Assay of enzymes forming AMP+PPi by the pyrophosphate determination based on the formation of 18-molybdopyrophosphate

The formation of 18-molybdopyrophosphate anion has been studied to develop a simple and rapid assay of the enzymatic reaction involving ATP→AMP+PPi(P(2)O(7)(4-)). By the addition of P(2)O(7)(4-) anion to an acidic acetonitrile-water solution containing MoO(4)(2-) anion, the colorless Mo(VI) solution immediately became yellow due to the formation of 18-molybdopyrophosphate anion. The absorbance of the P(2)O(7)(4-)-Mo(VI) mixture at, for example, 450nm was proportional to the analytical concentration of P(2)O(7)(4-) anion. Although the test Mo(VI) solution remained colorless by the addition of AMP, it gradually turned to yellow by ATP. The undesired color development is attributed to the formation of a yellow molybdophosphate species accompanied by the dissociation of PO(4)(3-) from the unstable ATP molecule. However, the color development became much slower when ethylenediaminetetraacetic acid was added into an assay mixture, where ATP may form a kinetically stable species. Thus, P(2)O(7)(4-) anion can be determined spectrophotometrically in the enzymatic reaction mixture containing ATP. By the addition of ascorbic acid, the yellow P(2)O(7)(4-)-Mo(VI) mixture turned to blue due to the reduction of the molybdopyrophosphate anion. Thus, P(2)O(7)(4-) anion can be detected colorimetrically by the blueness. The spectrophotometric and colorimetric methods could be applied advantageously to the assay of acetyl-CoA synthetase.     

Regeneration of Vocal Fold Mucosa Using Tissue-Engineered Structures with Oral Mucosal Cells

Objectives

Scarred vocal folds result in irregular vibrations during phonation due to stiffness of the vocal fold mucosa. To date, a completely satisfactory corrective procedure has yet to be achieved. We hypothesize that a potential treatment option for this disease is to replace scarred vocal folds with organotypic mucosa. The purpose of this study is to regenerate vocal fold mucosa using a tissue-engineered structure with autologous oral mucosal cells.

Study Design

Animal experiment using eight beagles (including three controls).

Methods

A 3 mm by 3 mm specimen of canine oral mucosa was surgically excised and divided into epithelial and subepithelial tissues. Epithelial cells and fibroblasts were isolated and cultured separately. The proliferated epithelial cells were co-cultured on oriented collagen gels containing the proliferated fibroblasts for an additional two weeks. The organotypic cultured tissues were transplanted to the mucosa-deficient vocal folds. Two months after transplantation, vocal fold vibrations and morphological characteristics were observed.

Results

A tissue-engineered vocal fold mucosa, consisting of stratified epithelium and lamina propria, was successfully fabricated to closely resemble the normal layered vocal fold mucosa. Laryngeal stroboscopy revealed regular but slightly small mucosal waves at the transplanted site. Immunohistochemically, stratified epithelium expressed cytokeratin, and the distributed cells in the lamina propria expressed vimentin. Elastic Van Gieson staining revealed a decreased number of elastic fibers in the lamina propria of the transplanted site.

Conclusion

The fabricated mucosa with autologous oral mucosal cells successfully restored the vocal fold mucosa. This reconstruction technique could offer substantial clinical advantages for treating intractable diseases such as scarring of the vocal folds.     

Purification and characterization of a non-S-RNase and S-RNases from styles of Japanese pear (Pyrus pyrifolia)

In this study we biochemically characterized stylar ribonucleases (RNases) of Japanese pear (Pyrus pyrifolia), which exhibits S-RNase-based gametophytic self-incompatibility. We separated the RNase fractions NS-1, NS-2, and NS-3 from stylar extracts of the cultivar Nijisseiki (S(2)S(4)). The RNase in each fraction was purified to homogeneity through a series of chromatographic steps. Chemical analysis of the proteins revealed that the basic RNases in the NS-2 and NS-3 fractions were the S(4)- and S(2)-RNases, respectively. Five additional S-RNases were purified from other cultivars. An acidic RNase in the NS-1 fraction was also purified from other cultivars, and identified as a non-S-allele-associated RNase (non-S-RNase). The non-S-RNase is composed of 203 amino acids, is non-glycosylated and is a N-terminal-pyroglutamylated enzyme of the RNase T(2) family. The substrate specificities and optimum pH levels of the non-S-RNase and S-RNases were similar. Interestingly, the specific activity of the non-S-RNase was 7.5-221-fold higher than those of the S-RNases when tolura yeast RNA was used as the substrate. The specific activity of the S(2)-RNase was 8.8-28.6-fold lower than those of the other S-RNases. These differences in specific activities among the stylar RNases are discussed.     

Ablation of Csk in neural crest lineages causes corneal anomaly by deregulating collagen fibril organization and cell motility

Src family kinases (SFKs) have been implicated in the regulation of cell motility. To verify their in vivo roles during development, we generated mutant mice in which Csk, a negative regulator of SFKs, was inactivated in neural crest lineages using the Protein zero promoter in a Cre-loxP system. Inactivation of Csk caused deformities in various tissues of neural crest origins, including facial dysplasia and corneal opacity. In the cornea, the stromal collagen fibril was disorganized and there was an overproduction of collagen 1a1 and several metalloproteases. The corneal endothelium failed to overlie the central region of the eye and the peripheral endothelium displayed a disorganized cytoskeleton. Corneal mesenchymal cells cultured from mutant mice showed attenuated cell motility. In these cells, p130 Crk-associated substrate (Cas) was hyperphosphorylated and markedly downregulated. The expression of a dominant negative Cas (CasΔSD) could suppress the cell motility defects. Fluorescence resonance energy transfer analysis revealed that activation of Rac1 and Cdc42 was depolarized in Csk-inactivated cells, which was restored by the expression of either Csk or CasΔSD. These results demonstrate that the SFKs/Csk circuit plays crucial roles in corneal development by controlling stromal organization and by ensuring cell motility via the Cas-Rac/Cdc42 pathways.     

Changes in the mutagenic and estrogenic activities of 17beta-estradiol after treatment with nitrite

We determined the changes in the mutagenic and estrogenic activities of 17beta-estradiol after a nitrite treatment. Nitrite-treated 17beta-estradiol showed mutagenic activities toward Salmonella typhimurium strains TA 100 and TA 98. We confirmed that nitrite-treated 17beta-estradiol generated radicals from the results of an analysis of electron spin resonance. By applying an instrumental analysis, we identified 2-nitro-17beta-estradiol to have been formed in the reaction mixture. 2-Nitro-17beta-estradiol did not exhibit mutagenic activities toward Salmonella typhimurium strains, suggesting that other mutagens might have been formed in the reaction mixture. The clastogenic properties of nitrite-treated 17beta-estradiol and 2-nitro-17beta-estradiol were analyzed by a micronucleus test with male ICR mice. Nitrite-treated 17beta-estradiol and 2-nitro-17beta-estradiol induced a significantly higher frequency of micronucleated reticulocytes in mice. The estrogenic activity of 2-nitro-17beta-estradiol was found to be lower than that of 17beta-estradiol. These data suggest that a daily oral intake of 17beta-estradiol and nitrite might induce the formation of mutagenic compounds in our body.     

Extracellular superoxide dismutase in tissues from obese (ob/ob) mice

We have examined the protein content and gene expression of three superoxide dismutase (SOD) isoenzymes in eight tissues from obese ob/ob mice, particularly placing the focus on extracellular-SOD (EC-SOD) in the white adipose tissue (WAT). Obesity significantly increased EC-SOD level in liver, kidney, testis, gastrocnemius muscle, WAT, brown adipose tissue (BAT), and plasma, but significantly decreased the isoenzyme level in lung. Tumor necrosis factor-α and interleukin-1β contents in WAT were significantly higher in obese mice than in lean control mice. Immunohistochemically, both WAT and BAT from obese mice could be stained deeply with anti-mouse EC-SOD antibody compared with those from lean mice. Each primary culture per se almost time-dependently enhanced EC-SOD production, and overtly expressed its mRNA. The loss of heparin-binding affinity of EC-SOD type C with high affinity for heparin occurred in kidney of obese mice. These results suggest that the physiological importance of this SOD isoenzyme in WAT may be a compensatory adaptation to oxidative stress.     

Extreme winter aggregation of invasive rainbow trout in small tributaries: implications for effective control

Because of their high economic and recreational value, salmonids have been extensively introduced worldwide and are responsible for significant impacts on native ecosystems. However, effective methods for controlling or eradicating introduced populations of salmonids are still limited, particularly in large river systems. Here, we demonstrated that invasive rainbow trout Oncorhynchus mykiss immigrated to and extremely aggregated in small tributaries during the winter, which can be utilized for effective population control. Seasonal census was conducted in 10 small tributaries of the Otofuke River in the Tokachi River basin, central Hokkaido, Japan. Winter abundance was 8–125 times higher than summer abundance in four of the tributaries where water velocities were low. Relatively large individuals (200–350 mm) aggregated in several pools at an unusually high density (>2–3 individuals/m² or 170–440 individuals per pool). Females were common in the small tributaries in the winter, but not in the summer. Therefore, removal of invasive rainbow trout may be best practised in such small tributaries during early winter. Because many stream fishes overwinter in specific, often limited, habitats, more attention should be paid to such winter habits in the effective management of non-native fish species.     

Proteomic identification of ZO-1/2 as a novel scaffold for Src/Csk regulatory circuit

To elucidate the regulatory mechanism of cell transformation induced by c-Src tyrosine kinase, we performed a proteomic analysis of tyrosine phosphorylated proteins that interact with c-Src and/or its negative regulator Csk. The c-Src interacting proteins were affinity-purified from Src transformed cells using the Src SH2 domain as a ligand. LC-MS/MS analysis of the purified proteins identified general Src substrates, such as focal adhesion kinase and paxillin, and ZO-1/2 as a transformation-dependent Src target. The Csk binding proteins were analyzed by a tandem affinity purification method. In addition to the previously identified Csk binding proteins, including Cbp/PAG, paxillin, and caveolin-1, we found that ZO-1/2 could also serve as a major Csk binding protein. ZO-2 was phosphorylated concurrently with Src transformation and specifically bound to Csk in a Csk SH2 dependent manner. These results suggest novel roles for ZO proteins as Src/Csk scaffolds potentially involved in the regulation of Src transformation.