Third Dentition Is the Main Cause of Premolar Supernumerary Tooth Formation.

While the prevalence of supernumerary teeth (ST) is high in permanent dentition, the etiology of ST in humans remains unclear. However, multiple murine models of ST have elaborated on dated mechanisms traditionally ascribed to ST etiology: one involves the rescue of rudimental teeth, and the second considers the contribution of odontogenic epithelial stem cells. It remains unclear whether these mechanisms of ST formation in mice are applicable to humans. The third dentition is usually regressed apoptotic-that is, the teeth do not completely form in humans. Recently, it was suggested that ST result from the rescue of regression of the third dentition in humans. The present investigation evaluates the proportion of collected general ST cases that evinced a third dentition based on the clinical definition of ST derived from the third dentition. We also investigated the contribution of SOX2-positive odontogenic epithelial stem cells to ST formation in humans. We collected 215 general ST cases from 15,008 patients. We confirmed that the general characteristics of the collected ST cases were similar to the results from previous reports. Of the 215 cases, we narrowed our analysis to the 78 patients who had received a computed tomography scan. The frequency of ST considered to have been derived from the third dentition was 26 out of 78 cases. Evidence of a third dentition was especially apparent in the premolar region, was more common in men, and was more likely among patients with ≥3 ST. SOX2-positive odontogenic epithelial stem cells within the surrounding epithelial cells of developing ST were observed in non-third dentition cases and not in third dentition cases. In conclusion, the third dentition is the main cause of ST in humans. The odontogenic epithelial stem cells may contribute to ST formation in cases not caused by a third dentition.

Importance of awareness of perioperative social and physical situations of living donors for liver transplantation.

INTRODUCTION: Transplantation in Japan still depends on living donors even after the new revised law. We must pay attention to protect living donors. PATIENTS AND METHODS: Perioperative qualities of life after living donation for liver transplantation were assessed with questionnaires including the Medical Outcomes Study 36-Item Short-Form Health Survey version 2 (SF36-v2). Nonparametric Mann-Whitney tests were used to determine statistical significance. P values<.05 were considered significant. RESULTS: Thirty-one among 33 donors answered questionnaires (93.9%). The 15 men and 16 women of average age of 39.7 years had a median hospital stay of 16 days and median duration after surgery of 78 months. Ten of 33 (35.7%) donors considered themselves to be the only possibility. The decision to a donor was established prior to informed consent in 23 donors (74.1%). Six months were required for them to experience a full recovery after donor surgery. Hamilton depression/anxiety score was significantly increased among donors who considered themselves to be the only possibility or those who had decided prior to informed consent. SF36-v2 revealed a significant decrease in social functioning among donors who did not have sufficient time to decide before surgery. General health was significantly decreased among donors who required more than 6 months for full recovery. Perioperative management of pain influenced general health, physical role, bodily pain, and physical functioning. CONCLUSION: We must pay attention to depression and anxiety among living donors. More care should be focused on pain control and sharing of information of postoperative courses.

Topical application of Aggregatibacter actinomycetemcomitans cytolethal distending toxin induces cell cycle arrest in the rat gingival epithelium in vivo.

BACKGROUND: Aggregatibacter actinomycetemcomitans is one of the etiological pathogens implicated in the onset of periodontal disease. This pathogen produces cytolethal distending toxin (CDT) that acts as a genotoxin to induce cell cycle arrest and cellular distension in cultured cell lines. Therefore, CDT is a possible virulence factor; however, the in vivo activity of CDT on periodontal tissue has not been explored. Here, CDT was topically applied into the rat molar gingival sulcus; and the periodontal tissue was histologically and immunohistochemically examined. MATERIALS AND METHODS: Recombinant purified A. actinomycetemcomitans CDT was applied to gingival sulcus of male Wistar rats and tissue samples were immunohistochemmically examined. RESULTS: One day after application, infiltration of neutrophils and dilation of blood vessels in the gingival connective tissue were found. At day three, desquamation and detachment of cells in the junctional epithelium was observed. This abrasion of junctional epithelium was not observed in rats treated with mutated CDT, in which a His274Ala mutation is present in the CdtB subunit. This indicates the tissue abrasion may be caused by the genotoxicity of CdtB. Expression of the proliferating cell nuclear antigen (PCNA), a marker for proliferating cells, was significantly suppressed using CDT treatment in the junctional epithelium and gingival epithelium. CONCLUSION: Using the rat model, these data suggest CDT intoxication induces cell cycle arrest and damage in periodontal epithelial cells in vivo.

Drug susceptibility and clonality of methicillin-resistant Staphylococcus epidermidis in hospitalized patients with hematological malignancies.

PURPOSE: To evaluate the clonal relatedness and drug susceptibility of Streptococcus epidermidis isolated from hematological patients. METHODS: All S. epidermidis isolated from hematological patients who developed bloodstream infections between June 2005 and December 2007 were included. The clonal relationship was tested by means of pulsed-field gel electrophoresis (PFGE) analysis. RESULTS: Fifteen methicillin-resistant S. epidermidis (MRSE) isolates were examined from patients' blood culture samples. Two subgroups that differed approximately by 40% in their PFGE banding were identified. In clinical practice, two cases were cured with cephalosporin only, thus demonstrating sensitivity of the strains to beta-lactam antibiotics. CONCLUSIONS: Our results represent two significant findings. One is the major capability of MRSE to colonize patients. The other is that some MRSE isolates proved to be sensitive to clindamycin, minocycline, and cephalosporin, so that using antibiotics to which MRSE is sensitive as first-line therapy can avoid the need for vancomycin in clinical settings.

Automutanolysin disrupts clinical isolates of cariogenic streptococci in biofilms and planktonic cells.

INTRODUCTION: Dental caries remains one of the most common chronic infectious diseases throughout the world. The formation of dental plaque is one of the caries risk factors. As a consequence, the removal of plaque may reduce the incidence of caries development. We identified an autolysin produced by Streptococcus mutans named auto-mutanolysin (Aml). Aml selectively lyses S. mutans and Streptococcus sobrinus. The specificity towards these cariogenic bacteria suggests that Aml may be used to prevent dental caries. Here, with the aim towards therapeutic application, we investigated the lytic activity of Aml against clinical isolates of S. mutans and S. sobrinus using planktonic cells and biofilms. METHODS: Planktonic cell suspensions and biofilms of clinically isolated streptococci were treated with Aml in the absence or the presence of Triton X-100. The lytic activity of Aml was monitored as the change in turbidity. The disruption of biofilms was evaluated by detecting the released DNA by polymerase chain reaction and observing the alteration of optical density of treated biofilms. RESULTS: Triton X-100 enhances the lytic ability of Aml. Using planktonic cells, Aml had various lysis levels against clinical strains. Repeated Aml treatment showed disruption of the biofilm using the representative clinical strains. CONCLUSION: Our study demonstrates that Aml has an ability to lyse planktonic and biofilm cells of clinically isolated mutans streptococci in the presence of Triton X-100. These results suggest the possibility of using Aml as an alternative or additional approach for caries prevention.

Living donor liver transplantation for a child with recurrent pediatric adult-type hepatocellular carcinoma.

INTRODUCTION: Pediatric hepatocellular carcinoma (HCC) is an uncommon disease with a poor prognosis. There are few reports about liver transplantation for pediatric adult-type HCC. We experienced a case of living donor liver transplantation (LDLT) for a child with recurrent pediatric adult-type HCC. CASE REPORT: A 12-year-old boy was admitted to the Department of Pediatrics in our institution due to HCC in May 2005. He underwent hepatectomy after 3 courses of chemotherapy in July 2005. After the operation, he had 2 more courses of the same chemotherapy. His posttheraputic course was uneventful for 1 year. However, his alpha-fetoprotein level increased and a computed tomography (CT) scan showed recurrent tumor in his remnant liver in October 2006. He underwent another chemotherapy session immediately. However, CT revealed multiple liver tumors after chemotherapy in December 2006. His mother requested to be an LDLT donor, which was performed on January 23, 2007. The donor operation was a right hepatic lobectomy. The postoperative course of the donor was unremarkable and she has now returned to work. The recipient's posttransplantation course was uneventful and he was discharged at postoperative day 53 and is currently doing well. CONCLUSION: Liver transplantation in conjunction with chemotherapy may have an increasing role in the management of pediatric HCC.

Cross-reactive adaptive immune response to oral commensal bacteria results in an induction of receptor activator of nuclear factor-kappaB ligand (RANKL)-dependent periodontal bone resorption in a mouse model.

INTRODUCTION: The present study examined whether induction of an adaptive immune response to orally colonizing non-pathogenic Pasteurella pneumotropica by immunization with the phylogenetically closely related bacterium, Actinobacillus actinomycetemcomitans, can result in periodontal bone loss in mice. METHODS: BALB/c mice harboring P. pneumotropica (P. pneumotropica(+) mice) in the oral cavity or control P. pneumotropica-free mice were immunized with fixed A. actinomycetemcomitans. The animals were sacrificed on day 30, and the following measurements were carried out: (i) serum immunoglobulin G and gingival T-cell responses to A. actinomycetemcomitans and P. pneumotropica; (ii) periodontal bone loss; and (iii) identification of receptor activator of nuclear factor-kappaB ligand (RANKL) -positive T cells in gingival tissue. RESULTS: Immunization with A. actinomycetemcomitans induced a significantly elevated serum immunoglobulin G response to the 29-kDa A. actinomycetemcomitans outer membrane protein (Omp29), which showed strong cross-reactivity with P. pneumotropica OmpA compared to results in the control non-immunized mice. The A. actinomycetemcomitans-immunized P. pneumotropica(+) mice developed remarkable periodontal bone loss in a RANKL-dependent manner, as determined by the abrogation of bone loss by treatment with osteoprotegerin-Fc. The T cells isolated from the gingival tissue of A. actinomycetemcomitans-immunized P. pneumotropica(+) mice showed an in vitro proliferative response to both A. actinomycetemcomitans and P. pneumotropica antigen presentation, as well as production of soluble(s)RANKL in the culture supernatant. Double-color confocal microscopy demonstrated that the frequency of RANKL(+) T cells in the gingival tissue of A. actinomycetemcomitans-immunized P. pneumotropica(+) mice was remarkably elevated compared to control mice. CONCLUSION: The induction of an adaptive immune response to orally colonizing non-pathogenic P. pneumotropica results in RANKL-dependent periodontal bone loss in mice.

Chromosomal instability by beta-catenin/TCF transcription in APC or beta-catenin mutant cells.

Adenomatous polyposis coli (APC/Apc) gene encodes a key tumor suppressor whose mutations activate beta-catenin/T-cell factor (TCF)-mediated transcription (canonical Wnt signaling). Here, we show that Wnt signaling can cause chromosomal instability (CIN). As an indicator of CIN, we scored anaphase bridge index (ABI) in mouse polyps and ES cells where Wnt signaling was activated by Apc or beta-catenin mutations. We found three to nine times higher ABI than in wild-type controls. Furthermore, karyotype analysis confirmed that the Wnt signal-activated ES cells produced new chromosomal aberrations at higher rates; hence CIN. Consistently, expression of dominant-negative TCFs in these cells reduced their ABI. We also found that Wnt signal activation increased phosphorylation of Cdc2 (Cdk1) that inhibited its activity, and suppressed apoptosis upon exposure of the cells to nocodazole or colcemid. The data suggest that Wnt signaling stimulates the cells to escape from mitotic arrest and apoptosis, resulting in CIN. In human gastric cancer tissues with nuclear beta-catenin, ABI was significantly higher than in those without. These results collectively indicate that beta-catenin/TCF-mediated transcription itself increases CIN through dysregulation of G2/M progression.

Innate immune peptide LL-37 displays distinct expression pattern from beta-defensins in inflamed gingival tissue.

Anti-microbial peptides produced from mucosal epithelium appear to play pivotal roles in the host innate immune defence system in the oral cavity. In particular, human beta-defensins (hBDs) and the cathelicidin-type anti-microbial peptide, LL-37, were reported to kill periodontal disease-associated bacteria. In contrast to well-studied hBDs, little is known about the expression profiles of LL-37 in gingival tissue. In this study, the anti-microbial peptides expressed in gingival tissue were analysed using immunohistochemistry and enxyme-linked immunosorbent assay (ELISA). Immunohistochemistry revealed that neutrophils expressed only LL-37, but not hBD-2 or hBD-3, and that such expression was prominent in the inflammatory lesions when compared to healthy gingivae which showed very few or no LL-37 expressing neutrophils. Gingival epithelial cells (GEC), however, expressed all three examined anti-microbial peptides, irrespective of the presence or absence of inflammation. Moreover, as determined by ELISA, the concentration of LL-37 in the gingival tissue homogenates determined was correlated positively with the depth of the gingival crevice. Stimulation with periodontal bacteria in vitro induced both hBD-2 and LL-37 expressions by GEC, whereas peripheral blood neutrophils produced only LL-37 production, but not hBD-2, in response to the bacterial stimulation. These findings suggest that LL-37 displays distinct expression patterns from those of hBDs in gingival tissue.

Dysregulated expression of P1 and P2 promoter-driven hepatocyte nuclear factor-4alpha in the pathogenesis of human cancer.

Hepatocyte nuclear factor-4alpha (HNF4alpha) exists in multiple isoforms that are generated by alternative promoter (P1 and P2) usage and splicing. Here we establish monoclonal antibodies (mAbs) for detecting P1 and P2 promoter-driven HNF4alpha, and evaluate their expression in normal adult human tissues and surgically resected carcinomas of different origins. Using immunohistochemical analysis, we demonstrate that, while P1 promoter-driven HNF4alpha is expressed in hepatocytes, small intestine, colon, kidney and epididymis, P2 promoter-driven HNF4alpha is expressed in bile duct, pancreas, stomach, small intestine, colon and epididymis. Altered expression patterns of P1 and P2 promoter-driven HNF4alpha were observed in gastric, hepatocellular and colorectal carcinomas. HNF4alpha was expressed in lung metastases from renal cell, hepatocellular and colorectal carcinoma but was not observed in lung tumours. The P1 and P2 promoter-driven HNF4alpha expression pattern of tumour metastases correlated with the primary site of origin. P1 promoter-driven HNF4alpha was also found in intestinal metaplasia of the stomach. These data provide evidence for the tissue distribution of P1 and P2 promoter-driven HNF4alpha at the protein level and suggest that HNF4alpha may be a novel diagnostic marker for metastases of unknown primary. We propose that the dysregulation of alternative promoter usage of HNF4alpha is associated with the pathogenesis of certain cancers.

Polymorphisms in PTCH1 affect the risk of ameloblastoma.

Ameloblastoma is the most common odontogenic tumor, but the genetic nature of the changes in the tumor cells has been unclear. Mutations of CTNNB1 or PTCH1 are observed in many human tumors. Both CTNNB1 and PTCH1 are important in tooth development and are expressed in ameloblastoma. The aim of this study was to investigate whether genetic alterations of CTNNB1 and PTCH1 are present in ameloblastoma. We investigated 14 cases of ameloblastoma. The polymorphisms found in the ameloblastoma patients were further examined in a subsequent case-control study. We found a CTNNB1 mutation in one case of plexiform-type ameloblastoma. CGG triplet repeat-number polymorphism (CGG7/CGG8) in the 5'-untranslated region of PTCH1 was observed. The proportion of CGG8 alleles was significantly higher in the ameloblastoma group. The results of this study indicate a possible relationship between the CGG8 allele in PTCH1 and the risk for ameloblastoma.

Cloning and sequencing of the gene, fmtC, which affects oxacillin resistance in methicillin-resistant Staphylococcus aureus.

Two Tn551 insertional mutants with reduced methicillin resistance were isolated from methicillin-resistant Staphylococcus aureus KSA8. These two mutants showed increased susceptibility to beta-lactam antibiotics and bacitracin, but not to fosfomycin and vancomycin. Tn551 in these mutants was inserted into the same gene, termed fmtC. The fmtC gene has an open reading frame of 840 amino acid residues with an estimated molecular mass of 96.9 kDa. The N-terminal half of the deduced FmtC protein is very hydrophobic, implying that this protein is a membrane-associated protein.

Expression of IL-1 beta and IL-8 by human gingival epithelial cells in response to Actinobacillus actinomycetemcomitans.

The interaction between epithelial cells and microorganisms is the most important step in bacterial infections. Epithelial cells in response to exposure to pathogenic bacteria produce cytokines that initiate inflammation. However, little is known about the cytokine response of gingival epithelial cells to periodontopathogenic bacteria. Actinobacillus actinomycetemcomitans is thought to play a significant role in the initiation of periodontitis because of its bacteriological characteristics. In the present study, we investigated the cytokine induction by human gingival epithelial cells (HGEC) following exposure to A. actinomycetemcomitans in comparison with human gingival fibroblasts (HGF) in culture. Northern blot analysis showed that mRNAs of interleukin 1beta (IL-1beta) and IL-8, but not IL-6, in HGEC were induced in response to A. actinomycetemcomitans. Secretion of IL-8 by HGEC was also increased following A. actinomycetemcomitans challenge, whereas production of IL-1beta could not be detected. The levels of IL-8 and its mRNA were increased depending on the concentration of A. actinomycetemcomitans. The co-culture with HGF and A. actinomycetemcomitans resulted in an increase in the levels of IL-6 and IL-8 mRNA in HGF. However, HGF exposed to A. actinomycetemcomitans, showed no expression of IL-1beta mRNA. These findings demonstrated that HGEC and HGF stimulated with A. actinomycetemcomitans have different profiles in cytokine mRNA expression. Furthermore, A. actinomycetemcomitans may play an important role in amplifying the local immune response and in initiating inflammatory reaction through release of IL-8 from gingival epithelial cells.

An NMR analysis of ubiquitin recognition by yeast ubiquitin hydrolase: evidence for novel substrate recognition by a cysteine protease.

Yeast ubiquitin hydrolase 1 (YUH1), a cysteine protease that catalyzes the removal of ubiquitin C-terminal adducts, is important for the generation of monomeric ubiquitin. Heteronuclear NMR spectroscopy has been utilized to map the YUH1 binding surface on ubiquitin. When YUH1 was titrated into a sample of ubiquitin, approximately 50% of the (1)H-(15)N correlation peaks of ubiquitin were affected to some degree, as a result of binding to YUH1. It is noteworthy that the amide resonances of the basic residues (Arg42, Lys48, Arg72, and Lys74) were highly perturbed. These positively charged basic residues may be involved in direct interactions with the negatively charged acidic residues on YUH1. In addition to the electrostatic surface, the hydrophobic surfaces on ubiquitin (Leu8, Ile44, Phe45, Val70, Leu71, and Leu73) and YUH1 are also likely to contribute to the binding interaction. Furthermore, the amide resonances of Ile13, Leu43, Leu50, and Leu69, the side chains of which are not on the surface, were also highly perturbed, indicating substrate-induced changes in the environments of these residues as well. These large changes, observed from residues located throughout the five-stranded beta-sheet surface and the C-terminus, suggest that substrate recognition by YUH1 involves a wider area on ubiquitin.

Expression of osteoprotegerin (osteoclastogenesis inhibitory factor) in cultures of human dental mesenchymal cells and epithelial cells.

Osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF) inhibits osteoclast differentiation, activity, and survival; therefore OPG/OCIF may regulate the resorption of dental hard tissues, such as alveolar bone, cementum, and dentin. To investigate this issue, reverse transcriptase-polymerase chain reaction using specific primers for OPG/OCIF was performed with total RNAs isolated from human gingival keratinocytes (HGKs), human gingival fibroblasts (HGFs), human periodontal ligament cells (HPDLs), and human pulp cells (HPCs) in culture. PCR products were found in HGFs, HPDLs, and HPCs, but not in HGKs, and the DNA sequence of these products was 100% identical to the reported sequence of the OPG gene. Northern blot analyses also showed that HGFs, HPDLs, and HPCs, but not HGKs, expressed OPG/OCIF transcripts of approximately 2.5 kb. Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) increased OPG/OCIF mRNA levels in a dose-and time-dependent manner in HPDL. After 12 h of treatment, IL-1beta at 3 ng/ml and TNF-alpha at 3 ng/ml increased OPG/OCIF mRNA expression by 190% and 110%, respectively, with a maximal effect. The stimulatory effects of IL-1beta and TNF-alpha were also seen in HPC. However, IL-6 and transforming growth factor-beta had little effect on OPG/OCIF mRNA levels in HPDL. These findings suggest that OPG/OCIF synthesized by dental mesenchymal cells locally regulates the resorption of dental hard tissues through cytokines.

Specific expression of activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in germinal center B cells.

We have identified a novel gene referred to as activation-induced deaminase (AID) by subtraction of cDNAs derived from switch-induced and uninduced murine B lymphoma CH12F3-2 cells, more than 80% of which switch exclusively to IgA upon stimulation. The amino acid sequence encoded by AID cDNA is homologous to that of apolipoprotein B (apoB) mRNA-editing enzyme, catalytic polypeptide 1 (APOBEC-1), a type of cytidine deaminase that constitutes a catalytic subunit for the apoB mRNA-editing complex. In vitro experiments using a glutathione S-transferase AID fusion protein revealed significant cytidine deaminase activity that is blocked by tetrahydrouridine and by zinc chelation. However, AID alone did neither demonstrate activity in C to U editing of apoB mRNA nor bind to AU-rich RNA targets. AID mRNA expression is induced in splenic B cells that were activated in vitro or by immunizations with sheep red blood cells. In situ hybridization of immunized spleen sections revealed the restricted expression of AID mRNA in developing germinal centers in which modulation of immunoglobulin gene information through somatic hypermutation and class switch recombination takes place. Taken together, these findings suggest that AID is a new member of the RNA-editing deaminase family and may play a role in genetic events in the germinal center B cell.

[Clinical efficacy of fosfomycin in combination with sulbactam/cefoperazone in the treatment of severe infections complicated to blood dyscrasia. Working Group of Kanto Combination Therapy for FOM + SBT/CPZ].

In the treatment of severe infections complicated to blood dyscrasia, the efficacy and usefulness of fosfomycin (FOM) in combination with sulbactam (SBT)/cefoperazone (CPZ) were compared between patients receiving FOM in the first followed by SBT/CPZ (Group A) and those receiving both drugs simultaneously (Group B). The following results were obtained. 1. The efficacy rate was 56.3% for Group A and 47.9% for Group B, with no significant difference. 2. The efficacy for patients suspected of the presence of septicemia, the efficacy rate was 57.9% for Group A and 54.3% for Group B, with no significant difference. 3. As for underlying disease, patients with acute myelogenous leukemia were most prevailing. In these patients, the efficacy rate was 57.1% for Group A and 27.3% for Group B, with no statistically significant difference. However, the efficacy rate tended to be higher in Group A. 4. The administration of antibiotics was effective to restore the neutrophil count to 501/microliters or higher in 77.8% and 45.5% of the cases for Groups A and B, respectively, with significantly higher efficacy for Group A. 5. In the safety evaluation a total of 115 cases were included. Side effects and laboratory abnormalities were seen in 3 cases each, but none of them were serious in degree. From these results, it was confirmed that the combination therapy consisting of administration of FOM followed by SBT/CPZ with some interval is effective for severe infections complicated to blood dyscrasia.

Isolation of differentially expressed genes upon immunoglobulin class switching by a subtractive hybridization method using uracil DNA glycosylase.

Immunoglobulin class switch recombination enables B lymphocytes to sequentially express antibodies that have identical specificities but that differ in class and effector function. Although several cis elements required for class switch recombination have been identified, few trans -acting factors which are directly related to class switching have been found. Previously we have developed an efficient in vitro class switching system using a cell line, CH12F3-2. To clarify the molecular mechanism of class switching, we intended to isolate genes induced with interleukin (IL)-4, transforming growth factor (TGF)-beta and CD40L using the in vitro class switching system. For that purpose, an improved method for making subtracted cDNA libraries, using uracil DNA glycosylase, has been developed. This method can overcome a general problem of subtraction, that rare cDNAs are easily lost. This new subtraction method was applied to the CH12F3-2 switching system to isolate genes induced by stimulations with IL-4, TGF-beta and CD40L, and cDNAs encoding deiodinase 1 and SS32, an alternatively spliced form of the muscle LIM protein, were obtained. Their expression patterns in response to various combinations of stimuli and the time courses of the induction supported the usefulness of this method.

Expression and purification of epidermal cell differentiation inhibitor (EDIN) from Bacillus subtilis.

The expression of staphylococcal epidermal cell differentiation inhibitor (EDIN), an ADP-ribosyltransferase targeting the small GTP-binding protein rho p21, was examined using Bacillus subtilis. A recombinant plasmid, containing B. licheniformis alpha-amylase promoter flanking either a beta-glucanase or a B. cereus sphingomyelinase signal sequence, and a DNA fragment corresponding to mature EDIN were constructed and used to transform B. subtilis KN2. Transformants were designated ED7 and ED8, respectively. ED7 extracellularly produced recombinant protein, which was purified to homogeneity through column chromatography using SP-Toyopearl 650 cation-exchange gel and the HA1000 hydroxyapatite HPLC column. ED8 did not grow in broth culture. Biochemical and biological studies of purified protein revealed that ED7 produced a correctly processed recombinant EDIN, indistinguishable from natural EDIN.