Comparative Study between Gas Chromatography and Ion-exchange Chromatography on Amino Acid Analysis of Foods

Lactobacillus brevis and Saccharomyces cerevisiae were completely sterilized by the supercritical (SC) CO2 micro-bubble method. Gaseous (G) and liquid (LQ) CO2 were used in a similar manner to compare the sterilizing effect. Among the three treatments, the microorganisms were only effectively sterilized by the SC CO2 treatment at 25 MPa and 35°C.     

Identification of catalytic residues of a very large NAD-glutamate dehydrogenase from Janthinobacterium lividum by site-directed mutagenesis

We previously found a very large NAD-dependent glutamate dehydrogenase with approximately 170?kDa subunit from Janthinobacterium lividum (Jl-GDH) and predicted that GDH reaction occurred in the central domain of the subunit. To gain further insights into the role of the central domain, several single point mutations were introduced. The enzyme activity was completely lost in all single mutants of R784A, K810A, K820A, D885A, and S1142A. Because, in sequence alignment analysis, these residues corresponded to the residues responsible for glutamate binding in well-known small GDH with approximately 50?kDa subunit, very large GDH and well-known small GDH may share the same catalytic mechanism. In addition, we demonstrated that C1141, one of the three cysteine residues in the central domain, was responsible for the inhibition of enzyme activity by HgCl₂, and HgCl₂ functioned as an activating compound for a C1141T mutant. At low concentrations, moreover, HgCl₂ was found to function as an activating compound for a wild-type Jl-GDH. This suggests that the mechanism for the activation is entirely different from that for the inhibition.     

Isolation of Escherichia coli B Mutant Deficient in Glutathione Biosynthesis

Certain edible large jellyfishes belonging to the order Rhizostomeae are consumed in large quantities in China and Japan. The exumbrella part of the edible jellyfish Stomolophus nomurai was cut and soaked in dilute hydrochloric acid solution (pH 3.0) for 12 h, and heated at 121 °C for 20 min. The immunostimulation effects of the jellyfish extract were examined. The jellyfish extract enhanced IgM production of human hybridoma HB4C5 cells 34-fold. IgM and IgG production of human peripheral blood lymphocytes (PBL) were also accelerated, 2.8- and 1.4-fold respectively. Moreover, production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α by human PBL was stimulated 100- and 17-fold respectively. Collagenase treatment inactivated the immunostimulation activity of the jellyfish extract. In addition, purified collagen from bovine Achilles’ tendon accelerated IgM production of hybridoma cells. These facts mean that collagen has an immunostimulation effect, and that the active substance in jellyfish extract is collagen.     

Purification and Some Properties of Debranching Enzyme of Germinating Rice Endosperm

A debranching enzyme was extracted from the endosperm of germinating rice seeds and purified through three steps, namely cyclohexaamylose-coupled Sepharose 6B, Ultrogel AcA-44 and Bio-Gel P-150 column chromatography. This disc-electrophoretically homogeneous enzyme showed a specific activity of 43 units/mg of protein (30°C) with a pH optimum of 5.5. The isoelectric point was 4.9, unlike that (pI 3.5) of debranching enzyme of ungerminated rice seeds. Our enzyme hydrolyzed pullulan rapidly, and glutinous rice starch and waxy corn starch moderately. The enzyme was also able to act on phytoglycogen and glycogen unlike debranching enzymes originating in some plants.     

Yamogenin in fenugreek inhibits lipid accumulation through the suppression of gene expression in fatty acid synthesis in hepatocytes

Yamogenin is a diastereomer of diosgenin, which we have identified as the compound responsible for the anti-hyperlipidemic effect of fenugreek. Here, we examined the effects of yamogenin on the accumulation of triacylglyceride (TG) in hepatocytes, because yamogenin is also contained in fenugreek. It was demonstrated that yamogenin also inhibited TG accumulation in HepG2 hepatocytes and suppressed the mRNA expression of fatty acid synthesis-related genes such as fatty acid synthase and sterol response element-binding protein-1c. Indeed, yamogenin also antagonized the activation of the liver X receptor (LXR) in luciferase ligand assay similar to diosgenin. However, yamogenin could not exert such effects in the presence of T0901713, a potent agonist of LXR. These findings indicate that the effects of yamogenin on TG accumulation would be weaker than those of diosgenin, suggesting that the structural difference between yamogenin and diosgenin would be important for the inhibition of LXR activation.     

Growth Profile of Crown Gall Cells of Tobacco in Suspension Culture

In order to obtain a basic information of plant cell suspension culture as a step toward the development of large scale culture, culture conditions of crown gall cells (auxin non-requiring cells) were investigated. Addition of yeast extract to culture medium was significantly effective for the growth and cell dispersion.

In experiments on the ability of the cultured cells to utilize sugars as the carbon source, it was observed that galactose, added to the culture medium, markedly inhibited the cell growth.

Pasteurization of the medium containing fructose as carbon source made it brownish by Maillard reaction and the medium apparently restrained the cell growth. However, the fructose medium sterilized by filtration was excellent for the cell growth as well as sucrose or glucose medium. In a jar fermentor, even the glucose medium became brownish by heat sterilization and the brown colored medium restrained the cell growth. Under optimum conditions, the doubling time was 1.1 day in exponential phase and 2.0 g of cell (dry weight) per 100 ml culture was obtained as the maximum yield.     

Validity of ultrasound muscle thickness measurements for predicting leg skeletal muscle mass in healthy Japanese middle-aged and older individuals

Background

The skeletal muscle mass of the lower limb plays a role in its mobility during daily life. From the perspective of physical resources, leg muscle mass dominantly decreases after the end of the fifth decade. Therefore, an accurate estimate of the muscle mass is important for the middle-aged and older population. The present study aimed to clarify the validity of ultrasound muscle thickness (MT) measurements for predicting leg skeletal muscle mass (SM) in the healthy Japanese middle-aged and older population.

Findings

MTs at four sites of the lower limb and the bone-free lean tissue mass (LTM) of the right leg were determined using brightness-mode ultrasonography and dual-energy X-ray absorptiometry (DXA), respectively, in 44 women and 33 men, 52- to 78-years old. LTM was used as a representative variable of leg skeletal muscle mass. In the model-development group (30 women and 22 men), regression analysis produced an equation with R² and standard error of the estimate (SEE) of 0.958 and 0.3 kg, respectively: LTM (kg) = 0.01464 × (MT_SUM×L) (cm²) - 2.767, where MT_SUM is the sum of the product of MTs at four sites, and L is length of segment where MT is determined. The estimated LTM (7.0 ± 1.7 kg) did not significantly differ from the measured LTM (7.0 ± 1.7 kg), without a significant systematic error on a Bland-Altman plot. The application of this equation for the cross-validation group (14 women and 11 men) did not yield a significant difference between the measured (7.2 ± 1.6 kg) or estimated (7.2 ± 1.6 kg) LTM and systematic error.

Conclusion

The developed prediction equation may be useful for estimating the lean tissue mass of the lower extremity for the healthy Japanese middle-aged and older population.     

Aberrant Glycogen Synthase Kinase 3β Is Involved in Pancreatic Cancer Cell Invasion and Resistance to Therapy

Background and Purpose

The major obstacles to treatment of pancreatic cancer are the highly invasive capacity and resistance to chemo- and radiotherapy. Glycogen synthase kinase 3β (GSK3β) regulates multiple cellular pathways and is implicated in various diseases including cancer. Here we investigate a pathological role for GSK3β in the invasive and treatment resistant phenotype of pancreatic cancer.

Methods

Pancreatic cancer cells were examined for GSK3β expression, phosphorylation and activity using Western blotting and in vitro kinase assay. The effects of GSK3β inhibition on cancer cell survival, proliferation, invasive ability and susceptibility to gemcitabine and radiation were examined following treatment with a pharmacological inhibitor or by RNA interference. Effects of GSK3β inhibition on cancer cell xenografts were also examined.

Results

Pancreatic cancer cells showed higher expression and activity of GSK3β than non-neoplastic cells, which were associated with changes in its differential phosphorylation. Inhibition of GSK3β significantly reduced the proliferation and survival of cancer cells, sensitized them to gemcitabine and ionizing radiation, and attenuated their migration and invasion. These effects were associated with decreases in cyclin D1 expression and Rb phosphorylation. Inhibition of GSK3β also altered the subcellular localization of Rac1 and F-actin and the cellular microarchitecture, including lamellipodia. Coincident with these changes were the reduced secretion of matrix metalloproteinase-2 (MMP-2) and decreased phosphorylation of focal adhesion kinase (FAK). The effects of GSK3β inhibition on tumor invasion, susceptibility to gemcitabine, MMP-2 expression and FAK phosphorylation were observed in tumor xenografts.

Conclusion

The targeting of GSK3β represents an effective strategy to overcome the dual challenges of invasiveness and treatment resistance in pancreatic cancer.     

Background Factors of Reflux Esophagitis and Non-Erosive Reflux Disease: A Cross-Sectional Study of 10,837 Subjects in Japan

Background

Despite the high prevalence of gastroesophageal reflux disease (GERD), its risk factors are still a subject of controversy. This is probably due to inadequate distinction between reflux esophagitis (RE) and non-erosive reflux disease (NERD), and is also due to inadequate evaluation of adjacent stomach. Our aim is therefore to define background factors of RE and NERD independently, based on the evaluation of Helicobacter pylori infection and gastric atrophy.

Methods

We analyzed 10,837 healthy Japanese subjects (6,332 men and 4,505 women, aged 20–87 years) who underwent upper gastrointestinal endoscopy. RE was diagnosed as the presence of mucosal break, and NERD was diagnosed as the presence of heartburn and/or acid regurgitation in RE-free subjects. Using GERD-free subjects as control, background factors for RE and NERD were separately analyzed using logistic regression to evaluate standardized coefficients (SC), odds ratio (OR), and p-value.

Results

Of the 10,837 study subjects, we diagnosed 733 (6.8%) as RE and 1,722 (15.9%) as NERD. For RE, male gender (SC = 0.557, OR = 1.75), HP non-infection (SC = 0.552, OR = 1.74), higher pepsinogen I/II ratio (SC = 0.496, OR = 1.64), higher BMI (SC = 0.464, OR = 1.60), alcohol drinking (SC = 0.161, OR = 1.17), older age (SC = 0.148, OR = 1.16), and smoking (SC = 0.129, OR = 1.14) are positively correlated factors. For NERD, HP infection (SC = 0.106, OR = 1.11), female gender (SC = 0.099, OR = 1.10), younger age (SC = 0.099, OR = 1.10), higher pepsinogen I/II ratio (SC = 0.099, OR = 1.10), smoking (SC = 0.080, OR = 1.08), higher BMI (SC = 0.078, OR = 1.08), and alcohol drinking (SC = 0.076, OR = 1.08) are positively correlated factors. Prevalence of RE in subjects with chronic HP infection and successful HP eradication denotes significant difference (2.3% and 8.8%; p<0.0001), whereas that of NERD shows no difference (18.2% and 20.8%; p = 0.064).

Conclusions

Significantly associated factors of NERD are considerably different from those of RE, indicating that these two disorders are pathophysiologically distinct. Eradication of Helicobacter pylori may have disadvantageous effects on RE but not on NERD.     

Establishment of laurel forest vegetation in adjacent secondary forest in central Japan

Distributions of lucidophyllous species are limited due to the fragmentation of laurel forest. On Komayama Hill in central Japan, we evaluated the colonization of typical lucidophyllous vascular plants from a 350-year-old laurel forest into adjacent abandoned secondary forest for conservation and restoration purposes. A total of 14 consecutive subplots were established along the vegetation border between the two forests (length, 30 m; width, 5 m), extending 70 m into the secondary forest; 18 quadrats of old-growth forest were surveyed. Edge effects of old-growth forest were found to play an important role in re-establishing lucidophyllous saplings and seedlings in the secondary forest. In particular, the abundances of the four dominant canopy species of the old-growth forest significantly decreased with increasing distance. Hence, they are expected to colonize further into the secondary forest and, ultimately, to dominate the canopy. However, the number of lucidophyllous species did not change with distance. Species such as Ficus nipponica, Damnacanthus indicus, Ilex integra, and Lemmaphyllum microphyllum were near-completely or completely limited to the old-growth forest. They are known to be negatively affected by forest fragmentation and were observed to be struggling to colonize the exterior of the old-growth forest even after 60 years of abandonment. Their absence highlighted the limited colonization capacities of some old-growth forest species and underlined the time required for habitat restoration following human disturbance. We conclude that it is important to consider the population dynamics of dominant canopy species and the colonization of these interior species when assessing the habitat expansion of lucidophyllous species and hence the restoration of degraded lands.