E. coli expression of TIMP-4 and comparative kinetic studies with TIMP-1 and TIMP-2: insights into the interactions of TIMPs and matrix metalloproteinase 2 (gelatinase A)

The inhibitory properties of TIMP-4 for matrix metalloproteinases (MMPs) were compared to those of TIMP-1 and TIMP-2. Full-length human TIMP-4 was expressed in E. coli, folded from inclusion bodies, and the active component was purified by MMP-1 affinity chromatography. Progress curve analysis of MMP inhibition by TIMP-4 indicated that association rate constants (k(on)) and inhibition constants (K(i)) were similar to those for other TIMPs ( approximately 10(5) M(-)(1) s(-)(1) and 10(-)(9)-10(-)(12) M, respectively). Dissociation rate constants (k(off)) for MMP-1 and MMP-3 determined using alpha(2)-macroglobulin to capture MMP dissociating from MMP-TIMP complexes were in good agreement with values deduced from progress curves ( approximately 10(-)(4) s(-)(1)). K(i) and k(on) for the interactions of TIMP-1, -2, and -4 with MMP-1 and -3 were shown to be pH dependent. TIMP-4 retained higher reactivity with MMPs at more acidic conditions than either TIMP-1 or TIMP-2. Molecular interactions of TIMPs and MMPs investigated by IAsys biosensor analysis highlighted different modes of interaction between proMMP-2-TIMP-2 (or TIMP-4) and active MMP-2-TIMP-2 (or TIMP-4) complexes. The observation that both active MMP-2 and inactive MMP-2 (with the active site blocked either by the propeptide or a hydroxamate inhibitor) have essentially identical affinities for TIMP-2 suggests that there are two TIMP binding sites on the hemopexin domain of MMP-2: one with high affinity that is involved in proMMP-2 or hydroxamate-inhibited MMP-2; and the other with low affinity involved in formation of the complex of active MMP-2 and TIMP-2. Similar models of interaction may apply to TIMP-4. The latter low-affinity site functions in conjunction with the active site of MMP-2 to generate a tight enzyme-inhibitor complex.     

Comparison of the expression of Bacillus thuringiensis full-length and N-terminally truncated vip3A gene in Escherichia coli

AIMS: Studies were performed to demonstrate the function of the putative signal peptide of Vip3A proteins in Escherichia coli. METHODS AND RESULTS: The full-length vip3A-S184 gene was isolated from a soil-isolated Bacillus thuringiensis, and the vip3AdeltaN was constructed by deleting 81 nucleotides at the 5'-terminus of vip3A-S184. Both were transformed and expressed in E. coli. About 19.2% of Vip3A-S184 proteins secreted soluble proteins and others formed inclusion bodies in the periplasmic space. In contrast, the Vip3AdeltaN was insoluble and formed inclusion bodies in the cytoplasm. Bioassay indicated that Vip3A-S184 showed different toxicity against Spodoptera exigua, Helicoverpa armigera and S. litura, but Vip3AdeltaN showed no toxicity to either of them because of the deletion of the first 27 amino acids at the N-terminus. CONCLUSIONS: The results suggest that the deleted N-terminal sequences were essential for the secretion of Vip3A-S184 protein in E. coli and might be required for toxicity. SIGNIFICANCE AND IMPACT OF THE STUDY: The function of the putative signal peptide of Vip3A protein in E. coli was investigated. These would be helpful to make clear the unknown secretion pathway of Vip3A protein in B. thuringiensis and determine the receptor-binding domain or toxic fragment of Vip3A-S184 protein.     

Oligonucleotide-based microarray for DNA methylation analysis: principles and applications

Gene silencing via promoter CpG island hypermethylation offers tumor cells growth advantages. This epigenetic event is pharmacologically reversible, and uncovering a unique set of methylation-silenced genes in tumor cells can bring a new avenue to cancer treatment. However, high-throughput tools capable of surveying the methylation status of multiple gene promoters are needed for this discovery process. Herein we describe an oligonucleotide-based microarray technique that is both versatile and sensitive in revealing hypermethylation in defined regions of the genome. DNA samples are bisulfite-treated and PCR-amplified to distinguish CpG dinucleotides that are methylated from those that are not. Fluorescently labeled PCR products are hybridized to arrayed oligonucleotides that can discriminate between methylated and unmethylated alleles in regions of interest. Using this technique, two clinical subtypes of non-Hodgkin's lymphomas, mantle cell lymphoma, and grades I/II follicular lymphoma, were further separated based on the differential methylation profiles of several gene promoters. Work is underway in our laboratory to extend the interrogation power of this microarray system in multiple candidate genes. This novel tool, therefore, holds promise to monitor the outcome of various epigenetic therapies on cancer patients.     

Adaptive diversification of bitter taste receptor genes in Mammalian evolution

The diversity and evolution of bitter taste perception in mammals is not well understood. Recent discoveries of bitter taste receptor (T2R) genes provide an opportunity for a genetic approach to this question. We here report the identification of 10 and 30 putative T2R genes from the draft human and mouse genome sequences, respectively, in addition to the 23 and 6 previously known T2R genes from the two species. A phylogenetic analysis of the T2R genes suggests that they can be classified into three main groups, which are designated A, B, and C. Interestingly, while the one-to-one gene orthology between the human and mouse is common to group B and C genes, group A genes show a pattern of species- or lineage-specific duplication. It is possible that group B and C genes are necessary for detecting bitter tastants common to both humans and mice, whereas group A genes are used for species-specific bitter tastants. The analysis also reveals that phylogenetically closely related T2R genes are close in their chromosomal locations, demonstrating tandem gene duplication as the primary source of new T2Rs. For closely related paralogous genes, a rate of nonsynonymous nucleotide substitution significantly higher than the rate of synonymous substitution was observed in the extracellular regions of T2Rs, which are presumably involved in tastant-binding. This suggests the role of positive selection in the diversification of newly duplicated T2R genes. Because many natural poisonous substances are bitter, we conjecture that the mammalian T2R genes are under diversifying selection for the ability to recognize a diverse array of poisons that the organisms may encounter in exploring new habitats and diets.     

Electron and hole transfer from DNA base radicals to oxidized products of guanine in DNA

An investigation of electron and hole transfer to oxidized guanine bases in DNA is reported. Guanine in DNA was preferentially oxidized by Br(2)(*-) at 298 K to 8-oxo-7,8-dihydro-guanine (8-oxo-G) and higher oxidation products. HPLC-EC analysis of irradiated DNA shows that the formation of 8-oxo-G could not be increased above the ratio of one 8-oxo-G to 127 +/- 6 bp regardless of dose. 8-oxo-G can be produced only at low levels because it is further oxidized to other species. These oxidation products of guanine have been extensively investigated and identified by others. Our electron spin resonance studies suggest that at 77 K 8-oxo-G is a trap for radiation-produced holes, but certain further oxidation products of 8-oxo-G (G(ox)) are found to be efficient electron traps. Gamma irradiation of oxidized DNA samples in frozen (D(2)O) aqueous ices and glassy 7 M LiBr solutions resulted in radicals formed by electron attachment to the G(ox) sites that were monitored by electron spin resonance spectroscopy (ESR) at 77 K. These ESR spectra suggest that those oxidation products of 8-oxo-G containing alpha-diketo groups account for the electron traps (G(ox)) in oxidized DNA with oxaluric acid a likely major trap. Electron transfer from DNA anion radicals to G(ox) was followed by monitoring of their ESR signals with time at 77 K. Using typical values for the tunneling constant beta estimates of the relative amount of G(ox) to base pairs were obtained. Radicals formed by UV photolysis of oxidized DNA in 8 M NaClO(4) glassy aqueous solutions were also investigated. The 8-oxo-G cation accounts for less than 10% of all the radicals observed after either gamma irradiation of oxidized DNA in frozen (D(2)O) aqueous solution or UV photolysis of oxidized DNA in 8 M NaClO(4) glassy aqueous solutions. We estimate hole transfer distances of about 7 +/- 1 bp at 1 min from G(*+) to 8-oxo-G.     

A recombinant anti-erbB2, scFv-Fc-IL-2 fusion protein retains antigen specificity and cytokine function

Elevated erbB2 expression is detected in many in situ and invasive human ductal carcinomas. Anti-erbB2 antibody directed at the extracellular domain of erbB2 can result in an antitumor response in some patients with tumors overexpressing erbB2 oncoprotein. By combining interleukin 2 (IL-2) activities with a tumor specific antibody, immunotherapy of tumors might be more effective in the future. In this study, a fusion protein consisting of erbB2 single chain antibody (scFv), Fc fragment of human IgG1 and IL-2 was constructed. The molecular weight of fusion proteins is 66 kDa, only one third of whole antibody-IL-2 fusion protein or 44% whole Ig molecule. The fusion proteins retained the activities of both antigen binding and IL-2. The scFv-Fc-IL-2 fusion protein may have advantages over whole antibody-IL-2 fusion proteins, such as smaller molecule, better activity of penetration, more favorable pharmacokinetic properties.     

New perspectives in arsenic-induced cell signal transduction

Although the carcinogenicity of arsenic has been well established, the underlying molecular mechanisms have not yet been fully identified. Accumulating evidence indicates that the alteration of cellular signal transduction is directly related to the carcinogenesis of arsenic. This review focuses on recent advances in arsenic-induced signal transduction, including reactive oxygen species (ROS) production, tyrosine phosphorylation, MAPK signaling, NF-kappaB activation, cell cycle arrest, and apoptosis.     

The Arabidopsis SOS5 locus encodes a putative cell surface adhesion protein and is required for normal cell expansion

Cell surface proteoglycans have been implicated in many aspects of plant growth and development, but genetic evidence supporting their function has been lacking. Here, we report that the Salt Overly Sensitive5 (SOS5) gene encodes a putative cell surface adhesion protein and is required for normal cell expansion. The sos5 mutant was isolated in a screen for Arabidopsis salt-hypersensitive mutants. Under salt stress, the root tips of sos5 mutant plants swell and root growth is arrested. The root-swelling phenotype is caused by abnormal expansion of epidermal, cortical, and endodermal cells. The SOS5 gene was isolated through map-based cloning. The predicted SOS5 protein contains an N-terminal signal sequence for plasma membrane localization, two arabinogalactan protein-like domains, two fasciclin-like domains, and a C-terminal glycosylphosphatidylinositol lipid anchor signal sequence. The presence of fasciclin-like domains, which typically are found in animal cell adhesion proteins, suggests a role for SOS5 in cell-to-cell adhesion in plants. The SOS5 protein was present at the outer surface of the plasma membrane. The cell walls are thinner in the sos5 mutant, and those between neighboring epidermal and cortical cells in sos5 roots appear less organized. SOS5 is expressed ubiquitously in all plant organs and tissues, including guard cells in the leaf.     

Synthesis of polymerized thin films for immobilized ligand display in proteomic analysis

We describe a new material for the display of biomolecular ligands for use in proteomic analysis. We report here on the construction of the first functionalized polymerized diacetylene thin films (PDTFs) for use in displaying immobilized ligands and their application in mass spectral proteomic analysis. Functionalized polymerized thin film surfaces were constructed with diacetylene-containing biotin lipid monomers designed for the capture of proteins (streptavidin) from a complex cellular lysate and detection with mass spectrometry (MS). These materials serve as a prototype for ligand-based spotted arrays amenable to high throughput screening. Functionalized PDTFs can be easily manufactured for customized microarrays and demonstrate high protein specificity and low nonspecific protein adsorption, and the resulting microarrays constructed from these materials are compatible with several different protein analysis platforms. Our results suggest that these materials have broad potential applications for use in mass spectral-based proteomic analysis.