Secretory immunoglobulin a from human milk catalyzes milk protein phosphorylation

This article presents evidence that protein kinase activity is an intrin sic property of secretory immunoglobulin A (sIgA) from milk of healthy human mothers. Polyclonal sIgA was purified by sequential chromatog raphy on protein A-Sepharose, DEAE-cellulose, and gel filtration on Toyopearl HW-55 and Sepharose 4B columns. Its purity was established by one-and two-dimensional SDS-PAGE. The protein kinase activity was inhibited by specific antibodies (Abs) against sIgA, and was stable to acidic and alkaline conditions. Catalytic sIgA showed optimal reaction conditions (pH and MgCl₂ concentration) and substrate specificity dif ferent from those of known protein kinases; i.e., sIgA phosphorylated the serine residues of various milk proteins in the presence of different γ-[³²P]nucleoside-and deoxynucleoside-5’-triphosphates. The homoge neous Fab fragment of sIgA also showed kinase activity. An ATP-binding activity of fractions of sIgA was demonstrated by affinity chromatog raphy on ATP-Sepharose and by covalent binding of an affinity analog of ATP; this activity was mediated by the L chain of sIgA. The authors believe these observations are the first example of the catalytic activity of IgA Abs and of natural catalytic Abs with synthetic activity. In addition, the findings suggest the likelihood that catalytic Abs are generated by the immune system of healthy mothers.     

Secretory immunoglobulin a from health y human mothers' milk catalyzes nucleic acid hydrolysis

The human milk secretory immune system is known to be the first line of protection for the newborn infant against various pathogens. Secretory IgA (sIgA), the typical immunoglobulin found in secretions, can fight infections through many mechanisms. Using different methods, we have shown that sIgA from the milk of healthy women possesses DNAse and RNAse activities. The catalytic center is localized in the light chain of catalytic sIgA, while the DNA-binding center is predominantly formed by its heavy chain. The enzymic properties and substrate specificity of catalytic sIgA distinguish it from other known DNases and RNases. It is reasonable to assume, that the milk DNA- and RNA-hydrolyzing antibodies are capable not only of neutralizing viral and bacterial nucleic acids by binding these antigens as well as by hydrolyzing them. The DNA-hydrolyzing activity of Abs raises the possibility that these catalytic Abs may provide protective functions for the newborn through the hydrolysis of viral and bacterial nucleic acids.     

Dialkylzinc Compounds as Chain Transfer Agents in Ethylene and Propylene Polymerizations Catalyzed by Metallocene Catalysts

Dialkylzinc compounds (ZnR₂) with the alkyl groups of different steric hindrance were used as chain transfer agents in ethylene and propylene polymerizations catalyzed by two conventional metallocene catalysts including rac-Et(Ind)₂ZrCl₂ and rac-Me₂Si[2-Me-4-Ph-Ind]₂ZrCl₂. In general, catalyst activities for ethylene polymerizations are barely affected by chain transfer agents, regardless of the R type; however, there are significant activity reductions in propylene polymerizations when the R in ZnR₂ is less hindered, and as R becomes bulkier, catalyst activities are gradually restored. ZnR₂ and metallocene catalyst active sites tend to form a reversible and catalytically inactive complex, thus, the geometry congested ZnR₂ would reduce complex formation tendency and hence, decrease its negative effect on catalyst activities.     

Dialkylzinc Compounds as Chain Transfer Agents in Ethylene and Propylene Polymerizations Catalyzed by Metallocene Catalysts

Dialkylzinc compounds (ZnR₂) with the alkyl groups of different steric hindrance were used as chain transfer agents in ethylene and propylene polymerizations catalyzed by two conventional metallocene catalysts including rac-Et(Ind)₂ZrCl₂ and rac-Me₂Si[2-Me-4-Ph-Ind]₂ZrCl₂. In general, catalyst activities for ethylene polymerizations are barely affected by chain transfer agents, regardless of the R type; however, there are significant activity reductions in propylene polymerizations when the R in ZnR₂ is less hindered, and as R becomes bulkier, catalyst activities are gradually restored. ZnR₂ and metallocene catalyst active site tend to form a reversible and catalytically inactive complex, thus the geometry congested ZnR₂ would reduce complex formation tendency and hence decreased its negative effect on catalyst activities.     

Production and characterization of monoclonal antibodies against urea derivatives

A panel of monoclonal antibodies was generated against the ureabased haptenN-(2-N-chloroacetylaminobenzyl)-N′-4-chlorophenylurea as a tool for building up sensitive immune assays to detect urea derivatives and to screen them for catalytic antibodies (Abs). Eleven hybridomas were obtained that produced Abs reactive to the hapten. All Abs were of IgG class. Crossreactivities of the Abs to different haptens were examined, especially to a possible transition-state analog. Only four of the hybridomas (R2-DA10/F7, R2-GE7/H2, R2-HC2/A5, R2-HD6/F7) produced Abs crossreactive with the transition-state analog. From the 11 hybridomas, hybridoma B76-BF5 was chosen for further characterization. Compared to the other Abs, B76-BF5 showed the strongest binding and had a rather restricted specificity. These Abs could be used to build up a sensitive enzyme immunoassay for the detection of the hapten. All Abs were screened for crossreactivity with the pesticides monuron and diuron. No reactivity could be detected. In addition, the nucleotide sequences of the variable light and heavy chain genes of the similarly reactive Abs B76-BF5, B76-BB3, R2-DA10/F7, and R2-GA6/G3 were determined to clarify whether structure and binding specificity of these Abs showed any correlation.     

DNA hydrolysis by monoclonal autoantibody BV 04-01

Monoclonal anti-DNA autoantibody BV 04-01 catalyzed hydrolysis of DNA in the presence of Mg²⁺. Catalysis was asssociated with BV 04-01 IgG, Fab, and single-chain-antibody (SCA) proteins. Cleavage of both ss and dsDNA was observed with efficient hydrolysis of the C-rich region of A₇C₇ATATAGCGCGT₂, as well as a preference for cleaving within CG-rich regions of dsDNA. Data nn specificity of ssDNA hydrolysis and kinetic data obtained from wild-type SCA, and two SCA mutants were used to model the catalytically active antibody site using the previously resolved X-ray structure of BV 04-01. The resulting model suggested that the target phosphodiester bond is activated by induction of conformational strain. In addition, the antibody-DNA complex contained a Mg²⁺ coordination site composed of the L32Tyr and L27d His side chains and a DNA 3′-phosphodiester group. Induction of strain along with the metal coordination could be part of the mechanism by which this antibody catalyzes DNA hydrolysis. Sequence data for BV 04-01 V_H and V_L genes suggested that the proposed catalytic-antibody active site was germline-encoded. This observation suggests that catalytic activity might represent an important—rarely examined—function for some antibody molecules.     

A Novel M8L6 Cubic Cage That Binds Tetrapyridyl Porphyrins: Cage and Solvent Effects in Cobalt-Porphyrin-Catalyzed Cyclopropanation Reactions

Confinement of a catalyst can have a significant impact on catalytic performance and can lead to otherwise difficult to achieve catalyst properties. Herein, we report the design and synthesis of a novel caged catalyst system Co−G@Fe₈(Zn−L ⋅ 1)₆ , which is soluble in both polar and apolar solvents without the necessity of any post-functionalization. This is a rare example of a metal-coordination cage able to bind catalytically active porphyrins that is soluble in solvents spanning a wide variety of polarity. This system was used to investigate the combined effects of the solvent and the cage on the catalytic performance in the cobalt catalyzed cyclopropanation of styrene, which involves radical intermediates. Kinetic studies show that DMF has a protective influence on the catalyst, slowing down deactivation of both [Co(TPP)] and Co−G@Fe₈(Zn−L ⋅ 1)₆ , leading to higher TONs in this solvent. Moreover, DFT studies on the [Co(TPP)] catalyst show that the rate determining energy barrier of this radical-type transformation is not influenced by the coordination of DMF. As such, the increased TONs obtained experimentally stem from the stabilizing effect of DMF and are not due to an intrinsic higher activity caused by axial ligand binding to the cobalt center ([Co(TPP)(L)]) . Remarkably, encapsulation of Co−G led to a three times more active catalyst than [Co(TPP)] (TOF_ini) and a substantially increased TON compared to both [Co(TPP)] and free Co−G . The increased local concentration of the substrates in the hydrophobic cage compared to the bulk explains the observed higher catalytic activities.     

Intracellular transport and localization of microsomal cytochrome P450

The cytochrome P450 (P450) enzymes are mainly localized to the endoplasmic reticulum (ER), where they function within catalytic complexes metabolizing xenobiotics and some endogenous substrates. However, certain members of families 1–3 were also found in other subcellular compartments, such as mitochondria, plasma membrane, and lysosomes. The physiological function of these enzymes in non-ER locations is not known, although plasma-membrane-associated P450s have been described to be catalytically active and to participate in immune-mediated reactions with autoantibody formation that can trigger drug-induced hepatitis. Several retention/retrieval mechanisms are active in the ER retention of the P450s and inverse integration of the translated P450 into the ER membrane appears to be responsible for transport to the plasma membrane. Furthermore, hydrophilic motifs in the NH₂-terminal part have been suggested to be important for mitochondrial import. Phosphorylation of P450s has been described to be important for increased rate of degradation as well as for targeting into mitochondria. It was also suggested that the mitochondria-targeted P450s from families 1–3 could be active in drug metabolism using an alternative electron transport chain. In this review we present an update of the field emphasizing studies concerning localization, posttranslational modification, such as phosphorylation, and intracellular transport of microsomal P450s.     

Development of an ICP-MS immunoassay for the detection of anti-erythropoietin antibodies

A sandwich-type immunoassay linked with inductively coupled plasma mass spectrometry (ICP-MS) has been developed for the detection of anti-erythropoietin antibodies (anti-EPO Abs). Recombinant human erythropoietin (rhEPO) was immobilized on the solid phase to capture anti-rhEPO Abs specifically. After the immunoreactions with Au-labeled goat-anti-rabbit IgG, a diluted HNO₃ (2%) was used to dissociate Au nanoparticles which was then introduced to the ICP-MS for measurements. Under the optimized conditions, the calibration graph for anti-EPO Abs was linear in the range of 35.6-500 ng mL⁻¹ with a detection limit of 10.7 ng mL⁻¹ (3σ, n = 9). The relative standard deviation (R.S.D.) for three replicate measurements of 30.9 ng mL⁻¹ of anti-EPO Abs was 8.43%. The recoveries of anti-EPO Abs in sera at the spiking level of 50, 100, 150, 200 and 400 ng mL⁻¹ were 99.2%, 101.5%, 95.0%, 94.0% and 102.9%, respectively. For the real sample analysis, 26 samples from healthy people and 53 samples from patients with rhEPO treatments were studied. One sample from patients showed significantly higher anti-EPO Abs from other samples, indicating a possibility of immune response of this patient.     

Stabilizing a Platinum1 Single‐Atom Catalyst on Supported Phosphomolybdic Acid without Compromising Hydrogenation Activity

In coordination chemistry, catalytically active metal complexes in a zero‐ or low‐valent state often adopt four‐coordinate square‐planar or tetrahedral geometry. By applying this principle, we have developed a stable Pt₁ single‐atom catalyst with a high Pt loading (close to 1 wt %) on phosphomolybdic acid(PMA)‐modified active carbon. This was achieved by anchoring Pt on the four‐fold hollow sites on PMA. Each Pt atom is stabilized by four oxygen atoms in a distorted square‐planar geometry, with Pt slightly protruding from the oxygen planar surface. Pt is positively charged, absorbs hydrogen easily, and exhibits excellent performance in the hydrogenation of nitrobenzene and cyclohexanone. It is likely that the system described here can be extended to a number of stable SACs with superior catalytic activities.     

具有甲状腺素脱碘酶活性的抗体酶的化学修饰

甲状腺素脱碘酶是一种膜硒蛋白[1], 它能够将甲状腺素降解为不同产物, 并对甲状腺素的生理功能起调控作用. 但是, 甲状腺素脱碘酶极易变性失活, 到目前为止仍未得到纯酶[2]. 近来, 此酶的人工模拟工作逐渐成为热点. 我们小组[3]首次以3,5,3′,5′-四碘甲状腺原氨酸五水钠盐(T4)为半抗原, 采用单克隆抗体技术和苯甲基磺酰氟及硒氢化钠修饰的方法, 成功地制备了具有甲状腺素脱碘酶活性的抗体酶.     

Molecular heterogeneity,detected by two electrophoretic micro procedures,of IgG in human cerebrospinal fluid (CSF)

Semi-automated electrophoretic procedures in the PhastSystem (Amersham Pharmacia Biotech) with micro polyacrylamide gels (PAGs) and SDS-PAG gradients were modified to analyze IgG in human cerebrospinal fluid (CSF) and matched serum samples with respect to the molecular IgG structure L-H-H-L. Isoelectric focusing (IEF) with specific immunofixation detected discrete IgG bands in CSF standing out against a polyclonal and monoclonal background pattern in CSF and serum; they were denoted oligoclonal bands (IgG OBs) (OB assay positive) indicating IgG synthesis in the central nervous system (CNS) of patients with subacute and chronic processes of inflammatory CNS disorders; assay was negative with identical (mirror) bands in CSF and serum for other CNS processes. IgG OBs were specified as lambda (kappa) IgG subfractions, precipitated with the anti-light (L) chains lambda (kappa) and anti-heavy (H) chain fragments (Fd, Fc, C(H)2) as well as with anti-F(ab')(2), and as duplex IgGs with kappa and lambda OBs at the same pI. With SDS-PAG gradient electrophoresis and specific immunofixation more than six IgG fractions were detected and classed according to apparent molecular weights of a S-sulfonated human IgG standard; they were characterized with the monospecific antibodies against the L and H chain fragments as 25, 50, 75, 100, 125 and 150 kD fractions containing combinations of L and H chains as well as mixtures of both L and H chain fragments of varying dimensions. Generally, this molecular IgG heterogeneity could not be connected with the IgG OB heterogeneity revealed by IEF; but single OBs in the strongly alkaline pH region of PAG may correspond to H fragments with basic pI. Nevertheless, evidence for the existence of both free L chains and the free H chain were revealed as specific OBs with IEF and with the anti-L and anti-H antibodies in the 25 kD and 50 kD fractions, respectively, of CSF samples of six patients with diverse CNS diseases. Further experiments are needed to elicit the origin of the molecular IgG heterogeneity during the immune response of subacute and chronic inflammatory processes in human CNS.     

Mechanism of the oxidative carbonylation of terminal alkynes at the ≡C-H bond in solutions of palladium complexes

The formation mechanism of the active catalyst in the oxidative carbonylation of terminal alkynes at the ≡C-H bond has been investigated for the catalytic system Pd(OAc)₂-PPh₃-p-benzoquinone (Q)-MeOH. It has been demonstrated by NMR spectroscopy, X-ray crystallography, and kinetic measurements that the catalytically active palladium is in the oxidation state 0 and is bound into complexes stabilized by p-benzoquinone (PdL₂Q, where L = PPh₃). A mechanism is suggested for the catalytic process, which includes the formation of the complex PdL₂Q, the oxidative addition of the alkyne to this complex at the ≡C-H bond, the insertion of CO into the Pd-C bond, and steps in which hydride hydrogen is intramolecularly transferred to the p-quinone.     

Generation of a Recombinant Full-Length Human Antibody Binding to Botulinum Neurotoxin A

In order to develop a recombinant full-length human anti-botulinum neurotoxin A (BoNT/A) antibody, human peripheral blood mononuclear cells (PBMC) were collected from three healthy volunteers and induced for BoNT/A-specific immune response by in vitro immunization. The genes encoding human Fd fragment, consisting of antibody heavy chain variable region and constant region 1 with the genes encoding antibody light chain, were cloned from the immunized PBMC. Afterwards, one combinatory human antigen-binding fragment (Fab) library was constructed using a lambda phage vector system. The size of the constructed library was approximately 10⁵ Escherichia coli transformants. After screening the library by BoNT/A antigen using a plaque lifting with immunostaining approach, 55 clones were identified as positive. The Fab gene of the most reactive clone exhibiting particularly strong BoNT/A binding signal was further subcloned into a full-length human IgG1 antibody gene template in an adenoviral expression vector, in which the heavy and light chains were linked by a foot-and-mouth-disease virus-derived 2A self-cleavage peptide under a single promoter. After the full-length human IgG1 was expressed in mammalian cells and purified with protein L column, sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the heavy and light chains of the antibody were cleaved completely. The affinity expressed as the dissociation constant (K _d) for the recombinant human antibody to bind to BoNT/A was determined by indirect enzyme-linked immunosorbent assay and results confirmed that the recombinant full-length human antibody retained BoNT/A-binding specificity with K _d value of 10⁻⁷ M.     

Selective alterations of the antibody response to HIV-1

HIV infection leads to progressive alterations of humoral immune functions, including B-cell hyperplasia, hypergammaglobulinemia, elevated autoantibody titers, a poor response to neoantigens and mitogens, polyclonal B-cell activation, monoclonal gammopathies, and a significant deterioration of the antigen-specific humoral response. There is also an important isotypic imbalance of the antibody (Ab) response in the systemic compartment and a profound modification of mucosal immune functions. These abnormalities may contribute to disease progression and development of opportunistic infections, despite the presence of serum-neutralizing anti-HIV Abs. Equally important are the abnormal selection mechanisms of the Ab repertoire that seem to be responsible for B-cell clonal deletions. The V_H3 gene family, which encodes for approx 50% of immunoglobulins expressed by peripheral B-cells from normal adults, is underrepresented in human monoclonal antibodies to HIV-1 and in the peripheral B-cells of AIDS patients. These abnormalities, together with features of germinal center alteration, could be responsible for the clonal elimination of a subset of B-cells, and could contribute to HIV pathogenesis.     

Antibody purification methods

Antibodies (Abs) from the sera of patients with autoimmune diseases are reported to have different catalytic functions. Their recovery by effi cient purification methods is, therefore, a crucial step. This article reviews different available methods for their recovery and emphasizes a new approach, namely adsorbents with immobilized histidine, which allows a good purification both in yield and purity of Abs, with the addi tional advantage of using gentle elution conditions. This, in turn, will ensure the recovery of intact (nondenatured) catalytically functional Abs, directly from the sera.     

Platinum Group Metal–free Catalysts for Hydrogen Evolution Reaction in Microbial Electrolysis Cells

Hydrogen gas is a green energy carrier with great environmental benefits. Microbial electrolysis cells (MECs) can convert low‐grade organic matter to hydrogen gas with low energy consumption and have gained a growing interest in the past decade. Cathode catalysts for the hydrogen evolution reaction (HER) present a major challenge for the development and future applications of MECs. An ideal cathode catalyst should be catalytically active, simple to synthesize, durable in a complex environment, and cost‐effective. A variety of noble‐metal free catalysts have been developed and investigated for HER in MECs, including Nickel and its alloys, MoS₂, carbon‐based catalysts and biocatalysts. MECs in turn can serve as a research platform to study the durability of the HER catalysts. This personal account has reviewed, analyzed, and discussed those catalysts with an emphasis on synthesis and modification, system performance and potential for practical applications. It is expected to provide insights into the development of HER catalysts towards MEC applications.     

Mechanism and functional role of antibody catalysis

The light (L) chain of a model antibody (Ab) was deduced to contain a serine protease-like catalytic site capable of cleaving peptide bonds. The catalytic site is encoded by a germline V_L gene. The catalytic activity can potentially be improved by somatic sequence diversification and pairing of the L chain with the appropriate heavy chain. Autoimmune disease is associated with increased synthesis of antigen (Ag)-specific Abs, but the reasons for this phenomenon are not known. Only recently has attention turned to the functional role of the catalytic function. Preliminary studies confirm that the catalytic cleavage of peptide bonds is a more potent means to achieve Ag neutralization, compared to reversible Ag binding. Administration of a monoclonal Ab to VIP in experimental animals induces an inflammatory response in the airways, suggesting that catalytic autoantibodies to this peptide found in airway disease and lupus are capable of causing airway dysfunction. The phenomenon of autoantibody catalysis can potentially be applied to isolate efficient catalysts directed against tumor or microbial Ags by exposing the autoimmune repertoire to such Ags or their analogs capable of recruiting the germline V_L gene encoding the catalytic site.     

Directed Evolution of a Designer Enzyme Featuring an Unnatural Catalytic Amino Acid

The impressive rate accelerations that enzymes display in nature often result from boosting the inherent catalytic activities of side chains by their precise positioning inside a protein binding pocket. Such fine‐tuning is also possible for catalytic unnatural amino acids. Specifically, the directed evolution of a recently described designer enzyme, which utilizes an aniline side chain to promote a model hydrazone formation reaction, is reported. Consecutive rounds of directed evolution identified several mutations in the promiscuous binding pocket, in which the unnatural amino acid is embedded in the starting catalyst. When combined, these mutations boost the turnover frequency (k_cat) of the designer enzyme by almost 100‐fold. This results from strengthening the catalytic contribution of the unnatural amino acid, as the engineered designer enzymes outperform variants, in which the aniline side chain is replaced with a catalytically inactive tyrosine residue, by more than 200‐fold.     

Efficient Synthesis of Polycyclic γ-Lactams by Catalytic Carbonylation of Ene-Imines via Nickelacycle Intermediates

The nickel(0)-catalyzed carbonylative cycloaddition of 1,5- and 1,6-ene-imines with carbon monoxide (CO) is reported. Key to this reaction is the efficient regeneration of the catalytically active nickel(0) species from nickel carbonyl complexes such as [Ni(CO)₃L]. A variety of tri- and tetracyclic γ-lactams were thus prepared in excellent yields with 100 % atom efficiency. Preliminary results on asymmetric derivatives promise potential in the synthesis of enantioenriched polycyclic γ-lactams.