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141.
Gelatinases have been purified from the exudate in the chronic-phase (day 7) of carrageenin-induced inflammation in rats. The day-7 exudate gelatinases gave two peaks on Sephadex G-150 gel filtration, the initial step of the purification. The molecular weights of the gelatinases corresponding to the two peaks were about 300 kDa (HMW fraction) and about 110 kDa (LMW fraction), respectively. The gelatinase in the HMW fraction has been purified to homogeneity; the purified gelatinase gave a single band corresponding to a molecular weight of 57 kDa on both SDS-polyacrylamide gel electrophoresis (PAGE) and SDS-gelatin PAGE. On the other hand, the gelatinase purified from the LMW fraction was found to consist of three species, with molecular weights of 66, 64, and 57 kDa, as judged on SDS-gelatin PAGE. Granulation tissue-derived fibroblasts in culture mainly produced the 64-kDa species, which was converted to a 57-kDa species on treatment with 4-amino-phenylmercuric acetate, while rat macrophages and polymorphonuclear leukocytes mainly secreted the 96-kDa species. These results suggest that exudate gelatinases are largely produced by fibroblasts in granulation tissue and that they bind to exudate proteins, resulting in the formation of complexes with molecular weights of about 300 kDa and about 110 kDa. The gelatinases purified from the HMW and LMW fractions are metalloproteinases, as judged from the results of inhibitor experiments. Both the gelatinases degraded gelatin, but showed to proteolytic activity toward alpha-casein or type I collagen. Type IV collagen was degraded at 35 degrees C by the gelatinases purified from the LMW fraction but not by that from the HMW fraction. 相似文献
142.
Poly(ethylene glycol)-conjugated human serum albumin including iron porphyrins: surface modification improves the O2-transporting ability 总被引:2,自引:0,他引:2
Artificial O2-carrying hemoprotein composed of human serum albumin including tetrakis(o-amidophenyl)porphinatoiron(II) (Fe4P or Fe3P) [HSA-FeXP] has been modified by maleimide- or succinimide-terminated poly(ethylene glycol) (PEG), and the formed PEG bioconjugates have been physicochemically characterized. 2-Iminothiolane (IMT) reacted with the amino groups of Lys to create active thiol groups, which bind to alpha-maleimide-omega-methoxy PEG [Mw: 2-kDa (PEG(M2)), 5-kDa (PEG(M5))]. On the other hand, alpha-succinimidyl-omega-methoxy PEG [Mw: 2-kDa (PEG(S2)), 5-kDa (PEG(S5))] directly binds to Lys residues. MALDI-TOF MS of the PEG-conjugated HSA-FeXP showed distinct molecular ion peaks, which provide an accurate number of the PEG chains. In the case of PEG(MY)(HSA-FeXP), the spectroscopic assay of the thiol groups also provided the mean of the binding numbers of the polymers, and the degree of the modification was controlled by the ratio of [IMT]/[HSA]. The viscosity and colloid osmotic pressures of the 2-kDa PEG conjugates (phosphate-buffered saline solution, [HSA] = 5 g dL(-1)) were almost the same as that of the nonmodified one, whereas the 5-kDa PEG binding increased the rheological parameters. The presence of flexible polymers on the HSA surface retarded the association reaction of O2 to FeXP and stabilized the oxygenated complex. Furthermore, PEG(MY)(HSA-FeXP) exhibited a long circulation lifetime of FeXP in rats (13-16 h). On the basis of these results, it can be concluded that the surface modification of HSA-FeXP by PEG has improved its comprehensive O2-transporting ability. In particular the PEG(MY)(HSA-FeXP) solution could be a promising material for entirely synthetic O2-carrying plasma expander as a red cell substitute. 相似文献
143.
Hiromi Maekawa Tomoko Nakagawa Yoko Uno Kenji Kitamura Chikashi Shimoda 《Molecular & general genetics : MGG》1994,244(5):456-464
When the fission yeastSchizosaccharomyces pombe is starved for nitrogen, the cells are arrested in the G1 phase, enter the G0 phase and initiate sexual development. Theste13 mutant, however, fails to undergo a G1 arrest when starved for nitrogen and since this mutant phenotype is not suppressed by a mutation in adenylyl cyclase (cyr1), it would appear thatste13
+ either acts independently of the decrease in the cellular cAMP level induced by starvation for nitrogen, or functions downstream of this controlling event. We have used functional complementation to clone theste13
+ gene from anS. pombe genomic library and show that its disruption is not lethal, indicating that, while the gene is required for sexual development, it is not essential for cell growth. Nucleotide sequencing predicts thatste13
+ should encode a protein of 485 amino acids in which the consensus motifs of ATP-dependent RNA helicases of the DEAD box family are completely conserved. Point mutations introduced into these consensus motifs abolished theste13
+ functions. The predicted Ste13 protein is 72% identical to theDrosophila melanogaster Me31B protein over a stretch of 391 amino acids. ME31B is a developmentally regulated gene that is expressed preferentially in the female germline and may be required for oogenesis. Expression of ME31B cDNA inS. pombe suppresses theste13 mutation. These two evolutionarily conserved genes encoding putative RNA helicases may play a pivotal role in sexual development. 相似文献
144.
Takayuki Aoyama Yasukazu Nakakita Masahira Nakagawa Heiichi Sakai 《Bioscience, biotechnology, and biochemistry》2013,77(9):2369-2371
Asymmetric hydrolysis of (dl)-1-acyloxy-2-halo-1-phenylethanes by lipoprotein lipase Amano P from Pseudomonas fluorescens and the lipase from Chromobacterium viscosum afforded the optically active (R) residual substrates and (S)-2-halo-1-hydroxy-1-phenylethanes in 100% enantiomeric excess (e.e.). The length of acyl residues from acetyl to octanoyl in the substrates did not influence the enantioselectivity.Both enantiomers of optically active styrene oxides were synthesized from the enzymatic products. 相似文献
145.
Chisato Kusunoki Liu Yang Takeshi Yoshizaki Fumiyuki Nakagawa Atsushi Ishikado Motoyuki Kondo Katsutaro Morino Osamu Sekine Satoshi Ugi Yoshihiko Nishio Atsunori Kashiwagi Hiroshi Maegawa 《Biochemical and biophysical research communications》2013,430(1):225-230
Oxidative stress is produced in adipose tissue of obese subjects and has been associated with obesity-related disorders. Recent studies have shown that omega-3 polyunsaturated fatty acid (ω3-PUFA) has beneficial effects in preventing atherosclerotic diseases and insulin resistance in adipose tissue. However, the role of ω3-PUFA on adipocytes has not been elucidated. In this study, 3T3-L1 adipocytes were treated with ω3-PUFA and its metabolites, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or 4-hydroxy hexenal (4-HHE). ω3-PUFA and its metabolites dose-dependently increased mRNA and protein levels of the anti-oxidative enzyme, heme oxygenase-1 (HO-1); whereas no changes in the well-known anti-oxidant molecules, superoxide dismutase, catalase, and glutathione peroxidase, were observed. Knockdown of nuclear factor erythroid 2-related factor 2 (Nrf-2) significantly reduced EPA, DHA or 4-HHE-induced HO-1 mRNA and protein expression. Also, pretreatment with ω3-PUFA prevented H2O2-induced cytotoxicity in a HO-1 dependent manner. In conclusion, treatment with EPA and DHA induced HO-1 through the activation of Nrf-2 and prevented oxidative stress in 3T3-L1 adipocytes. This anti-oxidant defense may be of high therapeutic value for clinical conditions associated with systemic oxidative stress. 相似文献
146.
Aniruddha Chatterjee Yuichi Ozaki Peter A Stockwell Julia A Horsfield Ian M Morison Shinichi Nakagawa 《Epigenetics》2013,8(9):979-989
Reduced representation bisulfite sequencing (RRBS) has been used to profile DNA methylation patterns in mammalian genomes such as human, mouse and rat. The methylome of the zebrafish, an important animal model, has not yet been characterized at base-pair resolution using RRBS. Therefore, we evaluated the technique of RRBS in this model organism by generating four single-nucleotide resolution DNA methylomes of adult zebrafish brain. We performed several simulations to show the distribution of fragments and enrichment of CpGs in different in silico reduced representation genomes of zebrafish. Four RRBS brain libraries generated 98 million sequenced reads and had higher frequencies of multiple mapping than equivalent human RRBS libraries. The zebrafish methylome indicates there is higher global DNA methylation in the zebrafish genome compared with its equivalent human methylome. This observation was confirmed by RRBS of zebrafish liver. High coverage CpG dinucleotides are enriched in CpG island shores more than in the CpG island core. We found that 45% of the mapped CpGs reside in gene bodies, and 7% in gene promoters. This analysis provides a roadmap for generating reproducible base-pair level methylomes for zebrafish using RRBS and our results provide the first evidence that RRBS is a suitable technique for global methylation analysis in zebrafish. 相似文献
147.
Mitsuru Ando Toshiie Nakamura Masayuki Nakagawa 《Bioscience, biotechnology, and biochemistry》2013,77(12):2451-2456
Excretion, distribution and metabolism of the fungicide, hymexazol, (3-hydroxy-5-methylisoxazole), labeled with carbon-14 were examined after administration of a single oral dose to Wistar-strain rats. Hymexazol was rapidly absorbed and distributed in the tissues. During 96 hr, 97% of the total radioactivity was excreted in the urine and 0.89% in the feces, and 0.86% was found in the expired gasses for 24 hr. Two metabolites were detected in the urine, whose chemical structures were determined as 3-(β-d-glucopyranuronosyloxy)-5- methylisoxazole and 5-methyl-3-isoxazolyl sulfate. 相似文献
148.
Qing-xin Hua Bin Xu Kun Huang Shi-Quan Hu Satoe Nakagawa Wenhua Jia Shuhua Wang Jonathan Whittaker Panayotis G. Katsoyannis Michael A. Weiss 《The Journal of biological chemistry》2009,284(21):14586-14596
A central tenet of molecular biology holds that the function of a protein
is mediated by its structure. An inactive ground-state conformation may
nonetheless be enjoined by the interplay of competing biological constraints.
A model is provided by insulin, well characterized at atomic resolution by
x-ray crystallography. Here, we demonstrate that the activity of the hormone
is enhanced by stereospecific unfolding of a conserved structural element. A
bifunctional β-strand mediates both self-assembly (within β-cell
storage vesicles) and receptor binding (in the bloodstream). This strand is
anchored by an invariant side chain (PheB24); its substitution by
Ala leads to an unstable but native-like analog of low activity. Substitution
by d-Ala is equally destabilizing, and yet the protein diastereomer
exhibits enhanced activity with segmental unfolding of the β-strand.
Corresponding photoactivable derivatives (containing l- or
d-para-azido-Phe) cross-link to the insulin receptor with
higher d-specific efficiency. Aberrant exposure of hydrophobic
surfaces in the analogs is associated with accelerated fibrillation, a form of
aggregation-coupled misfolding associated with cellular toxicity. Conservation
of PheB24, enforced by its dual role in native self-assembly and
induced fit, thus highlights the implicit role of misfolding as an
evolutionary constraint. Whereas classical crystal structures of insulin
depict its storage form, signaling requires engagement of a detachable arm at
an extended receptor interface. Because this active conformation resembles an
amyloidogenic intermediate, we envisage that induced fit and self-assembly
represent complementary molecular adaptations to potential proteotoxicity. The
cryptic threat of misfolding poses a universal constraint in the evolution of
polypeptide sequences.How insulin binds to the insulin receptor
(IR)2 is not well
understood despite decades of investigation. The hormone is a globular protein
containing two chains, A (21 residues) and B (30 residues)
(Fig. 1A). In
pancreatic β-cells, insulin is stored as Zn2+-stabilized
hexamers (Fig. 1B),
which form microcrystal-line arrays within specialized secretory granules
(1). The hexamers dissociate
upon secretion into the portal circulation, enabling the hormone to function
as a zinc-free monomer. The monomer is proposed to undergo a change in
conformation upon receptor binding
(2). In this study, we
investigated a site of conformational change in the B-chain
(PheB24) (arrow in Fig.
1A). In classical crystal structures, this invariant
aromatic side chain (tawny in Fig.
1B) anchors an antiparallel β-sheet at the dimer
interface (blue in Fig.
1C). Total chemical synthesis is exploited to enable
comparison of corresponding d- and l-amino acid
substitutions at this site, an approach designated “chiral
mutagenesis”
(3-5).
In the accompanying article, the consequences of this conformational change
are investigated by photomapping of the receptor-binding surface
(6). Together, these studies
redefine the interrelation of structure and activity in a protein central to
the hormonal control of metabolism.Open in a separate windowFIGURE 1.Sequence and structure of insulin. A, sequences of the
B-chain (upper) and A-chain (lower) with disulfide bridges
as indicated. The arrow indicates invariant PheB24. The
B24-B28 β-strand is highlighted in blue. B, crystal structure of
the T6 zinc insulin hexamer (Protein Data Bank code 4INS): ribbon
model (left) and space-filling model (right). The B24-B28
β-strand is shown in blue, and the side chain of
PheB24 is highlighted in tawny. The B-chain is otherwise
dark gray; the A-chain, light gray; and zinc ions,
magenta. Also shown at the left are the side chains of
HisB10 at the axial zinc-binding sites. C, cylinder model
of the insulin dimer showing the B24-B26 antiparallel β-sheet
(blue) anchored by the B24 side chain (tawny circle). The A-
and B-chains are shown in light and dark gray, respectively.
The protomer at the left is shown in the R-state, in which the central
α-helix of the B-chain is elongated (B3-B19 in the frayed Rf
protomer of T3Rf3 hexamers and B1-B19 in the
R protomer of R6 hexamers). The three types of zinc insulin
hexamers share similar B24-B26 antiparallel β-sheets as conserved
dimerization elements.The structure of an insulin monomer in solution resembles a
crystallographic protomer (Fig.
2A)
(7-9).
The A-chain contains an N-terminal α-helix, non-canonical turn, and
second helix; the B-chain contains an N-terminal segment, central
α-helix, and C-terminal β-strand. The β-strand is maintained
in an isolated monomer wherein the side chain of PheB24
(tawny in Fig.
2A), packing against the central α-helix of the
B-chain, provides a “plug” to seal a crevice in the hydrophobic
core (Fig. 2B).
Anomalies encountered in previous studies of insulin analogs suggest that
PheB24 functions as a conformational switch
(4,
7,
10-14).
Whereas l-amino acid substitutions at B24 generally impair activity
(even by such similar residues as l-Tyr)
(15), a seeming paradox is
posed by the enhanced activities of nonstandard analogs containing
d-amino acids (10-12).
Open in a separate windowaAffinities are given relative to wild-type insulin (100%).bLymphocytes are human, and hepatocytes are rat; CHO designates Chinese
hamster ovary.cStandard deviations are not provided in this reference.Open in a separate windowFIGURE 2.Role of PheB24 in an insulin monomer. A, shown
is a cylinder model of insulin as a T-state protomer. The C-terminal B-chain
β-strand is shown in blue, and the PheB24 side chain
is shown in tawny. The black portion of the N-terminal
A-chain α-helix (labeled buried) indicates a hidden
receptor-binding surface (IleA2 and ValA3). B,
the schematic representation of insulin highlights the proposed role of the
PheB24 side chain as a plug that inserts into a crevice at the edge
of the hydrophobic core. C and D, whereas substitution of
PheB24 by l-Ala (C) would only partially fill
the B24-related crevice, its substitution by d-Ala (D)
would be associated with a marked packing defect. An alternative conformation,
designated the R-state, is observed in zinc insulin hexamers at high ionic
strength (74) and upon binding
of small cyclic alcohols (75)
but has not been observed in an insulin monomer.Why do d-amino acid substitutions at B24 enhance the activity of
insulin? In this study, we describe the structure and function of insulin
analogs containing l-Ala or d-Ala at B24
(Fig. 2, C and
D). Our studies were conducted within an engineered
monomer (DKP-insulin, an insulin analog containing three substitutions in the
B-chain: AspB10, LysB28, and ProB29) to
circumvent effects of self-assembly
(16). Whereas the inactive
l-analog retains a native-like structure, the active
d-analog exhibits segmental unfolding of the B-chain. Studies of
corresponding analogs containing either l- or
d-photoactivable probes
(l-para-azido-PheB24 or
d-para-azido-PheB24 (l- or
d-PapB24), obtained from photostable
para-amino-Phe (Pmp) precursors
(17)) demonstrate specific
cross-linking to the IR. Although photo-contacts map in each case to the
N-terminal domain of the receptor α-subunit (the L1 β-helix),
higher cross-linking efficiency is achieved by the d-probe.
Together, this and the following study
(6) provide evidence that
insulin deploys a detachable arm that inserts between domains of the IR.Induced fit of insulin illuminates by its scope general principles at the
intersection of protein structure and cell biology. Protein evolution is
enjoined by multiple layers of biological selection. The pathway of insulin
biosynthesis, for example, successively requires (a) specific
disulfide pairing (in the endoplasmic reticulum), (b) subcellular
targeting and prohormone processing (in the trans-Golgi network),
(c) zinc-mediated protein assembly and microcrystallization (in
secretory granules), and (d) exocytosis and rapid disassembly of
insulin hexamers (in the portal circulation), in turn enabling binding of the
monomeric hormone to target tissues
(1). Each step imposes
structural constraints, which may be at odds. This study demonstrates that
stereospecific pre-detachment of a receptor-binding arm enhances biological
activity but impairs disulfide pairing and renders the hormone susceptible to
aggregation-coupled misfolding
(18). Whereas the classical
globular structure of insulin and its self-assembly prevent proteotoxicity
(3,
19), partial unfolding enables
receptor engagement. We envisage that a choreography of conformational change
has evolved as an adaptative response to the universal threat of toxic protein
misfolding. 相似文献
TABLE 1
Previous studies of insulin analogsAnalog | Affinitya | Assayb | Ref. |
---|---|---|---|
% | |||
d-PheB24-insulin | 180 | Lymphocytes | 10 |
l-AlaB24-insulin | 1 | Hepatocytes | 68 |
l-AlaB24-insulin | 3 | Lymphocytes | 69 |
d-PheB24-insulin | 140 ± 9 | Hepatocytes | 11 |
l-AlaB24-insulin | 1.0 ± 0.1 | Hepatocytes | 11 |
d-AlaB24-insulin | 150 ± 9 | Hepatocytes | 11 |
GlyB24-insulin | 78 ± 11 | Hepatocytes | 11 |
DKP-insulin | 200c | CHO cells | 12 |
d-PheB24-DKP-insulin | 180 | CHO cells | 12 |
l-AlaB24-DKP-insulin | 7 | CHO cells | 12 |
GlyB24-DKP-insulin | 50 | CHO cells | 12 |
149.
Inanobe A Nakagawa A Matsuura T Kurachi Y 《The Journal of biological chemistry》2010,285(49):38517-38523
Inward rectifier K(+) (Kir) channels are activated by phosphatidylinositol-(4,5)-bisphosphate (PIP(2)), but G protein-gated Kir (K(G)) channels further require either G protein βγ subunits (Gβγ) or intracellular Na(+) for their activation. To reveal the mechanism(s) underlying this regulation, we compared the crystal structures of the cytoplasmic domain of K(G) channel subunit Kir3.2 obtained in the presence and the absence of Na(+). The Na(+)-free Kir3.2, but not the Na(+)-plus Kir3.2, possessed an ionic bond connecting the N terminus and the CD loop of the C terminus. Functional analyses revealed that the ionic bond between His-69 on the N terminus and Asp-228 on the CD loop, which are known to be critically involved in Gβγ- and Na(+)-dependent activation, lowered PIP(2) sensitivity. The conservation of these residues within the K(G) channel family indicates that the ionic bond is a character that maintains the channels in a closed state by controlling the PIP(2) sensitivity. 相似文献
150.
Life span and reproductive cost explain interspecific variation in the optimal onset of reproduction 下载免费PDF全文
Emeline Mourocq Pierre Bize Sandra Bouwhuis Russell Bradley Anne Charmantier Carlos de la Cruz Szymon M. Drobniak Richard H. M. Espie Márton Herényi Hermann Hötker Oliver Krüger John Marzluff Anders P. Møller Shinichi Nakagawa Richard A. Phillips Andrew N. Radford Alexandre Roulin János Török Juliana Valencia Martijn van de Pol Ian G. Warkentin Isabel S. Winney Andrew G. Wood Michael Griesser 《Evolution; international journal of organic evolution》2016,70(2):296-313
Fitness can be profoundly influenced by the age at first reproduction (AFR), but to date the AFR–fitness relationship only has been investigated intraspecifically. Here, we investigated the relationship between AFR and average lifetime reproductive success (LRS) across 34 bird species. We assessed differences in the deviation of the Optimal AFR (i.e., the species‐specific AFR associated with the highest LRS) from the age at sexual maturity, considering potential effects of life history as well as social and ecological factors. Most individuals adopted the species‐specific Optimal AFR and both the mean and Optimal AFR of species correlated positively with life span. Interspecific deviations of the Optimal AFR were associated with indices reflecting a change in LRS or survival as a function of AFR: a delayed AFR was beneficial in species where early AFR was associated with a decrease in subsequent survival or reproductive output. Overall, our results suggest that a delayed onset of reproduction beyond maturity is an optimal strategy explained by a long life span and costs of early reproduction. By providing the first empirical confirmations of key predictions of life‐history theory across species, this study contributes to a better understanding of life‐history evolution. 相似文献