Imaging of protein layers with an optical microscope for the characterization of peptide microarrays.

Solid-phase assays play a crucial role today in biological studies. These assays are based on the immobilization of probe molecules on a surface, which are able to capture specifically soluble receptors. In particular, peptide microarrays have emerged as powerful tools in a variety of applications. In this context, optical techniques that allow imaging of nanometer-thick biomolecular films, and thereby the characterization of microarrays, are of great interest. For this purpose, we used a recently disclosed wide-field optical imaging technique of surface nanostructures called Sarfus, which is based on the use of a standard optical microscope and antireflection substrates. We demonstrate here that this technique allows the imaging of the protein layers that result from the specific capture of antibodies by arrayed peptide probes with a spatial resolution of 0.45 microm. The relationship between the thickness of the antibody layer and peptide or antibody concentration was examined.     

腺苷引起促黑素细胞膜电位超极化的离子机制

通过对青蛙垂体中叶促黑素细胞的实验已发现,腺苷激素Ａ１型腺苷受体后可引起细胞膜的超极化同时停止自发性动作电位的发放,但此现象所涉及的离子机制尚不清楚。本实验采用膜片箝技术的全细胞电流、电压记录和细胞吸附单通道电流记录方法,对此电流进行了探讨。实验结果表明：腺苷所致的细胞膜超极化是由于增加了非电压激活的钾离子通道的开放,而对超极化电压激活的内向阳离子电流（Ｉｈ）没有影响。     

Identification of a novel c-DNA overexpressed in Fanconi's anemia fibroblasts partially homologous to a putative l-3-phosphoserine-phosphatase

We applied the cDNA differential display technique (DDT) in a DNA-repair deficient cell model to isolate genes involved in dysregulation of cell proliferation and development of cancer. The comparative analysis of mRNA expression patterns of human diploid fibroblasts from Fanconi's amemia (FA) and normal phenotype led to the identification of a novel cDNA CO9. Northern blot analysis reveals that CO9 is significantly upregulated in FA fibroblasts but downregulated or absent in fibroblasts from normal donors. CO9 was also highly expressed in FA B-cells of complementation group A and in Raji cells. However, CO9 is not expressed in FA complementation groups B, C, D and E. The full-length cDNA is 840 bp long and contains an open reading frame of 216 bp (72 amino acids), which encodes for a 7.6-kDa protein. The lengths of the 5′ and 3′ untranslated region are 165 and 459 bp, respectively. The N-terminal and C-terminal nucleotide sequence of CO9 shows homology to a putative human l-3-phosphoserine phosphatase identified recently (HSPSPASE, EMBL Accession No. Y10275) but lacks a 476-bp stretch in the open reading frame. The loss of nucleotides within the open reading frame introduces a new termination codon in the CO9 cDNA along with a novel COOH terminus resulting in a new protein product. Database chromosome mapping localized CO9 to chromosome 7q 11.2. We hypothesize that CO9 represents a novel protein being a partial homologue to the l-3-phosphoserine phosphatase but with a different regulatory cell function.     

Use of a Hemoglobin-Trapping Approach in the Determination of Nitric Oxide in In Vitro and In Vivo Systems

We describe methods for measuring the release of nitric oxide (NO) derived from organic nitrates in vitro, using triple wavelength and difference spectrophotometry in the presence and absence of concentric microdialysis probes. These methods are based on the ability of NO to oxidize oxyhemoglobin (OxyHb) to methemoglobin (MetHb) quantitatively in aqueous solution. Isosorbide dinitrate (ISDN), a thiol-dependent organic nitrate, increased MetHb concentration in 45 min from 2.47 ± 0.47 to 4.15 ± 0.12 M (p < 0.05) and decreased OxyHb concentration from 2.13 ± 0.35 to 0.33 ± 0.26 M (p < 0.05) at 37°C. At 27°C, the OxyHb concentration was not significantly altered (2.04 ± 0.23 to 1.60 ± 0.04 M) by ISDN, nor was the MetHb concentration (from 2.68 ± 0.50 to 2.59 ± 0.25 M). Sodium nitroprusside (SNP), a thiol-independent organic nitrate, increased MetHb concentrations in 30 min from 4.21 ± 0.26 to 6.00 ± 0.56 M (p < 0.05) at 37°C, and from 4.23 ± 0.39 to 5.90 ± 0.43 M (p < 0.01) at 27°C. SNP also decreased OxyHb concentrations in 30 min from 1.99 ± 0.32 to 0.13 ± 0.12 M (p < 0.01) at 37°C, and from 2.25 ± 0.31 to 0.13 ± 0.09 M (p < 0.01) at 27°C. Difference spectrophometry indicated that 0.25-5 mM SNP significantly increased NO production in a dose-dependent fashion. This hemoglobin-trapping technique was also useful in quantifying the concentrations of NO released from SNP in aqueous solution in vitro, using concentric microdialysis probes. The NO concentration following exposure to SNP was 530 ± 50 nM, as determined using the difference spectrophotometric technique. To demonstrate the applicability of this technique to in vivo microdialysis, we implanted concentric microdialysis probes into hippocampus and cerebellum of conscious and anesthetized rats. Baseline NO concentrations in hippocampus of conscious and anesthetized rats were 11 ± 2 nM and 23 ± 9 nM, respectively, while in the cerebellum NO concentrations were 28 ± 9 nM and 41 ± 20 nM, respectively. These results demonstrate that microdialysis using a novel hemoglobin-trapping technique possesses adequate sensitivity to measure the NO levels produced from organic nitrates in aqueous solutions, and further document the applicability of this approach to in vivo systems.     

Plasmodium gallinaceum: mechanisms of anemia in infected chickens

Erythrocyte labeling by random and cohort techniques was used to study erythrocyte survival in normal chickens and chickens infected with Plasmodium gallinaceum. Occurrence of erythrocyte destruction during the prepatent period was apparent in infected chickens by both techniques. Treatment with the antimalarials chloroquine and quinacrine not only cleared the circulation of parasites promptly but brought about an equally prompt cessation of disease-related erythrocyte destruction. Plasmodium gallinaceum infection caused a transitory decrease in blood volume at the time of rapid decrease in packed cell volume. The blood volume returned to preinfection values before the packed cell volume returned to normal. Parasitized erythrocytes were present in capillaries of the spleen, liver, and bone marrow during the entire prepatent period of the infection, thus providing a reasonable explanation for erythrocyte destruction observed in the absence of parasitemia during the prepatent period.     

海洋固氮菌和解磷菌的分离鉴定及发酵条件优化

【目的】从西沙喜盐草根际沉积物中分离纯化得到具有高效固氮能力及解磷能力的菌株。优化其发酵培养条件,研究其制备海洋微生物菌剂的可能性。【方法】从形态学特征、生理生化、16S rDNA及功能基因水平进行鉴定,通过乙炔还原法、钼锑抗显色法检测菌株的固氮酶活性和解磷能力,单因素法和响应面法优化其发酵培养条件,溶血试验和急性毒性实验鉴定菌株的安全性。【结果】结果表明,菌株AZ16属于星箭头菌(Sagittula stellate),革兰氏阴性菌,选择性固氮培养基中菌落呈黄圆形黏稠状,固氮酶活性达34.63 nmol C2H2/(mL·h),最适生长条件为:盐度25‰、pH 7.5、温度33°C、接种量5.0%;菌株XT37为海洋芽孢杆菌(Bacillus sp.),革兰氏阳性菌,选择性固氮培养基中菌落呈深黄色圆形褶皱,植酸酶活性达239.49μg/L,最适合生长条件为:盐度25‰、pH 6.7、温度28°C、接种量5.0%。溶血实验和急性毒性实验证明两株菌属实际无毒级别。【结论】两株菌具有高效的固氮解磷功能,以及抗高盐、强碱等环境的能力,安全无毒,因此有潜力应用于多功能混合微生物菌剂的研制。     

Identification and quantification of blood–brain barrier transporters in isolated rat brain microvessels

The blood–brain barrier (BBB ) maintains brain homeostasis by tightly regulating the exchange of molecules with systemic circulation. It consists primarily of microvascular endothelial cells surrounded by astrocytic endfeet, pericytes, and microglia. Understanding the make‐up of transporters in rat BBB is essential to the translation of pharmacological and toxicological observations into humans. In this study, experimental workflows are presented in which the optimization of (a) isolation of rat brain microvessels (b) enrichment of endothelial cells, and (c) extraction and digestion of proteins were evaluated, followed by identification and quantification of BBB proteins. Optimization of microvessel isolation was indicated by 15‐fold enrichment of endothelial cell marker Glut1 mRNA , whereas markers for other cell types were not enriched. Filter‐aided sample preparation was shown to be superior to in‐solution sample preparation (10251 peptides vs. 7533 peptides). Label‐free proteomics was used to identify nearly 2000 proteins and quantify 1276 proteins in isolated microvessels. A combination of targeted and global proteomics was adopted to measure protein abundance of 6 ATP‐binding cassette and 27 solute carrier transporters. Data analysis using proprietary Progenesis and open access MaxQuant software showed overall agreement; however, Abcb9 and Slc22a8 were quantified only by MaxQuant, whereas Abcc9 and Abcd3 were quantified only by Progenesis. Agreement between targeted and untargeted quantification was demonstrated for Abcb1 (19.7 ± 1.4 vs. 17.8 ± 2.3) and Abcc4 (2.2 ± 0.7 vs. 2.1 ± 0.4), respectively. Rigorous quantification of BBB proteins, as reported in this study, should assist with translational modeling efforts involving brain disposition of xenobiotics.

     

Phototrophic cultivation of NaCl‐tolerant mutant of Spirulina platensis for enhanced C‐phycocyanin production under optimized culture conditions and its dynamic modeling

Commercial cultivation of Spirulina sp. is highly popular due to the presence of high amount of C‐phycocyanin (C‐PC ) and other valuable chemicals like carotenoids and γ‐linolenic acid. In this study, the pH and the concentrations of nitrogen and carbon source were manipulated to achieve improved cell growth and C‐PC production in NaCl‐tolerant mutant of Spirulina platensis . In this study, highest C‐PC (147 mg · L^?1) and biomass (2.83 g · L^?1) production was achieved when a NaCl‐tolerant mutant of S. platensis was cultivated in a nitrate and bicarbonate sufficient medium (40 and 60 mM, respectively) at pH 9.0 under phototrophic conditions. Kinetic study of wildtype S. platensis and its NaCl‐tolerant mutant was also done to determine optimum nitrate concentrations for maximum growth and C‐PC production. Kinetic parameter of inhibition (Haldane model) was fitted to the relationship between specific growth rate and substrate concentration obtained from the growth curves. Results showed that the maximum specific growth rate (μ_max) for NaCl‐tolerant mutant increased by 17.94% as compared to its wildtype counterpart, with a slight increase in half‐saturation constant (K_s), indicating that this strain could grow well at high concentration of NaNO₃. C‐PC production rate (C_max) in mutant cells increased by 12.2% at almost half the value of K_s as compared to its wildtype counterpart. Moreover, the inhibition constant (K_i) value was 207.85% higher in NaCl‐tolerant mutant as compared to its wildtype strain, suggesting its ability to produce C‐PC even at high concentrations of NaNO₃.     

论能源管理体系建设与医院后勤精细化管理的相关性

结合医院开展能源管理体系建设的工作要求,通过对能源管理体系建设和精细化管理各自特点以及两者之间相关性分析,找出共同点与相互依存的关系。继而以能源管理体系建设为抓手,分阶段实现医院后勤精细化管理的战略目标。