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991.
992.
The relationship between the structure of the N-terminal sequence of transthyretin (TTR) and the binding of thyroid hormone was studied. A recombinant human TTR and two derivatives of Crocodylus porosus TTRs, one with the N-terminal sequence replaced by that of human TTR (human/crocTTR), the other with the N-terminal segment removed (truncated crocTTR), were synthesized in Pichia pastoris. Subunit mass, native molecular weight, tetramer formation, cross-reactivity to TTR antibodies and binding to retinol-binding protein of these recombinant TTRs were similar to TTRs found in nature. Analysis of the binding affinity to thyroid hormones of recombinant human TTR showed a dissociation constant (Kd) for triiodothyronine (T3) of 53.26+/-3.97 nM and for thyroxine (T4) of 19.73+/-0.13 nM. These values are similar to those found for TTR purified from human serum, and gave a Kd T3/T4 ratio of 2.70. The affinity for T4 of human/crocTTR (Kd=22.75+/-1.89 nM) was higher than those of both human TTR and C. porosus TTR, but the affinity for T3 (Kd=5.40+/-0.25 nM) was similar to C. porosus TTR, giving a Kd T3/T4 ratio of 0.24. A similar affinity for both T3 (Kd=57.78+/-5.65 nM) and T4 (Kd=59.72+/-3.38 nM), with a Kd T3/T4 ratio of 0.97, was observed for truncated crocTTR. The obtained results strongly confirm the hypothesis that the unstructured N-terminal region of TTR critically influences the specificity and affinity of thyroid hormone binding to TTR. 相似文献
993.
The renal parasite Eimeria auritusi has caused several mortality events in double-crested cormorants (DCC; Phalacrocorax auritus) in the Midwest and southeastern United States. This parasite has only been detected during large-scale outbreaks, and its presence and prevalence in healthy populations of cormorants is unknown. In this study, 80 DCC were collected from the Chattahoochee River near Fort Gaines, Georgia, and examined for kidney and intestinal coccidia. Eighteen (22.5%) and 56 (70%) of the DCC were positive for E. auritusi and a new species of intestinal Eimeria, respectively. Oocysts of the new intestinal Eimeria species had a thin colorless wall, were ovoid with rare bumps on the outer surface, and measured 17.1 microm +/- 1.5 x 14.7 microm +/- 1.0 (16-18.5 x 13-17), with an average length:width ratio of 1.17 microm (1.03-1.29). A prominent micropyle (4-4.5 microm) was present, and a large oval-to-round polar body (2.5 microm) was located beneath the micropyle. Sporocysts were ovoid and measured 9.6 microm +/- 0.6 x 5.9 microm +/- 0.5 (8.5-10.5 x 5-6.5), with an average length:width ratio of 1.63 (1.3-1.82) with small stieda body present. Amplification and sequencing of a fragment of the 18S rRNA gene indicated that the 2 DCC Eimeria species and 2 Eimeria species from cranes were in a separate group from other Eimeriidae. These data indicate that E. auritusi and this new species of intestinal Eimeria are prevalent in this apparently healthy DCC population. The cause of renal coccidiosis outbreaks in other populations of cormorants is unknown but could be due to crowding or stress during the winter months or some other associated pathogen or immunosuppressor that might predispose individuals to clinical disease. 相似文献
994.
Justin F. Shaffer Robert W. Kensler Samantha P. Harris 《The Journal of biological chemistry》2009,284(18):12318-12327
Cardiac myosin-binding protein C (cMyBP-C) is a regulatory protein
expressed in cardiac sarcomeres that is known to interact with myosin, titin,
and actin. cMyBP-C modulates actomyosin interactions in a
phosphorylation-dependent way, but it is unclear whether interactions with
myosin, titin, or actin are required for these effects. Here we show using
cosedimentation binding assays, that the 4 N-terminal domains of murine
cMyBP-C (i.e. C0-C1-m-C2) bind to F-actin with a dissociation
constant (Kd) of ∼10 μm and a molar
binding ratio (Bmax) near 1.0, indicating 1:1 (mol/mol)
binding to actin. Electron microscopy and light scattering analyses show that
these domains cross-link F-actin filaments, implying multiple sites of
interaction with actin. Phosphorylation of the MyBP-C regulatory motif, or
m-domain, reduced binding to actin (reduced Bmax) and
eliminated actin cross-linking. These results suggest that the N terminus of
cMyBP-C interacts with F-actin through multiple distinct binding sites and
that binding at one or more sites is reduced by phosphorylation. Reversible
interactions with actin could contribute to effects of cMyBP-C to increase
cross-bridge cycling.Cardiac myosin-binding protein C
(cMyBP-C)2
is a thick filament accessory protein that performs both structural and
regulatory functions within vertebrate sarcomeres. Both roles are likely to be
essential in deciphering how a growing number of mutations found in the
cMyBP-C gene, i.e. MYBPC3, lead to cardiomyopathies and heart failure
in a substantial number of the world''s population
(1,
2).Considerable progress has recently been made in determining the regulatory
functions of cMyBP-C and it is now apparent that cMyBP-C normally limits
cross-bridge cycling kinetics and is critical for cardiac function
(3-5).
Phosphorylation of cMyBP-C is essential for its regulatory effects because
elimination of phosphorylation sites (serine to alanine substitutions)
abolishes the ability of protein kinase A (PKA) to accelerate cross-bridge
cycling kinetics and blunts cardiac responses to inotropic stimuli
(6). The substitutions further
impair cardiac function, reduce contractile reserve, and cause cardiac
hypertrophy in transgenic mice
(6,
7). By contrast, substitution
of aspartic acids at these sites to mimic constitutive phosphorylation is
benign or cardioprotective
(8).Although a role for cMyBP-C in modulating cross-bridge kinetics is
supported by several transgenic and knock-out mouse models
(6,
7,
9,
10), the precise mechanisms by
which cMyBP-C exerts these effects are not completely understood. For
instance, the unique regulatory motif or “m-domain” of cMyBP-C
binds to the S2 subfragment of myosin in vitro
(11) and binding is abolished
by PKA-mediated phosphorylation of the m-domain
(12). These observations have
led to the idea that (un)binding of the m-domain from myosin S2 mediates
PKA-induced increases in cross-bridge cycling kinetics. Consistent with this
idea, Calaghan and colleagues
(13) showed that S2 added to
transiently permeabilized myocytes increased their contractility, presumably
because added S2 displaced cMyBP-C from binding endogenous S2. However, other
reports indicate that cMyBP-C can influence actomyosin interactions through
mechanisms unrelated to S2 binding, because either purified cMyBP-C
(14) or recombinant N-terminal
domains of cMyBP-C (15)
affected acto-S1 filament sliding velocities and ATPase rates in the absence
of myosin S2. These results thus raise the possibility that interactions with
ligands other than myosin S2, such as actin or myosin S1, contribute to
effects of cMyBP-C on cross-bridge interaction kinetics.The idea that cMyBP-C interacts with actin to influence cross-bridge
cycling kinetics is supported by several studies that implicate the regulatory
m-domain or sequences near it in actin binding
(16-19).
cMyBP-C is a member of the immunoglobulin (Ig) superfamily of proteins and
consists of 11 repeating domains that bear homology to either Ig or
fibronectin-like folds. Domains are numbered sequentially from the N terminus
of cMyBP-C as C0 through C10. The m-domain, a unique sequence of ∼100
amino acids, is located between domains C1 and C2 and is phosphorylated on at
least 3 serine residues by PKA
(12). Although the precise
structure of the m-domain is not known, small angle x-ray scattering data
suggest that it is compact and folded in solution and is thus similar in size
and dimensions to the surrounding Ig domains
(20). Recombinant proteins
encompassing the m-domain and/or a combination of adjacent domains including
C0, C1, C2, and a proline-alanine-rich sequence that links C0 to C1 have been
shown to bind actin (16,
18,
19).The purpose of the present study was to characterize binding interactions
of the N terminus of cMyBP-C with actin and to determine whether interactions
with actin are influenced by phosphorylation of the m-domain. Results
demonstrate that the N terminus of cMyBP-C binds to F-actin and to native thin
filaments with affinities similar to that reported for cMyBP-C binding to
myosin S2 (11). Furthermore,
actin binding was reduced by m-domain phosphorylation, suggesting that
reversible interactions of cMyBP-C with actin could contribute to modulation
of cross-bridge kinetics. 相似文献
995.
Meghan E. McGee-Lawrence Samantha J. Wojda Lindsay N. Barlow Thomas D. Drummer Kevin Bunnell Janene Auger Hal L. Black Seth W. Donahue 《Journal of biomechanics》2009,42(10):1378-1383
Disuse typically uncouples bone formation from resorption, leading to bone loss which compromises bone mechanical properties and increases the risk of bone fracture. Previous studies suggest that bears can prevent bone loss during long periods of disuse (hibernation), but small sample sizes have limited the conclusions that can be drawn regarding the effects of hibernation on bone structure and strength in bears. Here we quantified the effects of hibernation on structural, mineral, and mechanical properties of black bear (Ursus americanus) cortical bone by studying femurs from large groups of male and female bears (with wide age ranges) killed during pre-hibernation (fall) and post-hibernation (spring) periods. Bone properties that are affected by body mass (e.g. bone geometrical properties) tended to be larger in male compared to female bears. There were no differences (p>0.226) in bone structure, mineral content, or mechanical properties between fall and spring bears. Bone geometrical properties differed by less than 5% and bone mechanical properties differed by less than 10% between fall and spring bears. Porosity (fall: 5.5±2.2%; spring: 4.8±1.6%) and ash fraction (fall: 0.694±0.011; spring: 0.696±0.010) also showed no change (p>0.304) between seasons. Statistical power was high (>72%) for these analyses. Furthermore, bone geometrical properties and ash fraction (a measure of mineral content) increased with age and porosity decreased with age. These results support the idea that bears possess a biological mechanism to prevent disuse and age-related osteoporoses. 相似文献
996.
Agier V Oliviero P Lainé J L'Hermitte-Stead C Girard S Fillaut S Jardel C Bouillaud F Bulteau AL Lombès A 《Biochimica et biophysica acta》2012,1822(10):1570-1580
997.
998.
999.
Roosild TP Castronovo S Villoso A Ziemba A Pizzorno G 《Journal of structural biology》2011,176(2):229-237
Uridine phosphorylase (UPP) catalyzes the reversible conversion of uridine to uracil and ribose-1-phosphate and plays an important pharmacological role in activating fluoropyrimidine nucleoside chemotherapeutic agents such as 5-fluorouracil and capecitabine. Most vertebrate animals, including humans, possess two homologs of this enzyme (UPP1 & UPP2), of which UPP1 has been more thoroughly studied and is better characterized. Here, we report two crystallographic structures of human UPP2 (hUPP2) in distinctly active and inactive conformations. These structures reveal that a conditional intramolecular disulfide bridge can form within the protein that dislocates a critical phosphate-coordinating arginine residue (R100) away from the active site, disabling the enzyme. In vitro activity measurements on both recombinant hUPP2 and native mouse UPP2 confirm the redox sensitivity of this enzyme, in contrast to UPP1. Sequence analysis shows that this feature is conserved among UPP2 homologs and lacking in all UPP1 proteins due to the absence of a necessary cysteine residue. The state of the disulfide bridge has further structural consequences for one face of the enzyme that suggest UPP2 may have additional functions in sensing and initiating cellular responses to oxidative stress. The molecular details surrounding these dynamic aspects of hUPP2 structure and regulation provide new insights as to how novel inhibitors of this protein may be developed with improved specificity and affinity. As uridine is emerging as a promising protective compound in neuro-degenerative diseases, including Alzheimer’s and Parkinson’s, understanding the regulatory mechanisms underlying UPP control of uridine concentration is key to improving clinical outcomes in these illnesses. 相似文献
1000.
Cloned ferrets produced by somatic cell nuclear transfer 总被引:10,自引:0,他引:10
Li Z Sun X Chen J Liu X Wisely SM Zhou Q Renard JP Leno GH Engelhardt JF 《Developmental biology》2006,293(2):439-448
Somatic cell nuclear transfer (SCNT) offers great potential for developing better animal models of human disease. The domestic ferret (Mustela putorius furo) is an ideal animal model for influenza infections and potentially other human respiratory diseases such as cystic fibrosis, where mouse models have failed to reproduce the human disease phenotype. Here, we report the successful production of live cloned, reproductively competent, ferrets using species-specific SCNT methodologies. Critical to developing a successful SCNT protocol for the ferret was the finding that hormonal treatment, normally used for superovulation, adversely affected the developmental potential of recipient oocytes. The onset of Oct4 expression was delayed and incomplete in parthenogenetically activated oocytes collected from hormone-treated females relative to oocytes collected from females naturally mated with vasectomized males. Stimulation induced by mating and in vitro oocyte maturation produced the optimal oocyte recipient for SCNT. Although nuclear injection and cell fusion produced mid-term fetuses at equivalent rates (approximately 3-4%), only cell fusion gave rise to healthy surviving clones. Single cell fusion rates and the efficiency of SCNT were also enhanced by placing two somatic cells into the perivitelline space. These species-specific modifications facilitated the birth of live, healthy, and fertile cloned ferrets. The development of microsatellite genotyping for domestic ferrets confirmed that ferret clones were genetically derived from their respective somatic cells and unrelated to their surrogate mother. With this technology, it is now feasible to begin generating genetically defined ferrets for studying transmissible and inherited human lung diseases. Cloning of the domestic ferret may also aid in recovery and conservation of the endangered black-footed ferret and European mink. 相似文献