1,2-Dioleoylglycerol method for pancreatic lipase catalytic activity in serum.

BACKGROUND: There is a need for a pancreatic lipase (LIP) reference assay to provide an accurate base to which routine methods can be traceable. METHODS: This study developed a novel LIP assay method in which 1,2-dioleoylglycerol (DODG) is the substrate and LIP activity is measured in a coupled enzymatic reaction from the increase in absorbance at 340 nm with production of NADPH. RESULTS: With this method, LIP activity was linear up to 440 U/L (8-times expected upper limit of physiological concentration). When assayed manually, the between-laboratory variation for six samples surveyed at five laboratories was 3.80-26.4% (CV) for samples containing about 20-290 U/L LIP activity; when assayed using an automated analyzer, the range was 1.86-4.86% (four laboratories). Interference by >5 mmol/L glycerol and low specificity with post-heparin samples were noted, but in practice these are avoidable. Precision analyzed by automated assay of 49 samples twice in random order produced a covariance of 2.27 U/L, which is comparable to routine methods, and good correlations were obtained with five routine methods. CONCLUSIONS: Although further studies are required, the DODG method may be likely applicable as one candidate reference method.

Antioxidant treatment during preservation of bovine ovaries increased the development potential of embryos.

The overnight preservation of bovine ovaries would be highly useful in the subsequent harvest of viable oocytes for reproductive study. The present study aimed to optimize conditions for overnight preservation of bovine ovaries by examining the effects of temperature, solution and supplementation. In Experiment 1, the rate of development to the blastocyst stage of oocytes derived from ovaries preserved at 15°C was higher than that at either 5 or 25°C (p < 0.05). In Experiment 2, the rate of development to the blastocyst stage of oocytes derived from ovaries preserved in University of Wisconsin solution was higher than when PBS or saline was used (p < 0.05). In Experiment 3, oocytes preserved in saline supplemented with 0.3 mM glutathione (GSH) exhibited an increase in the rate of blastocyst formation compared with oocytes supplemented with 0 or 3 mM GSH (p < 0.05). In Experiment 4, supplementation with 10 µM epigallocatechin gallate during ovary preservation increased the rate of blastocyst formation (p < 0.05). The blastocysts derived from ovaries stored in saline supplemented with GSH at 15°C for 24 h were shown to develop into normal offsprings following transfer to recipient heifers. Our studies indicate that bovine IVM/IVF embryos derived from ovaries preserved in saline supplemented with an antioxidant at 15°C for 24 h can successfully develop to the blastocyst stage and result in offspring.

Three-dimensional surface models of autopsied human brains constructed from multiple photographs by photogrammetry.

Virtual three-dimensional (3D) surface models of autopsied human brain hemispheres were constructed by integrating multiple two-dimensional (2D) photographs. To avoid gravity-dependent deformity, formalin-fixed hemispheres were placed on non-refractile, transparent acrylic plates, which allowed us to take 2D photographs from various different angles. Photogrammetric calculations using software (ReCap Pro cloud service, Autodesk, San Rafael, CA, USA) allowed us calculate the 3D surface of each brain hemisphere. Virtual brain models could be moved and rotated freely to allow smooth, seamless views from different angles and different magnifications. When viewing rotating 3D models on 2D screens, 3D aspects of the models were enhanced using motion parallax. Comparison of different brains using this method allowed us to identify disease-specific patterns of macroscopic atrophy, that were not apparent in conventional 2D photographs. For example, we observed frontal lobe atrophy in a progressive supranuclear palsy brain, and even more subtle atrophy in the superior temporal gyrus in amyotrophic lateral sclerosis-frontotemporal lobar degeneration. Thus, our method facilities recognition of gyral atrophy. In addition, it provides a much more powerful and suitable way of visualizing the overall appearance of the brain as a three-dimensional structure. Comparison of normal and diseased brains will allow us to associate different macroscopic changes in the brain to clinical manifestations of various diseases.

The expression of cell adhesion molecule 1 and its splicing variants in Sézary cells and cell lines from cutaneous T-cell lymphoma.

Cell adhesion molecule 1 (CADM1) is aberrantly expressed by T-cell neoplasms such as adult T-cell leukemia/lymphoma (ATLL) and mycosis fungoides (MF). We studied the expression of CADM1 and its splicing variants in Sézary syndrome (SS), MF, other cutaneous T-cell lymphoma (CTCL), and cell lines derived from T- and B-cell lymphomas. Soluble CADM1 was measured in the patients' sera. CADM1+ cells in the blood and skin lesions were examined by flow cytometry and immunostaining, respectively. Soluble CADM1 was measured by ELISA, and the splicing variants of CADM1 transcripts were determined by reverse transcriptase-polymerase chain reaction, followed by sequencing. As a result, circulating CADM1+ cells were significantly increased in seven out of 10 patients with SS, ranging from 7.9% to 74.5% of the CD3+CD4+ fractions (median 33.7%; cut-off value 6.5%). The percentages of CADM1+ cells were usually less than those of circulating Sézary cells. CADM1 was expressed, to various degrees, in six of nine T-cell lines derived from SS, MF, ATLL, and anaplastic large cell lymphoma (ALCL), but negative in B-cell lymphoma-derived cell lines. CADM1+ cells were present in the skin infiltrates of MF, SS, ATLL and ALCL. Serum levels of soluble CADM1 were not significantly elevated in SS/MF. Three major splicing variants of CADM1 expressed by neoplastic T-cells contained different combinations of the exons 7, 8, 9 and 11, including a putative oncogenic variant composed of exons 7-8-9-11. In conclusion, CADM1 is frequently expressed in Sézary cells and cell lines from CTCL.

Role of soybean oil fat emulsion in the prevention of hepatic xenobiotic transporter mRNA up- and down-regulation induced by overdose of fat-free total parenteral nutrition in infant rats.

Total parenteral nutrition (TPN) is associated with cholestasis and hepatic steatosis in human infants. The present study focused on the changes in hepatic xenobiotic transporters associated with overdose of fat-free or fat-containing TPN in infant rats. Three-week-old male Sprague-Dawley rats were divided into three groups: group 1 received an oral diet, group 2 received TPN without fat, and group 3 received TPN with 20% of its calories from fat (soybean oil emulsion). After TPN administration for 4 days, both serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, which are indicators of hepatic dysfunction, in group 2 were significantly higher (p<0.001) than those in the other groups, whereas there were no differences between groups 1 and 3 in either serum AST or ALT levels. The serum bilirubin concentration in group 2 was also markedly higher than that in the other groups. Mdr2, Bsep, Mrp2, Mrp6, Oct1, and Oat2 mRNA levels were decreased in group 2 (fat-free TPN) compared with those in group 1 (oral diet), whereas Mdr1b, Mrp1, and Mrp5 mRNA levels were increased. Specifically, the level of Mdr1b mRNA in group 2 was 16 times higher (p<0.001) than that in group 1. On the other hand, the changes in these mRNA expression levels in group 3 (fat-containing TPN) were smaller than those in group 2, and specifically, the expression levels of Mdr1b, Mrp1, Mrp5, Mrp6, and Oat2 mRNA in group 3 were not significantly different from those in group 1. The results of the present study indicate that including fat in the TPN regimen is very important in preventing the mRNA up- and down-regulation of xenobiotic transporters, which is considered to be the main factor responsible for the abnormal hepatic changes such as cholestasis associated with the excessive administration of fat-free TPN.

Clues to diagnosis for unusual mucosal pemphigus demonstrating undetectable anti-desmoglein 3 serum antibodies by routine tests.

Pemphigus is an autoimmune blistering disease caused by immunoglobulin (Ig)G autoantibodies against desmogleins (Dsg). In mucosal-dominant pemphigus vulgaris (PV), anti-Dsg3 antibodies play a critical role in acantholysis. We followed two mucosal-dominant PV cases who suffered from refractory oral mucosal erosions. In these cases, anti-Dsg3 serum antibodies were not detected by indirect immunofluorescence and enzyme-linked immunosorbent assay (ELISA). However, direct immunofluorescence showed the intercellular IgG deposition in the epidermis and histopathological findings revealed suprabasal acantholysis. In order to analyze the pathomechanisms in these cases, we first examined the Dsg3 expression patterns in lesional sites and compared them with those of typical mucosal-dominant PV cases. In typical PV cases, the alteration of Dsg3 distribution was observed in lesional sites by immunostaining. The aggregation of Dsg3, which is the characteristic change in PV mucosal lesions, was observed as the initial change prior to acantholysis. In our cases, a clustering of Dsg3 was observed at mucosal lesions, and the expression levels of Dsg3 in acantholytic lesions were decreased, as observed in typical mucosal-dominant PV cases. Although anti-Dsg3 serum antibodies could not be detected by routine tests, anti-Dsg3 serum antibodies were detected by Dsg3 ELISA using 10-times more concentrated sera (highly sensitive ELISA). Moreover, purified and concentrated PV IgG showed high pathogenicity when examined by dissociation assay. In conclusion, the detection of morphological changes in Dsg3 distribution and highly sensitive ELISA method could be useful for the early diagnosis of PV recurrence.

Present state of hepatitis E virus epidemiology in Tokyo, Japan.

Recently studies have reported the possibility that an indigenous hepatitis E virus (HEV) exists in Japan, but the epidemiological features of HEV in Japan are inadequate to make a judgment. In order to search the present state of HEV infection in Japan, we used ELISA to test 1033 sera from residents living in Tokyo and the Tokyo suburbs, for the presence of the antibody against HEV. The positive rate of anti-HEV IgG was 15.4% in all liver disease patients (68 of 440), 3% (6/200) in healthy individuals and 0.4% in infants (1/246), respectively (P<0.01). Anti-HEV IgG was seen in 17.6% (35/199) of liver disease patients of unknown etiology; 29.4% (5/17) of fulminant hepatitis, 17% of acute hepatitis (15/88) and 16% of chronic hepatitis (15/94). Anti-HEV IgG co-existed with hepatitis B virus and hepatitis C virus in 23.6% (21/89) and 7.9% (12/152), respectively. Furthermore, the prevalence of anti-HEV IgG was significantly higher in hemodialysis patients (18/60: 30%) and hospital workers (8/87: 9.2%) than in the healthy population (P<0.01). Anti-HEV IgM was detected in 0.1% of all samples tested (1/1033). The prevalence of anti-HEV IgG increased with age. No individuals with HEV antibody had a recent history of visiting countries where hepatitis E is endemic. These results indicate that generally 15.4% of Japanese patients with liver diseases had a history of HEV infection in the past. The routes of transmission of HEV require clarification in Japan.

Immune escape phenomenon in molluscum contagiosum and the induction of apoptosis.

Molluscum contagiosum (MC) may persist for many weeks, evading host immunity. We studied the mechanism of immune escape phenomenon in MC, and the possible inducer of apoptosis. Using tissue samples of MC, we examined the numbers of epidermal Langerhans cells (LC), the expression levels of macrophage inflammatory protein-3α (MIP-3α) and thymic stromal lymphopoietin (TSLP), and the apoptotic signals. After molluscum contagiosum virus (MCV) genotyping, we studied the expression of MCV-encoded MC148 mRNA and MC159 mRNA which correspond to viral antagonist for CCR8 and viral Fas-linked interleukin (IL)-1ß converting enzyme (FLICE)-like inhibitor protein (vFLIP), respectively. The nutlin-3-induced apoptosis in MC was observed ex vivo. The numbers of CD1a(+) or Langerin(+) epidermal LC and the expression levels of MIP-3α were markedly decreased in MC. The expression of TSLP was enhanced in the lesional epidermis of atopic dermatitis and human papillomavirus-induced warts, whereas the expression was observed locally in MC. All 14 MC samples examined harbored MCV type 1. The MC148 mRNA was detected in all 14 samples and the MC159 mRNA was detected in 13 samples. Apoptotic cells were absent or at a background level in the living layers of MC, but their numbers were increased in the molluscum bodies by overnight incubation with 5 µmol/L nutlin-3 in culture medium. In conclusion, molluscum bodies are protected from host immune responses and apoptotic signals by being surrounded by LC-depleted epidermal walls and viral immunosuppressive molecules, but could be eradicated by reagents inducing p53-dependent apoptosis.

Tgm2/Gh, Gbx1 and TGF-beta are involved in retinoic acid-induced transdifferentiation from epidermis to mucosal epithelium.

We previously demonstrated that retinoic acid (RA) induces epidermis to transdifferentiate to mucosal epithelium with goblet cells in chick embryonic cultured skin. To characterize the molecular mechanism of this transdifferentiation process, we used rat embryonic cultured skin and immunohistochemistry to confirm that RA-induced epidermal transdifferentiation accompanies the expression of markers of esophagus epithelium. Because Gbx1, TG2/Gh (transglutaminase2) and TGF-beta2 are reported individually to be induced by RA in cultures of chick embryonic skin, mouse epidermal cells and human hair follicles respectively, here, we investigated whether cooperative interplay of Gbx1, TG2/Gh and TGF-beta2 is required for the transdifferentiation of epidermal cells to mucosal cells. We have shown that expression of Gbx1, TG2/Gh and TGF-beta proteins were all upregulated in RA-induced transdifferentiated skin and that the former two were expressed in the epidermis, while TGF-beta was expressed in the dermis. Inhibitors of the TGF-beta signal pathway partially inhibited transdifferentiation. Overexpression of both hTG2/Gh and mGbx1 together in the epidermis by electroporation resulted in cuboidal cells in the upper cell layers of the epidermis without keratinized layers, although epidermal keratinization was observed in skin by overexpression of either of them. Labeling DNA with BrdU indicated that RA directly transdifferentiated transient amplifying epidermal cells, not stem cells, to mucosal cells. This study showed that coexpression of TG/2 and Gbx1 in the epidermis was required for esophagus-like mucosal transdifferentiation, and that increase in TGF-beta2 expression by RA in the dermis was essential to induce transdifferentiation through epithelial-mesenchymal interaction.

Soybean oil fat emulsion to prevent TPN-induced liver damage: possible molecular mechanisms and clinical implications.

Long-term total parenteral nutrition (TPN) is known to be associated with cholestasis and hepatic steatosis, which can be lethal in infants who cannot be fed orally. The present review focuses on the metabolic complications in the liver that may occur due to the excessive administration of fat-free TPN. We have recently developed an infant rat model of hepatic dysfunction and steatosis induced by overdose of fat-free TPN. In this model, plasma levels of liver enzymes in the fat-free TPN group were found to be significantly higher than in the other groups (i.e., the oral diet and fat-containing TPN groups). Pathological examination showed hepatomegaly and severe fatty changes without cholestasis in the liver of infant rats that received fat-free TPN. We clearly demonstrated that the addition of soybean oil emulsion to the TPN regimen prevented hepatic dysfunction and fatty changes. In the present review, we discuss the molecular mechanism of the hepatic dysfunction induced by fat-free TPN and the role of soybean oil fat emulsion in the TPN regimen. We also discuss the clinical implications of soybean oil-containing TPN solutions and point out the importance of including fat in the TPN regimen in order to prevent the hepatic abnormalities associated with the excessive administration of fat-free TPN.

Soybean oil in total parenteral nutrition maintains albumin and antioxidant enzyme mRNA levels.

Changes in albumin and antioxidant enzyme mRNA expression in infant rat liver following administration of total parenteral nutrition (TPN) with/without soybean oil emulsion were studied. Infant rats were divided into three groups: group 1=oral diet, group 2=TPN without fat, and group 3=TPN with 20% of calories from soybean oil emulsion. The period of TPN administration was 4 d. Serum aspartate aminotransferase and alanine aminotransferase levels were higher in group 2 than in the other groups, with similar levels seen in the other groups. Albumin, Cu, Zn-superoxide dismutase, and glutaredoxin 1 mRNA expression levels were lower in group 2 than in the other groups, with similar levels seen in the other groups. Catalase mRNA expression was higher in group 1 than in the other groups, with the lowest level seen in group 2. Soybean oil emulsion should be included in TPN regimens to prevent down-regulation of albumin and antioxidant enzyme mRNA expression.

Major role of soybean oil in preventing decrease in CYP4A1 mRNA content relative to PPARalpha mRNA down-regulation in infant rats.

Changes in the levels of CYP4A1, PPARalpha, and RXRalpha mRNA expression in the liver following overdose of fat-free or fat-containing total parenteral nutrition (TPN) were studied in 3-week-old male Sprague-Dawley rats. The rats were divided into three groups: group 1, oral diet; group 2, fat-free TPN; and group 3, TPN with 20% of calories from soybean oil emulsion. Levels of CYP4A1, PPARalpha, and RXRalpha mRNA in the fat-free TPN group were significantly lower than those in the other groups. Levels of CYP4A1 and PPARalpha mRNA were strongly correlated (r=0.849), and levels of CYP4A1 and RXRalpha mRNA were weakly correlated (r=0.618). This is the first report of a strong correlation between the levels of CYP4A1 and PPARalpha mRNA following overdose of fat-free or fat-containing TPN in infant rats. Our results also indicate that it is important to include fat in TPN regimens in order to prevent CYP4A1 mRNA down-regulation, which may be related to changes in PPARalpha mRNA levels in the liver.

Soybean oil fat emulsion prevents cytochrome P450 mRNA down-regulation induced by fat-free overdose total parenteral nutrition in infant rats.

Changes in the mRNA expression of hepatic cytochrome P450 (CYP) isoenzymes associated with overdose of fat-free or fat-containing total parenteral nutrition (TPN) were investigated in infant rats. Three-week-old male Sprague-Dawley rats were divided into three groups: group 1 received an oral diet, group 2 received TPN without fat, and group 3 received TPN with 20% of calories from fat (soybean oil emulsion). After TPN administration for 4 d, serum aspartate aminotransferase (AST) levels in group 2 were significantly increased (p<0.01) compared with the other groups. The mRNA expression of hepatic CYP isoenzymes in group 2 decreased to 0.76 to 31% of that in group 1 (p<0.01), but that in group 3 was maintained at 32 to 84% of that in group 1. These results indicate the importance of including fat in TPN regimens to prevent not only hepatic dysfunction but also mRNA down-regulation of liver CYP isoenzymes.