Synthesis and Biological Evaluation of Quinoxaline Derivatives for PET Imaging of the NMDA Receptor

Due to the biological complexity of the N‐methyl‐d ‐aspartate receptor (NMDAR ), the development of a positron emission tomography radiotracer for the imaging of NMDAR has met with limited success. Recent studies have established the presence of GluN2A subunit of the NMDAR in the heart and its role in the regulation of intracellular calcium levels. In our efforts to develop an imaging agent for the GluN2A subunit, we designed three new compounds based on a quinoxaline scaffold. The synthesis of the analogues was based on a two‐step Kabachnik–Fields reaction in sequence with Suzuki cross‐coupling and acid hydrolysis. They exhibited comparable high binding affinity values below 5 nm . A two‐step radiolabeling procedure was successfully developed for the synthesis of [¹⁸F] 1 . [¹⁸F] 1 was obtained in a modest overall radiochemical yield of 5.5 ± 4.2%, a good specific radioactivity of 254 ± 158 GBq/μmol, and a radiochemical purity > 99%. While compounds 2 and 3 showed comparable binding affinity towards NMDAR , sluggish radiolabeling, prevented their further evaluation. For [¹⁸F] 1 , in vitro autoradiography on rat heart slices demonstrated heterogeneous but unspecific accumulation, whereas for the brain a high in vitro specificity towards NMDAR , could be demonstrated.     

High-performance-liquid chromatography ofThermus aquaticus 50S and 30S ribosomal proteins

Summary The ribosomal 50S and 30S subunit proteins (r-proteins) ofThermus aquaticus have, for the first time, been characterized by size exclusion chromatography (SEC) and by reversed phase high performance liquid chromatography (RPC). To ensure that the best resolution in the RPC was obtained. the elution conditions, such as gradient time, flow rate, temperature, ionic strength of the eluent and the type of stationary phase were optimized. Correlation between experimentally found retention times and those predicted by DryLab G was better than 0.7% over 30 peaks. Protein fractions from RPC runs were desalted and processed by gel electrophoresis so that the ribosomal proteins could be identified by their position on SDS-polyacrylamide gels. The enhanced speed and quality of separation which has been achieved in this study is expected to bring advantaces in experimental work with ribosomal proteins as well as with other biopolymers. In our case the high resolution technique provides a basis for the preparation of a collection of individual ribosomal protein components for future rRNA-protein interaction studies.     

Efficient Cholera Toxin B Subunit‐Based Nanoparticles with MRI Capability for Drug Delivery to the Brain Following Intranasal Administration

Alzheimer's disease (AD) is an incurable neurodegenerative brain disorder that exhibits clear pathologic changes in the hippocampus. Traditional drug delivery systems are ineffective due to the existence of the blood–brain barrier (BBB). In this study, an efficient, stable, and easily constructed nanosystem (CB‐Gd‐Cy5.5) based on the cholera toxin B subunit (CB) is designed to improve the efficiency of drug delivery to the brain, especially the hippocampus. Through intranasal administration, CB‐Gd‐Cy5.5 is easily delivered to the brain without intervention by the BBB. The CB in CB‐Gd‐Cy5.5 is used for specifically combining with the monosialoganglioside GM1, which is widely found in the hippocampus. This nanosystem exhibits impressive performance in accumulating in the hippocampus. In addition, the good magnetic resonance imaging (MRI) capability of CB‐Gd‐Cy5.5 can satisfy the monitoring of AD in the different stages.     

The mass spectral analysis of isolated hops A-type proanthocyanidins by electrospray ionization tandem mass spectrometry

Comprehensive mass spectral fragmentation patterns have been established for sequencing chromatographically isolated A-type proanthocyanidins (PAs) using electrospray ionization tandem mass spectrometry (ESI-MS(n)) in the positive ion mode similar to those used for sequencing previously reported B-type PAs. Sequence-identifying fragmentations for A-type PAs include heterocyclic ring fission (HRF), retro-Diels-Alder (RDA) fission, benzofuran-forming (BFF) fission, and quinone methide (QM) fission. There is commonality in fragmentation patterns between A-type and B-type PAs, but distinguishing features in the mass spectral patterns between the two classes include 2-Da mass differences in the pseudo molecular ions, the propensity for the A-type PAs to undergo QM fissions and yield bis-quinoid ions as opposed to mono-quinoid ions in the upper unit of the sequence, and the reluctance of A-type linkages to undergo RDA, BFF, and BFF/H(2)O fissions from the upper unit. The positions of one or more A-type (C2-->O-->C7') ether linkages have been located in sequences of PAs ranging in chain lengths of two to five monomer units using ESI-MS(n) data. Using the fragmentation information from ESI-MS(n) experiments, a total of 17 PAs were structurally sequenced by systematic real time ESI-MS(n). Among them ten A-type and six B-type hop PAs are reported here for the first time.     

Specific and genotypic identification of Cryptosporidium from a broad range of host species by nonisotopic SSCP analysis of nuclear ribosomal DNA

The accurate identification of Cryptosporidium (Protozoa: Apicomplexa) species and genotypes is central to the understanding of the transmission and to the diagnosis and control of cryptosporidiosis. In this study, we demonstrate the effectiveness of nonisotopic SSCP analysis of a approximately 300 bp region of the small subunit (pSSU) of ribosomal DNA for the specific identification of and delineation among 18 different Cryptosporidium species and genotypes from a wide range of hosts. This mutation scanning approach allowed the rapid and reliable differentiation between species/genotypes differing by as little as 1.3% in the pSSU sequence, with the capacity to detect point mutations. The present findings confirm the usefulness of this tool for the rapid genetic screening of Cryptosporidium samples from any host species, providing a foundation for detailed systematic, epidemiological and ecological studies. Although applied herein to pSSU, this low cost approach should be applicable to a wide range of genetic loci for population genetic investigations of Cryptosporidium.     

Stable expression of foot-and-mouth disease virus protein VP1 fused with cholera toxin B subunit in the potato (Solanum tuberosum)

The expression vector, pBI121CTBVP1, containing the fusion of the foot and mouth disease virus (FMDV) VP1 gene and the cholera toxin B subunit (CTB) gene was constructed by fused PCR and transferred into potato (Solanum tuberosum L.) by Agrobacterium-mediated transformation. Transformed plants were obtained by selecting on kanamycin-resistant medium strictly and regenerated. The transgenic plantlets were identified by PCR, Southern-blot and the production of fused protein was confirmed and quantified by Western-blot and ELISA assays. The results showed that the fused genes were expressed stablely under the control of specific-tuber patatin promoter. The expressed fused proteins have a certain degree of immunogenicity.     

Subunit analyses of a novel thermostable glucose dehydrogenase showing different temperature properties according to its quaternary structure

We previously reported a novel glucose dehydrogenase (GDH) showing two peaks in the optimum temperature for the reaction at around 45°C and at around 75°C. Each peak derived from hetero-oligomeric enzyme, constructed from two distinct peptides with an α-subunit (MWs 67,000) and β-subunit (MWs 43,000), and a single peptide enzyme containing an α-subunit alone. The function of the two subunits in the thermostable co-factor binding GDH was investigated. The results of spectroscopic analyses indicated that the α-subunit contained an unknown co-factor showing specific fluorescence spectra like pyrroloquinoline quinone (PQQ), and the β-subunit was cytochrome c. Moreover, the results of a urea denaturation and reconstitution experiment suggested that the dissociation of the hetero-oligomeric complex to a single peptide was reversible. The kinetic parameter analyses for glucose and the electron mediator also suggested that the β-subunit was responsible for electron transfer from the catalytic center of the α-subunit to the electron mediator.     

电泳/质谱技术研究鲨鱼肝铁蛋白及亚基多聚体特性

改良鲨鱼肝铁蛋白(liver ferritin ofsphyrna zygaena,SZLF)分离技术,并结合非变性梯度聚丙烯酰胺凝胶电泳(NGPAGE)制备质谱纯SZLF,以维系铁蛋白分子和它的亚基之间的作用类型,供研究铁蛋白结构与功能。实验结果表明,SZLF由分子量约为20kDa的单类型亚基组成。SZLF和它的亚基均组成不同的聚合体。聚合体类型和聚合数目与铁蛋白亚基和分离介质有关。MALDI-TO F质谱仪的激光和基质协同能有效解吸SZLF中的亚基成为准分子离子,并供质量分析,其亚基特征质谱峰m/z值分别为10889.35和22030.45,确定为带双电荷[M 2H]2 和单电荷[M H] 的SZLF亚基分子量。SDS-PAGE和变性IEF技术研究指出,形成不同聚合态的SZLF,其分子之间的相互作用强度高于SZLF亚基自身,难以通过MALDI-TOF质谱技术测定其亚基的结构信息。尽管SZLF由单类型亚基组成,但它能通过聚合体和自身亚基之间的相互作用差异性发挥极其重要的生理功能。     

Comparison between the behavior of different hydrophobic peptides allowing membrane anchoring of proteins

Membrane binding of proteins such as short chain dehydrogenase reductases or tail-anchored proteins relies on their N- and/or C-terminal hydrophobic transmembrane segment. In this review, we propose guidelines to characterize such hydrophobic peptide segments using spectroscopic and biophysical measurements. The secondary structure content of the C-terminal peptides of retinol dehydrogenase 8, RGS9-1 anchor protein, lecithin retinol acyl transferase, and of the N-terminal peptide of retinol dehydrogenase 11 has been deduced by prediction tools from their primary sequence as well as by using infrared or circular dichroism analyses. Depending on the solvent and the solubilization method, significant structural differences were observed, often involving α-helices. The helical structure of these peptides was found to be consistent with their presumed membrane binding. Langmuir monolayers have been used as membrane models to study lipid–peptide interactions. The values of maximum insertion pressure obtained for all peptides using a monolayer of 1,2-dioleoyl-sn-glycero-3-phospho-ethanolamine (DOPE) are larger than the estimated lateral pressure of membranes, thus suggesting that they bind membranes. Polarization modulation infrared reflection absorption spectroscopy has been used to determine the structure and orientation of these peptides in the absence and in the presence of a DOPE monolayer. This lipid induced an increase or a decrease in the organization of the peptide secondary structure. Further measurements are necessary using other lipids to better understand the membrane interactions of these peptides.