Immunochromatographic assay for the detection of pseudojujubogenin glycosides

INTRODUCTION: Bacopa monnieri contains pseudojujubogenin glycosides as pharmacologically active compounds. In order to screen large numbers of plant samples for the presence of pseudojujubogenin glycosides, a rapid and simple assay system is required for application to small quantities of test materials. Immunoassays using monoclonal antibodies could be useful for the determination of small quantities of pseudojujubogenin glycosides in plant extracts. OBJECTIVE: The objective of this work was to develop a simple method for the detection of pseudojujubogenin glycosides by the immunochromatographic strip test using anti-bacopaside I monoclonal antibody. METHODOLOGY: The qualitative assay was based on a competitive immunoassay in which the detector reagent consisted of a colloidal gold particle coated with the respective anti-bacopaside I MAb. The capture reagent was a bacopaside I-human serum albumin conjugate immobilised onto a test strip membrane. RESULTS: The sample containing pseudojujubogenin glycosides and the detection reagent were incubated with the immobilised capture reagent. The glycosides in the sample competed in binding to the limited amount of antibodies in the detection reagent with the immobilised bacopaside I-HSA conjugates and, hence, positive samples showed no colour in the capture spot zone. The detection limit for the strip test was 125 ng/mL. CONCLUSION: The assay system was found to be useful as a rapid and simple screening method for the detection of pseudojujubogenin glycosides in plants.     

Change in Hair Cycle and Hair Length in Nude Mice by Administration of Deuterium Oxide

D₂O increased hair length in Balb/c nu/nu (nude) mice in our previous study although it has an antimitotic effect in cells. To investigate the mechanism of the effect on the hair length, we examined the change by the administration of D₂O in the duration of the hair cycle and the proliferating activity of the hair matrix in relation with hair length in nude mice. The results showed that 20 or 30% D₂O administration did not change the gross structure of the hairs, the proliferative activity and keratinization of the hair matrix cells, but elongated the hair cycle. The duration of the hair cycle increased by the administration of D₂O in a dose-dependent manner over the examined range and these effects were reversible by discontinuation of D₂O. The change in the hair length correlated with the change in the hair-existing phase particularly. We also showed that the mast cell density in the skin, which is related to the hair cycle, increased in the deuterated mice at anagen VI stage which nearly corresponds to the hair-existing phase. The increase in the mast cell density may be related to the increase in the hair cycle duration. These findings indicate that the increase in hair length may be due to the increase in the duration of the hair cycle, in particular, an increase in the hair-existing phase. This study thus suggests that D₂O slows not only short-term cycles such as circadian clock or ultradian clock, but also the hair cycle which is a long-term cycle.     

Production of β-xylosidase from <Emphasis Type="Italic">Trichoderma asperellum</Emphasis> KIF125 and its application in efficient hydrolysis of pretreated rice straw with fungal cellulase

On-site cellulase and hemicellulase production is a promising way to reduce enzyme cost in the commercialization of the lignocellulose-to-ethanol process. A hemicellulase-producing fungal strain suitable for on-site enzyme production was selected from cultures prepared using wet disc-milling rice straw (WDM-RS) and identified as Trichoderma asperellum KIF125. KIF125 hemicellulase showed uniquely high abundance of β-xylosidase in the xylanolytic enzyme system compared to other fungal hemicellulase preparations. Supplementation of Talaromyces cellulolyticus cellulase with KIF125 hemicellulase was more effective than that with the hemicellulases from other fungal sources in reducing the total enzyme loading for the improvement of xylose yield in the hydrolysis of ball-milling RS, due to its high β-xylosidase dominance. β-Xylosidase in KIF125 hemicellulase was purified and classified as a glycosyl hydrolase family 3 enzyme with relatively high specificity for xylobiose. The production of KIF125 β-xylosidase in the fermentor was estimated as 118 U/g-WDM-RS (2350 U/L culture) at 48 h. These results demonstrate that KIF125 is promising as a practical hemicellulase source to combine with on-site cellulase production using T. cellulolyticus.     

The Role of Sonic Hedgehog Signaling in Osteoclastogenesis and Jaw Bone Destruction

Sonic hedgehog (SHH) and its signaling have been identified in several human cancers, and increased levels of its expression appear to correlate with disease progression and metastasis. However, the role of SHH in bone destruction associated with oral squamous cell carcinomas is still unclear. In this study we analyzed SHH expression and the role played by SHH signaling in gingival carcinoma-induced jawbone destruction. From an analysis of surgically resected lower gingival squamous cell carcinoma mandible samples, we found that SHH was highly expressed in tumor cells that had invaded the bone matrix. On the other hand, the hedgehog receptor Patched and the signaling molecule Gli-2 were highly expressed in the osteoclasts and the progenitor cells. SHH stimulated osteoclast formation and pit formation in the presence of the receptor activator for nuclear factor-κB ligand (RANKL) in CD11b⁺ mouse bone marrow cells. SHH upregulated phosphorylation of ERK1/2 and p38 MAPK, NFATc1, tartrate-resistant acid phosphatase (TRAP), and Cathepsin K expression in RAW264.7 cells. Our results suggest that tumor-derived SHH stimulated the osteoclast formation and bone resorption in the tumor jawbone microenvironment.     

Monitoring oxygen consumption of single mouse embryos using an integrated electrochemical microdevice

Oxygen consumption (respiration activity) has been found to be the most remarkable criterion for determining the viability of an embryo produced in vitro. In this study, we propose an accurate, simple, and user-friendly device for measurement of the oxygen consumption of single mammalian embryos. An integrated electrode array was fabricated to determine the oxygen consumption of a single embryo, including the blastocyst stage, which has an inhomogeneous oxygen consumption rate, using a single measurement procedure. A single mouse embryo was positioned in a microwell at the center of an integrated electrode array, using a mouthpiece pipette, and immobilized by a cylindrical micropit with good reproducibility. The oxygen consumption of two-cell, morula, and blastocyst stages was measured amperometrically using the device. The recorded current profile was corrected to take into consideration transient background current during the measurement. A calculation method for oxygen consumption based on spherical diffusion centered on the defined point of the device was developed. This procedure is quite simple because it is not necessary to estimate the radius of the embryo being measured. The calculated values of oxygen consumption for two-cell, morula, and blastocyst stages were 1.36 ± 0.33 × 10⁻¹⁵ mol s⁻¹, 1.38 ± 0.58 × 10⁻¹⁵ mol s⁻¹, and 3.44 ± 2.07 × 10⁻¹⁵ mol s⁻¹, respectively. The increasing pattern of oxygen consumption from morula to blastocyst agreed well with measurements obtained using conventional scanning electrochemical microscopy (SECM).     

Using food network unfolding to evaluate food–web complexity in terms of biodiversity: theory and applications

Food–web complexity often hinders disentangling functionally relevant aspects of food–web structure and its relationships to biodiversity. Here, we present a theoretical framework to evaluate food–web complexity in terms of biodiversity. Food network unfolding is a theoretical method to transform a complex food web into a linear food chain based on ecosystem processes. Based on this method, we can define three biodiversity indices, horizontal diversity (D_H), vertical diversity (D_V) and range diversity (D_R), which are associated with the species diversity within each trophic level, diversity of trophic levels, and diversity in resource use, respectively. These indices are related to Shannon's diversity index (H′), where H′ = D_H + D_V ? D_R. Application of the framework to three riverine macroinvertebrate communities revealed that D indices, calculated from biomass and stable isotope features, captured well the anthropogenic, seasonal, or other within‐site changes in food–web structures that could not be captured with H′ alone.     

Genetic and biochemical analyses of Pfh1 DNA helicase function in fission yeast

The Schizosaccharomyces pombe pfh1⁺ gene (PIF1 homolog) encodes an essential enzyme that has both DNA helicase and ATPase activities and is implicated in lagging strand DNA processing. Mutations in the pfh1⁺ gene suppress a temperature-sensitive allele of cdc24⁺, which encodes a protein that functions with Schizosaccharomyces pombe Dna2 in Okazaki fragment processing. In this study, we describe the enzymatic properties of the Pfh1 helicase and the genetic interactions between pfh1 and cdc24, dna2, cdc27 or pol 3, all of which are involved in the Okazaki fragment metabolism. We show that a full-length Pfh1 fusion protein is active as a monomer. The helicase activity of Pfh1 displaced only short (<30 bp) duplex DNA regions efficiently in a highly distributive manner and was markedly stimulated by the presence of a replication-fork-like structure in the substrate. The temperature-sensitive phenotype of a dna2-C2 or a cdc24-M38 mutant was suppressed by pfh1-R20 (a cold-sensitive mutant allele of pfh1) and overexpression of wild-type pfh1⁺ abolished the ability of the pfh1 mutant alleles to suppress dna2-C2 and cdc24-M38. Purified Pfh1-R20 mutant protein displayed significantly reduced ATPase and helicase activities. These results indicate that the simultaneous loss-of-function mutations of pfh1⁺ and dna2⁺ (or cdc24⁺) are essential to restore the growth defect. Our genetic data indicate that the Pfh1 DNA helicase acts in concert with Cdc24 and Dna2 to process single-stranded DNA flaps generated in vivo by pol δ-mediated lagging strand displacement DNA synthesis.     

Normalization of phospholipids concentration of the gastric mucosa was observed in patients with peptic ulcer after eradication of Helicobacter pylori

Background. Phospholipids concentration in the gastric mucosa decreased in patients with Helicobacter pylori infection. The aim of this study is to examine the effects of eradication of H. pylori on decreasing the phospholipids concentration in the gastric mucosa in patients with gastric or duodenal ulcer. Materials and Methods. Phospholipids (phosphatidylcholine, phosphatidylethanolamine, and sphingonomyeline) were measured in biopsy specimens from the antrum and corpus using thin‐layer chromatography. In H. pylori positive patients with gastric ulcer (n = 26) and duodenal ulcer (n = 13), and H. pylori negative controls (n = 20), the biopsy specimens were obtained before and 3 months after eradication. Eradication was performed using lansoprazole, amoxycillin, and clarithromycin. Results. Compared with the H. pylori negative control group, the concentrations of phosphatidylcholine and phosphatidylethanolamine decreased significantly in the gastric ulcer group in both antrum and corpus mucosa, and in the duodenal ulcer group in antrum mucosa. This decrease returned to the control level after eradication. Conclusions. This study demonstrates that the eradication of H. pylori in patients with peptic ulcer normalized the decrease of phosphatidylcholine and phosphatidylethanolamine in the gastric mucosa.     

Establishment and characterization of cultured epithelial cells lacking expression of ZO-1

In well polarized epithelial cells, closely related ZO-1 and ZO-2 are thought to function as scaffold proteins at tight junctions (TJs). In epithelial cells at the initial phase of polarization, these proteins are recruited to cadherin-based spotlike adherens junctions (AJs). As a first step to clarify the function of ZO-1, we successfully generated mouse epithelial cell clones lacking ZO-1 expression (ZO-1-/- cells) by homologous recombination. Unexpectedly, in confluent cultures, ZO-1-/- cells were highly polarized with well organized AJs/TJs, which were indistinguishable from those in ZO-1+/+ cells by electron microscopy. In good agreement, by immunofluorescence microscopy, most TJ proteins including claudins and occludin appeared to be normally concentrated at TJs of ZO-1-/- cells with the exception that a ZO-1 deficiency significantly up- or down-regulated the recruitment of ZO-2 and cingulin, another TJ scaffold protein, respectively, to TJs. When the polarization of ZO-1-/- cells was initiated by a Ca2+ switch, the initial AJ formation did not appear to be affected; however, the subsequent TJ formation (recruitment of claudins/occludin to junctions and barrier establishment) was markedly retarded. This retardation as well as the disappearance of cingulin were rescued completely by exogenous ZO-1 but not by ZO-2 expression. Quantitative evaluation of ZO-1/ZO-2 expression levels led to the conclusion that ZO-1 and ZO-2 would function redundantly to some extent in junction formation/epithelial polarization but that they are not functionally identical. Finally, we discussed advantageous aspects of the gene knock-out system with cultured epithelial cells in epithelial cell biology.     

KIT (c-kit oncogene product) pathway is constitutively activated in human testicular germ cell tumors

We investigated the expression of KIT (product of c-kit oncogene), gain-of-function mutations, and activation of its downstream signal transduction in human testicular cancers. KIT was expressed in 88% (22/25) of seminomas and in 44.4% (4/9) of non-seminomas compared to adjacent normal testicular tissue. Nine of the KIT-expressing seminomas had mutations (40.9%; 9/22) in the c-kit gene; two cases in exon 11 and 7 cases in exon 17. Two of these mutations in exon 17 were novel, and the other seven mutations were identical to the already known gain-of-function mutations which cause activation of KIT without ligand stem cell factor. All of the mutant KIT and 53.8% (7/13) of wild-type KIT were phosphorylated (activated) and associated with phosphorylated phosphatidylinositol 3-kinase (PI3K). Akt was also phosphorylated in these seminomas, suggesting that the KIT-PI3K-Akt pathway is activated in seminoma. These findings suggest that the KIT-PI3K-Akt pathway is constitutively activated in testicular germ cell tumors, due to overexpression of KIT protein and/or gain-of-function mutations in the c-kit gene.