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Background
While the social, ethical, and legal implications of biobanking and large scale data sharing are already complicated enough, they may be further compounded by research on the human microbiome.Discussion
The human microbiome is the entire complement of microorganisms that exists in and on every human body. Currently most biobanks focus primarily on human tissues and/or associated data (e.g. health records). Accordingly, most discussions in the social sciences and humanities on these issues are focused (appropriately so) on the implications of biobanks and sharing data derived from human tissues. However, rapid advances in human microbiome research involve collecting large amounts of data on microorganisms that exist in symbiotic relationships with the human body. Currently it is not clear whether these microorganisms should be considered part of or separate from the human body. Arguments can be made for both, but ultimately it seems that the dichotomy of human versus non-human and self versus non-self inevitably breaks down in this context. This situation has the potential to add further complications to debates on biobanking.Summary
In this paper, we revisit some of the core problem areas of privacy, consent, ownership, return of results, governance, and benefit sharing, and consider how they might be impacted upon by human microbiome research. Some of the issues discussed also have relevance to other forms of microbial research. Discussion of these themes is guided by conceptual analysis of microbiome research and interviews with leading Canadian scientists in the field.4.
We investigated the features of the structural and functional organization of the left heart (ventricle—LV, atrium—LA) and the state of systemic hemodynamics at rest and in response to a single dose of cardioselective β1-blocker (BB) Egilok. We examined the patients with stage II (1–2 degrees) of arterial hypertension (AH); the study was performed in summer and winter in the northern regions of Russia. It was found that the process of adaptation to cold is accompanied by the inhibition of the pacemaker, a decrease in the rate of active diastolic blood filling of the LV and transaortic blood flow in the aortic root (VAo), an increase in the contractility of the LV posterior wall and interventricular septum (IVS). The negative chronotropic cardiac effect in these conditions results in the reduction of heart productivity per minute in 65% of cases. In winter we observed a more pronounced diastolic LV dysfunction and a decrease in the connectivity of active relaxation of LV posterior wall and LA walls with certain structural and functional cardiac parameters. In contrast to summer, in winter period BB causes a decrease in the active relaxation of LA walls and IVS and LA contractility, which leads to a decrease in the blood filling of passive and active LV. At the same time, LV systolic function (ejection fraction, VAo) and the rhythm and the performance of the heart (stroke volume and cardiac output) decreases; the hypotensive effect accompanied by an increase in peripheral vascular resistance is more pronounced. In winter, the effect of BB reduces the correlation between IVS and LV posterior wall contractions, but the feedback rate or passive to active LV diastolic hyperemia and after load increases. We suggest that in winter component “contractile apparatus” retains its the leading role in the organization of intracardiac response to the BB in patients with hypertension; in addition, new dominant components were formed: “contingency of the LV wall contraction with afterload” and “reverse contingency of early and late diastolic LV function.” 相似文献
5.
Nidhi Ahuja Dmitry Korkin Rachna Chaba Brent O. Cezairliyan Robert T. Sauer Kyeong Kyu Kim Carol A. Gross 《The Journal of biological chemistry》2009,284(8):5403-5413
The Escherichia coli envelope stress response is controlled by the
alternative sigma factor, σE, and is induced when unfolded
outer membrane proteins accumulate in the periplasm. The response is initiated
by sequential cleavage of the membrane-spanning antisigma factor, RseA. RseB
is an important negative regulator of envelope stress response that exerts its
negative effects onσE activity through its binding to RseA.
In this study, we analyze the interaction between RseA and RseB. We found that
tight binding of RseB to RseA required intact RseB. Using programs that
performed global and local sequence alignment of RseB and RseA, we found
regions of high similarity and performed alanine substitution mutagenesis to
test the hypothesis that these regions were functionally important. This
protocol is based on the hypothesis that functionally dependent regions of two
proteins co-evolve and therefore are likely to be sequentially conserved. This
procedure allowed us to identify both an N-terminal and C-terminal region in
RseB important for binding to RseA. We extensively analyzed the C-terminal
region, which aligns with a region of RseA coincident with the major RseB
binding determinant in RseA. Both allele-specific suppression analysis and
cysteine-mediated disulfide bond formation indicated that this C-terminal
region of similarity of RseA and RseB identifies a contact site between the
two proteins. We suggest a similar protocol can be successfully applied to
pairs of non-homologous but functionally linked proteins to find specific
regions of the protein sequences that are important for establishing
functional linkage.The Escherichia coli σE-mediated envelope stress
response is the major pathway to ensure homeostasis in the envelope
compartment of the cell
(1-3).
σE regulon members encode periplasmic chaperones and
proteases, the machinery for inserting β-barrel proteins into the outer
membrane and components controlling the synthesis and assembly of LPS
(4-6).
This pathway is highly conserved among γ-proteobacteria
(6).The σE response is initiated when periplasmic protein
folding and assembly is compromised
(7-9).
During steady state growth, σE is inhibited by its antisigma
factor, RseA, a membrane-spanning protein whose cytoplasmic domain binds to
σE with picomolar affinity
(10-13).
Accumulation of unassembled porin monomers serves as a signal to activate the
DegS protease to cleave RseA in its periplasmic domain
(14,
15). This initiates a
proteolytic cascade in which RseP cleaves periplasmically truncated RseA near
or within the cytoplasmic membrane to release the
RseAcytoplasmic-σE complex, and cytoplasmic
ATP-dependent proteases complete the degradation of RseA thereby releasing
active σE
(16-19).RseB, a second negative regulator of the envelope stress response
(11,
20,
21), binds to the periplasmic
domain of RseA with nanomolar affinity. RseB is an important regulator of the
response (2,
22,
23). It prevents RseP from
degrading intact RseA, thereby ensuring that proteolysis is initiated only
when the DegS protease is activated by a stress signal
(21). Additionally, RseB
prevents activated DegS from cleaving RseA, suggesting that interaction of
RseB with RseA must be altered before the signal transduction cascade is
activated (23).The goal of the present studies was to explore how RseB binds to RseA. The
interaction partner of RseB is the unstructured periplasmic domain of RseA
(RseA-peri). Within RseA-peri, amino acids ∼169-186 constitute a major
binding determinant to RseB
(23,
24). This peptide alone binds
RseB with 6 μm affinity, and deleting this region abrogates
binding to RseB (23).
Additional regions of RseA-peri also contribute to RseB binding, as intact
RseA-peri binds with 20 nm affinity to RseB
(23). Much less is known about
the regions of RseB required for interaction with RseA. RseB is homodimeric
two-domain protein, whose large N-terminal domain shares structural homology
with LolA, a protein that transports lipoproteins to outer membrane
(24,
25). The smaller C-terminal
domain is connected to the N-terminal domain by a linker, and the two domains
share a large interface, which may facilitate interdomain signaling.
Glutaraldehyde cross-linking studies indicate that the C-terminal domain
interacts with RseA, but the regions of interaction were not identified
(25).In the present report, we study the interaction of RseB and RseA. We
establish that both domains of RseB interact with RseA-peri. Using a global
sequence alignment, we discovered several regions in RseA and RseB that had
high sequence similarity, despite the low overall sequence similarity between
these two proteins, a finding that was independently confirmed by a local
sequence similarity algorithm. This suggested that these regions were
functionally dependent, and we performed a set of mutagenesis experiments
designed to test this idea. Our studies of the binding properties of these
mutants revealed that regions in both the N terminus and C terminus of RseB
modulate interaction with RseA. Moreover, genetic suppression analysis and
cysteine-mediated disulfide bond formation suggest that the region of RseA/B
with highest similarity (RseA residues 165-191 (major binding determinant in
RseA) and RseB residues 233-258) are interacting partners. 相似文献
6.
Intracellular β-Carbonic Anhydrase of the Unicellular Green
Alga Coccomyxa
: Cloning of the cDNA and Characterization of the Functional
Enzyme Overexpressed in Escherichia coli
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Thomas Hiltonen Harry Bj?rkbacka Cecilia Forsman Adrian K. Clarke G?ran Samuelsson 《Plant physiology》1998,117(4):1341-1349
Carbonic anhydrase (CA) (EC 4.2.1.1) enzymes catalyze the reversible hydration of CO2, a reaction that is important in many physiological processes. We have cloned and sequenced a full-length cDNA encoding an intracellular β-CA from the unicellular green alga Coccomyxa. Nucleotide sequence data show that the isolated cDNA contains an open reading frame encoding a polypeptide of 227 amino acids. The predicted polypeptide is similar to β-type CAs from Escherichia coli and higher plants, with an identity of 26% to 30%. The Coccomyxa cDNA was overexpressed in E. coli, and the enzyme was purified and biochemically characterized. The mature protein is a homotetramer with an estimated molecular mass of 100 kD. The CO2-hydration activity of the Coccomyxa enzyme is comparable with that of the pea homolog. However, the activity of Coccomyxa CA is largely insensitive to oxidative conditions, in contrast to similar enzymes from most higher plants. Fractionation studies further showed that Coccomyxa CA is extrachloroplastic. 相似文献
7.
The effects of FeCl3 and Fe–EDTA on the development of psoriasis were studied in the mouse model of vaginal epithelium and tail epidermis. The
mitoses of vaginal epithelial cell in female mice of their estrogenic stage and the formation of granular cell layers in male
mouse tail scale were observed. Mice were randomly divided into eight groups and treated with normal saline, methotrexate,
and different doses of two iron forms, FeCl3 and Fe–EDTA, respectively, for 10 days. To explore the influence of FeCl3 and Fe–EDTA on the excretion of Cu, Fe, Zn, Ca, Mg, Mn, and Se, the concentration of those elements in liver and kidney was
analyzed by atomic absorption spectrometry. The different doses of FeCl3 or Fe–EDTA could obviously inhibit the mitoses of vaginal epithelial cell (p< 0.05) and promote the formation of granular cell layers in mice tail scale (p < 0.05). No statistically significant results were found between the groups of FeCl3 and Fe–EDTA, and between experimental groups and methotrexate group acted as the positive control (p>0.05). Compared with the negative group, the concentrations of Cu, Fe, Zn, Ca, Mg, Mn, and Se in liver and kidney of experimental
groups and positive control group were not significantly changed (p > 0.05). FeCl3 and Fe–EDTA are as effective as methotrexate on inhibiting hyperplasia of epidermal cells and increasing the formation of
granular cell layers, and the concentrations of Cu, Fe, Zn, Ca, Mg, Mn, and Se in liver and kidney of experimental groups
and positive control group were not significantly changed compared with the negative group, possibly retarding the development
of psoriasis. 相似文献
8.
A comparative evaluation of the level of extracellular peroxidase activity and light-emission intensity of the mycelium of the luminescent basidiomycete Neonothopanus nambi in the presence of β-glucosidase was performed. The enzyme activity damages the hyphae of the fungus leading to osmotic imbalance, partial degradation of the mycelium, and release of extracellular peroxidases into the incubation medium. The presence of β-glucosidase reduces the time necessary to reach the maximum luminescence. Putative biochemical mechanisms that underlie the stimulation of reactive oxygen species formation (first and foremost, of hydrogen peroxide) in the N. nambi mycelium in the presence of β-glucosidase are proposed. 相似文献
9.
Yongmei Pu Susan H. Garfield Noemi Kedei Peter M. Blumberg 《The Journal of biological chemistry》2009,284(2):1302-1312
Classic and novel protein kinase C (PKC) isozymes contain two zinc finger
motifs, designated “C1a” and “C1b” domains, which
constitute the recognition modules for the second messenger diacylglycerol
(DAG) or the phorbol esters. However, the individual contributions of these
tandem C1 domains to PKC function and, reciprocally, the influence of protein
context on their function remain uncertain. In the present study, we prepared
PKCδ constructs in which the individual C1a and C1b domains were
deleted, swapped, or substituted for one another to explore these issues. As
isolated fragments, both the δC1a and δC1b domains potently bound
phorbol esters, but the binding of [3H]phorbol 12,13-dibutyrate
([3H]PDBu) by the δC1a domain depended much more on the
presence of phosphatidylserine than did that of the δC1b domain. In
intact PKCδ, the δC1b domain played the dominant role in
[3H]PDBu binding, membrane translocation, and down-regulation. A
contribution from the δC1a domain was nonetheless evident, as shown by
retention of [3H]PDBu binding at reduced affinity, by increased
[3H]PDBu affinity upon expression of a second δC1a domain
substituting for the δC1b domain, and by loss of persistent plasma
membrane translocation for PKCδ expressing only the δC1b domain,
but its contribution was less than predicted from the activity of the isolated
domain. Switching the position of the δC1b domain to the normal position
of the δC1a domain (or vice versa) had no apparent effect on the
response to phorbol esters, suggesting that the specific position of the C1
domain within PKCδ was not the primary determinant of its activity.One of the essential steps for protein kinase C
(PKC)2 activation is
its translocation from the cytosol to the membranes. For conventional
(α, βI, βII, and γ) and novel (δ, ε, η,
and θ) PKCs, this translocation is driven by interaction with the
lipophilic second messenger sn-1,2-diacylglycerol (DAG), generated
from phosphatidylinositol 4,5-bisphosphate upon the activation of
receptor-coupled phospholipase C or indirectly from phosphatidylcholine via
phospholipase D (1). A pair of
zinc finger structures in the regulatory domain of the PKCs, the
“C1” domains, are responsible for the recognition of the DAG
signal. The DAG-C1 domain-membrane interaction is coupled to a conformational
change in PKC, both causing the release of the pseudosubstrate domain from the
catalytic site to activate the enzyme and triggering the translocation to the
membrane (2). By regulating
access to substrates, PKC translocation complements the intrinsic enzymatic
specificity of PKC to determine its substrate profile.The C1 domain is a highly conserved cysteine-rich motif (∼50 amino
acids), which was first identified in PKC as the interaction site for DAG or
phorbol esters (3). It
possesses a globular structure with a hydrophilic binding cleft at one end
surrounded by hydrophobic residues. Binding of DAG or phorbol esters to the C1
domain caps the hydrophilic cleft and forms a continuous hydrophobic surface
favoring the interaction or penetration of the C1 domain into the membrane
(4). In addition to the novel
and classic PKCs, six other families of proteins have also been identified,
some of whose members possess DAG/phorbol ester-responsive C1 domains. These
are the protein kinase D (5),
the chimaerin (6), the munc-13
(7), the RasGRP (guanyl
nucleotide exchange factors for Ras and Rap1)
(8), the DAG kinase
(9), and the recently
characterized MRCK (myotonic dystrophy kinase-related
Cdc42-binding kinase) families
(10). Of these C1
domain-containing proteins, the PKCs have been studied most extensively and
are important therapeutic targets
(11). Among the drug
candidates in clinical trials that target PKC, a number such as bryostatin 1
and PEP005 are directed at the C1 domains of PKC rather than at its catalytic
site.Both the classic and novel PKCs contain in their N-terminal regulatory
region tandem C1 domains, C1a and C1b, which bind DAG/phorbol ester
(12). Multiple studies have
sought to define the respective roles of these two C1 domains in PKC
regulation, but the issue remains unclear. Initial in vitro binding
measurements with conventional PKCs suggested that 1 mol of phorbol ester
bound per mole of PKC
(13-15).
On the other hand, Stubbs et al., using a fluorescent phorbol ester
analog, reported that PKCα bound two ligands per PKC
(16). Further, site-directed
mutagenesis of the C1a and C1b domains of intact PKCα indicated that the
C1a and C1b domains played equivalent roles for membrane translocation in
response to phorbol 12-myristate 13-acetate (PMA) and (-)octylindolactam V
(17). Likewise, deletion
studies indicated that the C1a and C1b domains of PKCγ bound PDBu
equally with high potency (3,
18). Using a functional assay
with PKCα expression in yeast, Shieh et al.
(19) deleted individual C1
domains and reported that C1a and C1b were both functional and equivalent upon
stimulation by PMA, with either deletion causing a similar reduction in
potency of response, whereas for mezerein the response depended essentially on
the C1a domain, with much weaker response if only the C1b domain was present.
Using isolated C1 domains, Irie et al.
(20) suggested that the C1a
domain of PKCα but not those of PKCβ or PKCγ bound
[3H]PDBu preferentially; different ligands showed a generally
similar pattern but with different extents of selectivity. Using synthesized
dimeric bisphorbols, Newton''s group reported
(21) that, although both C1
domains of PKCβII are oriented for potential membrane interaction, only
one C1 domain bound ligand in a physiological context.In the case of novel PKCs, many studies have been performed on PKCδ
to study the equivalency of the twin C1 domains. The P11G point mutation of
the C1a domain, which caused a 300-fold loss of binding potency in the
isolated domain (22), had
little effect on the phorbol ester-dependent translocation of PKCδ in
NIH3T3 cells, whereas the same mutation of the C1b caused a 20-fold shift in
phorbol ester potency for inducing translocation, suggesting a major role of
the C1b domain for phorbol ester binding
(23). A secondary role for the
C1a domain was suggested, however, because mutation in the C1a domain as well
as the C1b domain caused a further 7-fold shift in potency. Using the same
mutations in the C1a and C1b domains, Bögi et al.
(24) found that the binding
selectivity for the C1a and C1b domains of PKCδ appeared to be
ligand-dependent. Whereas PMA and the indole alkaloids indolactam and
octylindolactam were selectively dependent on the C1b domain, selectivity was
not observed for mezerein, the 12-deoxyphorbol 13-monoesters prostratin and
12-deoxyphorbol 13-phenylacetate, and the macrocyclic lactone bryostatin 1
(24). In in vitro
studies using isolated C1a and C1b domains of PKCδ, Cho''s group
(25) described that the two C1
domains had opposite affinities for DAG and phorbol ester; i.e. the
C1a domain showed high affinity for DAG and the C1b domain showed high
affinity for phorbol ester. No such difference in selectivity was observed by
Irie et al. (20).PKC has emerged as a promising therapeutic target both for cancer and for
other conditions, such as diabetic retinopathy or macular degeneration
(26-30).
Kinase inhibitors represent one promising approach for targeting PKC, and
enzastaurin, an inhibitor with moderate selectivity for PKCβ relative to
other PKC isoforms (but still with activity on some other non-PKC kinases) is
currently in multiple clinical trials. An alternative strategy for drug
development has been to target the regulatory C1 domains of PKC. Strong proof
of principle for this approach is provided by multiple natural products,
e.g. bryostatin 1 and PEP005, which are likewise in clinical trials
and which are directed at the C1 domains. A potential advantage of this
approach is the lesser number of homologous targets, <30 DAG-sensitive C1
domains compared with over 500 kinases, as well as further opportunities for
specificity provided by the diversity of lipid environments, which form a
half-site for ligand binding to the C1 domain. Because different PKC isoforms
may induce antagonistic activities, inhibition of one isoform may be
functionally equivalent to activation of an antagonistic isoform
(31).Along with the benzolactams
(20,
32), the DAG lactones have
provided a powerful synthetic platform for manipulating ligand: C1 domain
interactions (31). For
example, the DAG lactone derivative 130C037 displayed marked selectivity among
the recombinant C1a and C1b domains of PKCα and PKCδ as well as
substantial selectivity for RasGRP relative to PKCα
(33). Likewise, we have shown
that a modified DAG lactone (dioxolanones) can afford an additional point of
contact in ligand binding to the C1b domain of PKCδ
(34). Such studies provide
clear examples that ligand-C1 domain interactions can be manipulated to yield
novel patterns of recognition. Further selectivity might be gained with
bivalent compounds, exploiting the spacing and individual characteristics of
the C1a and C1b domains (35).
A better understanding of the differential roles of the two C1 domains in PKC
regulation is critical for the rational development of such compounds. In this
study, by molecularly manipulating the C1a or C1b domains in intact
PKCδ, we find that both the C1a and C1b domains play important roles in
PKCδ regulation. The C1b domain is predominant for ligand binding and
for membrane translocation of the whole PKCδ molecule. The C1a domain of
intact PKCδ plays only a secondary role in ligand binding but stabilizes
the PKCδ molecule at the plasma membrane for downstream signaling. In
addition, we show that the effect of the individual C1 domains of PKCδ
does not critically depend on their position within the regulatory domain. 相似文献
10.
Sarecka-Hujar B Zak I Emich-Widera E Kopyta I Pilarska E Pienczk-Reclawowicz K 《Cell biochemistry and function》2010,28(7):591-596
Genes related to platelet and arterial endothelial function have been recently considered as independent risk factors for stroke. We aimed to analyze a relationship between the E‐selectin 98G > T polymorphism and stroke in children and to observe the transmission of E‐selectin alleles from heterozygous parents to their affected children. We studied 59 children after stroke, 112 parents, and 87 healthy children. The E‐selectin 98G > T polymorphism was analyzed with the polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) method. The frequency of the 98T allele in patients was almost twofold lower than in controls (5.1% vs. 9.8%, p = 0.145, odds ratios (OR) = 0.49) as well as carriers of the 98T allele (19.5% in controls vs. 8.5% in cases, p = 0.067, OR = 0.38). The G allele of the E‐selectin 98G > T polymorphism was more frequently transmitted to the children after stroke compared to the T allele (68% vs. 32%). In conclusion, we did not confirm the relationship between the 98G > T polymorphism of the E‐selectin gene and childhood ischemic stroke. There is still a need for further studies. Copyright © 2010 John Wiley & Sons, Ltd. 相似文献
11.
《Animal : an international journal of animal bioscience》2021,15(9):100324
The traditional production of the Iberian breed pig involves a long production cycle. It might be shortened by using Iberian pigs crossed with Duroc and by reducing the growing phase, but the age-related changes on productive performance and carcass quality should be addressed. Thus, productive performance, live measurements and carcass and primal cut traits were evaluated on Iberian × Duroc 50:50 crossed pigs according to animal age at the beginning of the free-range finishing phase (Montanera): 10, 12 and 14 months old (IBxD10 (n = 15), IBxD12 (n = 17) and IBxD14 (n = 18) animal batches, respectively) and gender (immunologically castrated female -consisted of the Improvac® vaccination- and surgically castrated males). During the growing period, animals were fed with restrictions; 1.49, 1.29 and 1.20 ± 0.023 (mean ± SEM) kg/day of commercial feeds to start Montanera with similar BW; 103.9, 102.9 and 102.1 ± 0.22 kg, for IBxD10, IBxD12 and IBxD14, respectively. IBxD14 animals yielded the highest average daily gain (ADG) and BW after Montanera, as well as larger rump height and croup width. In contrast, these animals had the lowest carcass yield. Although animals from IBxD10 yielded hams of inferior size, this could be of interest to the sector, as there is a certain segment of the market that demands hams of smaller size and, generally, this is difficult to obtain with the traditional Montanera production system. The gender had no major effects on performance and carcass and primal cut traits, so both immunologically castrated female and surgically castrated males are suitable for finishing in Montanera. 相似文献
12.
13.
Charles Robin Lisa M. J. Bardsley Chris Coppin John G. Oakeshott 《Journal of molecular evolution》2009,69(1):10-21
Here we analyze the molecular evolution of the β-esterase gene cluster in the Drosophila genus using the recently released genome sequences of 12 Drosophila species. Molecular evolution in this small cluster is noteworthy because it contains contrasting examples of the types and
stages of loss of gene function. Specifically, missing orthologs, pseudogenes, and null alleles are all inferred. Phylogenetic
analyses also suggest a minimum of 9 gene gain–loss events; however, the exact number and age of these events is confounded
by interparalog recombination. A previous enigma, in which allozyme loci were mapped to β-esterase genes that lacked catalytically
essential amino acids, was resolved through the identification of neighbouring genes that contain the canonical catalytic
residues and thus presumably encode the mapped allozymes. The originally identified genes are evolving with selective constraint,
suggesting that they have a “noncatalytic” function. Curiously, 3 of the 4 paralogous β-esterase genes in the D. ananassae genome sequence have single inactivating (frame-shift or nonsense) mutations. To determine whether these putatively inactivating
mutations were fixed, we sequenced other D. ananassae alleles of these four loci. We did not find any of the 3 inactivating mutations of the sequenced strain in 12 other strains;
however, other inactivating mutations were observed in the same 3 genes. This is reminiscent of the high frequency of null
alleles observed in one of the β-esterase genes (Est7/EstP) of D. melanogaster.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
14.
Raymond Counis Maithe Corbani Marc Poissonnier Marian Jutisz 《Biochemical and biophysical research communications》1982,107(3):998-1005
Poly(A+)RNA was prepared from anterior pituitary glands of ovariectomized (ovx) ewes and rats and the mRNAs were translated in a wheat-germ cell-free system in the presence of [35S]-labeled cysteine and methionine. Specific antisera raised against denaturated (RCXM) ovine FSHβ and α-subunits were used to demonstrate the in vitro synthesis of FSH subunits. Anti-RCXM FSHβ precipitated a single polypeptide, exhibiting a Mr ? 19,000 by SDS-polyacrylamide gel electrophoresis whether its synthesis was directed by ewe or rat mRNA. A Mr of 17,000–17,500 was found for the α-polypeptide. FSHβ-polypeptides represented about 0.015–0.019% of the total radioactivity incorporated in response to mRNA from ovx ewes and 0.046–0.050% in the case of mRNA from ovx rats. LHβ-polypeptides represented, under the same conditions, respectively, about 0.81% and 0.44% and α-polypeptides, 1.19% and 1.33%. Further, our results indicate that FSHβ is synthesized as a precursor with a size larger than the authentic apopeptide and that the β-subunits of either LH or FSH, as well as their common subunit α are encoded by distinct mRNAs. 相似文献
15.
L. A. Nesterova B. N. Manukhin 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2010,4(2):206-211
The effects of activation and inhibition of muscarinic cholinoceptors by carbachol and atropine on the binding of specific
nonselective α1-antagonist [3H]prazosine in synaptosomal membranes of rat cerebral cortex have been studied. It has been shown that the ligand-receptor
interaction of α1-adrenoceptors corresponds to the model suggesting the presence of a single receptor pool and the binding of two ligand molecules
to the receptor. The parameters of [3H]prazosine binding to α1-adrenoceptors were as follows: K
d = 1.56 ± 0.17 nM, B
max = 30.25 ± 1.78 fmol/mg protein, n = 2. Upon inhibition of muscarinic cholinoceptors by atropine or their activation by carbachol, the radiolabelled ligand
is bound to α1-adrenoceptors according to the same model but at n = 1. In the presence of atropine, the sensitivity of α1-adrenoceptors to [3H]prazosine decreases more than twofold (K
d = 3.52 ± 0.36 nM) and the concentration of the active receptors is 36% lower (B
max = 19.45 ± 1.46 fmol/mg protein). Carbachol does not reduce the affinity of adrenoceptors to the ligand, while the concentration
of active receptors decreases like in the case of atropine. It is supposed that α1-adrenoceptors in the membranes of rat cerebral cortex exist as dimers. The modulating effects of atropine and carbachol on
the binding of specific antagonist by α1-adrenoceptors are exhibited as changes in the general character of binding (monomerization of α1-adrenoceptors) and as inhibitory effect on the [3H]prazosine binding parameters. 相似文献
16.
Hong Cao Jing Chen Eugene W. Krueger Mark A. McNiven 《Molecular and cellular biology》2010,30(3):781-792
The mechanisms by which epithelial cells regulate clathrin-mediated endocytosis (CME) of transferrin are poorly defined and generally viewed as a constitutive process that occurs continuously without regulatory constraints. In this study, we demonstrate for the first time that endocytosis of the transferrin receptor is a regulated process that requires activated Src kinase and, subsequently, phosphorylation of two important components of the endocytic machinery, namely, the large GTPase dynamin 2 (Dyn2) and its associated actin-binding protein, cortactin (Cort). To our knowledge these findings are among the first to implicate an Src-mediated endocytic cascade in what was previously presumed to be a nonregulated internalization process.Iron is an essential element for all mammalian organisms that plays essential roles in hemoglobin and myoglobin production (23). Altered iron transport can lead to disease states such as hemochromatosis (23), anemia (5, 23), and neuronal disorders (23). The transferrin receptor (TfR) is an important component of iron regulation in cells. There are two distinct TfRs in humans sharing 45% identity that are homodimeric and bind iron-associated transferrin (Tf) at markedly different affinities (26). While significant attention has been paid toward understanding the basic endocytic machinery that supports the efficient internalization and recycling of the TfR1 and its associated iron-bound ligand, it has been assumed that this transport process is constitutive in nature. This is in direct contrast to the highly regulated internalization pathway used by members of the receptor tyrosine kinase family (RTKs) and the family of G-coupled protein receptors (GPCRs) that utilize phosphorylation and/or ubiquination as signaling modules to regulate internalization.To test if TfR1 internalization might be regulated in a similar fashion, we focused on two essential components of the endocytic machinery: the large GTPase Dyn2 that mediates endocytic vesicle scission (35) and Cort that binds to Dyn2 via an SH3-PRD interaction and has been postulated to regulate actin dynamics to facilitate vesicle invagination and release (36, 40). Both Dyn2 and Cort have shown to be phosphorylated in vivo and in vitro by a variety of kinases (51, 58). Dyn1 interacts with (17) and is phosphorylated by Src in neuronal cells and in other excitable cells in response to activation of GPCRs and epidermal growth factor (EGF) (1, 2). While the Src phosphorylation motifs of dynamin are conserved in the epithelial expressed form of Dyn2, it is unclear if Dyn2 is phosphorylated in response to ligands that induce clathrin-based endocytosis.Cort possesses a series of C-terminal tyrosines that are heavily Src-phosphorylated and implicated in regulating actin remodeling during cell motility (20). In this study, we demonstrate that addition of Tf to cultured epithelial cells results in an internalization of the TfR1 mediated by a Src kinase-dependent phosphoactivation of the Dyn2-Cort-based endocytic machinery. In support of these findings, dominant negative forms of c-Src kinase, when expressed in a hepatocyte-derived cell line (Clone 9), attenuate Tf internalization. Remarkably, cells exposed to Tf showed a 3- to 4-fold increase in Dyn2 and Cort phosphorylation compared to that shown by untreated cells, an increase exceeding that observed in cells treated with EGF. These findings provide new insights into the regulation of what was thought to be a constitutive endocytic process. 相似文献
17.
S. I. Bakholdina E. V. Sidorin V. A. Khomenko M. P. Isaeva N. Yu. Kim E. P. Bystritskaya E. A. Pimenova T. F. Solov’eva 《Russian Journal of Bioorganic Chemistry》2018,44(2):178-187
The effect of the concentration of an inducer (IPTG) and the time of induction at 37°С on the heterologous synthesis of the mature membrane protein phospholipase А1 (PldA) from Yersinia pseudotuberculosis in the form of inclusion bodies (IBs) and on the physicochemical and structural characteristics of IBs has been studied. The sizes, shape, stability (solubility in urea and detergents, resistance against proteolysis), the secondary structure of the protein of IBs, and the presence of amyloid structures have been determined by electron microscopy, dynamic light scattering, and optical spectroscopy. It was found that IBs have a shape close to spherical and a rough surface and are cleaved by proteinase K. The protein contained in IBs has an ordered secondary structure with a high content of β-structure. As the inducer concentration and the time of expression increase, the conformation of the recombinant protein in IBs undergoes changes, as indicated by an increase in the stability of IBs and a decrease in the enzymatic activity of the protein. When IBs are dissolved in 0.06% SDS and 5 M urea, the recombinant protein retains the secondary structure in a partially modified form, and the addition of a zwitterionic detergent at a micellar concentration does not transform the protein conformation into the native one. 相似文献
18.
Martin M Reyes SD Hiltner TD Givogri MI Tyszka JM Fisher R Campagnoni AT Fraser SE Jacobs RE Readhead C 《Neurochemical research》2007,32(2):159-165
Our objective was to follow the course of a dysmyelinating disease followed by partial recovery in transgenic mice using non-invasive
high-resolution (117 × 117 × 70 μm) magnetic resonance (μMRI) and evoked potential of the visual system (VEP) techniques.
We used JOE (for J37 golli overexpressing) transgenic mice engineered to overexpress golli J37, a product of the Golli–mbp gene complex, specifically in oligodendrocytes. Individual JOE transgenics and their unaffected siblings were followed from
21 until 75-days-old using non-invasive in vivo VEPs and 3D T2-weighted μMRI on an 11.7 T scanner, performing what we believe
is the first longitudinal study of its kind. The μMRI data indicated clear, global hypomyelination during the period of peak
myelination (21–42 days), which was partially corrected at later ages (>60 days) in the JOE mice compared to controls. These
μMRI data correlated well with [Campagnoni AT (1995) “Molecular biology of myelination”. In: Ransom B, Kettenmann H (eds)
Neuroglia—a Treatise. Oxford University Press, London, pp 555–570] myelin staining, [Campagnoni AT, Macklin WB (1988) Cellular
and molecular aspects of myelin protein gene-expression. Mol Neurobiol 2:41–89] a transient intention tremor during the peak
period of myelination, which abated at later ages, and [Lees MB, Brostoff SW (1984) Proteins in myelin. In: Morell (ed) Myelin.
Plenum Press, New York and London, pp 197–224] VEPs which all indicated a significant delay of CNS myelin development and
persistent hypomyelination in JOE mice. Overall these non-invasive techniques are capable of spatially resolving the increase
in myelination in the normally developing and developmentally delayed mouse brain.
Special Issue in honor of Anthony and Celia Campagnoni 相似文献
19.
Yu Wang Douglas C. Goodwin 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(1):362-371
Catalase–peroxidases (KatGs) have two peroxidase-like domains. The N-terminal domain contains the heme-dependent, bifunctional active site. Though the C-terminal domain lacks the ability to bind heme or directly catalyze any reaction, it has been proposed to serve as a platform to direct the folding of the N-terminal domain. Toward such a purpose, its I′-helix is highly conserved and appears at the interface between the two domains. Single and multiple substitution variants targeting highly conserved residues of the I′-helix were generated for intact KatG as well as the stand-alone C-terminal domain (KatGC). Single variants of intact KatG produced only subtle variations in spectroscopic and catalytic properties of the enzyme. However, the double and quadruple variants showed substantial increases in hexa-coordinate low-spin heme and diminished enzyme activity, similar to that observed for the N-terminal domain on its own (KatGN). The analogous variants of KatGC showed a much more profound loss of function as evaluated by their ability to return KatGN to its active conformation. All of the single variants showed a substantial decrease in the rate and extent of KatGN reactivation, but with two substitutions, KatGC completely lost its capacity for the reactivation of KatGN. These results suggest that the I′-helix is central to direct structural adjustments in the adjacent N-terminal domain and supports the hypothesis that the C-terminal domain serves as a platform to direct N-terminal domain conformation and bifunctionality. 相似文献
20.
W. B. Cheadle 《BMJ (Clinical research ed.)》1888,2(1456):1145-1148