayed with the synthetic peptide substrate Z-Leu-Leu-Glu-beta-naphthylamide (LLE-NA). N-tBoc-Ala-Ala-Asp-SBzl is also a substrate for the rat liver MCP, suggesting a broader specificity for cleavage on the carboxyl side of acidic residues than the peptidylglutamyl-peptide hydrolase activity previously reported. The pH optimum is in the range of pH 7.0-7.5. Studies of the dependence of velocity on LLE-NA concentration show (a) that there is a high-affinity site (LLE1) which obeys Michaelis-Menten kinetics with a Km value of approximately 100 microM and (b) that at higher substrate concentrations (LLE2) the curve is sigmoidal, suggesting either allosteric activation of the proteinase at a second site or the involvement of multiple catalytic sites which display positive cooperativity. Activity at the high-affinity site (LLE1) can be distinguished from that of the activity of the LLE2 component by the effect of inhibitors, divalent metal ions, and KCl, as well as by its response to heat treatment. The addition of 1 mM MnCl2 stimulates both LLE1 and LLE2 activities and also permits saturation of MCP with substrate at concentrations of LLE-NA below the solubility limit of this peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitation of psilocin in human plasma by high-performance liquid chromatography and electrochemical detection: comparison of liquid–liquid extraction with automated on-line solid-phase extraction

Two modifications of the HPLC–ED method with respect to extraction procedure used have been developed for psilocin, the active metabolite of psilocybin, in human plasma using either liquid–liquid extraction (LLE) or automated on-line solid-phase extraction (on-line SPE). Each type of the sample preparation required a different HPLC system followed by electrochemical detection at 650 to 675 mV. The limit of quantitation of both modifications was 10 ng/ml psilocin. There was no significant difference observable between the LLE and the on-line SPE in terms of method standard deviation (LLE 1.82%, on-line SPE 1.13%) and the analytical results. However, the advantages of on-line SPE in addition to different selectivity were less manual effort, smaller plasma volumes of 400 μl (LLE 2 ml) and a recovery of psilocin in human plasma of nearly 100% (LLE 88%). In contrast to a previous procedure both methods were rapid, simple and reliable and yielded high plasma recoveries. They were used successfully in the quantitation of psilocin in plasma samples obtained from healthy volunteers after p.o. administration of 0.2 mg psilocybin per kg body mass. Plasma concentration curves and pharmacokinetic parameters were calculated.     

Isolates of Microdochium nivale and M. majus Differentiated by Pathogenicity on Perennial Ryegrass (Lolium perenne L.) and in vitro Growth at Low Temperature

Pink snow mould is a serious disease on grasses and winter cereals in cold and temperate zones during winter. To better understand the basis for the variation in pathogenicity between different isolates of Microdochium nivale and M. majus and to simplify selection of highly pathogenic isolates to use when screening for resistance to pink snow mould in perennial ryegrass, we sought traits correlated with pathogenicity. Isolates of M. nivale were more pathogenic on perennial ryegrass than isolates of M. majus, as measured by survival and regrowth of perennial ryegrass after infection and incubation under simulated snow cover. Pathogenicity as measured by relative regrowth was highly correlated with fungal growth rate on potato dextrose agar (PDA) at 2°C. Measuring fungal growth on PDA therefore seems to be a relatively simple method of screening for potentially highly pathogenic isolates. In a study of a limited number of isolates, highly pathogenic isolates showed an earlier increase and a higher total specific activity of β‐glucosidase, a cell wall‐degrading enzyme, compared with less pathogenic isolates. None of the M. majus isolates was highly pathogenic on perennial ryegrass. Our results indicate biological differences between M. nivale and M. majus and thus strengthen the recently published sequence‐based evidence for the elevation of these former varieties to species status.     

Studies on the relationship between the pathogenicity of Paracoccidioides brasiliensis in mice and its growth rate under different oxygen atmospheres

We performed comparative studies of the pathogenicity of six strains of Paracoccidioides brasiliensis (Bt-9, Bt-4, Pb-9, Pb-18, Bt-7 and B-1183) for young adult male ddY mice and the growth rate of each strain under different oxygen atmospheres (aerobic, micro-aerobic and anaerobic atmospheres) at 37 °C. 10⁶ units of yeast cells were intravenously injected into each mouse. The pathogenicity of each isolate was determined by a scoring system based on organ culture and histopathological findings. The growth rates under different oxygen atmospheres were determined by a scoring system in which 300 fungal units per strain were counted. The strain Bt-9 showed the greatest pathogenicity, followed by Bt-4. Pb-9 and Pb-18 had on intermediate rank of pathogenicity. Bt-7 and B-1183 were the least pathogenic of the strains tested. Except for strain Bt-7 all strains showed an excellent growth under an aerobic atmosphere. Bt-4 and Bt-9 also showed excellent growth under a micro-aerobic atmosphere, followed by Pb-9, whereas the growth of Pb-18, Bt-7 and B-1183 was limited. There was a correlation between the growth rate under a micro-aerobic atmosphere and the pathogenicity of a strain. The growth rate of P. brasiliensis under a micro-aerobic atmosphere strongly correlated to its pathogenicity.