绿色糖单孢菌木质素过氧化物酶的分离纯化及鉴定

木质素过氧化物酶是一种重要的具有工业应用前景的木质素降解酶,但已报道真菌来源的木质素过氧化物酶只能在酸性低温条件下发挥作用,限制了其进一步的工业应用.通过培养一株耐热耐碱放线茵——绿色糖单孢茵发酵产酶,采用DEAE-Cellulose,CM-Cellulose和Superdex 75凝胶过滤层析等分离纯化方法,得到一种具有耐热耐碱特性的木质素过氧化物酶.经凝胶电泳检测其为单一蛋白,分子量为41 kD.最终纯化倍数达到20倍,活性回收率为6％.采用LTQ法对纯酶进行蛋白质归类鉴定,得到其部分氨基酸片段,为该酶的进一步分子生物学研究奠定基础.     

水稻蛋白质组双向电泳优化流程及方法

双向电泳是分析蛋白质混合物的一种有力手段,已在蛋白质组研究中得到广泛应用。水稻（Oryzasativa）作为重要的粮食作物,对其蛋白质组学研究开展较早。但由于技术复杂,对实验操作要求高,初学的研究者很难在较短的时间内掌握该实验技术。该文介绍了水稻研究中适合多个组织的双向电泳实验方法和优化流程。该优化流程能使新的研究者逐步优化实验条件,更快更好地完成双向电泳实验。同时详细介绍了实验关键环节的操作方法。     

水稻短根毛突变体Ossrh2的表型分析与基因定位

在粳稻品种中花11为遗传背景的T-DNA突变体库中筛选获得一个遗传稳定的水稻（Oryzasativa）短根毛突变体Ossrh2（Oryza sativa short root hair2）。突变体在苗期表现为根毛数量减少,为野生型的61.4%,根毛长度明显变短,只有野生型的22.8%,同时根毛增粗,根毛形态也发生了变异,局部扭曲膨胀和分叉,除此之外突变体的地上部和根部生长情况与野生型相比没有显著差异。遗传分析表明,该突变性状受1对隐性单基因控制。通过对突变体T2和F2代的分子检测发现,该突变体表型非T-DNA插入引起。利用Ossrh2纯合体和籼稻品种Kasalath杂交构建的F2群体对OsSRH2进行基因定位,发现其与第10号染色体短臂上的SSR（simple sequence repeat）标记RM6370和RM474连锁,遗传距离分别为1.1cM和3.0cM。通过在两标记间发展3个新的STS（sequence-taggedsite）标记,将OsSRH2基因定位于标记S1227和S1531之间,物理距离约为304kb,为进一步克隆OsSRH2打下了基础。     

Mannan Binding Lectin (MBL) genotypes coding for high MBL serum levels are associated with rheumatoid factor negative rheumatoid arthritis in never smokers

Introduction  

Previous studies have provided inconsistent results on whether variants in the MBL2 gene, coding for the complement-activating mannan-binding lectin (MBL) protein, associate with rheumatoid arthritis (RA). We re-evaluated this in context of the main environmental and genetic risk factors (smoking, HLA-DRB1 'shared epitope' (SE), PTPN22*620W), which predispose to rheumatoid factor (RF) and/or anti-citrullinated-protein antibody (ACPA)-positive RA.     

Horner syndrome after carotid sheath surgery in a pig: anatomic study of cervical sympathetic chain

In an experimental model, iatrogenic Horner syndrome developed after a right carotid sheath surgery in an infant pig (Sus scrofa). Horner syndrome is a classic clinical triad consisting of ipsilateral eyelid ptosis, pupil miosis, and facial anhydrosis. This syndrome results from cervical sympathetic chain (CSC) paresis and usually is acquired in humans. To determine whether the development of Horner syndrome in this situation could be attributed to pig anatomy, we compared the anatomy of the CSC in pigs and humans, by using 10 infant (age, 1 to 3 wk) pig cadavers. The CSC and cranial cervical sympathetic ganglion (CCG) were dissected bilaterally under a surgical microscope. These structures were consistently within the carotid sheaths of the pigs. In contrast, the CSC and CCG are outside the carotid sheath in humans. Awareness of the anatomic variation of the CSC and CCG within the carotid sheath in the pig and the possibility of the same variation in humans may help surgeons to identify and preserve important structures while performing cervical surgery in pigs and humans. Furthermore, this knowledge can aid in the diagnosis and prognosis of schwannoma.     

Optimum induction of recombinant thymidine phosphorylase and its application

Recombinant E. coli pDEOA was constructed and lactose can be used instead of IPTG to induce the expression of thymidine phosphorylase by pDEOA. The use of lactose at concentrations higher than 0.5 mmol/L had an induction effect similar to that of IPTG but resulted in a longer initial induction time and better cell growth. The thymidine phosphorylase induced by lactose was very stable at 50°C. Intact pDEOA cells induced by lactose can be used as a source of thymidine phosphorylase. Under standard reaction conditions, several deoxynucleosides were effectively produced from thymidine.     

Proteome dynamics and proteome function of cardiac 19S proteasomes

Myocardial proteasomes are comprised of 20S core particles and 19S regulatory particles, which together carry out targeted degradation of cardiac proteins. The 19S complex is unique among the regulators of proteasomes in that it affects both the capacity and specificity of protein degradation. However, a comprehensive molecular characterization of cardiac 19S complexes is lacking. In this investigation, we tailored a multidimensional chromatography-based purification strategy to isolate structurally intact and functionally viable 19S complexes from murine hearts. Two distinct subpopulations of 19S complexes were isolated based upon (1) potency of activating 20S proteolytic activity, and (2) molecular composition using a combination of immuno-detection, two-dimensional-differential gel electrophoresis, and MS-based approaches. Heat shock protein 90 (Hsp90) was identified to be characteristic to 19S subpopulation I. The physical interaction of Hsp90 with 19S complexes was demonstrated via multiple approaches. Inhibition of Hsp90 activity using geldanamycin or BIIB021 potentiated the ability of subpopulation I to activate 20S proteasomes in the murine heart, thus demonstrating functional specificity of Hsp90 in subpopulation I. This investigation has advanced our understanding of the molecular heterogeneity of cardiac proteasomes by identifying molecularly and functionally distinct cardiac 19S complexes. The preferential association of Hsp90 with 19S subpopulation I unveils novel targets for designing proteasome-based therapeutic interventions for combating cardiac disease.     

DeltAMT: a statistical algorithm for fast detection of protein modifications from LC-MS/MS data

Identification of proteins and their modifications via liquid chromatography-tandem mass spectrometry is an important task for the field of proteomics. However, because of the complexity of tandem mass spectra, the majority of the spectra cannot be identified. The presence of unanticipated protein modifications is among the major reasons for the low spectral identification rate. The conventional database search approach to protein identification has inherent difficulties in comprehensive detection of protein modifications. In recent years, increasing efforts have been devoted to developing unrestrictive approaches to modification identification, but they often suffer from their lack of speed. This paper presents a statistical algorithm named DeltAMT (Delta Accurate Mass and Time) for fast detection of abundant protein modifications from tandem mass spectra with high-accuracy precursor masses. The algorithm is based on the fact that the modified and unmodified versions of a peptide are usually present simultaneously in a sample and their spectra are correlated with each other in precursor masses and retention times. By representing each pair of spectra as a delta mass and time vector, bivariate Gaussian mixture models are used to detect modification-related spectral pairs. Unlike previous approaches to unrestrictive modification identification that mainly rely upon the fragment information and the mass dimension in liquid chromatography-tandem mass spectrometry, the proposed algorithm makes the most of precursor information. Thus, it is highly efficient while being accurate and sensitive. On two published data sets, the algorithm effectively detected various modifications and other interesting events, yielding deep insights into the data. Based on these discoveries, the spectral identification rates were significantly increased and many modified peptides were identified.     

Metabolomic study of interactive effects of phenol, furfural, and acetic acid on Saccharomyces cerevisiae

Metabolic profiling was carried out to investigate the interactive effects of three representative inhibitors (furfural, phenol, and acetic acid) in lignocellulosic hydrolysate on Saccharomyces cerevisiae during ethanol fermentation. Our results revealed that three inhibitors exhibited significantly synergistic effects on the growth, fermentation, and some metabolites of yeast. Acetic acid exerted the most severe effects on yeast in the combination of three inhibitors, enhancing amino acids metabolism and inhibiting central carbon metabolism. The effects on yeast cells by acetic acid were enhanced by the presence of phenol and furfural, which might be owing to the loss of membrane integrity and the inhibition on metabolism. Further investigation indicated that the combination of inhibitors also exhibited antagonistic effects mainly on threonine, cadaverine, inositol, and tryptophan, weakening or reversing the effects of individual inhibitor. It might be due to the more severe damage by the combined inhibitors, and different repairing mechanism of cells in the presence of individual and combined inhibitors. Better understanding of the synergistic and antagonistic effects of the inhibitors will be helpful for the improvement of tolerant strains and the optimization of lignocellulosic fermentation.     

The abnormal phenotypes of cartilage and bone in calcium-sensing receptor deficient mice are dependent on the actions of calcium, phosphorus, and PTH

Patients with neonatal severe hyperparathyroidism (NSHPT) are homozygous for the calcium-sensing receptor (CaR) mutation and have very high circulating PTH, abundant parathyroid hyperplasia, and severe life-threatening hypercalcemia. Mice with homozygous deletion of CaR mimic the syndrome of NSHPT. To determine effects of CaR deficiency on skeletal development and interactions between CaR and 1,25(OH)(2)D(3) or PTH on calcium and skeletal homeostasis, we compared the skeletal phenotypes of homozygous CaR-deficient (CaR(-/-)) mice to those of double homozygous CaR- and 1α(OH)ase-deficient [CaR(-/-)1α(OH)ase(-/-)] mice or those of double homozygous CaR- and PTH-deficient [CaR(-/-)PTH(-/-)] mice at 2 weeks of age. Compared to wild-type littermates, CaR(-/-) mice had hypercalcemia, hypophosphatemia, hyperparathyroidism, and severe skeletal growth retardation. Chondrocyte proliferation and PTHrP expression in growth plates were reduced significantly, whereas trabecular volume, osteoblast number, osteocalcin-positive areas, expression of the ALP, type I collagen, osteocalcin genes, and serum ALP levels were increased significantly. Deletion of 1α(OH)ase in CaR(-/-) mice resulted in a longer lifespan, normocalcemia, lower serum phosphorus, greater elevation in PTH, slight improvement in skeletal growth with increased chondrocyte proliferation and PTHrP expression, and further increases in indices of osteoblastic bone formation. Deletion of PTH in CaR(-/-) mice resulted in rescue of early lethality, normocalcemia, increased serum phosphorus, undetectable serum PTH, normalization in skeletal growth with normal chondrocyte proliferation and enhanced PTHrP expression, and dramatic decreases in indices of osteoblastic bone formation. Our results indicate that reductions in hypercalcemia play a critical role in preventing the early lethality of CaR(-/-) mice and that defects in endochondral bone formation in CaR(-/-) mice result from effects of the marked elevation in serum calcium concentration and the decreases in serum phosphorus concentration and skeletal PTHrP levels, whereas the increased osteoblastic bone formation results from direct effects of PTH.