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.     

Homologous and heterologous interactions between catalytic and regulatory subunits of Escherichia coli acetohydroxyacid synthaseⅠand Ⅲ

Homologous and heterologous interactions between acetohydroxyacid synthase (AHAS) I and Ⅲ from E. coli have been studied by surface plasmon resonance (SPR). The catalytic and regulatory subunits association for AHAS I (KD = 1.13 × 10-7 M) was stronger than that for AHAS Ⅲ (KD = 5.29 × 10-7 M). A strong heterologous association between regulatory and catalytic subunits and heterologous activation of catalytic subunits were observed. SPR results combined with enzyme kinetics indicate that the reconstituted he...     

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.     

Antibacterial Properties of Crotoxin from Crotalus durissus terrificus—Insight into the Mechanism of Action

The growing problem of antibiotic resistance among bacteria requires searching for new therapeutic agents with bacteriostatic and/or bactericidal properties. Crotoxin is a β-neurotoxin from the venom of the Crotalus durissus terrificus. It is composed of two subunits: CA (non-active) and CB (with phospholipase A₂ activity). It has already been shown that the isolated CB, but not the CA, subunit of crotoxin exhibits an antibacterial activity towards a variety of Gram-positive and Gram-negative bacterial species. However, no studies on the whole crotoxin complex have been carried out so far. We tested the antibacterial properties of crotoxin, as well as its isolated CB subunit, towards Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 6535, Micrococcus luteus ATCC 10240, Escherichia coli ATCC 25922, Escherichia coli ATCC 8739, and Pseudomonas aeruginosa ATCC 10145. Both toxins exhibited antibacterial properties only against Micrococcus luteus ATCC 10240. Crotoxin showed only bacteriostatic activity with a MIC of 46 µM, while the CB subunit acted as both a bacteriostatic and bactericidal agent with a MIC = MBC = 0.21 μM. The bacteriostatic effect of the toxins was independent of the enzymatic activity of the CB subunit. Bactericidal properties, however, require phospholipase A₂ activity. Both toxins reduced bacteria viability at the MIC by 72% and 85% for crotoxin- and CB-treated bacteria, respectively. The membrane permeability increased approximately three times within the first hour of incubation with toxins; afterwards, either no significant changes or a decrease of membrane permeability, compared to the control cells, were observed. We isolated a single, approximately 30 kDa bacterial wall protein which belongs to the NlpC/P60 family that interacts with crotoxin leading to the inhibition of bacterial growth. Neither crotoxin nor the CB subunit showed any cytotoxic properties to human fibroblasts at the MIC during the three-day incubation.     

应用全内反射单分子荧光成像研究蛋白复合物亚基组成

细胞的生化过程大都是由蛋白复合物完成的,研究蛋白复合物亚基的组成对于了解蛋白质的结构和生物学功能具有重要的意义,然而如何准确确定蛋白复合物中蛋白质亚基的数量（stoichiometry）仍然是一个挑战.近年来,活细胞体系单分子荧光成像技术的不断发展为原位实时动态地研究蛋白质的结构和性质提供了新的手段.本文主要介绍了应用活细胞全内反射单分子荧光成像技术表征细胞膜区蛋白复合物组成的3种方法,包括单分子漂白步数分析、荧光强度统计分布以及蛋白运动分析,并结合其基本原理介绍了这几种方法在活细胞体系膜蛋白研究中的应用.     

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.     

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.