Persistent protease-activated receptor 4 signaling mediates thrombin-induced microglial activation

We have previously reported that thrombin, the ultimate serine protease in the coagulation cascades, is a proinflammatory agent that causes proliferation and activation of brain microglial cells. However, participation of its principal receptor, the protease-activated receptor 1 (PAR1) appears to be limited to promoting microglial proliferation and not induction of inflammatory mediators. In the present study, we now report that thrombin action in promoting inflammatory mediators from brain microglia is mediated through another thrombin receptor, PAR4. Here we show that the PAR4 agonist peptide (PAR4AP, GYPGKF), but not the PAR1AP (TRAP, SFLLRN), induced tumor necrosis factor-alpha (TNF-alpha) production not only in cultured murine microglial cells in vitro but also in rat cortex in vivo. Down-regulation of PAR4 expression in microglial cultures by a specific antisense, but not a sense, oligonucleotide reduced PAR4AP-induced TNF-alpha. Mechanistic studies indicated that, in comparison with PAR1 signaling, prolonged increase of [Ca2+]i and phosphorylation of p44/42 mitogen-activated protein kinases, as well as NFkappaB activation may be responsible for PAR4AP-induced TNF-alpha production in microglia. Taken together, these results demonstrate that PAR4 activation mediates the potentially detrimental effects of thrombin on microglia, implying that perspectives of exploiting PAR1 as a potential anti-inflammatory target should be shifted toward PAR4 as a much more specific therapeutic target in brain inflammatory conditions associated with neurotrauma and neurodegenerations.     

Tandem heterocyclization activity of the multidomain 230 kDa HMWP2 subunit of Yersinia pestis yersiniabactin synthetase: interaction of the 1-1382 and 1383-2035 fragments.

The six-domain, 2035-amino acid subunit high-molecular weight protein 2 (HMWP2) activates salicylate and two cysteines and loads them covalently on its three carrier protein domains during assembly of the iron-chelating virulence factor, yersiniabactin of the plague bacterium Yersinia pestis. The 1-1382 fragment of HMWP2 (ArCP-Cy1-A), overproduced in Escherichia coli, contains the first three domains: the aryl carrier protein (ArCP) domain, the cysteine specific adenylation domain (A), and the first condensation/cyclization domain (Cy1). The ArCP can be posttranslationally phosphopantetheinylated on Ser52 and then loaded with a salicyl group on the phosphopantetheine (Ppant) thiol by action of the YbtE, a salicyl-AMP ligase. The HMWP2 1-1382 fragment can activate L-cysteine as Cys-AMP. The HMWP2 1383-2035 fragment contains the remaining three domains: two peptidyl carrier proteins (PCP1 and PCP2) separated by a second condensation/cyclization domain (Cy2). Phosphopantetheinylation of the HMWP2 1383-2035 fragment at Ser1439 (PCP1) and Ser1977 (PCP2) facilitates cysteinylation of both thiols by HMWP2 1-1382. When the holo 1-1382 and bis-holo 1383-2035 protein fragments are mixed with ATP, salicylate, and cysteine, four products are slowly released [salicylcysteine (Sal-Cys), (hydroxyphenylthiazolinyl)cysteine (HPT-Cys), HPT-Cys-Cys, and the bisheterocyclic HPTT-Cys], reflecting thiolytic rerouting by cysteine in solution of elongating acyl-S-enzyme intermediates tethered at ArCP, PCP1, and PCP2 carrier protein domains, respectively. Conducting the in trans reconstitution with the S1439A mutant of HMWP2 1383-2035 releases only Sal-Cys, while the S1977A mutant leads to HPT-Cys formation but not HPT-Cys-Cys or HPTT-Cys. These results suggest localization of particular acyl-S-enzyme intermediates to each of the three carrier protein regions and also establish the sequential action of Cy1 and Cy2, with the latter producing the tandem 4,2-bisheterocyclic hydroxyphenylthiazolinylthiazolinyl (HPTT) moiety characteristic of this class of siderophores.     

Effect of vitrification on mitochondrial distribution and membrane potential in mouse two pronuclear (2‐PN) embryos

The present study was designed to investigate the effect of vitrification on mitochondrial distribution, membrane potential (Δψ) and microtubule distribution in mouse 2‐PN embryos, as well as to document the relationship between mitochondrial distribution and developmental ability of those embryos. Mitochondrial distribution was examined by fluorescence microscopy technology. Results indicated that: (1) The rate of mitochondrial ring formation around pronuclei in vitrified 2‐PN embryos was significantly lower than in fresh ones (67.3 ± 3.0% vs. 84.9 ± 3.1%) (P < 0.05). (2) Blastocyst development rate of vitrified 2‐PN embryos without mitochondrial rings (61.7 ± 4.5%) was significantly lower than that of vitrified embryos with mitochondrial rings (82.1 ± 2.8%). (3) Following staining by 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethyl‐imidacarbo‐cyanine iodide (JC‐1), most red‐colored mitochondria (high Δψ) were distributed peripherally around pronuclei and along cell membranes of fresh 2‐PN embryos. Conversely, red‐colored mitochondria were greatly diminished in vitrified embryos, with green mitochondria (low Δψ) evenly distributed throughout the cytoplasm. The proportion of fresh 2‐PN embryos with obvious aggregation of high Δψ mitochondria (84.2 ± 2.2%) was significantly higher than that of vitrified embryos (26.7 ± 3.0%) (P < 0.05). (4) The proportion of fresh embryos with microtubules distributed around pronuclei (83.5 ± 3.4%) was similar to that of vitrified embryos (74.7 ± 2.5%). In conclusion, vitrification affected mitochondrial distribution and decreased the mitochondrial membrane potential in mouse 2‐PN embryos, events which may affect subsequent developmental viability of such embryos. Mol. Reprod. Dev. 76: 1056–1063, 2009. © 2009 Wiley‐Liss, Inc.     

In Vivo Availability of Pro-Resolving Lipid Mediators in Oxazolone Induced Dermal Inflammation in the Mouse

The activation and infiltration of polymorphonuclear neutrophils (PMN) are critical key steps in inflammation. PMN-mediated inflammation is limited by anti-inflammatory and pro-resolving mechanisms, including specialized pro-resolving lipid mediators (SPM). We examined the effects of 15-epi-LXA₄ on inflammation and the biosynthesis of pro-inflammatory mediators, such as prostaglandins, leukotriene B₄ and various hydroxyeicosatetraenoic acids and SPM, in an oxazolone (OXA)-induced hypersensitivity model for dermal inflammation. 15-epi-LXA₄ (100 μM, 5 μL subcutaneously injected) significantly (P < 0.05) reduced inflammation in skin, 24 hours after the OXA challenge, as compared to skin treated with vehicle. No significant influence on the biosynthesis of prostaglandins or leukotriene B₄ was observed, whereas the level of 15S-hydroxy-eicosatetraenoic acid was significantly (P < 0.05) lower in the skin areas treated with 15-epi-LXA₄. In spite of the use of a fully validated analytical procedure, no SPM were detected in the biological samples. To investigate the reason for the lack of analytical signal, we tried to mimic the production of SPM (lipoxins, resolvins, maresin and protectin) by injecting them subcutaneously into the skin of mice and studying the in vivo availability and distribution of the compounds. All analytes showed very little lateral distribution in skin tissue and their levels were markedly decreased (> 95%) 2 hours after injection. However, docosahexaenoic acid derivatives were biologically more stable than SPM derived from arachidonic acid or eicosapentaenoic acid.     

Long-distance propagation of forces in a cell

A fundamental question in the field of mechanotransduction is how forces propagate inside a cell. Recent experiments have shown that a force of a physiological magnitude, applied via a focal adhesion, can propagate a long distance into the cell. This observation disagrees with existing models that regard the cell as a homogeneous body. We show that this "action at a distance" results from the inhomogeneity in the cell: a prestressed and stiff actin bundle guides the propagation of forces over long distances. Our models highlight the enormous ratios of the prestress and the modulus of the actin bundle to the modulus of the cytoskeleton network. For a normal cell, the models predict that forces propagate over characteristic lengths comparable to the size of the cell. The characteristic lengths can be altered, however, by treatments of the cell. We provide experimental evidence and discuss biological implications.     

Eight-Shaped Hatching Increases the Risk of Inner Cell Mass Splitting in Extended Mouse Embryo Culture

Increased risk of monozygotic twinning (MZT) has been shown to be associated with assisted reproduction techniques, particularly blastocyst culture. Interestingly, inner cell mass (ICM) splitting in human ‘8’-shaped hatching blastocysts that resulted in MZT was reported. However, the underlying cause of MZT is not known. In this study, we investigated in a mouse model whether in vitro culture leads to ICM splitting and its association with hatching types. Blastocyst hatching was observed in: (i) in vivo developed blastocysts and (ii–iii) in vitro cultured blastocysts following in vivo or in vitro fertilization. We found that ‘8’-shaped hatching occurred with significantly higher frequency in the two groups of in vitro cultured blastocysts than in the group of in vivo developed blastocysts (24.4% and 20.4% versus 0.8%, respectively; n = 805, P < 0.01). Moreover, Oct4 immunofluorescence staining was performed to identify the ICM in the hatching and hatched blastocysts. Scattered and split distribution of ICM cells was observed around the small zona opening of ‘8’-shaped hatching blastocysts. This occurred at a high frequency in the in vitro cultured groups. Furthermore, we found more double OCT4-positive masses, suggestive of increased ICM splitting in ‘8’-shaped hatching and hatched blastocysts than in ‘U’-shaped hatching and hatched blastocysts (12.5% versus 1.9%, respectively; n = 838, P < 0.01). Therefore, our results demonstrate that extended in vitro culture can cause high frequencies of ‘8’-shaped hatching, and ‘8’-shaped hatching that may disturb ICM herniation leading to increased risk of ICM splitting in mouse blastocysts. These results may provide insights into the increased risk of human MZT after in vitro fertilization and blastocyst transfer.     

Inheritance and Variation of Cytosine Methylation in Three Populus Allotriploid Populations with Different Heterozygosity

DNA methylation is an epigenetic mechanism with the potential to regulate gene expression and affect plant phenotypes. Both hybridization and genome doubling may affect the DNA methylation status of newly formed allopolyploid plants. Previous studies demonstrated that changes in cytosine methylation levels and patterns were different among individual hybrid plant, therefore, studies investigating the characteristics of variation in cytosine methylation status must be conducted at the population level to avoid sampling error. In the present study, an F1 hybrid diploid population and three allotriploid populations with different heterozygosity [originating from first-division restitution (FDR), second-division restitution (SDR), and post-meiotic restitution (PMR) 2n eggs of the same female parent] were used to investigate cytosine methylation inheritance and variation relative to their common parents using methylation-sensitive amplification polymorphism (MSAP). The variation in cytosine methylation in individuals in each population exhibited substantial differences, confirming the necessity of population epigenetics. The total methylation levels of the diploid population were significantly higher than in the parents, but those of the three allotriploid populations were significantly lower than in the parents, indicating that both hybridization and polyploidization contributed to cytosine methylation variation. The vast majority of methylated status could be inherited from the parents, and the average percentages of non-additive variation were 6.29, 3.27, 5.49 and 5.07% in the diploid, FDR, SDR and PMR progeny populations, respectively. This study lays a foundation for further research on population epigenetics in allopolyploids.     

The Value of Blood Oxygenation Level-Dependent (BOLD) MR Imaging in Differentiation of Renal Solid Mass and Grading of Renal Cell Carcinoma (RCC): Analysis Based on the Largest Cross-Sectional Area versus the Entire Whole Tumour

Objectives

To study the value of assessing renal masses using different methods in parameter approaches and to determine whether BOLD MRI is helpful in differentiating RCC from benign renal masses, differentiating clear-cell RCC from renal masses other than clear-cell RCC and determining the tumour grade.

Methods

Ninety-five patients with 139 renal masses (93 malignant and 46 benign) who underwent abdominal BOLD MRI were enrolled. R2* values were derived from the largest cross-section (R2*_largest) and from the whole tumour (R2*_whole). Intra-observer and inter-observer agreements were analysed based on two measurements by the same observer and the first measurement from each observer, respectively, and these agreements are reported with intra-class correlation coefficients and 95% confidence intervals. The diagnostic value of the R2* value in the evaluation was assessed with receiver-operating characteristic analysis.

Results

The intra-observer agreement was very good for R2*_largest and R2*_whole (all > 0.8). The inter-observer agreement of R2*_whole (0.75, 95% confidence interval: 0.69~0.79) was good and was significantly improved compared with the R2*_largest (0.61, 95% confidence interval: 0.52~0.68), as there was no overlap in the 95% confidence interval of the intra-class correlation coefficients. The diagnostic value in differentiating renal cell carcinoma from benign lesions with R2*_whole (AUC=0.79/0.78[observer1/observer2]) and R2*_largest (AUC=0.75[observer1]) was good and significantly higher (p=0.01 for R2*_largest[observer2] vs R2*_whole[observer2], p<0.01 for R2*_whole[observer1] vs R2*_largest[observer2]) than R2*_largest for observer 2 (AUC=0.64). For the grading of clear-cell RCC, both R2*_whole and R2*_largest were good (all > 0.7) and were not significantly different (p=0.89/0.93 for R2*largest vs R2*whole[observer1/observer2], 0.96 for R2*whole[observer1] vs R2*largest[observer2] and 0.96 for R2*whole [observer2] vs R2*largest[observer1]).

Conclusions

BOLD MRI could provide a feasible parameter for differentiating renal cell carcinoma from benign renal masses and for predicting clear-cell renal cell carcinoma grading. Compared with the largest cross-section, assessing the whole tumour provides better inter-observer agreement in parameter measurement for differentiating renal cell carcinoma from benign renal masses.     

子宫内膜异位症患者血清可溶性B7-H4的水平及意义

目的:探讨子宫内膜异位症患者血清可溶性B7-H4(sB7-H4)的水平及其临床意义。方法:用ELISA夹心法检测43例子宫内膜异位症患者术前血清sB7-H4的水平及40例子宫内膜异位症患者术后血清sB7-H4的水平,同时选取30例体检健康妇女血清sB7-H4水平作为对照。结果:子宫内膜异位症患者血清sB7-H4水平为(36.23±5.67)μg/L,体检健康者血清sB7-H4水平为(31.24±4.56)μg/L,两者比较差异有统计学意义(P0.01)。手术前,子宫内膜异位症患者血清sB7-H4水平为(36.23±5.67)μg/L,明显高于术后(32.54±4.27)μg/L(P0.05)。子宫内膜异位症患者血清sB7-H4水平与CA125水平呈显著正相关(r=0.531,P0.01)。结论:血清可溶性B7-H4可能与子宫内膜异位症的发病有关,检测血清中可溶性B7-H4水平对内异症的辅助诊断和疗效观察可能具有一定的临床意义。