First Determination of the Level Structure of an sd-Shell Hypernucleus, _{Λ}

We report on the first observation of γ rays emitted from an sd-shell hypernucleus, _{Λ}^{19}F. The energy spacing between the ground state doublet, 1/2^{+} and 3/2^{+} states, of _{Λ}^{19}F is determined to be 315.5±0.4(stat)_{-0.5}^{+0.6}(syst) keV by measuring the γ-ray energy of the M1(3/2^{+}→1/2^{+}) transition. In addition, three γ-ray peaks are observed and assigned as E2(5/2^{+}→1/2^{+}), E1(1/2^{-}→1/2^{+}), and E1(1/2^{-}→3/2^{+}) transitions. The excitation energies of the 5/2^{+} and 1/2^{-} states are determined to be 895.2±0.3(stat)±0.5(syst) and 1265.6±1.2(stat)_{-0.5}^{+0.7}(syst) keV, respectively. It is found that the ground state doublet spacing is well described by theoretical models based on existing s- and p-shell hypernuclear data.

Visualization of viscoelastic behavior in skin equivalent using optical coherence tomography-based straingraphy.

BACKGROUND/PURPOSE: The relationships between the skin components and these mechanical roles are still unclear. To clarify these relationships, we investigated spatial mapping of the mechanical behavior of cultured skin equivalents (SEs) using optical coherence tomography (OCT)-based straingraphy. METHODS: We built a strain relaxation test system combined with OCT and developed an algorithm that could visualize a time-dependent strain distribution, named dynamic-optical coherence straingraphy (D-OCSA). Using this system, we analyzed how the spatial mechanical changes in the SEs depended on the culture duration. For quantitative analysis of viscoelastic behavior, we defined a relaxation attenuation coefficient of strain rate, which indicates the ratio of viscosity and elasticity in the Klevin-Voight model. RESULTS: By culturing for 4 days in comparison to culturing for 1 day, the strain relaxation attenuation coefficient of the whole skin, especially at the region of the dermal-epidermal junction (DEJ), significantly increased in the negative direction. In tissue slices taken for microscopy, several cracks were observed in the SEs cultured for 4 days. CONCLUSION: This study is the first to provide quantified evidence that the DEJ is a dynamically specialized region. An OCT-based straingraphy system (D-OCSA) would be beneficial for evaluating the quality of SEs, as well as functional analysis of their mechanics.

Analysis of the influence of collagen fibres in the dermis on skin optical reflectance by Monte Carlo simulation in a nine-layered skin model.

BACKGROUND: Collagen fibres in the dermis play an important structural role in the skin. Age-related changes to these fibres cause wrinkles and slackness of facial skin. However, it is not clear how dermal collagen fibres affect skin colour. The purpose of this study was to clarify the influence of altered collagen fibres on skin colour, using both experimental measurement of fibre density and Monte Carlo simulations in an optical model of skin. METHODS: Reflection spectra were measured from the cheeks of 12 Japanese women (22-65 years old) by spectral colorimeter. Two-dimensional autocorrelation functions were calculated from second harmonics generation (SHG) images acquired from the same locations and used to calculate collagen density indices. Monte Carlo simulations of light reflectance by skin were performed using a nine-layered model that precisely imitates skin structure. The relationship between dermal collagen fibre density and skin reflection spectra was analysed. RESULTS: A positive correlation was found between collagen density and skin brightness, as measured by the colour value, L* (using the L*a*b* colour space). In addition, collagen density showed a strong inverse correlation with age and with the optical absorption of dermis. The Monte Carlo simulations showed that the reflection spectrum of skin changes when the scattering coefficient of the dermis is altered. These changes were the same for simulated and experimentally measured reflection spectra. CONCLUSION: When collagen fibre density in the upper dermis is decreased with age, skin colour becomes less bright because light scattering in the skin is decreased.

Comparison of Residual Dimethyl Sulfoxide (DMSO) and Ethylene Glycol (EG) Concentration in Bovine Ovarian Tissue During Warming Steps Between Slow Freezing and Vitrification.

BACKGROUND: DMSO and EG have been used as cryoprotectants for human ovarian tissue cryopreservation, but residual cryoprotectants concentration and safety have rarely been reported. OBJECTIVE: We aimed to compare residual cryoprotectants (DMSO, EG) concentration in bovine ovarian tissue during warming steps between one kind of common slow freezing method and two kinds of vitrification methods, which are usually used for cryopreservation of human ovarian tissue in Japan. MATERIALS AND METHODS: In this study, we used five bovine ovaries with an average age of 24.2 months divided into three kinds of cryopreservation methods. All ovarian cortices cut to 1 mm thickness were cryopreserved in slow freezing and two kinds of vitrification methods. Residual cryoprotectants before, during and after warming of cryopreserved ovarian cortices were measured using GC-MS and compared. RESULTS: Concentrations of residual cryoprotectants in the ovarian tissue just before transplantation into the body after warming were high after both vitrification methods but almost zero with the slow freezing method. CONCLUSION: We are concerned about the residual cryoprotectants in ovarian tissue, and continue to study the safety of cryopreservation methods to the woman after reimplantation and her baby.

Response to Lefebvre et al.

Congenital scoliosis (CS) is a common vertebral malformation with incidence of up to 1 of 1000 births worldwide. Recently, TBX6 has been reported as the first disease gene for CS: about 10% of CS patients are compound heterozygotes of rare null mutations and a common haplotype composed by 3 SNPs in TBX6. Lefebvre et al in this journal reported that 2 patients with spondylocostal dysostosis (SCD), a rare skeletal dysplasia affecting spine and ribs also have TBX6 mutations: 1 carried the microdeletion and a rare missense variant, and another 2 rare missense variants. We investigated the pathogenicity of the 3 missense variants in SCD by a luciferase assay. The results were negative for the proposal of Lefebvre et al. We consider these 2 SCD patients are more probably compound heterozygotes of null mutations and a common risk haplotype just as CS patients with TBX6 mutations.

A transportation network for human ovarian tissue is indispensable to success for fertility preservation.

PURPOSE: The purpose of this study was to examine the efficacy of an ovarian tissue transportation network for fertility preservation (FP) for cancer patients in Japan. METHODS: PubMed was searched for papers on transportation of human ovarian tissue for FP. We analyzed population, area, number of cancer patients for ovarian tissue cryopreservation (OTC), quality control/assessment and safety, cost of a cryopreservation center for the building for 30 years, and medical fees of cancer patients (operation, cryopreservation, and storage of ovarian tissue). RESULTS: More than twenty babies have been born in Denmark and Germany through a transportation system. Up to 400 new patients a year need OTC. The fees for removal, cryopreservation, and storage for 5 years, and transplantation of ovarian tissue are around €5,000, €4,000, and €5,000, respectively. It costs more than €5 million to establish and maintain one cryopreservation center for 30 years. If we have a few cryopreservation centers in Japan, we can cryopreserve 400 patients' ovarian tissue per year by safer slow freezing and maintain quality control/assessment. We need to lighten the patients' burden for easy to use FP by a government subsidy and medical insurance coverage. CONCLUSIONS: This model has been termed the Danish model ("the woman stays - the tissue moves"). This is truly patient-centered medicine. We can have maximum effects with the minimum burden. A transportation network like those of Denmark and Germany is the best strategy for FP in Japan. It may be the best system for cancer patients, medical staff, and the Ministry of Health, Labor, and Welfare.

Inhibition of choroidal fibrovascular membrane formation by new class of RNA interference therapeutic agent targeting periostin.

Age-related macular degeneration (AMD) is a vision-threatening disease characterized by choroidal fibrovascular membrane (FVM) formation, choroidal neovascularization (CNV) and choroidal fibrosis. No safe and effective therapeutic method has been developed for the choroidal fibrosis, although anti-vascular endothelial growth factor therapy can partially shrink the CNV. We recently reported that periostin (POSTN), which is produced by retinal pigment epithelial cells, has an important role in the formation of preretinal FVMs, but its role in choroidal FVMs has not been determined. In this study, we used Postn knockout mice to investigate the role played by POSTN in choroidal FVM formation. In addition, we used a new class of RNA interference (RNAi) agent (NK0144) that targets POSTN and determined its effect on choroidal FVM development. Genetic ablation of Postn had an inhibitory effect not only on CNV formation but also on choroidal fibrosis in a mouse CNV model. NK0144 also had a greater inhibitory effect on both the CNV and choroidal fibrosis than control RNAi with no apparent adverse effects. These findings suggest a causal relationship between POSTN and choroidal FVM formation, and also a potential therapeutic role of intravitreal NK0144 for AMD.

Observation of Spin-Dependent Charge Symmetry Breaking in ΛN Interaction: Gamma-Ray Spectroscopy of _{Λ}

The energy spacing between the spin-doublet bound state of _{Λ}^{4}He(1^{+},0^{+}) was determined to be 1406±2±2 keV, by measuring γ rays for the 1^{+}→0^{+} transition with a high efficiency germanium detector array in coincidence with the ^{4}He(K^{-},π^{-})_{Λ}^{4}He reaction at J-PARC. In comparison to the corresponding energy spacing in the mirror hypernucleus _{Λ}^{4}H, the present result clearly indicates the existence of charge symmetry breaking (CSB) in ΛN interaction. By combining the energy spacings with the known ground-state binding energies, it is also found that the CSB effect is large in the 0^{+} ground state but is vanishingly small in the 1^{+} excited state, demonstrating that the ΛN CSB interaction has spin dependence.

Treatment of pressure ulcers in patients with declining renal function using arginine, glutamine and ß-hydroxy-ß-methylbutyrate.

The aim of this study is to examine the efficacy on healing pressure ulcers (PU) of using a supplement combination containing arginine, glutamine and ß-hydroxy-ß-methylbutyrate, which was given to two elderly patients with renal dysfunction. The PU was surgically opened, decompressed and treated by drugs. A half quantity of the defined dose of the supplement combination, with an enteral nutrition product, was administered to the patients twice a day. This combination improved the PUs, with no effect on renal function. This novel finding may provide a nutritional rationale of arginine, glutamine and ß-hydroxy-ß-methylbutyrate for PUs associated with renal dysfunction.

Living donor liver transplantation using a right liver graft with additional vein reconstructions for patient with situs inversus.

Living donor liver transplantation (LDLT) using a right liver graft with additional vein reconstructions has not been previously reported in a situs inversus (SI) patient. A 60-year-old man with SI was referred for LDLT for end-stage cirrhosis secondary to hepatitis B. The calculated regional volumes of the individual hepatic vein territories in the right liver graft suggested that the middle hepatic vein (MHV) tributaries and the inferior right hepatic veins (IRHVs) should be reconstructed in addition to the right hepatic vein (RHV). On the back-table, the recipient's recanalized umbilical vein graft was anastomosed to the V5 opening, and the other side of vein graft was anastomosed to the RHV and V8 opening to create a large single orifice. After total hepatectomy, the right liver graft was placed in the left subphrenic space at the reversed position. The common orifice of hepatic venous drainage from RHV, V8 and V5 was anastomosed to the anatomical RHV conduit of the recipient, followed by IRHV anastomosis to the inferior vena cava. Postoperative course was almost uneventful, and no vascular complications were experienced. Even for SI patients, LDLT using a right liver graft with reconstructions of the MHV tributaries and the IRHVs is feasible.

Hepatic arterial complications in adult living donor liver transplant recipients: a single-center experience of 673 cases.

BACKGROUND: Hepatic arterial reconstruction during living donor liver transplantation (LDLT) is a very delicate and technically complicated procedure. Post-LDLT hepatic arterial complications are associated with significant morbidity and mortality. METHODS: We retrospectively analyzed the details of post-operative hepatic arterial complications in 673 consecutive adult LDLT recipients between January 1996 and September 2009. RESULTS: Hepatic arterial complications occurred in 43 of 673 adult recipients (6.4%) within a median of 13 post-transplant days (range, 1-63). These included hepatic artery thrombosis (including anastomotic stenosis) in 33 cases, anastomotic bleeding in seven cases, and rupture of anastomotic aneurysm in three cases. To treat these complications, surgical re-anastomosis was performed in 26 cases, while the other 17 cases underwent conservative therapies, including four angioplasties by interventional radiology. Biliary complications after hepatic arterial complications occurred in 17 cases. The overall survival rate after LDLT was significantly lower in the hepatic arterial complication group compared with that in the non-complication group (60.7% vs. 80.1% at one yr, 44.3% vs. 74.2% at five yr, respectively; p < 0.001). Multivariate analysis showed that the extra-anatomical anastomosis (p = 0.011) was the only independent risk factor for hepatic arterial complications. CONCLUSION: Because hepatic arterial complications after LDLT are associated with poor patient survival, early diagnosis and immediate treatment are crucial. The anatomical anastomosis may be the first choice for the hepatic arterial reconstruction to the extent possible.

Impact of sarcopenia on survival in patients undergoing living donor liver transplantation.

Skeletal muscle depletion, referred to as sarcopenia, predicts morbidity and mortality in patients undergoing digestive surgery. However, the impact on liver transplantation is unclear. The present study investigated the impact of sarcopenia on patients undergoing living donor liver transplantation (LDLT). Sarcopenia was assessed by a body composition analyzer in 124 adult patients undergoing LDLT between February 2008 and April 2012. The correlation of sarcopenia with other patient factors and the impact of sarcopenia on survival after LDLT were analyzed. The median ratio of preoperative skeletal muscle mass was 92% (range, 67-130%) of the standard mass. Preoperative skeletal muscle mass was significantly correlated with the branched-chain amino acids to tyrosine ratio (r = -0.254, p = 0.005) and body cell mass (r = 0.636, p < 0.001). The overall survival rate in patients with low skeletal muscle mass was significantly lower than in patients with normal/high skeletal muscle mass (p < 0.001). Perioperative nutritional therapy significantly increased overall survival in patients with low skeletal muscle mass (p = 0.009). Multivariate analysis showed that low skeletal muscle mass was an independent risk factor for death after transplantation. In conclusion, sarcopenia was closely involved with posttransplant mortality in patients undergoing LDLT. Perioperative nutritional therapy significantly improved overall survival in patients with sarcopenia.

Pediatric liver transplantation using reduced and hyper-reduced left lateral segment grafts: a 10-year single-center experience.

Few studies have examined the long-term outcomes and prognostic factors associated with pediatric living living-donor liver transplantation (LDLT) using reduced and hyper-reduced left lateral segment grafts. We conducted a retrospective, single-center assessment of the outcomes of this procedure, as well as clinical factors that influenced graft and patient survival. Between September 2000 and December 2009, 49 patients (median age: 7 months, weight: 5.45 kg) underwent LDLT using reduced (partial left lateral segment; n = 5, monosegment; n = 26), or hyper-reduced (reduced monosegment grafts; n = 18) left lateral segment grafts. In all cases, the estimated graft-to-recipient body weight ratio of the left lateral segment was more than 4%, as assessed by preoperative computed tomography volumetry, and therefore further reduction was required. A hepatic artery thrombosis occurred in two patients (4.1%). Portal venous complications occurred in eight patients (16.3%). The overall patient survival rate at 1, 3 and 10 years after LDLT were 83.7%, 81.4% and 78.9%, respectively. Multivariate analysis revealed that recipient age of less than 2 months and warm ischemic time of more than 40 min affected patient survival. Pediatric LDLT using reduced and hyper-reduced left lateral segment grafts appears to be a feasible option with acceptable graft survival and vascular complication rates.

Effect of maintenance therapy with low-dose peginterferon for recurrent hepatitis C after living donor liver transplantation.

Approximately 30% of patients who have recurrent hepatitis C after liver transplantation achieve sustained virological response (SVR) by taking a combination therapy of pegylated interferon and ribavirin. For the remaining non-SVR patients, an effective management treatment has not yet been established. In this study, efficacy of long-term peginterferon maintenance therapy for non-SVR patients was evaluated. Forty patients who had previously received the combination therapy for hepatitis C after living donor liver transplantation were classified into one of the following three groups: the SVR group (n = 11); the non-SVR-IFN group (n =17), which received low-dose peginterferon maintenance therapy for non-SVR patients; and the non-SVR-Withdrawal group (n = 12), which discontinued the interferon treatment. We then compared histological changes among these three groups after 2 or more years follow-up. Activity grade of liver histology improved or remained stable in patients in the SVR and non-SVR-IFN groups, but deteriorated in half of the patients in the non-SVR-Withdrawal group. Fibrosis improved or remained stable in 10 of 11 SVR patients and in 13 of 17 non-SVR-IFN patients, but deteriorated in all non-SVR-Withdrawal patients. Mean changes in fibrosis stage between pretreatment and final liver biopsy were -0.18, +0.06 and +2.2 in the SVR, non-SVR-IFN and non-SVR-Withdrawal groups, respectively. Fibrosis stage deteriorated to F3 or F4 significantly more rapidly in the non-SVR-Withdrawal group than in the other two groups. In conclusion, continuing long-term maintenance therapy with peginterferon prevented histological progression of hepatitis C in patients who had undergone living donor liver transplantation.

Typing of O26 enterohaemorrhagic and enteropathogenic Escherichia coli isolated from humans and cattle with IS621 multiplex PCR-based fingerprinting.

AIMS: This study evaluated a typing method of O26:H11 enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) based on the variation in genomic location and copy numbers of IS621. METHODS AND RESULTS: Two multiplex PCRs, targeting either the left (5') or right (3') IS/chromosome junction of 12 IS621 insertion sites and one PCR specific of another truncated copy, were developed. Thirty-eight amplification profiles were observed amongst a collection of 69 human and bovine O26:H11 EHEC and EPEC. Seventy-one per cent of the 45 EHEC and EPEC with identical IS621 fingerprints within groups of two, three or four isolates had >85% pulsed field gel electrophoresis (PFGE) profile similarity, including four groups of epidemiologically related EHEC or EPEC, while most of the groups had <85% similarity between each others. Epidemiologically related EHEC from each of three independent outbreaks in Japan and Belgium also exhibited identical IS621 fingerprints and PFGE profiles. CONCLUSIONS: The IS621 fingerprinting and the PFGE are complementary typing assays of EHEC and EPEC; though, the former is less discriminatory. SIGNIFICANCE AND IMPACT OF THE STUDY: The IS621 printing method represents a rapid (24 h) first-line surveillance and typing assay, to compare and trace back O26:H11 EHEC and EPEC during surveys in farms, multiple human cases and outbreaks.

Hashimoto's encephalopathy after interferon therapy for hepatitis C virus in adult liver transplant recipient accompanied by post-transplant lymphoproliferative disorder related to Epstein-Barr virus infection.

A 55-year-old woman underwent living-donor liver transplantation (LDLT). She had no history of autoimmune diseases. Spleen was preserved. Steroids were withdrawn at 3 months after LDLT. Epstein-Barr virus (EBV) infection occurred at 3.5 years after LDLT. Recurrent hepatitis C virus infection was confirmed at 4.5 years after LDLT, and pegylated interferon was introduced. Diagnosis of EBV-positive post-transplant lymphoproliferative disorder (PTLD) was made at 4.8 years after LDLT, and tacrolimus (Tac) was stopped completely. Then, unconsciousness, convulsion, and cervical stiffness appeared suddenly. Electroencephalography, cerebrospinal fluid analysis, and image studies revealed normal or only nonspecific findings. The patient was in a state of exhaustion; therefore, steroid pulse therapy (SPT) was attempted. Surprisingly, her general condition, including consciousness disturbance, was improved markedly, and Hashimoto's encephalopathy (HE) was suspected, based on this reaction to SPT. Elevations of anti-thyroglobulin antibody and anti-thyroid peroxidase antibody were confirmed. After withdrawal of Tac, and treatment with acyclovir and steroids, EBV-positive PTLD and HE improved, although they recurred at 5.1 years after LDLT. SPT improved only neurological symptoms. Molecular-targeted therapy was given for recurrent PTLD, based on analysis of sampling specimens. This therapy was effective, but tumor lysis syndrome occurred, and the patient died at 5.3 years after LDLT.

Herpes simplex virus hepatitis after pediatric liver transplantation.

Herpes simplex virus (HSV) hepatitis has a fatal impact on the outcome of organ transplanted recipients. Here, we present a thought-provoking case of HSV hepatitis in a high-risk recipient after living-related liver transplantation (LRLT). A 1-month-old female newborn infant was affected by HSV encephalitis. Fulminant hepatic failure (FHF) of unknown etiology occurred suddenly at 4.4 years of age. Viral infections were ruled out as the cause of FHF. Intensive care including plasma exchange (PE) was started, and the preoperative treatments for ABO incompatibility were performed. Thereafter, LRLT was performed emergently. Although strong immunosuppression for ABO incompatibility was continued after LRLT, antibody-mediated rejection (AMR) occurred on postoperative day (POD) 4. PE was repeated and improvements were obtained. However, liver dysfunction appeared on POD 8. Histopathological findings of liver needle biopsy clearly revealed HSV hepatitis, although the results of HSV DNA and antibody titer in blood sample did not clearly indicate HSV infection. On POD 21, thrombotic microangiopathy (TMA) occurred and the plasma and immunoglobulin were replenished. Our pediatric recipient recovered successfully from AMR, HSV hepatitis, TMA, and repeated sepsis. We conclude that well considered therapy based on the real-time detection of HSV hepatitis is indispensable for the further improvements of outcome in HSV hepatitis after LRLT.

Significance of des-gamma-carboxy prothrombin in selection criteria for living donor liver transplantation for hepatocellular carcinoma.

Des-gamma-carboxy prothrombin (DCP) levels reportedly correlate with histological features of hepatocellular carcinoma (HCC). We examined serum DCP as a predictor of HCC recurrence in 144 patients who underwent living donor liver transplantation. Receiver operating characteristics (ROC) analysis revealed superiority of DCP and AFP over preoperative tumor size or number for predicting recurrence. Multivariate analysis revealed tumor size >5 cm, > or =11 nodules, and DCP >400 mAU/mL as significant independent risk factors for recurrence. Incidence of microvascular invasion (62% vs. 27%, p = 0.0003) and poor differentiation (38% vs. 16%, p = 0.0087) were significantly higher for patients with DCP >400 mAU/mL than for patients with DCP < or =400 mAU/mL. In ROC analysis for patients with < or =10 nodules all < or =5 cm to predict recurrence, area under the curve was much higher for DCP than for AFP (0.84 vs. 0.69). Kyoto criteria were thus defined as < or =10 nodules all < or =5 cm, and DCP < or =400 mAU/mL. The 5-year recurrence rate for 28 patients beyond-Milan but within-Kyoto criteria was as excellent as that for 78 patients within-Milan criteria (3% vs. 7%). The preoperative DCP level offers additional information regarding histological features, and thus can greatly improve patient selection criteria when used with tumor bulk information.

Portal vein complications in pediatric living donor liver transplantation using left-side grafts.

The aim of this report is to assess the rate of portal vein complications (PVCs), the success rate of treatment for PVCs and the prognosis of patients with PVCs for pediatric living donor liver transplantation (LDLT). Pre- and postoperative records of 521 pediatric LDLTs, using left-side grafts were retrospectively reviewed. The overall rate of PVC was 9%, with early PVC occurring in nine patients (1.7%) with a mortality rate of 67% and late PVC in 38 patients (7.3%). Fifteen of these patients with late PVC showed complete portal vein occlusion despite various treatments, and in six of them the graft was lost. Histological examination revealed fibrosis in portal areas in 13 patients, around the central veins associated with cholestasis in the parenchyma in 10, and hepatocyte ballooning in 12. Correction of portal vein flow or retransplantation is necessary for the rescue of patients with early PVCs. Graft loss in the long term may be high with the occurrence of liver failure or portal hypertension related causes, such as hepatopulmonary syndrome and gastrointestinal bleeding in patients with late PVCs. For the rescue of these patients, especially for patients with body weight < 6 kg, regular monitoring of portal vein flow is essential.

Plasmin induces degradation and dysfunction of laminin 332 (laminin 5) and impaired assembly of basement membrane at the dermal-epidermal junction.

BACKGROUND: The epidermal basement membrane (BM), located at the dermal-epidermal junction (DEJ), plays important roles not only in adhesion between epidermis and dermis, but also in controlling skin functions. In sun-exposed skin, the BM becomes disrupted and multilayered. In order to explore the impairment of BM assembly, we have used a skin-equivalent (SE) as a model of BM damage and previously clarified the involvement of matrix metalloproteinases (MMPs) in impairment of BM assembly. OBJECTIVES: In this work, we examined the role of urokinase-type plasminogen activator (uPA) and plasmin in impairment of BM assembly at the DEJ by using the SE, as ultraviolet irradiation to the skin increases uPA as well as MMPs. METHODS: SEs were used as a model of formation and damage of BM. Human uPA was detected by enzyme-linked immunosorbent assay and zymography, and gelatinases such as MMP-2 and MMP-9 were detected by zymography. Human plasminogen was added at 0.06 micromol L(-1) (about 3% of plasma level) to increase plasmin to a pathological level. N-terminal peptide sequence analysis of plasmin-treated laminin 332 was carried out to identify alpha3, beta3 and gamma2 chains of laminin 332 and their cleavage sites of each chain. Plasmin-treated laminin 332 was analysed in keratinocyte adhesion activity and binding to type VII collagen. RESULTS: Human uPA was detected in addition to MMP-2 and MMP-9, in conditioned medium of SE. Although the BM was well organized in the presence of an MMP inhibitor alone, the activated plasmin disorganized the BM even in the presence of the inhibitor. The impairment of BM assembly made the epidermis thinner as compared with that of a control cultured in the presence of MMP inhibitor, indicating that the BM affects the polarity and differentiation of the epidermis. The addition of aprotinin, a serine proteinase inhibitor, and tranexamic acid, a uPA-plasmin inhibitor, inhibited the plasmin-induced impairment of BM assembly and facilitated BM reorganization, thereby improving the epidermal structure. N-terminal peptide sequence analysis of plasmin-treated laminin 332 revealed the removal of a 5- or 10-kDa fragment, including the cell adhesion region, from the G3 domain of the alpha3 chain, and the LN domain, which binds to the noncollagenous 1 domain in type VII collagen, from the beta3 chain. Plasmin-treated laminin 332 showed lower keratinocyte adhesion activity and reduced binding to type VII collagen. CONCLUSIONS: These results suggest that uPA and plasmin are involved in the impairment of BM assembly and epidermal differentiation, and that these effects arise at least partly through direct degradation of laminin 332.