Near-UV Circular Dichroism and UV Resonance Raman Spectra of Individual Tryptophan Residues in Human Hemoglobin and Their Changes upon the Quaternary Structure Transition

The aromatic residues such as tryptophan (Trp) and tyrosine (Tyr) in human adult hemoglobin (Hb A) are known to contribute to near-UV circular dichroism (CD) and UV resonance Raman (RR) spectral changes upon the R → T quaternary structure transition. In Hb A, there are three Trp residues per αβ dimer: at α14, β15, and β37. To evaluate their individual contributions to the R → T spectral changes, we produced three mutant hemoglobins in E. coli; rHb (α14Trp→Leu), rHb (β15Trp→Leu), and rHb (β37Trp→His). Near-UV CD and UVRR spectra of these mutant Hbs were compared with those of Hb A under solvent conditions where mutant rHbs exhibited significant cooperativity in oxygen binding. Near-UV CD and UVRR spectra for individual Trp residues were extracted by the difference calculations between Hb A and the mutants. α14 and β15Trp exhibited negative CD bands in both oxy- and deoxy-Hb A, whereas β37Trp showed positive CD bands in oxy-Hb A but decreased intensity in deoxy-form. These differences in CD spectra among the three Trp residues in Hb A were ascribed to surrounding hydrophobicity by examining the spectral changes of a model compound of Trp, N-acetyl-l-Trp ethyl ester, in various solvents. Intensity enhancement of Trp UVRR bands upon the R → T transition was ascribed mostly to the hydrogen-bond formation of β37Trp in deoxy-Hb A because similar UVRR spectral changes were detected with N-acetyl-l-Trp ethyl ester upon addition of a hydrogen-bond acceptor.     

Iron overload inhibits calcification and differentiation of ATDC5 cells

There is a little information about the effects of iron overload on cartilage metabolism. In the present study, we examined the effects of excess iron on the differentiation and mineralization of cultured chondrocytes, ATDC5 cells. We used ferric ammonium citrate (FAC) as a ferric ion donor and desferrioxamine (DFO) as a ferric ion chelator. Neither chemical affected the production of proteoglycan, a marker of an early stage of ATDC5 differentiation. In contrast, FAC inhibited the deposition of calcium, a late-stage event in chondrocyte differentiation, by ATDC5 cells in a dose-dependent manner, and DFO accelerated it. Energy dispersive X-ray spectroscopy/scanning electron microscope analysis revealed that the levels of iron and calcium in cells treated with FAC were increased and decreased, respectively. Furthermore, FAC inhibited the expression of matrix metalloproteinase 13 mRNA, another marker of late-stage chondrocyte differentiation. In addition, we found that the heavy and light chains of ferritin were expressed specifically at a late stage of ATDC5 differentiation, and the levels of both proteins were enhanced by the addition of iron. These results suggest that iron overload might give rise to osteopenia and arthritis by inhibiting chondrocyte differentiation and mineralization.     

Learning to simulate others' decisions

A fundamental challenge in social cognition is how humans learn another person's values to predict their decision-making behavior. This form of learning is often assumed to require simulation of the other by direct recruitment of one's own valuation process to model the other's process. However, the cognitive and neural mechanism of simulation learning is not known. Using behavior, modeling, and fMRI, we show that simulation involves two learning signals in a hierarchical arrangement. A simulated-other's reward prediction error processed in ventromedial prefrontal cortex mediated simulation by direct recruitment, being identical for valuation of the self and simulated-other. However, direct recruitment was insufficient for learning, and also required observation of the other's choices to generate?a simulated-other's action prediction error encoded in dorsomedial/dorsolateral prefrontal cortex. These findings show that simulation uses a core prefrontal circuit for modeling the other's valuation to generate prediction and an adjunct circuit for tracking behavioral variation to refine prediction.     

Beneficial effects of edaravone, a novel antioxidant, in rats with dilated cardiomyopathy

Edaravone, a novel antioxidant, acts by trapping hydroxyl radicals, quenching active oxygen and so on. Its cardioprotective activity against experimental autoimmune myocarditis (EAM) was reported. Nevertheless, it remains to be determined whether edaravone protects against cardiac remodelling in dilated cardiomyopathy (DCM). The present study was undertaken to assess whether edaravone attenuates myocardial fibrosis, and examine the effect of edaravone on cardiac function in rats with DCM after EAM. Rat model of EAM was prepared by injection with porcine cardiac myosin 28 days after immunization, we administered edaravone intraperitoneally at 3 and 10 mg/kg/day to rats for 28 days. The results were compared with vehicle-treated rats with DCM. Cardiac function, by haemodynamic and echocardiographic study and histopathology were performed. Left ventricular (LV) expression of NADPH oxidase subunits (p47(phox), p67(phox), gp91(phox) and Nox4), fibrosis markers (TGF-β(1) and OPN), endoplasmic reticulum (ER) stress markers (GRP78 and GADD 153) and apoptosis markers (cytochrome C and caspase-3) were measured by Western blotting. Edaravone-treated DCM rats showed better cardiac function compared with those of the vehicle-treated rats. In addition, LV expressions of NADPH oxidase subunits levels were significantly down-regulated in edaravone-treated rats. Furthermore, the number of collagen-III positive cells in the myocardium of edaravone-treated rats was lower compared with those of the vehicle-treated rats. Our results suggest that edaravone ameliorated the progression of DCM by modulating oxidative and ER stress-mediated myocardial apoptosis and fibrosis.     

Delta-opioid augments cardiac contraction through β-adrenergic and CGRP-receptor co-signaling

Cardiac epinephrine and calcitonin gene-related peptide (CGRP) are produced by intrinsic cardiac adrenergic cells (ICA cells) residing in human and animal hearts. ICA cells are neuroparicine cells expressing δ-opioid receptors (DOR). We hypothesized that δ-opioid stimulation of ICA cells enhances epinephrine and CGRP release, which results in the augmentation of heart contraction. Rats were injected with DOR-agonist DPDPE (100 μg/kg) with or without 10-min pretreatment with either β-adrenergic receptor (β-AR) blocker propranolol (2mg/kg) or CGRP-receptor (CGRPR) blocker CGRP(8-37) (300 μg/kg), or their combination. Hemodynamics were monitored with echocardiogram and systolic blood pressure (SBP) was monitored via a tail arterial catheter. Changes in left ventricular fraction-shortening (LVFS) and heart rate (HR) were observed at 5-min after DPDPE infusion. At 5-min DPDPE induced a 36 ± 18% (p<0.001) increase of the LVFS, which continues to increase to 51 ± 24% (p<0.0001) by 10 min, and 68 ± 19% (p<0.001) by 20 min. The increase in LVFS was accompanied by the decrease of HR by 9±5% (p<0.01) by 5 min and 11 ± 6% (p<0.001) by 15 min post DPDPE infusion. This magnitude of HR reduction was observed for the remainder of the 20 min. Despite the HR-reduction, cardiac output was increased by 17 ± 8% (p<0.05) and 28±5% (p<0.001) by 5- and 20-min post DPDPE administration, respectively. There was a modest (9 ± 9%, p=0.03) decrease in SBP that was not apparent until 20 min post DPDPE infusion. The positive inotropism of DPDPE was abrogated in animals pretreated with propranolol, CGRP(8-37), or combined propranolol+CGRP(8-37). Furthermore, in whole animal and cardiomyocyte cell culture preparations, DPDPE induced myocardial protein-kinase A (PKA) activation which was abrogated in the animals pretreated with propranolol+CGRP(8-37). DOR agonists augment myocardial contraction through enhanced β-AR and CGRPR co-signaling.     

Improvement of the post-thaw qualities of Okinawan native Agu pig sperm frozen in an extender supplemented with antiapoptotic PTD-FNK protein

The technical establishment of boar sperm cryopreservation is indispensable for effective breeding of the scarce Okinawan native Agu pig. The objective was to determine whether an artificial anticell death protein (PTD-FNK protein) was capable of improving the quality of cryopreserved Agu sperm. Ejaculated Agu sperm frozen in an extender supplemented with 0, 100, 200, 300, or 400 nm PTD-FNK protein was thawed, and mitochondrial integrity and other sperm characteristics were evaluated. Treatment with 300 nm PTD-FNK protein had the most beneficial effect (P < 0.05) on mitochondrial integrity (45-59%) and sperm motility (56-67%) after freezing-thawing. In particular, the proportion of post-thaw sperm with activated caspase-9 and -3 but not caspase-8 was markedly reduced among sperm frozen in the presence of PTD-FNK protein (P < 0.05), implying protection against apoptotic-cell death in response to mitochondrial damage. There were high levels of intracellular ATP (9.4-10.5 nmol/10⁸ sperm) in post-thaw sperm treated with PTD-FNK protein, and the inhibitory effect of PTD-FNK protein on activation of caspases influenced the increase in the number of sperm with intact DNA (36-53%; P < 0.05). Furthermore, the addition of PTD-FNK protein to the freezing extender strongly preserved the ability of the sperm to penetrate to mature oocytes in all individuals (60-80%; P < 0.05). In conclusion, treatment with PTD-FNK protein in the freezing extender effectively improved post-thaw qualities of fragile Agu sperm through prevention of mitochondrial dysfunction leading to apoptotic-cell death during cryopreservation.     

Critical role of the Fc receptor gamma-chain on APCs in the development of allergen-induced airway hyperresponsiveness and inflammation

The FcR common gamma-chain (FcRgamma) is an essential component of the receptors FcepsilonRI, FcgammaRI, and FcgammaRIII, which are expressed on many inflammatory cell types. The role of these receptors in the initiation or maintenance of allergic inflammation has not been well defined. FcRgamma-deficient (FcRgamma(-/-)) and control (wild-type (WT)) mice were sensitized and subsequently challenged with OVA. Following sensitization and challenge to OVA, FcRgamma-deficient (FcRgamma(-/-)) mice developed comparable levels of IgE and IgG1 as WT mice. However, numbers of eosinophils, levels of IL-5, IL-13, and eotaxin in bronchoalveolar lavage fluid, and mononuclear cell (MNC) proliferative responses to OVA were significantly reduced, as was airway hyperresponsiveness (AHR) to inhaled methacholine. Reconstitution of FcRgamma(-/-) mice with whole spleen MNC from WT mice before sensitization restored development of AHR and the numbers of eosinophils in bronchoalveolar lavage fluid; reconstitution after sensitization but before OVA challenge only partially restored these responses. These responses were also restored when FcRgamma(-/-) mice received T cell-depleted MNC, T and B cell-depleted MNC, or bone marrow-derived dendritic cells before sensitization from FcR(+/+) or FcgammaRIII-deficient but not FcRgamma(-/-) mice. The expression levels of FcgammaRIV on bone marrow-derived dendritic cells from FcR(+/+) mice were found to be low. These results demonstrate that expression of FcRgamma, most likely FcgammaRI, on APCs is important during the sensitization phase for the development of allergic airway inflammation and AHR.     

Investigation of the microbial community in a microbiological additive used in a manure composting process

The objectives of this study were to investigate the fate of microorganisms by using cultivation methods as well as DNA analyses in a commercial microbiological additive (MA) in the course of the composting. Almost all the predominant species in the microbial succession during composting process determined by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) were in disagreement with those determined by the clone library method. None of the microbial species in the composting stages corresponded to the microorganisms identified in the MA either by the cultivation method or DNA analysis. The results in regard to predominant microorganisms of the MA detected from the liquid medium by the PCR-DGGE did not correspond with those detected from the MA itself and composting processes. Although no evidence was found that predominant species in the MA itself dominate in the composting process, predominant species diversity in the MA itself was markedly changed after culturing at different thermophilic temperatures. These results suggested that cultivable microorganisms in the MA did not become predominant in the composting process: however, some microorganisms that are detected from the MA itself by the DNA analysis may act effectively in the composting process.     

Human umbilical cord-derived cells can often serve as feeder cells to maintain primate embryonic stem cells in a state capable of producing hematopoietic cells

Clinical application of human embryonic stem (ES) cells will require the establishment of methods for their culture, either in the presence or absence of human-derived feeder cells. We have tested the ability of non-immortalized cultured cells derived from human umbilical cord (HUC cells) to support ES cell culture. A primate ES cell line that had been established and maintained with mouse embryonic fibroblasts was cultured on HUC cells for >3 months (HUC-maintained ES cells). These cells retained their expression of alkaline phosphatase, SSEA-4, Oct-3/4, and to a lesser extent Nanog, but did not express Rex-1. Nevertheless, HUC-maintained ES cells could produce ectoderm-, mesoderm- and endoderm-derived cells in teratomata that they formed in immunodeficient mice. We show that HUC-maintained ES cells could give rise to hematopoietic cells, although this ability of HUC cells varied among HUC cell populations derived from different neonates. HUC cells are promising as human material with which to maintain ES cells in a state that retains their ability to produce mature cells, including hematopoietic cells.     

Reciprocating-flow ATP amplification system for increasing the number of amplification cycles

We constructed a novel ATP amplification reactor using a reciprocating-flow system to increase the number of ATP amplification cycles without an increase in backpressure. We previously reported a continuous-flow ATP amplification system that effectively and quantitatively amplified ATP and increased the sensitivity of a quantitative bioluminescence assay. However, it was difficult to increase the number of amplification cycles due to backpressure in the system. Because addition of immobilized adenylate kinase (ADK) and pyruvate kinase (PK) columns increased backpressure, the maximum number of ATP amplification cycles within column durability was only 4. In this study, ATP amplification was performed using a reciprocating-flow system, and 10 cycles of ATP amplification could be achieved without an increase in backpressure. As a result, ATP was amplified more than 100-fold after 10 cycles of reciprocating flow. The gradient of ATP amplification was approximately 1.76^N. The backpressure on the columns was 0.03 MPa in 1–10 ATP amplification cycles, and no increases in backpressure were observed.