Highly conserved glycine 86 and arginine 87 residues contribute differently to the structure and activity of the mature HIV‐1 protease

The structural and functional role of conserved residue G86 in HIV‐1 protease (PR) was investigated by NMR and crystallographic analyses of substitution mutations of glycine to alanine and serine (PR_G86A and PR_G86S). While PR_G86S had undetectable catalytic activity, PR_G86A exhibited ～6000‐fold lower catalytic activity than PR. ¹H‐¹⁵N NMR correlation spectra revealed that PR_G86A and PR_G86S are dimeric, exhibiting dimer dissociation constants (K_d) of ～0.5 and ～3.2 μM, respectively, which are significantly lower than that seen for PR with R87K mutation (K_d > 1 mM). Thus, the G86 mutants, despite being partially dimeric under the assay conditions, are defective in catalyzing substrate hydrolysis. NMR spectra revealed no changes in the chemical shifts even in the presence of excess substrate, indicating very poor binding of the substrate. Both NMR chemical shift data and crystal structures of PR_G86A and PR_G86S in the presence of active‐site inhibitors indicated high structural similarity to previously described PR/inhibitor complexes, except for specific perturbations within the active site loop and around the mutation site. The crystal structures in the presence of the inhibitor showed that the region around residue 86 was connected to the active site by a conserved network of hydrogen bonds, and the two regions moved further apart in the mutants. Overall, in contrast to the role of R87 in contributing significantly to the dimer stability of PR, G86 is likely to play an important role in maintaining the correct geometry of the active site loop in the PR dimer for substrate binding and hydrolysis. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Effect of diabetic duration on serum concentrations of endogenous inhibitor of nitric oxide synthase in patients and rats with diabetes

This study was designed to investigate the effect of diabetic duration on serum concentrations of endogenous inhibitor of nitric oxide synthase N(G), N(G)-asymmetric dimethylarginine (ADMA) in patients and rats with diabetes, and to determine whether elevated endogenous ADMA is implicated in endothelial dysfunction or macroangiopathy in diabetes. Experimental diabetic model was induced by a single intraperitoneal injection of streptozotocin to male Sprague-Dawley rats and fed for 2-, 4- and 8-week, respectively. Type 2 diabetic patients with different diabetic duration were recruited from Xiangya Hospital. Plasma glucose and serum ADMA levels were measured in both patients and rats. Moreover, endothelium-dependent relaxation of thoracic aortas and some parameters of metabolic control were examined in rats. Serum ADMA concentrations were significantly elevated in type 2 diabetic patients compared with healthy subjects (3.44 +/- 0.40 vs 1.08 +/- 0.14 micromol/L, n = 50 in diabetic patients and n = 40 in healthy subjects, P < 0.01). The serum levels of ADMA in patients with macroangiopathy were higher than the patients without macroangiopathy (P < 0.01). But no difference was observed in serum ADMA concentrations between groups of patients with different diabetic duration. Similarly, serum levels of ADMA in diabetic rats were also significantly elevated at 2-week duration compared with duration-matched control (3.71 +/- 0.20 vs 1.04 +/- 0.23 micromol/L, n = 5 approximately 6, P < 0.01). This elevation of ADMA was retained to 4- and 8-week (3.54 +/- 0.76 vs 0.95 +/- 0.06 micromol/L for 4-week, 3.21 +/- 0.50 vs 1.03 +/- 0. 09 micromol/L for 8-week, n = 5 approximately 6, all P < 0.01) and remained unchanged among three diabetic groups. The elevation of ADMA was accompanied by impairment of endothelium-dependent relaxation and poor metabolic control in diabetic rat. These results first reveal that the extent of elevation in serum ADMA in both rats and patients with diabetes is not proportion with the length of their diabetic duration but rather with the metabolic control of this disease. Elevated endogenous ADMA may be implicated in diabetes-induced endothelial dysfunction and macroangiopathy. This study is helpful to prevention and treatment of diabetic-induced endothelial dysfunction or macroangiopathy.     

Predicting the suitable habitats of parasitic desert species based on a niche model with Haloxylon ammodendron and Cistanche deserticola as examples

Haloxylon ammodendron, an excellent tree species for sand fixation and afforestation in the desert areas of western China, is threatened by climate change and anthropogenic activities. The suitable habitat of this species is shrinking at a remarkable rate, although conservation measures have been implemented. Cistanche deserticola is an entirely parasitic herb that occurs in deserts, is a source of “desert ginseng” worldwide, and has extremely high medicinal value. Little is known about using niche models to simulate habitat suitability and evaluate important environmental variables related to parasitic species. In this study, we modeled the current suitable habitat of H. ammodendron and C. deserticola by MaxEnt based on occurrence record data of the distributions of these two species in China. We grouped H. ammodendron and C. deserticola into three groups according to the characteristics of parasitic species and modeled them with environmental factors. The results showed that bioclimate was the most important environmental parameter affecting the H. ammodendron and C. deserticola distribution. Precipitations, such as annual precipitation, precipitation seasonality, and precipitation in the driest quarter, were identified as the most critical parameters. The slope, diurnal temperature range, water vapor pressure, ground‐frost frequency, and solar radiation also substantially contributed to the distribution of the two species. The proportions of the most suitable areas for Groups 1, 2, and 3 were 1.2%, 1.3%, and 1.7%, respectively, in China. When combined with cultural geography, five hot spot conservation areas were determined within the distribution of H. ammodendron and C. deserticola. The comprehensive analysis indicated that by using MaxEnt to model the suitable habitat of parasitic species, we further improved the accuracy of the prediction and coupled the error of the distribution of a single species. This study provides a useful reference for the protection of H. ammodendron forests and the management of C. deserticola plantations.     

CRMP2 derived from cancer associated fibroblasts facilitates progression of ovarian cancer via HIF-1α-glycolysis signaling pathway

As the predominant stroma cells of tumor microenvironment (TME), cancer associated fibroblasts (CAFs) are robust tumor player of different malignancies. However, less is known about the regulatory mechanism of CAFs on promoting progression of ovarian cancer (OvCA). In the present study, the conditioned medium of primary CAFs (CAF-CM) from OvCA was used to culture cell lines of epithelial ovarian cancer (EOC), and showed a potent role in promoting proliferation, migration and invasion of cancer cells. Mass spectrum (MS) analysis identified that Collapsin response mediator protein-2 (CRMP2), a microtubule-associated protein involved in diverse malignancies, derived from CAFs was a key regulator responsible for mediating these cell events of OvCA. In vitro study using recombinant CRMP2 (r-CRMP2) revealed that the protein promoted proliferation, invasion, and migration of OvCA cells through activation of hypoxia-inducible factor (HIF)-1α-glycolysis signaling pathway. The CRMP2 was abundantly expressed in OvCA, with a well correlation with metastasis and poor prognosis, as analyzed from 118 patients’ samples. Inhibition of the CRMP2 derived from CAFs by neutralizing antibodies significantly attenuated the tumor size, weights, and metastatic foci numbers of mice in vivo. Our finding has provided a novel therapeutic clue for OvCA based on TME.Subject terms: Cancer, Tumour biomarkers     

枯草芽孢杆菌中bacilosarcin B对美人鱼发光杆菌的抑菌机理

【背景】美人鱼发光杆菌(Photobacteriumdamselae)是一种海洋条件致病菌,能够引起多种海洋生物和人类疾病。因此,探究美人鱼发光杆菌的生物防治技术具有重要意义。【目的】探究枯草芽孢杆菌(Bacillus subtilis)中bacilosarcin B对美人鱼发光杆菌的抑菌活性及其可能的抑菌机理。【方法】利用高效液相色谱法从枯草芽孢杆菌fmb60发酵液中制备bacilosarcin B,采用分光光度法测定bacilosarcinB对多种致病菌的最小抑菌浓度及其对美人鱼发光杆菌的时间-抑菌曲线。测定bacilosarcin B对美人鱼发光杆菌生物被膜、胞外核酸、蛋白质和胞内碱性磷酸酶含量的影响,结合荧光显微镜、扫描电镜和透射电镜检测美人鱼发光杆菌细胞膜通透性和细胞壁完整性,并研究bacilosarcin B对细菌运动能力和胞内DNA的作用。【结果】Bacilosarcin B对美人鱼发光杆菌最小抑菌浓度为8μg/mL。抑菌机理研究表明bacilosarcin B通过破坏细菌细胞壁和细胞膜的完整性使细胞膜通透性增强,造成细胞内成分渗出。此外,bacilosarcinB还可与...     

RNA interference of metastasis-associated gene 1 inhibits metastasis of B16F10 melanoma cells in a C57BL/6 mouse model

Background information. MTA1 (metastasis‐associated gene 1) has been reported to be overexpressed in cancers with high potential to metastasize. Studies of the molecular mechanisms revealed that MTA1 plays an important role in the process of metastasis of many types of cancer. However, the role of MTA1 in melanoma development is unclear. Results. We have investigated the therapeutic value of MTA1 in the B16F10 melanoma cell line with the C57BL/6 mouse model. Studies in vitro showed that MTA1 promoted the metastatic ability of B16F10 cancer cells. MTA1 down‐regulation by RNA interference greatly reversed the malignant phenotypes of cancer cells. Immunohistochemical staining of MTA1 in human melanoma samples confirmed the up‐regulation of MTA1 in the process of carcinogenesis. Studies in vivo confirmed down‐regulation of MTA1 suppressed the growth and experimental metastasis of B16F10 melanoma cells. Conclusions. MTA1 plays an important role in melanoma development and metastasis. It has a promising potential as a target for in cancer gene therapy or chemotherapy.     

Phosphoproteomic analysis reveals that PP4 dephosphorylates KAP-1 impacting the DNA damage response

Protein phosphatase PP4C has been implicated in the DNA damage response (DDR), but its substrates in DDR remain largely unknown. We devised a novel proteomic strategy for systematic identification of proteins dephosphorylated by PP4C and identified KRAB-domain-associated protein 1 (KAP-1) as a substrate. Ionizing radiation leads to phosphorylation of KAP-1 at S824 (via ATM) and at S473 (via CHK2). A PP4C/R3β complex interacts with KAP-1 and silencing this complex leads to persistence of phospho-S824 and phospho-S473. We identify a new role for KAP-1 in DDR by showing that phosphorylation of S473 impacts the G2/M checkpoint. Depletion of PP4R3β or expression of the phosphomimetic KAP-1 S473 mutant (S473D) leads to a prolonged G2/M checkpoint. Phosphorylation of S824 is necessary for repair of heterochromatic DNA lesions and similar to cells expressing phosphomimetic KAP-1 S824 mutant (S824D), or PP4R3β-silenced cells, display prolonged relaxation of chromatin with release of chromatin remodelling protein CHD3. Our results define a new role for PP4-mediated dephosphorylation in the DDR, including the regulation of a previously undescribed function of KAP-1 in checkpoint response.