6種類の血中膵酵素値に及ぼす加齢および性の影響

The serum levels of some pancreatic enzymes have been reported to be affected by age and gender. Currently, serum total amylase, pancreatic isoamylase (P-amylase) , lipase , trypsin (ogen) , pancreatic phospholipase A(2) (PLA(2)) , and elastase I are utilized in diagnosing pancreatic diseases. We here compared age and gender-related alterations of these six pancreatic enzymes in healthy subjects to delineate different properties among the enzymes. Subjects were 155 males and 172 females between ages 20 and 79 years who were apparently healthy, and were stratified by age and sex. PLA(2) and elastase I were assayed by RIA, trypsin (ogen) by EIA and others by activity. The pancreatic enzymes, except PLA(2), were significantly elevated with age, although they declined in males in the 260 age group. There were significant sex differences in total amylase and P-amylase. Total amylase was significantly higher in females than in males in the 260 age group, P-amylase in the 40-49 age group. Age should be considered in the valuation of serum enzymes except PLA(2), sex difference should be considered in the valuation of amylase (total and P-amylase).ある種の血中膵酵素値は加齢や性により影響を受けることが知られている。現在膵疾患の診断に利用されている6種類の膵酵素，アミラーゼ，P－アミラーゼ，リパーゼ，トリプシン，フォスフォリパーゼA(2)（PIA(2)），そしてエラスターゼⅠの血中値に及ぼす加齢および性の影響を比較検討した。健常者327名（男性：155名，女性：172名，年齢：20－79歳）を対象として，年齢および性により層別化して検討した。PLA(2)とエラスターゼⅠはRIA，トリプシンはEIA，そして他の酵素は活性を測定した。PLA(2)以外の血中月率酵素値は加齢とともに有意に上昇した。しかし男性では60代以降低下した。性差はアミラーゼとP－アミラーゼとに有意差が認められた。すなわち，アミラーゼは60代以降に，P－アミラーゼは40－49歳群でそれぞれ男性より女性において有意の高値を示した。PLA(2)以外の血中膵酵素値を評価する際には加齢による影響を考慮すべきであり，アミラーゼおよびP－アミラーゼの血中値を評価する際には性を考慮すべきである

Application of Microfluidics in Stem Cell Culture

In this chapter, we review the recent developments, including our studies on the microfabricated devices applicable to stem cell culture. We will focus on the application of pluripotent stem cells including embryonic stem cells and induced pluripotent stem cells. In the first section, we provide a background on microfluidic devices, including their fabrication technology, characteristics, and the advantages of their application in stem cell culture. The second section outlines the use of micropatterning technology in stem cell culture. The use of microwell array technology in stem cell culture is explored in the third section. In the fourth section, we discuss the use of the microfluidic perfusion culture system for stem cell culture, and the last section is a summary of the current state of the art and perspectives of microfluidic technologies in stem cell culture

Fluorescent Labeling of the Nuclear Envelope by Localizing Green Fluorescent Protein on the Inner Nuclear Membrane

The nuclear envelope (NE) is a double membrane that segregates nuclear components from the cytoplasm in eukaryotic cells. It is well-known that the NE undergoes a breakdown and reformation during mitosis in animal cells. However, the detailed mechanisms of the NE dynamics are not yet fully understood. Here, we propose a method for the fluorescent labeling of the NE in living cells, which enables the tracing of the NE dynamics during cell division under physiological conditions. In our method, labeling of the NE is accomplished by fixing green fluorescent protein carrying the nuclear localization signal on the inner nuclear membrane based on a unique biotinylation reaction from the archaeon Sulfolobus tokodaii. With this method, we observed HeLa cells during mitosis by confocal laser scanning microscopy and succeeded in clearly visualizing the difference in the timing of the formation of the NE and the nuclear lamina

Cytoskeletal inhibitors, anti-adhesion molecule antibodies, and lectins inhibit hepatocyte spheroid formation.

We investigated the role of cytoskeletons, adhesion molecules, membrane-glycosylations, and proteoglycans in forming the shape of adult rat hepatocyte spheroids. Isolated hepatocytes were cultured on dishes coated with chondroitin sulfate phosphatidyl ethanolamine (CS-PE). Spheroid-forming ability was observed after adding cytoskeletal inhibitors (cytochalasin D, colchicine, okadaic acid, mycalolide B), anti-adhesion molecule antibodies (anti-E-cadherin, anti-connexin 32, anti-zo-1), a glycosphingolipid synthetic inhibitor (N-butyldeoxynojirimycin), a proteoglycan synthetic inhibitor (p-nitrophenyl-beta-D-xylopyranoside), and several lectins. Localization of actin was studied using confocal microscopy after rhodamine-phalloidin staining. Adding cytoskeletal inhibitors on the initial day resulted in weakly clustered cell aggregates rather than smoothly formed spheroids. These effects disappeared at lower reagent concentrations. When reagents were added on day 3, after the formation of spheroids, only mycalolide B was associated with an irregular spheroid surface; the others had no effect. Adding the anti-E-cadherin, anti-connexin 32 on the initial day showed inhibition of spheroid formation, but anti-zo-1 and proteoglycan synthetic inhibitor had no effects. Among the several lectins, only Wheat Germ Agglutinin (WGA), Ricinus communis Agglutinin I (RCA-I), and Concanavalin A (ConA) showed inhibition. These results suggest that cytoskeletal conformation and some adhesion molecules are necessary to form spheroids. Based on the interactions between lectins and hepatocytes in the present study, hepatocytes appear to contain an N-linked complex or N-linked hybrid glycosylated chains

Biofilm deficiency in polysaccharide intercellular adhesin-negative variants of staphylococcus epidermidis selected by subminimal inhibitory concentrations of gentamicin

Staphylococcus epidermidis is a cause of orthopedic device-related infection, and to treat such infection, biofilms should be controlled. Polysaccharide intercellular adhesin (PIA) is associated with the biofilm-forming ability of staphylococcal strains. PIA in biofilm-positive staphylococcal strains can be detected by the Congo red agar (CRA) method. In this study, we used the CRA method to examine the effects of subminimal inhibitory concentrations (sub-MICs) of 11 antibacterial agents on PIA production by S. epidermidis. We found that the PIA-negative variants were selected only by sub- MICs of gentamicin (GM). This PIA-negative phenotype was maintained over several generations in the absence of GM. Such selection occurred in six of eight clinical isolates, as well as in the biofilm-positive control strain. No such selection occurred with aminoglycoside antibiotics except for GM. Most of the PIA-negative variants that were selected by GM showed a markedly lower biofilm-forming ability on stainless steel washers than their untreated parent strains. In conclusion, variants with lower biofilmforming ability may be selected by a sub-MIC of GM. Investigation of the reason why variants with reduced biofilm-forming ability can be selected in the presence of sub-MICs of GM may contribute to strategies against biofilm-related infections

Significance of Stabilometry for Assessing Postoperative Body Sway in Patients with Cervical Myelopathy

[Study Design] Prospective study. [Purpose] To examine the changes in body sway using stabilometry in patients who underwent cervical laminoplasty for cervical myelopathy. [Overview of Literature] Although the patients of cervical myelopathy complain body sway there are few report to examine body sway objectively. [Methods] Patients who received treatment for cervical myelopathy between October 2010 and February 2013 were included. Twenty-one patients underwent cervical laminoplasty (myelopathy group). Body sway was assessed using stabilometry, wherein patients stood on a stabilometer with their eyes closed for 30 seconds. The Romberg ratio, outer peripheral area (OPA) with eyes closed (cm2), and total locus length per unit area (L/A) with eyes closed (/cm) were examined. Examinations were performed preoperatively (at baseline) and at 8 weeks postoperatively. Examination results of patients in the myelopathy group were compared with those of 17 healthy individuals (control group). Clinical symptoms were evaluated using the Japanese Orthopaedic Association scale score (JOA score) and the timed up and go (TUG) test. [Results] In the myelopathy and control groups, the mean baseline Romberg ratio, OPA, and L/A were 2.3±1.2, 8.9±5.5 cm2, and 14.2±5.3/cm and 1.4±1.0, 4.3±2.8 cm2, and 23.7±10.1/cm, respectively. Eight weeks after laminoplasty, only L/A showed significant improvement from baseline in the myelopathy group (23.2±10.1 to 16.8±7.9; p=0.03). The Romberg ratio and OPA showed improvement in the myelopathy group, but the changes were not statistically significant. JOA scores and TUG test results in this group significantly improved from baseline to 8 weeks after laminoplasty (12.7 to 13.4 and 10.8 to 8.0 seconds, respectively; both p<0.05). [Conclusions] L/A is a useful parameter for measuring body sway to assess the recovery of body sway after laminoplasty

Functional promoter upstream p53 regulatory sequence of IGFBP3 that is silenced by tumor specific methylation

BACKGROUND: Insulin-like growth factor binding protein (IGFBP)-3 functions as a carrier of insulin-like growth factors (IGFs) in circulation and a mediator of the growth suppression signal in cells. There are two reported p53 regulatory regions in the IGFBP3 gene; one upstream of the promoter and one intronic. We previously reported a hot spot of promoter hypermethylation of IGFBP-3 in human hepatocellular carcinomas and derivative cell lines. As the hot spot locates at the putative upstream p53 consensus sequences, these p53 consensus sequences are really functional is a question to be answered. METHODS: In this study, we examined the p53 consensus sequences upstream of the IGFBP-3 promoter for the p53 induced expression of IGFBP-3. Deletion, mutagenesis, and methylation constructs of IGFBP-3 promoter were assessed in the human hepatoblastoma cell line HepG2 for promoter activity. RESULTS: Deletions and mutations of these sequences completely abolished the expression of IGFBP-3 in the presence of p53 overexpression. In vitro methylation of these p53 consensus sequences also suppressed IGFBP-3 expression. In contrast, the expression of IGFBP-3 was not affected in the absence of p53 overexpression. Further, we observed by electrophoresis mobility shift assay that p53 binding to the promoter region was diminished when methylated. CONCLUSION: From these observations, we conclude that four out of eleven p53 consensus sequences upstream of the IGFBP-3 promoter are essential for the p53 induced expression of IGFBP-3, and hypermethylation of these sequences selectively suppresses p53 induced IGFBP-3 expression in HepG2 cells