A novel approach for the detection of proteolytically activated transglutaminase 1 in epidermis using cleavage site-directed antibodies

It has been suggested that transglutaminase 1 is proteolytically activated upon the terminal differentiation of the keratinocyte, but the mechanisms are not well understood. We have established two mouse hybridoma cell lines producing monoclonal antibodies that specifically detect proteolytically cleaved transglutaminase 1. One detects the amino-terminus of the fragment produced by cleavage between Arginine 93 and Glycine 94, and the other detects the amino-terminus of the fragment produced by cleavage between Arginine 573 and Glycine 574. Using these two antibodies, immunohistochemical analyses of the epidermis revealed that the cleavages of the transglutaminase 1 protein occur early in the terminal differentiation of keratinocytes in the basal layer of the epidermis, that the cleavage between Arginine 573 and Glycine 574 (producing the 574G fragment) precedes the cleavage between Arginine 93 and Glycine 94 (producing the 94G fragment), that the 94G fragment is localized to the plasma membrane of keratinocytes and has cross-linking activity, whereas the 574G fragment is dispersed in the cytosol and does not have detectable levels of activity on in situ transglutaminase assay, and that 1-alpha-25-dihydroxycholecalciferol or all-trans retinoic acid treatment and ultraviolet B exposure disturb the localization of the transglutaminase 1 fragments with changes in the morphology of differentiating keratinocytes. All these results demonstrate that the antibodies generated in this work are useful to dissect the mechanism by which transglutaminase 1 is activated, and would provide us with novel insights into the biogenesis of the epidermis.     

Restoration of mechanical strength and morphological features of the periodontal ligament following orthodontic retention in the rat mandibular first molar

Biomechanical properties and morphological features of the periodontal ligament (PDL) in the rat mandibular molars were examined during orthodontic retention. Seventy-three male rats of the Wistar strain, 8 weeks of age, were used for biomechanical analysis and six rats for morphological analysis. An elastic band was inserted between the mandibular first and second molars for 4 days; after removal of the elastic band the interdental space was filled with resin for 4 and 8 days. The maximum shear stress, tangent modulus, and failure strain energy density of the PDL of the first molar in the experimental animals decreased markedly following application of an orthodontic force. They increased rapidly and were restored completely to the control levels by the 8th day after retention. Light microscopy showed severe compression and extension of the PDL in the experimental animals on the 8th day after retention. Birefringent collagen fibre bundles running across the compressed and expanded PDL were observed, although they appeared to be thinner with less insertions into the alveolar bone or cementum in the experimental animals than in the controls. This suggests that the periodontal collagen fibres were partially reorganized and rearranged during retention. The reorganization and rearrangement of periodontal collagen fibres seemed to be partly related to the restoration of mechanical strength of the rat molar PDL during the 8 days of retention.     

Motor evoked potentials in rats with congenital hydrocephalus

Motor evoked potential (MEP) by focal transcranial magnetic stimulation was used to test the functional integrity of the motor cortex in congenital hydrocephalic rats. Magnetic MEPs, using a figure-eight coil above the head, were recorded in the tibialis anterior muscle. The latency of transcranial magnetic MEP was 3.4 msec in nonhydrocephalic rats. In the hydrocephalic rats, the MEP had a lower threshold than in nonhydrocephalic rats, and showed two peaks. Latencies of early and late peaks were 3.9 msec and from 5.4 msec to 10.0 msec, respectively. Our findings suggest that hydrocephalus in rats is associated with changes in pyramidal cell excitability in the motor cortical area, probably induced by the fluctuations in cortical excitability and synaptic interaction in hydrocephalic rats.     

Role of macrophage inflammatory protein 2 in acute lung injury in murine peritonitis

BACKGROUND: Acute lung injury is a frequent extraabdominal complication of bacterial peritonitis, and neutrophil plays an important role in this lung damage. Macrophage inflammatory protein 2 (MIP-2) serves the same chemotactic function as IL-8 which is a potent neutrophil chemotactic factor in humans, and we investigated the role of MIP-2 associated with neutrophil recruitment in the lung of murine peritonitis. METHODS: Cecal ligation and puncture (CLP) were performed on mice. MIP-2 levels in blood and lung tissue, MIP-2 mRNA expression in lung tissue and bronchoalveolar lavage fluid (BALF), and CD11b expression on peripheral blood neutrophil and BALF cells were determined after CLP. In addition, we investigated the effect of anti-MIP-2 antibody on the lung injury associated with peritonitis. RESULTS: MIP-2 mRNA expression was observed in lung tissue after CLP and numerous neutrophils were accumulated in the lung under those conditions. Anti-MIP-2 antibody contributed to the inhibition of the CD11b expression and chemotaxis of pulmonary neutrophils, lung edema, and thus the reduction in peritonitis-related mortality. CONCLUSIONS: MIP-2 plays a pivotal role in neutrophil recruitment in the lung following peritonitis, and control of neutrophil accumulation in the lung by neutralizing MIP-2 is recommended as a new therapeutic approach to the lung damage associated with peritonitis.     

Hepatocyte growth factor protects functional and histological disorders of HgCl(2)-induced acute renal failure mice

Hepatocyte growth factor (HGF) enhances proliferation of renal epithelial cells as well as hepatocytes. HGF accelerates recovery from acute renal failure (ARF) in animal models. However, pharmacological profiles of HGF including its action mechanism has not been studied in detail. An HgCl(2)-induced ARF mouse was used in this study to evaluate the efficacy of HGF. Single administrations of recombinant human HGF or vehicle were given to ARF mice 30 min after HgCl(2) injection. Renal function was monitored by measuring serum creatinine, blood urea nitrogen and creatinine clearance. In the ARF mice, there was a deterioration of renal function biochemical parameters and histological evidence of renal damage including acute tubular necrosis of proximal tubules. These were both significantly ameliorated by a single HGF administration. The effect of HGF was noticeable in the early phase of ARF (1 day after onset) when there was no histological evidence of increased labeling indexes in renal tubular epithelial cells. Western blot analysis of the c-Met/HGF receptor showed that tyrosine phosphorylation was enhanced immediately after HGF administration indicating direct activation of renal epithelial cells. HGF prevented increase of apoptotic nuclei with DNA fragmentation in renal epithelial cells which suggests cytoprotective activity of HGF on renal epithelial cells in the ARF mice.     

Sarcomatoid carcinoma of the urinary bladder with a spontaneous perforation: a case report

A 65-year-old woman was referred to our clinic with gross hematuria. Cystoscopy revealed a non-papillary and non-pedunculated tumor on the left lateral wall of the bladder. A piece of necrotic tissue obtained from the bladder irrigation was histologically squamous cell carcinoma. A perforation at the left lateral wall of the bladder was found on the cystogram. Bone scintigraphy showed multiple metastases and computed tomography scans showed multiple lymph node metastases in the pelvic cavity. The clinical diagnosis was bladder carcinoma of T4N2M1 stage with an abscess due to a spontaneous perforation. Total cystectomy with bilateral ureterocutaneostomy was performed. She died due to sepsis 13 days after the operation. Histologically, the tumor was composed of carcinomatous and sarcomatous elements. The carcinomatous element was compatible with squamous cell carcinoma and the sarcomatous element was composed of undifferentiated malignant spindle cells. Immunohistochemical examination showed that the carcinomatous component was positive for keratin and human chorionic gonadotropin (HCG) and the spindle cell component positive for vimentin, desmin and HCG. Therefore, we diagnosed the tumor as sarcomatoid carcinoma. We reviewed 56 cases of carcinosarcoma of the bladder in Japan and discussed the clinicopathology of the disease.     

Impact of Prol2Ala variant in the peroxisome proliferator-activated receptor (PPAR) gamma2 on obesity and insulin resistance in Japanese Type 2 diabetic and healthy subjects

The peroxisome proliferator-activated receptor (PPAR) gamma is an important regulator of adipocyte differentiation and a modulator of intracellular insulin-signaling events. We examined the roles of the Pro12Ala variant of PPAR gamma2 in obesity and insulin resistance in 402 Japanese patients with type 2 diabetes and 116 control subjects. Among the diabetes subjects, the Pro12Pro homozygotes showed significantly higher body mass index (BMI) than those with the Pro12Ala variant (p = 0.020), while there was no association between genotype and BMI in the controls. Furthermore, diabetic subjects with Pro12Pro showed significantly higher fat body mass index (FBMI) than those with Pro12Ala (p = 0.016), while no association between genotype and lean body mass index (LBMI) was observed. Regarding insulin resistance, there was no difference in the HOMA index or in clamp index between Pro12Ala and Pro12Pro variants. These data suggest that the Pro12Ala polymorphism of PPAR gamma2 does not influence insulin resistance but body composition in Japanese diabetic subjects.     

Non-invasive assessment of language lateralization by transcranial near infrared optical topography and functional MRI

Near infrared optical topography (OT) is the simultaneous acquisition of hemoglobin absorption from an array of optical fibers on the scalp to construct maps of cortical activity. We demonstrate that OT can be used to determine lateralization of prefrontal areas to a language task that has been validated by functional MRI (fMRI). Studies were performed on six subjects using a visually presented language task. Laterality was quantified by the relative number of activated pixels in each hemisphere for fMRI, and the total hemoglobin responses in each hemisphere for OT. All subjects showed varying degrees of left hemisphere language dominance and the mean laterality indices for subjects who underwent both OT and fMRI were in good agreement. These studies demonstrate that OT gives predictions of hemispheric dominance that are consistent with fMRI. Due to the ease of use and portable nature of OT, it is anticipated that optical topography will be valuable tool for neurological examinations of cognitive function.