A clinical trial on a plate? The potential of 384-well format solid phase extraction for high-throughput bioanalysis using liquid chromatography/tandem mass spectrometry

The application of 384-well format solid phase extraction (SPE) for bioanalysis using liquid chromatography/tandem mass spectrometry (LC/MS/MS) is reported and a 384-well SPE method for the 5-HT agonist sumatriptan in human plasma described. Plasma samples were extracted on a prototype low-density polyethylene 384-well SPE block using a packed bed of 5 mg Oasistrade mark HLB. Liquid handling was automated by a combination of a robotic sampler processor and a 96/384 multi-channel dispensing station. Samples and SPE reagents were drawn through the SPE block by centrifugation. The extracts were analysed by LC/MS/MS with thermally and pneumatically assisted electrospray ionisation and selected reaction monitoring. The method is used to illustrate and discuss the feasibility and viability of sample preparation techniques in high-density microtitre plate format for routine bioanalysis.     

Anti-inflammatory and Antinociceptive Activity of Chitin-binding Lectin from Canna Limbata Seeds

Lectins are a structurally heterogeneous group of proteins or glycoproteins with at least one noncatalytic domain binding reversibly to a specific mono- or oligosaccharide. Monocot mannose-binding lectins are an extended superfamily of structurally and evolutionarily related proteins. In this study, we evaluated anti-inflammatory and antinociceptive effects of monocot lectin from the Canna limbata seeds (CLL). To accomplish this, CLL was purified and subjected to pharmacological assays: abdominal writhing induced by acetic acid, formalin, hot plate and Zymosan A-induced peritonitis tests. The CLL was purified by chromatographic chitin column, and the relative mass of 21 kDa observed in electrophoresis was confirmed by electrospray mass spectrometry, which also revealed that purified CLL consists of a dimer having a weight of 49,676 Da. The CLL showed nociceptive activity in the acetic acid test as well as peripheral antinociceptive response. The CLL also showed anti-inflammatory effect with the reduction of inflammation in the formalin test and neutrophil migration into the peritoneal cavity. This is the first report of anti-inflammatory activity for a monocot lectin, and it suggests a new pharmacological tool to understand inflammatory and antinociceptive processes mediated through lectins.     

Immobilization and Stabilization of Beta-Xylosidases from Penicillium janczewskii

β-Xylosidases are critical for complete degradation of xylan, the second main constituent of plant cell walls. A minor β-xylosidase (BXYL II) from Penicillium janczewskii was purified by ammonium sulfate precipitation (30% saturation) followed by DEAE-Sephadex chromatography in pH 6.5 and elution with KCl. The enzyme presented molecular weight (MW) of 301 kDa estimated by size exclusion chromatography. Optimal activity was observed in pH 3.0 and 70–75 °C, with higher stability in pH 3.0–4.5 and half-lives of 11, 5, and 2 min at 65, 70, and 75 °C, respectively. Inhibition was moderate with Pb⁺² and citrate and total with Cu⁺², Hg⁺², and Co⁺². Partially purified BXYL II and BXYL I (the main β-xylosidase from this fungus) were individually immobilized and stabilized in glyoxyl agarose gels. At 65 °C, immobilized BXYL I and BXYL II presented half-lives of 4.9 and 23.1 h, respectively, therefore being 12.3-fold and 33-fold more stable than their unipuntual CNBr derivatives (reference mimicking soluble enzyme behaviors). During long-term incubation in pH 5.0 at 50 °C, BXYL I and BXYL II glyoxyl derivatives preserved 85 and 35% activity after 25 and 7 days, respectively. Immobilized BXYL I retained 70% activity after 10 reuse cycles of p-nitrophenyl-β-D-xylopyranoside hydrolysis.

     

Crystallization and characterization of an inflammatory lectin purified from the seeds of Dioclea wilsonii

DwL, a lectin extracted from the seeds of Dioclea wilsonii, is a metalloprotein with strong agglutinating activity against rabbit and ABO erythrocytes, inhibited by glucose and mannose. DwL was purified by affinity chromatography on a Sephadex G-50 column and ion exchange chromatography on a HiTrap SP XL column. SDS-PAGE revealed three electrophoretic bands corresponding to the α (25,634 ± 2 Da), β (12,873 ± 2 Da) and γ (12,779 ± 2 Da) chains. Protein sequencing was done by Tandem Mass Spectrometry. The primary sequence featured 237 amino acids and was highly homologous to other reported Diocleinae lectins. A complete X-ray dataset was collected at 2.0 ? for X-Man-complexed DWL crystals produced by the vapor diffusion method. The crystals were orthorhombic and belonged to the space group I222, with the unit-cell parameters a = 59.6, b = 67.9 and c = 109.0 ?. DWL differed in potency from other ConA-like lectins and was found to induce neutrophil migration in rats, making it particularly useful in structural/functional studies of this class of proteins.     

直接键连原子间的NMR偶合常数的自然杂化轨道研究V.~(13)C-~(13)C和~(13)C-~(31)P核自旋偶合常数~1J_(CC)和~1J_(CP)

在前文工作的基础上，结合ＭＮＤＯ／ＥＨＭＯ分子轨道方法和自然杂化轨道方法，具体计算了ＣＣ键和ＣＰ键的核自旋偶合常数．计算结果表明，１ＪＣＣ和１ＪＣＰ主要由成键原子的轨道杂化作用和键极性这两种结构因素所决定．为从简单价键理论角度解释和计算１ＪＣＣ和１ＪＣＰ值提供了简便直观的方法．     

Identifying defective sets using queries of small size

We examine the following version of a classic combinatorial search problem introduced by Rényi: Given a finite set $X$ of $n$ elements we want to identify an unknown subset $Y$ of $X$, which is known to have exactly $d$ elements, by means of testing, for as few as possible subsets $A$ of $X$, whether $A$ intersects $Y$ or not. We are primarily concerned with the non-adaptive model, where the family of test sets is specified in advance, in the case where each test set is of size at most some given natural number $k$. Our main results are nearly tight bounds on the minimum number of tests necessary when $d$ and $k$ are fixed and $n$ is large enough.     

具有切向控制的局部四点插值曲线设计方法

Ｎｉｒａ Ｄｙｎ等提出的四点插值法是一种典型的自由曲线离散造型方法，但该方法不能控制插值点的切向。本文利用薄板样很可能 量的极小化原理给出了具有切向控制的四点分插值条件。用户可以方便地交互控制任一插值点的切向，使得四点插值法更为有效和实用。     

Light induced fading in the OSL response of Al2O3:C

We are reporting the results of an investigation designed to determine the magnitude of the light induced fading associated with the OSL response of Al₂O₃:C. Unlike previous studies where bare, radiation sensitive OSL elements were exposed directly to light, most of the experiments described here were conducted using sealed commercially available OSL dosimeters. During light exposure the OSL sensitive elements were kept inside a standard commercially available plastic badge. A commercial OSL system was used for these experiments in an attempt to simulate typical field use conditions. Both light induced signal and light induced fading were considered, however no measurable light induced signal could be identified. Light induced fading effects, however, were significant, up to 55% loss of OSL signal following daylight exposure of 45 days. The possibility that dose information may be easily erased, intentionally or accidentally, could impose significant restrictions on the ability of the US Navy to defend, if needed, the reported personnel dose levels.     

One-Step Immobilization and Stabilization of a Recombinant Enterococcus faecium DBFIQ E36 l-Arabinose Isomerase for d-Tagatose Synthesis

A recombinant l-arabinose isomerase from Enterococcus faecium DBFIQ E36 was immobilized onto multifunctional epoxide supports by chemical adsorption and onto a chelate-activated support via polyhistidine-tag, located on the N-terminal (N-His-L-AI) or on the C-terminal (C-His-L-AI) sequence, followed by covalent bonding between the enzyme and the support. The results were compared to reversible L-AI immobilization by adsorption onto charged agarose supports with improved stability. All the derivatives presented immobilization yields of above 75%. The ionic interaction established between agarose gels containing monoaminoethyl-N-aminoethyl structures (MANAE) and the enzyme was the most suitable strategy for L-AI immobilization in comparison to the chelate-activated agarose. In addition, the immobilized biocatalysts by ionic interaction in MANAE showed to be the most stable, retaining up to 100% of enzyme activity for 60 min at 60 °C and with K_m values of 28 and 218 mM for MANAE-N-His-L-AI and MANAE-C-His-L-AI, respectively.

     

Purification, Characterization, and Preliminary X-Ray Diffraction Analysis of a Lactose-Specific Lectin from Cymbosema roseum Seeds

The unique carbohydrate-binding property of lectins makes them invaluable tools in biomedical research. Here, we report the purification, partial primary structure, carbohydrate affinity characterization, crystallization, and preliminary X-ray diffraction analysis of a lactose-specific lectin from Cymbosema roseum seeds (CRLII). Isolation and purification of CRLII was performed by a single step using a Sepharose-4B-lactose affinity chromatography column. The carbohydrate affinity characterization was carried using assays for hemagglutination activity and inhibition. CRLII showed hemagglutinating activity toward rabbit erythrocytes. O-glycoproteins from mucine mucopolysaccharides showed the most potent inhibition capacity at a minimum concentration of 1.2 microg mL(-1). Protein sequencing by mass spectrometry was obtained by the digestion of CRLII with trypsin, Glu-C, and AspN. CRLII partial protein sequence exhibits 46% similarity with the ConA-like alpha chain precursor. Suitable protein crystals were obtained with the hanging-drop vapor-diffusion method with 8% ethylene glycol, 0.1 M Tris-HCl pH 8.5, and 11% PEG 8,000. The monoclinic crystals belong to space group P2(1) with unit cell parameters a = 49.4, b = 89.6, and c = 100.8 A.