Thrombin inhibition by serpins disrupts exosite II

Thrombin uses three principal sites, the active site, exosite I, and exosite II, for recognition of its many cofactors and substrates. It is synthesized in the zymogen form, prothrombin, and its activation at the end of the blood coagulation cascade results in the formation of the active site and exosite I and the exposure of exosite II. The physiological inhibitors of thrombin are all serpins, whose mechanism involves significant conformational change in both serpin and protease. It has been shown that the formation of the thrombin-serpin final complex disorders the active site and exosite I of thrombin, but exosite II is thought to remain functional. It has also been hypothesized that thrombin contains a receptor-binding site that is exposed upon final complex formation. The position of this cryptic site may depend on the regions of thrombin unfolded by serpin complexation. Here we investigate the conformation of thrombin in its final complex with serpins and find that in addition to exosite I, exosite II is also disordered, as reflected by a loss of affinity for the γ'-peptide of fibrinogen and for heparin and by susceptibility to limited proteolysis. This disordering of exosite II occurs for all tested natural thrombin-inhibiting serpins. Our data suggest a novel framework for understanding serpin function, especially with respect to thrombin inhibition, where serpins functionally "rezymogenize" proteases to ensure complete loss of activity and cofactor binding.     

The PEA-15 Protein Regulates Autophagy via Activation of JNK

PEA-15/PED (phosphoprotein enriched in astrocytes 15 kDa/phosphoprotein enriched in diabetes) is a death effector domain-containing protein which is known to modulate apoptotic cell death. The mechanism by which PEA-15 inhibits caspase activation and increases ERK (extracellular-regulated kinase) activity is well characterized. Here, we demonstrate that PEA-15 is not only pivotal in the activation of the ERK pathway but also modulates JNK (c-Jun N-terminal kinase) signaling. Upon overexpression of PEA-15 in malignant glioma cells, JNK is potently activated. The PEA-15-induced JNK activation depends on the phosphorylation of PEA-15 at both phosphorylation sites (serine 104 and serine 116). The activation of JNK is substantially inhibited by siRNA-mediated down-regulation of endogenous PEA-15. Moreover, we demonstrate that glioma cells overexpressing PEA-15 show increased signs of autophagy in response to classical autophagic stimuli such as ionizing irradiation, serum deprivation, or rapamycin treatment. In contrast, the non-phosphorylatable mutants of PEA-15 are not capable of promoting autophagy. The inhibition of JNK abrogates the PEA-15-mediated increase in autophagy. In conclusion, our data show that PEA-15 promotes autophagy in glioma cells in a JNK-dependent manner. This might render glioma cells more resistant to adverse stimuli such as starvation or ionizing irradiation.     

Total body nitrogen and total body carbon as indicators of body protein and body lipids in the melon fly Bactrocera cucurbitae: Effects of methoprene, a juvenile hormone analogue, and of diet supplementation with hydrolyzed yeast

The application of methoprene, and providing access to diet including hydrolyzed yeast, are treatments known to enhance mating success in the male melon fly Bactrocera cucurbitae Coquillett (Diptera: Tephritidae), supporting their use in mass rearing protocols for sterile males in the context of sterile insect technique (SIT) programmes. The objective of the present laboratory study was to investigate the effect of methoprene application and diet supplementation with hydrolyzed yeast (protein) on the turnover of body lipids and protein to confirm the feasibility of their application in melon fly SIT mass-rearing programmes. While females had access to a diet that included hydrolyzed yeast (protein), males were exposed to one of the following treatments: (1) topical application of methoprene and access to diet including protein (M+P+); (2) only diet including protein (M−P+); (3) only methoprene (M+P−) and (4) untreated, only sugar-fed, control males (M−P−). Total body carbon (TBC) and total body nitrogen (TBN) of flies were measured at regular intervals from emergence to 35 days of age for each of the different treatments. Nitrogen assimilation and turnover in the flies were measured using stable isotope (¹⁵N) dilution techniques. Hydrolyzed yeast incorporation into the diet significantly increased male body weight, TBC and TBN as compared to sugar-fed males. Females had significantly higher body weight, TBC and TBN as compared to all males. TBC and TBN showed age-dependent changes, increasing until the age of sexual maturity and decreasing afterwards in both sexes. Methoprene treatment did not significantly affect TBC or TBN. The progressive increase with age of TBC suggests that lipogenesis occurs in adult male B. cucurbitae, as is the case in other tephritids. Stable isotope dilution was shown to be an effective method for determining N uptake in B. cucurbitae. This technique was used to show that sugar-fed males rely solely on larval N reserves and that the N uptake rate in males with access to diet including hydrolyzed yeast was higher shortly after emergence and then stabilized. The implications of the results for SIT applications are discussed.     

胃癌组织中15-PGDH的表达特征及其临床意义

目的:探讨15-羟基前列腺素脱氢酶(15-PGDH)在胃癌组织和癌旁正常组织中的表达及其与临床病理特征的关系,初步评价15-PGDH在胃癌发生、发展中的作用及其意义。方法:随机收集60例胃癌手术病人的癌组织及相应癌旁正常组织,应用免疫组织化学检测15-PGDH表达特征并进行评分,进一步分析15-PGDH的表达与胃癌临床病理参数的关系。结果:胃癌组织中15-PGDH的表达水平显著降低甚至缺失(p<0.01)。结合胃癌的临床病理学特征统计分析表明,15-PGDH在低分化胃癌中明显低于高分化胃癌中的表达(p<0.05);15-PGDH在TNMⅢ期和Ⅳ期患者的胃癌组织中的染色明显低于在Ⅰ期和Ⅱ期组织中的表达(p<0.05);存在淋巴结转移的胃癌组织中15-PGDH表达显著低于无淋巴结转移胃癌组织中的表达(p<0.01)。结论:胃癌中15-PGDH的表达减少或缺失可能是胃癌发生、发展及浸润转移的重要机制之一,15-PGDH在胃癌中可能扮演着抑癌基因的角色。     

The cancer cell secretome: A good source for discovering biomarkers?

Cancer is a leading cause of death. Early detection is usually associated with better clinical outcomes. Recent advances in genomics and proteomics raised hopes that new biomarkers for diagnosis, prognosis or monitoring therapeutic response will soon be discovered. Proteins secreted by cancer cells, referred also as “the cancer cell secretome”, is a promising source for biomarker discovery. In this review we will summarize recent advances in cancer cell secretome analysis, focusing on the five most fatal cancers (lung, breast, prostate, colorectal, and pancreatic). For each cancer type we will describe the proteomic approaches utilized for the identification of novel biomarkers. Despite progress, identification of markers that are superior to those currently used has proven to be a difficult task and very few, if any, newly discovered biomarker has entered the clinic the last 10 years.     

A New Pregnane from Munronia delavayi Franc （Meliaceae）

To search for pharmacologically active constituents of folk medicine, a new pregnane, 2α,3α,15β-trihydroxy-20（S）-tigloyl-pregnane （compound 1）, and nine known compounds, geranylgeraniol （compound 2）, β-daucosterol （compound 3）, 6-hydroxystigmast-4oen-3-one （compound 4）, sitoindoside Ⅰ （compound 5）, sitoindoside Ⅱ （compound 6）, β-sitosterol （compound 7）, kaempferol （compound 8）, quercetin （compound 9）, and rutin （compound 10）, were isolated from the ethanol extract of whole plants of Munronia delavayi Franch using chromatographic methods. The structures of compounds 1-10 were elucidated on the basis of spectral data.     

Nuclear Magnetic Resonance Studies on Huwentoxin-Ⅺ from the Chinese Bird Spider Ornithoctonus huwena:~(15)N Labeling and Sequence-specific ~1H,~(15)N Nuclear Magnetic Resonance Assignments

Huwentoxin-XI purified from the Chinese bird spider Ornithoctonus huwena is a toxin withboth antiprotease activity and potassium channel blocking activity.To determine its solution structure,huwentoxin-Ⅺ was expressed in a yeast eukaryotic expression system and studied by NMR.The ~(15)N labelingstrategy was used to facilitate the process of resonance assignments.The nearly complete sequence-specificassignments of proton and nitrogen resonances were obtained by analyzing a series of two-dimensional(2D)and three-dimensional(3D)spectra,including DQF-COSY,TOCSY,NOESY,~(15)N-~1H HSQC,~(15)N-~1H HNHA,~(15)N-~1H HNHB,~(15)N-~1H TOCSY-HSQC and ~(15)N-~1H NOESY-HSQC spectra.Secondary structure analysis ofhuwentoxin-Ⅺ showed that it mainly contains an N-terminal 3_(10)-helix from Thr3 to Arg5 and a C-terminalα-helix from Gln45 to Cys52,plus a triple-stranded antiparallel β-sheet of Glul 8-Asn23,Thr26-Ile31 andAsn40-Lys41.These studies provide a solid basis for the final structure determination of huwentoxin-Ⅺ.     

Glycine origin of the methyl substituent on C7′-N of octodiose for the biosynthesis of apramycin

Apramycin is unique in the aminoglycoside family due to its octodiose moiety. However, either the biosynthesis process or the precursors involved are largely unknown. Addition of glycine, as well as serine or threonine, to the Streptomyces tenebrabrius UD2 fermentation medium substantially increases the production of apramycin with little effect on the growth of mycelia, indicat-ing that glycine and/or serine might be involved in the biosynthesis of apramycin. The ¹³C-NMR analysis of [2-¹³C] glycine-fed (25% enrichment) apramycin showed that glycine specifically and efficiently incorporated into the only N-CH₃ substituent of apramycin on the C7′ of the octodiose moiety. We noticed that the in vivo concentration of S-adenosyl methionine increased in parallel with the addition of glycine, while the addition of methione in the fermentation medium significantly decreased the productivity of apramycin. Therefore, the methyl donor function of glycine is proposed to be involved in the methionine cycle but methionine itself was proposed to inhibit the methylation and methyl transfer processes as previously reported for the case of rapamycin. The ¹⁵N NMR spectra of [2-¹³C,¹⁵N]serine labeled apramycin indicated that serine may also act as a limiting precursor contributing to the ―NH₂ substituents of apramycin.