Involvement of p38 MAP kinase during iron chelator-mediated apoptotic cell death

Iron is an essential element for the neoplastic cell growth, and iron chelators have been tested for their potential anti-proliferative and cytotoxic effects. To determine the mechanism of cell death induced by iron chelators, we explored the pathways of the three structurally related mitogen-activated protein (MAP) kinase subfamilies during apoptosis induced by iron chelators. We report that the chelator deferoxamine (DFO) strongly activates both p38 MAP kinase and extracellular signal-regulated kinase (ERK) at an early stage of incubation, but slightly activates c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) at a late stage of incubation. Among three MAP kinase blockers used, however, the selective p38 MAP kinase inhibitor SB203580 could only protect HL-60 cells from chelator-induced cell death, indicating that p38 MAP kinase serves as a major mediator of apoptosis induced by iron chelator. DFO also caused release of cytochrome c from mitochondria and induced activation of caspase 3 and caspase 8. Interestingly, treatment of HL-60 cells with SB203580 greatly abolished cytochrome c release, and activation of caspase 3 and caspase 8. Collectively, the current study reveals that p38 MAP kinase plays an important role in iron chelator-mediated cell death of HL-60 cells by activating downstream apoptotic cascade that executes cell death pathway.     

Immobilization of oligonucleotides on poly(ethylene glycol) brush-coated Si surfaces

High-density poly(ethylene glycol) (PEG) molecules are grafted onto Si surfaces in a brush-like configuration. We demonstrate that this surface is an excellent substrate for oligonucleotide immobilization. p-Maleimidophenyl isocyanate is used as a heterobifunctional cross-linker to tether thiol-modified oligonucleotides to terminal OH groups on the PEG brush. This approach gives excellent immobilization specificity and low background. The immobilized oligonucleotides show high sensitivity for the detection of complementary targets.     

Structural basis of the adaptive molecular recognition by MMP9

Matrix metalloproteinase (MMPs) are critical for the degradation of extracellular matrix components and, therefore, need to be regulated tightly. Almost all MMPs share a homologous C-terminal haemopexin-like domain (PEX). Besides its role in macromolecular substrate processing, the PEX domains appear to play a major role in regulating MMP activation, localisation and inhibition. One intriguing property of MMP9 is its competence to bind different proteins, involved in these regulatory processes, with high affinity at an overlapping recognition site on its PEX domain. With the crystal structure of the PEX9 dimer, we present the first example of how PEX domains accomplish these diverse roles. Blade IV of PEX9 mediates the non-covalent and predominantly hydrophobic dimerisation contact. Large shifts of blade III and, in particular, blade IV, accompany the dimerisation, resulting in a remarkably asymmetric homodimeric structure. The asymmetry provides a novel mechanism of adaptive protein recognition, where different proteins (PEX9, PEX1, and TIMP1) can bind with high affinity to PEX9 at an overlapping site. Finally, the structure illustrates how the dimerisation generates new properties on both a physico-chemical and functional level.     

Cytopathological effect of Bacillus thuringiensis israelensis endotoxins on the intestines of Aedes aegypti mosquito larvae

The dynamics of pathological changes in the intestine of Aedes aegypti larvae under the influence of toxins Cry11A and Cry4B produced by Bacillus thuringiensis israelensis was studied by means of electron microscope. Most significant ultrastructure changes in the intestine of the second instar larvae were observed in the midgut. The cytoplasm of cells disintegrated, and elongated lacunae appeared. The number of microvilli decreased, or they disappeared in the result of destruction. The peritrophic membrane displaced to the lumen of midgut. Any changes in epithelial cells and cuticle in time of foregut and hindgut were not observed in a comparison to control. The toxin Cry4B caused the most effective destruction of the midgut epithelium.     

A cell-free protein synthesis system as an investigational tool for the translation stop processes

Using Escherichia coli cell-free protein synthesis system and aminoacylated amber suppressor tRNA, we successfully inserted an unnatural amino acid S-(2-nitrobenzyl)cysteine into human erythropoietin. Three different types of translation stop suppression were observed and each of the three types was easily discerned with SDS-PAGE. Optimal conditions were established for correct stop and programmed suppressions. Since this system differentiates proteins produced by misreading of codons from those produced by programmed suppression, we conclude that this cell-free translation system that we describe in this paper will be of a great use for future investigations on translation stop processes.     

Identification of calmodulin isoform-specific binding peptides from a phage-displayed random 22-mer peptide library

Plants express numerous calmodulin (CaM) isoforms that exhibit differential activation or inhibition of CaM-dependent enzymes in vitro; however, their specificities toward target enzyme/protein binding are uncertain. A random peptide library displaying a 22-mer peptide on a bacteriophage surface was constructed to screen peptides that specifically bind to plant CaM isoforms (soybean calmodulin (ScaM)-1 and SCaM-4 were used in this study) in a Ca2+-dependent manner. The deduced amino acid sequence analyses of the respective 80 phage clones that were independently isolated via affinity panning revealed that SCaM isoforms require distinct amino acid sequences for optimal binding. SCaM-1-binding peptides conform to a 1-5-10 ((FILVW)XXX(FILV) XXXX(FILVW)) motif (where X denotes any amino acid), whereas SCaM-4-binding peptide sequences conform to a 1-8-14 ((FILVW)XXXXXX(FAILVW)XXXXX(FILVW)) motif. These motifs are classified based on the positions of conserved hydrophobic residues. To examine their binding properties further, two representative peptides from each of the SCaM isoform-binding sequences were synthesized and analyzed via gel mobility shift assays, Trp fluorescent spectra analyses, and phosphodiesterase competitive inhibition experiments. The results of these studies suggest that SCaM isoforms possess different binding sequences for optimal target interaction, which therefore may provide a molecular basis for CaM isoform-specific function in plants. Furthermore, the isolated peptide sequences may serve not only as useful CaM-binding sequence references but also as potential reagents for studying CaM isoform-specific function in vivo.     

PCR assays for specific and sensitive detection of Pseudomonas tolaasii,the cause of brown blotch disease of mushrooms

AIMS: The present study describes PCR assays to detect specifically Pseudomonas tolaasii from various samples. METHODS AND RESULTS: Two sets of PCR primers were developed to amplify genes required for tolaasin production. Only a PCR product of 449 bp or 249 bp was produced in PCR reactions with the Pt-1A/Pt-1D1 or Pt-PM/Pt-QM primer sets, respectively, and DNA and cells of Ps. tolaasii. Nested and immunocapture-nested PCR could detect to 3 cells of Ps. tolaasii and amplify the Ps. tolaasii-specific DNA from a sample containing 10 000 times more other bacterial cells than Ps. tolaasii, respectively. CONCLUSIONS: The PCR assays are simple, rapid and reliable methods for detection and identification of Ps. tolaasii. SIGNIFICANCE AND IMPACT OF THE STUDY: The protocols can effectively distinguish Ps. tolaasii from other bacteria and detect Ps. tolaasii from various samples for studying ecology of the bacterium and preventing the use of contaminated water or spawn or medium in mushroom cultivation.     

Reprogramming of the activity of the activator/dissociation transposon family during plant regeneration in rice

Many aspects of epigenetic phenomena have been elucidated via studies of transposable elements. An active transposable element frequently loses its ability to mobilize and goes into an inactive state during development. In this study, we describe the cyclic activity of a maize transposable element dissociation (Ds) in rice. In rice genome, Ds undergoes the spontaneous loss of mobility. However, an inactive state of Ds can be changed into an active state during tissue culture. The recovery of mobility accompanies not only changes in the methylation patterns of the terminal region of Ds, but also alteration in the steady state level of the activator (Ac) mRNA that is expressed by a constitutive CaMV 35S promoter. Furthermore, the Ds-reactivation process is not random, but stage-specific during plantlet regeneration. Our findings have expanded previous observations on Ac reactivation in the tissue culture of maize.     

Role of human organic anion transporter 4 in the transport of ochratoxin A

The purpose of this study was to investigate the characteristics of ochratoxin A (OTA) transport by multispecific human organic anion transporter 4 (hOAT4) using mouse proximal tubule cells stably transfected with hOAT4 (S(2) hOAT4). Immunohistochemical analysis revealed that hOAT4 protein was localized to the apical side of the proximal tubule. S(2) hOAT4 expressed hOAT4 protein in the apical side as well as basolateral side and the cells were cultured on the plastic dish for experiments. S(2) hOAT4 exhibited a time- and concentration-dependent, and a saturable increase in OTA uptake, with an apparent K(m) value of 22.9+/-2.44 microM. The OTA uptakes were inhibited by several substrates for the OATs. Probenecid, piroxicam, octanoate and citrinin inhibited OTA uptake by hOAT4 in a competitive manner (K(i)=44.4-336.4 microM), with the following order of potency: probenecid > piroxicam > octanoate >citrinin. The efflux of OTA by S(2) hOAT4 was higher than that by mock. Addition of OTA resulted in slight decrease in viability of S(2) hOAT4 compared with mock. These results indicate that hOAT4 mediates the high-affinity transport of OTA on the apical side of the proximal tubule, whereas the transport characteristics of OTA are distinct from those by basolateral OATs.