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The aldol reaction between benzaldehyde and acetone has been investigated using QM/MM Monte Carlo calculations and free-energy perturbation theory to determine the origin of the enhanced rates and enantioselectivities (% ee) derived from an enamine-based catalytic antibody 33F12 and a chiral organocatalyst. Electrostatic stabilization of the general acid/base TyrL36 by TrpH103, SerH100, and AsnL34 enabled the 33F12 active site to exclusively adopt an si-face benzaldehyde orientation for C-C bond formation with the LysH93-enamine. Whereas preorganization was responsible for the exclusive (S)-aldol product in the antibody, the organocatalyst featuring a chiral diphenyl amino alcohol moiety instead derived its preferred (R)-aldol product from an interplay between sterics and electronic stabilization. The si-face benzaldehyde conformation had unfavorable interactions with the organocatalyst in contrast to the re-face. Gas-phase calculations predicted a 73% ee; however, solution boosted the % ee values despite similar reaction geometries. An "on water" environment, defined as a reaction that proceeds in an aqueous organic emulsion, yielded a computed 94% ee (exptl 93% ee) compared to a calculated 87% ee in "neat" acetone (exptl 85% ee). Specific hydrogen bonding between the interfacial waters and an amide oxygen on the catalyst was found to control the % ee. A more compact si-face transition structure reduced solvent accessibility to the amide oxygen with a "closed state" steric barrier compared to an "open state" for the re-face. New insight into the synthetically important aldol reaction and state-of-the-art methodology is presented herein.

The photocatalytic degradation of synthetic dye solution of Reactive Blue 2 was investigated using UV irradiation in aqueous suspension of nanotitania as photocatalyst and H2O2 as electron acceptor in a slurry photoreactor. To determine the optimum condition of decolorization, the influential parameters including initial dye concentration, catalyst quantity, hydrogen peroxide concentration, pH value and UV light intensity were evaluated using the Taguchi statistical method. The results indicated that compared to the other parameters, UV light intensity was the most effective. The theoretically predicted value of decolorization efficiency (95.48%) was confirmed by the experimental value (95.22%). The mineralization was considered significant with regard to the obtained values of total organic carbon (TOC) analysis (92.52%) and chemical oxygen demand (COD) analysis (94.05%) in the optimum condition. Investigations were also carried out to determine the appropriate adsorption isotherm and kinetic model in the optimum condition. The obtained optimum condition for dye removal was finally examined applying a real sample of textile wastewater containing the dye.

ZnO1-x/graphene hybrid photocatalyst was prepared via a facile in-situ reduction of graphene oxide and ZnO1-x surface defect oxide. The hybrid photocatlayst showed enhanced photocatalytic activity for the photodegradation of methylene blue. The photocorrosion of ZnO1-x was successfully inhibited by graphene hybridation. ZnO1-x/graphene hybrid photocatalyst with 1.2 wt % graphene showed the optimized photocatalytic activity. The photocatalytic activity of ZnO1-x/graphene-1.2 wt % under visible and UV light was about 4.6 and 1.2 times that of ZnO1-x sample, respectively. The photocurrent intensity of ZnO1-x under visible and UV light irradiation can be enhanced by 2 and 3.5 times by graphene hybridization. The enhancement of photocatalytic activity and photocurrent intensity in ZnO1-x/graphene was attributed to the synergistic effect between graphene and ZnO1-x for high separation efficiency of photoinduced electron-hole pairs mainly resulting from the promotion of HOMO orbit of graphene and the Oi″ defect level of ZnO1-x in ZnO1-x/graphene.

Human myelin basic protein (hMBP)-hydrolyzing activity was recently shown to be an intrinsic property of antibodies from systemic lupus erythematosus (SLE) patients. Here, we present the first evidence demonstrating a significant diversity of different fractions of polyclonal IgGs (pIgGs) from SLE patients in their affinity for hMBP and in the ability of pIgGs to hydrolyze hMBP at different optimal pH values (5.3-9.5); the pH profiles of IgG1, IgG2, IgG3 and IgG4 were unique. IgGs containing the λ-type of light chains demonstrated higher relative activities (RAs) in the hydrolysis of hMBP and its oligopeptides (OPs) than κ-IgGs. IgGs of all four subclasses were catalytically active; their RAs in the hydrolysis of hMBP increased in the following order: IgG4 < IgG2 < IgG3 < IgG1. Metal-dependent proteolytic activity of λ-IgG, IgG1, IgG2 and IgG3 was higher than their serine protease-like activity, while these activities of κ-IgG were comparable. Phenylmethylsulfonylfluoride had almost no effect on the activity of IgG4, while EDTA significantly suppressed its activity. The RAs of λ-IgG in the hydrolysis of four OPs corresponding to different cleavage sites of hMBP were remarkably higher than those for κ-IgGs. IgG1-IgG4 demonstrated different RAs and patterns of hydrolysis of these four OPs. Although combination of Ca²⁺ plus Mg²⁺ was the best in the activation of IgG1 and IgG2, IgG3 and IgG4 demonstrated the highest activity in the presence of Ca²⁺ plus Co²⁺. The ratio of the RAs of λ-IgG, κ-IgG and IgG1-IgG4 preparations in all analyzed cases was individual for each preparation.

Specific entities of naturally-occurring DNA hydrolytic/cytotoxic antibodies (abzymes) are linked to autoimmune and lymphoproliferative disorders. Suggested sequence of underlying activities conform to such entities penetrating the living cells, trans-locating to nucleus and recognising specific binding sites within single- or double-stranded DNA. Their origin is unknown since corresponding immunogens are unidentified. These anti-DNA antibodies could be the organism's immune response to microbial attack. Their structure, function and pathogenicity were investigated in wet-lab and via bioinformatics in context of Rational Vaccine Designs. This paper offers a comprehensive critical review on the subject in the light of known and newly proposed concepts.

Malaria is a devastating disease that kills millions of people every year, yet there has been little progress in controlling this disease. Mosquitoes are obligatory vectors for the disease and this part of the parasite cycle represents a potential weak link in transmission. Therefore, control of parasite development in the mosquito has considerable promise as a new approach in the fight against malaria. In recent year, methods for the genetic modification of mosquitoes have been developed, and effector genes whose products interfere with Plasmodium development in the mosquito are beginning to be identified. Here we review strategies to alter mosquito vector competence and consider issues related to translating this knowledge to field applications.

Advances in methods for conformational prediction, structural analysis and site-directed mutagenesis of proteins and peptides have contributed to the understanding of their structure and function. However, with the exception of a few successes, the generation of practical functional molecules solely by rational design remains a difficult challenge. The aim of our study is to investigate molecular design relying on evolutionary processes, called as "directed evolution", to generate a novel class of biofunctional molecules. This evolutionary approach consists of three steps; 1) constructions of protein/peptide libraries based on structural information, 2) expressions of the libraries on phage particles, and 3) selections with investigator-imposed selective pressures. In this work, we study on directed evolution with antibody libraries. We have succeeded in generating highly active catalytic antibodies in phage-displayed antibody (Fab) libraries. To evolve catalytic antibodies toward higher catalytic activity, we have mimicked an enzyme-evolutional process, in which an enzyme has evolved their ability to use binding energies for catalysis by increasing the affinity for the transition state of a reaction and decreasing the affinity for the ground state. Thus, phage-displayed libraries derived from an original catalytic antibody were selected against a newly-devised TSA, which was programmed to optimize the differential affinity for the transition state relative to the ground state, to provide variants with improved reaction rates (k(cat)). The in vitro evolution has great potential for generating novel catalysts as well as for providing opportunities to examine the evolutionary dynamics of enzymes.

Natural antibodies possessing catalytic activity present a new group of biologically active substances that are found in various autoimmune diseases such as autoimmune thyroiditis, myocarditis, multiple sclerosis, and system lupus erythematosus. Presently, an interconnection between the activity of these antibodies and the extent of organic and tissue lesion in autoaggression have been revealed. Clinical use of catalytic antibodies as a diagnostic criterion to evaluate the severity of disease, a prognostic criterion of the risk of invalidization, and as a pathogenetic basis for medicamentous treatment of autoimmune process is a promising directions of study of the role of catalytic antibodies.

Autoantibodies toward myelin basic protein (MBP) evidently emerge in sera and cerebrospinal fluid of the patients with multiple sclerosis (MS), as well as in a MS rodent model, i.e., experimental autoimmune encephalomyelitis (EAE). The studies of the last two decades have unveiled somewhat controversial data on the diagnostic applicability of anti-MBP autoantibodies as a disease' marker. Here, we present the results of new functional analysis of the anti-MBP autoantibodies isolated from MS (in patients) and EAE (in mice) sera, based on their proteolytic activity against the targeted autoantigen. The activity was shown to be the intrinsic property of the IgG molecule. No activity was found in the sera-derived antibody fraction of healthy donors and control mice. Sera of 24 patients with clinically proven MS at different stages of the disease, and 20 healthy controls were screened for the anti-MBP antibody-mediated proteolytic activity. The activity correlated with the scores on the MS expanded disability status scale (EDSS) (r(2)=0.85, P<0.001). Thus, the anti-MBP autoantibody-mediated proteolysis may be regarded as an additional marker of the disease progression.

Immunoglobulins have initially been illustrated as proteins produced by the immune system for binding and neutralizing foreign molecules potentially harmful to the organism. The number of V(H), D(H), J(H), V(L) and J(L) genes that encode the variable regions of immunoglobulins and the junctional diversity that occurs at the time of somatic rearrangement determine the extent of the repertoire of antibodies that may be potentially produced by an organism. This potential repertoire includes antibodies the antigen binding site of which may recognize external as well as autologous antigens, or may structurally resemble the active site of enzymes and be endowed with enzymatic activity. Under physiological conditions, B cell clones that produce antibodies naturally endowed with catalytic activity are negatively regulated and subjected to apoptosis. Catalytic antibodies are expressed only following active immunization, or if the physiological regulatory mechanisms that control the expression of catalytic antibody-producing B cell clones are perturbed, e.g. in the context of pregnancy or in the course of autoimmune diseases.

The challenging field of de novo enzyme design is beginning to produce exciting results. The application of powerful computational methods to functional protein design has recently succeeded at engineering target activities. In addition, efforts in directed evolution continue to expand the transformations that can be accomplished by existing enzymes. The engineering of completely novel catalytic activity requires traversing inactive sequence space in a fitness landscape, a feat that is better suited to computational design. Optimizing activity, which can include subtle alterations in backbone conformation and protein motion, is better suited to directed evolution, which is highly effective at scaling fitness landscapes towards maxima. Improved rational design efforts coupled with directed evolution should dramatically improve the scope of de novo enzyme design.

An antibody against a transition state analog (TSA) may share some common features with an enzyme that produces such a transition state. SIC172 antibody binds specifically to Neu2en5Ac, a TSA of Neu5Ac in the sialidase reaction, but has no catalytic activity. To understand how the antibody recognizes Neu2en5Ac and to find out if it is possible to convert it to a catalytic antibody, we made and sequenced the SIC172 ScFv, and constructed a 3-D model of it. The VH-CDR3 contains a unique sequence with Cys at H95. The 3-D model showed that Cys-H95 is exposed inside the antigen-binding cavity. After affinity docking, 4 types emerged. In type I, the carboxyl group of Neu2en5Ac is located near the Cys-H95 and neighboring positively charged residues. The change of Cys-H95 to Asp by site-directed mutation decreased the binding activity, while a change to Arg did not. These and other mutation experiments, and further modeling of mutant Fv, support the 3-D model and docking type I. A comparison with sialidase indicates that SIC172 antibody appears to have some groups of residues that are conserved at the active site of the enzyme. The possibility of Neu2en5Ac-binding antibody being converted to a catalytic antibody is discussed., (Copyright 2001 Wiley-Liss, Inc.)

The preparative scale kinetic resolution of racemic aldols 1-4 using aldolase antibodies 38C2 (Aldrich no. 47995-0) and 84G3 (Aldrich no. 52785-8) is described. These reactions use a biphasic aqueous/organic solvent system that allows the catalyst to be reused. Reaction scales range from miligrams to grams, with 0.0086 to 0.12 mol% of antibody binding sites. Because antibodies 38C2 and 84G3 have opposite enantioselectivities, both aldol product enantiomers are accessible by kinetic resolution.

The combination of the tools and principles of chemistry, together with the tools of modern molecular biology, allow us to create complex synthetic and natural molecules, and processes with novel biological, chemical and physical properties. This article illustrates the tremendous opportunity that lies at this interface of chemistry and biology by describing a number of examples, ranging from efforts to expand the genetic code of living organisms to the use of combinatorial methods to generate biologically active synthetic molecules.

The purpose of this study was to assess the delivery to the lungs and the short-term safety of recombinant human deoxyribonuclease (rhDNase, Pulmozyme) in children with cystic fibrosis younger than 5 years of age compared with older children. Patients between the ages of 3 months and 10 years had bronchoscopic examination with bronchoalveolar lavage (BAL) after administration of an aerosol dose of 2.5 mg of rhDNase. After recovery from the procedure, patients were discharged home for an additional 13 days of rhDNase therapy. During this time adverse events were recorded to assess short-term safety. A total of 98 patients were enrolled, 65 (66%) aged 3 months to 5 years and 33 (34%) aged 5 years to 10 years. Deoxyribonuclease concentrations in BAL fluid were variable (interquartile range, 752 to 3943 micrograms/mL epithelial lining fluid [ELF]) and did not depend on patient age, weight, or height or differ when delivered through a mouthpiece or mask. The median value for the BAL DNA concentration in the younger group was 432 micrograms/mL ELF compared with 703 micrograms/mL ELF in the older patients. This study demonstrates the value of bronchoscopy and BAL for assessing nebulized medication delivery in young children and shows that aerosolized medications can be delivered to and are present in comparable amounts in the lower airways of younger and older children. Exposure to rhDNase appears to be safe over 2 weeks in infants and young children with cystic fibrosis.

The variable-region peptide sequence and steady-state kinetic behavior are compared for a family of catalytic antibodies that arose from the same immune response to a transition-state analog. The crystal structure of the most catalytically active member of the family (17E8) has been solved to 2.5 A resolution and shows that the antibody active site contains a SerH99-HisH35 (H = heavy chain) catalytic dyad analogous to the Ser-His-Asp catalytic triad of serine proteases. The variable-region peptide sequence of the next most active antibody (29G11) differs from that of 17E8 by nine heavy-chain point mutations, and results from computer modeling suggest that the three-dimensional structure of 29G11 is similar to that of 17E8. In addition, 29G11 is an efficient catalytic antibody; it possesses 26% of the hydrolytic activity of 17E8. There is one active-site mutation in 29G11 compared to 17E8; position 99 of the heavy chain of 29G11 contains a glycine residue in place of the nucleophilic serine at this position in 17E8. Consistent with this mutation, results from pH-rate studies and hydroxylamine partitioning experiments indicate that in contrast to the catalytic mechanism of 17E8, the mechanism of 29G11-catalyzed esterolysis does not feature nucleophilic catalysis.

Antibodies capable of decarboxylating orotate were sought by immunization with a hapten designed to elicit antibodies with combining sites that resemble the orotate-binding and catalytic portion of the active site of the enzyme orotidine 5'-monophosphate (OMP) decarboxylase (orotidine-5'-monophosphate carboxy-lyase, EC 4.1.1.23). Active recombinant antibody fragments (Fabs) were selected from a combinatorial cDNA library by complementation of a pyrF strain of Escherichia coli and growth of the library-expressing cells on pyrimidine-free medium. In this biological screen, a sufficiently active antibody from the library would decarboxylate orotate to produce uracil, a pyrimidine source for the auxotroph, and would provide the cells with a growth advantage compared to cells without an active antibody. Six recombinant Fabs yielded identifiable colonies in a screen of 16,000 transformants. To enhance its stability and expression level, one of the six positive fragments was converted into single-chain form. In this form, the antibody fragment conferred a definite growth advantage to the auxotroph that was eliminated when the hapten was included in the medium. The purified single-chain antibody displayed orotate decarboxylase activity in vitro, as determined by a 14CO2 displacement assay. The specific activity of the antibody is approximately 10(-7) times that of naturally occurring OMP decarboxylase, but this antibody-catalyzed rate is estimated to be 10(8) times the background rate. The results offer the potential to use these methods to obtain catalytic antibodies for other biosynthetic reactions as well as to assess the effectiveness of the hapten transition state or active site analog in eliciting antibody catalysts.

The first catalytic antibodies (abzymes) were reported in 1986. Since then abzymes catalyzing various types of reactions have been reported. There have been a number of reviews on the abzymes, so in this review I intended not to overlap them, but briefly described (i) the background of abzyme production, (ii) the catalytic mechanism of the enzymic reaction, and (iii) some examples of monoclonal and polyclonal abzymes. Recent advancements as to abzyme studies are the invention of an assay system for catalytic activity during cloning, the estimation of the solvent effect on abzyme-catalyzed decarboxylation, the production of abzymes catalyzing disfavored chemical reactions and cocaine hydrolysis, and the feasibility of the usage of polyclonal abzymes. Several advantages of polyclonal abzyme studies were also pointed out.