Simultaneous acquisition of [13C,15N]- and [15N,15N]-separated 4D gradient-enhanced NOESY spectra in proteins

The simultaneous acquisition of a 4D gradient-enhanced and sensitivity-enhanced [13C,15N]/[15N,15N]-separated NOESY is presented for the 74-residue [13C,15N]-labeled N-terminal SH3 domain of mGrb2 complexed with a peptide fragment from mSOS-2 in 90% H2O. The method readily accommodates different 13C and 15N spectral widths, but requires that the same number of increments be collected for both 13C and 15N in the simultaneous dimension (F2). For purposes of display and analysis, the two 4D spectra can be deconvolved during the processing stage by the appropriate linear combination of separately stored FIDs. Compared to collecting each of these two 4D data sets separately, the presented method is a factor (2)1/2 more efficient in sensitivity per unit acquisition time. The interleaved nature of this method may also lead to improved peak registration between the two 4D spectra.     

NMR investigations of recombinant 15N/13C/2H-labeled bradykinin bound to a Fab mimic of the B2 receptor

NMR spectroscopy has been used to obtain structural information on the bioactive conformation of the nonapeptide hormone bradykinin (Arg-Pro-Pro-Gly-Ser-Pro-Phe-Arg, BK) bound to the Fab-fragment of an antibody that mimics the hormone binding site of the natural bradykinin B2-receptor. Using 15N or 15N,13C, 60% 2H isotope labelled bradykinin, complete 1H, 13C and 15N assignments for bradykinin bound to the Fab-fragment have been obtained. Preliminary interpretation of 15N edited NOE spectra indicates that the conformation of bradykinin bound to the model receptor differs substantially from previous computer models of the bioactive conformation of bradykinin.     

Simple, efficient protocol for enzymatic synthesis of uniformly 13C, 15N-labeled DNA for heteronuclear NMR studies

The use of uniformly 13C,15N-labeled RNA has greatly facilitated structural studies of RNA oligonucleotides by NMR. Application of similar methodologies for the study of DNA has been limited, primarily due to the lack of adequate methods for sample preparation. Methods for both chemical and enzymatic synthesis of DNA oligonucleotides uniformly labeled with 13C and/or 15N have been published, but have not yet been widely used. We have developed a modified procedure for preparing uniformly 13C,15N-labeled DNA based on enzymatic synthesis using Taq DNA polymerase. The highly efficient protocol results in quantitative polymerization of the template and approximately 80% incorporation of the labeled dNTPs. Procedures for avoiding non-templated addition of nucleotides or for their removal are given. The method has been used to synthesize several DNA oligonucleotides, including two complementary 15 base strands, a 32 base DNA oligonucleotide that folds to form an intramolecular triplex and a 12 base oligonucleotide that dimerizes and folds to form a quadruplex. Heteronuclear NMR spectra of the samples illustrate the quality of the labeled DNA obtained by these procedures.     

Photoimmunodetection: a nonradioactive labeling and detection method for DNA

We describe a simple detection system for DNA based on antibody detection of UV-induced photoproducts. It includes a convenient and inexpensive labeling procedure, which is completed in 5-10 min. The only equipment required is a UV source such as an ordinary transilluminator or a DNA crosslinker. Using a monoclonal antibody specific for thymine dimers, coupled to horseradish peroxidase, we are able to detect subpicogram amounts of UV-irradiated DNA directly, and approximately 10 pg homologues DNA indirectly by hybridization with an irradiated probe.     

A PCR-based strategy for extensive mutagenesis of a target DNA sequence

A mixed population of mutagenic oligonucleotide primers was used to generate a set of point mutations in a short region of a retroviral gene by PCR amplification. The mixed population of mutagenic primers was generated by incorporating a mixture of A, G, C and T at specific sites during oligonucleotide synthesis. With the proportions of mutagenic nucleotides used for our experiments, 47 percent of the 213 clones analyzed had one or more point mutation in the target DNA sequence. In addition, unpredicted mutations were observed that contributed to the mutagenic complexity of the population. We have found this approach to be an efficient means for extensive mutagenesis of a defined target DNA sequence.     

A noninvasive method for measuring urea kinetics with a single dose of [15N15N]urea in free-living humans

A noninvasive method is described in which the endogenous rate of urea production can be determined in normal, free-living adults. A single dose of [15N15N]urea was given orally, and the amount of label excreted as [15N15N]urea and [15N14N]urea in urine over the subsequent 48 h was measured. From the rates of excretion of labeled and unlabeled urea the rate of urea production was derived. Using this single-dose protocol the rate of urea production was 207 +/- 56 (mean +/- SD) mg N/(kg.d) in six normal adult men consuming 74 g protein/d. These results were not different when compared with rates of urea production obtained with a prime/intermittent protocol in an earlier study in the same individuals [199 +/- 20 mg N/(kg.d)]. We conclude that urea kinetics can be measured noninvasively with a single dose of [15N15N]urea and that this method may be suitable for use in free-living individuals to determine urea production rates for habitual dietary intakes.     

Rotational diffusion anisotropy of proteins from simultaneous analysis of 15N and 13C alpha nuclear spin relaxation

Current methods of determining the rotational diffusion tensors of proteins in solution by NMR spectroscopy exclusively utilize relaxation rate constants for backbone amide 15N spins. However, the distributions of orientations of N-H bond vectors are not isotropic in many proteins, and correlations between bond vector orientations reduce the accuracy and precision of rotational diffusion tensors extracted from 15N spin relaxation data. The inclusion of both 13C alpha and 15N spin relaxation rate constants increases the robustness of the diffusion tensor analysis because the orientations of the C alpha-H alpha bond vectors differ from the orientations of the N-H bond vectors. Theoretical and experimental results for calbindin D9k, granulocyte colony stimulating factor, and ubiquitin, three proteins with different distributions of N-H and C alpha-H alpha bond vectors, are used to illustrate the advantages of the simultaneous utilization of 13C alpha and 15N relaxation data.     

The Stimulated Raman Spectrum of Symmetric 13C Cyanogen, 13C2N2

BACKGROUND: The authors examined the interaction of ketamine with recombinant mu, kappa, and delta opioid receptors and recombinant orphan opioid receptors expressed in Chinese hamster ovary cells (CHO-mu, CHO-kappa, CHO-delta, and CHO(ORL1), respectively). METHODS: CHO-mu, CHO-kappa, and CHO-delta membranes were incubated with the opioid receptor radioligand [3H]diprenorphine at room temperature. Ketamine (racemic, R(-) and S(+)) was included at concentrations covering the clinical range. CHO(ORL1) membranes were incubated with [125I]Tyr(14)nociceptin and racemic ketamine at room temperature. The effects of racemic ketamine and selective opioid receptor agonists (mu: [D-Ala2, MePhe4, Gly(ol)5] enkephalin (DAMGO); kappa: spiradoline or delta: [D-pen2, D-pen5] enkephalin (DPDPE)) on forskolin-stimulated cyclic adenosine monophosphate formation also were examined. Data are mean +/- SEM. RESULTS: Racemic ketamine increased the radioligand equilibrium dissociation constant for [3H]diprenorphine from 85+/-5 to 273+/-11, 91+/-6 to 154+/-16, and 372+/-15 to 855+/-42 pM in CHO-mu, CHO-kappa, and CHO-delta, respectively. The concentration of radioligand bound at saturation was unaffected. In CHO-mu and CHO-kappa cells, racemic ketamine did not slow the rate of naloxone-induced [3H]diprenorphine dissociation. Ketamine and its isomers also displaced [3H]diprenorphine binding to mu, kappa, and delta receptors in a dose-dependent manner, with pKi values for racemic ketamine of 4.38+/-0.02, 4.55+/-0.04, and 3.57+/-0.02, respectively. S(+)-ketamine was two to three times more potent than R(-)-ketamine at mu and kappa receptors. Racemic ketamine displaced [125I]Tyr(14)nociceptin with an estimated affinity constant of 0.5 mM. Racemic ketamine inhibited the formation of cyclic adenosine monophosphate (naloxone insensitive) in a dose-dependent manner (concentration producing 50% inhibition approximately 2 mM) in all cell lines, including untransfected CHO cells. Ketamine (100 microM) reversed DAMGO (mu) and spiradoline (kappa) inhibition of formation of cyclic adenosine monophosphate. CONCLUSIONS: Ketamine interacts stereoselectively with recombinant mu and kappa opioid receptors.     

A modified PCR-based method for rapid non-radioactive detection of clinically important pathogens

We have devised a sensitive and rapid method for the detection of several bacterial pathogens in clinical specimens using PCR. This method has been named Direct Labeling and Detection Procedure (DLDP) and is based on the direct incorporation of a nonradioactive digoxigenin label (DIG-11-dUTP) into a microbial species-specific gene fragment during amplification. Following amplification, the resulting PCR products are cleansed of nonincorporated DIG-11-dUTP, spotted onto a nylon membrane, fixed by UV-crosslinking and the labeled DNA is visualized by digoxigenin detection reagents. Using cultivated reference bacteria (Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa) we were able to demonstrate a rapid and sensitive detection of < 20 CFU of bacteria in human secretions (sputum, urine, mucous). The present study suggests that DLDP can be used as a reliable method for indication of bacteria in clinical or environmental specimens with the proviso that the selected corresponding oligonucleotide primers provide amplification of strong species-specific genes.     

1H, 13C, 15N nuclear magnetic resonance backbone assignments and secondary structure of human calcineurin B

The calmodulin- and calcium-stimulated protein phosphatase calcineurin, PP2B, consists of two subunits: calcineurin B, which binds Ca2+, and calcineurin A, which contains the catalytic site and a calmodulin binding site. Heteronuclear 3D and 4D NMR experiments were carried out on a recombinant human calcineurin B which is a 170-residue protein of molecular mass 19.3 kDa, uniformly labeled with 15N and 13C. The nondenaturing detergent CHAPS was used to obtain a monomeric form of calcineurin B. Three-dimensional triple resonance experiments yielded complete sequential assignment of the backbone nuclei (1H, 13C, and 15N). This assignment was verified by a 4D HN(COCA)NH experiment carried out with 50% randomly deuteriated and uniformly 15N- and 13C-enriched calcineurin B. The secondary structure of calcineurin B has been determined on the basis of the 13C alpha and 13C beta secondary chemical shifts, J(HNH alpha) couplings, and NOE connectivities obtained from 3D 15N-separated and 4D 13C/15N-separated NOESY spectra. Calcineurin B has eight helices distributed in four EF-hand, helix-loop-helix [Kretsinger, R. H. (1980) CRC Crit. Rev. Biochem. 8, 119-174] calcium binding domains. The secondary structure of calcineurin B is highly homologous to that of calmodulin. In comparison to calmodulin, helices B and C are shorter while helix G is considerably longer. As was observed for calmodulin in solution, calcineurin B does not have a single long central helix; rather, helices D and E are separated by a six-residue sequence in a flexible nonhelical conformation.     

一种面向网络长文本的话题检测方法

提出了一种面向网络长文本的话题检测方法.针对文本表示的高维稀疏性和忽略潜在语义的问题,提出了Word2vec&LDA（latent dirichlet allocation）的文本表示方法.将LDA提取的文本特征词隐含主题和Word2vec映射的特征词向量进行加权融合既能够进行降维的作用又可以较为完整的表示出文本信息.针对传统话题发现方法对长文本输入顺序敏感问题,提出了基于文本聚类的Single-Pass&HAC（hierarchical agglomerative clustering）的话题发现方法,在引入时间窗口和凝聚式层次聚类的基础上对于文本的输入顺序具有了更强的鲁棒性,同时提高了聚类的精度和效率.为了评估所提出方法的有效性,本文从某大学社交平台收集了来自真实世界的多源数据集,并基于此进行了大量的实验.实验结果证明,本文提出的方法相对于现有的方法,如VSM（state vector space model）、Single-Pass等拥有更好的效果,话题检测的精度提高了10%~20%.      

一种面向网络长文本的话题检测方法

提出了一种面向网络长文本的话题检测方法.针对文本表示的高维稀疏性和忽略潜在语义的问题,提出了Word2vec&LDA (latent dirichlet allocation)的文本表示方法.将LDA提取的文本特征词隐含主题和Word2vec映射的特征词向量进行加权融合既能够进行降维的作用又可以较为完整的表示出文本信息.针对传统话题发现方法对长文本输入顺序敏感问题,提出了基于文本聚类的Single-Pass&HAC (hierarchical agglomerative clustering)的话题发现方法,在引入时间窗口和凝聚式层次聚类的基础上对于文本的输入顺序具有了更强的鲁棒性,同时提高了聚类的精度和效率.为了评估所提出方法的有效性,本文从某大学社交平台收集了来自真实世界的多源数据集,并基于此进行了大量的实验.实验结果证明,本文提出的方法相对于现有的方法,如VSM (state vector space model)、Single-Pass等拥有更好的效果,话题检测的精度提高了10%～20%.     

Assignment of the 1H, 15N and 13C resonances of the calcium-free and calcium-bound forms of the first C2-domain of synaptotagmin I

Two cases of sigmoid perforation and fistula occurred as late complications after insertion of a nonresorbable prosthesis by the open preperitoneal inguinal route. These infrequent complications are favoured by peritoneal defects and use of materials which can cause extensive sclerous reactions. Indications for this type of mesh are increasingly common with the intraperitoneal laparoscopic approach, so that careful peritoneal dissection and closure are required.