Identifying specific matrix metalloproteinase-2-inhibiting peptides through phage display-based subtractive screening

Gelatinases A and B, which are members of the matrix metalloproteinase (MMP) family, play essential roles in cancer development and metastasis, as they can break down basal membranes. Therefore, the determination and inhibition of gelatinases is essential for cancer treatment. Peptides that can specifically block each gelatinase may, therefore, be useful for cancer treatment. In this study, subtractive panning was carried out using a 12-mer peptide library to identify peptides that block gelatinase A activity (MMP-2), which is a key pharmacological target. Using this method, 17 unique peptide sequences were determined. MMP-2 inhibition by these peptides was evaluated through zymogram analyses, which revealed that four peptides inhibited MMP-2 activity by at least 65%. These four peptides were synthesized and used for in vitro wound healing using human umbilical vein endothelial cells, and two peptides, AOMP12 and AOMP29, were found to inhibit wound healing by 40%. These peptides are, thus, potential candidates for MMP-2 inhibition for cancer treatment. Furthermore, our findings suggest that our substractive biopanning screening method is a suitable strategy for identifying peptides that selectively inhibit MMP-2.     

The properties of bioengineered chondrocyte sheets for cartilage regeneration

Background  

Although the clinical results of autologous chondrocyte implantation for articular cartilage defects have recently improved as a result of advanced techniques based on tissue engineering procedures, problems with cell handling and scaffold imperfections remain to be solved. A new cell-sheet technique has been developed, and is potentially able to overcome these obstacles. Chondrocyte sheets applicable to cartilage regeneration can be prepared with this cell-sheet technique using temperature-responsive culture dishes. However, for clinical application, it is necessary to evaluate the characteristics of the cells in these sheets and to identify their similarities to naive cartilage.     

FolX and FolM Are Essential for Tetrahydromonapterin Synthesis in Escherichia coli and Pseudomonas aeruginosa

Tetrahydromonapterin is a major pterin in Escherichia coli and is hypothesized to be the cofactor for phenylalanine hydroxylase (PhhA) in Pseudomonas aeruginosa, but neither its biosynthetic origin nor its cofactor role has been clearly demonstrated. A comparative genomics analysis implicated the enigmatic folX and folM genes in tetrahydromonapterin synthesis via their phyletic distribution and chromosomal clustering patterns. folX encodes dihydroneopterin triphosphate epimerase, which interconverts dihydroneopterin triphosphate and dihydromonapterin triphosphate. folM encodes an unusual short-chain dehydrogenase/reductase known to have dihydrofolate and dihydrobiopterin reductase activity. The roles of FolX and FolM were tested experimentally first in E. coli, which lacks PhhA and in which the expression of P. aeruginosa PhhA plus the recycling enzyme pterin 4a-carbinolamine dehydratase, PhhB, rescues tyrosine auxotrophy. This rescue was abrogated by deleting folX or folM and restored by expressing the deleted gene from a plasmid. The folX deletion selectively eliminated tetrahydromonapterin production, which far exceeded folate production. Purified FolM showed high, NADPH-dependent dihydromonapterin reductase activity. These results were substantiated in P. aeruginosa by deleting tyrA (making PhhA the sole source of tyrosine) and folX. The ΔtyrA strain was, as expected, prototrophic for tyrosine, whereas the ΔtyrA ΔfolX strain was auxotrophic. As in E. coli, the folX deletant lacked tetrahydromonapterin. Collectively, these data establish that tetrahydromonapterin formation requires both FolX and FolM, that tetrahydromonapterin is the physiological cofactor for PhhA, and that tetrahydromonapterin can outrank folate as an end product of pterin biosynthesis.Pterins contain the bicyclic pteridine ring with an amino group in the 2-position and an oxo group in the 4-position; they can be reduced through the dihydro forms to the tetrahydro forms, which are active as cofactors (Fig. ​(Fig.1A).1A). Tetrahydropterins are known to be the cofactors for phenylalanine hydroxylases from Pseudomonas and Chromatium species as well as for mammalian aromatic amino acid hydroxylases and other mammalian enzymes (13, 17, 38, 41) (Fig. ​(Fig.1B).1B). Although the identity of the mammalian tetrahydropterin cofactor, tetrahydrobiopterin (H₄-BPt), is firmly established (38), the same is not true for bacteria, and the biosynthesis of bacterial tetrahydropterins is not well understood.Open in a separate windowFIG. 1.Tetrahydropterin structure, cofactor role, and biosynthesis. (A) The pterin nucleus, its levels of reduction, and the structures of compounds relevant to this study. (B) The requirement for a tetrahydropterin (H₄-pterin) cofactor for phenylalanine hydroxylase (PAH) and the cofactor regeneration cycle involving pterin-4a-carbinolamine dehydratase (PCD) and quinonoid dihydropterin (q-H₂-pterin) reductase (q-DHPR; EC 1.5.1.34). (C) The established steps in tetrahydrobiopterin (H₄-BPt) biosynthesis and possible routes for tetrahydromonapterin (H₄-MPt) biosynthesis in relation to the folate pathway. H₄-BPt is formed by the sequential action of 6-pyruvoyltetrahydropterin (P-H₄-Pt) synthase (PTPS-II) and sepiapterin reductase (SR). H₄-MPt could originate via the FolX-catalyzed epimerization of dihydroneopterin triphosphate (H₂-NPt-P₃) to dihydromonapterin triphosphate (H₂-MPt-P₃), followed by dephosphorylation to dihydromonapterin (H₂-MPt) and reduction by a dihydropterin reductase (EC 1.5.1.33), putatively FolM. H₄-MPt also could come from the FolB-mediated epimerization of dihydroneopterin (H₂-NPt) followed by reduction. FolB also mediates the side chain cleavage of H₂-NPt or H₂-MPt to give 6-hydroxymethyldihydropterin (H₂-HMPt); the H₂-MPt cleavage is omitted for simplicity. Other abbreviations: P-ase, phosphatase; H₂-HMPt-P₂, 6-hydroxymethyldihydropterin diphosphate; pABA, p-aminobenzoate; H₂-pteroate, dihydropteroate; H₂-folate, dihydrofolate; H₄-folate, tetrahydrofolate.While a few bacterial taxa, such as Cyanobacteria and Chlorobia, produce H₄-BPt, most do not, as judged directly from pterin analysis and indirectly from the rarity of H₄-BPt biosynthesis genes 6-pyruvoyltetrahydropterin synthase II (PTPS-II) and sepiapterin reductase (SR) (Fig. ​(Fig.1C)1C) among sequenced genomes (12, 25). As bacteria lacking H₄-BPt include Pseudomonas and many others with phenylalanine hydroxylase genes, it is clear that bacterial phenylalanine hydroxylases generally must use a cofactor other than H₄-BPt. The most prominent candidate is tetrahydromonapterin (H₄-MPt), which occurs in Escherichia coli (21) and almost certainly also in Pseudomonas species (11, 17). H₄-MPt could be derived from the dihydropterin intermediates of folate biosynthesis via two different routes (Fig. ​(Fig.1C).1C). These are (i) the conversion of dihydroneopterin triphosphate (H₂-NPt-P₃) to dihydromonapterin triphosphate (H₂-MPt-P₃) by H₂-NPt-P₃ epimerase (FolX) followed by dephosphorylation and reduction to the tetrahydro level, and (ii) the conversion of dihydroneopterin (H₂-NPt) to dihydromonapterin (H₂-MPt) by the epimerase action of dihydroneopterin aldolase (FolB) and then reduction. FolB is a fairly well-understood enzyme of folate synthesis (9), but FolX has no known biological role and a folX deletant has no obvious phenotype (19). folX genes apparently are confined to Gammaproteobacteria (9).Although the epimerase activities of FolX and FolB have been demonstrated amply in vitro (1, 5, 19), no genetic evidence links either enzyme to H₄-MPt formation in vivo. The situation with the reduction of H₂-MPt to H₄-MPt is even less clear, because this activity has not been investigated experimentally. A candidate enzyme for this step nevertheless can be proposed on bioinformatic grounds: the somewhat mysterious FolM protein (9). FolM belongs to a subset of the short-chain dehydrogenase/reductase (SDR) family having the characteristic motif TGX₃RXG (in place of TGX₃GXG, which typifies other SDRs). The archetype of this subset is Leishmania pteridine reductase 1 (PTR1), which reduces various dihydropterins to the tetrahydro state (15). E. coli FolM has low dihydrofolate (H₂-folate) and dihydrobiopterin (H₂-BPt) reductase activities in vitro (14), but neither of these is likely to be its physiological function, since H₂-folate reduction normally is mediated by FolA and E. coli lacks H₄-BPt. folM genes occur in many Gram-negative organisms, including Chlamdiae, Chloroflexi, Cyanobacteria, Acidobacteria, Planctomycetes, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria (9).We report here comparative genomic and genetic evidence that FolX and FolM are required for H₄-MPt synthesis in E. coli and P. aeruginosa, the bacteria in which H₄-MPt has been most studied, and biochemical evidence that FolM has high H₂-MPt reductase activity. We also point out gaps in the understanding of pterin metabolism that our data bring sharply into focus.     

Reproductive isolating barriers between colour‐differentiated populations of an African annual killifish,Nothobranchius korthausae (Cyprinodontiformes)

Allopatric populations separated by vicariance events are expected to evolve reproductive isolating mechanisms as a result of disparate selection pressures and genetic drift. The appearance of reproductive isolating mechanisms may vary across taxa with differences in the opportunity for mate choice, and may be asymmetrical. In addition, premating barriers may be affected by individual mating experience. We used choice and no‐choice experiments to investigate reproductive isolation between two allopatric (island and mainland) and colour‐differentiated populations of an African annual fish, Nothobranchius korthausae. Assortative mating under experimental conditions was limited and asymmetrical. Preference for sympatric males was only expressed in nonvirgin females from one population. Virgin fish from both populations mated indiscriminately. No difference in the number of eggs laid, fertilization rate and hatching success was detected in no‐choice experiments. All mating combinations produced viable offspring and no postmating barriers were detected in terms of the performance and fertility of F1 hybrids. Overall, we found little evidence for significant reproductive isolation, which is in contrast with the related killifish taxa in which assortative mating can be strong, even among allopatric populations with no colour differentiation. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 62–72.     

Effect of the lipophilic/hydrophilic character of cationic triarylmethane dyes on their selective phototoxicity toward tumor cells

The observation that enhanced mitochondrial transmembrane potential is a prevalent tumor cell phenotype has provided the conceptual basis for the development of mitochondrial targeting as a novel therapeutic strategy for both chemo- and photochemotherapy of neoplastic diseases. Because the plasma transmembrane potential is negative on the inner side of the cell and the mitochondrial transmembrane potential is negative on the inner side of this organelle, extensively conjugated cationic molecules (dyes) displaying appropriate structural features are driven electrophoretically through these membranes and tend to accumulate inside energized mitochondria. As a result of the higher mitochondrial transmembrane potential typical of tumor cells, a number of cationic dyes preferentially accrue and are retained for longer periods in the mitochondria of these cells compared to normal cells. This differential in both drug loading and retention brings about the opportunity to attack and destroy tumor cells with a high degree of selectivity. Only a small subset of the cationic dyes known to accumulate in energized mitochondria mediate the destruction of tumor cells with a high degree of selectivity, and the lack of a reliable model to describe the structural determinants of this tumor specificity has prevented mitochondrial targeting from becoming a more reliable therapeutic strategy. We describe here a systematic study of how the molecular structure of closely related cationic triarylmethanes affects the selectivity with which these dyes mediate the photochemical destruction of tumor cells. Based on our observations of how the lipophilic/hydrophilic character of these dyes affects tumor selectivity, we propose a simple model to assist in the design of new drugs tailored specifically for imaging and selective destruction of neoplastic tissue via mitochondrial targeting.     

Experimental evolution of a facultative thermophile from a mesophilic ancestor

Experimental evolution via continuous culture is a powerful approach to the alteration of complex phenotypes, such as optimal/maximal growth temperatures. The benefit of this approach is that phenotypic selection is tied to growth rate, allowing the production of optimized strains. Herein, we demonstrate the use of a recently described long-term culture apparatus called the Evolugator for the generation of a thermophilic descendant from a mesophilic ancestor (Escherichia coli MG1655). In addition, we used whole-genome sequencing of sequentially isolated strains throughout the thermal adaptation process to characterize the evolutionary history of the resultant genotype, identifying 31 genetic alterations that may contribute to thermotolerance, although some of these mutations may be adaptive for off-target environmental parameters, such as rich medium. We undertook preliminary phenotypic analysis of mutations identified in the glpF and fabA genes. Deletion of glpF in a mesophilic wild-type background conferred significantly improved growth rates in the 43-to-48°C temperature range and altered optimal growth temperature from 37°C to 43°C. In addition, transforming our evolved thermotolerant strain (EVG1064) with a wild-type allele of glpF reduced fitness at high temperatures. On the other hand, the mutation in fabA predictably increased the degree of saturation in membrane lipids, which is a known adaptation to elevated temperature. However, transforming EVG1064 with a wild-type fabA allele had only modest effects on fitness at intermediate temperatures. The Evolugator is fully automated and demonstrates the potential to accelerate the selection for complex traits by experimental evolution and significantly decrease development time for new industrial strains.