Y10W beta(1-40) fluorescence reflects epitope exposure in conformers of Alzheimer's beta-peptide

The Alzheimer's beta-peptide in neutral aqueous solution is characterized variously as a random coil or a heterogeneous mixture of conformers. Under conditions of lowered pH characteristic of intracellular compartments such as endosomes or lysosomes, a different conformation is favored, which is reflected in the biophysical and biological properties of the peptide. The reactivity of the epitope of the monoclonal antibody 6F/3D, encompassing residues 9-14, is drastically reduced. The fluorescence of human sequence beta(1-40) with the tyrosine at position 10 substituted with tryptophan (Y10W beta(1-40)) is quenched nearly 50% when the peptide is shifted to pH 4.6. The exposure of the 6F/3D epitope parallels Y10W beta(1-40) fluorescence changes induced by a variety of perturbations. The linkage of the sensitivity of immunological detection with the potential for monitoring rapid changes by fluorescence offers convergence of biology and biophysics in the study of beta-amyloid peptide conformation.     

Characteristics of <Emphasis Type="Italic">Pseudomonas aurantiaca</Emphasis> DNA supramolecular complexes at various developmental stages

Differences in viscoelasticity (η) and molecular mass (M) values, as well as in the fatty acid profile of lipids in DNA supramolecular complexes (SC), isolated from Pseudomonas aurantiaca cultures at the exponential and stationary growth phases, were established for the first time. Typical characteristics of DNA SC from actively growing cells were the following: η = 315 ± 15 dl/g, M_DNA = 39 × 10⁶ Da, C_16:0 > C_18:0 > C_18:1 present as basic fatty acids (FA) in a pool of loosely DNA-bound lipids; the tightly DNA-bound lipid fraction consisted of only two acids C_18:0 > C_16:0. Significantly higher values of viscoelasticity η = 779 ± 8 dl/g and M_DNA = 198 × 10⁶ Da were observed for DNA SC of the stationary phase cells; one more FA, C_14:0, was detected in the loosely bound lipid fraction, while lipids tightly bound to DNA contained mainly C_16:0 > C_18:1 > C_18:0 > C_14:0 FA. The content of saturated FA in the DNA-bound lipids in the stationary phase cells was twice as high than in the exponential phase cells. The fraction of tightly bound lipids from the stationary phase cells contained nine times more unsaturated fatty acids than the fraction from proliferating cells. These differences in FA composition of DNA-bound lipids demonstrate the importance of lipids for the structural organization and functioning of genomic DNA during bacterial culture development.     

A rapid approach for characterization of thiol-conjugated antibody–drug conjugates and calculation of drug–antibody ratio by liquid chromatography mass spectrometry

We present the demonstration of a rapid “middle-up” liquid chromatography mass spectrometry (LC–MS)-based workflow for use in the characterization of thiol-conjugated maleimidocaproyl-monomethyl auristatin F (mcMMAF) and valine-citrulline-monomethyl auristatin E (vcMMAE) antibody–drug conjugates. Deconvoluted spectra were generated following a combination of deglycosylation, IdeS (immunoglobulin-degrading enzyme from Streptococcus pyogenes) digestion, and reduction steps that provide a visual representation of the product for rapid lot-to-lot comparison—a means to quickly assess the integrity of the antibody structure and the applied conjugation chemistry by mass. The relative abundance of the detected ions also offer information regarding differences in drug conjugation levels between samples, and the average drug–antibody ratio can be calculated. The approach requires little material (<100 μg) and, thus, is amenable to small-scale process development testing or as an early component of a complete characterization project facilitating informed decision making regarding which aspects of a molecule might need to be examined in more detail by orthogonal methodologies.     

Analysis of spectral shape in the barn owl auditory system

In a behavioral experiment, we investigated how efficiently barn owls (Tyto alba) could detect changes in the spectral profile of multi-component auditory signals with stochastic envelope patterns. Signals consisted of one or five bands of noise (bandwidth 4, 16, or 64 Hz each; center frequencies 1.02, 1.43, 2.0, 2.8, 3.92 kHz). We determined increment thresholds for the 2 kHz component for three conditions: single-band condition (only the 2 kHz component), all five noise bands with the envelope fluctuations of the bands being either correlated or uncorrelated. Noise bandwidth had no significant effect on increment detection. Increment thresholds for the different conditions, however, differed significantly. Thresholds in correlated conditions were generally the lowest of all conditions, whereas, thresholds in uncorrelated conditions mostly resulted in the highest thresholds. This can be interpreted as evidence for comodulation masking release in barn owls. If the increment in the 2 kHz component is balanced by decrementing the four flanking bands in amplitude, increment detection thresholds are not affected. The data suggest that the barn owls used information from simultaneous spectral comparison across different frequency channels to detect spectral changes in multi-component noise signals rather than sequential comparison of overall stimulus levels.     

Effect of Sargassum polycystum (Phaeophyceae)-sulphated polysaccharide extract against acetaminophen-induced hyperlipidemia during toxic hepatitis in experimental rats

The effect of Sargassum polycystum crude extract on lipid metabolism was examined against acetaminophen-induced (800 mg/kg body wt., intraperitoneally) hyperlipidemia during toxic hepatitis in experimental rats. The animals intoxicated with acetaminophen showed significant elevation in the levels of cholesterol, triglycerides and free fatty acid in both serum and liver tissue. The levels of tissue total lipids and serum LDL-cholesterol were also elevated with depleted levels of serum HDL-cholesterol and tissue phospholipid. The acetaminophen-induced animals showed significant alterations in the activities of lipid metabolizing enzymes serum lecithin cholesterol acyl transferase (LCAT) and hepatic triglyceride lipase (HTGL). The levels of liver tissue fatty acids (saturated, mono and polyunsaturated) such as palmitic acid, stearic acid, oleic acid, linoleic acid, arachidonic acid and linolenic acid monitored by gas chromatography were considerably altered in acetaminophen intoxicated animals when compared with control animals. The prior oral administration of Sargassum polycystum (200 mg/kg body wt./day for a period of 15 days) crude extract showed considerable prevention in the severe disturbances of lipid profile and metabolizing enzymes triggered by acetaminophen during hepatic injury. Liver histology also showed convincing supportive evidence regarding their protective nature against fatty changes induced during acetaminophen intoxication. Thus the present study indicates that the protective nature of Sargassum polycystum extract may be due to the presence of active compounds possessing antilipemic property against acetaminophen challenge. (Mol Cell Biochem 276: 89–96, 2005)     

凋亡和周期基因芯片研究As2O3对NB4细胞凋亡相关基因表达谱的影响

目的:利用细胞凋亡和周期基因芯片研究As₂O₃作用前后NB4细胞基因表达谱的差异性,寻找As₂O₃诱导NB4细胞凋亡的相关基因并分析其可能机制。方法:流式细胞仪检测细胞凋亡率,抽提对照及诱导组细胞的mRNA,通过逆转录将As₂O₃处理前后的NB4细胞cDNA进行生物素标记,用含269个目的基因的细胞凋亡和周期基因芯片进行杂交,GEArray软件分析,筛选出As₂O₃诱导前后表达有差异的基因。芯片结果用荧光定量聚合酶链反应(realtimepolymerasechainreaction)进行验证。结果:筛选出As₂O₃作用前后表达有差异的基因共100条(占芯片基因总数的37.2%),其中97条(97/100,97%)基因表达上调,3条(3/100,3%)基因表达下调。表达上调的基因主要包括肿瘤坏死因子配体和受体家族、bcl2家族、半胱氨酸家族、DNA损伤检测和P53途径以及细胞分裂周期蛋白和激酶等基因。结论:As₂O₃主要通过上调促凋亡基因表达来诱导NB4细胞凋亡,其中TNFSF15、Apaf1、Caspase3和p16等基因可能参与As₂O₃诱导的NB4细胞凋亡,As₂O₃诱导NB4细胞凋亡的可能机制主要涉及TNF途径、线粒体途径、Caspase途径、细胞周期抑制途径和P53途径等。     

GABAA Receptor Subtypes Expressed in Cerebellar Granule Cells: A Developmental Study

Abstract: The developmental properties of primary rat cerebellar granule cells have been characterised with respect to their expression of GABA_A receptor subtypes using both an immunological approach and radioligand binding assays. At day 1 in culture, the GABA_A receptor α1 subunit was detectable in immunoblots and increased in level up to day 9. The GABA_A receptor α6 subunit was not detectable at day 1; however, at days 3–5, a specific M_r 58,000 anti-α6 1–16 Cys immunoreactive species was present which further increased in level up to 9 days in culture. Similar qualitative results were obtained for the expression of the GABA_A receptor α6 subunit in age-matched rat cerebellar membranes. In parallel studies, it was found that although there was an overall increase in [³H]Ro 15–4513 binding sites with days in culture, the relative contributions of diazepam-sensitive and diazepam-in-sensitive [³H]Ro 15–4513 binding changed. A time-dependent enrichment of the diazepam-insensitive binding site up to a maximum of 74% of total [³H]Ro 15–4513 sites was found. This was concomitant with the appearance of the GABA_A receptor α6 subunit. These results are in agreement with the pharmacology described for α6βγ2 cloned receptors. They suggest a developmentally regulated expression of the GABA_A receptor α6 subunit gene at a time that is correlated in vivo with establishment of neuronal connections.     

Establishment of a signal peptide with cross-species compatibility for functional antibody expression in both Escherichia coli and Chinese hamster ovary cells

Signal peptides are short peptides located at the N-terminus of secreted proteins. They characteristically have three domains; a basic region at the N-terminus (n-region), a central hydrophobic core (h-region) and a carboxy-terminal cleavage region (c-region). Although hundreds of different signal peptides have been identified, it has not been completely understood how their features enable signal peptides to influence protein expression. Antibody-derived signal peptides are often used to prepare recombinant antibodies expressed by eukaryotic cells, especially Chinese hamster ovary (CHO) cells. However, when prokaryotic Escherichia coli (E. coli) are utilized in drug discovery processes, such as for phage display selection or antibody humanization, signal peptides have been selected separately due to the differences in the expression systems between the species. In this study, we successfully established a signal peptide that enables a functional antibody to be expressed in both prokaryotic and eukaryotic cells by focusing on the importance of having an Ala residue in the c-region of the signal sequence. We found that changing Ser to Ala at only two positions significantly augmented the anti-HER2 antigen binding fragment (Fab) expression in E. coli. In addition, this altered signal peptide also retained the ability to express functional anti-HER2 antibody in CHO cells. Taken together, the present findings indicate that the signal peptide can promote functional antibody expression in both prokaryotic E. coli and eukaryotic CHO cells. This finding will contribute to the understanding of signal peptides and accelerate therapeutic antibody research.