High affinity binding of albicidin phytotoxins by the AlbA protein from Klebsiella oxytoca

Albicidins are a family of phytotoxins and antibiotics which play an important role in the pathogenesis of sugarcane leaf scald disease. The albA gene from Klebsiella oxytoca encodes a protein which inactivates albicidin by heat-reversible binding. Albicidin ligand binding to a recombinant AlbA protein, purified by means of a glutathione S-transferase gene fusion system, is an almost instant and saturable reaction. Kinetic and stoichiometric analysis of the binding reaction indicated the presence of a single high affinity binding site with a dissociation constant of 6.4 x 10(-8) M. The AlbA-albicidin complex is stable from 4 to 40 degrees C, from ph 5 to 9 and in high salt solutions. Treatment with protein denaturants released all bound albicidin. These properties indicate that AlbA may be a useful affinity matrix for selective purification of albicidin antibiotics. AlbA does not bind to p-nitrophenyl butyrate or alpha-naphthyl butyrate, the substrates of the albicidin detoxification enzyme AlbD from Pantoea dispersa. The potential exists to pyramid genes for different mechanisms in transgenic plants to protect plastid DNA replication from inhibition by albicidins.

Bacterial expression of two human aryl sulfotransferases

The effect of replacing a single codon in the N-terminal of human aryl sulfotransferase (HAST) 1 and 3 with one that is more commonly found in E. coli genes was assessed. The pKK233-2 E. coli expression vector was employed and the polymerase chain reaction (PCR) was used to introduce the 5' nucleotide substitution, at the same time maintaining the fidelity of the amino acid sequence. The data indicates that this change had a minimal effect on protein production, subcellular localization or, in the case of HAST3, catalytic activity. In general, the pKK233-2 E. coli vector has been less than optimal for expressing human sulfotransferase cDNAs. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.

A protein with protective properties against the cellular defense reactions in insects

The molecular mechanism of how insects recognize intruding microorganisms and parasites and distinguish them from own body structures is not well known. We explored evolutionary adaptations in an insect parasitoid host interaction to identify components that interfere with the recognition of foreign objects and cellular encapsulation. Because some parasitoids provide protection for the developing wasp in the absence of an overt suppression of the insect host defense, we analyzed the surface of eggs and symbiotic viruses for protective properties. Here we report on the molecular cloning of a 32-kDa protein (Crp32) that is one of the major protective components. It is produced in the calyx cells of the female wasp ovaries and attached to the surface of the egg and other particles including polydnaviruses. The recombinant protein confers protection to coated objects in a cellular encapsulation assay suggesting that a layer of Crp32 may prevent cellular encapsulation reactions by a local inactivation of the host defense system.

Analysis and application of an equilibrium model for in vitro bioassay systems with three components: Receptor, hormone and hormone-binding-protein

A simple theoretical framework is presented for bioassay studies using three component in vitro systems. An equilibrium model is used to derive equations useful for predicting changes in biological response after addition of hormone-binding-protein or as a consequence of increased hormone affinity. Sets of possible solutions for receptor occupancy and binding protein occupancy are found for typical values of receptor and binding protein affinity constants. Unique equilibrium solutions are dictated by the initial condition of total hormone concentration. According to the occupancy theory of drug action, increasing the affinity of a hormone for its receptor will result in a proportional increase in biological potency. However, the three component model predicts that the magnitude of increase in biological potency will be a small fraction of the proportional increase in affinity. With typical initial conditions a two-fold increase in hormone affinity for its receptor is predicted to result in only a 33% increase in biological response. Under the same conditions an Ii-fold increase in hormone affinity for receptor would be needed to produce a two-fold increase in biological potency. Some currently used bioassay systems may be unrecognized three component systems and gross errors in biopotency estimates will result if the effect of binding protein is not calculated. An algorithm derived from the three component model is used to predict changes in biological response after addition of binding protein to in vitro systems. The algorithm is tested by application to a published data set from an experimental study in an in vitro system (Lim et al., 1990, Endocrinology 127, 1287-1291). Predicted changes show good agreement (within 8%) with experimental observations. (C) 1998 Academic Press Limited.

Type specific and genotype cross reactive B epitopes of the L1 protein of HPV16 defined by a panel of monoclonal antibodies

Mouse monoclonal antibodies (mAbs) were raised against the major capsid protein, L1, of human papillomavirus type 16 (HPV16), produced in Escherichia coil with the expression plasmid pTrcL1. Epitope specificity could be assigned to 11 of these 12 antibodies using a series of linear peptides and fusion proteins from HPV16. One mAb (MC53) recognized a novel linear epitope that appears to be unique to the HPV16 genotype. A further 11 mAbs were characterized as recognizing novel and previously defined linear and conformational epitopes shared among more than one HPV genotype. The apparently genotype specific mAb could be useful for the development of diagnostic tests for vegetative virus infection in clinical specimens. (C) 1998 Academic Press.

Dragmacidins: New protein phosphatase inhibitors from a southern Australian deep-water marine sponge, Spongosorites sp.

A Spongosorites sp. collected during trawling operations off the southern coast of Australia returned the new alkaloid dragmacidin E (3), the structure of which was secured by detailed spectroscopic analysis. Dragmacidin E (3), and its co-metabolite dragmacidin D (1) have been identified as potent inhibitors of serine-threonine protein phosphatases.

Crystallization of a trimeric human T cell leukemia virus type 1 gp21 ectodomain fragment as a chimera with maltose-binding protein

We present a novel protein crystallization strategy, applied to the crystallization of human T cell leukemia virus type 1 (HTLV-1) transmembrane protein gp21 lacking the fusion peptide and the transmembrane domain, as a chimera with the Escherichia coli maltose binding protein (MBP). Crystals could not be obtained with a MBP/gp21 fusion protein in which fusion partners were separated by a flexible linker, but were obtained after connecting the MBP C-terminal alpha-helix to the predicted N-terminal alpha-helical sequence of gp21 via three alanine residues. The gp21 sequences conferred a trimeric structure to the soluble fusion proteins as assessed by sedimentation equilibrium and X-ray diffraction, consistent with the trimeric structures of other retroviral transmembrane proteins. The envelope protein precursor, gp62, is likewise trimeric when expressed in mammalian cells. Our results suggest that MBP may have a general application for the crystallization of proteins containing N-terminal alpha-helical sequences.

Small molecular probes for G-protein-coupled C5a receptors: Conformationally constrained antagonists derived from the C terminus of the human plasma protein C5a

Activation of the human complement system of plasma proteins in response to infection or injury produces a 4-helix bundle glycoprotein (74 amino acids) known as C5a. C5a binds to G-protein-coupled receptors on cell surfaces triggering receptor-ligand internalization, signal transduction, and powerful inflammatory responses. Since excessive levels of C5a are associated with autoimmune and chronic inflammatory disorders, inhibitors of receptor activation may have therapeutic potential. We now report solution structures and receptor-binding and antagonist activities for some of the first small molecule antagonists of C5a derived from its hexapeptide C terminus. The antagonist NMe-Phe-Lys-Pro-D-Cha-Trp-D-Arg-CO2H (1) surprisingly shows an unusually well-defined solution structure as determined by H-1 NMR spectroscopy. This is one of the smallest acyclic peptides found to possess a defined solution conformation, which can be explained by the constraining role of intramolecular hydrogen bonding. NOE and coupling constant data, slow deuterium exchange, and a low dependence on temperature for the chemical shift of the D-Cha-NH strongly indicate an inverse gamma turn stabilized by a D-Cha-NH ... OC-Lys hydrogen bond. Smaller conformational populations are associated with a hydrogen bond between Trp-NH ... OC-Lys, defining a type II beta turn distorted by the inverse gamma turn incorporated within it. An excellent correlation between receptor-affinity and antagonist activity is indicated for a limited set of synthetic peptides. Conversion of the C-terminal carboxylate of 1 to an amide decreases antagonist potency 5-fold, but potency is increased up to 10-fold over 1 if the amide bond is made between the C-terminal carboxylate and a Lys/Orn side chain to form a cyclic analogue. The solution structure of cycle 6 also shows gamma and beta turns; however, the latter occurs in a different position, and there are clear conformational changes in 6 vs 1 that result in enhanced activity. These results indicate that potent C5a antagonists can be developed by targeting site 2 alone of the C5a receptor and define a novel pharmacophore for developing powerful receptor probes or drug candidates.

Simultaneous detection of multiple antigens with triple immunolabeling

Immunolabeling is commonly used to localize antigens within frozen or paraffin tissue sections. We modified existing immunolabeling techniques to allow the detection of three antigens simultaneously within the one tissue section. The approach relies on the use of three monoclonal antibodies in sequential immunoperoxidase staining steps, each with colored substrates, resulting in the deposition of black, brown, and rose stains. The method is rapid and does not require novel techniques or materials. In this report, we demonstrate the colocalization of mast cell tryptase, neurofilament protein, and CD31 (platelet-endothelial cell adhesion molecule) or laminin in normal human skin and normal buccal mucosa, as an illustration of the power and simplicity of the multiple antigen localization technique.

The relationship between hetero-oligomer formation and function of the topological specificity domain of the Escherichia coli MinE protein

MinE is an oligomeric protein that, in conjunction with other Min proteins, is required for the proper placement of the cell division site of Escherichia coli. We have examined the self-association properties of MinE by analytical ultracentrifugation and by studies of hetero-oligomer formation in non-denaturing polyacrylamide gets. The self-association properties of purified MinE predict that cytoplasmic MinE is likely to exist as a mixture of monomers and dimers. Consistent with this prediction, the C-terminal MinE(22-88) fragment forms hetero-oligomers with MinE(+) when the proteins are co-expressed. In contrast, the MinE(36-88) fragment does not form MinE(+)/MinE(36-88) hetero-oligomers, although MinE36-88 affects the topological specificity of septum placement as shown by its ability to induce minicell formation when co-expressed with MinE(+) in wild-type cells. Therefore, hetero-oligomer formation is not necessary for the induction of mini-celling by expression of MinE(36-88) in wild-type cells. The interference with normal septal placement is ascribed to competition between MinE(36-88),nd the corresponding domain in the complete MinE protein for a component required for the topological specificity of septal placement.

Murine DEP-1, a receptor protein tyrosine phosphatase, is expressed in macrophages and is regulated by CSF-1 and LPS

The spectrum of protein tyrosine phosphatases (PTPs) expressed in bone marrow-derived murine macrophages (BMMs) was examined using reverse transcriptase-polymerase chain reaction. Ten different PTP cDNAs were isolated and in this study we focus on mDEP-1, a type III receptor PTP. Three mDEP-1 transcripts were expressed in primary macrophages and macrophage cell lines and were induced during macrophage differentiation of M1 myeloid leukemia cells. A valiant mRNA Tvas identified that encodes an alternate carboxyl-terminus and 3' UTR. The expression of mDEP-1 was down-regulated by CSF-1 (macrophage colony-stimulating factor) and up-regulated by bacterial lipopolysaccharide, an important physiological regulator of macrophage function that opposes CSF-1 action. Whole mount irt situ hybridization, and immunolocalization of the protein, confirmed that mDEP-1 is expressed by a subset of embryonic macrophages in the liver and mesenchyme. mDEP-1 was also detected in the eye and peripheral nervous system of the developing embryo. Attempts to express mDEP-1 constitutively in the macrophage cell line RAW264 were unsuccessful, with results suggesting that the gene product inhibits cell proliferation.

Comparison of carbohydrate-deficient transferrin, immunoglobulin A antibodies reactive with acetaldehyde-modified protein and acetaldehyde-modified albumin with conventional markers of alcohol consumption

Carbohydrate-deficient transferrin (CDT) has emerged as the best new marker for alcohol abuse. Recently plasma immunoglobulin A (IgA) reactivity with acetaldehyde (AcH)-modified proteins, or the modified proteins per se, have been proposed as a markers for high levels of alcohol consumption. In this study, we have compared CDT, IgA reactivity with AcH adducts (IgA ASR), and AcH-modified albumin with conventional markers of high alcohol intake in groups with well-defined drinking histories, The plasma activity of ALT, AST, and gamma-glutamyltransferase increased steadily with increasing alcohol consumption, CDT and AcH-modified albumin showed a similar pattern, whereas IgA ASR appeared only to be elevated after a threshold level of consumption had been reached, Neither CDT IgA ASR or AcH-modified albumin correlated strongly with any of the conventional markers or each other. This study shows that CDT, IgA ASR, AcH-modified albumin, and the conventional markers are not related, but suggests that the concurrent use of CDT and IgA ASR may lead to better identification of high alcohol intake.

Activation of beta(2)-adrenergic receptors hastens relaxation and mediates phosphorylation of phospholamban, troponin I, and C-protein in ventricular myocardium from patients with terminal heart failure

Background-Catecholamines hasten cardiac relaxation through beta-adrenergic receptors, presumably by phosphorylation of several proteins, but it is unknown which receptor subtypes are involved in human ventricle. We assessed the role of beta(1)- and beta(2)-adrenergic receptors in phosphorylating proteins implicated in ventricular relaxation. Methods and Results-Right ventricular trabeculae, obtained from freshly explanted hearts of patients with dilated cardiomyopathy (n=5) or ischemic cardiomyopathy (n=5), were paced at 60 bpm. After measurement of the contractile and relaxant effects of epinephrine (10 mu mol/L) or zinterol (10 mu mol/L), mediated through beta(2)-adrenergic receptors, and of norepinephrine (10 mu mol/L), mediated through beta(1)-adrenergic receptors, tissues were freeze clamped. We assessed phosphorylation of phospholamban, troponin I, and C-protein, as well as specific phosphorylation of phospholamban at serine 16 and threonine 17, Data did not differ between the 2 disease groups and were therefore pooled. Epinephrine, zinterol, and norepinephrine increased contractile force to approximately the same extent, hastened the onset of relaxation by 15+/-3%, 5+/-2%, and 20+/-3%, respectively, and reduced the time to half-relaxation by 26+/-3%, 21+/-3%, and 37+/-3%. These effects of epinephrine, zinterol, and norepinephrine were associated with phosphorylation (pmol phosphate/mg protein) of phospholamban 14+/-3, 12+/-4, and 12+/-3, troponin I 40+/-7, 33+/-7, and 31+/-6; and C-protein 7.2+/-1.9, 9.3 +/- 1.4, and 7.5 +/- 2.0. Phosphorylation of phospholamban occurred at both Ser16 and Thr17 residues through both beta(1)- and beta(2)-adrenergic receptors. Conclusions-Norepinephrine and epinephrine hasten human ventricular relaxation and promote phosphorylation of implicated proteins through both beta(1)- and beta(2)-adrenergic receptors, thereby potentially improving diastolic function.

Chemical treatment of Escherichia coli: 3. Selective extraction of a recombinant protein from cytoplasmic inclusion bodies in intact cells

In previous parts of this study we developed procedures for the high-efficiency chemical extraction of soluble and insoluble protein from intact Escherichia coli cells. Although high yields were obtained, extraction of recombinant protein directly from cytoplasmic inclusion bodies led to low product purity due to coextraction of soluble contaminants. In this work, a two-stage procedure for the selective extraction of recombinant protein at high efficiency and high purity is reported. In the first stage, inclusion-body stability is promoted by the addition of 15 mM 2-hydroxyethyldisulfide (2-HEDS), also known as oxidized P-mercaptoethanol, to the permeabil ization buffer (6 M urea + 3 mM ethylenediaminetetra-acetate [EDTA]). 2-HEDS is an oxidizing agent believed to promote disulfide bond formation, rendering the inclusion body resistant to solubilization in 6 M urea. Contaminating proteins are separated from the inclusion-body fraction by centrifugation. in the second stage, disulfide bonds are readily eliminated by including reducing agent (20 mM dithiothreitol [DTT]) into the permeabilization buffer. Extraction using this selective two-stage process yielded an 81% (w/w) recovery of the recombinant protein Long-R-3-IGF-I from inclusion bodies located in the cytoplasm of intact E. coli, at a purity of 46% (w/w). This was comparable to that achieved by conventional extraction (mechanical disruption followed by centrifugation and solubilization). A pilot-scale procedure was also demonstrated using a stirred reactor and diafiltration. This is the first reported study that achieves both high extraction efficiency and selectivity by the chemical treatment of cytoplasmic inclusion bodies in intact bacterial cells. (C) 1999 John Wiley & Sons, Inc.