Mutational analysis of an antigenic peptide shows recognition in a loop conformation

We have analyzed the recognition between an antigenic undecapeptide and a monoclonal antibody through a mutational approach. Antibody mAb164 is directed against the native form of the TrpB2 subunit of Escherichia coli tryptophan synthase. It recognizes a synthetic peptide, P11, constituted of residues 273-HGRVGIYFGMK-283 of TrpB with high affinity. P11 was fused with a carrier protein, MalE, to facilitate its manipulation. The affinities between mAb164 and the MalE-P11 hybrids were measured by competition enzyme-linked immunosorbent assay (ELISA). The changes of the P11 residues into progressively shorter residues, the comparison of changes into Pro and Ala, and the study of double mutants showed the following. Four hydrophobic residues of P11, Val276, Ile278, Tyr279, and Phe280, were predominant in the interaction. For some residues, e.g., Tyr279, most groups of the side chain contributed to the interaction. For others, only some groups played a significant role, e.g., the Cdelta group of Ile278 or the Cbeta group of Phe280. The lack of side chain in position Gly281 and a tertiary interaction between the side chains of Ile278 and Lys283 were important. P11 was recognized in a loop conformation, close to that of residues 273-283 of TrpB in the crystal structure of the complete tryptophan synthase, TrpA2TrpB2. Comparison of our mutational data with NMR data on the conformation of the isolated peptide P11 and with kinetic data on its interaction with mAb164 indicate that mAb164 selects a conformer of P11 that represents only a small minority of the molecules. Our results provide useful information on the mechanisms by which linear epitopes and unconstrained peptides are recognized by receptors.     

A proposal for the conformation of loop E in Escherichia coli 5S rRNA

A proposal is advanced for the conformation of the loop E region of prokaryotic 5S rRNAs based on spectroscopic data obtained from pAD3 RNA, a construct that includes helix IV, helix V, and loops D and E from Escherichia coli 5S rRNA. Even though loop E juxtaposes bases that cannot form Watson-Crick base pairs, it resembles an A-form double helix; its nucleotides relate to each other spectroscopically in a helix-like way and are in the anti conformation. The ends of loop E, which is palindromic, have the same conformation. Working in from either end towards the center of the loop, a closing GC is followed by a side-by-side GA and then by a reversed Hoogsteen AU, a pattern resembling that found at one end of eukaryotic loop E. The center of the loop consists of three nucleotide pairs, which appear to be an asymmetric GG pair, a Watson-Crick-like AG, and a GU stabilized by a single hydrogen bond.     

Extended helical conformation newly observed in protein folding

A new secondary structure, which shows regularity within the experimental error, is noticed in alpha-chymotrypsin, and considering its extended nature, the name epsilon-helix has been suggested for the same. The average observed values of phi and psi for this conformation are -93 degrees and +146 degrees, respectively. The helical parameters turn out to be n = 2.7 and h = 3.3 angstroms.     

Preservation of native conformation during aluminium-induced aggregation of tau protein

Aluminium exposure has been shown to result in aggregation of microtubule-associated protein tau in vitro. In the light of recent observations that the native random structure of tau protein is maintained in its monomeric and dimeric states as well as in the paired helical filaments characteristic of Alzheimer's disease, it is likely that factors playing a causative role in neurofibrillary pathology would not drastically alter the native conformation of tau protein. We have studied the interaction of tau protein with aluminium using circular dichroism (CD) and 27Al NMR spectroscopy. The CD studies revealed a five-fold increase in the observed elipticity of the tau-aluminium assembly. The increase in elipticity was not associated with a change in the general conformation of the protein and was most likely due to an aggregation of the tau protein induced by aluminium. 27Al NMR spectroscopy confirmed the binding of aluminium to tau protein. Hyperphosphorylation of tau in Alzheimer's disease is known to be associated with defective microtubule assembly in this condition. Abnormally phosphorylated tau exists in a polymerized form in the paired helical filaments (PHF) which constitute the neurofibrillary tangles found in Alzheimer's disease. While it is hypothesized that its altered biophysical characteristics render abnormally phosphorylated tau resistant to proteolysis, causing the formation of stable deposits, the sequence of events resulting in the polymerization of tau are little understood, as are the additional factors or modifications required for this process. Based on the results of our spectroscopic studies, a model for the sequence of events occurring in neurofibrillary pathology is proposed.     

A study into the effects of protein binding on nucleotide conformation

In this study, we examine the effects of binding to protein upon nucleotide conformation, by the comparison of X-ray crystal structures of free and protein-bound nucleotides. A dataset of structurally non-homologous protein-nucleotide complexes was derived from the Brookhaven Protein Data Bank by a novel protocol of dual sequential and structural alignments, and a dataset of native nucleotide structures was obtained from the Cambridge Structural Database. The nucleotide torsion angles and sugar puckers, which describe nucleotide conformation, were analysed in both datasets and compared. Differences between them are described and discussed. Overall, the nucleotides were found to bind in low energy conformations, not significantly different from their 'free' conformations except that they adopted an extended conformation in preference to the 'closed' structure predominantly observed by free nucleotide. The archetypal conformation of a protein-bound nucleotide is derived from these observations.     

Determining residual urine volumes using a portable ultrasonographic device

To evaluate the effects of long-term treatment antihypertensive with the dihydropyridine calcium antagonist amlodipine on insulin sensitivity, plasma insulin, and lipoprotein metabolism in obese hypertensive patients. We measured the insulin sensitivity index (SI), determined by the Minimal Model Method of Bergman, fasting plasma insulin and glucose concentrations, serum total triglyceride and lipoprotein cholesterol fractions, and blood pressure in 20 obese, non-diabetic patients with essential hypertension before and after 6 weeks of placebo and again after 6 months of amlodipine. Ten patients [mean body mass index (BMI) 30.2 kg.m-2] had been on prior treatment with a thiazide diuretic in low dosage and/or a beta-adrenoceptor blocker (group A), and 10 matched patients [BMI 31.8 kg.m-2] had been previously untreated (group B). Amlodipine was started in a dose of 5 mg and was increased to 10 mg once daily in 14 patients who were hypertensive after 8 weeks on the lower dosage. At entry (before placebo), SI was slightly but not significantly lower in group A than B [2.7 vs. 3.6 x 10(-4) ml.microU-4.min-1]; fasting plasma insulin was 13.6 vs. 12.9 microU.ml-1. After 6 weeks on placebo, S1 averaged 3.7 in group A and 4.4 x 10(-4) microU.ml-1.min-1 in group B; fasting plasma insulin was 14.6 vs. 15.1 microU.ml-1, and glucose 5.5 vs. 5.5 mmol.l-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

水杨基荧光酮荧光法测定羟自由基的探讨

建立了一种新的测定Fenton反应所产生的羟自由基的方法,经过条件的研究,得出最佳实验条件,同时,通过清除率实验反证了该方法的可靠性,利用顺磁共振法测定SAF加入前后的波谱变化来进一步证实该方法的正确性,化学测定过程均在国产仪器下完成。     

Automatic tumor segmentation using knowledge-based techniques

A system that automatically segments and labels glioblastoma-multiforme tumors in magnetic resonance images (MRI's) of the human brain is presented. The MRI's consist of T1-weighted, proton density, and T2-weighted feature images and are processed by a system which integrates knowledge-based (KB) techniques with multispectral analysis. Initial segmentation is performed by an unsupervised clustering algorithm. The segmented image, along with cluster centers for each class are provided to a rule-based expert system which extracts the intracranial region. Multispectral histogram analysis separates suspected tumor from the rest of the intracranial region, with region analysis used in performing the final tumor labeling. This system has been trained on three volume data sets and tested on thirteen unseen volume data sets acquired from a single MRI system. The KB tumor segmentation was compared with supervised, radiologist-labeled "ground truth" tumor volumes and supervised k-nearest neighbors tumor segmentations. The results of this system generally correspond well to ground truth, both on a per slice basis and more importantly in tracking total tumor volume during treatment over time.     

Mutations can cause large changes in the conformation of a denatured protein

Deletion of 13 amino acids from the carboxyl terminus of staphylococcal nuclease (WTSNase delta) results in a denatured, partially unfolded molecule that lacks significant persistent secondary structure but is relatively compact and monomeric under physiological conditions [Shortle & Meeker (1989) Biochemistry 28, 936-944; Flanagan et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 748-752]. Because of these and other properties of the SNase delta polypeptide, it is a useful model system for investigating the conformation of the denatured state of a protein without using extreme temperature or solvent conditions. Moreover, since the modification is a carboxyl-terminal deletion, SNase delta may also resemble a transient state of the polypeptide chain as it emerges from a ribosome prior to its folding. In the present study, we have examined the sizes and conformations of mutated forms of SNase delta, using small-angle X-ray scattering and circular dichroism spectroscopy. Seven mutated forms were studied: four with single substitutions, two with double substitutions, and one triple substitution. When present in the full-length SNase, each of these mutated forms exhibited unusual behavior upon solvent or thermal denaturation. In the case of the truncated form (SNase delta), the small-angle scattering curves of the mutated forms fall into two classes: one resembling the scattering curve of compact native nuclease and the other having features consistent with those expected for an expanded coil-like polymer. In contrast, the scattering curve of WT SNase delta exhibits features intermediate between those observed for globular proteins and random polymers. The amino acid substitutions that gave rise to compact, native-like versions of SNase delta were all of the m--type (m-substitutions are predicted to decrease the size of the denatured state). Those which gave rise to versions of SNase delta that were more extended and coil-like than WT SNase delta were of the m+ type (m+ substitutions are predicted to increase the size of the denatured state). Estimates of the residual secondary structure present in WT SNase delta, as well as both the m+ and m-substituted versions of SNase delta, as determined by CD, suggest that the formation of secondary structure and compaction of the polypeptide chain occur concurrently. Our results show that single amino acid substitutions can radically alter the conformational distribution of a partially condensed polypeptide chain.(ABSTRACT TRUNCATED AT 400 WORDS)     

Determining protein-protein interactions by oxidative cross-linking of a glycine-glycine-histidine fusion protein

The Ni(II) complex of the tripeptide NH2-glycine-glycine-histidine-COOH (GGH) mediates efficient protein-protein cross-linking in the presence of oxidants such as oxone and monoperoxyphthalic acid (MMPP). Here we demonstrate that GGH fused to the amino terminus of a protein can still support cross-linking. The tripeptide was expressed at the amino terminus of ecotin, a dimeric macromolecular serine protease inhibitor found in the periplasm of Escherichia coli. In the presence of Ni(OAc)2 and MMPP, GGH-ecotin is cross-linked to give a species that has an apparent molecular mass of a GGH-ecotin dimer with no observable protein degradation. The cross-linking reaction occurs between two ecotin proteins in a dimer complex. Furthermore, GGH-ecotin can be cross-linked to a serine protease target, trypsin, and the reaction is specific for proteins that interact with ecotin. The cross-linking reaction has been carried out on small peptides, and the reaction products have been analyzed by matrix-assisted laser desorption/ionization mass spectrometry. The target of the reaction is tyrosine, and the product is bityrosyl cross-links. The yield of the cross-linking is on the order of 15%. However, the reaction efficiency can be increased 4-fold by a single amino acid substitution in the carboxy terminus of ecotin that places an engineered tyrosine within 5 A of a naturally occurring tyrosine. This cross-linking methodology allows for the protein cross-linking reagent to be encoded for at the DNA level, thus circumventing the need for posttranslational modification.     

利用模糊综合评价法确定矿山生产规模

矿山生产规模是矿山开发过程中一项非常重要的决策要素。文中利用模糊综合评价方法综合考虑矿山生产的多种因素，对矿山生产规模的确定作了有益的探索，并建立了焦家金矿技术改造生产规模确定的模糊综合评价模型。     

Determining the optimal mix of exercise activities using mathematical programming

A 56K protein co-purified with bovine milk fat globule membrane (MFGM) proteins bound to Wisteria floribunda agglutinin (WFA) like most MFGM glycoproteins. Treatment with N-glycanase or beta-N-acetylhexosaminidase abolished the lectin binding to the protein. Amino acid sequence and immunoblot analyses revealed that the 56K protein is an IgG heavy chain. Lectin column chromatography of the oligosaccharides released by hydrazinolysis from the purified IgG heavy chains revealed that 0.08% of the total N-linked sugar chains bind to a WFA-agarose column, suggesting that they contain the beta-N-acetylgalactosaminylated structure.     

Pedicle screw placement using image guided techniques

Clinical evaluation of a computer assisted spine surgical system is presented. Eighty pedicle screws were inserted using computer assisted technology in thoracic and lumbar vertebrae for treatment of different types of disorders including fractures, spondylolisthesis, and scoliosis. Fifty-two patients with severe fractures, spondylolisthesis, or pseudoarthrosis of T10 to L5 were treated using a computer assisted technique on 1/2 the patients and performing the screw insertion manually for the other 1/2. At the same time, 28 pedicle screws were inserted in T12 to L4 vertebrae for scoliosis with the help of the computer assisted technique. Surgery was followed in all cases (66 vertebrae; 132 pedicle screws) by postoperative radiographs and computed tomographic examination, on which measurements of screw position relative to pedicle position could be done. For fractures, spondylolisthesis, or pseudarthrosis, comparison between the two groups showed that four screws in 52 (8%) vertebrae had incorrect placement with computer assisted technique whereas 22 screws in 52 (42%) vertebrae had incorrect placement with manual insertion. In patients with scoliosis, four screws in 28 (14%) vertebrae had incorrect placement. In all of the patients (132 pedicle screws) there were no neurologic complications. These results show that a computer assisted technique is much more accurate and safe than manual insertion.     

Advancement of the midface using distraction techniques

Fourteen patients underwent Le Fort III midface advancement using distraction techniques. Six have cephalometric documentation extending beyond 1 year postoperatively, and the positions of cephalometric points A and orbitale over time are reported here. Excellent stability of advancement at the occlusal level and some relapse at the level of orbitale are documented. Elimination or diminution of obstructive sleep apnea occurred in all patients so affected, and one of two patients with tracheostomy has been decannulated. Speech effects have been mild or transient. No untoward effects on extraocular muscle function have occurred.     

Kinetic analyses of mutations in the glycine-rich loop of cAMP-dependent protein kinase

The conserved glycines in the glycine-rich loop (Leu-Gly50-Thr-Gly52-Ser-Phe-Gly55-Arg-Val) of the catalytic (C) subunit of cAMP-dependent protein kinase were each mutated to Ser (G50S, G52S, and G55S). The effects of these mutations were assessed here using both steady-state and pre-steady-state kinetic methods. While G50S and G52S reduced the apparent affinity for ATP by approximately 10-fold, substitution at Gly55 had no effect on nucleotide binding. In contrast to ATP, only mutation at position 50 interfered with ADP binding. These three mutations lowered the rate of phosphoryl transfer by 7-300-fold. The combined data indicate that G50 and G52 are the most critical residues in the loop for catalysis, with replacement at position 52 being the most extreme owing to a larger decrease in the rate of phosphoryl transfer (29 vs 1.6 s-1 in contrast to 500 s-1 for wild-type C). Surprisingly, all three mutations lowered the affinity for Kemptide by approximately 10-fold, although none of the loop glycines makes direct contact with the substrate. The inability to correlate the rate constant for net product release with the dissociation constant for ADP implies that other steps may limit the decomposition of the ternary product complex. The observations that G52S (a) selectively affects ATP binding and (b) significantly lowers the rate of phosphoryl transfer without making direct contact with either the nucleotide or the peptide imply that this residue serves a structural role in the loop, most likely by positioning the backbone amide of Ser53 for contacting the gamma-phosphate of ATP. Energy-minimized models of the mutant proteins are consistent with the observed kinetic consequences of each mutation. The models predict that only mutation of Gly52 will interfere with the observed hydrogen bonding between the backbone and ATP.     

Percutaneous iliosacral screw placement using image guided techniques

A computer assisted technique of iliosacral screw placement that is applicable to unstable pelvic ring fractures is proposed. The goals are to operate noninvasively with a percutaneous procedure to decrease the complications of surgical exposure and to provide greater accuracy in locating the close neurovascular structures. Preoperative computed tomographic images of the pelvis are provided and a computed tomography three-dimensional model is built. In this model, the optimal trajectories for the drilling are planned. An ultrasound based registration is performed intraoperatively. This registration is the most original part of this work. After performing the passive drilling guidance step, the surgeon places the screws. The accuracy of the ultrasound based registration is checked by comparison with a standard surface based registration at the end of the test experiment. Each screw position is verified by a computed tomographic examination. Four human anatomic specimen pelves were tested with three screw insertions for each pelvis (12 screws). All of the screws were considered to be placed correctly. The method is safe and encourages the start of clinical application.