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1.
N J Beekman W M Schaaper J P Langeveld R S Boshuizen R H Meloen 《The journal of peptide research》2001,58(3):237-245
The influence of the nature of the bond between a peptide and a (lipidic) carrier molecule on the immunogenicity of that construct was investigated. As types of bonds a thioester-, a disulfide-, an amide- and a thioether bond were investigated. As carrier molecules a peptide, an N-palmitoylated peptide or a C(16)-hydrocarbon chain were used. The biostability of the bond between peptide and carrier molecule is thioether > amide > disulfide > thioester. However, the immunogenic potency of the constructs used was found to be thioester > disulfide > amide > thioether. In conclusion, a construct with a bond between peptide and (lipidic) carrier molecule that is more susceptible to biological degradation is more immunogenic when used in a peptide-based vaccine than a bond that is less susceptible to biological degradation. 相似文献
2.
High level of divergence of male-reproductive-tract proteins, between Drosophila melanogaster and its sibling species, D. simulans 总被引:1,自引:0,他引:1
We compared male-reproductive-tract polypeptides of Drosophila melanogaster
and D. simulans by using two-dimensional gel electrophoresis. Approximately
64% of male-reproductive-tract polypeptides were identical between two
randomly chosen isofemale lines from these two species, compared with 83%
identity for third-instar imaginal wing-disc polypeptides. Qualitatively
similar differences were found between reproductive tracts and imaginal
discs when D. sechellia was compared with D. melanogaster and with D.
simulans. When genic polymorphism was taken into account, approximately 10%
of male- reproductive-tract polypeptides were apparently fixed for
different alleles between D. melanogaster and D. simulans; this proportion
is the same as that found for soluble enzymes by one-dimensional gel
electrophoresis. Strikingly, approximately 20% of male-reproductive- tract
polypeptides of either D. melanogaster or D. simulans had no detectable
homologue in the other species. We propose that proteins of the Drosophila
male reproductive tract may have diverged more extensively between species
than have other types of proteins and that much of this divergence may
involve large changes in levels of polypeptide expression.
相似文献
3.
RS Fisher 《The Journal of general physiology》1977,69(5):571-604
When the outer surface of short-circuited frog skin was penetrated with microelectrodes, stable negative potentials that averaged near -100 mV were recorded consistently, confirming the results of Nagel (W. Nagel. 1975. Abstracts of the 5th International Biophysics Congress, Copenhagen. P-147.). The appearance of these stable potentials, V(O), concurrent with the observations that (a) a high resistance outer barrier R(O) accounting for approximately 75 percent or more of the transcellular resistance of control skins had been penetrated and that (b) 10(-5) M amiloride and reduced [Na] outside caused the values of V(O) to increase towards means value near -130 mV while the values of percent R(O) increased to more than 90 percent. It was of relationships were the same as the values of E(1) observed in studies of the current-voltage relationships were the same as the values of E’(1) defined as the values of voltage at the inner barrier when the V(O) of the outer barrier was reduced to zero by voltage clamping of the skins. Accordingly, these data are interpreted to mean that the values of E(1), approximately 130 mV, represent the E(Na) of the sodium pump at the inner barrier. 2,4-DNP was observed to decrease the values of transepithelial voltage less than E(1) the V(O) was negative. These data can be interpreted with a simple electrical equivalent circuit of the active sodium transport pathway of the frog skin that includes the idea that the outer membrane behaves as an electrical rectifier for ion transport. 相似文献
4.
EFFECTS OF HUMAN ACTIVITIES ON STRUCTURE AND COMPOSITION OF WOODY SPECIES OF THE NOKREK BIOSPHERE RESERVE OF MEGHALAYA,NORTHEAST INDIA 下载免费PDF全文
Aims Our study was conducted in the Nokrek Biosphere Reserve (NBR) in the Garo hills districts of Meghalaya, Northeast India. Our aim was to assess the effects of human activities on plant diversity,population structure and regeneration.Methods We selected a representative 1.2 hm2 stand in both the core and buffer zones of NBR. Structure and composition were determined by randomly sampling square quadrats, population structure was assessed by determining age structure, and regeneration was assessed by measuring densities of seedling, sapling and adult trees.Important findings More woody species were recorded from the core zone than the buffer zone (87 vs. 81 species), and there were a large number of tropical, temperate, and Sino-Himalayan, Burma-Malaysian and Malayan elements, primitive families and primitive genera. The trees were distributed in three distinct strata,canopy, subcanopy and sapling. Subcanopy and sapling layers had the highest species richness (81% -88% ). Lauraceae and Euphorbiaceae were the dominant families in terms of the number of species, and a large number of families were represented by single species. Most woody species (57 % - 79 % ) were contagiously distributed and had low frequency ( < 20% ). Although stand density was high in the buffer zone, its basal area was low compared to the stand in the core zone. Low similarity and high β-diversity indicate marked differences in species composition of the stands. Shannon diversity index was high in both the stands, while Simpson dominance index was low. The diameter-class distribution for dominant species revealed that the most had a large number of young individuals in their populations. Preponderance of tree seedlings, followed by a steep decline in population density of saplings and adult trees, indicated that the seedling to sapling stage was the most critical in the life cycle of the tree populations. Most species (42 % - 48 % ) had no regeneration,25 % - 35 % had good/fair regeneration, and the rest had poor regeneration or reoccurred as immigrants. 相似文献
5.
6.
George O. Agogo Hilko van der Voet Pieter van’t Veer Pietro Ferrari Max Leenders David C. Muller Emilio Sánchez-Cantalejo Christina Bamia Tonje Braaten Sven Knüppel Ingegerd Johansson Fred A. van Eeuwijk Hendriek Boshuizen 《PloS one》2014,9(11)
In epidemiologic studies, measurement error in dietary variables often attenuates association between dietary intake and disease occurrence. To adjust for the attenuation caused by error in dietary intake, regression calibration is commonly used. To apply regression calibration, unbiased reference measurements are required. Short-term reference measurements for foods that are not consumed daily contain excess zeroes that pose challenges in the calibration model. We adapted two-part regression calibration model, initially developed for multiple replicates of reference measurements per individual to a single-replicate setting. We showed how to handle excess zero reference measurements by two-step modeling approach, how to explore heteroscedasticity in the consumed amount with variance-mean graph, how to explore nonlinearity with the generalized additive modeling (GAM) and the empirical logit approaches, and how to select covariates in the calibration model. The performance of two-part calibration model was compared with the one-part counterpart. We used vegetable intake and mortality data from European Prospective Investigation on Cancer and Nutrition (EPIC) study. In the EPIC, reference measurements were taken with 24-hour recalls. For each of the three vegetable subgroups assessed separately, correcting for error with an appropriately specified two-part calibration model resulted in about three fold increase in the strength of association with all-cause mortality, as measured by the log hazard ratio. Further found is that the standard way of including covariates in the calibration model can lead to over fitting the two-part calibration model. Moreover, the extent of adjusting for error is influenced by the number and forms of covariates in the calibration model. For episodically consumed foods, we advise researchers to pay special attention to response distribution, nonlinearity, and covariate inclusion in specifying the calibration model. 相似文献
7.
Patients with systemic autoimmune diseases usually produce high levels of antibodies to self-antigens (autoantigens). The
repertoire of common autoantigens is remarkably limited, yet no readily understandable shared thread links these apparently
diverse proteins. Using computer prediction algorithms, we have found that most nuclear systemic autoantigens are predicted
to contain long regions of extreme structural disorder. Such disordered regions would generally make poor B cell epitopes
and are predicted to be under-represented as potential T cell epitopes. Consideration of the potential role of protein disorder
may give novel insights into the possible role of molecular mimicry in the pathogenesis of autoimmunity. The recognition of
extreme autoantigen protein disorder has led us to an explicit model of epitope spreading that explains many of the paradoxical
aspects of autoimmunity – in particular, the difficulty in identifying autoantigen-specific helper T cells that might collaborate
with the B cells activated in systemic autoimmunity. The model also explains the experimentally observed breakdown of major
histocompatibility complex (MHC) class specificity in peptides associated with the MHC II proteins of activated autoimmune
B cells, and sheds light on the selection of particular T cell epitopes in autoimmunity. Finally, the model helps to rationalize
the relative rarity of clinically significant autoimmunity despite the prevalence of low specificity/low avidity autoantibodies
in normal individuals. 相似文献
8.
Paula H Suss Luiz Guilherme A Capriglione Fabiane Barchiki Lye Miyague Danielle Jackowski Letícia Fracaro Andressa V Schittini Alexandra C Senegaglia Carmen LK Rebelatto Márcia Olandoski Alejandro Correa Paulo RS Brofman 《Experimental biology and medicine (Maywood, N.J.)》2015,240(7):969-978
The development of new therapeutic strategies is necessary to reduce the worldwide social and economic impact of cardiovascular disease, which produces high rates of morbidity and mortality. A therapeutic option that has emerged in the last decade is cell therapy. The aim of this study was to compare the effect of transplanting human umbilical cord-derived stromal cells (UCSCs), human umbilical cord blood-derived endothelial cells (UCBECs) or a combination of these two cell types for the treatment of ischemic cardiomyopathy (IC) in a Wistar rat model. IC was induced by left coronary artery ligation, and baseline echocardiography was performed seven days later. Animals with a left ventricular ejection fraction (LVEF) of ≤40% were selected for the study. On the ninth day after IC was induced, the animals were randomized into the following experimental groups: UCSCs, UCBECs, UCSCs plus UCBECs, or vehicle (control). Thirty days after treatment, an echocardiographic analysis was performed, followed by euthanasia. The animals in all of the cell therapy groups, regardless of the cell type transplanted, had less collagen deposition in their heart tissue and demonstrated a significant improvement in myocardial function after IC. Furthermore, there was a trend of increasing numbers of blood vessels in the infarcted area. The median value of LVEF increased by 7.19% to 11.77%, whereas the control group decreased by 0.24%. These results suggest that UCSCs and UCBECs are promising cells for cellular cardiomyoplasty and can be an effective therapy for improving cardiac function following IC. 相似文献
9.
Rotavirus enterotoxin NSP4 binds to the extracellular matrix proteins laminin-beta3 and fibronectin 总被引:1,自引:0,他引:1 下载免费PDF全文
Boshuizen JA Rossen JW Sitaram CK Kimenai FF Simons-Oosterhuis Y Laffeber C Büller HA Einerhand AW 《Journal of virology》2004,78(18):10045-10053
Rotavirus is the most important cause of viral gastroenteritis and dehydrating diarrhea in young children. Rotavirus nonstructural protein 4 (NSP4) is an enterotoxin that was identified as an important agent in symptomatic rotavirus infection. To identify cellular proteins that interact with NSP4, a two-hybrid technique with Saccharomyces cerevisiae was used. NSP4 cDNA, derived from the human rotavirus strain Wa, was cloned into the yeast shuttle vector pGBKT7. An intestinal cDNA library derived from Caco-2 cells cloned into the yeast shuttle vector pGAD10 was screened for proteins that interact with NSP4. Protein interactions were confirmed in vivo by coimmunoprecipitation and immunohistochemical colocalization. After two-hybrid library screening, we repeatedly isolated cDNAs encoding the extracellular matrix (ECM) protein laminin-beta3 (amino acids [aa] 274 to 878) and a cDNA encoding the ECM protein fibronectin (aa 1755 to 1884). Using deletion mutants of NSP4, we mapped the region of interaction with the ECM proteins between aa 87 and 145. Deletion analysis of laminin-beta3 indicated that the region comprising aa 726 to 875 of laminin-beta3 interacts with NSP4. Interaction of NSP4 with either laminin-beta3 or fibronectin was confirmed by coimmunoprecipitation. NSP4 was present in infected enterocytes and in the basement membrane (BM) of infected neonatal mice and colocalized with laminin-beta3, indicating a physiological interaction. In conclusion, two-hybrid screening with NSP4 yielded two potential target proteins, laminin-beta3 and fibronectin, interacting with the enterotoxin NSP4. The release of NSP4 from the basal side of infected epithelial cells and the subsequent binding to ECM proteins localized at the BM may signify a new mechanism by which rotavirus disease is established. 相似文献
10.
Ronald S. Boshuizen Veronica Schulz Michela Morbin Giulia Mazzoleni Rob H. Meloen Johannes P. M. Langedijk 《The Journal of biological chemistry》2009,284(19):12809-12820
Fibrils play an important role in the pathogenesis of amyloidosis; however,
the underlying mechanisms of the growth process and the structural details of
fibrils are poorly understood. Crucial in the fibril formation of prion
proteins is the stacking of PrP monomers. We previously proposed that the
structure of the prion protein fibril may be similar as a parallel left-handed
β-helix. The β-helix is composed of spiraling rungs of parallel
β-strands, and in the PrP model residues 105–143 of each PrP
monomer can contribute two β-helical rungs to the growing fibril. Here we
report data to support this model. We show that two cyclized human PrP
peptides corresponding to residues 105–124 and 125–143, based on
two single rungs of the left-handed β-helical core of the human
PrPSc fibril, show spontaneous cooperative fibril growth in
vitro by heterologous stacking. Because the structural model must have
predictive value, peptides were designed based on the structure rules of the
left-handed β-helical fold that could stack with prion protein peptides
to stimulate or to block fibril growth. The stimulator peptide was designed as
an optimal left-handed β-helical fold that can serve as a template for
fibril growth initiation. The inhibiting peptide was designed to bind to the
exposed rung but frustrate the propagation of the fibril growth. The single
inhibitory peptide hardly shows inhibition, but the combination of the
inhibitory with the stimulatory peptide showed complete inhibition of the
fibril growth of peptide huPrP-(106–126). Moreover, the unique strategy
based on stimulatory and inhibitory peptides seems a powerful new approach to
study amyloidogenic fibril structures in general and could prove useful for
the development of therapeutics.Transmissible spongiform encephalopathies are neurodegenerative disorders
in a wide range of mammalian species, including Creutzfeldt-Jacob disease in
man, scrapie in sheep, and bovine spongiform encephalopathy in cattle. The
deposition of aggregated prion protein fibrils on and in neurons is regarded
to be the source of these neurodegenerative diseases and is frequently
associated with occurrence of Congo red positivity
(1–3).
The fibrils are formed by the conformational change of the prion protein
(PrPc)2
into the scrapie form (PrPSc). The misfolded conformer of the prion
protein (PrPSc) is considered as the causative agent in these
diseases according to the protein-only hypothesis
(4). Studies have shown the
toxicity of fibrils of the full-length recombinant mammalian prion protein as
well as soluble β-rich oligomers to cultured cells and primary neurons
(5).It is still unknown how much of the whole PrPSc molecule is
involved in the fibril growth. It is shown that the N-terminal part of PrP,
specifically residues 112–141, can go through conformational changes
involving β-strand formation, which subsequently triggers fibril growth
(6–8),
and solid state NMR studies showed that residues 112–141 are part of the
highly ordered core of huPrP-(23–144)
(9). It was previously shown
that peptides based on the 89–143 region of the human PrP protein can
form fibrils rich in β-sheet structure which are biologically active in
transgenic mice (10). Within
this region it is the huPrP-(106–126) peptide that is the smallest known
region of PrP that forms fibrils that are toxic and resemble the physiological
properties of PrPSc
(11–16).
The formation of PrPSc is considered to be a two-step event; first,
there is the binding between PrPc and PrPSc and
subsequently the conformational conversion from PrPc into
PrPSc occurs. Mutation studies in a prion-infected neuroblastoma
cell line showed that in mouse PrP the regions 101–110 and 136–158
are crucial for the binding and conversion events, respectively
(17). Because prevention of
fibril growth is the prime therapeutic target, detailed structural knowledge
of the fibril is essential for understanding the mechanism of fibril growth.
However, structural analysis of amyloid fibrils is hampered by insolubility,
isomorphism, and aggregation. X-ray diffraction of several amyloid fibrils
revealed a so-called cross-β diffraction pattern which indicates that the
fibrils contain β-strands perpendicular to the fibril axis and hydrogen
bonds in parallel (18,
19). Thus, for fibril growth
the β-strands have to stack on top of each other. Several structures have
been suggested to explain the structure of the stacked β-strands;
e.g. a parallel in register organization of stacked β hairpins
(24) or the comparable dry
steric zipper structure (25).
Previously, we and other groups suggested that the β-sheet structures in
the PrPSc fibril may be similar to the topologically most simple
class of β-sheets; that is, the parallel left-handed β-helix
(Fig. 1A)
(6,
20,
21). The left-handed β
helix is formed by triangular progressive coils (rungs) of 18–20
residues. Each rung is formed by three hexapeptide motifs, which results in an
approximate 3-fold symmetry. Backbone-backbone hydrogen bonding and stacking
of the side chains in adjacent rungs contribute to the folding of
β-helical rungs. We suggested that each PrPSc monomer
contributes two left-handed β-helical rungs to the fibril, comprising
residues 105–124 and 125–143
(Fig. 1A). This
two-rung structural model was recently confirmed for amyloid fibrils of the
HET-s prion by NMR analysis
(22). In contrast to fibrils
which are composed of homologous stacks of identical peptides, e.g.
the Aβ peptide (23), the
PrPSc fibril is more complex because it is composed of heterologous
stacks of at least two peptides. For homologous stacking of two identical
peptides, the complementarity issue is relatively simple because the identical
side chains are in register (e.g. Ile-Ile, Val-Val stacking, and Asn
ladders). However, in the case of heterologous stacking, the side chains of
the additional heterologous peptide needs to be complementary with the other
peptide to allow fibril growth.Open in a separate windowFIGURE 1.A, theoretical model of the fibrillogenic core of
PrPSc. In the PrPSc model based on the left-handed
β-helix structure, each PrPSc monomer contributes two stacked
rungs to the fibril (different color for each monomer). The protofibril is
formed by consecutive stacking of the two windings. The stack of two rungs
provides enough elevation to accommodate the remaining part (residues ∼
146–253) of the PrPSc molecule
(20). B, the
left-handed β-helix structure of LpxA-based on x-ray crystallography. In
the left-handed β-helix structure of LpxA (PDB code 1LXA) rungs 6 and 7
are indicated (red) that were used for the heterologous stacking
studies. Linear and cyclized peptides based on rung 6 and rung 7 were modified
to satisfy the ideal left-handed β-helix motif (see “LpxA
Peptides” under “Results”) and tested for their intrinsic
and cooperative fibrillogenicity. C, left-handed β-helical rung
based on rung 6 of LpxA. The rung is formed by three hexapeptide motifs, which
results in an approximate 3-fold symmetry. A left-handed β-helical rung
can be cyclized by a disulfide bridge after the introduction of a cysteine at
position 2 of the first hexapeptide and position 1 of the fourth hexapeptide
(according to the numbering used for the hexapeptide repeats in the
left-handed β-helix).To investigate whether the suggested rungs 105–123 and 125–143
from human PrP could be complementary
(20), we studied the
homologous stacking and the heterologous stacking of linear and cyclized prion
protein peptides comprising the huPrP-(105–143) region
(KTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGS). Qualitative and semiquantitative
analysis were done by electron microscopy and Congo red staining. The
quantification of the fibril formation was assessed by thioflavin S staining,
in which the addition of polyanions (e.g. heparin) enhance the
β-sheet formation of peptides comprising the 82–143 region of PrP
and improve the reproducibility of the fibril growth
(24). This study provides
first evidence of heterologous stacking by two isolated putative β-strand
layers (or rungs) of the human prion protein with fibril formation as a
result. The left-handed β-helix structure provided insight for the
“stack-and-stop” approach. With this approach a mix of a
stimulatory peptide and an inhibitory peptide could completely block fibril
formation. The stimulatory peptide was based on the 125–143 region that
was optimized to serve as a folding template for the consecutive stacking of
the 106–126 peptide. This cooperative fibril growth was completely
inhibited by the inhibitory peptide based on peptides 106–126 with
strategic d-amino acid and/or proline substitutions. The findings
in this study support models in which the sequential strands in a fibril must
somehow spiral up- or downward along the fibril axis, e.g. like the
hypothetical left-handed β-helical structure of PrPSc fibrils
(20). Furthermore, it allows
the development of well defined small protein modules which can be used for
structure studies of the 82–143 domain of PrPSc and the
development of therapeutics. 相似文献