Structural and Functional Analysis of a Platelet-Activating Lysophosphatidylcholine of Trypanosoma cruzi

Background

Trypanosoma cruzi is the causative agent of the life-threatening Chagas disease, in which increased platelet aggregation related to myocarditis is observed. Platelet-activating factor (PAF) is a potent intercellular lipid mediator and second messenger that exerts its activity through a PAF-specific receptor (PAFR). Previous data from our group suggested that T. cruzi synthesizes a phospholipid with PAF-like activity. The structure of T. cruzi PAF-like molecule, however, remains elusive.

Methodology/Principal findings

Here, we have purified and structurally characterized the putative T. cruzi PAF-like molecule by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Our ESI-MS/MS data demonstrated that the T. cruzi PAF-like molecule is actually a lysophosphatidylcholine (LPC), namely sn-1 C18:1(delta 9)-LPC. Similar to PAF, the platelet-aggregating activity of C18:1-LPC was abrogated by the PAFR antagonist, WEB 2086. Other major LPC species, i.e., C16:0-, C18:0-, and C18:2-LPC, were also characterized in all T. cruzi stages. These LPC species, however, failed to induce platelet aggregation. Quantification of T. cruzi LPC species by ESI-MS revealed that intracellular amastigote and trypomastigote forms have much higher levels of C18:1-LPC than epimastigote and metacyclic trypomastigote forms. C18:1-LPC was also found to be secreted by the parasite in extracellular vesicles (EV) and an EV-free fraction. A three-dimensional model of PAFR was constructed and a molecular docking study was performed to predict the interactions between the PAFR model and PAF, and each LPC species. Molecular docking data suggested that, contrary to other LPC species analyzed, C18:1-LPC is predicted to interact with the PAFR model in a fashion similar to PAF.

Conclusions/Significance

Taken together, our data indicate that T. cruzi synthesizes a bioactive C18:1-LPC, which aggregates platelets via PAFR. We propose that C18:1-LPC might be an important lipid mediator in the progression of Chagas disease and its biosynthesis could eventually be exploited as a potential target for new therapeutic interventions.     

Pulmonary Toxocariasis Mimicking Invasive Aspergillosis in a Patient with Ulcerative Colitis

A 45-year-old-male who had underlying ulcerative colitis and presented with fever and dry cough. Initially, the patient was considered to have invasive aspergillosis due to a positive galactomannan assay. He was treated with amphotericin B followed by voriconazole. Nevertheless, the patient deteriorated clinically and radiographically. The lung biopsy revealed eosinophilic pneumonia, and ELISA for Toxocara antigen was positive, leading to a diagnosis of pulmonary toxocariasis. After a 10-day treatment course with albendazole and adjunctive steroids, the patient recovered completely without any sequelae. Pulmonary toxocariasis may be considered in patients with subacute or chronic pneumonia unresponsive to antibiotic agents, particularly in cases with eosinophilia.     

Tumorigenesis of Papillary Thyroid Cancer Is Not BRAF-Dependent in Patients with Acromegaly

Introduction

Several studies have reported a high frequency of papillary thyroid cancer (PTC) in patients with acromegaly. The aim of this study was to determine the prevalence and predictors of thyroid cancer in patients with acromegaly and to investigate the frequency of the BRAF ^V600E mutation in PTC patients with and without acromegaly.

Materials and Methods

We conducted a retrospective study of 60 patients with acromegaly. Thyroid ultrasonography (US) and US-guided fine needle aspiration were performed on nodules with sonographic features of malignancy. We selected 16 patients with non-acromegalic PTC as a control group. The BRAF ^V600E mutation was analyzed in paraffin-embedded surgical specimens of PTC by real-time polymerase chain reaction, and tumor specimens from patients with PTC were stained immunohistochemically with an antibody against insulin-like growth factor-1 receptor β (IGF-1Rβ).

Results

Thyroid cancer was found in 15 (25.0%) patients. No differences in age, sex, initial growth hormone (GH) and IGF-1 percentage of the upper limit of normal values or treatment modalities were observed between patients with and without PTC. Acromegaly was active in 12 of 15 patients at the time of PTC diagnosis; uncontrolled acromegaly had a significantly higher frequency in the PTC group (60%) than in the non-PTC group (28.9%) (p = 0.030). The BRAF ^V600E mutation was present in only 9.1% (1/11) of PTC patients with acromegaly, although 62.5% (10/16) of control patients with PTC had the mutation (p = 0.007). IGF-1Rβ immunostaining showed moderate-to-strong staining in all malignant PTC cells in patients with and without acromegaly. Significantly less staining for IGF-1Rβ was observed in normal adjacent thyroid tissues of PTC patients with acromegaly compared with those without (p = 0.014).

Conclusion

The prevalence of PTC in acromegalic patients was high (25%). An uncontrolled hyperactive GH-IGF-1 axis may play a dominant role in the development of PTC rather than the BRAF ^V600E mutation in patients with acromegaly.     

Positional relationships between photoperiod response QTL and photoreceptor and vernalization genes in barley

Winterhardiness has three primary components: photoperiod (day length) sensitivity, vernalization response, and low temperature tolerance. Photoperiod and vernalization regulate the vegetative to reproductive phase transition, and photoperiod regulates expression of key vernalization genes. Using two barley mapping populations, we mapped six individual photoperiod response QTL and determined their positional relationship to the phytochrome and cryptochrome photoreceptor gene families and the vernalization regulatory genes HvBM5A, ZCCT-H, and HvVRT-2. Of the six photoreceptors mapped in the current study (HvPhyA and HvPhyB to 4HS, HvPhyC to 5HL, HvCry1a and HvCry2 to 6HS, and HvCry1b to 2HL), only HvPhyC coincided with a photoperiod response QTL. We recently mapped the candidate genes for the 5HL VRN-H1 (HvBM5A) and 4HL VRN-H2 (ZCCT-H) loci, and in this study, we mapped HvVRT-2, the barley TaVRT-2 ortholog (a wheat flowering repressor regulated by vernalization and photoperiod) to 7HS. Each of these three vernalization genes is located in chromosome regions determining small photoperiod response QTL effects. HvBM5A and HvPhyC are closely linked on 5HL and therefore are currently both positional candidates for the same photoperiod effect. The coincidence of photoperiod-responsive vernalization genes with photoperiod QTL suggests vernalization genes should also be considered candidates for photoperiod effects.     

Fetal lamb 3 beta, 20 alpha-hydroxysteroid oxidoreductase: dual activity at the same active site examined by affinity labeling with 16 alpha-(bromo[2'-14C]acetoxy)progesterone

3 beta,20 alpha-Hydroxysteroid oxidoreductase was purified to homogeneity from fetal lamb erythrocytes. The Mr 35,000 enzyme utilizes NADPH and reduces progesterone to 4-pregnen-20 alpha-ol-3-one [Km = 30.8 microM and Vmax = 0.7 nmol min-1 (nmol of enzyme)-1] and 5 alpha-dihydrotestosterone to 5 alpha-androstane-3 beta, 17 beta-diol [Km = 74 microM and Vmax = 1.3 nmol min-1 (nmol of enzyme)-1]. 5 alpha-Dihydrotestosterone competitively inhibits (Ki = 102 microM) 20 alpha-reductase activity, suggesting that both substrates may be reduced at the same active site. 16 alpha-(Bromoacetoxy)progesterone competitively inhibits 3 beta- and 20 alpha-reductase activities and also causes time-dependent and irreversible losses of both 3 beta-reductase and 20 alpha-reductase activities with the same pseudo-first order kinetic t1/2 value of 75 min. Progesterone and 5 alpha-dihydrotestosterone protect the enzyme against loss of the two reductase activities presumably by competing with the affinity alkylating steroid for the active site of 3 beta,20 alpha-hydroxysteroid oxidoreductase. 16 alpha-(Bromo[2'-14C]acetoxy) progesterone radiolabels the active site of 3 beta,20 alpha-hydroxysteroid oxidoreductase wherein 1 mol of steroid completely inactivates 1 mol of enzyme with complete loss of both reductase activities. Hydrolysis of the 14C-labeled enzyme with 6 N HCl at 110 degrees C and analysis of the amino acid hydrolysate identified predominantly N pi-(carboxy[2'-14C]methyl)histidine [His(pi-CM)].(ABSTRACT TRUNCATED AT 250 WORDS)     

凝血接触系统对人血管内皮细胞炎症活性的影响

内源性凝血途径的起始部分称为接触系统,包括高分子量激肽原、前激肽释放酶、XII因子和XI因子。以接触系统成分及激活产物分别刺激人血管内皮细胞,检测了其NF-κB活性、细胞间黏附分子1(ICAM-1)表达和炎性细胞因子分泌的变化。结果显示:与对照组相比较,只有游离XI因子和激活的XI因子可以使内皮细胞NF-κB活性升高,并具有统计学差异(P<0.01);而激活的XI因子能够进一步使内皮细胞的ICAM-1和细胞因子分泌显著增加(P<0.01)。其余各组与对照组相比没有统计学差异。这些观察结果提示接触系统可以直接活化血管内皮细胞,说明内源性凝血途径也参与了炎症的发展过程。     

Bemerkungen zur Variabilität vonSaxifraga marginata

Saxifraga marginata Sternb. is divided into three subspecies:S. m. subsp.marginata, S. m. subsp.bubakii (Rohlena)Chrtek etSoják,S. m. subsp.karadzicensis (Degen etKo?anin)Chrtek etSoják (environments of Skopje, Macedonia).     

二氧化硫熏气对小麦叶片中1-氨基环丙烷-1-羧酸、1-(丙二酰氨基)环丙烷-1-羧酸含量的影响及与乙烯产生的关系

在SO_2熏气9h过程中,小麦叶片中乙烯先上升,约6h达高峰,后下降;ACC含量则随熏气时间的延长而上升。停止熏气,乙烯继续下降,ACC含量也明显降低。MACC含量从熏气3h后不断上升,脱离接触后仍继续增加。6-BA预处理对SO_2引起的乙烯和ACC上升有促进作用,但对MACC含量无明显影响。SO_2熏气提高了乙烯形成酶活性。6-BA预处理对SO_2伤害有保护作用。对逆境乙烯的产生与调节作用进行了讨论。     

大肠杆菌青霉素G酰化酶基因及其邻近区域的核苷酸全序列

Some of microorganisms have been known to possess penicillin G acylase activity. The E. coli derived penicillin G acylase (PGA) can catalyze the conversion of penicillin G into phenylacetic acid and 6-amino-penicillanic acid, the latter is used as the starting compound for the industrial formation of semi-synthetic penicillins. Apart from its industrial importance, the enzyme PGA displays a number of interesting properties. Catalytically active enzyme is localized in the periplasmic space of E. coli cells and composed of two dissimilar subunits. The two subunits are apparently produced from a precursor protein, via a processing pathway hitherto unique in its features for a prokaryotic enzyme. The studies on processing of the precursor and on the relationship between structure and function of the mature enzyme are important theoretically. Previously we cloned a 3.5 kb DNA fragment from a strain (E. coli AS 1.76), which displays PGA activity. In this paper, we report a nucleotide sequence of the 3.5 kb DNA fragment containing PGA gene. After insertion of the DNA fragment into EcoR I and Hind III sites in pWR 13, pPGA 20 had been obtained. We subcloned the Hind III and Bg1 II treated fragment of 1.6 kb in length from pPGA 20 into Hind III and BamH I sites of pWR 13 to get a pPGA 1.6, and Bg1 II and EcoR I treated fragment of 1.9 kb in length into BamH I and EcoR I sites of pWR 13 to get a pPGA 1.9. The linearized pPGA 1.9 which were digested with appropriate restriction enzymes were progressively shortened from both ends respectively by digestion with Bal 31 nuclease, followed by cleavage of shortened target DNA off vector DNA molecules with appropriate restriction enzymes. The series of the DNA fragments shortened from EcoR I end were then cloned into plasmid pWR 13 which had previously digested with Hind III and Sma I enzymes (Fig. 1). The DNA fragment cloned in pWR 13 were directly sequenced on the resulted plasmids by using primer I and primer II. Thus we have obtained the complete nucleotide sequence of the 3.5 kb DNA fragment. The 3.5 kb fragment contains an intact PGA gene which is 2.6 kb.(ABSTRACT TRUNCATED AT 400 WORDS)