A novel mechanism in maggot debridement therapy: protease in excretion/secretion promotes hepatocyte growth factor production

Maggot debridement therapy (MDT) is effective for treating intractable wounds, but its precise molecular mechanism, including the association between MDT and growth factors, remains unknown. We administered MDT to nine patients (66.3 ± 11.8 yr, 5 male and 4 female) with intractable wounds of lower extremities because they did not respond to conventional therapies. Significant increases of hepatocyte growth factor (HGF) levels were observed in femoral vein blood during 48 h of MDT (P < 0.05), but no significant change was found for vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor-β1 (TGF-β1), or tumor necrosis factor-α (TNF-α). We conducted NIH-3T3 cell stimulation assay to evaluate the relation between HGF and protease activity in excretion/secretion (ES) derived from maggots. Compared with the control group, HGF was significantly higher in the 0.05 μg/ml ES group (P < 0.01). Furthermore, protease inhibitors suppressed the increase of HGF (P < 0.05). The HGF expression was increased in proportion to the ES protein concentration of 0.025 to 0.5 μg/ml. In fact, ES showed stronger capability of promoting HGF production and less cytotoxicity than chymotrypsin or bromelain. HGF is an important factor involved in cutaneous wound healing. Therefore, these results suggest that formation of healthy granulation tissue observed during MDT results from the increased HGF. Further investigation to identify molecules enhancing HGF expression by MDT will contribute greatly to drug target discovery for intractable wound healing therapy.     

Bacterial cysteine desulfurases: versatile key players in biosynthetic pathways of sulfur-containing biofactors

Cysteine desulfurases are pyridoxal 5′-phosphate-dependent homodimeric enzymes that catalyze the conversion of L-cysteine to L-alanine and sulfane sulfur via the formation of a protein-bound cysteine persulfide intermediate on a conserved cysteine residue. The enzymes are capable of donating the persulfide sulfur atoms to a variety of biosynthetic pathways for sulfur-containing biofactors, such as iron–sulfur clusters, thiamin, transfer RNA thionucleosides, biotin, and lipoic acid. The enormous advances in biochemical and structural studies of these biosynthetic pathways over the past decades provide an opportunity for detailed understanding of the nature of the excellent sulfur transfer mechanism of cysteine desulfurases.     

Physiological stress exacerbates murine colitis by enhancing proinflammatory cytokine expression that is dependent on IL-18

Psychological stress is an environmental factor considered to be a precipitating factor of inflammatory bowel disease. Interleukin (IL)-18 plays a role in stress-induced aggravation in some diseases. The aim of this study was to establish a model of murine colitis exacerbated by psychological stress and to clarify the role of IL-18 in this model. Male C57Bl/6 mice and IL-18(-/-) mice were used for this study. The mice received dextran sulfate sodium (DSS) for induction of colitis. Some mice were exposed to psychological stress using a communication box. Body weight, colonic length, and histological inflammation were measured for assessment of colitis. Tumor necrosis factor (TNF)-α and IL-18 expression in the colon and IL-18 expression in the adrenal gland were analyzed using real-time PCR. The effect of anti-IL-18 antibody was also investigated. Effects of TNF-α and IL-18 on cytokine expressions were studied using the colonic epithelial cell line LS174T. Induction of psychological stress in DSS-treated wild-type mice significantly exacerbated colitis with enhanced expression of proinflammatory cytokines and IL-18. However, induction of psychological stress in DSS-treated IL-18(-/-) mice did not aggravate colitis compared with that in the IL-18(-/-) group given only DSS treatment. Stress-induced aggravation of colitis was ameliorated significantly by anti-IL-18 antibody treatment. IL-18 did not enhance TNF-α-induced expression of intercellular adhesion molecule-1 or IL-8 in LS174T. We established a model of colitis exacerbated by psychological stress. Psychological stress enhanced IL-18 expression and plays a proinflammatory role in stress-induced aggravation of colitis.     

Fission yeast Pot1 and RecQ helicase are required for efficient chromosome segregation

Pot1 is a single-stranded telomere-binding protein that is conserved from fission yeast to mammals. Deletion of Schizosaccharomyces pombe pot1(+) causes immediate telomere loss. S. pombe Rqh1 is a homolog of the human RecQ helicase WRN, which plays essential roles in the maintenance of genomic stability. Here, we demonstrate that a pot1Δ rqh1-hd (helicase-dead) double mutant maintains telomeres that are dependent on Rad51-mediated homologous recombination. Interestingly, the pot1Δ rqh1-hd double mutant displays a "cut" (cell untimely torn) phenotype and is sensitive to the antimicrotubule drug thiabendazole (TBZ). Moreover, the chromosome ends of the double mutant do not enter the pulsed-field electrophoresis gel. These results suggest that the entangled chromosome ends in the pot1Δ rqh1-hd double mutant inhibit chromosome segregation, signifying that Pot1 and Rqh1 are required for efficient chromosome segregation. We also found that POT1 knockdown, WRN-deficient human cells are sensitive to the antimicrotubule drug vinblastine, implying that some of the functions of S. pombe Pot1 and Rqh1 may be conserved in their respective human counterparts POT1 and WRN.     

Triceps surae muscle-tendon unit length changes as a function of ankle joint angles and contraction levels: the effect of foot arch deformation

The purpose of this study was to clarify how foot deformation affects the relationship between triceps surae muscle-tendon unit (MTU) length and ankle joint angle. For six women and six men a series of sagittal magnetic resonance (MR) images of the right foot were taken, and changes in MTU length (the displacement of the calcaneal tuberosity), foot arch angle, and ankle joint angle were measured. In the passive session, each subject's ankle joint was secured at 10° dorsiflexed position, neutral position (NP), and 10° and 20° plantar flexed positions while MR images were acquired. In the active session, each subject was requested to perform submaximal isometric plantar flexions (30%, 60%, and 80% of voluntary maximum) at NP. The changes in MTU length in each trial were estimated by two different formulae reported previously. The changes of the measured MTU length as a function of ankle joint angles observed in all trials of the active session were significantly (p<0.05) larger than corresponding values in the passive session and by the estimation formulae. In the passive session, MTU length changes were significantly smaller than the estimated values when the ankle was plantar flexed. The foot arch angle increased as the contraction level increased from rest (117 ± 4°) to 80% (125 ± 3°), and decreased as the ankle was positioned further into plantar flexion in the passive session (115 ± 3°). These results indicate that foot deformation profoundly affects the triceps surae MTU length-ankle joint angle relationship during plantar flexion.     

Evaluation of the protein interfaces that form an intermolecular four-helix bundle as studied by computer simulation

Rational design of protein surface is important for creating higher order protein structures, but it is still challenging. In this study, we designed in silico the several binding interfaces on protein surfaces that allow a de novo protein–protein interaction to be formed. We used a computer simulation technique to find appropriate amino acid arrangements for the binding interface. The protein–protein interaction can be made by forming an intermolecular four-helix bundle structure, which is often found in naturally occurring protein subunit interfaces. As a model protein, we used a helical protein, YciF. Molecular dynamics simulation showed that a new protein–protein interaction is formed depending on the number of hydrophobic and charged amino acid residues present in the binding surfaces. However, too many hydrophobic amino acid residues present in the interface negatively affected on the binding. Finally, we found an appropriate arrangement of hydrophobic and charged amino acid residues that induces a protein–protein interaction through an intermolecular four-helix bundle formation.