Significance of the hydrophobic residues 225–230 of apoA-I for the biogenesis of HDL

We studied the significance of four hydrophobic residues within the 225–230 region of apoA-I on its structure and functions and their contribution to the biogenesis of HDL. Adenovirus-mediated gene transfer of an apoA-I[F225A/V227A/F229A/L230A] mutant in apoA-I^−/− mice decreased plasma cholesterol, HDL cholesterol, and apoA-I levels. When expressed in apoA-I^−/− × apoE^−/− mice, approximately 40% of the mutant apoA-I as well as mouse apoA-IV and apoB-48 appeared in the VLDL/IDL/LDL. In both mouse models, the apoA-I mutant generated small spherical particles of pre-β- and α4-HDL mobility. Coexpression of the apoA-I mutant and LCAT increased and shifted the-HDL cholesterol peak toward lower densities, created normal αHDL subpopulations, and generated spherical-HDL particles. Biophysical analyses suggested that the apoA-I[225–230] mutations led to a more compact folding that may limit the conformational flexibility of the protein. The mutations also reduced the ability of apoA-I to promote ABCA1-mediated cholesterol efflux and to activate LCAT to 31% and 66%, respectively, of the WT control. Overall, the apoA-I[225–230] mutations inhibited the biogenesis of-HDL and led to the accumulation of immature pre-β- and α4-HDL particles, a phenotype that could be corrected by administration of LCAT.     

Characteristics of Malignant Tumors in 230 Husband–Wife Pairs

The aim of this study is to obtain a reference point for early detection of tumors in individuals whose spouses were diagnosed with malignant tumors. Data from 230 husband and wife pairs with malignant tumors were collected and analyzed from the family history records of 15,000 people who came to the Department of Cancer Prevention, Cancer Institute/Hospital, Chinese Academy of Medical Sciences for cancer screening between January 2009 and May 2012. The median diagnosis age was 67 years for husbands and 65 years for wives. A total of 214 cases (46.5 %) had digestive system malignancies. Respiratory system cancers were diagnosed in 64 husbands, of whom 20 (31.3 %) had spouses also with respiratory system cancer. Lung cancer ranked first for the females. The total number of lung cancer and commonly seen female-specific cancers (breast, ovarian, uterine, and cervical) was 127 (55.2 %). The difference in age at diagnosis between spouses was less than 10 years in 134 couples (58.3 %), while 77 (33.5 %) couples had an age difference less than 5 years. A family history of malignant tumors in first-degree relatives was documented in 48.3 % of the husbands and 48.7 % of the wives. The occurrence of cancer in both spouses of the couples studied resulted from an interaction between genetic and environmental factors. Nonhereditary factors such as diet, smoking, passive smoking, and air pollution also contributed to the development of cancers. It is recommended that when husband is diagnosed with cancer, the wife should be screened focusing on lung, breast, and gynecological cancers. If the wife was diagnosed with malignant disease, then screening for lung and digestive system cancers should be emphasized in the husband.     

Anchorless 23–230 PrPC Interactomics for Elucidation of PrPC Protective Role

Accumulation of conformationally altered cellular proteins (i.e., prion protein) is the common feature of prions and other neurodegenerative diseases. Previous studies demonstrated that the lack of terminal sequence of cellular prion protein (PrP^C), necessary for the addition of glycosylphosphatidylinositol lipid anchor, leads to a protease-resistant conformation that resembles scrapie-associated isoform of prion protein. Moreover, mice overexpressing the truncated form of PrP^C showed late-onset, amyloid deposition, and the presence of a short protease-resistant PrP fragment in the brain similar to those found in Gerstmann–Sträussler–Scheinker disease patients. Therefore, the physiopathological function of truncated_/anchorless 23–230 PrP^C (Δ23–230 PrP^C) has come into focus of attention. The present study aims at revealing the physiopathological function of the anchorless PrP^C form by identifying its interacting proteins. The truncated_/anchorless Δ23–230 PrP^C along with its interacting proteins was affinity purified using STrEP-Tactin chromatography, in-gel digested, and identified by quadrupole time-of-flight tandem mass spectrometry analysis in prion protein-deficient murine hippocampus (HpL3-4) neuronal cell line. Twenty-three proteins appeared to interact with anchorless Δ23–230 PrP^C in HpL3-4 cells. Out of the 23 proteins, one novel protein, pyruvate kinase isozymes M1/M2 (PKM2), exhibited a potential interaction with the anchorless Δ23–230 form of PrP^C. Both reverse co-immunoprecipitation and confocal laser-scanning microscopic analysis confirmed an interaction of PKM2 with the anchorless Δ23–230 form of PrP^C. Furthermore, we provide the first evidence for co-localization of PKM2 and PrP^C as well as PrP^C-dependent PKM2 expression regulation. In addition, given the involvement of PrP^C in the regulation of apoptosis, we exposed HpL3-4 cells to staurosporine (STS)-mediated apoptotic stress. In response to STS-mediated apoptotic stress, HpL3-4 cells transiently expressing 23–230-truncated PrP^C were markedly less viable, were more prone to apoptosis and exhibited significantly higher PKM2 expressional regulation as compared with HpL3-4 cells transiently expressing full-length PrP^C (1–253 PrP^C). The enhanced STS-induced apoptosis was shown by increased caspase-3 cleavage. Together, our data suggest that the misbalance or over expression of anchorless Δ23–230 form of PrP^C in association with the expressional regulation of interacting proteins could render cells more prone to cellular insults-stress response, formation of aggregates and may ultimately be linked to the cell death.     

Do cysteine 230 and lysine 238 of biotin carboxylase play a role in the activation of biotin?

Biotin carboxylase from Escherichia coli catalyzes the ATP-dependent carboxylation of biotin and is one component of the multienzyme complex acetyl-CoA carboxylase, which catalyzes the committed step in long-chain fatty acid synthesis. For the carboxylation of biotin to occur, biotin must be deprotonated at its N1' position. Kinetic investigations, including solvent isotope effects and enzyme inactivation by N-ethylmaleimide, suggested a catalytic role for a cysteine residue and led to the proposal of a mechanism for the deprotonation of biotin. The proposed pathway suggests a catalytic base removes a proton from a nearby cysteine residue, forming a thiolate anion, which then abstracts the proton from biotin. Inactivation studies of pyruvate carboxylase, which has an analogous mode of action to biotin carboxylase, suggests the catalytic base in this reaction is a lysine residue. Using the crystal structure of biotin carboxylase, cysteine 230 and lysine 238 were identified as the likely active-site residues that act as this acid-base pair. To test the hypothesis that cysteine 230 and lysine 238 act as an acid-base pair to deprotonate biotin, site-directed mutagenesis was used to mutate cysteine 230 to alanine (C230A) and lysine 238 to glutamine (K238Q). Mutations at either residue resulted in a 50-fold increase in the K(m) for ATP. The C230A mutation had no effect on the formation of carboxybiotin, indicating that cysteine 230 does not play a role in the deprotonation of biotin. However, the K238Q mutation resulted in no formation of carboxybiotin, which showed that lysine 238 has a role in the carboxylation reaction. N-Ethylmaleimide was found to inactivate the C230A mutant but not the K238Q mutant, suggesting that N-ethylmaleimide is reacting with lysine 238 and not cysteine 230. The pH dependence of N-ethylmaleimide inactivation revealed that the pK value for lysine 238 was 9.4 or higher, suggesting lysine 238 is not a catalytic base. Thus, the results suggest that cysteine 230 and lysine 238 do not act as an acid-base pair in the deprotonation of biotin.     

Integrin α6β4 Recognition of a Linear Motif of Bullous Pemphigoid Antigen BP230 Controls Its Recruitment to Hemidesmosomes

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Novel variant of p230 trans-Golgi network protein identified by serum from Sjögren's syndrome patient

Trans-Golgi network (TGN) protein p230 is a peripheral membrane protein associated with the cytoplasmic face of the TGN. TGNp230 is an extensively coiled-coil protein with flexible amino- and carboxyl-terminal ends, associates with non-clathrin-coated vesicles arising from the TGN, and is implicated in vesicle biogenesis. Here we used an autoimmune serum from a patient with S ogren's syndrome to clone partial cDNAs from a human hepatoma HepG2 expression library. The partial cDNAs encoded a novel amino-terminal splice variant of TGNp230. Specific reactivity of the autoimmune serum for p230 is supported by immunofluorescene staining of the Golgi apparatus, immunoblotting of a > 200-kDa HeLa cell protein, and reactivity with a bacterially expressed GST-p230 fusion protein. The alternative splicing occurs within the first proline-rich domain of p230. It comprises a deletion of 30 bp followed immediately by an additional 66 bp absent in the published sequence. RT-PCR analysis indicated that the splicing occurs independently of previously reported carboxyl-terminal splicing, and that this novel splice variant is more frequent than the previously reported p230. The novel splice variant of p230 is also located at the TGN. We propose that p230 splice variants may be implicated in selection of cargo molecules for vesicles arising from the TGN.     

美生物技术产品销售将超过230亿美元

美生物技术产品销售将超过２３０亿美元据一项对生物技术工业的新的分析预测,美国生物技术产品的销售在２００４年将超过２３０亿美元,较互９９４年６０亿美元的基数平均年增长率为１５ｑｏ。生物技术工业６９主要部分包括人用治疗药品（有时称做生物药品）、人用诊断制...     

北京市市售230件谷物制品重金属分析

目的：分析2015—2016年北京市六个区县小麦粉、玉米面重金属检测结果,为预防和控制提供科学依据。方法：按照国标方法对食品样品进行镉、铅、汞、砷4个项目的检测。结果：230件样品中,汞、砷均未检出;小麦粉中镉的检出率（83.33%）高于在玉米面中的检出率（48.08%）（P＜0.05）,铅的检出率（77.00%）高于在玉米面中的检出率（60.58%）（P＜0.05）;产地不同的样品中铅的检出率不同（P＜0.05）。结论：北京市六个区市售小麦粉、玉米面均有不同程度的镉、铅污染。     

Feasibility study on the use of 230 MeV proton cyclotron in proton therapy centers as a spallation neutron source for BNCT

AimThe feasibility of using 230 MeV proton cyclotrons in proton therapy centers as a spallation neutron source for Boron Neutron Capture Therapy (BNCT) was investigated.BackgroundBNCT is based on the neutron irradiation of a ¹⁰B-containing compound located selectively in tumor cells. Among various types of neutron generators, the spallation neutron source is a unique way to generate high-energy and high-flux neutrons.Materials and MethodsNeutron beam was generated by a proton accelerator via spallation reactions and then the produced neutron beam was shaped to be appropriate for BNCT. The proposed Beam Shaping Assembly (BSA) consists of different moderators, a reflector, a collimator, as well as thermal and gamma filters. In addition, the simulated Snyder head phantom was utilized to evaluate the dose distribution in tumor and normal tissue due to the irradiation by the designed beam. MCNPX2.6 Monte Carlo code was used to optimize BSA as well as evaluate dose evaluation.ResultsA BSA was designed. With the BSA configuration and a beam current of 104 nA, epithermal neutron flux of 3.94 × 10⁶ [n/cm²] can be achieved, which is very low. Provided that we use the beam current of 5.75 μA, epithermal neutron flux of 2.18 × 10⁸ [n/cm²] can be obtained and the maximum dose of 38.2 Gy-eq can be delivered to tumor tissue at 1.4 cm from the phantom surface.ConclusionsResults for 230 MeV protons show that with proposed BSA, proton beam current about 5.75 μA is required for this purpose.     

230例糖尿病足部溃疡感染病原菌分布及药物敏感性分析

分析近2 a糖尿病足患者足部感染常见病原菌分布及抗菌药物敏感性,为临床合理选择抗菌药物提供参考依据。回顾性分析了2011年6月至2012年12月天津中医药大学第二附属医院门诊及住院糖尿病足感染患者病例资料。全部细菌中革兰阴性菌138株(59.0%),革兰阳性菌94株(40.2%),真菌2株(0.8%)。培养阳性率为95.22%,占前3位的细菌为金黄色葡萄球菌(下称金葡菌)、奇异变形杆菌、肺炎克雷伯菌。产ESBL(超广谱β-内酰胺酶)菌株共9株,占全部革兰阴性菌的6.52%,肠杆菌科细菌对亚胺培南、美罗培南均敏感,铜绿假单胞菌对阿米卡星、头孢吡肟、头孢哌酮/舒巴坦均敏感。金葡菌中MRSA(甲氧西林耐药金葡菌)占19.6%,克林霉素诱导实验(D-实验)阳性的金葡菌的占27.5%。在粪肠球菌中,高水平庆大霉素耐药菌株(HLGR)和高水平链霉素耐药菌株(HLSR)分别占50%及20%。万古霉素与利奈唑胺对所感染的阳性球菌均敏感。结果表明,糖尿病足部溃疡患者应及时进行微生物学检查、耐药类型鉴定和药敏实验,对耐药菌株给予高度重视,并根据药敏结果合理应用抗菌药物以减少耐药菌的产生。     

恶性疟原虫重组pfs230蛋白在不同表达系统中表达的研究进展

恶性疟原虫pfs230蛋白是恶性疟疾传播阻断疫苗主要的抗原之一,可影响雌雄配子的受精过程,这种疫苗对阻断恶性疟疾的传播至关重要。鉴于pfs230蛋白分子较大,以及独特的富含半胱氨酸的结构特征,体内表达有活性的全长重组pfs230蛋白存在困难。现就pfs230蛋白的生物学特征和恶性疟疾传播阻断型疫苗的活性评价方法,用大肠埃希菌、小麦胚芽无细胞系统、毕赤酵母和昆虫细胞中表达pfs230蛋白的进展,以及在不同表达系统中优化恶性疟疾pfs230蛋白表达的策略作一概述。     

NMR experiments for resonance assignments of 13C, 15N doubly-labeled flexible polypeptides: Application to the human prion protein hPrP(23–230)

A combination of three heteronuclear three-dimensional NMR experiments tailored for sequential resonance assignments in uniformly ¹⁵N, ¹³C-labeled flexible polypeptide chains is described. The 3D (H)N(CO-TOCSY)NH, 3D (H)CA(CO-TOCSY)NH and 3D (H)CBCA(CO-TOCSY)NH schemes make use of the favorable ¹⁵N chemical shift dispersion in unfolded polypeptides, exploit the slow transverse ¹⁵N relaxation rates of unfolded polypeptides in high resolution constant-time [¹H, ¹⁵N]-correlation experiments, and use carbonyl carbon homonuclear isotropic mixing to transfer magnetization sequentially along the amino acid sequence. Practical applications are demonstrated with the 100-residue flexible tail of the recombinant human prion protein, making use of spectral resolution up to 0.6 Hz in the ¹⁵N dimension, simultaneous correlation with the two adjacent amino acid residues to overcome problems associated with spectral overlap, and the potential of the presently described experiments to establish nearest-neighbor correlations across proline residues in the amino acid sequence.     

棕色固氮菌(Azotobacter vinelandii230)中氢酶与固氮酶关系的探讨

自Stephensen和Stickland发现氢酶之后,氢酶已在许多类型的微生物中被发现。然而固氮菌中氢酶的存在却引起了生物固氮工作者的普遍重视。Wilson及其同事对自生固氮菌中氢酶的性质作了一系列的报导。其中最有意义的发现是氢酶参与氧的吸收(尽管高浓度的氧能抑制氢酶)。Hyndman等发现氢酶参与氧化磷酸化的作用。Cixon报导氢酶可能有三种功能:(1)通过氢的被氧化除氧;(2)解除氢对固氮的抑制作用;(3)作为再循环的部分补充由于     

Cloning,Expression, Purification,and Characterization of Cold-Adapted α-Amylase from <Emphasis Type="Italic">Pseudoalteromonas arctica</Emphasis> GS230

A cold-adapted α-amylase (ParAmy) gene from Pseudoalteromonas arctica GS230 was cloned, sequenced, and expressed as an N-terminus His-tag fusion protein in E. coli. A recombinant protein was produced and purified with DEAE-sepherose ion exchange chromatography and Ni affinity chromatography. The molecular weight of ParAmy was estimated to be 55 KDa with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). With an optimum temperature for activity 30 °C, ParAmy showed 34.5% of maximum activity at 0 °C and its activity decreased sharply at above 40 °C. ParAmy was stable in the range of pH 7–8.5 at 30 °C for 1 h. ParAmy was activated by Mn²⁺, K⁺ and Na⁺, and inhibited by Hg²⁺, Cu²⁺, and Fe³⁺. N-Bromosuccinimid showed a significant repressive effect on enzyme activity. The K _m and V _max values of the α-amylase for soluble starch were 7.28 mg/mL and 13.07 mg/mL min, respectively. This research suggests that Paramy has a good potential to be a cold-stable and alkalitolerant amylase in detergent industry.     

Modification of mouse A2M B (620-792) and A2M N (168-230) by malondialdehyde and acetaldehyde attenuates the proteinase and TGF-β1 binding ability of A2MB

Acetaldehyde and malondialdehyde react covalently with cellular proteins forming protein-malondialdehyde-acetaldehyde adducts thus modulating their biochemical functions. Alpha-2 macroglobulin, an acute phase protein produced by liver binds to cytokines, growth factors and neutralizes proteinases. In this study we examined the formation of MAA adducts of N-terminal and bait region of mouse A2M and their effect on modulating its proteinase and TGF-β1 binding activities. Adduct formation abrogated the binding of bait region with TGF-β1, trypsin, and elastase. TGF-β1 induced NO production was also suppressed. Acetaldehyde and MDA adduction of A2M may have physiological consequences in alcoholic patients.