Calpain-dependent Cleavage of N-cadherin Is Involved in the Progression of Post-myocardial Infarction Remodeling

Enzymatic proteolysis by calpains, Ca²⁺-dependent intracellular cysteine proteases, has been implicated in pathological processes such as cellular degeneration or death. Here, we investigated the role of calpain activation in the hearts subjected to myocardial infarction. We produced myocardial infarction in Cast^−/− mice deficient for calpastatin, the specific endogenous inhibitory protein for calpains, and Cast^+/+ mice. The activity of cardiac calpains in Cast^+/+ mice was not elevated within 1 day but showed a gradual elevation after 7 days following myocardial infarction, which was further pronounced in Cast^−/− mice. Although the prevalence of cardiomyocyte death was indistinguishable between Cast^−/− and Cast^+/+ mice, Cast^−/− mice exhibited profound contractile dysfunction and chamber dilatation and showed a significant reduction in survival rate after myocardial infarction as compared with Cast^+/+ mice. Notably, immunofluorescence revealed that at 28 days after myocardial infarction, calpains were activated in cardiomyocytes exclusively at the border zone and that Cast^−/− mice showed higher intensity and a broader extent of calpain activation at the border zone than Cast^+/+ mice. In the border zone of Cast^−/− mice, pronounced activation of calpains was associated with a decrease in N-cadherin expression and up-regulation of molecular markers for cardiac hypertrophy and fibrosis. In cultured rat neonatal cardiomyocytes, calpain activation by treatment with ionomycin induced cleavage of N-cadherin and decreased expression levels of β-catenin and connexin 43, which was attenuated by calpain inhibitor. These results thus demonstrate that activation of calpains disassembles cell-cell adhesion at intercalated discs by degrading N-cadherin and thereby promotes left ventricular remodeling after myocardial infarction.     

Asp-52 in Combination with Asp-398 Plays a Critical Role in ATP Hydrolysis of Chaperonin GroEL

The Escherichia coli chaperonin GroEL is a double-ring chaperone that assists protein folding with the aid of GroES and ATP. Asp-398 in GroEL is known as one of the critical residues on ATP hydrolysis because GroEL(D398A) mutant is deficient in ATP hydrolysis (<2% of the wild type) but not in ATP binding. In the archaeal Group II chaperonin, another aspartate residue, Asp-52 in the corresponding E. coli GroEL, in addition to Asp-398 is also important for ATP hydrolysis. We investigated the role of Asp-52 in GroEL and found that ATPase activity of GroEL(D52A) and GroEL(D52A/D398A) mutants was ∼20% and <0.01% of wild-type GroEL, respectively, indicating that Asp-52 in E. coli GroEL is also involved in the ATP hydrolysis. GroEL(D52A/D398A) formed a symmetric football-shaped GroEL-GroES complex in the presence of ATP, again confirming the importance of the symmetric complex during the GroEL ATPase cycle. Notably, the symmetric complex of GroEL(D52A/D398A) was extremely stable, with a half-time of ∼150 h (∼6 days), providing a good model to characterize the football-shaped complex.     

Inhibition of transforming growth factor-β signaling potentiates tumor cell invasion into collagen matrix induced by fibroblast-derived hepatocyte growth factor

Interaction between tumor cells and stromal fibroblasts plays essential roles in tumor progression. However, its detailed molecular mechanism remains unclear. To understand the mechanism, we investigated molecules mediating this interaction using the three-dimensional (3D) co-culture system of Panc-1 pancreatic carcinoma cells with normal fibroblasts. When the two kinds of cells were placed on the top of collagen gel, the tumor cells scattered into the fibroblast layer, apparently undergoing epithelial‐mesenchymal transition. When fibroblasts were placed within collagen gel, Panc-1 cells actively invaded into the collagen gel, extending a microtubule-based long protrusion. Although transforming growth factor-β (TGF-β) and hepatocyte growth factor (HGF) individually stimulated the tumor cell invasion into collagen gel without fibroblasts, TGF-β signaling inhibitors (SB431542 and LY2157299) significantly enhanced the Panc-1 cell invasion in the 3D co-culture with fibroblasts. Experiments with HGF/Met signaling inhibitors or with the fibroblast conditioned medium revealed that HGF was a major invasion-promoting factor secreted from fibroblasts and SB431542 increased the HGF secretion by blocking the HGF-suppressing activity of cancer cell-derived TGF-β. These results indicate that HGF and TGF-β are critical regulators for both tumor–stroma interaction and tumor invasion. The results also suggest that TGF-β signaling inhibitors may promote tumor progression under some pathological conditions.     

Mycorrhizal symbiosis and Lygodium microphyllum Invasion in South Florida—a biogeographic comparison

Lygodium microphyllum (Old World Climbing Fern) is one of the most problematic weeds in south Florida, invading numerous habitats from mangroves to pine flatwoods natural ecosystems. Much of the research efforts on L. microphyllum has been focused on reproductive potential, spore release, growth under different environmental conditions, belowground rhizome dormancy and survival strategies that describes its invasiveness. However, the role of an important mutualistic association with arbuscular mycorrhizal fungi (AMF) in the competitive ability and successful invasion of L. microphyllum by enhancing nutrient uptake has not been previously considered. Analysis of field root and soil samples from the ferns introduced and native range as well as a 7-week growth chamber experiment were done to determine the level of mycorrhizal colonization in the roots of L. microphyllum and the dependency on mycorrhizal fungi for growth and phosphorus (P) uptake. The field root samples showed that L. microphyllum was heavily colonized by AMF in relatively drier conditions, which are commonly found on some Florida sites compared to more common wetter sites where the fern is found in its native Australia. The results from the growth chamber experiment showed that the mycorrhizal treatment plants had significantly higher relative growth rate and biomass compared to the non-mycorrhizal plants. Similarly, L. microphyllum was highly dependent on the mycorrhizal fungi for growth and P uptake. Our results suggest that AMF play a significant role in vegetative reproduction and likely enhance the invasiveness of L. microphyllum in south Florida natural areas.     

Difference in cesium accumulation among rice cultivars grown in the paddy field in Fukushima Prefecture in 2011 and 2012

After the accident of the Fukushima 1 Nuclear Power Plant in March 2011, radioactive cesium was released and paddy fields in a wide area including Fukushima Prefecture were contaminated. To estimate the levels of radioactive Cs accumulation in rice produced in Fukushima, it is crucial to obtain the actual data of Cs accumulation levels in rice plants grown in the actual paddy field in Fukushima City. We herein conducted a two-year survey in 2011 and 2012 of radioactive and non-radioactive Cs accumulation in rice using a number of rice cultivars grown in the paddy field in Fukushima City. Our study demonstrated a substantial variation in Cs accumulation levels among the cultivars of rice.     

Draft Sequences of the Radish (Raphanus sativus L.) Genome

Radish (Raphanus sativus L., n = 9) is one of the major vegetables in Asia. Since the genomes of Brassica and related species including radish underwent genome rearrangement, it is quite difficult to perform functional analysis based on the reported genomic sequence of Brassica rapa. Therefore, we performed genome sequencing of radish. Short reads of genomic sequences of 191.1 Gb were obtained by next-generation sequencing (NGS) for a radish inbred line, and 76,592 scaffolds of ≥300 bp were constructed along with the bacterial artificial chromosome-end sequences. Finally, the whole draft genomic sequence of 402 Mb spanning 75.9% of the estimated genomic size and containing 61,572 predicted genes was obtained. Subsequently, 221 single nucleotide polymorphism markers and 768 PCR-RFLP markers were used together with the 746 markers produced in our previous study for the construction of a linkage map. The map was combined further with another radish linkage map constructed mainly with expressed sequence tag-simple sequence repeat markers into a high-density integrated map of 1,166 cM with 2,553 DNA markers. A total of 1,345 scaffolds were assigned to the linkage map, spanning 116.0 Mb. Bulked PCR products amplified by 2,880 primer pairs were sequenced by NGS, and SNPs in eight inbred lines were identified.     

Pilot-scale experiment of down-flow hanging sponge for direct treatment of low-strength municipal wastewater in Bangkok,Thailand

A pilot-scale experiment of a down-flow hanging sponge (DHS) reactor for treatment of low-strength municipal wastewater was conducted over 1 year in Bangkok, Thailand, to establish an appropriate method for treatment under tropical climate conditions. Municipal wastewater with an average BOD of 19 mg/L was fed directly into the DHS reactor. Superior effluent quality (5.1 ± 3.4 mg/L TSS, 21.1 ± 9.0 mg/L COD, 2.8 ± 1.4 mg/L BOD, and 4.1 ± 1.0 mg/L TN) was achieved at a hydraulic retention time (HRT) of 1 h under an average temperature of 30 °C. The DHS reactor reached an actual HRT of 19.0 min, indicating good contact efficiency between wastewater and retained sludge. The DHS reactor retained dense sludge at 15.3–26.4 g VSS/L based on the sponge media volume. The sludge activity in terms of specific oxygen uptake rate was good. Excess sludge was produced as 0.051 g TSS/g COD removed (0.11 g TSS/g BOD removed), and a good SVI of 28 mL/g was observed. The sufficient performance was attributed to dense sludge with high activity, regardless of the low-strength wastewater. Overall, the DHS was advantageous owing to its simple operation, lack of operational problems, and low power consumption.     

Relationship between stable cesium concentration and body size of Japanese flounder Paralichthys olivaceus and the effect of a size-dependent shift in diet

To understand the relationship between the radioactive cesium (Cs) concentration in muscle of Japanese flounder Paralichthys olivaceus and the species' biological characteristics (size, sex, and age) under conditions of ecological equilibrium (i.e., distributed among ecosystem components over sufficient time, and with nearly constant ratios of Cs concentration in organisms to the concentration in water) as existed before the accident at the Fukushima Dai-ichi Nuclear Power Station (FDNPS), Japan, in 2011, we examined stable Cs, as it is thought to exist in equilibrium in the environment and behave similarly to radioactive Cs in aquatic animals. The concentration of stable Cs in 241 P. olivaceus (range 216–782 mm total length [TL]) collected in Sendai Bay, approximately 90 km north of the FDNPS, in June–July 2015 was expressed as an exponential function with size as an independent variable; the results show the concentration of stable Cs doubled with an increase in TL of 442 mm. Next, to evaluate the cause of the size-dependent change in stable Cs concentration, we examined 909 individuals (200–770 mm TL) collected in September 2013–July 2015 to determine their feeding habit based on size. Analysis of the frequency of occurrence of prey organisms in stomach contents showed that sand lance Ammodytes japonicus (55–180 mm standard length [SL]) was the most consistently consumed across size classes. Analysis on a wet-mass basis showed that A. japonicus and anchovy Engraulis japonicus (65–130 mm SL) were the main food of P. olivaceus sized 200–599 mm TL, whereas chub mackerel Scomber japonicus (120–230 mm SL) and two species of flatfishes (180–205 mm SL) were abundant in the diet of P. olivaceus sized ≥600 mm TL. All these prey items were presumed to have similar concentrations of stable Cs. Based on the above, the effect of diet on the relationship between stable Cs in muscle and fish size was considered negligible. That the diet of P. olivaceus largely did not change with size was also confirmed by C and N stable isotope ratios in P. olivaceus and their prey species. Therefore, the Cs–size relationship is probably determined by changes in the balance between the rate of Cs intake from food and seawater and the excretion rate during growth, both of which change as functions of body mass. Values of stable Cs concentrations among environmental components and animals appear to be a valid indicator for understanding the radioactive Cs distribution in the marine environment and aquatic animals under the equilibrium state, as existed before the 2011 nuclear accident.