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A novel DBU-promoted tandem cyclization reaction of ynones with diazo compounds as the N-terminal electrophiles has been developed. The reaction provides a simple and efficient method for the synthesis of fused eight-membered oxocino[2,3- c ] pyrazoles from readily available acyclic starting materials in a single step. This reaction allows the formation of four new bonds and two rings in a highly regio- and diastereoselective manner, where two adjacent stereocenters are created simultaneously in an atom-economic manner.

The key to gene therapy is the design of biocompatible and efficient delivery systems. In this work, a glutathione (GSH)-activated aggregation-induced-emission (AIE) cationic amphiphilic lipid, termed QM-SS-KK, was prepared for nonviral gene delivery. QM-SS-KK was composed of a hydrophilic biocompatible lysine tripeptide headgroup, a GSH-triggered disulfide linkage, and a hydrophobic AIE fluorophore QM-OH (QM: quinoline-malononitrile) tail. The peptide moiety could not only efficiently compact DNA but also well modulate the dispersion properties of QM-SS-KK, leading to the fluorescence-off state before GSH treatment. The cleavage of disulfide in QM-SS-KK by GSH generated AIE signals in situ with a tracking ability. The liposomes consisted of QM-SS-KK, and 1,2-dioleoylphosphatidylethanolamine (DOPE) (QM-SS-KK/DOPE) delivered plasmid DNAs (pDNAs) into cells with high efficiency. In particular, QM-SS-KK/DOPE had an enhanced transfection efficiency (TE) in the presence of 10% serum, which was two times higher than that of the commercial transfection agent PEI25K. These results highlighted the great potential of peptide and QM-based fluorescence AIE lipids for gene delivery applications.

Background: With the increasing incidence of mobile phone addiction, the potential risk factors of mobile phone addiction have attracted more and more researchers' attention. Although various personality trait factors have been proven to be significant predictors of mobile phone addiction, limited attention has been paid to preference for solitude. Considering the adverse impacts of preference for solitude in the context of collectivistic societies and its possible negative effect on mobile phone addiction, this study was designed to examine the relationship between preference for solitude and mobile phone addiction, and to test the mediating role of psychological distress and the moderating role of mindfulness in this relationship. Methods: Data were collected through convenience sampling from a comprehensive university in China. A total of 927 Chinese college students (371 males and 556 females), aged from 16 to 24 ( M age = 19.89 years, SD = 1.22), participated in this study. Their preference for solitude, psychological distress, mindfulness, and mobile phone addiction were measured using well-validated self-report questionnaires. Results: Correlational analyses, sobel test, SPSS macro PROCESS (Model 8) and simple slopes analyses were used for major data analysis. Results showed that preference for solitude was significantly and positively associated with mobile phone addiction, and this link could be mediated by psychological distress. Moreover, the indirect effect of psychological distress in this link was moderated by mindfulness, with this effect being stronger for college students with lower levels of mindfulness. However, mindfulness can not moderate the direct relation between preference for solitude and mobile phone addiction. Conclusion: The present study broadened our knowledge of how and when (or for whom) preference for solitude is related to mobile phone addiction. Education professionals and parents should pay special attention to the psychological distress and mobile phone addiction of college students with high levels of preference for solitude, particularly for those with lower levels of mindfulness., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chen, Yan, Yuan, Zhu, Zhang and Lian.)

Objective: We aimed at investigating the expression level of vascular endothelial growth factor-A (VEGF-A) in patients with primary Sjögren's Syndrome (pSS) and evaluating the relationship between serum VEGF-A and the laboratory indicators that are associated with it in pSS., Methods: The VEGF-A levels were measured by ELISA in a total of 88 participants, including 58 patients with pSS and 30 healthy people. The VEGF-A levels between two groups were analyzed., Results: The serum levels of VEGF-A in pSS and control groups were 175.50 (112.00,296.50) pg/mL and 181.50 (155.25,288.50) pg/mL, without statistically significant difference. The associations were found between serum levels of VEGF-A with C reactive protein (CRP) (r=0.265, P <0.05), white blood cells (WBC) (r=0.302, P<0.05), neutrophils (NEUT) (r=0.349, P <0.05), platelets(PLT) (r=0.276, P <0.05), complement 3 (C3) (r=0.477, P<0.05), complement 4 (C4) (r=0.387, P <0.05) and CA19-9 (r=0.392, P <0.05). Among these laboratory indicators, VEGF-A was correlated with platelets and complement 4 in patients with pSS., Conclusions: In patients with pSS, the levels of VEGF-A were independently influenced by the levels of platelet and complement 4, which indicated the intermodulation between the growth factor and immune system in the autoimmune disease., (© 2020 by the Association of Clinical Scientists, Inc.)

Fermentative caproate production from wastewater is attractive but is currently limited by the low product purity and concentration. In this work, continuous, selective production of caproate from acetate and ethanol, the common products of wastewater anaerobic fermentation, was achieved in an anaerobic membrane bioreactor (AnMBR). The reactor was continuously operated for over 522 days without need for chemical cleaning. With an ethanol-to-acetate ratio of 3.0, the effluent caproate concentration was 2.62 g/L on average and the caproate ratio in liquid products reached 74%. Further raising the influent ethanol content slightly increased the effluent caproate level but lowered the product selectivity and resulted in microbial inhibition. The Clostridia (the major caproate-producing bacteria) and Methanobacterium species (which consume hydrogen to alleviate microbial inhibition) was significantly enriched in the acclimated sludge. Our results imply a great potential of utilizing AnMBR to recover caproate from the effluent of wastewater acidogenic fermentation process., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Nickel hydroxide (Ni(OH) 2 )-based electrocatalysts are promising for the oxygen evolution reaction (OER) due to their low cost, but their activity and durability still need substantial improvement to meet practical application. Here, we report a sulfate-functionalized Ni(OH) 2 nanobelt (S-Ni(OH) 2 ) electrocatalyst, which exhibited self-enhanced OER activity due to its self-renewed surface during anodic oxidation. The S-Ni(OH) 2 was in situ grown on the nickel foam (NF) surface in potassium peroxydisulfate solution through one-step hydrothermal treatment. This material outperformed all the existing electrocatalysts in the intensity and duration of the OER activity enhancement. An overpotential drop of 70 mV is shown by the S-Ni(OH) 2 /NF electrode during 110 h reaction at a current density of 100 mA cm -2 , and the overpotential remains as low as 358 mV at a current density of 200 mA cm -2 . Such activity enhancement during OER is mainly ascribed to the formation of a highly active NiOOH/Ni(SO 4 ) 0.3 (OH) 1.4 composite on the S-Ni(OH) 2 surface as a result of gradual sulfate release. Given the facile and environmentally benign fabrication process (without external addition of a Ni source and surfactant) and good electrochemical properties (high activity and long lifetime), the S-Ni(OH) 2 holds great potential for practical OER application. The surface self-renewal strategy developed here might also be expanded to other electrocatalysts and electrochemical processes.

An aldo-keto reductase AKR5C3 from Gluconobacter oxydans (designated as Gox0644) is a useful enzyme with various substrates, including aldehydes, diacetyl, keto esters, and α-ketocarbonyl compounds. The crystal structures of AKR5C3 in apoform in complex with NADPH and the D53A mutant (AKR5C3(-D53A) ) in complex with NADPH are presented herein. Structure comparison and site-directed mutagenesis combined with biochemical kinetics analysis reveal that the conserved Asp53 in the AKR5C3 catalytic tetrad has a crucial role in securing active pocket conformation. The gain-of-function Asp53 to Ala mutation triggers conformational changes on the Trp30 and Trp191 side chains, improving NADPH affinity to AKR5C3, which helps increase catalytic efficiency. The highly conserved Trp30 and Trp191 residues interact with the nicotinamide moiety of NADPH and help form the NADPH-binding pocket. The AKR5C3(-W30A) and AKR5C3(-W191Y) mutants show decreased activities, confirming that both residues facilitate catalysis. Residue Trp191 is in the loop structure, and the AKR5C3(-W191Y) mutant does not react with benzaldehyde, which might also determine substrate recognition. Arg192, which is involved in the substrate binding, is another important residue. The introduction of R192G increases substrate-binding affinity by improving hydrophobicity in the substrate-binding pocket. These results not only supplement the AKRs superfamily with crystal structures but also provide useful information for understanding the catalytic properties of AKR5C3 and guiding further engineering of this enzyme., (© 2014 The Protein Society.)

Aldehyde dehydrogenase (ALDH) catalyzes the oxidation of aldehydes to carboxylic acids. Cyanobacterium Synechococcus contains one ALDH enzyme (Sp2771), together with a novel 2-oxoglutarate decarboxylase, to complete a non-canonical tricarboxylic acid cycle. However, the molecular mechanisms for substrate selection and cofactor preference by Sp2771 are largely unknown. Here, we report crystal structures of wild type Sp2771, Sp2771 S419A mutant and ternary structure of Sp2771 C262A mutant in complex with NADP(+) and SSA, as well as binary structure of Gluconobacter oxydans aldehyde dehydrogenase (Gox0499) in complex with PEG. Structural comparison of Sp2771 with Gox0499, coupled with mutational analysis, demonstrates that Ser157 residue in Sp2771 and corresponding Pro159 residue in Gox0499 play critical structural roles in determining NADP(+) and NAD(+) preference for Sp2771 and Gox0499, respectively, whereas size and distribution of hydrophobic residues along the substrate binding funnel determine substrate selection. Hence, our work has provided insightful structural information into cofactor and substrate selection by ALDH., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Airway hyperresponsiveness (AHR) is the characteristic functional abnormality of asthma, and previous studies have shown the potential for AHR to be influenced by psychological factors, yet the relationship between anxiety and/or depression and AHR remains unclear in patients with asthma., Objective: To explore the relationship between psychological status and AHR in asthma patients., Methods: In a cross-sectional study, 168 adult subjects were recruited with physician-diagnosed uncontrolled asthma and a positive result for AHR in methacholine (Mch) challenge test. Psychological status, asthma control, and asthma quality of life were assessed using Zung self-rating anxiety/depression scale, asthma control test (ACT), and asthma quality of life questionnaire (AQLQ), respectively. AHR severity was evaluated and quantified by the provocative concentration of Mch, which evoked a given decrease of 20% in FEV(1)., Results: A total of 70.23% of recruited patients (n = 118) met the diagnostic criteria for anxiety and/or depression. There was a trend between negative psychological status and AHR in asthma patients that did not reach statistical significance, but no independent effects of negative mood states (anxiety, depression, or both) on AHR were established. Further, analyses revealed that only anxiety is associated with worse asthma control (p = 0.029), and a significant interaction effect of depression and anxiety accounted for lower asthma-related quality-of-life scores (p < 0.001)., Conclusions: AHR and psychological status are loosely related to each other even if in uncontrolled asthma.

RNA silencing refers to small regulatory RNA-mediated processes that repress endogenous gene expression and defend hosts from offending viruses. As an anti-host defense mechanism, viruses encode suppressors that can block RNA silencing pathways. Cucumber mosaic virus (CMV)-encoded 2b protein was among the first suppressors identified that could inhibit post-transcriptional gene silencing (PTGS), but with little or no effect on miRNA functions. The mechanisms underlying 2b suppression of RNA silencing are unknown. Here, we demonstrate that the CMV 2b protein also interferes with miRNA pathways, eliciting developmental anomalies partially phenocopying ago1 mutant alleles. In contrast to most characterized suppressors, 2b directly interacts with Argonaute1 (AGO1) in vitro and in vivo, and this interaction occurs primarily on one surface of the PAZ-containing module and part of the PIWI-box of AGO1. Consistent with this interaction, 2b specifically inhibits AGO1 cleavage activity in RISC reconstitution assays. In addition, AGO1 recruits virus-derived small interfering RNAs (siRNAs) in vivo, suggesting that AGO1 is a major factor in defense against CMV infection. We conclude that 2b blocks AGO1 cleavage activity to inhibit miRNA pathways, attenuate RNA silencing, and counter host defense. These findings provide insight on the molecular arms race between host antiviral RNA silencing and virus counterdefense.

Argonaute proteins are key components of the RNA-induced silencing complex (RISC). They provide both architectural and catalytic functionalities associated with small interfering RNA (siRNA) guide strand recognition and subsequent guide strand-mediated cleavage of complementary mRNAs. We report on the 3.0 A crystal structures of 22-mer and 26-mer siRNAs bound to Aquifex aeolicus Argonaute (Aa-Ago), where one 2 nt 3' overhang of the siRNA inserts into a cavity positioned on the outer surface of the PAZ-containing lobe of the bilobal Aa-Ago architecture. The first overhang nucleotide stacks over a tyrosine ring, while the second overhang nucleotide, together with the intervening sugar-phosphate backbone, inserts into a preformed surface cavity. Photochemical crosslinking studies on Aa-Ago with 5-iodoU-labeled single-stranded siRNA and siRNA duplex provide support for this externally bound siRNA-Aa-Ago complex. The structure and biochemical data together provide insights into a protein-RNA recognition event potentially associated with the RISC-loading pathway.

Objective: To investigate the bronchial hyperreactivity (BHR) and pulmonary function in patients suffering from endobronchial tuberculosis (EBTB), and therefore to provide clues for early and correct diagnosis of EBTB., Methods: Twenty-four patients with EBTB, proved by fiberoptic bronchoscopy, chest computerized tomography and biopsy, received measurements of pulmonary ventilatory function, airway resistance (Raw), and bronchial responsiveness test. The results were analyzed by t test and correlations., Results: As many as 41.7% of the patients with EBTB were found to have BHR, which had never been recognized before. The patients with EBTB usually had severe cough (100%, 24/24), shortness of breath (54%, 13/24), and wheezing, but bloody sputum was found in only 21% (5/24), and so the patients tended to be misdiagnosed as having asthma, especially cough variant asthma. FEV(1%) in the group of EBTB with BHR was significantly higher than that in the group of EBTB without BHR (t = 2.345, P < 0.05). But there was no significant difference of FEV(1)/FVC%, MMEF%, V(75%) and Raw between the two groups. In the group of EBTB with BHR, FEV(1%) showed a negative correlation with BHR (r = -0.61, P < 0.05), but there was no remarkable correlation between the other pulmonary function parameters with BHR., Conclusion: BHR is present in a considerable number of patients with EBTB, and therefore attention should be paid to the differential diagnosis of EBTB and cough variant asthma.

The nuclear phosphoprotein La was identified as an autoantigen in patients with systemic lupus erythematosus and Sjogren's syndrome. La binds to and protects the UUU(OH) 3' terminii of nascent RNA polymerase III transcripts from exonuclease digestion. We report the 1.85 angstroms crystal structure of the N-terminal domain of human La, consisting of La and RRM1 motifs, bound to r(U1-G2-C3-U4-G5-U6-U7-U8-U9OH). The U7-U8-U9OH 3' end, in a splayed-apart orientation, is sequestered within a basic and aromatic amino acid-lined cleft between the La and RRM1 motifs. The specificity-determining U8 residue bridges both motifs, in part through unprecedented targeting of the beta sheet edge, rather than the anticipated face, of the RRM1 motif. Our structural observations, supported by mutation studies of both La and RNA components, illustrate the principles behind RNA sequestration by a rheumatic disease autoantigen, whereby the UUU(OH) 3' ends of nascent RNA transcripts are protected during downstream processing and maturation events.

We outline structure-function contributions from our laboratories on protein-RNA recognition events that monitor siRNA length, 5 -phosphate and 2-nucleotide 3 overhangs, as well as the architecture of Argonaute, its externally bound siRNA complex, and Argonaute-based models involving guide-strand-mediated mRNA binding, cleavage, and release.

Argonaute (Ago) proteins constitute a key component of the RNA-induced silencing complex (RISC). We report the crystal structure of Aquifex aeolicus Ago (Aa-Ago) together with binding and cleavage studies, which establish this eubacterial Ago as a bona fide guide DNA strand-mediated site-specific RNA endonuclease. We have generated a stereochemically robust model of the complex, where the guide DNA-mRNA duplex is positioned within a basic channel spanning the bilobal interface, such that the 5' phosphate of the guide strand can be anchored in a basic pocket, and the mRNA can be positioned for site-specific cleavage by RNase H-type divalent cation-coordinated catalytic Asp residues of the PIWI domain. Domain swap experiments involving chimeras of human Ago (hAgo1) and cleavage-competent hAgo2 reinforce the role of the PIWI domain in "slicer" activity. We propose a four-step Ago-mediated catalytic cleavage cycle model, which provides distinct perspectives into the mechanism of guide strand-mediated mRNA cleavage within the RISC.

RNA interference (RNAi) is a conserved sequence-specific gene regulatory mechanism mediated by the RNA-induced silencing complex (RISC), which is composed of a single-stranded guide RNA and an Argonaute protein. The PIWI domain, a highly conserved motif within Argonaute, has been shown to adopt an RNase H fold critical for the endonuclease cleavage activity of RISC. Here we report the crystal structure of Archaeoglobus fulgidus Piwi protein bound to double-stranded RNA, thereby identifying the binding pocket for guide-strand 5'-end recognition and providing insight into guide-strand-mediated messenger RNA target recognition. The phosphorylated 5' end of the guide RNA is anchored within a highly conserved basic pocket, supplemented by the carboxy-terminal carboxylate and a bound divalent cation. The first nucleotide from the 5' end of the guide RNA is unpaired and stacks over a conserved tyrosine residue, whereas successive nucleotides form a four-base-pair RNA duplex. Mutation of the corresponding amino acids that contact the 5' phosphate in human Ago2 resulted in attenuated mRNA cleavage activity. Our structure of the Piwi-RNA complex, and that determined elsewhere, provide direct support for the 5' region of the guide RNA serving as a nucleation site for pairing with target mRNA and for a fixed distance separating the RISC-mediated mRNA cleavage site from the anchored 5' end of the guide RNA.

Metabolite-sensing mRNAs, or "riboswitches," specifically interact with small ligands and direct expression of the genes involved in their metabolism. Riboswitches contain sensing "aptamer" modules, capable of ligand-induced structural changes, and downstream regions, harboring expression-controlling elements. We report the crystal structures of the add A-riboswitch and xpt G-riboswitch aptamer modules that distinguish between bound adenine and guanine with exquisite specificity and modulate expression of two different sets of genes. The riboswitches form tuning fork-like architectures, in which the prongs are held in parallel through hairpin loop interactions, and the internal bubble zippers up to form the purine binding pocket. The bound purines are held by hydrogen bonding interactions involving conserved nucleotides along their entire periphery. Recognition specificity is associated with Watson-Crick pairing of the encapsulated adenine and guanine ligands with uridine and cytosine, respectively.

ATP-binding cassettes represent the motor domains in ABC transporters, a superfamily of integral membrane-protein pumps that couple the hydrolysis of ATP to transmembrane solute translocation. A crystal of a Mg-ADP complex of the MJ1267 ATP-binding cassette was obtained that produced a diffraction pattern characterized by pathological streaking of the spots in the a* x b* plane. While the Laue symmetry of the diffraction pattern was P3;1m, the crystal was determined to be twinned based on intensity statistics, molecular-replacement analysis and difference Fourier analysis of an engineered single-site methylmercury derivative. The unit cell contains three similar 3(1) fibers, with two of them related by primarily translational non-crystallographic symmetry (NCS) and the third related to the first two by approximate twofold screw operations whose rotational components are very similar to the twinning operator. The promiscuous packing of these 3(1) fibers, which make both parallel and antiparallel interactions in the primary crystal lattice, can explain the twinning tendency based on the ability of the twin-related lattices to interact with one another while making only one slightly sub-optimal intermolecular contact per unit cell in the boundary region. The promiscuous fiber packing can also explain the streaking in the diffraction pattern based on the ability to form a variety of different lattices with similar inter-fiber packing interactions. The crystal structure was refined as a twin in space group P3(1) using the program CNS, yielding a free R factor of 28.9% at 2.6 A and a refined twin fraction of 0.50. The structure shows a rigid-body rotation of the ABC-transporter-specific alpha-helical subdomain (ABCalpha subdomain) in MJ1267 compared with the conformation observed for the same protein in a C2 crystal lattice; this observation suggests that the ABCalpha subdomain is flexibly attached to the F1-type ATP-binding core of the ATP-binding cassette when Mg-ADP is bound at the active site.

The crystal structure of the MJ0796 ATP-binding cassette, a member of the o228/LolD transporter family, has been determined at 2.7-A resolution with MgADP bound at its active site. Comparing this structure with that of the ATP-bound form of the HisP ATP-binding cassette (Hung, L. W., Wang, I. X., Nikaido, K., Liu, P. Q., Ames, G. F., and Kim, S. H. (1998) Nature 396, 703-707) shows a 5-A withdrawal of a phylogenetically invariant glutamine residue from contact with the gamma-phosphate of ATP in the active site. This glutamine is located in a protein segment that links the rigid F(1)-type ATP-binding core of the enzyme to an ABC transporter-specific alpha-helical subdomain that moves substantially away from the active site in the MgADP-bound structure of MJ0796 compared with the ATP-bound structure of HisP. A similar conformational effect is observed in the MgADP-bound structure of MJ1267 (Karpowich, N., et al. (2001) Structure, in press), establishing the withdrawal of the glutamine and the coupled outward rotation of the alpha-helical subdomain as consistent consequences of gamma-phosphate release from the active site of the transporter. Considering this subdomain movement in the context of a leading model for the physiological dimer of cassettes present in ABC transporters indicates that it produces a modest mechanical change that is likely to play a role in facilitating nucleotide exchange out of the ATPase active site. Finally, it is noteworthy that one of the intersubunit packing interactions in the MJ0796 crystal involves antiparallel beta-type hydrogen bonding interactions between the outermost beta-strands in the two core beta-sheets, leading to their fusion into a single extended beta-sheet, a type of structural interaction that has been proposed to play a role in mediating the aggregation of beta-sheet-containing proteins.

Background: ATP binding cassette (ABC) transporters are ubiquitously distributed transmembrane solute pumps that play a causative role in numerous diseases. Previous structures have defined the fold of the ABC and established the flexibility of its alpha-helical subdomain. But the nature of the mechanical changes that occur at each step of the chemical ATPase cycle have not been defined., Results: Crystal structures were determined of the MJ1267 ABC from Methanococcus jannaschii in Mg-ADP-bound and nucleotide-free forms. Comparison of these structures reveals an induced-fit effect at the active site likely to be a consequence of nucleotide binding. In the Mg-ADP-bound structure, the loop following the Walker B moves toward the Walker A (P-loop) coupled to backbone conformational changes in the intervening "H-loop", which contains an invariant histidine. These changes affect the region believed to mediate intercassette interaction in the ABC transporter complex. Comparison of the Mg-ADP-bound structure of MJ1267 to the ATP-bound structure of HisP suggests that an outward rotation of the alpha-helical subdomain is coupled to the loss of a molecular contact between the gamma-phosphate of ATP and an invariant glutamine in a segment connecting this subdomain to the core of the cassette., Conclusions: The induced-fit effect and rotation of the alpha-helical subdomain may play a role in controlling the nucleotide-dependent change in cassette-cassette interaction affinity believed to represent the power-stroke of ABC transporters. Outward rotation of the alpha-helical subdomain also likely facilitates Mg-ADP release after hydrolysis. The MJ1267 structures therefore define features of the nucleotide-dependent conformational changes that drive transmembrane transport in ABC transporters.

Beta-Momorcharin (Mr approximately 29 kDa) is a single-chained ribosome-inactivating protein (RIP) with a branched hexasaccharide bound to Asn51. The crystal structure of beta-momorcharin has been determined using the molecular-replacement method and refined to 2. 55 A resolution. The final structural model gave an R factor of 17. 2% and root-mean-square deviations of 0.016 A and 1.76 degrees from ideal bond lengths and bond angles, respectively. beta-Momorcharin contains nine alpha-helices, two 310 helices and three beta-sheets, and its overall structure is similar to those of other single-chained RIPs. Residues Tyr70, Tyr109, Glu158 and Arg161 are expected to define the active site of beta-momorcharin as an rRNA N-glycosidase. The oligosaccharide is linked to the protein through an N-glycosidic bond, beta-GlcNAc-(1-N)-Asn51, and stretches from the surface of the N-terminal domain far from the active site, which suggests that it should not play a role in enzymatic function. The oligosaccharide of each beta-momorcharin molecule interacts with the protein through hydrogen bonds, although in the crystals most of these are intermolecular interactions with the protein atoms in an adjacent unit cell. This is the first example of an RIP structure which provides information about the three-dimensional structure and binding site of the oligosaccharide in the active chains of RIPs.

The mechanism of ester hydrolysis has been extensively studied; however, the precise function of active-site residues in promoting catalysis is unclear. We describe here the structural models for the complex of a catalytic antibody Fv fragment with a phosphonate transition-state analogue, constructed by using gene cloning, sequencing and molecular modeling, mainly based on a known X-ray structure of a catalytic antibody. Hydrophobic and electrostatic analyses of the Fv/analog and Fv/substrate interaction suggest the hydrolysis mechanism: Tyr L91 and Tyr H97 play important roles to stabilize the beta-naphthyl group of hapten through pi-stack; His H35 donates a pair of free electrons at the atom NE2 to an active water and let it to be a partial hydroxide, which attacks the carbon atom of the carbonyl group of the substrate. Both His H35 and Arg L96 can form hydrogen bonds and stabilize the anionic tetrahedral intermediate formed during turnover. This mechanism emphasizes that an active water bridge may be formed during hydrolysis process.

Recently we constructed a murine IgE phage surface display library and screened out two IgE (Fab) clones with specific binding activity to Trichosanthin (TCS). In this work, the V epsilon and V kappa genes of the two clones were sequenced and their putative germline gene usages were studied. On the basis of the known 3D structure of Trichosanthin and antibody, molecular modeling was carried out to study the antigen-antibody interaction. The possible antigenic determinant sites on the surface of TCS recognized by both the clones were analyzed, and the reaction forces between TCS and two Fab fragments were also analyzed respectively.

Monoclonal antibodies were raised against a beta-naphthyl phosphonate hapten (1) to elicit antibodies capable of catalyzing the hydrolysis of beta-naphthyl acetate (3). After cell fusion, potential catalytic antibody-producing hybridomas were selected, by use of a competitive inhibition assay on the basis of the binding activity for a short transition-state analogue (inhibitor 5), followed by use of high-performance liquid chromatography analysis for the hybridoma supernatants to screen the antibodies processing catalytic activities. It was shown that supernatants of 12 wells had high binding activity with inhibitor and of them, 7 had catalytic activities. After cloning by limiting dilution, we got two hybridoma clones producing monoclonal antibodies which catalyzed the hydrolysis of beta-naphthyl acetate. This combination of competitive inhibition assay with high-performance liquid chromatography analysis represents an improved rapid approach for the screening of potential catalytic antibodies and significantly increases the possibility of obtaining efficient catalytic monoclonal antibodies. Further study of the catalytic antibodies revealed significant rate enhancement (Kcat/K(uncat) approximately 10(6) and specificity.

This paper analyzes the actual values of FEV1 and MBC in 240 normal subjects, 147 patients with COPD and 85 cases of cancer tumors, and shows that the actual values of MBC in both COPD patients and cancerous cases are much lower than the calculated values from FEV1. The authors believe that the ratio between the actual values of MBC and its calculated values may be used to reflect the strengths or abilities of the respiratory muscles efforted, called "Effort Index". The formula of MBC calculated from FEV1 is based on the linear regression equation formulated from the actual values of FEV1 and MBC in 240 normal subjects (aged 16-80 yrs). The "Effort Index" is equal to the actual value of MBC/the calculated value of MBC. The "Effort Index" in 240 normal subjects is 1.00 +/- 0.14 (M +/- SD), in mild, moderate and severe cases of COPD being 0.907, 0.807 and 0.626 respectively, and in markedly wasted cancer cases, 0.861 +/- 0.130. The results show that the formula of MBC calculated from FEV1 is useful only for the subjects with normal respiratory muscular abilities; while in COPD patients and markedly emaciated cancerous cases, owing to the chronically tired-out or exhausted respiratory muscular strength, the actual values of MBC are actually lower than those calculated from FEV1, hence the decreased "Effort Index".