Growth condition and bacterial community for maximum hydrolysis of suspended organic materials in anaerobic digestion of food waste-recycling wastewater

This paper reports the effects of changing pH (5–7) and temperature (T, 40–60 °C) on the efficiencies of bacterial hydrolysis of suspended organic matter (SOM) in wastewater from food waste recycling (FWR) and the changes in the bacterial community responsible for this hydrolysis. Maximum hydrolysis efficiency (i.e., 50.5% reduction of volatile suspended solids) was predicted to occur at pH 5.7 and T = 44.5 °C. Changes in short-chain volatile organic acid profiles and in acidogenic bacterial communities were investigated under these conditions. Propionic and butyric acids concentrations increased rapidly during the first 2 days of incubation. Several band sequences consistent with Clostridium spp. were detected using denaturing gel gradient electrophoresis. Clostridium thermopalmarium and Clostridium novyi seemed to contribute to butyric acid production during the first 1.5 days of acidification of FWR wastewater, and C. thermopalmarium was a major butyric acid producer afterward. C. novyi was an important propionic acid producer. These two species appear to be important contributors to hydrolysis of SOM in the wastewater. Other acidogenic anaerobes, Aeromonas sharmana, Bacillus coagulans, and Pseudomonas plecoglossicida, were also indentified.     

c-Cbl-mediated degradation of TRAIL receptors is responsible for the development of the early phase of TRAIL resistance

We previously reported two modes of development of acquired TRAIL resistance: early phase and late phase [1]. In these studies, we observed that greater Akt activity and the expression of Bcl-xL were related mainly to the late phase of acquired TRAIL resistance.Recently we became aware of a possible mechanism of early phase TRAIL resistance development through internalization and degradation of TRAIL receptors (DR4 and DR5). Our current studies demonstrate that TRAIL receptors rapidly diminish at the membrane as well as the cytoplasm within 4 h after TRAIL exposure, but recover completely after one or two days. Our studies also reveal that Cbl, a ubiquitously expressed cytoplasmic adaptor protein, is responsible for the rapid degradation of TRAIL receptors; Cbl binds to them and induces monoubiquitination of these receptors concurrent with their degeneration soon after TRAIL exposure, creating the early phase of acquired TRAIL resistance.     

Thymosin Beta4 Regulates Cardiac Valve Formation Via Endothelial-Mesenchymal Transformation in Zebrafish Embryos

Thymosin beta4 (TB4) has multiple functions in cellular response in processes as diverse as embryonic organ development and the pathogeneses of disease, especially those associated with cardiac coronary vessels. However, the specific roles played by TB4 during heart valve development in vertebrates are largely unknown. Here, we identified a novel function of TB4 in endothelialmesenchymal transformation (EMT) in cardiac valve endocardial cushions in zebrafish. The expressions of thymosin family members in developing zebrafish embryos were determined by whole mount in situ hybridization. Of the thymosin family members only zTB4 was expressed in the developing heart region. Cardiac valve development at 48 h post fertilization was defected in zebrafish TB4 (zTB4) morpholino-injected embryos (morphants). In zTB4 morphants, abnormal linear heart tube development was observed. The expressions of bone morphogenetic protein (BMP) 4, notch1b, and hyaluronic acid synthase (HAS) 2 genes were also markedly reduced in atrio-ventricular canal (AVC). Endocardial cells in the AVC region were stained with anti-Zn5 antibody reactive against Dm-grasp (an EMT marker) to observe EMT in developing cardiac valves in zTB4 morphants. EMT marker expression in valve endothelial cells was confirmed after transfection with TB4 siRNA in the presence of transforming growth factor β (TGFβ) by RT-PCR and immunofluorescent assay. Zn5-positive endocardial AVC cells were not observed in zTB4 morphants, and knockdown of TB4 suppressed TGF-β-induced EMT in ovine valve endothelial cells. Taken together, our results demonstrate that TB4 plays a pivotal role in cardiac valve formation by increasing EMT.     

Heat shock augments myosin phosphatase target-subunit phosphorylation

Our previous study demonstrated that heat shock augmented vascular contraction. In the present study, we hypothesized that heat shock augments myosin phosphatase target-subunit (MYPT1) phosphorylation resulting in augmented vascular contraction. Endothelium-denuded rat aortic rings were mounted in organ baths, exposed to heat shock (42 degrees C for 45 min), and subjected to contraction 4 h after the heat shock followed by Western blot analysis for MLC(20) (the 20 kDa light chains of myosin II) or MYPT1. The contractile responses in both control and heat shock-treated aorta were inhibited by Y27632, an inhibitor of Rho-kinase. The level of the MLC(20) and MYPT1(Thr855) phosphorylation in response to KCl was higher in heat shock-treated aorta than that in timed-control. The increased MYPT1(Thr855) phosphorylation was inhibited by Y27632 (1.0 microM) in parallel with inhibition of MLC(20) phosphorylation and vascular contraction. These results indicate that heat shock augments MYPT1 phosphorylation resulting in augmented vascular contraction.     

Alternative SAIL-Trp for robust aromatic signal assignment and determination of the χ<Subscript>2</Subscript> conformation by intra-residue NOEs

Tryptophan (Trp) residues are frequently found in the hydrophobic cores of proteins, and therefore, their side-chain conformations, especially the precise locations of the bulky indole rings, are critical for determining structures by NMR. However, when analyzing [U–¹³C,¹⁵N]-proteins, the observation and assignment of the ring signals are often hampered by excessive overlaps and tight spin couplings. These difficulties have been greatly alleviated by using stereo-array isotope labeled (SAIL) proteins, which are composed of isotope-labeled amino acids optimized for unambiguous side-chain NMR assignment, exclusively through the ¹³C–¹³C and ¹³C–¹H spin coupling networks (Kainosho et al. in Nature 440:52–57, 2006). In this paper, we propose an alternative type of SAIL-Trp with the [ζ2,ζ3-²H₂; δ1,ε3,η2-¹³C₃; ε1-¹⁵N]-indole ring ([¹²C_γ, ¹²C_ε2] SAIL-Trp), which provides a more robust way to correlate the ¹H_β, ¹H_α, and ¹H_N to the ¹H_δ1 and ¹H_ε3 through the intra-residue NOEs. The assignment of the ¹H_δ1/¹³C_δ1 and ¹H_ε3/¹³C_ε3 signals can thus be transferred to the ¹H_ε1/¹⁵N_ε1 and ¹H_η2/¹³C_η2 signals, as with the previous type of SAIL-Trp, which has an extra ¹³C at the C_γ of the ring. By taking advantage of the stereospecific deuteration of one of the prochiral β-methylene protons, which was ¹H_β2 in this experiment, one can determine the side-chain conformation of the Trp residue including the χ₂ angle, which is especially important for Trp residues, as they can adopt three preferred conformations. We demonstrated the usefulness of [¹²C_γ,¹²C_ε2] SAIL-Trp for the 12 kDa DNA binding domain of mouse c-Myb protein (Myb-R2R3), which contains six Trp residues.     

Tau and GSK3β Dephosphorylations are Required for Regulating Pin1 Phosphorylation

Pin1 binds mitotically phosphorylated Thr231–Pro232 and Thr212–Pro213 sites on tau, and a Pin1 deficiency in mice leads to tau hyperphosphorylation. The aim of this study was to determine if the dephosphorylation or inhibition of tau and GSK3β phosphorylation induces the Pin1 phosphorylation. To test this, human SK-N-MC cells were stably transfected with a fusion gene containing neuron-specific enolase (NSE)-controlled APPsw gene(NSE/APPsw), to induce Aβ-42. The stable transfectants were then transiently transfected with NSE/Splice, lacking human tau (NSE/Splice), or NSE/hTau, containing human tau, into the cells. The NSE/Splice- and NSE/hTau-cells were then treated with lithium. We concluded that (i) there was more C99-β APP accumulation than C83-βAPP in APPsw-tansfectant and thereby promoted Aβ-42 production in transfectants. (ii) the inhibition of tau and GSK3β phosphorylations correlated with increase in Pin1 activation in NSE/hTau- cells. Thus, these observations suggest that Pin1 might have an inhibitive role in phosphorylating tau and GSK3β for protecting against Alzheimer’s disease.     

The snpA, a temperature-sensitive suppressor of npgA1, encodes the eukaryotic translation release factor, eRF1, in Aspergillus nidulans

The npgA1 mutation causes defects in the outer layer of the cell wall resulting in a colorless colony. In this study, a temperature-sensitive suppressor of npgA1 named snpA was isolated by UV mutagenesis. The suppressing mutant showed pleiotropic phenotypes in cellular structure and developmental processes when incubated at a temperature of 37 degrees C or above. At 37 degrees C, multiple germ tubes emerged from germinating conidia. Moreover, at 42 degrees C conidia germination was delayed more than 12h and hyphal growth was strongly inhibited. The suppressor allele, snpA6, is recessive and maps to the linkage group III. A gene complementing the mutation was identified employing the chromosome III-specific cosmid library. Sequencing analysis revealed that the snpA gene encodes the eukaryotic polypeptide release factor, eRF1. The snpA6 allele contains a G-A mutation resulting in SnpA(E117K), which may allow read-through of the nonsense mutation in the npgA1 allele in a similar manner to the yeast omni-potent suppressor SUP45 and SUP35.