Solution structure of the plant defensin VrD1 from mung bean and its possible role in insecticidal activity against bruchids

Vigna radiata plant defensin 1 (VrD1) is the first reported plant defensin exhibiting insecticidal activity. We report herein the nuclear magnetic resonance solution structure of VrD1 and the implication on its insecticidal activity. The root-mean-square deviation values are 0.51 +/- 0.35 and 1.23 +/- 0.29 A for backbone and all heavy atoms, respectively. The VrD1 structure comprises a triple-stranded antiparallel beta-sheet, an alpha-helix, and a 3(10) helix stabilized by four disulfide bonds, forming a typical cysteine-stabilized alphabeta motif. Among plant defensins of known structure, VrD1 is the first to contain a 3(10) helix. Glu26 is highly conserved among defensins; VrD1 contains an arginine at this position, which may induce a shift in the orientation of Trp10, thereby promoting the formation of this 3(10) helix. Moreover, VrD1 inhibits Tenebrio molitor alpha-amylase. Alpha-amylase has an essential role in the digestion of plant starch in the insect gut, and expression of the common bean alpha-amylase inhibitor 1 in transgenic pea imparts complete resistance against bruchids. These results imply that VrD1 insecticidal activity has its basis in the inhibition of a polysaccharide hydrolase. Sequence and structural comparisons between two groups of plant defensins having different specificity toward insect alpha-amylase reveal that the loop between beta2 and beta3 is the probable binding site for the alpha-amylase. Computational docking experiments were used to study VrD1-alpha-amylase interactions, and these results provide information that may be used to improve the insecticidal activity of VrD1.     

The ensemble folding kinetics of the FBP28 WW domain revealed by an all-atom Monte Carlo simulation in a knowledge-based potential

In this work, we apply a detailed all‐atom model with a transferable knowledge‐based potential to study the folding kinetics of Formin‐Binding protein, FBP28, which is a canonical three‐stranded β‐sheet WW domain. Replica exchange Monte Carlo simulations starting from random coils find native‐like (Cα RMSD of 2.68 Å) lowest energy structure. We also study the folding kinetics of FBP28 WW domain by performing a large number of ab initio Monte Carlo folding simulations. Using these trajectories, we examine the order of formation of two β‐hairpins, the folding mechanism of each individual β‐hairpin, and transition state ensemble (TSE) of FBP28 WW domain and compare our results with experimental data and previous computational studies. To obtain detailed structural information on the folding dynamics viewed as an ensemble process, we perform a clustering analysis procedure based on graph theory. Further, a rigorous P_fold analysis is used to obtain representative samples of the TSEs showing good quantitative agreement between experimental and simulated Φ values. Our analysis shows that the turn structure between first and second β strands is a partially stable structural motif that gets formed before entering the TSE in FBP28 WW domain and there exist two major pathways for the folding of FBP28 WW domain, which differ in the order and mechanism of hairpin formation. Proteins 2011. © 2011 Wiley‐Liss, Inc.     

Foreign gene expression (beta-galactosidase) during the cell cycle phases in recombinant CHO cells

Recombinant mammalian cultures for heterologous gene expression typically involve cells traversing the cell cycle. Studies were conducted to characterize rates of accumulation of intracellular foreign protein in single cells during the cell cycle of Chinese hamster ovary (CHO) cells transfected with an expression vector containing the gene for dihydrofolate reductase (dhfr) and the lacZ gene for bacterial beta-galactosidase (a nonsecreated protein). The lacZ gene was under the control of the constitutive cytomegalovirus promoter. These normally attachment-grown cells were adapted to suspension culture in 10(-7) M methotrexate, and a dual-laser flow cytometer was used to simultaneously determine the DNA and foreign protein (beta-galactosidase) content of single living cells. Expression of beta-galactosidase as a function of cell cycle phase was evaluated for cells in the exponential growth phase, early plateau phase, and inhibited traverse of the cell cycle during exponential growth. The results showed that the beta-galactosidase production rate is higher in the S phase than that in the G1 or G2/M phases. Also, when cell cycle progression was stopped at the S phase by addition of aphidicolin, beta-galactosidase content in single cells was higher than that in exponential phase or plateau phase cells and increased with increasing culture time. Although the cells did not continue to divide after aphidicolin addition, the production of beta-galactosidase per unit volume of culture was very similar to that in normal exponential growth. (c) 1993 John Wiley & Sons, Inc.     

Characteristics of chimeric enzymes constructed between Thermotoga maritima and Agrobacterium tumefaciens β-glucosidases: Role of C-terminal domain in catalytic activity

The importance of C-terminal domain of β-glucosidase (family 3 glycosidase) from Thermotoga maritima, a hyper-thermophilic bacterium was investigated by gene shuffling. The amino acid sequences of β-glucosidases from T. maritima and A. tumefaciens share high degree of homology (approximately 40%). However, despite such a high homology, both enzymes exhibited quite distinct characteristics in terms of their pH and temperature profile and substrate specificities. To investigate the functional role of the C-terminal domains of T. maritima and A. tumefaciens β-glucosidases, three chimeric genes were constructed by shuffling at three selected regions. Out of the three chimeric enzymes, only two (Tm533/626At and Tm630/727At) were catalytically active. Parental and the chimeric enzymes were subsequently characterized for the substrate specificities and their response towards pH and temperature. Our results revealed that C-terminal domain was catalytically important. The study clearly establishes the significance of gene shuffling in probing the structure and function relationship in hyper-thermophilic bacterium and evolving enzymes with altered features.     

Biodegradable cationic polyester as an efficient carrier for gene delivery to neonatal cardiomyocytes

Viral-mediated gene delivery has been explored for the treatment and protection of cardiomyocytes, but so far there is only one report using cationic polymer for gene delivery to cardiomyocytes in spite of many advantages of polymer-mediated gene delivery. In this study, a cationic poly(beta-amino ester) (PDMA) with a degradable backbone and cleavable side chains was synthesized by Michael addition reaction. The toxicity of PDMA to neonatal mouse cardiomyocytes (NMCMs) was significantly lower than that of polyethyleneimine (PEI). PDMA formed stable polyplexes with pEGFP. The dissociation of the polyplexes could be triggered by PDMA degradation, and the dissociation time was tunable via the polymer/pEGFP ratio. In vitro transfection showed that PDMA was an effective and low toxic gene delivery carrier for NMCMs. The PDMA/pEGFP polyplexes transfected EGFP gene to NMCMs with about 28% efficiency and caused little death. In contrast, a significant portion of cardiomyocytes cultured with PEI/pEGFP died.     

Structural malleability of plasticins: preorganized conformations in solution and relevance for antimicrobial activity

Plasticins (23 long-residue glycine-leucine-rich dermaseptin-related peptides produced by the skin of South American hylids) have very similar amino acid sequences, hydrophobicities, and amphipathicities, but differ in their membrane-damaging properties and structurations (i.e. destabilized helix states, beta-hairpin, beta-sheet, and disordered states) at anionic and zwitterionic membrane interfaces. Structural malleability of plasticins in aqueous solutions together with parameters that may govern their ability to fold within beta-hairpin like structures were analyzed through circular dichroism and FTIR spectroscopic studies completed by molecular dynamics simulations in polar mimetic media. The goal of this study was to probe to which extent pre-existent peptide conformations, i.e. intrinsic "conformational landscape", may be responsible for variability in bioactive conformation and antimicrobial/hemolytic mechanisms of action of these peptides in relation with their various membrane disturbing properties. All plasticins present a turn region that does not always result in folding into a beta-hairpin shaped conformation. Residue at position 8 plays a major role in initiating the folding, while position 12 is not critical. Conformational stability has no major impact on antimicrobial efficacy. However, preformed beta-hairpin in solution may act as a conformational lock that prevents switch to alpha-helical structure. This lock lowers the antimicrobial efficiency and explains subtle differences in potencies of the most active antimicrobial plasticins.     

Stereochemistry of tissue distribution of racemic propranolol in the dog

Only limited information is available on the stereochemistry of the in vivo distribution of beta-receptor-blocking drugs. In this study we determined the levels of the propranolol enantiomers in plasma, cerebrospinal fluid (CSF) and central nervous system (CNS), and peripheral tissues in the dog following an intravenous dose of a deuterium-labeled pseudoracemate. The appearance of the propranolol enantiomers in the CSF was rapid and nonstereoselective, with maximum concentrations reached at 15 min after dosing. The levels of the enantiomers in both CSF and plasma then declined in a parallel biphasic fashion, with a terminal t1/2 of about 125 min. Except for an early high CSF/plasma concentration ratio of 0.35, the CSF propranolol levels corresponded to the unbound concentration in plasma, CSF/plasma 0.20. All areas of the brain showed a similar uptake of propranolol, with a tissue concentration exceeding that in plasma about 10-fold during the terminal phase of elimination. The uptake of propranolol by peripheral tissues varied widely, ranging from a 50-fold accumulation by the lungs compared to plasma to no accumulation by adipose tissue. However, as for the CSF, there was no evidence of stereoselective uptake of propranolol by any CNS or peripheral tissue except for the liver. A significantly higher level of (+)-vs. (-)-propranolol in liver tissue presumably was a reflection of stereoselective hepatic metabolism of (-)-propranolol by this tissue. The slight stereoselectivity in plasma binding of propranolol known to exist in the dog had no significant influence on tissue or CSF distribution.     

Influence of various biological matrices (plasma, blood microdialysate) on chromatographic performance in the determination of β-blockers using an alkyl-diol silica precolumn for sample clean-up

A HPLC column-switching system with LiChrospher RP-8 ADS precolumn was applied for the determination of beta-blockers (atenolol, pindolol, propranolol) in human plasma. The influence of biological matrices on the changes of the chromatographic parameters such as retention time, peak symmetry, area and selectivity were investigated. After injection of 5 ml plasma a decrease of retention times of the analytes was observed of up to 25% and an increase of asymmetry factors of up to 5%. Peak areas and selectivities were not changed. The observed effect could indicate changes of chromatographic performance caused by contributions of the analytical column or the ADS precolumn. The experiments with microdialysis excluded the contribution of the analytical column. A detailed investigation of experiments have been discussed in this paper.     

Immobilization of beta-galactosidase for application in organic chemistry using a chelating peptide

The strong interaction of hexa-histidine fusion proteins with metal chelate adsorbents was utilized to immobilize beta-galactosidase with a hexa-histidine peptide at the N-terminus to the Ni(2+)-nitrilotriacetic acid adsorbent. The fusion protein was cloned and expressed in Escherichia coli. The purified soluble fusion protein showed the same specific activity as the purified beta-galactosidase and retained 64 percent of its beta-galactosidase activity when bound to the adsorbent. To demonstrate the potential of the immobilized beta-galactosidase in organic chemistry, allyl-beta-D-galactosidase was synthesized from lactose and allyl alcohol on a gram scale. The same enzyme preparation was reused in three subsequent batches to prepare the model compound with high yield. (c) 1993 John Wiley & Sons, Inc.     

Comparison of prostate gene expression and tissue weight changes as monitors of antiandrogen activity in GNRH-inhibited rats

BACKGROUND: The Hershberger assay for antiandrogens and modifiers of steroid biosynthesis uses surgically‐castrated rats. We described an adaptation of the assay using the GnRH inhibitor Antarelix in place of surgical castration [Ashby J, Lefevre PA, Deghenghi R, Wallis N. Regulatory Toxicology and Pharmacology 34:188–203, 2001], and concomitantly described changes in expression of the androgen‐dependent prostatic genes PBP C3, TRPM‐2, and ODC as a possible complement to gravimetric analysis of the sex accessory tissues (SAT) [Nellemann C, Vinggaard AM, Dalgaard M, Hossaini A, Larsen J‐J. Toxicology 163:29–38, 2001]. METHODS: The present study describes the results of combining these two modifications into a single assay. During the course of these experiments it was shown that SD rats gave similar results to AP rats and that the higher stimulatory dose of testosterone propionate (TP) used in our experiments gave stronger assay responses to FLU than the lower dose of TP used by some earlier investigators. The potent antiandrogen flutamide (FLU) and the weak antiandrogen DDE were used to evaluate this modified assay. RESULTS: For all parameters studied (SAT weights and changes in expression of the 3 prostatic genes) FLU gave the expected positive results. The weak antiandrogen DDE gave variable and mainly non‐reproducible responses. Use of DDE as a weak antiandrogen accelerated assessment of the new assay. CONCLUSIONS: Possible reasons for this failure to detect DDE are discussed, and it is concluded that the modified assay is unsuitable for use in its present form. The use of gene expression analyses together with evaluation of SAT weights is a promising tool as an early and sensitive marker of antiandrogen action, but more work is needed on the choice of time frame as well as the selection of genes to monitor. Birth Defects Res B 68:344–354, 2003. © 2003 Wiley‐Liss, Inc.     

Structure of the human glycogen-associated protein phosphatase 1 regulatory subunit hGM: homology modeling revealed an (alpha/beta)8-barrel-like fold in the multidomain protein

Protein phosphatase 1 (PP1) is widely distributed among tissues and species and acts as a regulator of many important cellular processes. By targeting the catalytic part of PP1 (PP1C) toward particular loci and substrates, regulatory subunits constitute key elements conferring specificity to the holoenzyme. Here, we report the identification of an (alpha/beta)8-barrel-like structure within the N-ter stretch of the human PP1 regulatory subunit hGM, which is part of the family of diverse proteins associated with glycogen metabolism. Protein homology modeling gave rise to a three-dimensional (3D) model for the 381 N-ter residue stretch of hGM, based on sequence similarity with Streptomyces olivochromogenes xylose isomerase, identified by using FASTA. The alignment was subsequently extended by using hydrophobic cluster analysis. The homology-derived model includes the putative glycogen binding area located within the 142-230 domain of hGM as well as a structural characterization of the PP1C interacting domain (segment 51-67). Refinement of the latter by molecular dynamics afforded a topology that is in agreement with previous X-ray studies (Egloff et al., 1997). Finite difference Poisson-Boltzmann calculations performed on the interacting domains of PP1C and hGM confirm the complementarity of the local electrostatic potentials of the two partners. This work highlights the presence of a conserved fold among distant species (mammalian, Caenorhabditis elegans, yeast) and, thus, emphasizes the involvement of PP1 in crucial basic cellular functions.     

Use of nfsB, encoding nitroreductase, as a reporter gene to determine the mutational spectrum of spontaneous mutations in Neisseria gonorrhoeae

Background  

Organisms that are sensitive to nitrofurantoin express a nitroreductase. Since bacterial resistance to this compound results primarily from mutations in the gene encoding nitroreductase, the resulting loss of function of nitroreductase results in a selectable phenotype; resistance to nitrofurantoin. We exploited this direct selection for mutation to study the frequency at which spontaneous mutations arise (transitions and transversions, insertions and deletions).     

Purification, characterization and synergistic activity of β-1,3-glucanase and antibiotic extract from an antagonistic Bacillus subtilis NSRS 89-24 against rice blast and sheath blight

Antifungal compounds in the culture filtrate from Bacillus subtilis NSRS 89-24 that inhibited the growth of Pyricularia grisea and Rhizoctonia solani were mainly heat stable as the filter sterilized culture filtrate showed higher activity than an autoclaved one. The heat stable and labile components were due to an antibiotic and a β-1,3-glucanase, respectively. This β-1,3-glucanase was purified and characterized. Glucanase activity in the culture medium of B. subtilis NSRS 89-24 was inducible in the presence of 0.3% chitin, reaching a maximum on day 5. After purification, activity was associated with a protein of molecular mass of approximately 95.5 kDa by both gel filtration and native PAGE. Two major bands of M_r 64.6 and 32.4 kDa were revealed by SDS–PAGE. The enzyme had a K_m of 0.9 mg/ml, and V_max of 0.11 U, the optimal pH was 6.5–9.5 and was stable up to 50 °C. Both the pure enzyme and the antibiotic extract from the culture filtrate of the B. subtilis separately inhibited R. solani and P. grisea with MIC values of 12.5 and 6.25 mU/ml and 3.13 and 1.56 μg/ml, respectively. The glucanase enzyme in combination with the antibiotic showed a strong synergistic inhibitory effect on the hyphal growth of both fungi.