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101.
Iimura S Umezaki T Takeuchi M Mizuguchi M Yagi H Ogasahara K Akutsu H Noda Y Segawa S Yutani K 《Biochemistry》2007,46(12):3664-3672
The cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from a hyperthermophile, Pyrococcus furiosus, can be trapped in the denatured state under nondenaturing conditions, corresponding to the denatured structure that exists in equilibrium with the native state under physiological conditions. The denatured state is the initial state (D1 state) in the refolding process but differs from the completely denatured state (D2 state) in the concentrated denaturant. Also, it has been found that the D1 state corresponds to the heat-denatured state. To elucidate the structural basis of the D1 state, H/D exchange experiments with PCP-0SH were performed at pD 3.4 and 4 degrees C. The results indicated that amide protons in the C-terminal alpha6-helix region hardly exchanged in the D1 state with deuterium even after 7 days, suggesting that the alpha6-helix (from Ser188 to Glu205) of PCP-0SH was stably formed in the D1 state. In order to examine the role of the alpha6-helix in folding and stability, H/D exchange experiments with a mutant, A199P, at position 199 in the alpha6-helix region were performed. The alpha6-helix region of A199P in the D1 state was partially unprotected, while some hydrophobic residues were protected against the H/D exchange, although these hydrophobic residues were unprotected in the wild-type protein. These results suggest that the structure of A199P in the D1 state formed a temporary stable denatured structure with a non-native hydrophobic cluster and the unstructured alpha6-helix. Both the stability and the refolding rate decreased by the substitution of Pro for Ala199. We can conclude that the native-like helix (alpha6-helix) of PCP-0SH is already constructed in the D1 state and is necessary for efficient refolding into the native structure and stabilization of PCP-0SH. 相似文献
102.
Yuusuke Maruyama Toshihiko Ogura Kazuhiro Mio Kenta Kato Takeshi Kaneko Shigeki Kiyonaka Yasuo Mori Chikara Sato 《The Journal of biological chemistry》2009,284(20):13676-13685
The Ca2+ release-activated Ca2+ channel is a
principal regulator of intracellular Ca2+ rise, which conducts
various biological functions, including immune responses. This channel,
involved in store-operated Ca2+ influx, is believed to be composed
of at least two major components. Orai1 has a putative channel pore and
locates in the plasma membrane, and STIM1 is a sensor for luminal
Ca2+ store depletion in the endoplasmic reticulum membrane. Here we
have purified the FLAG-fused Orai1 protein, determined its tetrameric
stoichiometry, and reconstructed its three-dimensional structure at 21-Å
resolution from 3681 automatically selected particle images, taken with an
electron microscope. This first structural depiction of a member of the Orai
family shows an elongated teardrop-shape 150Å in height and 95Å in
width. Antibody decoration and volume estimation from the amino acid sequence
indicate that the widest transmembrane domain is located between the round
extracellular domain and the tapered cytoplasmic domain. The cytoplasmic
length of 100Å is sufficient for direct association with STIM1. Orifices
close to the extracellular and intracellular membrane surfaces of Orai1 seem
to connect outside the molecule to large internal cavities.Ca2+ is an intracellular second messenger that plays important
roles in various physiological functions such as immune response, muscle
contraction, neurotransmitter release, and cell proliferation. Intracellular
Ca2+ is mainly stored in the endoplasmic reticulum
(ER).2 This ER system
is distributed through the cytoplasm from around the nucleus to the cell
periphery close to the plasma membrane. In non-excitable cells, the ER
releases Ca2+ through the inositol 1,4,5-trisphosphate
(IP3) receptor channel in response to various signals, and the
Ca2+ store is depleted. Depletion of Ca2+ then induces
Ca2+ influx from outside the cell to help in refilling the
Ca2+ stores and to continue Ca2+ rise for several
minutes in the cytoplasm (1,
2). This Ca2+ influx
was first proposed by Putney
(3) and was named
store-operated Ca2+ influx. In the immune system, store-operated
Ca2+ influx is mainly mediated by the Ca2+
release-activated Ca2+ (CRAC) current, which is a highly
Ca2+-selective inwardly rectified current with low conductance
(4,
5). Pathologically, the loss of
CRAC current in T cells causes severe combined immunodeficiency
(6) where many Ca2+
signal-dependent gene expressions, including cytokines, are interrupted
(7). Therefore, CRAC current is
necessary for T cell functions.Recently, Orai1 (also called CRACM1) and STIM1 have been physiologically
characterized as essential components of the CRAC channel
(8–12).
They are separately located in the plasma membrane and in the ER membrane;
co-expression of these proteins presents heterologous CRAC-like currents in
various types of cells (10,
13–15).
Both of them are shown to be expressed ubiquitously in various tissues
(16–18).
STIM1 senses Ca2+ depletion in the ER through its EF hand motif
(19) and transmits a signal to
Orai1 in the plasma membrane. Although Orai1 is proposed as a regulatory
component for some transient receptor potential canonical channels
(20,
21), it is believed from the
mutation analyses to be the pore-forming subunit of the CRAC channel
(8,
22–24).
In the steady state, both Orai1 and STIM1 molecules are dispersed in each
membrane. When store depletion occurs, STIM1 proteins gather into clusters to
form puncta in the ER membrane near the plasma membrane
(11,
19). These clusters then
trigger the clustering of Orai1 in the plasma membrane sites opposite the
puncta (25,
26), and CRAC channels are
activated (27).Orai1 has two homologous genes, Orai2 and Orai3
(8). They form the Orai family
and have in common the four transmembrane (TM) segments with relatively large
N and C termini. These termini are demonstrated to be in the cytoplasm,
because both N- and C-terminally introduced tags are immunologically detected
only in the membrane-permeabilized cells
(8,
9). The subunit stoichiometry
of Orai1 is as yet controversial: it is believed to be an oligomer, presumably
a dimer or tetramer even in the steady state
(16,
28–30).Despite the accumulation of biochemical and electrophysiological data,
structural information about Orai1 is limited due to difficulties in
purification and crystallization. In this study, we have purified Orai1 in its
tetrameric form and have reconstructed the three-dimensional structure from
negatively stained electron microscopic (EM) images. 相似文献
103.
l-Glutamic acid was formed from d-, l-, and dl-PCA with cell-free extract of Pseudomonas alcaligenes ATCC-12815 grown in the medium containing dl-PCA as a sole source of carbon and nitrogen. The enzyme(s) involved in this conversion reaction was distributed in the soluble fraction within the cell and in 0.5 saturated fraction at the fractionation procedure with the saturation of ammonium sulfate. Optimum pH of this enzyme(s) lied at pH 8.5 and optimum temperature was 30°C. Cu (5 × 10?3 m) inhibited the reaction considerably while Ca or Fe accelerated it. PALP (1×10?3 m) also gave an enhanced activity to some extent. The enzyme preparation converted dextro-rotatory enan-thiomorph of PCA to its laevo-rotatory one which in turn was not converted to the opposite rotation direction by this enzyme. Furthermore, the preparation did not, if any, show d-glutamic acid racemase activity. Isotopic experiments with using dl-PCA-1-14C revealed that l-glutamic acid-1-14C was formed by the cleavage of –CO–NH– bond of pyrrolidone ring of PCA. It was concluded that dl-PCA when assimilated by the present bacterium is at first transformed to l-PCA by the optically isomerizing enzyme and subsequently is cleaved to l-glutamic acid probably by the PCA hydrolysing enzyme. 相似文献
104.
Kobayashi I Shidara K Okuno S Yamada S Imanishi Y Mori K Ishimura E Shoji S Yamakawa T Inaba M 《Life sciences》2012,90(5-6):212-218
AimsHigher serum alkaline phosphatase predicts lower mortality in chronic kidney disease and hemodialysis patients without liver dysfunction because it reflects high bone turnover. The purpose of our study was to compare the significance of serum bone alkaline phosphatase (BAP) with that of other bone markers in prediction of all-cause mortality(ACM) in male hemodialysis patients.Main methodsThe study was performed for 5 years. Serum BAP, intact osteocalcin (iOC), ß-CrossLaps (CTX), and intact parathyroid hormone (iPTH) were measured in 196 male hemodialysis patients without radiographic fracture. Their day-to-day variation during 5 consecutive days and diurnal variation were determined in 13 healthy males.Key findingsThe patients were divided into higher and lower groups based on serum levels of bone markers(mean ± SD: iPTH 218.6 ± 214.5 pg/ml, BAP 23.6 ± 12.2U/L, iOC 42.8 ± 45.2 ng/ml, CTX 1.71 ± 1.23 nmol/L BCE). In Kaplan–Meier analysis, the higher BAP group had significantly higher ACM than the lower BAP group (P = 0.013), whereas mortality did not differ between the higher and lower groups in other markers. Cox regression hazard analysis identified higher log BAP as a significant independent predictor [hazard ratio(HR) 8.32(95%CI:1.18–58.98)] for ACM after adjustment for various factors including pre-existing cardiovasucular disease, presence of DM. The significant association of mortality with serum BAP alone, in contrast with other markers including CTX [HR0.64 (95%CI:0.16–2.47)], iOC [HR0.97(95%CI:0.36–2.64)], iPTH [HR0.84(95%CI:0.44–1.60)],it may be due to the narrower day-to-day variation and the absence of diurnal variation in serum BAP compared to other markers.SignificanceHigher serum BAP may be a predictor of ACM in male hemodialysis patients. 相似文献
105.
Masahiro Tsuchiya Fukie Niijima-Yaoita Hiroyuki Yoneda Ko Chiba Shinobu Tsuchiya Yoshihiro Hagiwara Keiichi Sasaki Shunji Sugawara Yasuo Endo Koichi Tan-No Makoto Watanabe 《Life sciences》2014
Aims
Dietary habits are crucial factors affecting metabolic homeostasis. However, few animal experiments have addressed the effects of long-term feeding with soft food on parameters reflecting systemic health.Main methods
Using mice, we compared the effects of short (3 days) and long (17 weeks from weaning) feeding periods between powdered food and normal pellet food on the levels of blood glucose, serum levels of insulin, catecholamines, and corticosterone, blood pressure, and/or social interaction behaviors. In addition, the effects of a human glucagon-like peptide-1 analog, liraglutide (a new drug with protective effects against neuronal and cardiovascular diseases), were compared between the powder and pellet groups.Key finding
(i) Powdered food, even for such a short period, resulted in a greater glycemic response than pellet food, consistent with powdered food being more easily digested and absorbed. (ii) Long-term feeding on powdered food induced hyperglycemia and related systemic signs of illness, including increases in serum adrenaline, noradrenaline, and corticosterone, higher blood pressures (especially diastolic), and increased social interaction behaviors. (iii) Liraglutide, when administered subcutaneously for the last 2 weeks of the 17-week period of feeding, improved these changes (including those in social interaction behaviors).Significance
The hyperglycemia associated with long-term powdered-food feeding may lead to certain systemic illness signs, such as elevations of blood glucose, hypertension, and abnormal behaviors in mice. Mastication of food of adequate hardness may be very important for the maintenance of systemic (physical and mental) health, possibly via reduction in the levels of blood glucose and/or adrenal stress hormones (catecholamines and glucocorticoids). 相似文献106.
Akira Yano Sayaka Kikuchi Yoshihisa Yamashita Yuichi Sakamoto Yuko Nakagawa Yasuo Yoshida 《Applied microbiology and biotechnology》2010,86(2):615-623
Mushrooms contain large quantities of α-glucans. Shiitake (Lentinula edodes), Japan’s most popular edible mushroom, has been reported to contain about 6% (weight/dried weight) of α-(1,3)-glucan. This
glucan is one of the major components of oral biofilm formed by the cariogenic bacteria Streptococcus mutans and Streptococcus sobrinus. We found that extracts from shiitake and other edible mushrooms could reduce preformed biofilms of S. mutans and S. sobrinus in the presence of dextranase. We also investigated the α-glucanase activities of shiitake mushroom extracts and their effects
on biofilm formation. The extracts possessed α-glucanase activity and degraded water-insoluble glucans from mutans streptococci.
The extracts strongly inhibited the sucrose-dependent formation of biofilms by S. mutans and S. sobrinus in the presence of dextranase. Our results suggest that some components of mushrooms, including α-glucanases, might inhibit
the sucrose-induced formation of oral biofilms. 相似文献
107.
108.
Ko-ichiro Miyamoto Parida Yamada Ryo-taro Yamaguchi Takami Muto Ayumi Hirano Yasuo Kimura Michio Niwano Hiroko Isoda 《Cytotechnology》2007,55(2-3):143-149
In this study, we report on an in situ monitoring system of living cultured cells using infrared absorption spectroscopy in
the geometry of multiple internal reflections (MIR-IRAS). In order to observe living cultured cells, the temperature in the
sample chamber of a FT-IR spectrometer was maintained at 37 °C and a humidified gas mixture containing 5% CO2 was introduced into the sample chamber. Human breast cell line MCF-7 cultured on Si MIR prisms were placed in the sample
chamber and infrared spectra of MCF-7 cells were collected for 5 h. It was found that the adhesion and metabolism of MCF-7
cells could be monitored by the absorption intensity of amide-II protein band (1,545 cm−1) and also by the absorption intensities of CH
x
bands (2,700–3,100 cm−1). These results suggest that our system is useful for a nondestructive and non-label monitoring of cell viability. Our method
based on infrared absorption spectroscopy has a potential for bioscreening application. 相似文献
109.
Kajiya M Hirota M Inai Y Kiyooka T Morimoto T Iwasaki T Endo K Mohri S Shimizu J Yada T Ogasawara Y Naruse K Ohe T Kajiya F 《American journal of physiology. Heart and circulatory physiology》2007,292(6):H2737-H2744
Pulmonary hypertension (PH) causes right ventricular (RV) hypertrophy and, according to the extent of pressure overload, eventual heart failure. We tested the hypothesis that the mechanical stress in PH-RV impairs the vasoreactivity of the RV coronary microvessels of different sizes with increased superoxide levels. Five-week-old male Sprague-Dawley rats were injected with monocrotaline (n=126) to induce PH or with saline as controls (n=114). After 3 wk, coronary arterioles (diameter = 30-100 microm) and small arteries (diameter = 100-200 microm) in the RV were visualized using intravital videomicroscopy. We evaluated ACh-induced vasodilation alone, in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME), in the presence of tetraethylammonium (TEA) or catalase with or without L-NAME, and in the presence of SOD. The degree of suppression in vasodilation by L-NAME and TEA was used as indexes of the contributions of endothelial nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF), respectively. In PH rats, ACh-induced vasodilation was significantly attenuated in both arterioles and small arteries, especially in arterioles. This decreased vasodilation was largely attributable to reduced NO-mediated vasoreactivity, whereas the EDHF-mediated vasodilation was relatively robust. The suppressive effect on arteriolar vasodilation by catalase was similar to TEA in both groups. Superoxide, as measured by lucigenin chemiluminescence, was significantly elevated in the RV tissues in PH. SOD significantly ameliorated the impairment of ACh-induced vasodilation in PH. Robust EDHF function will play a protective role in preserving coronary microvascular homeostasis in the event of NO dysfunction with increased superoxide levels. 相似文献
110.
Removal and recovery of phosphorous from swine wastewater by demonstration crystallization reactor and struvite accumulation device 总被引:10,自引:0,他引:10
Suzuki K Tanaka Y Kuroda K Hanajima D Fukumoto Y Yasuda T Waki M 《Bioresource technology》2007,98(8):1573-1578
A demonstration crystallization reactor and struvite accumulation device for the removal and recovery of phosphorous was constructed and their performance was evaluated using actual swine wastewater for 3.5 years. The wastewater pH was increased by aeration, and the concentrations of total P and soluble PO(4)-P were reduced by a struvite crystallization reaction induced under a high pH condition. A 30% MgCl(2) addition was effective in enhancing the struvite crystallization reaction. The concentrations of suspended solids, total Zn and total Cu, were also decreased by the settling function of the reactor. On removing the efficiencies of these components, no noticeable seasonal fluctuation in performance was observed during the 3.5-year operation. In terms of maximum yield, 171g struvite was obtained from 1m(3) swine wastewater by the demonstration accumulation device for struvite recovery. The recovered struvite needed only air-drying before use since it was approximately 95% pure even without washing. 相似文献