The gene encoding an extracellular chitinase from marine Alteromonas sp. strain 0-7 was cloned in Escherichia coli JM109 by using pUC18. The chitinase produced was not secreted into the growth medium but accumulated in the periplasmic space. A chitinase-positive clone of E. coli produced two chitinases with different molecular weights from a single chitinase gene. These proteins showed almost the same enzymatic properties as the native chitinase ofAlteromonas sp. strain 0-7. The N-terminal sequences of the two enzymes were identical. The nucleotide sequence of the 3,394-bp SphI-HindIII fragment that included the chitinase gene was determined. A single open reading frame was found to encode a protein consisting of 820 amino acids with a molecular weight of 87,341. A putative ribosome-binding site, promoter, and signal sequence were identified. The deduced amino acid sequence of the cloned chitinase showed sequence homology with chitinases A (33.4%) and B (15.3%) from Serratia marcescens. Regardless of origin, the enzymes of the two bacteria isolated from marine and terrestrial environments had high homology, suggesting that these organisms evolved from a common ancestor.Chitin is a major component of the exoskeletons of insects and crustaceans, and it is abundantly distributed throughout nature. This polysaccharide is also an important nutrient source of both carbon and nitrogen in the marine environment. Yu et al. (38) pointed out that the oceans would be completely depleted of carbon and nitrogen in a relatively short time if chitin could not be returned to the ecosystem in a biologically usable form. However, marine sediment contains relatively little chitin, despite the production of huge amounts of this insoluble polysaccharide by crustaceans. ZoBell and Rittenberg (39) reported that chitinolytic bacteria, which are abundant and widely distributed in the sea, play an important role in converting insoluble chitin into a biologically usable form. Chitinases (EC 3.2.1.14) are enzymes which degrade chitin, and they have been detected in various microorganisms (3,25,28,30), plants (1, 18), insects (5, 13), and crustaceans (19). Alteromonas sp. strain 0-7 is a gram-negative, flagellated, motile, and aerobic rod-shaped bacterium of marine origin. This strain excretes chitinase into the growth medium in the presence of chitin (32). We have already reported the purification, properties, and partial amino acid sequence of the enzyme from this strain (33). Recently, bacterial chitinase genes from terrestrial and marine bacteria such as Serratia marcescens (6, 10), Bacillus circulans (35, 36), Vibrio harveyi (28), and Vibno vulnificus (37) have been cloned and sequenced. However, the mechanism of hydrolysis, the relationship between structure and function, and the regulatory system involved in enzyme induction are still unclear. In this report, we describe the cloning of a gene coding for chitinase and the purification of the gene products. The nucleotide sequence of the gene was determined, and the deduced amino acid s...

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Low-energy extracorporeal shock wave (LE-ESW) treatment has been shown to accelerate wound repair; however, the mechanisms of treatment remain unclear. In the present study, we addressed the role of endothelial nitric oxide synthase (eNOS). A single LE-ESW treatment accelerated the healing of wounds in diabetic mice caused by the injection of streptozotocin. This accelerated healing was accompanied by the increased expression of eNOS and vascular endothelial growth factor (VEGF) and the generation of new vessels at the wound tissues. These results raised the possibility that eNOS may be involved in the beneficial effects of LE-ESW treatment. To address this possibility, we compared the effects of this treatment between mice with a genetic disruption of eNOS knockout (eNOS-KO mice) and wild-type (WT) control mice. Interestingly, the LE-ESW-induced acceleration of wound closure and the increase in VEGF expression and neovascularization was significantly attenuated in eNOS-KO mice compared with WT mice. Considered collectively, these results showed that eNOS was induced at the wound tissues by LE-ESW treatment and played a critical role in the therapeutic effects of this treatment by accelerating the wound healing by promoting VEGF expression and neovascularization.

Wound healing is promoted by the presence of replicating microorganisms adhering to the wounded tissue, but the precise mechanism is not fully understood. In the present study, using a rat model with full-thickness dermal wounds, we examined the effect of Pseudomonas aeruginosa inoculation on wound healing and the role of neutrophils infiltrating the wound site. Within 3 days, inoculation with this bacterium had accelerated re-epithelialization, epidermal cell proliferation, and neo-vascularization, as well as the local infiltration of neutrophils, which reached a peak at 24 hours. Tumor necrosis factor (TNF)-α was detected in the wound tissues on the mRNA and protein levels within 24 hours. Flow cytometry and immunohistochemical analyses detected higher levels of TNF-α in the infiltrating neutrophils in rats inoculated with P. aeruginosa than in uninoculated rats. Neutropenic rats treated with anti-neutrophil mAb or cyclophosphamide exhibited significant attenuation in re-epithelialization, epidermal cell proliferation, neo-vascularization, and TNF-α synthesis compared with control; administration of TNF-α reversed these attenuations. These wound-healing responses were decelerated in rats treated with anti-TNF-α mAb, as was the infiltration of neutrophils. These results indicate that inoculation with P. aeruginosa promotes wound healing by inducing the infiltration of neutrophils, which play a critical role as a major source of TNF-α.

In the wound healing process, neutrophils are the first inflammatory cells to move to the wound tissues. They sterilize wounds by killing microbes, and they stimulate other immune cells to protect the host from infection. In contrast, neutrophil-derived proteases cause damage to host tissues, so neutrophils play dual opposite roles in wound healing. Interleukin-17A (IL-17A) is a proinflammatory cytokine that promotes the recruitment of these cells. The role of this cytokine in the wound healing process is not fully clarified. In the present study, therefore, we examined how defect in IL-17A production affected the wound healing in skin. IL-17A-knockout (KO) mice showed promoted wound closure, myofibroblast differentiation and collagen deposition and decreased the neutrophil accumulation compared with wild-type (WT) mice. In contrast, the administration of recombinant IL-17A led to delayed wound closure, low collagen deposition and accelerated neutrophilic accumulation. In addition, the treatment of IL-17A-administered mice with a neutrophil elastase inhibitor improved the wound repair to the same level as that of WT mice. These results indicated that IL-17A hampered the wound healing process and suggested that neutrophilic inflammation caused by IL-17A may be associated with impaired wound healing in skin.