Mechanistic study of proton transfer and hysteresis in catalytic antibody 16E7 by site-directed mutagenesis and homology modeling

Antibody 16E7 catalyzes the carbon protonation of enol ether 2 to hemiacetal 3, and the carbon deprotonation of benzisoxazole 7 to phenol 8. This antibody shows an extreme case of hysteresis, requiring several hours to reach full activity. Antibody 16E7 was expressed as recombinant chimeric Fab in Escherichia coli. A model for the three-dimensional structure was produced by homology modeling and used for a docking procedure to obtain models for antibody-ligand complexes. Site-direct mutagenesis of GluL39, identified as a possible catalytic residue by the model, to either glutamine or alanine abolished catalysis, showing that both the protonation reaction of enol ether 2 and the deprotonation of benzisoxazole 7 are promoted by the same residue. The model furthermore suggested that substrate access to the catalytic site might be hindered by a flexible HCDR3 loop held in closed position by a hydrogen bond between SerH99 and GluL39, which could explain the observed hysteresis effect. In agreement with this model, mutagenesis of SerH99 to alanine, or deletion of this residue, was found to reduce hysteresis by approximately 50%.     

Bystander effect of normal fibroblasts for macrophages co-cultured with susceptible transformed target cells

Macrophages attack and kill pathologically changed, transformed and tumor cells. However, in some cases they may also support tumor growth, modulate the action of anticancer drugs, and even facilitate the development of drug resistance in tumor cells. Here we present data that bystander fibroblasts NIH3T3 were not only resistant to murine macrophages J774.2 but also blocked their killing action towards murine transformed fibroblasts L929. Macrophages were isolated from mixed cultures by means of CD11b specific immunomagnetic beads, and changes induced by their former co-culturing were studied using DNA microarray technology and other tests. An expression of candidate genes coding for cytokines and for signal transduction pathway proteins was estimated in macrophages in different variants of their co-culture with target cells. Changes in expression of mRNA for interleukin 1beta, NFkappaB, IkappaBalpha, gadd45, and CD5 were detected as the most prominent in the macrophages co-cultured with the transformed cells. Bystander NIH3T3 fibroblasts abolished these changes in the macrophages J774.2, and the level of expression of the above mentioned genes was close to the level seen in the macrophages which did not exert cytotoxicity towards the target fibroblasts. Potential implications and research perspectives of using the macrophage-target cell co-cultures with different bystander cellular partners are discussed.     

The gene ENHANCER OF PINOID controls cotyledon development in the Arabidopsis embryo

During Arabidopsis embryo development, cotyledon primordia are generated at transition stage from precursor cells that are not derived from the embryonic shoot apical meristem (SAM). To date, it is not known which genes specifically instruct these precursor cells to elaborate cotyledons, nor is the role of auxin in cotyledon development clear. In laterne mutants, the cotyledons are precisely deleted, yet the hypocotyl and root are unaffected. The laterne phenotype is caused by a combination of two mutations: one in the PINOID (PID) gene and another mutation in a novel locus designated ENHANCER OF PINOID (ENP). The expression domains of shoot apex organising genes such as SHOOT MERISTEMLESS (STM) extend along the entire apical region of laterne embryos. However, analysis of pid enp stm triple mutants shows that ectopic activity of STM does not appear to cause cotyledon obliteration. This is exclusively caused by enp in concert with pid. In pinoid embryos, reversal of polarity of the PIN1 auxin transport facilitator in the apex is only occasional, explaining irregular auxin maxima in the cotyledon tips. By contrast, polarity of PIN1:GFP is completely reversed to basal position in the epidermal layer of the laterne embryo. Consequently auxin, which is believed to be essential for organ formation, fails to accumulate in the apex. This strongly suggests that ENP specifically regulates cotyledon development through control of PIN1 polarity in concert with PID.     

Evaluation of the effect of GHRH(1-44)NH2 on the secretion of interleukin-2 (IL-2) and soluble IL-2 receptor alpha (sIL-2Ralpha) from human peripheral blood mononuclear cells in vitro

OBJECTIVE: The bidirectional communication between the neuroendocrine and the immune systems is now a subject of an intensive investigation. Somatoliberin (GHRH) is a hypothalamic hormone that enhances the synthesis and the release of growth hormone (GH) from the anterior pituitary cells. Few recent reports demonstrate also role of the neuropeptide in the regulation of the immune system function. AIM: The aim of the study was to examine the influence of GHRH on IL -2 as well as sIL -2Ralpha secretion from mitogen-stimulated peripheral blood mononuclear cells. MATERIAL AND METHOD: Mononuclear cells (PBMC) were isolated from the peripheral blood of healthy adults according to the technique described by B?yum. Cells, cultured 24 hours at 37 degrees C in humidified atmosphere of 95% air and 5% CO2, were stimulated with phytohemaglutinin (PHA; 10 microg/ml), and then GHRH(1-44)NH2 at the final concentrations 10(-12), 10(-10), 10(-8) and 10(-6) M was added to appropriate wells. ELISA kits were used to measure IL-2 and sIL-2Ralpha concentrations in the supernatants of cultured cells. Comparisons between tested groups were made by U Mann-Whitney test. The differences were considered significant if p < 0.05. RESULTS: GHRH stimulated the secretion of IL -2 into the supernatants acting significantly at the concentration of 10(-12) M (p < 0.001). Moreover, GHRH at concentrations 10(-10) M and 10(-8) M significantly increased the secretion of sIL-2Ralpha as well (p < 0.001). Strong positive correlation between tested GHRH concentrations and sIL-2Ralpha levels in the supernatants was demonstrated (r = 0.8664; p <0.001). CONCLUSIONS: The results demonstrate the potential involvement of GHRH in the regulation of T lymphocytes secretory function.     

Origin of the sequence-dependent polyproline II structure in unfolded peptides

Recent studies have indicated that the unfolded states of polypeptides contain a substantial amount of polyproline type II (P(II)) structure. This energetically and structurally preorganized state may contribute to the reduction of the folding search, as well as to the recognition of intrinsically unstructured proteins and polyproline ligands. Using Monte Carlo simulations of natively unfolded peptides in the presence of explicit aqueous solvation, we observe that residue-specific P(II) conformational propensity is the result of the modulation of polypeptide backbone hydration by a proximal side-chain. Such a mechanism may be unique among those that contribute to the modulation of secondary structures in proteins. The calculated conformational propensities should prove useful for the development of a configurational P(II) scale necessary for the prediction and design of natural-like polypeptides.     

A major histocompatibility complex-peptide-restricted antibody and t cell receptor molecules recognize their target by distinct binding modes: crystal structure of human leukocyte antigen (HLA)-A1-MAGE-A1 in complex with FAB-HYB3

Antibodies with T cell receptor-like specificity possess a considerable diagnostic and therapeutic potential, but the structural basis of the interaction between an antibody and an histocompatibility antigen has so far not been determined. We present here the crystal structure (at 2.15 A resolution) of the recombinant, affinity-matured human antibody fragment Fab-Hyb3 bound to the tumor-associated human leukocyte antigen (HLA)/peptide complex HLA-A1.MAGE-A1. Fab-Hyb3 employs a diagonal docking mode resembling that of T cell receptors. However, other than these natural ligands, the antibody uses only four of its six complementarity-determining regions for direct interactions with the target. It recognizes the C-terminal half of the MAGE-A1 peptide, the HLA-A1 alpha1-helix, and N-terminal residues of the alpha2-helix, accompanied by a large tilting angle between the two types of molecules within the complex. Interestingly, only a single hydrogen bond between a peptide side chain and Fab-Hyb3 contributes to the interaction, but large buried surface areas with pronounced shape complementarity assure high affinity and specificity for MAGE-A1. The HLA-A1.MAGE-A1.antibody structure is discussed in comparison with those of natural ligands recognizing HLA.peptide complexes.     

gamma-Glutamyl dipeptides in Petiveria alliacea

Three gamma-glutamyl dipeptides have been isolated from Petiveria alliacea L. roots. These dipeptides include (S(C2)R(C7))-gamma-glutamyl-S-benzylcysteine together with two diastereomeric sulfoxides, namely (S(C2)R(C7)R(S))- and (S(C2)R(C7)R(S))-gamma-glutamyl-S-benzylcysteine S-oxides (gamma-glutamyl-petiveriins A and B, respectively). Their structures and absolute configurations have been determined by NMR, MALDI-HRMS, IR and CD spectroscopy, and confirmed by comparison with authentic compounds obtained by synthesis.     

Spatiotemporal distribution of insects and mites in horizontally stored wheat

Samples were taken from a flat storage facility located in central Greece, filled with approximately 45 tons of hard wheat, to assess the spatiotemporal distribution of stored-product insects and mites. The wheat was stored in a 1.5-m-deep bulk from June 2001 until March 2002. The samples were taken with a partitioned grain trier during the entire storage period, at 10-d intervals. The trier samples were examined separately for the upper, medial, and lower 0.5 m of the bulk. The spatial distribution of the insect and mite species found was examined by contour analysis based on the numbers of individuals in the trier samples. Nine insect and 20 mite taxa were found during the sampling period. The most abundant insect species were Tribolium castaneum (Herbst), Cryptolestes ferrugineus (Stephens), and Rhyzopertha dominica (F.); the most abundant mite species were Lepidoglyphus destructor (Schrank), Acarus siro L., and the predator Cheyletus malaccensis Oudemans. The highest population densities for the majority of the insect and mite species were recorded during autumn. The majority of the individuals of the most abundant insect and mite species were found in the upper 0.5 m of the bulk, with the exception of C. malaccensis, which was equally distributed in the upper and medial 0.5 m of the bulk. The spatiotemporal distribution during the entire experimental period was notably varied according to the insect and mite species.     

Persistence and efficacy of three diatomaceous earth formulations against Sitophilus oryzae (Coleoptera: Curculionidae) on wheat and barley

The insecticidal and residual efficacy of three diatomaceous earth (DE) formulations, Insecto, PyriSec, and SilicoSec, against Sitophilus oryzae (L.) on barley and wheat was assessed. For this purpose, 4-kg lots of barley and wheat were treated with the above-mentioned DE formulations, in three dose rates (0.75, 1, and 1.5 g/kg grain) and stored at 26 degrees C. Samples of these lots were taken at the day of storage, and every 45 d, until the completion of a 450-d period of storage. Bioassays were conducted by exposing S. oryzae adults to these samples, at 26 degrees C and 57% RH. In these bioassays, the DE efficacy was evaluated by recording adult mortality after 24 h, 48 h, 7 d, and 14 d of exposure on the treated grains. After the 14-d count, all adults were removed, and the samples were left at the same conditions for an additional 45 d, to evaluate the capacity for progeny production in the treated grains. Adult mortality after 14 d of exposure was exponentially decreased with time. During the first 270 d of storage, mortality was > 90%, and progeny production was < 1 adult per sample, whereas after 270 d a gradual decrease in adult mortality occurred, with a resulting increase in progeny production. Generally, the three DE formulations tested were equally effective against S. onyzae adults. During the first 270 d of storage, the DE formulations were equally effective on both grains tested, but from 315 d of storage and on, S. oryzae mortality was higher on barley than on wheat. At this interval, progeny production was gradually increased, especially on grains treated with the lowest DE dose rate. However, even this rate caused a satisfactory level of mortality (> 90% after 14 d of exposure) during the first 270 d of storage.     

Proteomics-grade de novo sequencing approach

The conventional approach in modern proteomics to identify proteins from limited information provided by molecular and fragment masses of their enzymatic degradation products carries an inherent risk of both false positive and false negative identifications. For reliable identification of even known proteins, complete de novo sequencing of their peptides is desired. The main problems of conventional sequencing based on tandem mass spectrometry are incomplete backbone fragmentation and the frequent overlap of fragment masses. In this work, the first proteomics-grade de novo approach is presented, where the above problems are alleviated by the use of complementary fragmentation techniques CAD and ECD. Implementation of a high-current, large-area dispenser cathode as a source of low-energy electrons provided efficient ECD of doubly charged peptides, the most abundant species (65-80%), in a typical trypsin-based proteomics experiment. A new linear de novo algorithm is developed combining efficiency and speed, processing on a conventional 3 GHz PC, 1000 MS/MS data sets in 60 s. More than 6% of all MS/MS data for doubly charged peptides yielded complete sequences, and another 13% gave nearly complete sequences with a maximum gap of two amino acid residues. These figures are comparable with the typical success rates (5-15%) of database identification. For peptides reliably found in the database (Mowse score > or = 34), the agreement with de novo-derived full sequences was >95%. Full sequences were derived in 67% of the cases when full sequence information was present in MS/MS spectra. Thus the new de novo sequencing approach reached the same level of efficiency and reliability as conventional database-identification strategies.