Nupr1 deficiency downregulates HtrA1, enhances SMAD1 signaling,and suppresses age-related bone loss in male mice

Nuclear protein 1 (NUPR1) is a stress-induced protein activated by various stresses, such as inflammation and oxidative stress. We previously reported that Nupr1 deficiency increased bone volume by enhancing bone formation in 11-week-old mice. Analysis of differentially expressed genes between wild-type (WT) and Nupr1-knockout (Nupr1-KO) osteocytes revealed that high temperature requirement A 1 (HTRA1), a serine protease implicated in osteogenesis and transforming growth factor-β signaling was markedly downregulated in Nupr1-KO osteocytes. Nupr1 deficiency also markedly reduced HtrA1 expression, but enhanced SMAD1 signaling in in vitro-cultured primary osteoblasts. In contrast, Nupr1 overexpression enhanced HtrA1 expression in osteoblasts, suggesting that Nupr1 regulates HtrA1 expression, thereby suppressing osteoblastogenesis. Since HtrA1 is also involved in cellular senescence and age-related diseases, we analyzed aging-related bone loss in Nupr1-KO mice. Significant spine trabecular bone loss was noted in WT male and female mice during 6−19 months of age, whereas aging-related trabecular bone loss was attenuated, especially in Nupr1-KO male mice. Moreover, cellular senescence-related markers were upregulated in the osteocytes of 6−19-month-old WT male mice but markedly downregulated in the osteocytes of 19-month-old Nupr1-KO male mice. Oxidative stress-induced cellular senescence stimulated Nupr1 and HtrA1 expression in in vitro-cultured primary osteoblasts, and Nupr1 overexpression enhanced p16^ink4a expression in osteoblasts. Finally, NUPR1 expression in osteocytes isolated from the bones of patients with osteoarthritis was correlated with age. Collectively, these results indicate that Nupr1 regulates HtrA1-mediated osteoblast differentiation and senescence. Our findings unveil a novel Nupr1/HtrA1 axis, which may play pivotal roles in bone formation and age-related bone loss.     

Fermentation of wheat: effects of backslopping different proportions of pre-fermented wheat on the microbial and chemical composition

The objective of the study was to examine effect of backslop on the chemical and microbiological characteristics of fermented wheat (FW). Coarsely ground wheat was mixed with water (1:3 wt/wt) and inoculated with 6 log cfu ml(-1) each of an overnight culture of Lactobacillus plantarum and Pediococcus pentosaceus. Four fermentation treatments were conducted in 45 1, closed, PVC containers over 48 hours. Three treatments investigated the benefits of the addition of previously fermented wheat (backslopping, BSL) at different proportions (0.20, 0.33 or 0.42 kg) to freshly prepared wheat. The control treatment contained no addition of BSL. Elimination of coliforms from the FW within 48 h was only achieved through backslopping; where coliform bacteria counts decreased from approximately 6.5 log10 cfu ml(-1) to less than 3 log10 cfu ml(-1). There was no apparent advantage in increasing the backslop proportion above 0.20. However, the exclusion of coliform bacteria required the pH to remain below 4.0 for at a minimum of 24 h. The results of these studies indicate that fermentation of wheat has the potential to reduce the risk of feed-borne colibacillosis and provides a practical alternative to producers that cannot ferment multiple diets or have limited fermentation capacity.     

Calcium occlusion in plasma membrane Ca2+-ATPase

In this work, we set out to identify and characterize the calcium occluded intermediate(s) of the plasma membrane Ca(2+)-ATPase (PMCA) to study the mechanism of calcium transport. To this end, we developed a procedure for measuring the occlusion of Ca(2+) in microsomes containing PMCA. This involves a system for overexpression of the PMCA and the use of a rapid mixing device combined with a filtration chamber, allowing the isolation of the enzyme and quantification of retained calcium. Measurements of retained calcium as a function of the Ca(2+) concentration in steady state showed a hyperbolic dependence with an apparent dissociation constant of 12 ± 2.2 μM, which agrees with the value found through measurements of PMCA activity in the absence of calmodulin. When enzyme phosphorylation and the retained calcium were studied as a function of time in the presence of La(III) (inducing accumulation of phosphoenzyme in the E(1)P state), we obtained apparent rate constants not significantly different from each other. Quantification of EP and retained calcium in steady state yield a stoichiometry of one mole of occluded calcium per mole of phosphoenzyme. These results demonstrate for the first time that one calcium ion becomes occluded in the E(1)P-phosphorylated intermediate of the PMCA.     

实验藏酋猴日粮蛋白质营养水平研究

目的本试验旨在研究不同蛋白水平对实验藏酋猴生长性能和血液生化指标的影响并确定幼年实验藏酋猴的蛋白营养需要量。方法选用15只幼年实验藏酋猴随机分为5个处理组,每个处理3个重复,分别饲喂蛋白水平为11．1％、16．1％、20．5％、24．5％和29．8％的日粮,试验期90d。结果随着日粮中蛋白水平的提高,实验藏酋猴的增重、血清白蛋白、球蛋白、天门冬氨酸氨基转移酶、丙氨酸氨基转移酶和尿素氮含量显著增加（P〈0．05）,而血清总蛋白、甘油三酯、γ-谷氨酰胺转移酶和碱性磷酸酶无显著差异。应用折线法确定幼年实验藏酋猴的蛋白营养需要量为23．69％。结论日粮蛋白水平对实验藏酋猴生长性能有显著影响,从生物学和经济学角度考虑,初步认为在本试验条件下,未成年实验藏酋猴日粮蛋白水平以23．69％最合适。     

Expression of baculovirus anti-apoptotic genes p35 and op-iap in cotton (Gossypium hirsutum L.) enhances tolerance to verticillium wilt

Background

Programmed cell death plays an important role in mediating plant adaptive responses to the environment such as the invasion of pathogens. Verticillium wilt, caused by the necrotrophic pathogen Verticillium dahliae, is a serious vascular disease responsible for great economic losses to cotton, but the molecular mechanisms of verticillium disease and effective, safe methods of resistance to verticillium wilt remain unexplored.

Methodology/Principal Findings

In this study, we introduced baculovirus apoptosis inhibitor genes p35 and op-iap into the genome of cotton via Agrobacterium-mediated transformation and analyzed the response of transgenic plants to verticillium wilt. Results showed that p35 and op-iap constructs were stably integrated into the cotton genome, expressed in the transgenic lines, and inherited through the T₃ generation. The transgenic lines had significantly increased tolerance to verticillium wilt throughout the developmental stages. The disease index of T₁–T₃ generation was lower than 19, significantly (P<0.05) better than the negative control line z99668. After treatment with 250 mg/L VD-toxins for 36 hours, DNA from negative control leaves was fragmented, whereas fragmentation in the transgenic leaf DNA did not occur. The percentage of cell death in transgenic lines increased by 7.11% after 60 mg/L VD-toxin treatment, which was less than that of the negative control lines''s 21.27%. This indicates that p35 and op-iap gene expression partially protects cells from VD-toxin induced programmed cell death (PCD).

Conclusion/Significance

Verticillium dahliae can trigger plant cells to die through induction of a PCD mechanism involved in pathogenesis. This paper provides a potential strategy for engineering broad-spectrum necrotrophic disease resistance in plants.     

The solvent in CNBr cleavage reactions determines the fragmentation efficiency of ketosteroid isomerase fusion proteins used in the production of recombinant peptides

Abnormal fragmentation during cyanogen bromide polypeptide cleavage rarely occurs, although parallel side reactions are known to typically accompany normal cleavage. We have observed that cyanogen bromide cleavage of highly hydrophobic fusion proteins utilized for production of recombinant peptides results in almost complete abolishment of the expected reaction products when the reaction is carried out in 70% trifluoroacetic acid. On the basis of mass spectrometric analysis of the reaction products, we have identified a number of fragments whose origin can be attributed to incomplete fragmentation of the fusion protein, and to unspecific degradation affecting the carrier protein. Substituting the solvent in the reaction media with 70% formic acid or with a matrix composed of 6M guanidinium hydrochloride in 0.1M HCl, however, was found to alleviate polypeptide cleavage. We have attributed the poor yields of the CNBr cleavage carried out in 70% TFA to the increased hydrophobicity of our particular fusion proteins, and to the poor solubilizing ability of this reaction medium. We propose the utilization of chaotropic agents in the presence of diluted acids as the preferred cyanogen bromide cleavage medium of fusion proteins in order to maximize cleavage efficiency of hydrophobic sequences and to prevent deleterious degradation and structural modifications of the target peptides.