Purification and Properties of Orotidine-5′-phosphate Pyrophosphorylase in Micrococcus glutamicus.

A coupled enzyme system of orotidine-5′-phosphate pyrophosphorylase and orotidine-5′-phosphate decarboxylase has been purified approximately 30-fold from cell-free extract of Micrococcus glutamicus 534 Co–147 by means of acid treatment and fractionations with ammonium sulfate and ethanol addition, and properties of the enzyme system have been studied.

Optima of pH, temperature and substrate concentrations for the activity of the purified enzyme system have been investigated, and compared with those of the same enzyme system from dried brewer’s yeast. Furthermore, effects of various inhibitors on the enzyme activity have been examined and it has become evident that the enzyme system is completely inactivated by addition of chelating agent such as EDTA, and regenerated by further addition of magnesium ion.     

Overexpression of the NADP+-specific isocitrate dehydrogenase gene (icdA) in citric acid-producing Aspergillus niger WU-2223L

In the tricarboxylic acid (TCA) cycle, NADP⁺-specific isocitrate dehydrogenase (NADP⁺-ICDH) catalyzes oxidative decarboxylation of isocitric acid to form α-ketoglutaric acid with NADP⁺ as a cofactor. We constructed an NADP⁺-ICDH gene (icdA)-overexpressing strain (OPI-1) using Aspergillus niger WU-2223L as a host and examined the effects of increase in NADP⁺-ICDH activity on citric acid production. Under citric acid-producing conditions with glucose as the carbon source, the amounts of citric acid produced and glucose consumed by OPI-1 for the 12-d cultivation period decreased by 18.7 and 10.5%, respectively, compared with those by WU-2223L. These results indicate that the amount of citric acid produced by A. niger can be altered with the NADP⁺-ICDH activity. Therefore, NADP⁺-ICDH is an important regulator of citric acid production in the TCA cycle of A. niger. Thus, we propose that the icdA gene is a potentially valuable tool for modulating citric acid production by metabolic engineering.     

A New Metabolite,Parasiticolide A,from Aspergillus parasiticus

Two β-glucosidases, G1 and G2, were purified from the culture supernatant of Penicillium herquei Banier and Sartory. Both the purified enzymes were homogeneous on polyacrylamide disc gel electrophoresis. The molecular weights of G1 and G2 were estimated to be 125,000 and 122,000, respectively, by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. G1 and G2 contained 12.7% and 16.1% carbohydrate as glucose, and had isoelectric points of 5.02 and 5.24, respectively. Both enzymes had optimum pHs of 4.0~4.5 and optimum temperatures at 60°C, but pH - and thermo-stabilities of G1 were higher than those of G2. Both enzymes were active not only on p-nitrophenyl β-d-glucopyranoside, salicin, and the p-glucobioses tested but also on laminarin. CM-Cellulose was a very poor substrate for both enzymes. The activities of G1 toward the substrates except for laminarin and CM-cellulose were apparently higher than those of G2. Both enzymes acted on cellobiose to produce a transfer product.     

P2X7 Integrates PI3K/AKT and AMPK-PRAS40-mTOR Signaling Pathways to Mediate Tumor Cell Death

Background

Extracellular adenosine triphosphate (ATP) functions as a novel danger signal that boosts antitumor immunity and can also directly kill tumor cells. We have previously reported that chronic exposure of tumor cells to ATP provokes P2X7-mediated tumor cell death, by as yet incompletely defined molecular mechanisms.

Methodology/Principal Findings

Here, we show that acute exposure of tumor cells to ATP results in rapid cytotoxic effects impacting several aspects of cell growth/survival, leading to inhibition of tumor growth in vitro and in vivo. Using agonist and antagonist studies together with generation of P2X7 deficient tumor cell lines by lentiviral shRNA delivery system, we confirm P2X7 to be the central control node transmitting extracellular ATP signals. We identify that downstream intracellular signaling regulatory networks implicate two signaling pathways: the known P2X7-PI3K/AKT axis and remarkably a novel P2X7-AMPK-PRAS40-mTOR axis. When exposed to high levels of extracellular ATP, these two signaling axes perturb the balance between growth and autophagy, thereby promoting tumor cell death.

Conclusions

Our study defines novel molecular mechanisms underpinning the antitumor actions of P2X7 and provides a further rationale for purine-based drugs in targeted cancer therapy.     

Environmental DNA as a ‘Snapshot’ of Fish Distribution: A Case Study of Japanese Jack Mackerel in Maizuru Bay,Sea of Japan

Recent studies in streams and ponds have demonstrated that the distribution and biomass of aquatic organisms can be estimated by detection and quantification of environmental DNA (eDNA). In more open systems such as seas, it is not evident whether eDNA can represent the distribution and biomass of aquatic organisms because various environmental factors (e.g., water flow) are expected to affect eDNA distribution and concentration. To test the relationships between the distribution of fish and eDNA, we conducted a grid survey in Maizuru Bay, Sea of Japan, and sampled surface and bottom waters while monitoring biomass of the Japanese jack mackerel (Trachurus japonicus) using echo sounder technology. A linear model showed a high R² value (0.665) without outlier data points, and the association between estimated eDNA concentrations from the surface water samples and echo intensity was significantly positive, suggesting that the estimated spatial variation in eDNA concentration can reflect the local biomass of the jack mackerel. We also found that a best-fit model included echo intensity obtained within 10–150 m from water sampling sites, indicating that the estimated eDNA concentration most likely reflects fish biomass within 150 m in the bay. Although eDNA from a wholesale fish market partially affected eDNA concentration, we conclude that eDNA generally provides a ‘snapshot’ of fish distribution and biomass in a large area. Further studies in which dynamics of eDNA under field conditions (e.g., patterns of release, degradation, and diffusion of eDNA) are taken into account will provide a better estimate of fish distribution and biomass based on eDNA.     

Associations among Erythroferrone and Biomarkers of Erythropoiesis and Iron Metabolism,and Treatment with Long-Term Erythropoiesis-Stimulating Agents in Patients on Hemodialysis

Background

We aimed to identify associations between erythroferrone (ERFE), a regulator of hepcidin 25, and biomarkers of erythropoiesis and iron metabolism. We also aimed to determine the effects of erythropoiesis-stimulating agents (ESA), continuous erythropoietin receptor activator (CERA) and darbepoetin-α (DA) on ERFE production in patients on hemodialysis (HD).

Methods

Blood samples were obtained from 59 patients before HD sessions on day 0 (baseline). Twenty patients who were injected with either CERA (N = 10) or DA (N = 10) at the end of the dialysis week (day 0), who had ferritin ≥ 100 ng/mL and/or transferrin saturation ≥ 20%, and hemoglobin > 9 g/dL were selected from among the 59 patients. Blood was sampled serially before HD sessions on days 3, 5, 7 from patients on DA and on the same days plus day 14 from those on CERA.

Results

Levels of ERFE correlated inversely with those of hepcidin 25 and ferritin, and positively with those of soluble transferrin receptor. The hepcidin 25: ERFE ratio and hepcidin 25 levels positively correlated with ferritin levels. Levels of ERFE significantly increased from day 3 of treatment with DA and CERA and decreased by days 7 and 14, respectively. Erythropoiesis-stimulating agents concomitantly decreased levels of hepcidin 25 as those of ERFE increased.

Conclusion

We identified a novel association between ESA and ERFE in patients on HD. Both DA and CERA increased levels of ERFE that regulated hepcidin 25 and led to iron mobilization from body stores during erythropoiesis.     

(Pro)renin receptor accelerates development of sarcopenia via activation of Wnt/YAP signaling axis

To extend life expectancy and ensure healthy aging, it is crucial to prevent and minimize age‐induced skeletal muscle atrophy, also known as sarcopenia. However, the disease's molecular mechanism remains unclear. The age‐related Wnt/β‐catenin signaling pathway has been recently shown to be activated by the (pro)renin receptor ((P)RR). We report here that (P)RR expression was increased in the atrophied skeletal muscles of aged mice and humans. Therefore, we developed a gain‐of‐function model of age‐related sarcopenia via transgenic expression of (P)RR under control of the CAG promoter. Consistent with our hypothesis, (P)RR‐Tg mice died early and exhibited muscle atrophy with histological features of sarcopenia. Moreover, Wnt/β‐catenin signaling was activated and the regenerative capacity of muscle progenitor cells after cardiotoxin injury was impaired due to cell fusion failure in (P)RR‐Tg mice. In vitro forced expression of (P)RR protein in C2C12 myoblast cells suppressed myotube formation by activating Wnt/β‐catenin signaling. Administration of Dickkopf‐related protein 1, an inhibitor of Wnt/β‐catenin signaling, and anti‐(P)RR neutralizing antibody, which inhibits binding of (P)RR to the Wnt receptor, significantly improved sarcopenia in (P)RR‐Tg mice. Furthermore, the use of anti‐(P)RR neutralizing antibodies significantly improved the regenerative ability of skeletal muscle in aged mice. Finally, we show that Yes‐associated protein (YAP) signaling, which is coordinately regulated by Wnt/β‐catenin, contributed to the development of (P)RR‐induced sarcopenia. The present study demonstrates the use of (P)RR‐Tg mice as a novel sarcopenia model, and shows that (P)RR‐Wnt‐YAP signaling plays a pivotal role in the pathogenesis of this disease.     

Sexual system in the tanaidacean Falsapseudes bowmani (Crustacea: Malacostraca: Peracarida)

With more than 40,000 species, Malacostraca is the most diverse crustacean class. Most malacostracans are gonochoristic, but simultaneous hermaphrodites are also known. Tanaidacea is one of two malacostracan orders that includes simultaneously hermaphroditic species; so far, simultaneous hermaphroditism has been confirmed externally and internally in only two tanaidacean species, both in the genus Apseudes (Apseudidae). Here we show, through external and internal morphological observations of fixed specimens, that the apseudid Falsapseudes bowmani is a simultaneous hermaphrodite, making Falsapseudes the second tanaidacean genus in which simultaneous hermaphroditism has been confirmed both externally and internally. In this species, the epistome (a projection on the clypeus) was thick and elongate in large specimens but was thin and spiniform in smaller specimens; the brooding of eggs or embryos was observed only in thin‐epistome individuals, although a pair of ovaries was confirmed in both thick‐ and thin‐epistome individuals. This suggests that individuals with a thick epistome may act as males while also retaining the female reproductive organs.     

Possible requirement of intercellular adhesion molecule-1 for invasion of gingival epithelial cells by Treponema medium

Oral treponemes are members of the spirochete family of bacteria associated with periodontal diseases. In the present study, we demonstrate that intercellular adhesion molecule-1 (ICAM-1) on human gingival epithelial cells (HGEC) contributed to the invasion of Treponema medium, a medium-sized oral Treponema, into those cells. The quantity of T. medium in HGEC was found to peak at 2 h after inoculation and then decreased gradually. Immunofluorescence microscopy findings showed that the bacteria were colocalized with ICAM-1 on HGEC. Furthermore, knockdown of ICAM-1 in HGEC resulted in inhibition of T. medium invasion by RNA interference, whereas that of Toll-like receptor 2 did not. These results suggest that ICAM-1 may be required for the invasion of T. medium into HGEC, and they indicate that the molecule plays a principal role in the primary stages of the development and progression of chronic periodontitis.